Comparison of behavioral responses to a novel environment between three teleosts,bluegill <Emphasis Type="Italic">Lepomis macrochirus</Emphasis>, crucian carp <Emphasis Type="Italic">Carassius langsdorfii</Emphasis>, and goldfish <Emphasis Type="Italic">Carassius auratus</Emphasis>

To investigate the emotional reactivity of fish in a novel environment, the swimway test was developed. The swimway apparatus consists of a shaded start chamber and an open, illuminated swimway. Fish were first introduced and habituated to the start chamber. A door partitioning the start chamber from the swimway was then opened, and behavioral responses of the fish in the apparatus were measured. By using the swimway test, behavioral responses to a novel environment of bluegill Lepomis macrochirus, crucian carp Carassius langsdorfii, and goldfish Carassius auratus were quantified and compared. The emotional reactivities in blue gill were found to be the lowest and crucian carp the highest, indicating bluegill are relatively active or ‘bold’, and that the crucian carp are relatively passive or ‘shy’, in a novel environment. It is suggested that the swimway test is applicable to examning inter-species differences in relative emotional reactivity or boldness in a simplified novel situation.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Plasma biochemical responses of the omnivorous crucian carp (<Emphasis Type="Italic">Carassius auratus</Emphasis>) to crude cyanobacterial extracts

Healthy crucian carp (Carassius auratus) were treated by intraperitoneal (i.p.) injection of crude cyanobacterial extracts at two doses, 50 and 200 μg MC-LR equiv kg⁻¹ BW. High mortality (100%) was observed within 60 h post injection in the high-dose group. In the treated fish, activities of four plasma enzymes, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), all showed substantial increases, with both dose and time-dependent effects. These increases of enzyme activity indicate severe impairment occurred in the liver of crucian carp over time. Plasma concentrations of energy-related biomolecules including glucose (GLU), cholesterol (CHO), triglyceride (TG), and total protein (TP) showed marked changes in the high-dose group, possibly a nutritional imbalance correlated with the liver injury caused by intraperitoneal exposure to crude cyanobacterial extracts.     

Seasonal distribution of adult crucian carp nigorobuna <Emphasis Type="Italic">Carassius auratus grandoculis</Emphasis> and gengoroubuna <Emphasis Type="Italic">Carassius cuvieri</Emphasis> in Lake Biwa,Japan

Seasonal habitat use by nigorobuna Carassius auratus grandoculis Temminck et Schlegel and gengoroubuna Carassius cuvieri (Temminck et Schlegel) in Lake Biwa was investigated using acoustic telemetry. Twenty-three nigorobuna and 11 gengoroubuna specimens caught using set-nets in the lake’s south basin were surgically fitted with acoustic transmitters and then released. Signals from the fishes were recorded by 23 receivers installed around the lake. Between April and June 2007, the first spawning season after release, signals were received from all tagged fishes. Thereafter, until the second spawning season, signals were collected from 26 and 45% of released nigorobuna and gengoroubuna individuals, respectively. Seasonal habitat preferences for these species were studied by distance-based analysis. The analysis revealed that nigorobuna tended to stay near their spawning area in the south basin of the lake throughout the year, whilst gengoroubuna tended to show a seasonal migration pattern between the north and south basins. After the spawning season, the latter species migrated to the north basin where it remained until the next spawning season, when it returned to the south basin. This is the first report of seasonal migration of nigorobuna and gengoroubuna in Lake Biwa.     

