Stable minihairpin structures forming at minisatellite DNA isolated from yellow fin sea bream Acanthopagrus latus

The lengths of simple repeat sequences are generally unstable or polymorphic (highly variable with respect to the numbers of tandem repeats). Previously we have isolated a family of minisatellite DNA (GenBank accession AF422186) that appears specifically and abundantly in the genome of yellow fin sea bream Acanthopagrus latus but not in closely-related red sea bream Pagrus major, and found that the numbers of tandem arrays in the homologous loci are polymorphic. This means that the minisatellite sequence has appeared and propagated in A. latus genome after speciation. In order to understand what makes the minisatellite widespread within the A. latus genome and what causes the polymorphic nature of the number of tandem repeats, the structural features of single-stranded polynucleotides were analyzed by electrophoresis, chemical modification, circular dichroism (CD), differential scanning calorimetry (DSC) and electron microscopy. The results suggest that a portion of the repeat unit forms a stable minihairpin structure, and it can cause polymerase pausing within the minisatellite DNA.     

Comparison of UV-B tolerance between wild and hatchery-reared juveniles of red sea bream (Pagrus major) and black sea bream (Acanthopagrus schlegeli)

The amount of ultraviolet (UV)-B radiation reaching the sea surface has increased due to ozone depletion. Several laboratory studies have highlighted the negative impacts of UV radiation on fish using hatchery-reared specimens. However, potential differences in UV tolerance between wild and hatchery-reared fish have been given little consideration. Wild and reared juveniles of red sea bream and black sea bream were exposed to one of four different UV-B radiation levels (1.8; 1.1; 0.4; 0?W/m²) for 4?h. Survival rate was measured every 2?h for a period of 24?h (red sea bream) or 48?h (black sea bream) following exposure. Wild and reared juvenile red sea bream were characterized by similar survival rate, with survival declining to almost 0?% 24?h after exposure at the 1.1 and 1.8?W/m² levels. In black sea bream, wild individuals showed significantly higher survival than reared fish in levels 1.1 and 1.8?W/m². Melanophore density was also measured since melanin absorbs UV radiation. Wild black sea bream showed higher melanophore density compared to reared individuals, while no such difference was observed in red sea bream. We conclude that wild black sea bream juveniles acquire higher UV tolerance partly by increasing melanophore density through exposure to UV radiation. Our results indicate that the predicted impacts of UV radiation on fish populations solely based on experimentation with hatchery-reared specimens may be overestimated for some species.     

Sexuality, sex change and maturation patterns in the yellowfin seabream, Acanthopagrus latus (Teleostei: Sparidae) (Houttuyn, 1782)

Macroscopic and histological studies were carried out to describe the reproductive styles and sex reversal and to follow gonadal changes in captive yellowfin seabream during the second year of life. Four reproductive styles are found in Acanthopagrus latus (Houttuyn, 1782): (i) males and females (gonochorism), (ii) functional males, (iii) transitionals and (iv) functional females. The species is a protandric hermaphrodite and begins life as a functional male with testicular zone undergoing active spermatogenesis, while the ovarian zone is arrested at the primary growth (perinucleolar) phase. Males and females were encountered in virtually all size‐classes. Functional males outnumbered the functional females in all size‐classes in which they were encountered. Sex reversal begins in the transitionals from July to August, after spawning in the functional males, at 14.9–20.2 cm standard length (SL) and, by November, maturation of the ovarian tissue begins. A. latus in cages in Kuwait waters spawns from January to April with a peak in February for males, and a peak in March for females and transitionals. Spawning begins in the 18.3–20.2 cm size‐range fish, peaking in the 20.3–22.2 cm size‐range in both males and females and also in the transitionals, although a few of the latter spawn from 14.9 cm SL. In relation to age, spawning begins at 20 months in males and peaks at 21 months. Females begin to spawn at 21 months with a peak at 22 months, while transitionals generally begin to spawn at 20 months, although a few 14–15‐month‐old sex‐changing individuals were encountered. Temperature, either alone or in combination with other unknown factors, triggers spawning in A. latus.     

