Genomic prediction for testes weight of the tiger pufferfish, Takifugu rubripes, using medium to low density SNPs.

Aquaculture production is expected to increase with the help of genomic selection (GS). The possibility of performing GS using only a small number of SNPs has been examined in order to reduce genotyping costs; however, the practicality of this approach is still unclear. Here, we tested whether the effects of reducing the number of SNPs impaired the prediction accuracy of GS for standard length, body weight, and testes weight in the tiger pufferfish (Takifugu rubripes). High values for predictive ability (0.563-0.606) were obtained with 4000 SNPs for all traits under a genomic best linear unbiased predictor (GBLUP) model. These values were still within an acceptable range with 1200 SNPs (0.554-0.588). However, predictive abilities and prediction accuracies deteriorated using less than 1200 SNPs largely due to the reduced power in accurately estimating the genetic relationship among individuals; family structure could still be resolved with as few as 400 SNPs. This suggests that the SNPs informative for estimation of genetic relatedness among individuals differ from those for inference of family structure, and that non-random SNP selection based on the effects on family structure (e.g., site-FST, principal components, or random forest) is unlikely to increase the prediction accuracy for these traits.

Research on the drag reduction property of puffer (Takifugu flavidus) spinal nonsmooth structure surface.

Puffers show good drag reduction performance during migration. It is worth noting that spines which are different from ordinary fish scales are densely distributed on the puffer skin. Here, the special morphological structure of puffer spines was observed using microscopy techniques, accurate contour models were established based on image processing techniques and curve fitting, then feature sizes were obtained. Based on the results, the nonsmooth surface was established by orthogonal test to simulate the flow field. In addition, the influence of spinal structure on boundary layer flow field and the drag reduction property of nonsmooth surface were further analyzed. The nonsmooth surface formed by spinal structure elements can effectively reduce the wall shear stress and Reynolds stress, and there was a special "climbing vortex" phenomenon, so as to reduce the surface viscous friction resistance and achieve drag reduction. Compared with the smooth surface, the drag reduction rate of the nonsmooth surface was 12.94% when the inflow velocity was 5 m/s, which revealed and verified the drag reduction performance of the spines of puffer skin. The results lay a foundation for further research and optimization of drag reduction ability of nonsmooth surface of bionic spines. HIGHLIGHTS: The contour of the spinous process was accurately reflected by the Fourier function. The spines of puffer skin have good drag reduction effect. There was a special "climbing vortex" phenomenon to explain the drag reduction property.

Effects of acute temperature and salinity changes, body length and starvation on the critical swimming speed of juvenile tiger puffer, Takifugu rubripes.

The critical swimming speed (U crit, cm s-1) of juvenile tiger puffer Takifugu rubripes was determined under different temperatures (15, 21, 25 and 30 °C), salinities (5, 10, 20, 32 and 40), body lengths (3.32, 4.08, 5.06 and 5.74 cm) and starvation days (1, 3, 6 and 9 days). Acute temperature change, body length and starvation significantly influenced the U crit of tiger puffers, whereas acute salinity change had no significant effect. The U crit increased as the temperature increased from 15 to 30 °C. The U crit increased as the body length increased from 3.32 to 5.74 cm, whereas relative critical swimming speed (U crit', body length s-1) decreased. The relationship between the body length (l, cm) and U crit or U crit' can be described by the quadratic model as U crit = - 1.4088 l 2 + 16.976 l - 11.64, R 2 = 0.9698 (P < 0.01) or U crit' = - 0.1937 l 2 + 0.9504 l + 7.7666, R 2 = 0.9493 (P < 0.01). The U crit decreased as starvation days increased from 1 to 9 days. Low temperature and starvation can reduce the swimming ability of juvenile tiger puffers. Results can be of value in evaluating the swimming ability of juvenile tiger puffers, understanding ecological processes and improving the population enhancement of tiger puffers.

The genome of the largest bony fish, ocean sunfish (Mola mola), provides insights into its fast growth rate.

