Yongshuang Xiao

Germ line specific expression of a vasa homologue gene in turbot (Scophthalmus maximus): evidence for vasa localization at cleavage furrows in euteleostei

Specification of primordial germ cells during early embryogenesis is a critical biological issue in reproduction and development. Yet, little is known in marine economic fish species. Vasa, a component of germ plasm, is the most‐documented germ cell marker in teleosts. We isolated a full‐length vasa cDNA (Smvas) from turbot (Scophthalmus maximus), a marine Euteleostei species, and investigated its expression patterns by RT‐PCR and in situ hybridization during embryogenesis and gametogenesis to identify the germ cell lineage in this species. The deduced amino acid sequence of the isolated cDNA shared typical characteristics of Vasa protein and high identity to Vasa homologues in medaka (76.9%) and zebrafish (68.5%). The Smvas transcripts were exclusively detected in germ cells of testis and ovary, and exhibited an interesting dynamic localization pattern during oogenesis. The distribution pattern of Smvas during embyogenesis in this Euteleostei closely resembled the pattern observed in zebrafish (belonging to Osteriophysans) rather than medaka (belonging to Euteleostei). Thus, it is concluded that Smvas isolated in this study is a germ cell specific molecular marker in turbot. Furthermore, we hypothesize that Euteleostei could localize vasa mRNA by a special mode. The results not only facilitate the germ cell manipulation of the turbot, but also improve our understanding of germline development and evolution of vasa localization in teleost. Mol. Reprod. Dev. 79: 803–813, 2012.

The dnd RNA Identifies Germ Cell Origin and Migration in Olive Flounder (Paralichthys olivaceus)

The present study obtained a germ cell-specific marker dead end (dnd) in olive flounder (Paralichthys olivaceus) named Podnd. The tissue-specific expressions of Podnd transcripts were present in testis and ovary but were not detectable in other somatic tissues detected. SISH showed that Podnd expressed only in germ cells at different developmental stages but not in surrounding somatic cells. The expression of Podnd during embryonic development at 16 different stages revealed that the relative expression of Podnd transcript fluctuated at a high level in the cleavage stages, gradually decreased through subsequent development, and reached the lowest at late gastrula stage till it was nearly undetectable. The Podnd transcripts localization and migration were similar to zebrafish. Further research on the specification migration mechanism of PGCs and the role of germ cell during gonadal development in olive flounder would improve our understanding of germline development.

Germline-specific and sexually dimorphic expression of a dead end gene homologue in turbot (Scophthalmus maximus)

Germ cells are indispensable for gonadal development and fertility. However, the physiological mechanisms regulating germ cell development in marine fish are poorly understood due to a lack of germ cell markers. The dead end (dnd) gene is a vertebrate-specific component of germplasm crucial for primordial germ cells (PGCs) migration and development in teleosts. In this study, we identified a dnd homologue (Smdnd) in turbot (Scophthalmus maximus) and investigated its expression pattern during embryogenesis and gonadal development. The deduced amino acid sequence of Smdnd shared several conserved motifs of Dnd homologues as well as high identity to other Dnd proteins. Phylogenetic analysis revealed that the SmDnd was closely related to its teleost counterparts. Reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization revealed that Smdnd transcripts could be exclusively detected in germ cells, including presumptive PGC and adult male and female germ cells. In addition, an interesting sexually dimorphic expression of Smdnd during gonadal development was observed by real-time PCR. Female turbot showed greater (P < 0.05) Smdnd expression than male before sex maturation. This difference reduced gradually due to the upregulation of Smdnd in the male during the period corresponding to spermatogonia proliferation and meiosis. These results indicate that Smdnd can be used as a germ cell marker in turbot. In addition, the temporal and sex differences in Smdnd expression indicate that this gene may play different roles in gonadal development in both sexes.

Skeletal development and abnormalities of the vertebral column and of the fins in hatchery-reared turbot Scophthalmus maximus

