Arachidonic acid metabolism in gill homogenate and isolated gill cells from rainbow trout,Oncorhynchus mykiss: the effect of osmolality,electrolytes and prolactin

An assay method based on thin layer chromatography to study the arachidonic acid (AA) metabolism in gill tissues was optimized and the effect of osmotically different incubation mediums on AA metabolism was evaluated. Rainbow trout gill tissues metabolize AA into PGE₂ in highest concentration followed by PGD₂, PGF₂ and 6-keto-PGF₁ (the stable metabolite of PGI₂) among the prostanoids tested. Approximately 40% of PGE₂ is synthesized within the first minute of incubation and is directly dependent on the substrate concentration (AA). As in mammalian tissues, PGE₂ synthesis in fish gills is inhibited by the cyclooxygenase inhibitor indomethacin. PGE₂ synthesis in gill homogenate and isolated gill cells incubated in trout Ringer was 0.45 and 1.9 ng/mg protein, respectively, and increased to 8.9 and 4.3 ng/mg protein, respectively, when incubated in KPO₄ buffer, due to a ten-fold increase in the free AA. The hydroxy acid synthesis of the gill homogenate was higher (13%), and that of the isolated gill cells incubated in KPO₄ buffer was lower (44%) compared to gill homogenate and cells incubated in trout Ringer. Gill homogenate incubated in 50 mM phosphate buffer with increasing sodium or potassium concentrations (up to 250 mM) exhibited a concentration-dependent increase in PGE₂ synthesis (220% and 72%, respectively). Prolactin stimulated the PGE₂ synthesis up to 30% while PGD₂, PGF₂ and 6-keto-PGF₁ synthesis was not affected. This effect of prolactin was maximal when PGE₂ synthesis was estimated 30 minutes after prolactin addition and diminished after two hours. These results suggest that rainbow trout gills possess the ability to metabolize AA through the cyclooxygenase and lipoxygenase pathways. PGE₂ synthesis may be under the influence of ion balance and prolactin availability, indicating the probable involvement of AA metabolites in the regulation of ion balances across the gill membrane.     

Sex organ extracts and artificial hormonal compounds as sex pheromones to attract broodfish and to induce spawning of Chinese black sleeper (Bostrichthys sinensis Lacépède)

Ovarian, testicular and seminal vesicle extracts of mature Chinese black sleeper (Bostrichthys sinensis Lacepede), synthetic steroids 17α‐progesterone (17α‐P), 17α,20β‐dihydroxy‐4‐pregnen‐3one (17α,20β‐P) and prostaglandins PGE₂ and PGF_2α were used in an attempt to attract broodfish and to induce spawning. Furthermore, 17α‐P, PGE₂ and PGF_2α levels were determined in extracts, and PGE₂ and PGF_2α levels were determined in holding and spawning waters. The results showed that ovarian extracts attracted more males than females to enter artificial nests, while testicular and seminal vesicle extracts attracted more females than males. The highest percentage of spawned nests were found when 17α,20β‐P and PGE₂ were administered to the nests, while the largest egg numbers and the mean highest fertilization rate were observed when PGE₂ was added. In ovarian extract, 17α‐P level was higher than levels in testicular or seminal vesicle extracts. The PGE₂ levels were significantly (P<0.05) higher than PGF_2α in the ovarian, testicular and seminal vesicle extracts. The PGE₂ and PGF_2α levels in spawning waters were higher than those in holding waters. The PGE₂ levels were greatly significantly (P<0.01) higher than PGF_2α in spawning and in holding waters. The results suggest that the sex organs of B. sinensis contain 17α‐P, 17α, 20β‐P, PGE₂ and PGF_2α and that 17α‐P, 17α,20β‐P and PGE₂ may act as sex pheromones in this species, attracting both male and female conspesifics to spawning sites and inducing spawning. Among the compounds tested, PGE₂ was the most effective sex pheromone for induction of spawning in B. sinensis.     

