Inflexibility of vertebral number in chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis>

Chum salmon Oncorhynchus keta eggs from the Chitose and Shiriuchi rivers in Hokkaido, northern Japan, were incubated under identical conditions with the aim of comparing vertebral number (V _N) to that reported archived data from the 1950s. The comparison revealed that mean V _N of salmon from the Shiriuchi River has remained unchanged for 50 years and is higher than that from the Chitose River. The V _N in the Chitose River salmon was found to be significantly higher than that recorded in the archival data. The variation in the mean V _N of chum salmon fry collected during different seasons and years in the Chitose River falls within a range that has remained constant, suggesting that the difference between the values obtained in our study and the archival data may be due to differences in the timing of egg collection. These results indicate that the mean V _N of chum salmon has been stable for a long period. Incubation experiments under identical conditions are a useful tool for examining changes in genetic structure and detecting the essential properties of populations.     

Immunostimulatory effect of salmon prolactin on expression of Toll-like receptors in <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> infected with <Emphasis Type="Italic">Piscirickettsia salmonis</Emphasis>

In aquaculture, antibiotics are the traditional treatment used against bacterial infections. However, their use has increasingly come into question given their effects on fish and, possibly, on human health. Consequently, there is interest in developing alternative treatments aimed at stimulating the innate immune response of fish, which is the first line of defense against pathogens. In relation to this, the Toll-like receptors (TLR) aid in the selective identification of pathogens. The present study evaluated immunostimulatory activity of prolactin (PRL) hormone on expression levels of TLR1, 9, and 22, MyD88, and IL-1β during in vitro infection with the fish pathogen Piscirickettsia salmonis, in primary cultures of Oncorhynchus mykiss head kidney cells. Results indicated that PRL increased expression of TLRs and MyD88 during the first hours of bacterial infection, while a constant increase in expression was found for IL-1β. These findings suggest that PRL indirectly modulates expression of TLRs by activating expression of suppressors of cytokine signaling, thereby regulating immune response over long periods of time during bacterial infection.     

Differences in longitudinal distribution patterns along a Honshu stream of brown trout <Emphasis Type="Italic">Salmo trutta</Emphasis>, white-spotted charr <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis> and masu salmon <Emphasis Type="Italic">Oncorhynchus masou</Emphasis>

Brown trout Salmo trutta were first introduced into Japan in 1892, and they currently naturally reproduce in several rivers in Honshu and Hokkaido, Japan. Although negative impacts of brown trout introductions on native salmonid fishes have been documented in some Hokkaido rivers, studies of ecological interactions between brown trout and native salmonid fishes on Honshu are limited. In this study, we describe the longitudinal distribution patterns of introduced brown trout, white-spotted charr Salvelinus leucomaenis and masu salmon Oncorhynchus masou in a 4 km stretch of a stream in central Honshu. Underwater observations were conducted in all pools within upstream, middle and downstream sections (190–400 m in length) of this stretch in order to estimate the densities of these species. Only white-spotted charr was observed in the upstream section, while brown trout and masu salmon were observed in the middle and downstream sections. Masu salmon densities, however, were much lower than brown trout densities. In the downstream section, white-spotted charr was absent. These results are consistent with results from previous studies of Hokkaido rivers, where it was found that white-spotted charr in low-gradient areas tend to be displaced by brown trout.     

Quantitative analysis of <Emphasis Type="Italic">Ichthyobodo salmonis</Emphasis> an ectoparasitic flagellate infecting juvenile chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> in hatcheries

Ichthyobodosis caused by the ectoparasitic flagellate Ichthyobodo salmonis is a significant cause of mortality in juvenile chum salmon Oncorhynchus keta reared in hatcheries of northern Japan. The present study established a real-time quantitative polymerase chain reaction assay (qPCR) of I. salmonis ribosomal DNA (rDNA) using SYBR Green. This assay allows monitoring of parasite infections for the epidemiological study and control of ichthyobodosis in hatcheries. qPCR showed high reproducibility for measurements between 1.0 and 1.0 × 10⁸ rDNA copy/μl. There was a significant positive correlation between the number of parasites and the amount of I. salmonis rDNA. A survey using the qPCR assay indicated that infection by I. salmonis was present in 23 of 87 hatcheries; parasite loads were estimated to be between 50 and 750 parasites/g juvenile body weight. These results demonstrate that our qPCR assay enables the surveying of juvenile chum salmon reared in hatcheries for infection by I. salmonis.     

