Microsatellite and mitochondrial DNA analyses reveal no genetic difference between two pufferfish species torafugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and karasu <Emphasis Type="Italic">T. chinensis</Emphasis>

It has been reported that the nucleotide sequences of the mitochondrial and nuclear genes of Takifugu pufferfish torafugu T. rubripes and karasu T. chinensis show 99–100% sequence identity, indicating a very close relationship between the two species. To further investigate this genetic relationship, we compared genetic variation at four microsatellite loci and at the mitochondrial control region (CR) (561 bp) between groups of T. rubripes caught at two locations [TrG, caught in the Genkai Sea off Tsushima Island in 2003 (n = 50); TrS, caught in the Suwo Sea off Kita-Kyushu in 2008 (n = 50)] and T. chinensis caught at one location (TcK, caught off the east coast of Korea in 2004; n = 50). Analyses using microsatellite loci showed that genetic diversity index values of the TrG, TrS and TcK groups were 0.9505, 0.9350 and 0.9335, respectively, while values of genetic distance and genetic differentiation between TrG and TcK (0.0543 and 0.0189, respectively) were smaller than those between TrG and TrS (0.0857 and 0.0194, respectively). Analyses using CR for the same specimens showed that genetic distances were consistent with those obtained using microsatellite loci. These results, together with our previous observations, suggest that T. rubripes and T. chinensis are very closely related and possibly can be regarded as the same species.     

Low-salinity tolerance of juvenile fugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis>

ABSTRACT:   To study the adaptability of juvenile fugu Takifugu rubripes to low-salinity environments, fish were transferred from full-strength seawater (100% SW) to freshwater (FW) and 25, 50, 75 and 100% SW, and checked for mortality over 3 days. No mortality was observed in 25–100% SW, whereas all fish died in FW. In fish transferred to 25–100% SW, blood osmolality was maintained within a physiological range. To further explore the lower limit of salinity that fugu could tolerate, fish were transferred from 100% SW to FW and 1, 5, 10, 15 and 25% SW. All fish survived in 5–25% SW, but fish died in FW and 1% SW. In fish surviving transfer to FW and 1 and 5% SW, blood osmolality was decreased to a near sublethal level of approximately 300 mOsm/kg·H₂O. Therefore, the lower limit of salinity tolerance is estimated to lie between 5 and 10% SW. Preacclimation in 25% SW for 7 days did not essentially affect the survival salinity range. Although survival rates and blood osmolality were slightly improved by preacclimation in 25% SW, blood osmolality was markedly decreased in salinities less than 10% SW, as was seen in the direct transfer. Neither chloride cell morphology nor sodium-potassium adenosinetriphosphatase activity in the gills showed a significant change following transfer to low salinities. These findings indicate that fugu can be adapted to hypoosmotic environments to some extent, exerting hyperosmoregulatory ability, although chloride cells are less likely to absorb ions in hypoosmotic environments.     

Random sequencing of genomic DNA of <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis>

ABSTRACT:   Random sequencing of the genomic DNA of Photobacterium damselae subsp. piscicida was conducted. The sequences were assembled into 930 contigs. The total length of these contigs accounts for 37.5% of the genome of P. damselae subsp. piscicida. The contigs contain 2055 open reading frames (ORFs) that have homology with genes in the GenBank database. Furthermore, some of the ORFs have homology with reported virulence related genes, and are classified as encoding colonization factors, exotoxin, and lipopolysaccharide (LPS). Thirty-seven ORFs have homology with colonization factors. In those colonization factors, 27, three, and four ORFs have homology with polar flagellar-related genes, capsule-related genes, and others (accessory colonization factors, superoxide dismutase (Cu-Zn), MshA biogenesis protein, and heme receptor genes), respectively. Five ORFs have homology with exotoxin-related genes (2 hemolysin, phospholipase, hyaluronidase, lysophospholipase L2 genes). Further, six ORFs have homology with LPS-related genes.     

Natural hybridization between <Emphasis Type="Italic">Pinctada fucata</Emphasis> and <Emphasis Type="Italic">Pinctada maculata</Emphasis> inferred from internal transcribed spacer regions of nuclear ribosomal RNA genes

ABSTRACT:   Genetic evidence of the occurrence of natural hybridization between female Pinctada fucata and male Pinctada maculata among wild pearl oysters ( n  = 20) collected for use as the mother shell for private pearl farming in the Oshima Strait at Amami-o-shima, Kagoshima Prefecture, Japan, were obtained. A polymerase chain reaction-based species identification method for Pinctada was developed using polymorphisms in the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA (rRNA) gene. This method enabled the amplification of the ITS regions using a primer set specific for P. maculata and P. fucata . However, 10 of 20 individuals morphologically identified as P. fucata had sequences specific to both P. maculata and P. fucata in the ITS region. These putative hybrids showed sequences of a maternally inherited mitochondrial 16S rRNA gene, identical to that of P. fucata . Shells of the putative hybrids were difficult to discriminate from those of P. fucata exhibiting similar taxonomic traits. Moreover, the hybrids exhibited slower growth than P. fucata but faster growth than P. maculata .     

