<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.     

Osmoregulation in juvenile Chinese sturgeon (<Emphasis Type="Italic">Acipenser sinensis</Emphasis> Gray) during brackish water adaptation

The osmoregulation capabilities of 7-month-old juvenile Chinese sturgeon (Acipenser sinensis Gray) (128.8 ± 15 g) transferred directly from fresh water (0‰, 46 mOsmol kg⁻¹) to brackish water (10‰, 273 mOsmol kg⁻¹) were studied over a 20-day period. Changes in serum osmolarity, chloride (Cl⁻), sodium (Na⁺), potassium (K⁺) and calcium (Ca²⁺) ion concentrations, as well as gill and spiral valve Na⁺,K⁺-ATPase activities were measured at 3, 12, 24, 72, 216 and 480 h after transfer to BW. The serum osmolarity and ion concentrations (Na⁺, Cl⁻ and Ca²⁺) increased immediately after the transference to BW, reaching maximum at 24 h and returned to a new steady state at 216 h, while the FW control group maintained basal levels which showed lower (P < 0.05) than the BW group. Gill Na⁺,K⁺-ATPase activity of BW group exhibited an abrupt decrease in the first 3 h after transfer, but began to increase at 3 h, reaching a peak value at 24 h, and returned to a new steady state at 216 h. The differences between gill Na⁺,K⁺-ATPase activity of BW and FW fish were significant (P < 0.05) after 12 h. In contrast, Na⁺,K⁺-ATPase activity of the spiral valve showed transient increase after transference from FW to BW, and then decreased rapidly at 3 h, reaching the lowest at 24 h after transference. At 216 h after exposure to BW, Na⁺,K⁺-ATPase activities of the spiral valve increased slowly to the levels of FW control. The results of our study indicate the existence of hyposmoregulatory adaptive mechanisms in 7-month-old juvenile Chinese sturgeon which enable this fish to acclimate itself successfully to brackish water.     

Repeatable immersion infection with <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis> reproducing clinical signs and moderate mortality

Pseudotuberculosis is a bacterial septicaemia caused by Photobacterium damselae subsp. piscicida in several marine fish species. Yellowtail Seriola quinqueradiata is the most sensitive fish species to this disease. The internal organs of naturally infected yellowtail exhibit whitish spots, tubercle-like tissue structures, consisting of bacterial accumulations. There have been many trials for experimental infection, however adequate method of infection that reproduces moderate mortality and primary clinical signs has not yet established. Present investigation evaluated an immersion infection method by using logarithmic culture-phase bacteria resulting in higher mortality than that using stationary culture-phase bacteria. Typical white spots on the spleen and kidney were also observed constantly in dead fish. Transmission electron microscopy and fluorescent antibody microscopy showed bacterial clusters not only in the spleen and kidney but also in the blood channels in the secondary gill filaments. These results were confirmed repeatedly by plural experiments. The use of logarithmic-phase bacteria in immersion infection is an appropriate technique to reproduce moderate mortality and primary clinical signs, which will be a reliable infection method also for the challenge test of pseudotuberculosis vaccine.     

Effects of temperature and salinity on survival and growth of juvenile ark shell <Emphasis Type="Italic">Anadara broughtonii</Emphasis>

We investigated the combined effects of temperature (23, 26, 29 and 32 °C) and salinity [15, 18, 21, 24, 27 and 30 practical salinity units (PSU)] on the growth and survival of juvenile ark shell Anadara broughtonii under hatchery conditions. Mortality, shell length and shell height were monitored for a period of 25 days in all exposure groups. Survival greater than 98% was observed in all treatment groups with no significant difference among treatment combinations. Absolute growth and specific shell length and height growth rate were significantly influenced by temperature and salinity. Growth of juvenile A. broughtonii increased with higher salinity and peaked at an intermediate temperature (26 °C). Optimal specific growth rates of 4.64 ± 0.04% day^?1 by shell length and 4.76 ± 0.11% day^?1 by shell height were observed at a combination of 26 °C and 30 PSU. This study enhances our understanding of the biology of A. broughtonii and determines ideal environmental conditions for pre-planting culture operations.     