BACKGROUND: The ocean sunfish (Mola mola), which can grow up to a length of 2.7 m and weigh 2.3 tons, is the world's largest bony fish. It has an extremely fast growth rate and its endoskeleton is mainly composed of cartilage. Another unique feature of the sunfish is its lack of a caudal fin, which is replaced by a broad and stiff lobe that results in the characteristic truncated appearance of the fish. RESULTS: To gain insights into the genomic basis of these phenotypic traits, we sequenced the sunfish genome and performed a comparative analysis with other teleost genomes. Several sunfish genes involved in the growth hormone and insulin-like growth factor 1 (GH/IGF1) axis signalling pathway were found to be under positive selection or accelerated evolution, which might explain its fast growth rate and large body size. A number of genes associated with the extracellular matrix, some of which are involved in the regulation of bone and cartilage development, have also undergone positive selection or accelerated evolution. A comparison of the sunfish genome with that of the pufferfish (fugu), which has a caudal fin, revealed that the sunfish contains more homeobox (Hox) genes although both genomes contain seven Hox clusters. Thus, caudal fin loss in sunfish is not associated with the loss of a specific Hox gene. CONCLUSIONS: Our analyses provide insights into the molecular basis of the fast growth rate and large size of the ocean sunfish. The high-quality genome assembly generated in this study should facilitate further studies of this 'natural mutant'.

Neural basis of the stress response in a pufferfish, Takifugu obscurus.

When faced with danger, pufferfish react with both a fast-start escape response and inflation behavior. The neural basis of these stress responses in the pufferfish has not been described. The present study reveals that during inflation behavior, the buccal cavity expands and compresses as a pump to control the direction of water flow and draws water in and out. The inflation involves a series of major anatomical modifications of the head. The greatly enlarged first branchiostegal ray and its associated hyohyoideus abductor muscle are the key mechanisms responsible for this behavior. The nerve branch innervating the hyohyoideus abductor muscle is highly developed, and its central origin at the motor nucleus of the VIIth cranial nerve was revealed by tract-tracing using the carbocyanine dye DiI. The central connections from its origin were found to be several motor nuclei in the medulla and the spinal cord, the nuclei of cranial nerve III and IV in the mesencephalon, and the pretectalis superficialis and periventricular preoptic nuclei in the diencephalon. Both the sympathetic cells and the parvocellular part of the periventricular preoptic nucleus might play a neuro-endocrine role in the rapid movement of the buccal cavity during puffing behavior in this species of pufferfish. The central circuit revealed by this study is hypothesized to mediate the C-start escape behavior and puffing behavior.

Identification and proximal tubular localization of the Mg²âº transporter, Slc41a1, in a seawater fish.

The second most abundant cation in seawater (SW), Mg²âº, is present at concentrations of ~53 mM. Marine teleosts maintain plasma Mg²âº concentration at 1-2 mM by excreting Mg²âº into the urine. Urine Mg²âº concentrations of SW teleosts exceed 70 mM, most of which is secreted by the renal tubular epithelial cells. However, molecular mechanisms of the Mg²âº secretion have yet to be clarified. To identify transporters involved in Mg²âº secretion, we analyzed the expression of fish homologs of the Slc41 Mg²âº transporter family in various tissues of SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus). Takifugu genome contained five members of Slc41 genes, and only Slc41a1 was highly expressed in the kidney. Renal expression of Slc41a1 was markedly elevated when mefugu were transferred from fresh water (FW) to SW. In situ hybridization analysis and immunohistochemistry at the light and electron microscopic levels revealed that Slc41a1 is localized to vacuoles in the apical cytoplasm of the proximal tubules. These results suggest that pufferfish Slc41a1 is a Mg²âº transporter involved in renal tubular transepithelial Mg²âº secretion by mediating Mg²âº transport from the cytosol to the vacuolar lumen, and support the hypothesis that Mg²âº secretion is mediated by exocytosis of Mg²âº-rich vacuoles to the lumen.

Histological demonstration of glucose transporters, fructose-1,6-bisphosphatase, and glycogen in gas gland cells of the swimbladder: is a metabolic futile cycle operating?

Luminal surface of the swimbladder is covered by gas gland epithelial cells and is responsible for inflating the swimbladder by generating O(2) from Root-effect hemoglobin that releases O(2) under acidic conditions. Acidification of blood is achieved by lactic acid secreted from gas gland cells, which are poor in mitochondria but rich in the glycolytic activity. The acidic conditions are locally maintained by a countercurrent capillary system called rete mirabile. To understand the regulation of anaerobic metabolism of glucose in the gas gland cells, we analyzed the glucose transporter expressed there and the fate of ATP generated by glycolysis. The latter is important because the ATP should be immediately consumed otherwise it strongly inhibits the glycolysis rendering the cells unable to produce lactic acid anymore. Expression analyses of glucose transporter (glut) genes in the swimbladder of fugu (Takifugu rubripes) by RT-PCR and in situ hybridization demonstrated that glut1a and glut6 are expressed in gas gland cells. Immunohistochemical analyses of metabolic enzymes demonstrated that a gluconeogenesis enzyme fructose-1,6-bisphosphatase (Fbp1) and a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Gapdh) are highly expressed in gas gland cells. The simultaneous catalyses of glycolysis and gluconeogenesis reactions suggest the presence of a futile cycle in gas gland cells to maintain the levels of ATP low and to generate heat that helps reduce the solubility of O(2).