Dietary Arachidonic Acid Alters Tissue Fatty Acid Profile, Whole Body Eicosanoid Production and Resistance to Hypersaline Challenge in Larvae of the Temperate Marine Fish, Striped Trumpeter (Latris lineata)

We determined the effect of dietary arachidonic acid (20:4n-6, ARA) on tissue ratios of ARA/eicosapentaenoic acid (20:5n-3, EPA) and subsequent whole body production of the eicosanoids, prostaglandin F_2α (PGF_2α) and E₂ (PGE₂) in the marine larvae of striped trumpeter, Latris lineata. Larvae were also subjected to a hypersaline challenge (55 ppt) with an aim to determine possible relationships between tissue fatty acid profiles, prostanoid production, and their tolerance to osmotic challenge. From 5 to 23 days post-hatch (dph) larvae were fed live food, rotifers (Brachionus plicatilis), that had been enriched with one of five experimental emulsions containing increasing concentrations of ARA and constant EPA and 22:6n-3 (docosahexaenoic acid, DHA). Final ARA concentrations in the rotifers were 1.33, 3.57, 6.21, 8.21 and 11.22 mg g⁻¹ DM. Larval growth and survival was unaffected by dietary ARA. Tissue fatty acid concentrations generally corresponded with dietary fatty acids and final tissue ratios of ARA/EPA ranged from 0.9 to 4.9. At 18 and 23 dph whole body concentrations of PGF_2α and PGE₂ generally increased as more dietary ARA was provided in a dose-response manner, and a significant elevation in both PGF_2α and PGE₂ in larvae fed the highest dietary ARA concentration was recorded at 23 dph compared to larvae receiving the lowest concentration. At 18 dph, the highest cumulative inactivity following a hypersaline challenge occurred in larvae fed 8.21 or 11.22 mg ARA g⁻¹ DM, which was significantly greater than those receiving 3.57 mg ARA g⁻¹ DM. At 23 dph no relationship between inactivity of larvae subjected to a hypersaline challenge to dietary ARA was evident. In summary, dietary ARA altered tissue ARA/EPA ratios, prostanoid production and resistance to a hypersaline challenge in larval striped trumpeter. While increasing dietary ARA generally resulted in elevation of prostanoids as well as increasing the number of inactive larvae following a hypersaline challenge at 18 dph, similar trends between prostanoids and larval inactivity were not evident at 23 dph, suggesting the exact mechanisms and relationships between eicosanoids and larval osmoregulation warrants further investigation. Nevertheless the study provides preliminary data on the effect of dietary ARA on the prostaglandin production in marine fish larvae.     

PGF<Subscript>2α</Subscript> and gonadal steroid plasma levels of successful and unsuccessful spawning <Emphasis Type="Italic">Piaractus mesopotamicus</Emphasis> (Teleostei,Characiformes) females

Gonadal steroid and prostaglandin F₂α (PGF_2α) plasma levels were evaluated in successfully (SP) and unsuccessfully ovulated (UN) female Piaractus mesopotamicus. Forty-one females were injected with crude carp pituitary extract (0.6 and 5.4 mg kg^?1 with a 24-h interval between the doses) and sampled to determine the plasma concentration of 17β-estradiol (E₂), 17α-hydroxyprogesterone (17α-OHP), 17α,20β-dihydroxy-4-pregnen-3-one (DHP), PGF_2α, and testosterone (T) after each injection (first—A1 and second—A2), and at the time of ovulation for SP and UN. Two clusters were obtained using multivariate analysis: 1—composed of all A1, all A2, and some UN; and 2—composed of all SP and some UN. Median values of E₂ plasma levels were similar between clusters; however, plasma levels of T, 17α-OHP, DHP, and PGF_2α of cluster 2 (predominantly formed by SP) were higher than those of cluster 1. Since cluster 2 contained all SP and females of this cluster presented higher levels of PGF_2α, T, 17α-OHP, and DHP, here we evidently shown in an unprecedented manner that concomitant increased levels of these substances were associated with successful ovulation in this species, but such an increase was not determinant for successful ovulation due to the presence of some UN females in the same cluster 2. These findings highlight the unexplored potential of PGF_2α to be used as an accessory tool for inducing successful ovulation for fish farming purposes.     