Molecular cloning of CD4 from ginbuna crucian carp <Emphasis Type="Italic">Carassius auratus langsdorfii</Emphasis>

ABSTRACT:   The clonal triploid ginbuna crucian carp Carassius auratus langsdorfii , a naturally occurring gynogenetic fish, is a useful model for studying T-cell-mediated immunity. CD4, a T-cell receptor (TCR) coreceptor, is a membrane-bound glycoprotein found on helper T-cells, and assists in the binding of major histocompatibility complexes. In the present study, full-length cDNAs encoding the CD4 molecule from the S3n strain of ginbuna crucian carp were cloned and characterized. 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE yielded two distinct cDNA clones of CD4 homolog from the ginbuna, and these sequences share 95% identity at the amino acid level. These ginbuna CD4 molecules consisted of a signal peptide, immunoglobulin superfamily (IgSf) like domains, a transmembrane domain, and a cytoplasmic domain similar to other known CD4. A tyrosine protein kinase p56^lck binding motif is conserved in the cytoplasmic tail of ginbuna CD4. Phylogenetic tree analysis indicated that ginbuna CD4 sequences are closely related to CD4L-1 from other fish species. Expression of ginbuna CD4 mRNA was detected in the gill, thymus, head kidney, trunk kidney and peripheral blood leukocytes, indicating that its expression pattern is similar to that of ginbuna TCRβ mRNA. The results suggest that ginbuna CD4 sequences are useful as molecular probes for helper T-cells.     

Establishment and characterization of a heart-derived cell line from goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25 °C in M199 medium supplemented with 10–20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 10^5.0, 10^4.5, and 10^5.0 TCID₅₀/0.1 mL, respectively, within 7 days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.     

Cloning and characterization of <Emphasis Type="Italic">Bax1</Emphasis> and <Emphasis Type="Italic">Bax2</Emphasis> genes of <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> and evaluation of transcript expression in response to grass carp reovirus infection

Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.     

Evidence that <Emphasis Type="Italic">ghrelin</Emphasis> may be associated with the food intake of gibel carp (<Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>)

Ghrelin, a non-amidated peptide hormone, is a potent anorectic neuropeptide implicated in feeding regulation in mammals and non-mammalian vertebrates. However, the involvement of ghrelin in the feeding behavior of teleosts has not been well understood. To better understand the role of ghrelin in the regulation of appetite in fish, in this study, we cloned the cDNAs encoding ghrelin and investigated their mRNA distributions in gibel carp tissues. We also assessed the effects of different nutritional status on ghrelin mRNA abundance. Ghrelin mRNAs were ubiquitously expressed in ten tissues (intestine, liver, brain, mesonephron, head kidney, spleen, skin, heart, muscle, gill and pituitary gland), and relatively high expression levels were detected in the gut. Postprandial studies analysis revealed a significant postprandial decrease in ghrelin mRNA expression in the gut (1 and 3 h after the regular feeding time). In addition, ghrelin mRNA expression in the gut significantly increased at day 7 after fasting and declined sharply after refeeding, which suggested that ghrelin might be involved in the regulation of appetite in gibel carp. Overall, our result provides basis for further investigation into the regulation of feeding in gibel carp.     

<Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> from tilapia (<Emphasis Type="Italic">Oreochromis</Emphasis> sp.) transmitted to a new host,bighead carp (<Emphasis Type="Italic">Aristichthys nobilis</Emphasis>), in China

Several outbreaks of Streptococcus agalactiae infection of bighead carp (Aristichthys nobilis) were observed in China. The molecular epidemiology and pathogenicity of S. agalactiae in bighead carp and tilapia (Oreochromis sp.) is poorly understood. In the present study, we identified S. agalactiae strains isolated from diseased bighead carp using the API 20 Strep kit and 16S rDNA sequencing and determined whether these strains came from tilapia. Of the 46 identified S. agalactiae strains, 24 strains were successfully isolated from diseased bighead carps, 20 S. agalactiae strains were isolated from tilapia, and two S. agalactiae strains were isolated from tiger frog (Hoplobatrachus chinensis). The results of molecular typing, including multilocus sequence typing, molecular serotyping, surface protein gene detection, and virulence-related gene detection showed that the 44 strains from bighead carp and tilapia were highly similar, whereas different from tiger frog GBS strains. Remarkably, the bighead carp strain Hn1404 showed high virulence in bighead carp and zebrafish. Moreover, this strain was pathogenic to Nile tilapia (Oreochromis niloticus). In addition, comparative genomic analysis showed that isolate Hn1404 had a close relationship with the bighead carp and tilapia S. agalactiae strains. All the analyses of the genetic characteristics of bighead carp and tilapia strains showed that tilapia S. agalactiae strains could be transmitted to other fish species such as bighead carp.     

