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Testis-specific serine/threonine kinase 1-like (TSSK1-like), which plays important roles in late-phase spermatogenesis and male fertility, was characterized in Pacific abalone Haliotis discus hannai, an important commercial marine gastropod. Further, its expression patterns were assessed in diploid and induced triploid males showing differential degrees of testis maturation. Abalone TSSK1-like shared conserved structural features with mammalian TSSK1s and other potential metazoan orthologs, especially regarding the catalytic STKc domain. Phylogenetically, abalone TSSK1-like displayed a genetic affiliation with its molluscan TSSK1-like orthologs and human TSSK1. Additionally, abalone TSSK1-like gene showed a tetrapartite exon-intron organization, unlike the intronless structure of most amniotic tetrapodian TSSK1s. Molecular phylogenetic analysis in the metazoan lineage suggested a possible revision in the origin of the earliest ancestral TSSK1. Further, abalone TSSK1-like showed testis-predominant expression, which was significantly influenced by both age and seasonal reproductive cycles. Comparative expression analyses between diploid and triploid abalone males suggested that robust TSSK1-like expression occurred primarily at the post-meiotic stage. Additionally, RT-PCR assay indicates that mature abalone sperms retain TSSK1-like transcripts after release. Taken together, this study provides useful insights for further studies to assess male reproduction and sterility and/or partial fertility of induced male triploidy in abalone species. [ABSTRACT FROM AUTHOR]

The optimal transfection conditions for efficient transgene delivery into a specific cell type should be empirically determined, particularly in cases involving unusual cell types. We compared the conditions for effective introduction of transgenes into Siberian sturgeon (Acipenser baerii) cell lines by evaluating the cytotoxicity and transfection efficiency of three commercially available transfection reagents: Lipofectamine 2000, X-tremeGENE HP DNA Transfection Reagent, and GeneJuice Transfection Reagent. Plasmid vectors containing the gene encoding enhanced green fluorescent protein were mixed with each of the transfection reagents using reagent-to-plasmid ratios of 1:1, 2:1, and 4:1. Then, the complexes were used to transfect three Siberian sturgeon cell lines derived from the heart, head kidney, and gonad. Cytotoxicity and transfection efficiency were measured via flow cytometry after propidium iodide staining. No significant cytotoxicity was observed at the optimal treatment conditions in all cases, with the exception of Lipofectamine 2000-treated gonad-derived cells. Although the transfection efficiencies in A. baerii cells were generally low, X-tremeGENE HP DNA Transfection Reagent showed the highest transfection efficiency at ratios of 2:1 or 4:1, depending on the cell type. Hence, X-tremeGENE HP DNA Transfection Reagent can be used to effectively transfer foreign genes into three A. baerii cell lines. [ABSTRACT FROM AUTHOR]

An anesthetic protocol was optimized for microinjection-related handling of Siberian sturgeon (Acipenser baerii; Acipenseriformes) prolarvae, an extant primitive fish species commonly grown in aquaculture. Comparative examinations of three selected anesthetics (clove oil, lidocaine, and MS-222) with a dosage regime of 50, 100, 200, and 400 mg/L indicated that MS-222 was the most efficient agent for Siberian sturgeon prolarvae, as evidenced by the fast induction of anesthesia with quick and uniform recovery. Meanwhile, clove oil should be avoided, due to prolonged recovery times varying widely between individuals. None of the tested anesthetics significantly affected prolarval viability at any of the dosage regimes tested in this study. Based on an analysis of the duration of an unconscious state in air, we recommend a dose of 200 mg/L MS-222 for microinjection. Recovery time after use of this dose was influenced by the prolarval age and the development of gills, in which prolarvae older than 3 days after hatching required longer recovery times than did younger prolarvae. Post-recovery behavioral assessment showed no apparent difference between MS-222-anesthetized and non-anesthetized prolarvae in their swimming behavior and phototactic responses. Applicability of currently developed anesthetic protocol using MS-222 in larval microinjection was demonstrated with the injection of a visible dye to the anesthetized prolarvae, followed by the analysis of post-recovery viability. Taken together, the present anesthetic protocol based on 200 mg/L of MS-222 could provide researchers with practical usefulness with good safety margins for the micromanipulation and other related handlings of Siberian sturgeon prolarvae. [ABSTRACT FROM AUTHOR]