Cloning and expression of prostaglandin E2 receptor subtype 1 (ep 1 ) in Bostrichthys sinensis

Our previous studies suggested that prostaglandin E₂ (PGE₂) is a putative sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. We found immunoreactivities of PGE₂ receptor subtypes (Ep_1–3) expressed in the olfactory sac, but only Ep₁ presented higher density of immunoreactivity in mature fish than that in immature fish in both sexes. To gain a better understanding of the underlying molecular mechanism for the detection of PGE₂ in the olfactory system, we cloned an ep ₁ cDNA from the adult olfactory sac. The open-reading frame of the ep ₁ consisted of 1,134-bp nucleotides that encoded a 378-amino acid-long protein with a seven-transmembrane domain, typical for the G protein-coupled receptors superfamily. Expression of ep ₁ mRNA was observed in all tissues examined, with higher levels obtained in the olfactory sacs and testes. The expression of ep ₁ mRNA in the olfactory sacs and gonads was significantly higher in both sexes of mature fish than in those of immature ones. Taken together, our results suggested that Ep₁, which is highly expressed in the olfactory sacs and gonads of mature fish, is important for the control of reproduction and may be involved in PGE₂-initiated spawning behavior in B. sinensis.     

Effects of different dietary arachidonic acid : docosahexaenoic acid ratios on phospholipid fatty acid compositions and prostaglandin production in juvenile turbot (Scophthalmus maximus)

Five purified diets containing AA (20:4n-6) at 0.02–0.78% dry weight and DHA (22:6n-3) at 0.93–0.17% dry weight were fed to duplicate groups of juvenile turbot (Scophthalmus maximus) of initial weight 0.87 g for a period of 11 weeks. The dietary DHA:AA ratio ranged from 62 to 0.2. Incorporation of AA into liver phospholipids increased with increasing dietary AA input. Phospholipids from fish fed diets containing 0.02, 0.06 and 0.11% of dry weight as AA generally contained less AA compared to fish fed fish oil while those fed diets containing 0.35 and 0.78% of dry weight as AA had higher AA levels in their phospholipids. The highest levels of AA were found in PI but the greatest percentage increase in AA incorporation was in PE and PC. Brain phospholipid fatty acid compositions were less altered by dietary treatment than those of liver but DHA content of PC and PE in brain was substantially lower in fish fed 0.93% pure DHA compared to those fed fish oil. This suggests that dietary DHA must exceed 1% of dry weight to satisfy the requirements of the developing neural system in juvenile turbot. In both tissues, (20:5n-3) concentration was inversely related to both dietary and tissue PI AA concentration. Similar dietary induced changes in AA, EPA and DHA concentrations occurred in the phospholipids of heart, gill and kidney. PGE₂ and 6-ketoPGF₁ were measured in homogenates of heart, brain, gill and kidney. In general, fish fed the lowest dietary AA levels had reduced levels of prostaglandins in their tissue homogenates while those fed the highest level of AA had increased prostaglandin levels, compared to fish fed fish oil. In brains, the PGE₂ concentration was only significantly increased in fish fed the highest dietary AA.Abbreviations AA arachidonic acid - DHA docosahexaenoic acid - EFA essential fatty acid - EPA eicosapentaenoic acid - HPTLC high performance thin-layer chromatography - HUFA highly unsaturated fatty acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PGE prostaglandin E - PGE prostaglandin E - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acid - TLC thin-layer chromatography     

Metabolism of 18:4n-3 (stearidonic acid) and 20:4n-3 in salmonid cells in culture and inhibition of the production of prostaglandin F2α (PGF2α) from 20:4n-6 (arachidonic acid)