Estimation of heritability of vertebral number in chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis>

Hereditary causes of variation in the vertebral number of chum salmon were analyzed to estimate heritability for populations in the Chitose and Shikiu Rivers, Hokkaido Island, northern Japan. A total of 70 families were produced by diallel cross mating and then heritability was estimated using two statistical tests: a two-way ANOVA and a parent-offspring regression. Heritability estimates ranged from 0.26 to 1.91 and showed a strong correlation between the values from the two different tests. Estimates from female components were substantially higher than those from male components, which can be attributed to the maternal effect. Heritability in the Shikiu River population was shown to be higher than that in the Chitose River population. These results indicate that the heritability of vertebral number in chum salmon is generally high, and also that vertebral number is controlled by hereditary causes.     

Farming evaluation of the brownbow triploid hybrid (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis><Emphasis Type="Italic">Salmo trutta</Emphasis>)

Triploid hybrids between female rainbow trout (Oncorhynchus mykiss Walbaum) and male brown trout (Salmo trutta L.) were tested for farming performances, with reference to parental species. The main drawback of hybrids lay in embryonic and larval mortalities, amounting to 60% on average, and displaying a large variability between spawns. Further survival was inferior to that of diploid, but similar to that of triploid rainbow trout. Hybrid body weight was intermediate between weights of rainbow and brown trout of the same age, mainly as a consequence of differences in precocious growth. Analysis of relative growth rates from 6 to 18 months showed that hybrids were surpassed by rainbow controls in common rearing, but not in separate rearing. Hybrid behaviour was similar to that of rainbow trout. These results are discussed in the scope of providing fisheries managers with original and sterile game fishes. An erratum to this article can be found at .     

Purification and properties of digestive lipases from Chinook salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) and New Zealand hoki (<Emphasis Type="Italic">Macruronus novaezelandiae</Emphasis>)

Lipases were purified from delipidated pyloric ceca powder of two New Zealand-sourced fish, Chinook salmon (Oncorhynchus tshawytscha) and hoki (Macruronus novaezelandiae), by fractional precipitation with polyethylene glycol 1000, followed by affinity chromatography using cholate-Affi-Gel 102, and gel filtration on Sephacryl S-300 HR. For the first time, in-polyacrylamide gel activity of purified fish lipases against 4-methylumbelliferyl butyrate has been demonstrated. Calcium ions and sodium cholate were absolutely necessary both for lipase stability in the gel and for optimum activity against caprate and palmitate esters of p-nitrophenol. A single protein band was present in native polyacrylamide gels for both salmon and hoki final enzyme preparations. Under denaturing conditions, electrophoretic analysis revealed two bands of 79.6 and 54.9 kDa for salmon lipase. It is proposed that these bands correspond to an uncleaved and a final form of the enzyme. One band of 44.6 kDa was seen for hoki lipase. pI values of 5.8 ± 0.1 and 5.7 ± 0.1 were obtained for the two salmon lipase forms. The hoki lipase had a pI of 5.8 ± 0.1. Both lipases had the highest activity at 35°C, were thermally labile, had a pH optimum of 8–8.5, and were more acid stable compared to other fish lipases studied to date. Both enzymes were inhibited by the organophosphate paraoxon. Chinook salmon and hoki lipases showed good stability in several water-immiscible solvents. The enzymes had very similar amino acid composition to mammalian carboxyl ester lipases and one other fish digestive lipase. The salmon enzyme was an overall better catalyst based on its higher turnover number (3.7 ± 0.3 vs. 0.71 ± 0.05 s⁻¹ for the hoki enzyme) and lower activation energy (2.0 ± 0.4 vs. 7.6 ± 0.8 kcal/mol for the hoki enzyme) for the hydrolysis of p-nitrophenyl caprate. The salmon and hoki enzymes are homologous with mammalian carboxyl ester lipases.     

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.     