Correlation with larval body size of mRNA levels of growth hormone, growth hormone receptor I and insulin-like growth factor I in larval torafugu Takifugu rubripes.

The full-length of insulin-like growth factor (IGF) complementary (c)DNAs encoded by igf-I and igf-II from torafugu pufferfish Takifugu rubripes were cloned in the present study. The deduced amino acid sequences of the two genes showed c. 80% identity each with those of Igf-I and Igf-II from other teleosts, respectively. Two growth hormone (GH) receptors, ghr1 and ghr2, were also cloned in silico using the T. rubripes Fugu genome database. The transcripts of T. rubripes igf-I were detected in slow muscle, heart, skin, gill, liver and intestine but not in fast muscle, spleen and testis of adult fish, whereas those of igf-II were found in all tissues examined. Subsequently, the accumulated messenger (m)RNA levels of igf-I and igf-II were investigated in an F(2) population derived from a male of an apparent fast-growing T. rubripes strain and a wild female T. rubripes together with those of other growth-related genes encoding Gh, Ghr1 and Ghr2, and with those of prolactin (Prl) and leptin (Lep) previously reported. The accumulated mRNA levels of igf-I, gh and ghr1 were significantly correlated to growth rate at larval stages in the population, but not for those of igf-II, prl, ghr2 and lep. Although it is unclear whether or not this phenotype is directly related to the heredity of the fast-growing strain, the findings suggest that the expression of igf-I, gh and ghr1 is involved in the regulation of growth rate at larval stages in T. rubripes.

Adaptation of pineal expressed teleost exo-rod opsin to non-image forming photoreception through enhanced Meta II decay.

Photoreception by vertebrates enables both image-forming vision and non-image-forming responses such as circadian photoentrainment. Over the recent years, distinct non-rod non-cone photopigments have been found to support circadian photoreception in diverse species. By allowing specialization to this sensory task a selective advantage is implied, but the nature of that specialization remains elusive. We have used the presence of distinct rod opsin genes specialized to either image-forming (retinal rod opsin) or non-image-forming (pineal exo-rod opsin) photoreception in ray-finned fish (Actinopterygii) to gain a unique insight into this problem. A comparison of biochemical features for these paralogous opsins in two model teleosts, Fugu pufferfish (Takifugu rubripes) and zebrafish (Danio rerio), reveals striking differences. While spectral sensitivity is largely unaltered by specialization to the pineal environment, in other aspects exo-rod opsins exhibit a behavior that is quite distinct from the cardinal features of the rod opsin family. While they display a similar thermal stability, they show a greater than tenfold reduction in the lifetime of the signaling active Meta II photoproduct. We show that these features reflect structural changes in retinal association domains of helices 3 and 5 but, interestingly, not at either of the two residues known to define these characteristics in cone opsins. Our findings suggest that the requirements of non-image-forming photoreception have lead exo-rod opsin to adopt a characteristic that seemingly favors efficient bleach recovery but not at the expense of absolute sensitivity.

[Functional morphology of puffing behavior in pufferfish (Takifugu obscurus)].

As a defensive behavior of escaping from the predators, sudden inflating ("puffing") has been found in all members of the pufferfish sister taxa Diodontidae and Tetraodontidae. However, the mechanism of inflation is largely unknown. To further understand the inflation behavior of Tetraodontidae, morphological dissection, X-ray and staining methods were conducted to investigate morphological variations and functional adaptation during the inflation in Takifugu obscurus. The results indicated that after receiving stimulation, Takifugu obscurus quickly and frequently swallows water or/and air into the flexible but tough sac, which is a distinct portion of the specialized esophagus. For adapting the inflation, the anterior and posterior joints of the sac and the digestive duct are tightened by special musculature; and the structure and arrangement of the skin of the body, as well as the abdomen muscle bundles are particularly modified to be elasticated; more over, even the backbone and the nerve are ready for flexible positioning change. This study provides some important information about the inflation mechanism of the Takifugu obscurus in sucking and expelling water/air during the puffing behavior, which would be helpful to further understand the neural control mechanism of pufferfish inflation.