Arachidonic acid (AA; 20:4n-6) is the precursor of a range of highly biologically active derivatives, collectively termed eicosanoids, including prostaglandins, thromboxanes, leukotrienes and lipoxins, that act as autocrine hormones regulating many physiological processes including haemostasis, reproduction, immune and inflammatory responses. Eicosapentaenoic (EPA; 20:5n-3) and dihomo-γ-linolenic (20:3n-6) acids modulate eicosanoid metabolism by both inhibiting the conversion of AA to eicosanoids whilst simultaneously being converted to eicosanoids with different, often attenuated, properties compared to their AA homologues. Eicosatetraenoic acid (20:4n-3) is a naturally occurring C₂₀ polyunsaturated fatty acid (PUFA), present in fish oil at levels of around 1–2%, that has been suggested to be the active metabolite responsible for the anti-inflammatory effects of plant oils containing stearidonic acid (18:4n-3). However, the biochemical properties of 20:4n-3 in terms of cellular biology have rarely been investigated, partly due to difficulties in obtaining the fatty acid in high purity. In this paper, we describe methods for the medium scale laboratory preparation of high purity 20:4n-3, and investigate its metabolism in fish cell culture systems which normally contain significant amounts of n-3 PUFA. Thus the incorporation and metabolism of 18:4n-3 and 20:4n-3, and their distribution in phospholipid classes was studied in an established cell line from Atlantic salmon (Salmo salar) (AS), and the effects of 20:4n-3 on eicosanoid production studied in freshly isolated macrophages from rainbow trout (Oncorhynchus mykiss). Both 18:4n-3 and 20:4n-3 were preferentially esterified into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in contrast with the accumulation of AA in phosphatidylinositol. Incorporated 18:4n-3 was readily converted to 20:4n-3, and both fatty acids were further desaturated and elongated to EPA and 22:5n-3 but not 22:6n-3. Supplementation with 20:4n-3 decreased the conversion of AA into prostaglandins, as demonstrated by the decreased levels of PGF_2α produced in trout macrophages supplemented with 20:4n-3 and AA compared to cells supplemented with AA alone. In addition, 20:4n-3 was converted into eicosanoids in fish cells as indicated by the presence of Δ^17,18 12-HETE, Δ^17,18 PGE1 and Δ^17,18 PGF_1α in extracts from rainbow trout macrophages incubated with 20:4n-3.     

Diets with different n − 6/n − 3 fatty acid ratio in diets for juvenile Atlantic salmon, effects on growth, body composition, bone development and eicosanoid production

Vegetable oils, typically high in n − 6 fatty acids, are necessary alternatives in fish feed production. Such oils have been tested in salmonid diets with good growth results, but with major changes in lipid composition. The aim of this study was to provide information regarding the effect of the higher dietary n − 6/n − 3 fatty acid ratio caused by vegetable oil on growth, chemical composition and bone development, on eicosanoid production and on formation and mineralization of bone.A feeding experiment was carried out in juvenile Atlantic salmon (Salmo salar). Groups of fish (1.28 g) were fed diets added fish oil (FO) or soybean oil (SO) as the main lipid source through the 174 days long experimental period with sampling points at days 0, 31, 53, 96, 136 and 174 up to smoltification. Subsequently, fish performance was observed after 2, 8 and 18 months in sea water.Weight was significantly higher in the fish oil group (95.4 g) than in the soybean oil group (87.4 g) after the 174 days experimental period. This difference developed during short day treatment prior to smoltification, and the fish oil group had a significantly higher growth rate (TGC) during this period (1.62 vs 1.45). Whole body mineral analysis displayed low levels of Ca (2400 and 3500 mg kg−1) and P (3200 and 3900 mg kg−1) in all samples. The corresponding Ca:P ratio ranged from 0.74 to 0.91. On day 136 the level of Ca, and Ca/P ratio, were significantly higher in the fish oil group than in the soybean oil group. There was a significant increase in n − 6 fatty acids and decrease in n − 3 fatty acids in the soybean oil group compared to the fish oil group for both TAG and PL in vertebrae. Individual variation was large in plasma prostaglandin E₂ (PGE₂), and at day 174 the trend was towards a higher level in the soybean oil group. Radiography analyses demonstrated a distinctive pattern of development of spinal pathology with time and fish size, similar in both groups. Hyper dense vertebrae dominated at earlier stages, fusion-type malformations dominated at harvest size. The histological examination of the vertebrae revealed no differences between treatments.The results suggest that vegetable oil as a replacement for fish oil influence the arachidonic acid level in phospholipids of vertebrae, may slightly increase production of PGE₂ in blood, and reduce vertebrae mineralization around the sensitive smoltification phase. These trends did not lead to any detectable skeletal malformations. Differences in growth and vertebrae mineralization observed around smoltification disappeared during the seawater phase.     

