Effects of Large-Scale Releases on the Genetic Structure of Red Sea Bream (Pagrus major,Temminck et Schlegel) Populations in Japan

Large-scale hatchery releases are carried out for many marine fish species worldwide; nevertheless, the long-term effects of this practice on the genetic structure of natural populations remains unclear. The lack of knowledge is especially evident when independent stock enhancement programs are conducted simultaneously on the same species at different geographical locations, as occurs with red sea bream (Pagrus major, Temminck et Schlegel) in Japan. In this study, we examined the putative effects of intensive offspring releases on the genetic structure of red sea bream populations along the Japanese archipelago by genotyping 848 fish at fifteen microsatellite loci. Our results suggests weak but consistent patterns of genetic divergence (F_ST = 0.002, p < 0.001). Red sea bream in Japan appeared spatially structured with several patches of distinct allelic composition, which corresponded to areas receiving an important influx of fish of hatchery origin, either released intentionally or from unintentional escapees from aquaculture operations. In addition to impacts upon local populations inhabiting semi-enclosed embayments, large-scale releases (either intentionally or from unintentional escapes) appeared also to have perturbed genetic structure in open areas. Hence, results of the present study suggest that independent large-scale marine stock enhancement programs conducted simultaneously on one species at different geographical locations may compromise native genetic structure and lead to patchy patterns in population genetic structure.     

Inference of Potential Genetic Risks Associated with Large-Scale Releases of Red Sea Bream in Kanagawa Prefecture, Japan Based on Nuclear and Mitochondrial DNA Analysis

Since 1978, millions of hatchery-reared red sea bream (Pagrus major) juveniles have been released in Sagami Bay and Tokyo Bay in Kanagawa Prefecture, Japan. The stock enhancement program has contributed to total catch; however, no information regarding the genetic interactions with wild counterparts is available. Here, we combined 15 microsatellite loci and mitochondrial D-loop sequencing to characterize the genetic resources of red sea bream in Sagami Bay and Tokyo Bay and to elucidate the potential harmful genetic effects associated with fish releases. Both types of markers evidenced higher levels of genetic diversity in wild samples (SB and TB) compared with offspring before stocking (H07 and H08) as well as a hatchery-released sample recaptured in Sagami Bay (HR). Microsatellite F _ST estimates and Bayesian clustering analysis found significant genetic differences among samples (F _ST?=?0.013–0.054), except for the two wild samples (F _ST?=?0.002) and HR vs. H07 (F _ST?=?0.007). On the other hand, mitochondrial-based Ф _ST suggested haplotypic similarity between SB, H07, and HR. The low effective number of females contributing to the offspring over multiple generations may be responsible for the lack of haplotypic differentiation. Moreover, the putative hatchery origin to three fish (8 %) without deformity in the inter-nostril epidermis was inferred for the first time. Our results showed the usefulness of combining nuclear and mitochondrial markers to elucidate genetic interactions between hatchery-released and wild red sea bream and warned about potential harmful genetic effects should interbreeding takes place.     

Chalimus stages of Caligus latigenitalis (Copepoda:Caligidae) parasitic on blackhead seabream from Japanese waters, with discussion of terminology used for developmental stages of caligids

The first and third chalimus stages and chalimus adult (previously known as young adult) of Caligus latigenitalis are described based on new material collected from the body surface of a heavily infected wild blackhead seabream Acanthopagrus schlegelii schlegelii from Hiroshima Bay, Japan. The second and fourth chalimus stages of the same species are redescribed. Adults of C. latigenitalis are characterized by possessing 2 stout marginally indented processes at the base of 2 terminal spines at distal exopodal segment of leg 4. The chalimi were identified to stage based on the structure of the frontal filament and the discrete ranges in body length. Sexual dimorphism is first observed at the third chalimus stage in the shape of the distal segment of the antenna. The total number of postnaupliar stages of C. latigenitalis is 6, including 4 semaphoronts, i.e., 1 copepodid stage consisting of 1 infective copepodid and the chalimus copepodid, 4 chalimus stages, and 1 adult stage with 1 chalimus adult and 1 mobile adult. New terminology for the developmental stages of caligid copepods is proposed herein by amending the definition of chalimus as the postnaupliar stages, as well as semaphoronts having a frontal filament for host attachment.     

