Recovery of gonadal development in tiger puffer Takifugu rubripes after exposure to 17β-estradiol during early life stages

The aim of the present study was to investigate the long-term effects of 17β-estradiol(E2) exposure on gonadal development in the tiger puffer( Takifugu rubripe s), which has a genetic sex determination system of male homogametic XY-XX. Tiger puffer larvae were exposed to 1, 10 and 100 μg/L E2 from 15 to 100 days post-hatch(dph) and then maintained in clean seawater until 400 dph. Changes in sex ratio, gonadal structure and gonadosomatic index(GSI) were monitored at 100, 160, 270 and 400 dph. Sex-associated single nucleotide polymorphism(SNP) markers were used to analyze the genetic sex of samples, except those at 100 dph. Exposure had a positive effect on the conversion of genetically male gonads into phenotypically female gonads at 100 dph. However, gonads from 60% of genetic XY males in the 1-μg/L E2 group and 100% in the 10-μg/L E2 group developed intersexual gonads at 160 dph; gonads of all genetic XY males in the two treatment groups reverted to testis by 270 dph. While 38%, 57% and 44% of gonads of XY fish in the 100-μg/L E2 group reverted to intersexual gonads at 160, 270 and 400 dph, respectively, none reverted to testis after E2 treatment. In addition, E2 exposure inhibited gonadal growth of both genetic sexes, as indicated by the clear dose-dependent decrease in GSI at 270 and 400 dph. The results showed that exposure to E2 during the early life stages of tiger puffer disrupted gonadal development, but that fish recovered after migration to clean seawater. The study suggests the potential use of tiger puffer as a valuable indicator species to evaluate the effects of environmental estrogens on marine fish, thereby protecting valuable fishery resources.     

Temperature-control-induced masculinization in tiger puffer Takifugu rubripes

This study used tiger puffer Takifugu rubripes to explore new, simple methods for lowtemperature-induced masculinization in a cultured fish without the use of ultraviolet irradiation or sex hormones. An orthogonal test L_9(3~4) design was used to consider three factors at three levels: treatment starting times(days post-hatch, dph: factor A) of 20, 50 and 80 dph; treatment temperatures(factor B)of 13℃, 15℃ and 17℃; and treatment durations(factor C) of 30, 45 and 60 days. A control group was reared at 21±1℃. The experiments were repeated twice. At 230 dph, the gonads were removed from thirty randomly sampled fish in each group. Histological observations and analysis of single nucleotide polymorphisms(SNP) were used to identified pseudo males, which biological sex was male and genetic sex were female(XX). Treatment group 4(A2B1C2) resulted in the highest proportion of males(75%).According to the intuitive analysis of the orthogonal-array experiments, the optimal combination of lowtemperature-induced masculinization of T. rubripes was A2B1C2. The population sex ratio depended on the three factors in the sequence B→A→C. A comparison of the daily increases in length and weight during and after the low-temperature conditions showed that the absolute daily increases in weight and length were significantly less during treatment than after treatment. Daily increases in weight and length did not significantly differ between the treatment groups and the controls(P0.05), demonstrating that the growth rate could return to normal after the low-temperature conditions. This study establishes a low-temperatureinduced masculinization technology for T. rubripes and demonstrates that although the growth rate(length and weight) decreased in an array of nine treatment groups during the processing time, it returned to a normal level after processing. The results should serve as a guide for achieving the masculinization of T.rubripes in production.     