Rh glycoprotein expression is modulated in pufferfish (Takifugu rubripes) during high environmental ammonia exposure.

Rhesus (Rh) protein involvement in ammonia transport processes in freshwater fish has received considerable attention; however, parallel investigations in seawater species are scant. We exposed pufferfish to high environmental ammonia (HEA; 1 and 5 mmol l(-1) NH(4)HCO(3)) and evaluated the patterns of ammonia excretion and gill Rh mRNA and protein expression. Gill H(+)-ATPase, NHE1, NHE2, NHE3, Na(+)/K(+)-ATPase (NKA), Na(+)/K(+)/2Cl(-) co-transporter (NKCC1) mRNA, H(+)-ATPase activity, NKA protein and activity, were also quantified. Activation of NKA by NH(4)(+) was demonstrated in vitro. The downregulation of Rhbg mRNA and simultaneous upregulations of Rhcg1, H(+)-ATPase, NHE3, NKA, NKCC1 mRNA, H(+)-ATPase activity, and NKA protein and activity levels suggested that during HEA, ammonia excretion was mediated mainly by mitochondria-rich cells (MRCs) driven by NKA with basolateral NH(4)(+) entry via NKA and/or NKCC1, and apical NH(3) extrusion via Rhcg1. Reprotonation of NH(3) by NHE3 and/or H(+)-ATPase would minimise back flux through the Rh channels. Downregulated Rhbg and Rhag mRNA observed in the gill during HEA suggests a coordinated protective response to minimise the influx of external ammonia via the pavement cells and pillar cells, respectively, while routing ammonia excretion through the MRCs. Exposure to hypercapnia (1% CO(2) in air) resulted in downregulated gill and erythrocyte Rhag mRNA. Surprisingly, Rhag, Rhbg, Rhcg1 and Rhcg2 proteins responded to both hypercapnia and HEA with changes in their apparent molecular masses. A dual NH(3)/CO(2) transport function of the pufferfish Rh proteins is therefore suggested. The results support and extend an earlier proposed model of pufferfish gill ammonia excretion that was based on immunolocalisation of the Rh proteins. Passive processes and/or Rhbg and Rhcg2 in the pavement cells may maintain basal levels of plasma ammonia but elevated levels may require active excretion via NKA and Rhcg1 in the MRCs.

Comparative proteomics analysis of cardiac muscle samples from pufferfish Takifugu rubripes exposed to excessive fluoride: initial molecular response to fluorosis.

Comparative proteomics was performed to identify proteins in the cardiac muscle of Takifugu rubripes in initial response to excessive fluoride. Sixteen fish were randomly divided into a control group and an experimental group. The control group was raised in softwater alone (F(-) = 0.4 mg/L), and the experimental group was raised in the same water with sodium fluoride at a high concentration of 35 mg/L. After 3 days, proteins were extracted from the fish cardiac muscle and then were subjected to 2D PAGE analysis. The matrix assisted laser desorption ionization time of flight mass spectrometry (Maldi TOF MS) was applied to identify the proteins that were differentially expressed from the two groups of fish. Average of 318 and 275 proteins detected in the control and the experimental group, respectively, 167 spots were matched and 21 highly differentially expressed proteins were further analyzed by Maldi TOF-TOF MS, and 15 were identified by MASCOT. These 15 proteins include Telomerase reverse transcriptase, 4SNc-Tudor domain protein, protein disulfide isomerase ER-60, Tuba1 protein, mitogen-activated protein kinase 10, and SMC4 protein. Consistent with their previously known functions, these identified proteins seem to be involved in apoptosis and other functions associated with fluorosis. These results will contribute to our understanding for the effects of fluoride exposure on the physiological and biochemical functions of takifugu, and the toxicological mechanism of fluoride causing fluorosis in both fish and human.

Fugu (Takifugu rubripes) sexual differentiation: CYP19 regulation and aromatase inhibitor induced testicular development.