Review of the Acanthopagrus latus complex (Perciformes: Sparidae) with descriptions of three new species from the Indo‐West Pacific Ocean

Acanthopagrus latus, long considered a single valid Indo‐West Pacific Ocean species, characterized by having yellow pelvic, anal and caudal fins, is reviewed and separated into A. latus (east Asian shelf) and Acanthopagrus longispinnis (Bengal Bay), and three new species: Acanthopagrus morrisoni sp. nov. (north‐western Australia), Acanthopagrus arabicus sp. nov. [Middle East (except for the Red Sea) to coasts of Iran and Pakistan, and western Indian coast] and Acanthopagrus sheim sp. nov. (The Gulf). Although A. latus as redefined considerably varies in morphology and colouration, it can be recognized as a discrete east Asian endemic, with the following nominal species being junior synonyms: Chrysophrys auripes, Chrysophrys xanthopoda, Chrysophrys rubroptera and Sparus chrysopterus. Chrysophrys novaecaledoniae, known only from the holotype (type locality: Nouméa, New Caledonia), is a questionable junior synonym of A. latus, the lack of subsequent collections suggesting that the type locality is erroneous. Acanthopagrus longispinnis is differentiated from the other species in the complex by consistently having 12 dorsal‐fin spines and a much larger second anal‐fin spine, 21–26% (mean 23%) of standard length (L_S) (v. 14–24%, mean 18–21% in the other four species). Acanthopagrus morrisoni sp. nov. has the entire caudal fin yellow with a wide black posterior margin (persisting in preserved specimens) and consistently 3 ½ scale rows between the fifth dorsal‐fin spine base and the lateral line. Acanthopagrus sheim sp. nov. has the pelvic, anal and lower caudal fins vivid yellow, with two (rarely three) small black blotches on the lower inter‐radial membranes between the spinous and soft dorsal‐fin rays. Acanthopagrus arabicus sp. nov. consistently has 4 ½ scale rows between the fifth dorsal‐fin spine base and the lateral line, whereas A. latus always has black streaks proximally on the inter‐radial membranes between the yellow anal‐fin rays. A neotype and lectotye, respectively, are designated for A. latus and A. longispinnis. The p‐distance (net nucleotide substitutions per site) of partial mitochondrial 16s ribosomal RNA genes (538 bp) among the above species (except A. longispinnis) and three other congeners (Acanthopagrus berda, Acanthopagrus pacificus and Acanthopagrus bifasciatus) strongly indicates that each is a distinct species. A key is provided for the 20 species of Acanthopagrus currently known from the Indo‐West Pacific Ocean.     

Isolation and characterization of eight microsatellite loci from Picnic seabream (Acanthopagrus berda)

Acanthopagrus berda (Sparidae) is abundant in estuaries and inshore waters of the tropical Indo‐Pacific oceans. Because of the drastic decrease in the species’ wild populations, there is an urgent need to understand its population structure. Eight microsatellite loci were isolated from A. berda. An analysis of 50 individuals from Tapeng Lagoon, southwestern Taiwan demonstrated a low degree of diversity. The allelic numbers ranged from nine to 20. The observed heterozygosity (H_O) ranged from 0.28 to 0.64 (mean, 0.45). We also investigated the prospective value of the microsatellite primers to examine whether they were applicable to related taxa.     

Genetic evidence for the mating system and reproductive success of black sea bream (Acanthopagrus schlegelii)

Understanding the mating system and reproductive success of a species provides evidence for sexual selection. We examined the mating system and the reproductive success of captive adult black sea bream (Acanthopagrus schlegelii), using parentage assignment based on two microsatellites multiplex PCR systems, with 91.5% accuracy in a mixed family (29 sires, 25 dams, and 200 offspring). Based on the parentage result, we found that 93.1% of males and 100% of females participated in reproduction. A total of 79% of males and 92% of females mated with multiple partners (only 1 sire and 1 dam were monogamous), indicating that polygynandry best described the genetic mating system of black sea bream. For males, maximizing the reproductive success by multiple mating was accorded with the sexual selection theory while the material benefits hypothesis may contribute to explain the multiple mating for females. For both sexes, there was a significant correlation between mating success and reproductive success and the variance in reproductive success of males was higher than females. Variation in mating success is the greatest determinant to variation in reproductive success when the relationship is strongly positive. The opportunity for sexual selection of males was twice that of females, as well as the higher slope of the Bateman curve in males suggested that the intensity of intrasexual selection of males was higher than females. Thus, male–male competition would lead to the greater variation of mating success for males, which caused greater variation in reproductive success in males. The effective population number of breeders (N_b) was 33, and the N_b/N ratio was 0.61, slightly higher than the general ratio in polygynandrous fish populations which possibly because most individuals mated and had offspring with a low variance. The relatively high N_b contributes to the maintenance of genetic diversity in farmed black sea bream populations.     