Genetic differentiation of white sea bream within the Lion's Gulf and the Ligurian Sea (Mediterranean Sea)

An allozymic study was conducted on 190 individuals of white sea bream, Diplodus sargus sampled on five locations throughout the Lion's Gulf and the Ligurian Sea (Mediterranean Sea), in order to identify genetic structure. Electrophoretic surveys carried out on muscle and liver tissues identified 25 loci of which 12 were polymorphic ( P 0.95). G -test analysis shows significant differences on allelic frequencies between the five stations at six loci. Examination of the spatial structure was performed using Nei's distances and F statistics, and indicated significant genetic differences between three groups. A group which clustered Blanes (Spain), Marseille (France) and Livorno (Italy) where the absence of genetic difference can be explained by migration of larvae and adults along a coastal 'continuum'. The Elba sample (Italy) shows genetic divergence from other samples and this difference may result from isolation due to limited migration of larvae and adults. Banyuls (France), differs from all other stations. Several hypotheses are proposed to explain genetic patterns including local current systems, larval dispersal, geographic isolation and historical effects, and variation in the size classes of sampled individuals between sites.     

Patterns of gastric evacuation, digesta characteristics and pH changes along the gastrointestinal tract of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.)

A comparative study of gastric evacuation rates (GERs) and digesta content, moisture and pH values along the gastrointestinal tract was performed between gilthead sea bream and European sea bass. In order to distinguish species-specific differences from diet-elicited effects, all parameters were determined under either a fishmeal diet or a carob seed germ meal diet that contained high levels of total and soluble non-starch polysaccharides. GERs were significantly different between species and they were not affected by diet. Similarly, species-specific patterns were revealed in the distribution of digesta and water content along the gastrointestinal tract. In sea bream, stomach digesta and water content decreased with time, whereas in sea bass stomach retained the highest digesta and water content throughout the sampling period. The anterior and distal intestine exhibited the lowest accommodating capacities of digesta and water in either species. Overall, sea bream performed stomach digestion at lower hydration levels and higher pH compared with sea bass. Diet affected stomach moisture in both species and pH of stomach digesta in sea bass and of all intestinal sections in sea bream. The results obtained indicated that water and inorganic ion exchanges through the gut may differentiate between the species and warrant further investigation.     

Sorting grid trials to improve size selectivity of red mullet (Mullus barbatus) and annular sea bream (Diplodus annularis) in Turkish bottom trawl fishery

Sorting grids with two different bar spacings (12 mm and 14 mm) were tested to improve size selectivity of the commercially important fish species, red mullet (Mullus barbatus) and annular sea bream (Diplodus annularis), in Turkish bottom trawl fishery. Fishing trials were carried out with R/V ‘Egesüf’ between April and May 2003 in Izmir Bay, Aegean Sea, using a traditional bottom trawl. Selectivity data were collected by the top cover method and analyzed by means of a logistic equation with the maximum likelihood method. Selectivity parameters for individual hauls were obtained with the software program cc 2000 . Mean selectivity was also estimated and compared using the EC Model software that takes between‐haul variations into account. The codend catch size as an additional explanatory variable was used in the comparison. The Kolmogorov–Smirnov (K–S) test was also applied to detect differences between length‐frequency distributions in the upper and lower bags of the 12 and 14 mm bar spacings for red mullet and annular sea bream. The mean L₅₀ values of red mullet were 8.7 and 10.0 cm with the 12 and 14 mm grids, respectively. The L₅₀ value of 14 mm was comparable with the L₅₀ value of the codend mesh size; however, the 12 mm value was rather low. The mean L₅₀ values of 12 and 14 mm bar spacings were 8.8 and 10.4 cm for annular sea bream, respectively; the value of 14 mm bar spacing was very close to 50% size at sexual maturity of the species (10.5 cm). The K–S test indicated length distributions of red mullet and annular sea bream in the 12 and 14 mm upper and lower bags as significantly different (P < 0.05). These results show that improving the size selectivity in a multi‐species fishery using a single selective device is rather difficult. However, higher size selectivity can be obtained when considering the minimum landing size or the 50% sexual maturity size for a given species.     