Administration of tetrodotoxin protects artificially raised juvenile tiger puffer <Emphasis Type="Italic">Takifugu rubripes</Emphasis> from predators

We examined the effects of tetrodotoxin (TTX) administration on artificially raised tiger puffer Takifugu rubripes juvenile survival after release into a mesocosm with predators to clarify the ecological significance of TTX. Pellets containing three different concentrations of TTX [0 as the control, 7 and 14 mouse units (MU)/g diet] were fed to non-toxic artificially raised T. rubripes juveniles for 10 days. TTX accumulation in the various tissues of fish was detected except for in the control diet group. TTX administration did not affect survival or growth of the fish. One hundred fish from each diet group were released together after TTX administration into a salt pond mesocosm (2650 m²) with predators (Lateolabrax sp.) for 5 days. Survival after release was significantly higher in both the fish fed with the 7 MU TTX/g diet (62%) and the 14 MU TTX/g diet (74%) than in the control fish (32%).     

Identification of razor clams <Emphasis Type="Italic">Ensis arcuatus</Emphasis> and <Emphasis Type="Italic">Ensis siliqua</Emphasis> by PCR-RFLP analysis of ITS1 region

ABSTRACT:   Ensis arcuatus and Ensis siliqua are the most economically important species of razor clams in the European Union. Due to similarities between their shell morphology, and the differing retail value, these species are often misidentified. Therefore, it is necessary to develop an appropriate protocol to allow accurate differentiation between these species of razor clam in order to protect consumer rights, avoid commercial fraud (whether intentional or unintentional), and to enforce labeling and safety regulations. With the aim of developing a rapid and reliable method of differentiation, individuals of E. arcuatus and of E. siliqua were examined by polymerase chain reaction restriction fragment polymorphism (PCR–RFLP) using the internal transcribed spacer region 1 (ITS1). A species-specific restriction endonuclease pattern was found with the enzyme Ksp I for both species, allowing their exact identification. Thus, this work provides a simple, reliable and rapid protocol for accurate discrimination between E. arcuatus and E. siliqua , which proves useful for traceability and enabling the enforcement of labeling regulations.     

Effect of dietary fatty acid composition on the growth of the tiger puffer <Emphasis Type="Italic">Takifugu rubripes</Emphasis>

Effects of dietary fatty acid composition on the growth of the tiger puffer Takifugu rubripes were examined. Eight experimental diets were formulated with fish meal and casein as the major ingredients, providing 45.0–48.2% crude protein. Pollack and squid liver oils were used for the control diet while experimental diets contained three levels of EPA-DHA concentrated (C-HUFA) oils, soybean oil, linseed oil, and combinations of them, providing 0.5–5.5% n-3 HUFA and 5.0–11.1% crude lipid. Triplicate groups of fish with a mean body weight of 18.7 g per dietary treatment were fed the diets twice daily to apparent satiation, 6 days per week for 8 weeks. The final body weight and weight gain of the fish fed the soybean oil diet without C-HUFA oils were significantly lower than those fed the control diet. Both parameters were not statistically different among the other dietary groups, although the values obtained for the linseed oil diet without C-HUFA oils were lower. Similar trends were seen for SGR and feed efficiency. However, significantly lower PER values compared to the control were observed for the lower C-HUFA and the vegetable oil without C-HUFA oil groups. These results indicate that tiger puffer can be produced successfully with diets containing more than 1.5% n-3 HUFA.     

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.     

Species identification of <Emphasis Type="Italic">Pinctada imbricata</Emphasis> using intergenic spacer of nuclear ribosomal RNA genes and mitochondrial 16S ribosomal RNA gene regions

ABSTRACT:   For pearl production, pearl oyster seeds from foreign pearl oysters as well as hybrids between native and such foreign pearl oysters are produced in Japanese hatcheries. However, it is very difficult to identify these pearl oysters and hybrids based on morphological measurements. Thus, a molecular identification method for distinguishing Atlantic pearl oysters Pinctada imbricata from the Indian-Pacific pearl oyster group including P. martensii and P. fucata , was developed. The polymerase chain reaction (PCR) products of the partial intergenic spacer (IGS) of nuclear ribosomal RNA (rRNA) genes exhibited length polymorphism between P. imbricata (590 bp) and the other two species (427 bp). The restriction fragment length polymorphism analysis of the PCR products (PCR-RFLP) cleaved with Mse  I observed in the IGS of nuclear rRNA genes also gave different profiles between P. imbricata and the other two species. The difference in PCR-RFLP using Alu  I was also detected in the mitochondrial 16S rRNA gene regions between P. imbricata and the other two species. Thus, the method developed enables the distinction of P. imbricata from P. martensii and P. fucata .     