Albinism, a congenital genetic mutation, is rarely found in natural populations of cyprinid loach Misgurnus anguillicaudatus (Cypriniformes; Cobitidae). The entire mitogenome of albino M. anguillicaudatus (16,647 bp), which is found in a tributary of Korean river, represents a structural organization typical of a vertebrate mtDNA comprising of 13 protein coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNA (tRNA) genes, one replication origin (OL), and one control region (D-loop). Phylogenetic analysis suggests that albino cyprinid loach in this study corresponds to the “introgressed” mitochondrial type of M. anguillicaudatus based on the mtDNA introgression hypothesis in genus Misgurnus. Mitogenome information from this study could be a useful basis for conservation and phylogenetics of the natural albino cyprinid loaches. [ABSTRACT FROM AUTHOR]

We investigated the effects of hydrogen peroxide on mucous cells and lysozymes in gill tissue of olive flounder. Morphological analysis revealed significant changes in the number of mucous cells after exposure to 300 and 500 mg L −1 hydrogen peroxide; however, there was no detectable difference in the 100 mg L −1 group. Lysozyme activities declined gradually in the gill mucus, but in contrast activities in serum increased significantly following the completion of treatment. The hepatic c- and g-type lysozyme levels showed significant responses, indicating that the liver is a major lysozyme producer in response to stress condition; however, in the head kidney, there were no significant changes in the g-type lysozyme level in all the groups, while the only detectable difference with the c-type gene in the 300 mg L −1. While the treatments showed no significant changes compared with the control for either branchial lysozyme, there were significant differences in c-type lysozyme between the 3rd treatment and 5 day. Taken together, our data indicate that hydrogen peroxide treatment may lead to modulation of the innate immune response of olive flounder; therefore, the application of hydrogen peroxide in a field system will require more attention to dose and timing effects. [ABSTRACT FROM AUTHOR]

Background and aims: Here, we present the full-length mitogenome sequences of Hemibarbus labeo, Hemibarbus longirostris, and Hemibarbus mylodon - three fish species belonging to the subfamily Gobioninae (Teleostei, Cypriniformes, and Cyprinidae). H. mylodon is endemic to the Korean Peninsula and a critically endangered freshwater species because of various anthropogenic activities around its natural habitat. Results: Overall characteristics of mitogenomes of the three Hemibarbus species were identical to previously known typical vertebrate mitogenomes. Bayesian inference of phylogeny recovered the monophyly of the subfamily Gobioninae with high posterior probability support, with the three Hemibarbus species forming a strongly supported monophyletic group. H. mylodon was placed at the basal position, and H. longirostris was separated from two monophyletic H. labeo types. Conclusion: Our findings are congruent with the previous taxonomic appraisal based on morphological analysis. [ABSTRACT FROM AUTHOR]

A 75-day study was conducted to determine the effect of starvation on classical and truss parameters in Rhynchocypris oxycephalus (Sauvage and Dabry). Truss dimensions of almost the entire head and trunk region as well as the abdomen were increased significantly through feeding or starvation (P < 0.05). Truss dimensions of the caudal region generally decreased through feeding or starvation, particularly those dimensions at the hind part of the trunk. There were some significant decreases in classical dimensions of the head region during feeding, in relation to body depth characteristics in the trunk and caudal region during starvation, whereas there was only one decreasing classical dimension in the caudal region during feeding. The results of this study indicate that application of the truss network as a character set enforces classical coverage across the body form, discrimination among experimental groups thus being enhanced. Considering that the dimension of the lower part of the head and some truss and classical dimensions were least affected by feeding and starvation, these dimensions may then be useful as a taxonomical indicator to discriminate the species of Rhynchocypris sp. The value of trunk region dimensions with a large component of body depth in R. oxycephalus is most likely to be compromised by variability related to differences in feeding regimes of fish in different habitats. [ABSTRACT FROM AUTHOR]