Demographic and competition studies on <Emphasis Type="Italic">Brachionus ibericus</Emphasis> and <Emphasis Type="Italic">Proales similis</Emphasis> in relation to salinity and algal (<Emphasis Type="Italic">Nannochloropsis oculata</Emphasis>) density

We isolated the rotifers Brachionus ibericus and Proales similis from the sediment of shrimp tanks and studied their individual demographic characters and competition between them at two food levels (0.25?×?10⁶, 1.00?×?10⁶ cells ml^?1 of Nannochloropsis oculata at 25 °C) and salinities ranging from 10 to 30‰. Our hypothesis was that growth rates would be higher with increasing food levels and salinities. Observations were taken twice a day for life table studies and daily once for population growth experiments. Using survivorship and fecundity data, we derived various life history variables. Although the average life span (7.6?±?0.4 days) and gross reproductive rate (33.8?±?2.9 neonate female^?1 day^?1) of B. ibericus were higher than those of P. similis (average life span 5.4?±?0.6 days and gross reproductive rate 13.0?±?0.6 neonate female^?1 day^?1), the population growth experiments showed that P. similis had higher r values (0.32?±?0.005 day^?1) than B. ibericus (0.23?±?0.002 day^?1) at 1.0?×?10⁶ cells ml^?1 of N. oculata. The rotifer P. similis was more adversely affected due to the presence of B. ibericus than vice versa. The data are important for developing techniques for a large-scale culture of these rotifers as food in aquaculture.     

Effects of soy sauce <Emphasis Type="Italic">koji</Emphasis> and lactic acid bacteria on the fermentation of fish sauce from freshwater silver carp <Emphasis Type="Italic">Hypophthalmichthys molitrix</Emphasis>

The effects of soy sauce koji and the lactic acid bacterium, Tetragenococcus halophilus, were studied on the fermentation of fish sauce prepared from Chinese silver carp. The fish sauce prepared without koji and the lactic acid bacterium contained low levels of organic materials, total nitrogen, and organic acids. The use of koji was effective in increasing these qualitative parameters and further improved the amino acid score of the fish sauce. Addition of T. halophilus had an effect on lowering the pH value during the initial period of fermentation when the soy sauce koji was also supplemented. In contrast, T. halophilus-like bacteria were found to be predominant for all tanks fermented under the different starting conditions. Although it was not examined whether the T. halophilus-like bacteria observed after fermentation were the same as the starter-bacterium or not, it was suggested that T. halophilus plays an important role in the successful fermentation of silver carp fish sauce. Sensory evaluation conducted with Japanese and Chinese panelists also suggested the superiority of the use of koji for fermentation of silver carp fish sauce.     

温度和盐度对橄榄蚶耗氧率和排氨率的影响

测定不同温度(15℃、20℃、25℃、30℃)和盐度(16、21、26、31、36)对橄榄蚶(Estellarca olivacea)呼吸和排泄的影响。结果表明,温度和盐度对橄榄蚶的耗氧率和排氨率都有显著影响(P<0.05)。在15~30℃范围内,橄榄蚶单位软体干质量耗氧率(OR)、排氨率(NR)都随温度的升高而升高,变化范围分别为0.37~0.65 mg/(g.h)和1.74~3.15μmol/(g.h);耗氧率和排氨率与温度(t)之间的相关方程分别为:ORT[mg/(g.h)]=0.323 4e0.1765t(R2=0.9755,P<0.05);NRT[μmol/(g.h)]=1.3788e0.1986t(R2=0.9784,P<0.05)。在16~36盐度范围内,橄榄蚶单位软体干质量耗氧率和排氨率开始时随盐度的升高而降低,在盐度26时达到最小值,然后随盐度的升高而升高,变化范围分别为0.43~0.60 mg/(g.h)和1.75~3.26μmol/(g.h);耗氧率和排氨率与盐度(s)之间的相关方程分别为:ORS[mg/(g.h)]=0.0014s2-0.0751s 1.4312(R2=0.9455,P<0.05,n=30);NRS[μmol/(g.h)]=0.0105s2-0.5728s 9.7114(R2=0.9285,P<0.05,n=30)。     

温度与盐度对云龙石斑鱼幼鱼耗氧率和排氨率的影响

为了研究饱食和饥饿状态下温度(24、27、30和33℃)、盐度(15、20、25、30和33)对云龙石斑鱼幼鱼(Epinephelus groupers)耗氧率(OCR)和排氨率(AER)的影响,采用密闭流水式呼吸实验方法对OCR和AER进行了测定。结果显示:饱食和饥饿状态下,温度、盐度对云龙石斑鱼幼鱼OCR和AER影响均显著(P0.05);当温度为24℃~33℃时,饱食状态下的OCR和AER分别比饥饿状态的提高30.29%~131.84%和131.82%~316.19%。饱食状态下,氧氮比(O∶N)、蛋白质供能比(PSR)、Q10(呼吸)、Q10(代泄)分别为12.54~20.18、27.74%~48.48%、1.21、1.60;而在饥饿状态下分别为25.77~35.90、19.49%~27.19%、1.87、3.41;当盐度为15~33时,饱食状态下OCR和AER分别比饥饿状态的提高100.41%~128.21%、182.19%~382.83%。饱食状态下,O∶N、PSR分别为13.32~20.72、39.47%~49.42%,而在饥饿状态下分别为29.18~30.98、22.59%~23.98%。研究表明,云龙石斑鱼幼鱼的最适生长温度为24℃~27℃,最适生长盐度为30~33。     

Induction of diploid gynogenesis in turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> with left-eyed flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig’s effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm⁻². At 15°C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3°C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1°C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.     