Expression profiles of sex-related genes in gonads of genetic male Takifugu rubripes after 17β-estradiol immersion

Estradiol treatment during early life stages of tiger puffer Takifugu rubripes induces feminization in genetic males. However, the ovaries in genetic males may revert to testes once estradiol treatment is halted.Therefore studies should investigate molecular mechanisms underlying ovary-to-testis recovery in genetic males after treatment. In the present study, tiger puffer were exposed to 10, and 100 μg/L 17 p-estradiol(E_2)from 15 to 100 days post-hatching(dph), then gonad phenotypes and expression profiles of six sex-related genes(cyp19a,foxl2, dmrtl, amh, sox9a, and sox9b) were characterized after the exposure. Results showed that both 10 and 100 μg/L E_2 induced ovarian development in genetic males at 100 dph. However, all ovaries induced by 10 μg/L E_2 first developed into intersexual gonads and subsequently reverted to testes after the exposure. As for treatment of 100 pg/L E_2, while the rest of the ovaries maintained morphological stability,percentages of intersexual gonads reached 38%-57%, and none were reverted to testes. Increased mRNA levels of cyp19 a,foxl2 and sox9b and decreased mRNA levels of dmrtl, amh, and sox9a were observed during the ovarian development in genetic males. While contrary gene expression profiles were detected during ovary-to-testis transformation. The mRNA levels of all the six genes were increased during the development of intersexual gonads. These results indicated that up-regulation of dmrtl, amh and sox9a is associated with initial ovary-to-intersexual transformation, and suppression of foxl2, cypl9a and sox9b is essential for complete ovary-to-testis recovery in genetic males. This research will help to trace the molecular processes underlying gonadal transformation in teleosts.     

ANNUAL CHANGES IN PLASMA LEVELS OF CORTISOL AND SEX STEROID HORMONES IN MALE RAINBOW TROUT, ONCORHYNCHUS MYKISS

The profiles of cortisol, testosterone, ll-ketotestosterone and 17α， 20β- dihydroxy-4-pregnene-3-one in mule rainbow trout reared under constant water temperature and natural photoperiod were determined by radioimmunoassay. Gonads of male rainbow trout reached maturity when the fish were two years old. Changes in the plasma levels of both sex steroid hormones and cortisol were closely related to the GSI. Plasma levels of testosterone, 11-ketotestostemne and 17a, 2013-dihydroxy 4-pregnene-3-one showed a clear peak in the annual breeding season, when the GSI reached their maxima. Plasma cortisol levels also showed clearly seasonal changes in both two- and three-year-old fish. The results suggest that the elevated plasma levels of cortisol may not just be due to stresses during the breeding season but have certain physiological functions in the reproduction of rainbow trout.     

Body composition and compensatory growth in Nile tilapia Oreochromis niloticus under different feeding intervals

We investigated the growth and body composition of Nile tilapia under five different feeding regimes. A control group was fed to satiation twice daily for 185 days; four treatment groups were fed at intervals of 2, 3, 4 or 7 days(dietary ‘restricted' period, days 0–80) and then fed to satiation(‘refeeding' period, days 80–185). Compensatory growth in weight and length of the feed-restricted groups was observed during the refeeding period. However, the growth of none of the restricted groups caught up with that of the control group over the experimental period. Feed intake upon refeeding increased with the duration of deprivation. There were no significant differences in feed efficiency between the restricted and control groups during the refeeding stage, suggesting that hyperphagia was the mechanism responsible for the increased growth rates during this period. Tilapia preferentially used n-3 polyunsaturated fatty acids and nonessential amino acids during the restricted-feeding period. Higher production was achieved by higher feed consumption. We suggest that if attainment of market size in minimum time is required, fish should be consistently fed to satiation, while taking care to avoid the possible negative consequences of overfeeding.     

Compensatory Growth of Juvenile Brown Flounder Paralichthys olivaceus Following Low Temperature Treatment for Different Periods