Exploring neutral and adaptive processes in expanding populations of gilthead sea bream, Sparus aurata L., in the North-East Atlantic

Recent studies in empirical population genetics have highlighted the importance of taking into account both neutral and adaptive genetic variation in characterizing microevolutionary dynamics. Here, we explore the genetic population structure and the footprints of selection in four populations of the warm-temperate coastal fish, the gilthead sea bream (Sparus aurata), whose recent northward expansion has been linked to climate change. Samples were collected at four Atlantic locations, including Spain, Portugal, France and the South of Ireland, and genetically assayed using a suite of species-specific markers, including 15 putatively neutral microsatellites and 23 expressed sequence tag-linked markers, as well as a portion of the mitochondrial DNA (mtDNA) control region. Two of the putatively neutral markers, Bld-10 and Ad-10, bore signatures of strong directional selection, particularly in the newly established Irish population, although the potential 'surfing effect' of rare alleles at the edge of the expansion front was also considered. Analyses after the removal of these loci suggest low but significant population structure likely affected by some degree of gene flow counteracting random genetic drift. No signal of historic divergence was detected at mtDNA. BLAST searches conducted with all 38 markers used failed to identify specific genomic regions associated to adaptive functions. However, the availability of genomic resources for this commercially valuable species is rapidly increasing, bringing us closer to the understanding of the interplay between selective and neutral evolutionary forces, shaping population divergence of an expanding species in a heterogeneous milieu.     

Genetic integrity of black sea bream (Acanthopagrus schlegeli) sperm following cryopreservation

Although semen cryopreservation has been applied successfully in many fish species, extensive variation in post‐thaw semen quality exists between species and individuals. AFLP (amplified restriction fragment length polymorphism) is a powerful method for detecting DNA polymorphisms at the individual, population, and species levels. The method has been successfully applied to boars (Sus domestica, Suidae, Artiodactyla, Mammalia) to detect and evaluate differences in DNA sequences that correspond with semen integretiy after employing various freezing techniques. Freezing and thawing of semen has also an effect of selecting for freezing‐resistant (or intact) and eliminating non‐viable or defective sperm. Only the fully intact and functional sperm, despite potential compromise by adverse freezing and osmotic stresses, retain fertility after thawing. The objective of this study was to use AFLP to assess any genetic changes associated with the effect of employed cryo‐methodology on the genetic integrity of sperm of the black sea bream (Acanthopagrus schlegeli) under different cryopreservation treatments. The cryopreservation protocols had no significant effect on sperm motility or survival rate of fertilized ova regardless of using fresh (% motile sperm 89.6 ± 3.0; % embryonic survival rate 54.4 ± 2.9) and frozen‐thawed semen (% motile sperm 80.2 ± 2.0; % embryonic survival rate 51.8 ± 2.0). The post‐thaw sperm motility and survival rates were not significantly different among the sperm samples of the five black sea bream males examined. In the present study, the remaining black sea bream sperm that survive the cryopreservation limit the power of AFLP to trace the genetic markers which correlate with the differences in the sensitivity of sperm to cryo‐injury. It is also possible that point mutations outside the AFLP priming sites may not have been detected. More thorough investigations are needed to determine whether such DNA fingerprints would be found in fish species.     

Feeding by marine fish larvae: developmental and functional responses

Synopsis The relationship between prey consumption rate and prey concentration (functional response), and its change with growth (developmental response) were examined in the laboratory for three species of marine fish larvae: bay anchovy Anchoa mitchilli (Engraulidae), sea bream Archosargus rhomboidalis (Sparidae) and lined sole Achirus lineatus (Soleidae). The major objective was to determine relative predatory abilities of the larvae by fitting feeding rate data to developmental and functional response models. Feeding success, prey capture success, attack rates, handling times and search rates were estimated. Prey consumption rates and attack rates of bay anchovy usually were highest, but at the lowest prey level (50 per liter) first-feeding sea bream larvae had the highest consumption rate. Sea bream could consume prey at near-maximum rates at prey levels lower than those required by the other species. As larvae grew, time searching per attack decreased rapidly for all species, especially at low prey levels. Handling time also decreased, but most rapidly for bay anchovy. Search rates were highest for bay anchovy and lowest for lined sole. Bay anchovy had the best apparent predation ability, but when previous results on larval growth rates, survival rates and growth efficiencies were considered, sea bream larvae were the most efficient predators and the least likely of the three species to be limited by low prey levels.     