Comparative time-course study on pituitary and branchial response to salinity challenge in Mozambique tilapia (<Emphasis Type="Italic">Oreochromis mossambicus</Emphasis>) and Nile tilapia (<Emphasis Type="Italic">O</Emphasis>. <Emphasis Type="Italic">niloticus</Emphasis>)

The physiological response of Mozambique and Nile tilapia transferred from fresh to brackish (15 ppt) water was compared during a one-week time course. Response in the pituitary was measured by the gene expression pattern of prolactin (PRL I), growth hormone (GH), and calcium-sensing receptor (CaSR), while the response in the gills was measured by the gene expression pattern of the prolactin receptor (PRL-R), Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) and Na⁺/Cl⁻ cotransporter (NCC), and by activity and expression of Na⁺/K⁺-ATPase (NKA). The time-course curves of plasma osmolality levels indicate a rapid elevation 24 h after transfer, which later decreased and maintained at stable level. PRL I expression decreased in both species, but with stronger response in the Nile tilapia, while no differences were found in the slightly elevated levels of GH mRNA. The branchial response demonstrated a faster up-regulation of NKA and NKCC in the Mozambique tilapia, but similar levels after a week, while Nile tilapia had stronger and constant down-regulation of NCC. The time-course response of the measured osmoregulatory parameters indicate that 24 h after transfer is a critical time point for brackish-water adaptation. The differences in responses to saltwater challenge between Mozambique and Nile tilapia shown in this study may be associated with the differences in saltwater tolerance between these two tilapiine species.     

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.     

Recent expansion of Northeast Atlantic and Mediterranean populations of <Emphasis Type="Italic">Melicertus</Emphasis> (<Emphasis Type="Italic">Penaeus</Emphasis>) <Emphasis Type="Italic">kerathurus</Emphasis> (Crustacea: Decapoda)

We analysed the genetic diversity of Melicertus kerathurus (Penaeidae), a commercially valuable penaeid shrimp that is distributed in the Mediterranean Sea and eastern Atlantic Ocean. We examined the polymorphism of a 494 bp DNA segment of the mitochondrial COI region in 173 individuals, sampled in nine Mediterranean and two Atlantic samples, covering the whole range of the species from the tropical waters of the Gulf of Guinea to the eastern part of the Mediterranean Sea. The mean nucleotide and haplotype diversities were π = 0.00275 and h = 0.718, respectively, for the global data set, with the highest values occurring in the African samples and the lowest in the Adriatic Sea. A clear sample differentiation was found (F _st = 0.194), but this did not reflect a geographical pattern and there were only faint traces of an Atlantic–Mediterranean subdivision. Mismatch analysis and a high significant negative value of Tajima’s D suggested that M. kerathurus is not at mutation drift-equilibrium, but underwent a recent expansion after a period of low effective sample size. A postglacial recolonisation of the Mediterranean from an Atlantic refuge could be hypothesised based on these data.     

Growth and survival of grouper <Emphasis Type="Italic">Epinephelus</Emphasis><Emphasis Type="Italic">coioides</Emphasis> (Hamilton) larvae fed free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> at first feeding

The free-living nematode, Panagrellus redivivus, was tested as live food for grouper Epinephelus coioides larvae during the first feeding stage. A series of experiments were conducted to determine the acceptability of the free-living nematodes in grouper larvae at first feeding, the optimum nematode density and the response of the larvae to nutritionally enriched nematode. All experiments were conducted in 200-L conical tanks filled with 150-L filtered seawater and stocked at 15 larvae L⁻¹. Duration of feeding experiments was up to day 21 (experiment 1) and 14 days (experiment 2 and 3). Brachionus plicatilis and Artemia (experiment 1) and Brachionus plicatilis alone (experiment 2 & 3) was used as the control treatment. Observations indicated that the grouper larvae readily fed on free-living nematodes as early as 3 days posthatching, the start of exogenous feeding. Optimum feeding density for the larvae was 75 nematodes ml⁻¹. The enrichment of cod liver oil or sunflower oil influenced the total lipids and n-3 highly unsaturated fatty acids of P. redivivus, which in turn influenced those of the grouper larvae, however, growth and survival of the larvae were not affected (P > 0.05). The results from this investigation showed that the nematode, P. redivivus, can be used as first live food for grouper larvae from the onset of exogenous feeding until they could feed on Artemia nauplii.     

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.