We investigated the effects of low temperature(8.5℃) on the growth and feeding rates and feed conversion efficiency of juvenile P.olivaceus with an average initial weight of 3.87 ± 0.06 g(mean ± SE).Fish were exposed to 8.5℃ for 0(control),1,2,3 and 4 weeks,and then to 20℃ for 10,9,8,7 and 6 weeks,respectively.Low temperature clearly led to growth depression.The weight of fish exposed to low temperature for 1 week was restored to that of control,while that of fish exposed to low temperature longer was significantly decreased(P 0.05).During the entire low-temperature period,specific growth rate,feeding rate and feed conversion efficiency of the fish were significantly lower(P 0.05) than those of control,while in the whole recovery period,specific growth and average feeding rate were markedly higher(P 0.05) than those of control.At the end of experiment,only the feeding rate of the fish exposed to low temperature for 1 week was not significantly different from that of control(P 0.05).Feeding rate and feed conversion efficiency were reduced at low temperature in juvenile P.olivaceus.The compensatory growth of juvenile P.olivaceus may therefore be attributed to the improvement of feeding rate.Our results suggested that growth depression occurs when juvenile P.olivaceus are exposed to low temperature for more than one week.     

Tissue-specific bioaccumulation and oxidative stress responses in juvenile Japanese flounder (Paralichthys olivaceus) exposed to mercury

To understand mercury (Hg) toxicity in marine fish, we measured Hg accumulation in juvenile Japanese flounder (Paralichthys olivaceus) and assessed the effects on growth and antioxidant responses. After Hg exposure (control, 5, 40, and 160 μg/L Hg) for 28 d, fish growth was significantly reduced. The accumulation of Hg in fish was dose-dependent and tissue-specific, with the maximum accumulation in kidney and liver, followed by gills, bone, and muscle. Different antioxidants responded differently to Hg exposure to cope with the induction of lipid peroxidation (LPO), which was also tissue-specific and dosedependent. As Hg concentration increased, superoxide dismutase (SOD) and catalase (CAT) activities increased significantly, whereas glutathione S-transferase (GST) activity and glutathione (GSH) levels decreased significantly in the gills. SOD and glutathione peroxidase (GPx) activities and the GSH level increased significantly in the liver. SOD activity and GSH levels increased significantly, but CAT activity decreased significantly with an increase in Hg concentration in the kidney. LPO was induced significantly by elevated Hg in the gills and kidney but was least affected in the liver. Therefore, oxidative stress biomarkers in gills were more sensitive than those in the liver and kidney to Hg exposure. Thus, the gills have potential as bioindicators for evaluating Hg toxicity in juvenile flounder.     

Effects of supplemental coated or crystalline methionine in low-fishmeal diet on the growth performance and body composition of juvenile cobia Rachycentron canadum （Linnaeus）

We evaluated the effects of supplemental coated and crystalline methionine （Met） on the growth performance and feed utilization of juvenile cobia （Rachycentron canadum Linnaeus） in a 60-d feeding trial. Fish groups were fed one of six isonitrogenous and isolipidic diets： 1） fishmeal control; 2） un- supplemented experimental （low-fish-meal diet deficient in Met）; or 3） one of four Met diets supplemented with crystalline L-Met, cellulose-acetate-phthalate coated L-Met, acrylic-resin coated L-Met, or tripalmitin- polyvinyl alcohol coated L-Met. The test diets were fed to triplicate groups of cobia （initial body weight 5.40-4-0.07 g） twice a day. The weight gain and specific growth rate of the fish fed the RES diet were highest among the Met-supplemented groups and were 23.64% and 7.99%, respectively, higher than those of the fish fed with the un-supplemented experimental diet （P〈0.05）. The protein efficiency ratio of the fish fed the MET diet was significantly higher than that of the fish fed the un-supplemented experimental diet and the fish in the other methionine supplementation groups （P〈0.05）. Our results suggest that supplementation of crystalline Met in low-fish-meal diets promotes the growth performance of juvenile cobia.     