Relationship between dietary lipid source, oxidative stress, and the physiological response to stress in sub-yearling chinook salmon (Oncorhynchus tshawytscha)

Relationships between dietary lipid source, stress, and oxidative stress were examined in juvenile chinook salmon (Oncorhynchus tshawytscha). Four different experimental diets were used: menhaden oil (MHO; elevated 20:5n-3 and 22:6n-3), soybean oil (SBO; elevated 18:2n-6), linseed oil (LSO; elevated 18:3n-3), and a mixture of 55% linseed oil and 45% soybean oil (MIX; approximately equal levels of 18:2n-6 and 18:3n-3). Juvenile salmon (initial body weight of 16.0 g) were fed experimental diets for 12 weeks (early March to early June). At the end of feeding, fish subjected to a low-water stressor for 96 h had greater liver and brain lipid peroxidation compared to unstressed controls; peroxidation was not influenced by diet. Diet and stress affected plasma cortisol levels. Stressed fish fed SBO had the greatest cortisol concentrations, followed by MIX, MHO, and LSO (mean concentrations for the SBO and LSO diets differed significantly). The cortisol response to stress may have been influenced by the ratio of prostaglandin 1- and 2-series to prostaglandin 3-series precursor fatty acids provided by the different diets. The results of this study suggest a connection between the physiological response to stress, dietary lipid quality, and oxidative stress. This is the first evidence of such a relationship in fish. Abbreviations: AA - arachidonic acid; ACTH - adrenocorticotropin; BHT - butylated hydroxytoluene; BLPO - brain lipid peroxidation; dGLA - dihomo-γ-linolenic acid; DHA - docosahexanoic acid; EPA - eicosapentanoic acid; FER - feed efficiency ratio; FOX - ferrous oxidation-xylenol orange; GLA -γ-linolenic acid; LA - linoleic acid; LCO3 - long-chain n-3 polyunsaturated fatty acids; LLPO - liver lipid peroxidation; LN - linolenic acid; LPO - lipid peroxidation; LSO - linseed oil; MHO - menhaden oil; MIX - 55% linseed oil + 45% soybean oil; PC - plasma cortisol; PG - prostaglandin(s); PGE₂- prostaglandin E₂; PUFA - polyunsaturated fatty acid; SBO - soybean oil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Incorporation and metabolism of14C-labelled polyunsaturated fatty acids in wild-caught juveniles of golden grey mullet,Liza aurata,in vivo

The incorporation, and the capacity for desaturation and elongation in vivo, of intraperitoneally-injected,¹⁴C-labelled n-3 and n-6 C₁₈ and C₂₀ polyunsaturated fatty acids (PUFA) were investigated in juvenile golden grey mullet,Liza aurata. The results indicate that juvenile mullet have only limited ability to convert C₁₈ polyunsaturated fatty acids to C₂₀ and C₂₂ highly unsaturated fatty acids (HUFA)in vivo. This suggests that juvenile golden grey mullet require the provision of preformed C_20/22 HUFA, such as eicosapentaenoic and docosahexaenoic acids, in the diet. The impairment in the desaturase/elongase pathway was similar to that found in turbot,Scophthalmus maximus, and gilthead sea bream,Sparus aurata, being primarily at the level of Δ5-desaturase. The data from the largely herbivorous golden grey mullet juveniles are consistent with the hypothesis that marine fish in general, irrespective of dietary habits, have limited capacity for the desaturation and elongation of C₁₈ PUFA. The defect in Δ5-desaturase activity combined with the consistent finding that arachidonic acid is selectively incorporated and retained in membrane phosphatidylinositol suggests that, like turbot and gilthead sea bream, golden grey mullet may also have a requirement for preformed arachidonic acid in the diet.     

The optimum dietary docosahexaenoic acid level based on growth and non‐specific immune responses in juvenile rock bream,Oplegnathus fasciatus

A feeding trial was conducted to determine the optimum level and effect of incremental dietary levels of docosahexaenoic acid (DHA, 22:6n‐3) on growth and non‐specific immune responses in juvenile rock bream, Oplegnathus fasciatus. A basal diet without DHA supplementation was used as a control, and six other diets were prepared by supplementing with 4, 8, 12, 16, 20 or 40 g kg^?1 DHA. These diets included no eicosapentaenoic acid and/or arachidonic acid contents. The actual DHA concentrations of the diets were 1, 4.8, 8.9, 13.1, 17.6, 21.2 and 41.4 g kg^?1 diet (DHA_1.0, DHA_4.8, DHA_8.9, DHA_13.1, DHA_17.6, DHA_21.2 and DHA_41.4 respectively). At the end of feeding trial, final body weight, weight gain, specific growth rate and feed efficiency of fish fed the DHA_13.1, DHA_17.6, DHA_21.2 and DHA_41.4 diets were significantly higher than those fed the other diets (P < 0.05). The broken‐line analysis of weight gain indicates that the optimum dietary DHA level is 11.9 g kg^?1. Fish fed DHA_1.0 had the highest hepatosomatic index, an increase in plasma cholesterol, triglyceride, low‐density lipoprotein and aspartate aminotransferase levels, as well as a decrease in high‐density lipoprotein. Superoxide dismutase activity of fish fed DHA_13.1 and DHA_17.6 diets was significantly higher than that of fish fed DHA_1.0, DHA_4.8 and DHA_8.9 diets. Fish fed the DHA_17.6, DHA_21.2 and DHA_41.4 diets showed significantly higher lysozyme activity than those fish fed DHA_1.0, DHA_4.8 and DHA_8.9 diets. Therefore, the optimum dietary DHA level could be greater than 11.9 g kg^?1 but less than 13.1 g kg^?1 in diet.     