Identification and characterization of simple sequence repeat (SSR) markers from Fragaria×ananassa expressed sequences

The availability of expressed sequence data derived from gene discovery programmes enables mining for simple sequence repeats (SSRs), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study reports on the development and characterization of expressed sequence tag (EST)–SSR markers in the cultivated strawberry, Fragaria×ananassa. Fourteen primer pairs were assessed for polymorphism in 13 F.×ananassa genotypes. The markers show reliable amplification and considerable polymorphism, demonstrating the utility of EST–SSRs for genetic analysis of commercial strawberry germplasm.     

Novel polymorphic microsatellite loci for the protogynous hermaphrodite slinger sea bream (Chrysoblephus puniceus, Sparidae)

As a result of the global decline of fish stocks, an increasing number of fish species are becoming targets of heavy exploitation, often concomitantly with a lack of biological knowledge on their structure and demographics. Here we present 11 new polymorphic microsatellite loci, isolated from the slinger sea bream (Chrysoblephus puniceus, Sparidae), a relatively recent target of coastal fisheries in eastern South Africa. Levels of genetic diversity were assessed in 39 individuals collected from the KwaZulu-Natal coast (Park Rynie, South Africa). Observed and expected heterozygosities varied between 0.39 and 0.97 and between 0.53 and 0.96, respectively. One locus (SL35) showed significant heterozygote deficiency and linkage disequilibrium was detected between SL35 and SL1. Importantly, five of these microsatellites cross-amplify in Cheimerius nufar, a sympatric species also subjected to exploitation.     

Identification and characterization of simple sequence repeat markers from Brassica napus expressed sequences

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)‐SSR markers from Brassica napus and their cross‐amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST‐SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.     

Isolation and characterization of Pseudomonas sp. KUMS3 from Asian sea bass (Lates calcarifer) with fin rot

Bacteria were isolated from Asian Sea bass, Lates calcarifer kept in a farm, on the South-east coast of India. During an outbreak of fin rot, the affected fish had hemorrhages at the base of fins, mouth and skin muscles and faded pigments. Pure colonies were isolated on NA and ZMA from internal organs of the fish and the bacterial morphology was identified as gram-negative rod-shaped bacteria. Based on different biochemical tests and sequence of 16S rDNA, the causative bacteria were identified as Pseudomonas sp. KUMS3. Bacterial cells were isolated from liver and kidney of all artificially infected moribund fish and confirmed as Pseudomonas sp. KUMS3 by morphological and biochemical characteristics. During the experimental infection, the first incidence of dead fish was observed on 2nd day after exposure to Pseudomonas sp. KUMS3 and no fish died after 12 days post exposure and the cumulative percent of mortality was 70. Histological lesions were observed in the spleen, liver and kidney of the infected fish. Pseudomonas sp. KUMS3 could be considered as an opportunistic pathogen, which can survive on the fish surface or in water or in the gut and may cause disease when unfavorable conditions develop.     

Characterization of eight microsatellite markers in the white sea bream, Diplodus sargus (Teleostei, Sparidae)

The white sea bream, Diplodus sargus (Teleostei, Sparidae), is a species with a high commercial importance in Mediterranean aquaculture. There is currently little information available about the genetic characteristics of cultured populations. In this survey, we have developed eight polymorphic microsatellites for the white sea bream using an enriched genome library protocol. All of them were polymorphic in the 67 individuals tested, 32 of which were wild specimens, and 35 were individuals from a captive F(1) broodstock. These markers can potentially be useful tools for use in population genetic studies.     