In silico mining and characterization of simple sequence repeats from gilthead sea bream (Sparus aurata) expressed sequence tags (EST-SSRs); PCR amplification, polymorphism evaluation and multiplexing and cross-species assays

We screened for simple sequence repeats (SSRs) found in ESTs derived from an EST-database development project ('Marine Genomics Europe' Network of Excellence). Different motifs of di-, tri-, tetra-, penta- and hexanucleotide SSRs were evaluated for variation in length and position in the expressed sequences, relative abundance and distribution in gilthead sea bream (Sparus aurata). We found 899 ESTs that harbor 997 SSRs (4.94%). On average, one SSR was found per 2.95 kb of EST sequence and the dinucleotide SSRs are the most abundant accounting for 47.6% of the total number. EST-SSRs were used as template for primer design. 664 primer pairs could be successfully identified and a subset of 206 pairs of primers was synthesized, PCR-tested and visualized on ethidium bromide stained agarose gels. The main objective was to further assess the potential of EST-SSRs as informative markers and investigate their cross-species amplification in sixteen teleost fish species: seven sparid species and nine other species from different families. Approximately 78% of the primer pairs gave PCR products of expected size in gilthead sea bream, and as expected, the rate of successful amplification of sea bream EST-SSRs was higher in sparids, lower in other perciforms and even lower in species of the Clupeiform and Gadiform orders. We finally determined the polymorphism and the heterozygosity of 63 markers in a wild gilthead sea bream population; fifty-eight loci were found to be polymorphic with the expected heterozygosity and the number of alleles ranging from 0.089 to 0.946 and from 2 to 27, respectively. These tools and markers are expected to enhance the available genetic linkage map in gilthead sea bream, to assist comparative mapping and genome analyses for this species and further with other model fish species and finally to help advance genetic analysis for cultivated and wild populations and accelerate breeding programs.     

Genetic relationships among populations of the Argentine ant Linepithema humile introduced into Japan

Since some colonies of Argentine ant Linepithema humile were discovered in Japan in 1993, populations of this invasive alien ant species have been expanding their distribution. To resolve the number of invasions and the genetic structure in the early stages of introduction, we inferred the genetic structure and relationships among colonies from eight localities, from analyses of eight nuclear microsatellite DNA markers. F _ST analysis, principal component analysis and assignment test showed that at least three highly genetically differentiated groups of Argentine ants are present in Japan. Populations from Hiroshima Bay were grouped together (Hiroshima, Hatsukaichi, Otake, Iwakuni Central and Iwakuni Kuroiso), while those from Kobe and Yanai were both genetically distant from each other and from the Hiroshima Bay group. Hatsukaichi and Kobe are international seaports, suggesting that the by-ship invasion occurred at least twice. The invasion route of the Yanai population is unknown at this moment. The Aichi population was genetically distant from that of the Hiroshima Bay group by the difference in allele frequencies, and it was plausible that the Aichi population was introduced from the Hiroshima Bay group by human-mediated jump dispersal.     

Demographic and genetic structures of white sea bream populations (Diplodus sargus, Linnaeus, 1758) inside and outside a Mediterranean marine reserve

We studied the white sea bream (Diplodus sargus), a protandrous hermaphroditic fish, in two protected and unprotected areas in southwestern France. We observed a significant difference in the demographic structure between the two areas. Females were present in two different age distributions inside and outside the marine reserve with younger females outside. This suggests plasticity in the age of sexual inversion in the case of an exploited population. Genetic differentiation was weak and apparent at only one locus of 26 surveyed (FST = 0.007, p = 0.04). Our data suggest that gene flow between the two areas is important, or the separation between the two sites is recent. Our data on the white sea bream show that fishes inside and outside the marine reserve are very similar genetically, which means that the 'reserve effect' is truly a demographic one, not the result of genetic differences.     

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.     

广东湛江湾可培养丝状真菌分布状况的初步调查

通过一次航行对广东湛江湾19个站位的表层和底层海水进行采样。经过对样品的分离纯化,共获得253株丝状真菌菌株。通过测序获得了121个rDNA-ITS序列,并已提交给GenBank。经统计分析发现,湛江湾表层和底层海水真菌数量总体上表现为由湾内向湾口逐渐减少的水平分布格局;湛江湾表层丝状真菌的数量略多于底层,在海水表层和底层中真菌种类和数量的分布规律相似。根据形态特征及rDNA-ITS的Blast同源性分析,这些菌株分属于18个属和32个分类单元,其中包括7个海洋真菌新记录属。结果表明,湛江湾丝状真菌多样性Shannon指数达2.75,物种优势度变化范围为30.90%–0.02%。枝孢属Cladosporium的优势度最高,为湛江湾优势种群,其次是青霉属Penicillium、侧齿霉属Engyodontium、曲霉属Aspergillus、枝顶孢属Acremonium等。湛江湾表层和底层海水真菌群落的Jaccard相似系数为0.42。     

Isolation and characterization of polymorphic microsatellite markers in Pagellus bogaraveo,and cross‐species amplification in Sparus aurata and Dicentrarchus labrax

Several new fish species are currently being included in breeding programmes. However, as specific molecular markers have not yet been developed, this represents a commercial handicap with respect to traditional aquaculture species such as gilthead sea bream or Atlantic salmon. In the present study, 12 new microsatellite loci were developed for blackspot sea bream (Pagellus bogaraveo) that show high levels of polymorphism, especially useful in parentage assignment and individual identification. In addition, cross‐amplification was obtained for two important species for Spanish aquaculture, gilthead sea bream and sea bass.     