Impacts of mariculture practices on the temporal distribution of macrobenthos in Sandu Bay,South China

Sandu Bay is located in the East China Sea and is characterized by high-density fish farming and kelp culture. Despite this, little is known about the impacts of these different mariculture practices on the local environment. We investigated the temporal variation in macrobenthos and environmental conditions at three sites in the bay (fish farming site, kelp culture site, and a control site). We collected water and sediment samples during nine cruises between May 2009 and February 2010. The density of macrobenthos peaked at the fish farming site in July (655 ind./m2) whereas density did not fluctuate as widely at the other two sites. Biomass varied significantly at both the control and kelp culture sites, but had only a single peak at the fish farming site in June (21.90 g/m2). The dominant species varied throughout the study period at the control and kelp culture sites, whereas a single terebellid species (Lysilla pacifica) dominated the macrobenthos at the fish farming site. The diversity index H’ increased at the control site beginning in February then decreased after May, whereas H’ was low at the other sites in December. The mean dissolved oxygen level was highest at the control site (6.59 mg/L) and lowest at the fish farming site (5.54 mg/L). DO levels were lowest at all sites in summer (July and August). The sediment acid volatile sulfide content was higher at the fish farming site (1.46 mg/g dry weight) than those at the kelp culture and control sites (1.22 and 0.14 mg/g, respectively). Our results suggest that mariculture practices have a clear impact on the benthic environment/ community in Sandu Bay.     

Effect of Bioactive Peptides(BPs) on the Development of Pacific White Shrimp(Litopenaeus vannamei Boone, 1931)

The present study was conducted to evaluate the feasibility of replacing fish meal(FM) with bioactive peptides(BPs) in diet of white shrimp(Litopenaeus vannamei). The changes in growth performance, body composition, non-specific immunity, and water quality were examined after the shrimp were fed four diets, in which 0%(control), 33.3%, 66.7% and 100% of FM was replaced by BPs, respectively. The groups were designated as Con, 1/3BPs, 2/3BPs, and 3/3BPs. A total of 720 shrimp with an initial body weight of 1.46 ± 0.78 g were fed the experimental diets for 56 days. The results revealed that: 1) the weight gain rate(WGR) in 1/3BPs, 2/3BPs, and 3/3BPs was significantly higher than that in Con(P 0.05), while no significant difference was found on survival rate and feed conversion ratio(FCR); 2) the whole-body crude protein(CP) and crude lipids(CL) were significantly different among groups, while there was no significant difference between crude ash and phosphorus contents; 3) the levels of acid phosphatase(ACP), lysozyme(LZM), superoxide dismutase(SOD), phenol oxidase(PO) and bactericidal activity increased significantly with the inclusion of BPs; 4) in terms of water quality, no significant difference was found in p H and dissolved oxygen among diets during the whole experimental period. Moreover, even though nitrite and ammonium levels tended to increase with time, there was no significant difference among groups. The results indicated that BPs is an applicable alternative of protein source, which can substitute FM in the diets of L. vannamei; it is able to effectively promote growth performance and improve immunity. Moreover, BPs in the diets had no negative impact on water quality.     

Effects of Dietary Protein and Lipid Levels on Growth Performance,Muscle Composition,Immunity Index and Biochemical Index of the Greenfin Horse-Faced Filefish(Thamnaconus septentrionalis) Juvenile

The effects of different dietary protein and lipid levels on juvenile greenfin horse-faced filefish(Thamnaconus septentrionalis) was experimentally evaluated in a 56-day study. Fish(initial weight 6.47 ± 0.56 g) were fed with nine different diet combinations containing three varying levels of protein(37%, 43%, 49%) and lipids(4%, 7%, 10%) in triplicates, where the protein-to-energy(P/E) ratios of the diets varied in the range of 19.17–26.99 mg kJ-1. Results showed that fish fed diets containing 49%protein and 10% lipid exhibited significantly lower feed intake than those fed other diets. The weight gain of fish fed diets with 43%protein was significantly higher than that of fish fed 37% or 49% protein diets. While no significant change in weight gain was observed when lipid content was increased from 4% to 7%, weight gain decreased significantly when lipid increased from 7% to 10%.The protein efficiency ratio and feed efficiency were highest when the fish were fed the diet with 49% protein and 7% lipids. Muscle crude lipid content was positively correlated with dietary protein level regardless of lipid level. The diet of 43% protein or 7% lipids significantly reduced the activity of alanine aminotransferase and aspartate aminotransferase in liver. The contents of aspartate aminotransferase, superoxide dismutase and malondialdehyde were significantly increased from 4% to 10% lipids in plasma. The plasma total cholesterol and triglyceride contents were positively correlated with dietary lipid level at the same protein level. Our comprehensive analysis concluded that a diet with 43% protein and 7% lipid, with a P/E ratio of 23.07 mg kJ-1, significantly promote the growth performance, feed utilization and health of juvenile greenfin horse-faced filefish.     