Effects of low-voltage constant direct current on plasma biochemical profiles and gene expression levels in crucian carp <Emphasis Type="Italic">Carassius carassius</Emphasis>

This study investigated the plasma biochemical profiles and gene expression levels of crucian carp Carassius carassius subjected to anesthetization with low-voltage constant direct current (DC) and tricaine methanesulfonate (MS222). Fish in the MS222 treatments and the DC treatments were exposed to either two concentrations of MS222 (100 and 200 mg/l) or constant DC with a voltage of 26 V, at a voltage gradient of 0.68 V/cm, respectively. The results showed that low-voltage constant DC immobilized fish more quickly, resulting in significant elevations of plasma cortisol levels compared to exposure of high or low doses of MS222. Electronarcosis led to an increased expression of heat shock proteins 70 (HSP70) as well as HSP90 compared to the treatment group with MS222. Also, electronarcosis enhanced mitochondrial respiratory chain gene (atp6, cox1 and nd5) and antioxidant enzyme gene (CAT, SOD and GSH-px1) expression levels. In conclusion, low-voltage constant DC can disturb fish plasma biochemical profiles and gene expression levels.     

Isolation and expression analyses of the <Emphasis Type="Italic">Sox9a</Emphasis> gene in triploid crucian carp

To investigate the evolutional significance of Sox9 in fish, we isolated and characterized Sox9a cDNA and genomic clones in triploid crucian carp. The cDNA encoded a protein of 457 amino acids with an HMG box and showed more than 60% amino acid sequence identity with known vertebrate Sox9 proteins. Triploid crucian carp and vertebrate Sox9s showed similar gene structure, and two introns in the coding region were located at conserved positions. On the basis of the amino acid sequences, Sox9a can be categorized into the same subgroup of Sox-E proteins as Sox8, 9, and 10. Interestingly, the expression of triploid crucian carp Sox9a was predominantly observed not in the ovary but in the testis by Northern blot and RT-PCR analysis. The expression analysis of Sox9a suggested that it may seldom contribute to the formation of normal functions of spermatozoa, but it may play an important role in the development of testicular tubules. Besides the testicular expression, Sox9a was also shown to be expressed in many other tissues including the brain, kidney, and heart of triploid crucian carp, indicating that Sox9 may have unique functions in some specific tissues during development.     

Homogeneity of <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> from farmed amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> in Japan

Streptococcus dysgalactiae strains have been isolated from cultured amberjack Seriola dumerili and yellowtail Seriola quinqueradiata in Japan. To characterize the fish isolates, we performed genetic analysis and compared the biochemical properties of these isolates with those of the S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis strains isolated from mammals. The genetic analysis revealed that the fish isolates were genetically very similar to each other with high DNA–DNA relatedness (>95.4%) and sequence homology. Meanwhile, the DNA relatedness between mammalian isolates and the fish isolates was 73.4–82.6%. In biased sinusoidal gel electrophoresis (BSFGE) analysis, the restriction patterns of mammalian isolates were different from those of fish isolates. The fish isolates did not show streptokinase activity in plasminogen obtained from mammals. These characteristics enabled us to distinguish between the fish isolates and the Sdd and Sde strains isolated from mammals. In order to obtain epidemiological information on the fish isolates, BSFGE patterns from 284 S. dysgalactiae strains from fish in Japan were examined. Based on the results of BSFGE analysis, the fish isolates were classified into 16 groups (AP1–AP16) with restriction enzyme ApaI. The dendrogram based on BSFGE analysis indicated that all fish isolates using in this study were closely related.     