Effects of the different levels of dietary vitamin C on growth and tissue ascorbic acid changes in parrot fish (Oplegnathus fasciatus)

An 11-week feeding trial was conducted to study the effects of the different levels of dietary vitamin C on growth, tissue ascorbic acid concentrations and histopathological changes in parrot fish. Casein and gelatin-based diets were formulated to contain 0, 60, 120, 240, 480 and 2000 mg l-ascorbic acid (AA) per kg diets on AA equivalent basis in the form of l-ascorbyl-2-monophosphate (AMP), 60 and 240 mg AA per kg diet as l-ascorbic acid. However, the analyzed AA levels were 0, 50, 100, 205, 426 and 1869 mg AA per kg diet in AMP supplemented diets; 36 and 149 mg AA per kg diet in l-ascorbic acid supplemented diets. Thus, the diets were designated as AA-free, AMP₅₀, AMP₁₀₀, AMP₂₀₅, AMP₄₂₆, AMP₁₈₆₉, AA₃₆ and AA₁₄₉. Fish averaging 3.9±0.06 g (mean±S.D.) randomly were fed the experimental diets in triplicate groups for 11 weeks. Weight gain (WG) of fish fed AMP₅₀ and AA₃₆ diets were significantly lower than those of the other groups, and fish fed AMP₁₈₆₉ diet showed a higher WG than did fish fed AMP₅₀, AMP₁₀₀, AMP₂₀₅, AA₃₆ and AA₁₄₉ diets (P<0.05). However, there was no significant difference in WG between fish fed diets AMP₄₂₆ and AMP₁₈₆₉, and among fish fed AMP₁₀₀, AMP₂₀₅, AMP₄₂₆ and AA₁₄₉ diets. Fish fed the AA-free diet began to show deficiency signs, such as retarded growth, darkening, anorexia and high mortality, after 3 weeks of feeding trial. After 6 weeks of the feeding trial, liver from fish fed the AA-free diet showed severe atrophy. By the end of the seventh week, all fish fed the AA-free diet were dead. Liver AA concentration from fish fed AMP₅₀ diet was significantly lower than that from fish fed AMP₂₀₅, AMP₄₂₆, AMP₁₈₆₉ and AA₁₄₉ diets, and fish fed AMP₅₀, AMP₁₀₀, AA₃₆ diets showed a significant lower AA concentration in liver than did fish fed AMP₂₀₅, AMP₄₂₆ and AMP₁₈₆₉ diets.Broken line analysis of weight gain indicated that the dietary vitamin C requirement of parrot fish is 118±12 mg AA per kg diet in the form of l-ascorbyl-2-monophosphate for maximum growth.     