Application of hydroacoustics to investigate the distribution,diel movement,and abundance of fish on Zhubi Reef,Nansha Islands,South China Sea

A combination of traditional fish sampling methods(hand-line and gill net) and modern hydroacoustic techniques were used to study fish community structure,distribution,and diel movements of fish on Zhubi Reef to enhance understanding of the ecosystem.We collected 126 individuals from 29 species,20 genera,17 families,three orders,and two classes using traditional gear.Perciforms were the dominant group in terms of species richness and biomass.The acoustic data indicated that very small(target strength[TS],dB) -60 dB) and small(-60 dB≤TS-45 dB) fish contributed the most to abundance and species richness on the coral reef,and that the proportion of medium-sized(-45 dB≤TS-35 dB) and largesized(-35 dB≤TS) fish increased gradually as depth increased.The single-target detection method revealed two distinct size classes during the day in the 12-16 and 16-20-m layers.One group consisted of very smallsized fish(TS-60 dB) and the other consisted of medium and large-sized fish(TS-55 dB).The number of single-target detections was significantly higher during the night than during the day(P0.05).The singletarget TS frequency distribution during the day was significantly different than during the night at depths of4-8,8-12,12-16,and 16-20 m.Significant differences were observed among the 4-8,8-12,12-16,and16-20-m-depth layers during day and night.Diel vertical movement was evidenced as fish began to spread and move upward just before sunset and began to assemble and descend shortly(15 min) after sunrise.     

Effects of diet and body size on phosphorus utilization of Liza haematocheila T. ＆ S.

A 21-d laboratory experiment was conducted to study, the phosphorus （P） utilization of two different diets by redlip mullet Liza haematocheila T. ＆ S. Sand-filtered water in salinity 30 and temperatare 25℃ was used. Twenty-nine fish individuals were divided into three groups： 11 to group 1 （G1） fed on diet 1, 11 to group 2 （G2） fed on diet 2, and 7 to contrast group. Diet 1 was a commercial feed, more valuable in nutrition than diet 2 that similar to natural detritus. The results show the intake phosphorus （IP） of G1 was significantly higher than that of G2, and both increased linearly with body size at a certain amount of diet. The retention phosphorus （RP） in fish of G1 was lower than G2. The relationship between retention phosphorus and body size was positive and stronger in G2. Significant difference in faecal phosphorus （FP） was found between G1 and G2. Body size significantly impacted the excretion phosphorus （EP） in G1 but G2. The loss of intake phosphorus in G1 was 10.83-20.27 mg per g fish weight gain, higher than that in G2 for 6.63-9.56. Of the phosphorus, about 10% was allocated into growth, 50% in faeces, and the rest lost in excretion. The main part of phosphorus was lost in faeces but excretion. The phosphorus budget of the fish could be described as 100IP = 7.40RP ＋ 47.39FP ＋ 36.63EP （Diet 1） or 100IP = 11.93RP ＋ 56.64FP ＋ 21.76EP （Diet 2）.     