Purification and characterization of the amylase from a small abalone <Emphasis Type="Italic">Haliotis sieboldii</Emphasis>

ABSTRACT:   Amylase, with MW of 59 kDa, was purified from small abalone Haliotis sieboldii by ammonium sulfate fractionation, CM Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The optimal pH and temperature of purified amylase were 6.0 and 37°C, respectively. The purified enzyme was stable at pH 6.0–8.0 and low temperatures. It was activated by Ba²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, K⁺, Ag⁺, Na⁺ and Li⁺, but completely or partially inhibited by Al³⁺, Cu²⁺, Cd²⁺, Hg²⁺ and Zn²⁺. EDTA could completely inhibit, while iodoacetamide, N-ethylmaleimide and urea partially inhibit the purified amylase. According to the digestion mode of various polysaccharides, the purified enzyme was considered to be an α-amylase.     

Diel cyclic hypoxia alters plasma lipid dynamics and impairs reproduction in goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

Diel cyclic hypoxia occurs with varying frequency and duration in freshwater habitats, yet little is known about its effects on reproduction of freshwater fishes. The present study shows that long-term exposure of goldfish (Carassius auratus) to cyclic hypoxia (0.8 ± 0.2 mg/l dissolved oxygen) for 9 h or more, per day, altered plasma lipid and sex steroid profiles, which in turn directly or indirectly suppressed ovarian growth and viable spermatozoa production. Hypoxia decreased total cholesterol and high density lipoprotein (HDL p < 0.05) and elevated triglycerides (TG; p < 0.05) in both sexes. Plasma steroid concentrations particularly of 17α-hydroxyprogesterone (17-HP), estradiol (E2), testosterone (T) in females, and T and 11-ketotestosterone (11-KT) in males were attenuated under diel hypoxic conditions. Intriguingly, both diel and continuous hypoxia elevated plasma E2 and vitellogenin levels in males. However, neither lipid nor steroid profiles recorded any variation in a dose-dependent manner in response to diel hypoxia. The reduced GSI, decreased number of tertiary oocytes, and motile spermatozoa in hypoxic fish clearly indicate suppression of gametogenesis. Thereby, prolonged diel cyclic hypoxia may affect valuable fishery resources and fish population structure by impairing reproductive performances and inducing estrogenic effects in males.     

Characterization of <Emphasis Type="Italic">kiss2</Emphasis> and <Emphasis Type="Italic">kissr2</Emphasis> genes and the regulation of kisspeptin on the HPG axis in <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic–pituitary–gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5′-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.     

Effect of prebiotic xylooligosaccharides on growth performances and digestive enzyme activities of allogynogenetic crucian carp (<Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>)

The effect of prebiotic xylooligosaccharides (XOS) on the growth performance and digestive enzyme activities of the allogynogenetic crucian carp, Carassius auratus gibelio, was investigated. XOS was added to fish basal semi-purified diets at three concentrations by dry feed weight: diet 1, 50 mg kg⁻¹; diet 2, 100 mg kg⁻¹; diet 3, 200 mg kg⁻¹, respectively. Twelve aquaria (n = 20) with three replicates for each treatment group (diets 1–3) and control treated without XOS were used. Weights of all collected carp from each aquarium were determined at the initial phase and at the end of the experiment, and the carp survival was also determined by counting the individuals in each aquarium. After 45 days, there were significant differences (P < 0.05) in the relative gain rate (RGR), and daily weight gain (DWG) of diets 1–3 were compared with the control. However, the survival rate was not affected (P > 0.05) by the dietary treatments. For enzymatic analysis, dissection produced a crude mixture of intestine and hepatopancreas of each segment to measure. The protease activity in the intestine and hepatopancreas content of fish in diet 2 (487.37 ± 20.58 U g⁻¹ and 20.52 ± 1.93 U g⁻¹) were significantly different (P < 0.05) from that in the control (428.13 ± 23.26 U g⁻¹ and 12.81 ± 1.52 U g⁻¹) and diet 3 (428.00 ± 23.78 U g⁻¹ and 14.04 ± 1.59 U g⁻¹). Amylase activity in the intestine was significantly higher for diet 2 compared to diet 1 and the control. As for amylase in the hepatopancreas, assays showed higher activity in diet 2 (P < 0.05) compared to the rest.