To describe the skeletal development and abnormalities in turbot Scophthalmus maximus, samples were collected every day from hatching to 60 days after hatching (DAH). A whole-mount cartilage and bone-staining technique was used. Vertebral ontogeny started with the formation of anterior haemal arches at 5·1 mm standard length (L(S) ) c. 11 DAH, and was completed by the full attainment of parapophyses at 16·9 mm L(S) c. 31 DAH. Vertebral centra started to develop at 6·3 mm L(S) c. 16 DAH and ossification in all centra was visible at 11·0 mm L(S) c. 25 DAH. The caudal fin appeared at 5·1 mm L(S) c. 11 DAH and ossification was visible at 20·6 mm L(S) c. 37 DAH. The onset of dorsal and anal fin elements appeared at 5·8 mm L(S) c. 15 DAH and 6·3 mm L(S) c. 16 DAH, respectively. Ossifications of both dorsal fin and anal fin were visible at 20·6 mm L(S) c. 37 DAH. The pectorals were the only fins present before first feeding, their ossifications were completed at 23·5 mm L(S) c. 48 DAH. Pelvic fins began forming at 7·2 mm L(S) c. 19 DAH and calcification of the whole structure was visible at 19·8 mm L(S) c. 36 DAH. In the present study, 24 types of skeletal abnormalities were observed. About 51% of individuals presented skeletal abnormalities, and the highest occurrence was found in the haemal region of the vertebral column. As for each developmental stage, the most common abnormalities were in the dorsal fin during early metamorphic period (stage 2), vertebral fusion during climax metamorphosis (stage 3) and caudal fin abnormality during both late-metamorphic period (stage 4) and post-metamorphic period (stage 5). Such research will be useful for early detection of skeletal malformations during different growth periods of reared S. maximus.

Analysis of the early development in first and backcross generations between Paralichthys olivaceus and Paralichthys dentatus

This study investigated the performance difference of reciprocal hybrids and backcrosses between Paralichthys olivaceus and Paralichthys dentatus. The fertilization and hatching rates, combined fitness measure, early developmental characteristics and chromosome number were analyzed. The crosses of P. olivaceus female x P. dentatus male (F1), F1 female x P. olivaceus male and F1 female x P. dentatus male could normally fertilize (the fertilization rate: 93.2 to 97.7%), hatch (the hatching rate: 84.4 to 93.1%) and develop during pre-larvae stage. However, the fertilization rate (41.7%) and combined fitness measure (0.327) of P. dentatus female x P. olivaceus male was significantly reduced compared with other crosses. And the embryos of P. dentatus female x P. olivaceus male hatched out with flexural spine and poor vitality and died within 3 days post hatching. In addition, the result of cytogenetics analysis showed two chromosomes were missing in P. dentatus female x P. olivaceus male, which provided evidence of postzygotic barriers in the two species. The results would be useful for better understanding the genetic phenomena of the distant hybridization in fish species.

Pepsinogen A and C genes in turbot (Scophthalmus maximus): characterization and expression in early development.

We characterized the expression patterns of pepsinogen A (tPGA) and pepsinogen C (tPGC) in turbot (Scophthalmus maximus). Quantitative expression analysis showed that tPGC was preferentially expressed in early developmental stages, and that the tPGA mRNA expression level was higher in adult fish. Full-length cDNA constructs of tPGA and tPGC were 1307 bp (from which 377 amino acids were deduced); and 1430 bp (from which 385 amino acids were deduced), respectively. The deduced proteins of tPGA and tPGC possessed signal peptides of 17 amino acids and 20 amino acids respectively. The initial transcripts of tPGA and tPGC were detected at 22 days post hatching (dph), well after the formation of gastric glands (16 dph). This suggested that the morphologic development of gastric glands was not synchronous with their functional development. In addition, tPGA and tPGC mRNAs were also expressed in muscle and ovary at much lower levels than in stomach and esophagus. The distribution of tPGA and tPGC in the turbot was investigated using in-situ hybridization, and tPGA and tPGC were first detected in the esophagus and cardiac region of the stomach, and then throughout the stomach.

Significant population genetic structure detected in the small yellow croaker Larimichthys polyactis inferred from mitochondrial control region

Abstract The population genetic structure of the small yellow croaker (Larimichthys polyactis) between China and Korea was further estimated by broad-scale sampling locations (Gulf of Bohai, Yellow Sea including Korea). One hundred and seventeen individuals from eight localities from coastal waters of China and Korea were analyzed based on mtDNA control region sequences (5′ mtDNA CR). A total of 97 polymorphic sites were checked, which defined 136 haplotypes. A pattern with high levels of haplotype diversity (h = 0.994 ± 0.002) and nucleotide diversity (л = 0.020 ± 0.010) was detected in the examined range, and the genetic diversity of Korea populations was higher than that of China populations. Population genetic structure analyses (MDS, AMOVA, Fst, Barrier) showed that significant genetic differentiation existed between China and Korea populations. The migration analysis indicated asymmetry migration also existed among populations, which was consistent with the result of population genetic structure. Using a variety of phylogenetic methods, coalescent reasoning, and molecular dating interpreted in conjunction with paleoclimateic and physiographic evidence, we inferred that the genetic make-up of extant populations of L. polyactis was shaped by Pleistocene environmental impacts on the historical demography of this species. Coalescent analyses (Neutrality tests, Mismatch distribution analysis, Bayesian skyline analyses) showed that the species along coastline of China and Korea has experienced population expansions originated in its most recent history at about 32–196 kya and 166–662 kya before present, respectively.