In order to assess the involvement of aromatase CYP19 isoforms and endogenous sex steroids in gonadal sex differentiation and development of the Japanese fugu (Takifugu rubripes), an aromatase inhibitor (AI, fadrozole) was administered to developing fishes from the 'first feeding' till the 100th day after hatching. It was observed that ovarian cavity formation was inhibited by fadrozole at doses of 500 and 1000 microg/g diet, which was followed by testicular differentiation in all treated fugu. In the non-treated fugu, CYP19A was predominantly expressed in the ovary and CYP19B in the brain (in both sexes), although both were expressed interchangeably at low levels. An exceptionally high expression of CYP19B was also evident in testis throughout the study period. Both forms of CYP19 mRNA showed low levels of expression in brain and gonad with no significant differences between the two AI treatments. AI treatment inhibited CYP19A mRNA in trunk during the crucial period of ovarian cavity formation and CYP19B in gonad and brain by the end of gonadal sex differentiation. An elevation of testosterone and 11-ketotestosterone was observed which can be associated with the down-regulation of the circulating 17beta-estradiol production during the AI treatment period. After stopping AI treatment, both circulating estrogen and androgen were normalized. The current results suggest that suppression of CYP19A before and during morphological sex differentiation inhibits ovarian cavity formation in fugu. Furthermore, non-detectable limits of 17beta-estradiol and high testosterone levels by the end of the gonadal differentiation period can be ascribed to inhibition of CYP19B, suggesting that conversion of 17beta-estradiol from testosterone is plausibly regulated by CYP19B, and that this factor (CYP19B) may play an important role in AI-induced testicular development after gonadal sex differentiation through regulation of the testosterone-17beta-estradiol balance in fugu.

Takifugu obscurus is a euryhaline fugu species very close to Takifugu rubripes and suitable for studying osmoregulation.

BACKGROUND: The genome sequence of the pufferfish Takifugu rubripes is an enormously useful tool in the molecular physiology of fish. Euryhaline fish that can survive both in freshwater (FW) and seawater (SW) are also very useful for studying fish physiology, especially osmoregulation. Recently we learned that there is a pufferfish, Takifugu obscurus, common name "mefugu" that migrates into FW to spawn. If T. obscurus is indeed a euryhaline fish and shares a high sequence homology with T. rubripes, it will become a superior animal model for studying the mechanism of osmoregulation. We have therefore determined its euryhalinity and phylogenetic relationship to the members of the Takifugu family. RESULTS: The following six Takifugu species were used for the analyses: T. obscurus, T. rubripes, T. niphobles, T. pardalis, T. poecilonotus, and T. porphyreus. When transferred to FW, only T. obscurus could survive while the others could not survive more than ten days in FW. During this course of FW adaptation, serum Na+ concentration of T. obscurus decreased only slightly, but a rapid and large decrease occurred even in the case of T. niphobles, a peripheral fresh water species that is often seen in brackish river mouths. Phylogenetic analysis using nucleotide sequences of the mitochondrial 16S ribosomal RNA gene of each species indicated that the six Takifugu species are very closely related with each other. CONCLUSION: T. obscurus is capable of adapting to both FW and SW. Its genomic sequence shares a very high homology with those of the other Takifugu species such that the existing Takifugu genomic information resources can be utilized. These properties make "mefugu", which has drawn little attention from animal physiologists until this study, a useful model animal for studying the molecular mechanism of maintaining body fluid homeostasis.

Two ribeye genes in teleosts: the role of Ribeye in ribbon formation and bipolar cell development.

Ribeye is the only known protein specific to synaptic ribbon, but its function is unclear. We show that the teleost fish, Fugu and zebrafish, have two ribeye genes, ribeye a and ribeye b. Whole-mount in situ hybridization revealed that ribeye a is expressed in tissues containing synaptic ribbons, including the pineal gland, inner ear, and retina. Ribeye b is absent in the pineal gland. In the retina, ribeye a is expressed in both photoreceptors and bipolar cells, whereas ribeye b is detected only in photoreceptors. To study the function of Ribeye a in retina, we depleted it by morpholino antisense oligos. Fish deficient in Ribeye a lack an optokinetic response and have shorter synaptic ribbons in photoreceptors and fewer synaptic ribbons in bipolar cells. Their bipolar cells still target Syntaxin-3 proteins to the inner plexiform layer and have abundant vsx1 mRNA. However, they lack large synaptic terminals and show increased apoptosis. Rod bipolar cells are fewer in number and/or deficient in PKCalpha. Recovery of Ribeye a levels rescues the optokinetic response, increases the number of PKCalpha-positive bipolar cells, and stops apoptosis. We conclude that Ribeye a is important for late steps in bipolar cell development.