SUMMARY:A stable homozygous line of transgenic mud loach (Misgurnus mizolepis) carrying a CAT reporter construct was developed. The founder transgenic mud loach was produced by microinjection of a CAT reporter gene driven by a carp beta-actin promoter and regulatory region (pFV4CAT) into fertilized eggs. The presumptive homozygous transgenic fish were generated by sister–brother mating between two heterozygous transgenic F2 siblings. Out of 28 transgenic individuals tested, five fish transferred the transgene into their progeny at a rate of 100%, four fish at 80–91%, and remaining 19 individuals inherited transgenic construct at a rate of approximately 50%. The level of expressed CAT enzyme in tissues (liver and spleen) from homozygous transgenic fish was higher than in those from heterozygous transgenic fish, although the increased amounts of CAT expression were slightly variable among homozygous individuals. The average level of CAT expression in homozygous fish was 1.8-fold (for liver) and 1.6-fold (for spleen), that of heterozygous fish. [ABSTRACT FROM AUTHOR]

Three distinct superoxide dismutases (SODs)—copper/zinc-SOD (SOD1), manganese-SOD (SOD2), and extracellular copper/zinc-SOD (SOD3)—were identified from a primitive chondrostean fish, Acipenser baerii, enabling the comparison of their transcriptional regulation patterns during development, prelarval ontogeny, and immune stimulation. Each A. baerii SOD isoform (AbSOD) shared conserved structural features with its vertebrate orthologs; however, phylogenetic analyses hypothesized a different evolutionary history for AbSOD3 relative to AbSOD1 and AbSOD2 in the vertebrate lineage. The AbSOD isoforms showed different tissue distribution patterns; AbSOD1 was predominantly expressed in most tissues. The expression of the AbSOD isoforms showed isoform-dependent dynamic modulation according to embryonic development and prelarval ontogenic behaviors. Prelarval microinjections revealed that lipopolysaccharide only induced AbSOD3 expression, while Aeromonas hydrophila induced the expression of AbSOD2 and AbSOD3. In fingerlings, the transcriptional response of each AbSOD isoform to bacterial infection was highly tissue-specific, and the three isoforms exhibited different response patterns within a given tissue type; AbSOD3 was induced the most sensitively, and its induction was the most pronounced in the kidneys and skin. Collectively, these findings suggest isoform-dependent roles for the multigene SOD family in antioxidant defenses against the oxidative stress associated with development and immune responses in these endangered sturgeon fish. [ABSTRACT FROM AUTHOR]

Maternal genes are important in directing early development and determining egg quality in fish. We here report the de novo transcriptome from four tissue libraries of the cyprinid loach, Misgurnus anguillicaudatus, and for the first time identified maternal gene transcripts in unfertilized eggs and suggest their immune system involvement. Expression profiles and functional enrichment revealed a total 24,116 transcripts were expressed as maternal transcripts in unfertilized eggs, which were involved in a wide range of biological functions and pathways. Comparison expression profiles and analysis of tissue specificity revealed that the large numbers of maternal transcripts were stored in unfertilized eggs near the late phase of ovarian maturation and before ovulation. Functional classification showed a total of 279 maternal immune-related transcripts classified with immune system process GO term and immune system KEGG pathway. qPCR analysis showed that transcript levels of identified maternal immune-related candidate genes were dynamically modulated during development and early ontogeny of M. anguillicaudatus. Taken together, this study could not only provide knowledge on the protective roles of maternal immune-related genes during early life stage of M. anguillicaudatus but could also be a valuable transcriptomic/genomic resource for further analysis of maternally provisioned genes in M. anguillicaudatus and other related teleost fishes. [ABSTRACT FROM AUTHOR]

Development of advanced cell culture methods has gained increasing attention because it allows for efficient genetic engineering and precise regulation of animal reproduction on a cellular basis. Numerous studies have attempted to develop an advanced cell culture method. Previous studies have altered cell culture media and pretreated culture plates with functional molecules. Among them, a mussel-inspired polymer coating has been extensively utilized owing to its wide applicability. For instance, adhesion of human embryonic stem cells and neuronal cells on solid surfaces has been improved. Despite the excellent capability of the mussel-inspired polymer coating, most studies have primarily focused on mammalian cells. However, the efficacy of these coatings on the adhesion of other cell lines is yet unclear. This study aimed to assess the potential of the mussel-inspired polymer coating in the regulation of the adhesion of fish ovarian germline stem cells on solid surfaces. Solid surfaces were coated by polydopamine and poly-L-lysine, and the effect of the coatings on cellular behaviors was investigated. [ABSTRACT FROM AUTHOR]