Pronounced population genetic differentiation in the rock bream Oplegnathus fasciatus inferred from mitochondrial DNA sequences.

Abstract The population genetic structure of the rock bream (Oplegnathus fasciatus) along the coastal waters of China was estimated based on three mtDNA fragments (D-loop, COI, and Cytb). A total of 112 polymorphic sites were checked, which defined 63 haplotypes. A pattern with high levels of haplotype diversity (hCOI = 0.886 ± 0.034, hCytb = 0.874 ± 0.023) and low levels of nucleotide diversity (лCOI = 0.009 ± 0.005, лCytb = 0.006 ± 0.003) was detected based on the COI and Cytb fragments, and high levels of genetic diversity (hD-loop = 0.995 ± 0.007, лD-loop = 0.021 ± 0.011) were detected from the mtDNA D-loop. The population genetic diversity of O. fasciatus in south China was significantly higher than those of north China. Three genealogical clades were checked in the O. fasciatus populations based on the NJ and MST analyses of mtDNA COI gene sequence, and the genetic distances among the clades ranged from 0.018 to 0.025. Significant population genetic differentiation was also checked based on the Fst (0.331, p = 0.000) and exact p (0.000) test analyses. No significant population differentiations were checked based on mtDNA D-loop and Cytb fragments. Using a variety of phylogenetic methods, coalescent reasoning, and molecular dating interpreted in conjunction with paleoclimatic and physiographic evidences, we inferred that the genetic make-up of extant populations of O. fasciatus was shaped by Pleistocene environmental impacts on the historical demography of this species. Coalescent analyses (neutrality tests, mismatch distribution analysis, and Bayesian skyline analyses) showed that the species along coastline of China has experienced population expansions originated in its most recent history at about 169–175 kya before present.

Germ cells and fertilization differences among Japanese flounder Paralichthys olivaceus, summer flounder Paralichthys dentatus and their first and second generations

The morphology of gametes and the fertilization biology of Japanese flounder Paralichthys olivaceus (Po), summer flounder Paralichthys dentatus (Pd) and their hybrids were examined. Multiple generations (two hybrids: Po♀× Pd♂ (F1) and Pd♀× Po♂; two backcrosses: F1♀× Po♂ and F1♀× Pd♂) were obtained by artificial insemination. Comparison of egg ultrastructure among Po, Pd and F1 showed the morphology of micropyle region and the distribution density of pores were species specific. There were c. 100-200 accessory openings around the micropyle in Po, but not in Pd and F1. The zona radiata thickness and number of parallel bands were similar between F1 and Po, which were different from Pd. Comparison of spermatozoa ultrastructure revealed a close relationship between Po and Pd. Cytologically, the six crosses obeyed normal fertilization and cleavage processes, and only one male pronucleus was observed in a fertilized egg, indicating a monospermic fertilization pattern. Analysis of the time distribution from fertilization to first cleavage revealed an obvious delay at pronucleus fusion in the Pd × Po cross. The delay might indicate some cytoplasmic-nuclear incompatibility during the process of fertilization.

Histological change and heat shock protein 70 expression in different tissues of Japanese flounder Paralichthys olivaceus in response to elevated temperature

High temperature influences the homeostasis of fish. We investigated the effects of elevated temperature on tissues of Japanese flounder (Paralichthys olivaceus) by analyzing the histology and heat shock protein 70 (hsp70) expression of fish reared in warm conditions. In this study, temperature was increased at 1±0.5°C/day starting at 24±0.5°C, and was kept at that temperature for 5 days before the next rise. After raising temperature at the rate up to 32±0.5°C, tissue samples from midgut, spleen, stomach, liver, muscle, gill, heart, trunk kidney and brain were collected for histological analysis and mRNA assay. Almost all the tissues showed changes in morphological structure and hsp70 level at 32±0.5°C. Histological assessment of the tissues indicated that the gill had the most serious damage, including highly severe epithelial lifting and edema, curved tips and hyperemia at the ending of the lamellars, desquamation and necrosis. The next most severe damage was found in liver and kidney. The hsp70 levels in all the tissues first increased and then decreased. The gut, stomach, muscle, heart, and brain had the highest expressions in 6 h, whereas the spleen, liver, gill and kidney had the highest expressions in 2 h. Therefore, tissues with the most significant lesions (especially gill and liver) responded much earlier (2 h) in hsp70 expression than other tissues, and these tissues demonstrated the most marked histological disruption and elevated mRNA levels, making them ideal candidates for further studies on the thermal physiology of this species.