The relationship between the position of the retinal area centralis and feeding behaviour in juvenile black bream Acanthopagrus butcheri (Sparidae: Teleostei)

The topography of the neurons in the retinal ganglion cell layer of juvenile black bream Acanthopagrus butcheri changes during development. The region of high cell density the area centralis (AC), relocates from a temporal (central) to a dorsal (peripheral) position within the dorso-temporal retinal quadrant. To ascertain whether the differences in the position of the AC during development are related to feeding behaviour, we monitored fishes that were given a choice of food. A range of feeding behaviour patterns was recorded in individual fishes. The smallest fishes (8-15 mm standard length (SL)) took live food from the water column. Following weaning onto pellets, fishes exhibited a preference for taking food from either the substrate or the surface (but not both). When greater than 20 mm SL, a number of individuals then divided their time between surface and substrate feeding before all fishes became exclusive benthic feeders at a stage between 50 and 80 mm SL. Three individual fishes, for which behaviour patterns were categorized, were killed and the topography of the retinal ganglion cell layer analysed. A range of positions for the AC was found with the smallest fish (12 mm SL) possessing a region of high cell density in the temporal retina. In a larger fish (70 mm SL), feeding from both the substrate and the surface, the AC was found in an intermediate dorso-temporal position. The AC of a fish (51 mm SL) preferentially taking food from the substrate was located in a dorsal position.     

Isolation and characterization of nine polymorphic microsatellite markers in the two‐banded sea bream (Diplodus vulgaris) and cross‐species amplification in the white sea bream (Diplodus sargus) and the saddled bream (Oblada melanura)

We have developed nine new microsatellite markers for the two‐banded sea bream (Diplodus vulgaris) from an enriched genome library protocol. All these loci are polymorphic, with mean allelic diversity of 13 (range 5 –21), and expected and observed heterozygosities from 0.641 to 0.932 and 0.428 to 0.914, respectively. Cross‐species tests in two close‐related species of the genus Diplodus (D. sargus and O. melanura) revealed successful amplifications at 8 out of 9 loci, with mean allele number of 4.75 (range 2–8) and 5.50 (range 3 –10), respectively. These results are consistent with the close phylogenetic relationships between the three species, indicating this set of primers might prove useful for studying the levels of genetic diversity and population differentiation in these three species and in other phylogenetically close species of the genus Diplodus and Sparus.     

Fine structure of spermatozoa in the gilthead sea bream (Sparus aurata Linnaeus, 1758) (Perciformes, Sparidae)

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the sparid fish Sparus aurata L. The mature spermatozoon of gilthead sea bream belongs, like that of the other sparid fish, to a "type I" as defined by Mattei (1970). It has a spherical head which lacks an acrosome, a short, irregularly-shaped midpiece and a long cylindrical tail. The nucleus reveals a deep invagination (nuclear fossa) in which the centriolar complex is located. The two centrioles are approximately perpendicular to each other and show a conventional "9+0" pattern. The proximal centriole is associated with a cross-striated cylindrical body lying inside a peculiar satellite nuclear notch which appears as a narrow invagination of the nuclear fossa. The distal centriole is attached to the nuclear envelope by means of a lateral plate and radial fibres made of an electron-dense material. The short midpiece houses one mitochondrion. The flagellum is inserted perpendicularly into the base of the nucleus and contains the conventional 9+2 axoneme.     

Identification and characterization of simple sequence repeat (SSR) markers derived in silico from Brassica oleracea genome shotgun sequences

The availability of sequence data derived from shotgun sequencing programs enables mining for simple sequence repeats (SSRs), providing useful genetic markers for crop improvement. This study presents the development and characterization of 40 SSR markers from Brassica oleracea shotgun sequence and their cross‐amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of SSRs for genetic analysis of commercial Brassica germplasm.     

Morphometric characterization of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa using computer-assisted spermatozoa analysis (ASMA)