Digestive enzymes and in-vitro digestibility of different species of phytoplankton for culture of the freshwater pearl mussel, <Emphasis Type="Italic">Hyriopsis</Emphasis> (<Emphasis Type="Italic">Hyriopsis</Emphasis>) <Emphasis Type="Italic">bialatus</Emphasis>

Hyriopsis (Hyriopsis) bialatus has been cultured during the mussel life cycle from glochidia to the adult stage with a low total survival of 6% up to 130-day-old juveniles. The main digestive enzymes (amylase and proteinases) were not detectable in one-day-old juveniles, and increased during development. The stomach, including digestive glands, was the major digestive organ for both carbohydrate and protein. The optimum conditions for amylase activity were 40°C and pH 7; for acidic proteinases they were 60°C and pH 5. Two main alkaline proteinases were found in the intestine, with optimum conditions of 30°C and pH 8 and 60°C and pH 8. To improve mussel survival by finding suitable phytoplankton species and age as food for juveniles and adults, an in-vitro digestibility test was performed on ten algal species three and seven days old using amylase and proteinases in crude enzyme extracts from different mussel life stages. Among the phytoplankton selected, the three most efficiently digested by juveniles were seven-day-old Chlorella sp.2, seven-day-old Chlorococcum sp. and seven-day-old Kirchneriella incurvata, in the ratio 1:1:3 for 30-day-old juveniles and 3:1:1 for 130-day-old juveniles. For the adult mussel, three-day-old Chlorella sp.2, seven-day-old Coccomyxa sp., and seven-day-old Monoraphidium sp., in the ratio 3:1:1, were the most digestible phytoplankton. Levels of in-vitro digestibility were related to the quality (not the concentrations) of carbohydrate and protein in the phytoplankton mixtures, and protein digestibility seemed to be the key factor determining food quality for the mussel.     

Comparison of larval thermal maxima between <Emphasis Type="Italic">Fundulus heteroclitus</Emphasis> and <Emphasis Type="Italic">F. grandis</Emphasis>

Fundulus heteroclitus and F. grandis are resident salt marsh fishes that overlap in distribution over a narrow range in northeastern Florida. The objective of the present study was to examine whether the limits of the species’ ranges could be explained by differences in thermal tolerance. Two populations of each species were collected and then spawned in the laboratory, and 9-day-old larvae were used for critical thermal maxima trials. Mean LOE temperatures of larvae ranged from 43.04 to 43.65°C and showed little difference between species. Therefore, differences in high temperatures experienced cannot account for the differences of the distributions of the two species. Condition-specific competition may play a greater role in determining the observed range of the two species.     

Rapid identification of eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">Anguilla anguilla</Emphasis> by polymerase chain reaction with single nucleotide polymorphism-based specific probes

ABSTRACT:   Standard molecular techniques, such as sequencing and restriction fragment length polymorphism analysis after polymerase chain reaction (PCR) amplification are relatively complicated, and species identification can take a long time when using such techniques. We established a quick method, using PCR with species-specific TaqMan Minor Groove Binder (MGB) probes based on single nucleotide polymorphism (SNP) to distinguish the two eel species Anguilla japonica and Anguilla anguilla . This method can be used in processed products. Partial sequences of the mitochondrial 16S rRNA gene were compared between A. japonica and A. anguilla to design a primer pair common to both A. japonica and A. anguilla and probes specific to A. japonica and A. anguilla . Different fluorescence intensities were produced in two PCR microtubes each containing A. japonica - and A. anguilla -specific probes for one target sample. We observed the fluorescence intensity of PCR products in microtubes under ultraviolet transillumination, with similar results to those obtained by real-time PCR. Therefore, SNP-based PCR is a powerful tool for identifying materials of processed foods from either A. japonica or A. anguilla .     

Development of nuclear DNA markers to characterize genetically diverse groups of <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis> and its closely related species

Repetitive DNA sequences, ManDra and ManBgl, were isolated from the DraI and BglII digests of the genomic DNA of Misgurnus anguillicaudatus, respectively. A primer set of ManDra distinguished two genetically different groups (A and B) of M. anguillicaudatus by specific electrophoretograms. A primer set of ManBgl amplified the DNA of M. anguillicaudatus and M. mizolepis. The individuals of M. anguillicaudatus were divided into two groups depending on the fragment sizes, in which the groups A and B (B-1 and B-2) showed 400 and 460 bp, respectively. M. mizolepis was distinguished by a different pattern (400-, 460-, and 510-bp fragments). PCR–RFLP analyses of recombination activating gene 1 gave a clear difference between A or B-2 (443-bp fragment) and B-1 groups (296- and 147-bp fragments). Clonal lineages and hybrids between B-1 and B-2 groups could be identified by appearance of three fragments (443, 296, and 147 bp). The combined analyses using the above three nuclear markers discriminated among nuclear genomes of genetic groups (A, B-1 and B-2) of M. anguillicaudatus and M. mizolepis. In several localities, natural hybridizations between the group B-1 and B-2 loaches and introgressions of clonal mitochondrial genomes into the group B-1 loaches were detected.     

Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

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.