Otolith Shape Analysis as a Tool to Identify Two Pacific Saury(Cololabis saira) Groups from a Mixed Stock in the High-Seas Fishing Ground

The Pacific saury Cololabis saira is a commercially important pelagic species with wide distribution in the North Pacific Ocean. Previous studies identified two geographical groups by comparing the radius of otolith annual rings(ROA) of the fish collected during the pre-fishing season. Moreover, understanding the group composition in the mixed catch from the fishing ground is essential for sustainable exploitation of this species. Using samples collected during the fishing season(July–November) from 2016 to 2018, the potential of applying otolith shape analysis as a tool to identify these two mixed groups was tested in this research. Two otolith morphotypes that belong to the eastern group and western group were identified by K-means cluster analysis using Fourier coefficients and ROA. The high classification success of random forest further confirmed the effectiveness of otolith shape analysis. The western group accounted for the majority of catch in July and August at 160?E. With the passing of sampling time and fishing fleets moving westward, the proportion of eastern group gradually increased and finally reached more than 60% of the whole catch in November at 150°E. This result suggests that the eastern and western groups are well mixed during spawning migration. In addition, the temporal and spatial dynamics of their catch composition provide information for their migration route. This study is helpful for improving fisheries management and understanding the short-term stock fluctuation of Pacific saury.     

Effects of Calanoid Copepod Schmackeriapoplesia as a Live Food on the Growth,Survival and Fatty Acid Composition of Larvae and Juveniles of Japanese Flounder,Paralichthys olivaceus

Zooplankton constitutes a major part of the diet for fish larvae in the marine food web, and it is generally believed that copepods can meet the nutritional requirements of fish larvae. In this study, calanoid copepod Schmackeria poplesia, rotifer Brachionus plicatilis and anostraca crustacean Artemia sp. were analyzed for fatty acid contents, and were used as live food for cul- turing larval Japanese flounder, Paralichthys olivaceus. The total content of three types of HUFAs （DHA, EPA and ARA） in S. po- plesia was significantly higher than that in the other two live foods （P〈O.O1）. Three live organisms were used for raising larvae and juveniles of Paralichthys olivaceus respectively for 15 and 10 d. Then the growth, survival and fatty acid composition of the larvae and juveniles were investigated. The results showed that the larvae and juveniles fed with copepods （S. poplesia） had significantly higher growth rate than those fed with the other two organisms （P〈0.01）. The survival of the flounder larvae fed with copepods was significantly higher than that of the others （P〈0.01）, and the survival of the juvenile fish fed with copepods was higher than that fed with Artemia （P〈0.05）. The contents of three types of HUFAs （DHA, EPA and ARA） and the ratio of DHA/EPA in larval and juve- nile flounder P. olivaceus were analyzed. The results showed that the contents of DHA, EPA and ARA in the larvae and juveniles fed with S. poplesia were higher than those fed with a mixed diet orArtemia only, and the ratio of EPA/ARA in larvae and juveniles of P. olivaceus fed with S. poplesia was lower than that in the case of feeding with a mixed diet or Artemia only. The present data showed that copepod is the best choice for feeding the larvae and juveniles of fish considering its effects on the survival, growth and nutrition composition of the fish.     

Effect of Dietary Vitamin E on the Sperm Quality of Turbot(Scophthalmus maximus)

A 3-month feeding experiment was conducted in an in-door seawater system to investigate the effect of dietary vitamin E(Ve) on the sperm quality of turbot(Scophthalmus maximus). D-α-tocopherol acetate was supplemented to the basal(control) diet(65.14 mg kg–1 Ve) to obtain low and high levels of dietary Ve(244.60 mg kg–1, LVe; 721.60 mg kg–1, HVe). Compared with the control, sperm concentration was significantly increased in Ve-supplemented groups(LVe and HVe); while relative sperm volume and testis-somatic index were significantly increased in group HVe only. Sperm motility duration was significantly longer in group HVe than in the control, but no significant difference was observed in percent motility among groups. Sperm size, the uniformity of mitochondrial size, and the integrity of mitochondria cristae and plasma membrane were improved by dietary Ve, especially in HVe. The content of Ve in testis and liver as well as polyunsaturated fatty acids in sperm increased with dietary Ve. These results suggested that dietary Ve, especially at the high level(721.60 mg kg–1), significantly improved sperm concentration and motility duration and maintained normal sperm morphology of turbot.