弓背青鳉早期胚胎色素细胞发生及相关基因表达规律分析

体表色素具有保护鱼类免受紫外线辐照伤害等重要功能。依照自然选择理论,不同群体的体色形成过程可能受到光照等环境因素的选择作用而发生遗传分化。为了检验这一假说,本研究以相同条件下人工繁育的不同纬度的弓背青鳉(Oryzias curvinotus)群体（饶平、高桥、三亚）后代（F6）为材料,使用荧光倒置显微镜对早期胚胎的黑色素细胞和虹彩色素细胞进行观察统计,并利用实时荧光定量PCR（qRT-PCR）分析4种色素细胞发生关键基因（黑色素形成的限速酶基因tyr、虹彩色素细胞相关基因alk、sox10和pax3）在高桥群体后代早期发育（6-10,12,14肌节期）过程中的表达加以验证。结果表明,高纬度饶平群体早期胚胎的黑色素细胞和虹彩色素细胞分别集中出现于8肌节期和10肌节期,占比为81.9%和52.1%;相应地,高桥群体早期胚胎的黑色素细胞和虹彩色素细胞分别集中出现于7和11肌节期,占比47.7%和42.1%;而三亚群体早期胚胎的黑色素细胞和虹彩色素细胞分别集中出现于8和12肌节期,占比 44.0%和62.2%。对高桥F6代胚胎样本的色素细胞发生相关基因qRT-PCR检测确定：tyr表达水平从6肌节期开始上调,至10肌节期趋于平稳;而抑制黑色素形成通路并启动虹彩细胞分化通路的sox10和pax3基因与下游的虹彩色素细胞相关基因alk在10-14肌节期均呈现一致的单峰表达模式。综上,本研究支持弓背青鳉早期发育阶段的黑色素细胞和虹彩色素发生模式与鱼类经典理论相符,受tyr、alk、sox10、pax3等基因的有序表达的调控,且证实了发生阶段存在由遗传因素决定的群体分化,低纬度群体的虹彩细胞发生更晚,其具体机制仍待进一步研究。     

Deficient melanin production contributes to the absence of melanophores in early development of red carp

Red carp and red crucian carp are ornamental fish with a red body color. Unlike in red crucian carp, no melanophores are observed in red carp embryos or larvae. To explore the roles of the mitfa gene in body color formation in red carp, we investigated the structural characteristics and physicochemical properties of the mitfa gene in 16 kinds of fish. The mitfa amino acid sequence similarity between red carp and red crucian carp was 95.6%, and this was 91.5% similar between carp and zebrafish. Compared with red crucian carp, red carp showed lower tyrp1 messenger ribonucleic acid (mRNA) expression but similar mitfa mRNA expression in the body pigment stage of the embryo. Moreover, mitfa⁺ cells as well as melanocytes could be observed in cultured embryo cells derived from red carp and red crucian carp. Our data show that the absence of melanophores in red carp is not the result of mitfa gene deletion or mutation, increasing our understanding of the molecular and genetic mechanisms of coloration in cyprinid fish.     

Organic acids blend as dietary antibiotic replacer in marine fish olive flounder,Paralichthys olivaceus

The present study was conducted to evaluate the efficacy of organic acid blends as dietary antibiotic replacer in marine fish olive flounder, Paralichthys olivaceus. Fish averaging 3.5 ± 0.05 g (mean ± SD) were fed one of the four experimental diets: (1) without antibiotic or organic acid (Control/CON); (2) with antibiotic—50 mg oxytetracycline per kg body weight per day (OTC); (3) with organic acid blend A—4 g/kg diet (OA_A); and (4) with organic acid blend B—4 g/kg diet (OA_B), for 10 weeks. At the end of the experiment, total intestinal bacterial counts in fish‐fed OA_A, OA_B and OTC were significantly lower than that of fish‐fed CON diet (p < 0.05). Further, the group of fish‐fed organic acid blends (OA_A, OA_B) or antibiotic (OTC)‐supplemented diets exhibited lower intestinal Vibrio sp. counts compared with fish‐fed CON diet. Disease challenge test with bacteria Edwardsiella tarda showed significantly lower cumulative mortality rates for the group of fish‐fed OA_A, OA_B or OTC than that of fish‐fed CON diet (p < 0.05). There were no negative effects on the growth, serological characteristics and proximate composition among the group of fish‐fed different experimental diets. Therefore, the present experiment demonstrates that blends of organic acid could be a promising alternative to dietary antibiotics for the preventive and/or curative health management in marine fish olive flounder aquaculture.     

Formation process of staining-type hypermelanosis in Japanese flounder juveniles revealed by examination of chromatophores and scales

Staining-type hypermelanosis, defined as blind-side melanosis occurring after completion of metamorphosis, reduces commercial value in hatchery-produced flatfishes. Detailed characterization was performed on the stained area of juvenile Japanese flounder Paralichthys olivaceus to physiologically understand this phenomenon. From 80 to 120 days after hatching, juveniles were reared in sandy and sandless tanks. By classifying the staining degree into 7 levels, about 2 times higher occurrence of middle-level staining was reconfirmed in sandless tank (about 80 %) than in sandy tank (about 40 %). In the stained area, we found 3 types of chromatophores (melanophore, xanthophore, and iridophore) and ctenoid scales, which would be typically observed on the normal ocular side. Detailed examination on the melanophores revealed further similarity between the stained area and the normal ocular side, in terms of the distribution at 2 layers (shallower and deeper than scale), and the densities in both layers (about 1000 cells/mm² above scale and 200 cells/mm² beneath scale). These results strongly suggest that the staining is a status change in the body surface conditions from the blind side to that on the ocular side, and not a simple darkening caused by disordered proliferation of melanophores on the blind side.     