Trawl codend design (44 mm diamond PE mesh) and the effect on selectivity for Pagellus erythrinus and Pagellus acarne, two species with different morphometrics

The effects of different body shapes on size selectivity were analysed. Relationships between total length and fork length, height, width and girth were estimated and the selectivity parameters of a 44 mm PE diamond mesh codend determined for common pandora (Pagellus erythrinus) and axillary sea bream (Pagellus acarne). Two data sets were collected during demersal trawl trials carried out in Izmir Bay (Aegean Sea) between May 1996 and February 1997. Regression analyses were used to determine the relationships between total length and other measured dimensions of the species. However, selectivity parameters were estimated from pooled data by using the logistic equation with the maximum likelihood method. Fork length, height, width and girth were found to increase linearly with total length for both species (R² > 0.90, except TL–W for axillary sea bream, 0.76). Ratios of average body thickness (W/H) were 0.45 (±0.002) for common pandora and 0.52 (±0.002) for axillary sea bream. L₅₀ and SR values were found as 12.4 (±0.44) and 2.2 (±0.51) from the eight valid hauls for common pandora and as 13.6 (±0.13) and 1.9 (±0.26) from the three valid hauls for axillary sea bream, respectively. The difference between the size selectivity of the two species in the same family can be explained by the body shape and fish behaviour distinctions.     

Lateral line deformities in wild and farmed sea bass (Dicentrarchus labrax,L.) and sea bream (Sparus aurata,L.)

The lateral line of aquaculture fishes has rarely been studied although it is a very important anatomical organ that could serve as an inexpensive and easy tool to distinguish farmed from wild individuals. In the present study, lateral line deformities were examined in both wild and farmed sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) specimens to try to detail all possible differences between them. In order to do so, the morphology of the trunk lateral line in wild and farmed adults was examined whereby two major deformities were observed in both species: the ‘scale pocket’ deformity (14–40% incidence in all groups) where the specialized scales are missing but the canal underneath is present and the scale print is obvious, and the ‘somatic scales’ deformity (14–56% incidence in farmed individuals only) where the missing lateral line is covered with normal somatic scales. Histological examination confirmed the macroscopic observations in that the lateral line mechanism was present – although damaged – beneath the scale pocket deformity and completely absent beneath the somatic scales deformity. It is argued that the scale pocket deformity is a result of an accident during the life of the fish whereas the somatic scales deformity is an actual deformity in development.     

A comparison of fatty acid,cholesterol and vitamin composition in sea bass [Dicentrarchus labrax (Linnaeus, 1758)] and sea bream [Sparus aurata (Linnaeus, 1758)] from three cage farm areas: Antalya and Muğla (Turkey) and İskele (Northern Cyprus)

In the present study the fatty acids, cholesterol and vitamin composition in farmed sea bass (8 fish per species per farm; weight range: 389.6–395.8 g, total length range: 297–316 mm) and sea bream (8 fish per species per farm; weight range: 386.8–391.7 g, total length range: 263–268 mm) from three cage farms (?skele in northern Cyprus, Antalya and Mu?la in Turkey) were compared during the harvesting period in June–July 2011. The results showed that the muscles of D. labrax and S. aurata farmed fish were rich in n‐3 fatty acids, but with important differences. For example, the muscles of sea bass farmed in ?skele were rich in docosahexaenoic acid (DHA) and n‐3 polyunsaturated fatty acids (PUFA). Palmitic acid (C16:0) was the primary saturated fatty acid, and oleic acid (C18:1 n‐9) the primary monounsaturated fatty acid in the muscle and liver samples of the cage‐farmed sea bass and sea bream. There were no significant differences in the cholesterol content in the muscles of sea bream farmed in ?skele, Antalya or Mu?la. In conclusion, the n‐3/n‐6 ratio in the muscle of farmed S. aurata and D. labrax is within the recommended limits for a healthy human diet, being very suitable for human nutrition.