Preparation of biologically active monomeric recombinant zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) and rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) recombinant growth hormones

Fish growth hormones (GHs) play an important role in regulating growth, metabolism, reproduction, osmoregulation, and immunity and have thus garnered attention for their application in aquaculture. Zebrafish GH (zGH) cDNA or rainbow trout GH (rtGH) cDNA was cloned into the pMon3401 vector, expressed in MON105-competent Escherichia coli and purified to homogeneity. Their biological activity was evidenced by their ability to interact with ovine GH receptor extracellular domain and stimulate GH receptor-mediated proliferation in FDC-P1-3B9 cells stably transfected with rabbit GH receptor. The relative affinity of zGH and rtGH, estimated by IC₅₀, was about 38-fold and 512-fold lower, respectively, than ovine GH. This is likely the reason for the low biological activity in cells with rabbit GH receptor, ~?36-fold lower for zGH and ~?107-fold lower for rtGH than for human GH. This was not due to improper refolding, as evidenced by circular dichroism analysis. Predicting the activity of fish GHs is problematic as there is no one single optimal in vitro bioassay; heterologous assays may be ambiguous, and only homologous assays are suitable for measuring activity.     

Anesthesia induces stress in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>), Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>) and Atlantic halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

Stress in response to anesthesia with benzocaine, MS-222, metomidate and isoeugenol was studied in Atlantic salmon (Salmo salar), Atlantic halibut (Hippoglossus hippoglossus), and Atlantic cod (Gadus morhua) with no concomitant stress from handling or confinement in association with anesthesia or sampling. All of the anesthetics tested induced a stress response in all species, displayed by a release of cortisol to the water. MS-222 anesthesia elicited the highest cortisol release rates, reaching maximum levels 0.5 h post-exposure and returning to basal levels after 3–4 h. Benzocaine anesthesia caused a bimodal response where the initial peak in cortisol release rate was followed by a second increase lasting towards the end of the trial (6 h). This bimodality was more profound in Atlantic salmon than in Atlantic halibut and Atlantic cod. Metomidate anesthesia induced the lowest release of cortisol of the agents tested in both Atlantic halibut and Atlantic cod, but resulted in a bimodal response in Atlantic salmon where the initial increase in cortisol release was followed by a larger increase peaking at 2–2.5 h post exposure before returning to basal levels after 5 h. The stress induced in Atlantic salmon by isoeugenol anesthesia resembled that of MS-222, but did not reach the same elevated level. Overall, the cortisol release was most profound in Atlantic salmon followed by Atlantic halibut and Atlantic cod.     

Probiotic bacteria <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> influences the blood profile in rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> (Walbaum)

This paper reports the effect of feeding probiotic diets on blood profiles in rainbow trout. Two experiments were performed: in the first, fish of average weight 75 g were offered either a commercial feed or the same incorporated with 10⁹ CFU g⁻¹ of lactic acid bacteria Lactobacillus rhamnosus for 30 days; in the second study performed for a similar duration, fish of average weight 126 g were offered formulated diets that either contained the same bacteria in heat-killed or freeze-dried form (nearly 10¹¹ CFU g⁻¹), or the basal diet without the bacteria. Blood samples were collected at different times after commencement of probiotic feeding to determine the total cholesterol, triglyceride contents, the plasma alkaline phosphatase activity, plasma protein and hematocrit value. The plasma cholesterol significantly increased upon probiotic feeding in the first experiment. A significant elevation (P < 0.05) of plasma cholesterol and triglyceride and alkaline phosphatase activity level was found in the freeze-dried probiotic fed groups at 20 and 30 days postfeeding. This was concomitant with the increased plasma protein and hematocrit values in FD group at 20 and 30 days. Likewise, the heat-killed probiotic fed group registered significantly high values of triglycerides, alkaline phosphatase activity, and plasma protein compared to the control diet fed groups after 20 days of feeding. Thus, alterations in the blood profiles could serve as supplementary information when examining the benefits of probiotics for fish.     

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.     