Evolutionary assembly rules for fish life histories

We revisit the empirical equation of Gislason et al. (2010, Fish and Fisheries 11 :149–158) for predicting natural mortality (M, year^?1) of marine fish. We show it to be equivalent to , where L_∞ (cm) and K (year^?1) are the von Bertalanffy growth equation (VBGE) parameters, and L (cm) is fish length along the growth trajectory within the species. We then interpret K in terms of the VBGE in mass , and show that the previous equation is itself equivalent to a ?? power function rule between M and the mass at first reproduction (W_α); this new ?? power function emerges directly from the life history that maximizes Darwinian fitness in non‐growing populations. We merge this M, W_α power function with other power functions to produce general across‐species scaling rules for yearly reproductive allocation, reproductive effort and age at first reproduction in fish. We then suggest a new way to classify habitats (or lifestyles) as to the life histories they should contain, and we contrast our scheme with the widely used Winemiller–Rose fish lifestyle classification.     

Detection of prostaglandin E<Subscript>2</Subscript> in polychaete <Emphasis Type="Italic">Perinereis</Emphasis> sp. and its effect on <Emphasis Type="Italic">Penaeus monodon</Emphasis> oocyte development in vitro

Prostaglandins are involved in the reproductive processes in a variety of animals, including crustaceans. It was found that polychaetes, the best maturation diet for shrimp broodstock, possessed the greatest variation of prostaglandin E₂ (PGE₂) when compared with other live feeds. The level of PGE₂ varied according to sizes, feed intake, sources and type of polychaete. The matured and also larger sand polychaete Perinereis sp. contained higher PGE₂ levels than younger and smaller sand polychaetes (18.16 ± 5.82 ng PGE₂ mg⁻¹ protein for polychaetes at an average length of 10 cm up to 160.8 ± 37.09 ng PGE₂ mg⁻¹ protein for polychaetes at an average length of 17 cm). The PGE₂ levels in ovaries and haemolymph of female shrimp fluctuated with the developmental stage of the ovaries. The highest concentration of PGE₂ in haemolymph was at stage 3 of ovarian development, whereas the highest concentration of PGE₂ in shrimp ovaries was at stage 4. In vitro incubation of Penaeus monodon pre-vitellogenic oocytes with polychaete extract and synthetic PGE₂ demonstrated that both PGE₂s enhanced oocyte development, especially during late development and ovulation. The putative role of PGE₂ from polychaetes or the presence of PGE₂ in polychaetes may be a factor in their role as a dietary constituent required for shrimp oocyte development.     

The influence of dietary arachidonic acid on the immune response and performance of Pacific whiteleg shrimp,Litopenaeus vannamei,at high stocking density

Stocking shrimp at high densities increases yield during culture, but growth is generally compromised and weakened immune response associated with poor water quality has also been reported. Therefore, we tested if supplying more arachidonic acid (ARA) in the diet, a precursor of eicosanoids such as prostaglandins E from the series II (PGE₂) that enhance immune response can counteract the negative effect of stocking shrimp at high densities. The effect of physical crowding was separated from the effect of water quality, both a result of high density, by using tanks divided by a hard plastic net that allowed water flow between two density conditions. Crowding reduced weight gain by 8.3%, although the effect was more evident with deteriorated water quality from combined effects of high total ammonia and low dissolved oxygen levels (18.4%), but no effect on survival was found. A clear food imprinting of ARA levels in hemocytes was observed, but ARA did not clearly counteract the negative effects of high density on overall performance. However, ARA could minimize stress response of sampling and enhance some effectors of the immune system, such as clotting and respiratory burst. The increase in PGE₂ metabolite in shrimp fed with the high‐ARA diet was not consistent, and thus, the effects of ARA were not necessarily mediated by these eicosanoids.