As part of a larger study on sperm quality and cryopreservation methods, the present study characterized the head morphometry of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa, using both scanning electron microscopy (SEM) and computer‐assisted morphology analysis (ASMA). The latter method has been used rarely in fish and this is its first application on sharpsnout sea bream and gilthead sea bream spermatozoa. Results obtained using SEM are expensive and time‐consuming, while ASMA provides a faster and automated evaluation of morphometric parameters of spermatozoa head. For sharpsnout sea bream spermatozoa, similar head measurement values were obtained using both ASMA and SEM, having a mean ± standard error length of 2.57 ± 0.01 μm vs 2.54 ± 0.02 μm, width of 2.22 ± 0.02 μm vs 2.26 ± 0.04 μm, surface area of 4.44 ± 0.02 μm² vs 4.50 ± 0.04 μm² and perimeter of 7.70 ± 0.02 μm vs 7.73 ± 0.04 μm using ASMA and SEM, respectively. Although gilthead sea bream spermatozoa were found to be smaller than those of sharpsnout sea bream, spermatozoal head morphometry parameters were also found to be similar regardless of evaluation method, having a mean head length of 1.97 ± 0.01 μm vs 1.94 ± 0.02 μm, head width of 1.80 ± 0.01 μm vs 1.78 ± 0.02 μm, surface area of 3.16 ± 0.03 μm² vs 3.18 ± 0.06 μm² and perimeter of 6.52 ± 0.04 μm vs 6.56 ± 0.08 μm using ASMA and SEM, respectively. The results demonstrate that ASMA can be considered as a reliable technique for spermatozoal morphology analysis, and can be a useful tool for studies on fish spermatozoa, providing quick and objective results.     

Cloning and sequencing of the cDNA encoding the pituitary gonadotropin II β-subunit of yellowfin porgy (Acanthopagrus latus)

The complementary DNA (cDNA) encoding the gonadotropic hormone (GTH) II pre-β-subunit of yellowfin porgy ( Acanthopagrus latus ) was isolated from a pituitary gland cDNA phage library. The cDNA insert, 598 base pairs (bp), contained a 411 bp open reading frame with 35 bp and 152 bp flanking regions at the 5'- and 3'-ends, respectively. The deduced amino acid sequence revealed a putative signal peptide of 24 amino acid residues and a 113 amino acid mature β-subunit of GTH polypeptide. This pre-β-subunit polypeptide of the yellowfin porgy GTH II showed 86% sequence identity with that of bonito GTH II-β 73% with killifish GTH II-β, 63% with African catfish GTH II-β 61% with pike eel GTH-β, 60% with silver carp GTH-β, 59% with chum salmon GTH 11-β, 58% with common carp GTH-β 57% with chinook Pacific salmon GTH-β, and 53% with European eel GTH II-β.     

Age,growth and reproduction of the black sea bream Spondyliosoma cantharus (Linnaeus, ) (Sparidae) in the Gulf of Annaba (Algeria)

The objective of this study was to determine the basic population‐specific parameters necessary for fish stock assessment in the Gulf of Annaba and to compare these with data from other Mediterranean regions. Black sea bream Spondyliosoma cantharus (Linnaeus, 1758) (N = 501) were collected monthly from January to December 2008 along the Algerian eastern coasts. More than 22 fish were collected each month and ranged in size from 13.4 to 40 cm total length, weighing from 36 to 1080 g eviscerated weight. Biological sampling included weighing and measuring the fish, gonad weighing, sex and maturity stage determination, and age estimation through otolith readings. Validity of the otolith readings for estimating age and growth was supported using the back‐calculation method. Estimated parameters of the von Bertalanffy model are: L_∞ = 33.54 cm, W_∞ = 633.46 g, k = 0.52 year^?1 and t_o = ?0.04 year. The growth performance index (φ) is: 2.76. The length‐weight relationship is: EW = 4.4.10^?6 TL^3.23. The spawning period occurred from February to May, while the gamete emission peaked in April. Females reached sexual maturity at 19.3 cm (2 years) and males at 21.3 cm (3 years). Sexual inversion occurs at approximately 24.3 cm. Spondyliosoma cantharus was characterized as being a protogynic hermaphrodite.     

Development and characterization of simple sequence repeat (SSR) markers in the water lotus (Nelumbo nucifera)

Simple sequence repeat (SSR) markers were developed in the water lotus (Nelumbo nucifera Gaertn.) from an SSR-enriched genomic library. Of the SSR markers tested, 11 primer pairs produced clearly distinguishable DNA banding patterns. Forty-three alleles were detected with the 11 markers. The allele number per locus ranged from 2 to 5 with an average of 3.9. Polymorphism values ranged from 0.11 to 0.66 with an average of 0.51. These primers were also applicable to another Nelumbo species, Nelumbo lutea (Willd.) Pers. (American lotus) and hybrids between N. nucifera and N. lutea. These results indicate that the SSR markers developed in this study are informative and will be useful for genetic analysis in Nelumbo species.