Detection of hybrids between masu salmon <Emphasis Type="Italic">Oncorhynchus masou masou</Emphasis> and amago salmon <Emphasis Type="Italic">O. m. ishikawae</Emphasis> occurred in the Jinzu River using a random amplified polymorphic DNA technique

Identification of masu salmon Oncorhynchus masou masou and amago salmon O. m. ishikawae was accomplished using a random amplified polymorphic DNA (RAPD) technique. Of 80 primers screened, three primers amplified subspecies-specific fragments (OPA11-1095 ^*, OPB5-1618^* and OPD5-2038 ^*). Based on fragment patterns, 33 hybrids were detected among 150 upstream-migrating individuals in the Jinzu River, central Japan, a long-time habitat of masu salmon, to which amago salmon were recently introduced. All of the individuals examined in the 2000–2002 cohorts were identified as F ₁ and F _x as well as pure masu and amago forms. These results indicated continuing hybridization, the genetic pollution of the native masu salmon population by amago salmon possibly being a serious problem in the Jinzu River. Both standard length and body weight in F ₁ hybrids tended to be less than in pure masu salmon. However, F _x hybrids showed similar body sizes to masu salmon.     

<Emphasis Type="Italic">Neoheterobothrium hirame</Emphasis> (Monogenea) alters the feeding behavior of juvenile olive flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We monitored feeding behavior and survival of starved juvenile olive flounder experimentally infected with the gill monogenean Neoheterobothrium hirame. Infected flounder increased amount of the time spent in the water column by 117% when trying to capture live mysids, Neomysis sp. They also showed different feeding patterns from those of uninfected fish and made fewer attacks towards prey during one feeding attempt. Although the average numbers of mysids captured by individuals were similar between infected and uninfected fish, heavily infected fish tended to catch less prey. These results indicate that N. hirame reduces the feeding efficiency of the host for capturing live prey and possibly makes them more vulnerable to predation during feeding. We could not detect any obvious difference in survival rates between uninfected, lightly and heavily infected fish during 3 months of starvation. There was no evidence that starvation makes fish more susceptible to N. hirame. The present study provides first experimental evidence that N. hirame affects feeding behavior of juvenile olive flounder and supports the idea that this parasite indirectly reducing the host’s survival and may be responsible for the recent reduction of the flounder population in Japan.     

Effect of <Emphasis Type="Italic">Cratoxylum formosum</Emphasis> on innate immune response and disease resistance against <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> in tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The effect of aqueous extract of Cratoxylum formosum on innate immune response and disease resistance in tilapia Oreochromis niloticus was investigated. The fish were fed diets containing 0% (control), 0.5% (diet 1), 1% (diet 2), and 1.5% (diet 3) of C. formosum aqueous extract for 30 days. At the end of the experimental period, parameters of innate immune response including phagocytosis of blood leukocytes, lysozyme activity in plasma, and respiratory bust activity were examined. Feeding the fish with diet 2 and diet 3 for 20 days enhanced phagocytic activity and for 30 days stimulated lysozyme and respiratory burst activities. Diet 1 increased the phagocytic activity at 30 days, but did not affect the other measured parameters. All parameters were not significantly changed (P > 0.05) in the control group throughout the experiment. Following 30 days of feeding, fish were infected with S. agalactiae. The cumulative mortalities of bacterial-infected tilapia that were fed diet 1, diet 2, and diet 3 were 56, 12, and 10%, respectively, compared with 85% in the control group. These results indicate that the aqueous extract of C. formosum may elevate the innate immune response and enhance disease resistance in tilapia.     

Effect of riverbed conditions on survival of planted eyed eggs in chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis>

Wild stocks of chum salmon Oncorhynchus keta are supplemented by hatchery fry enhancement programs in northern Honshu, Japan. To maintain these programs, there is a need to reduce expenses and labor. Eyed egg planting is more cost effective than hatchery production of fry. Therefore, we evaluated the effect of environmental conditions on survival of chum salmon eyed eggs planted using Whitlock–Vibert boxes. We measured the percent cumulative weight of fine sediments, Fredle index (FI) as a measure of permeability, vertical hydraulic gradient, water depth (WD), and flow velocity at planting locations. Egg-to-fry survival averaged 92.7% (range: 57.2–100%) in 2013 (N = 19) and 71.5% (range: 6.4–100%) in 2014 (N = 23). Survival was significantly positively correlated with FI and flow velocity, negatively associated with percent cumulative weight of fine sediments and WD. Vertical hydraulic gradient had no effect on survival. Our results suggest that a higher FI (i.e., low amount of fine material and larger particle size), higher flow velocity, and shallower WD reduce the mortality of planted chum salmon eyed eggs. This is likely a result of increased permeability in the substrate and restriction of fine sediment intrusion into the incubation zone.