Disease resistance and immune‐relevant gene expression in golden mandarin fish,Siniperca scherzeri Steindachner,infected with infectious spleen and kidney necrosis virus‐like agent

Infectious spleen and kidney necrosis virus (ISKNV), family Iridoviridae, genus Megalocytivirus, may cause high mortality rates such as those seen in mandarin fish, Siniperca chuatsi. ISKNV has attracted much attention due to the possible environmental threat and economic losses it poses on both cultured and wild populations. We have investigated the pathogenicity of ISKNV‐like agent Megalocytivirus, isolated from infected pearl gourami, in golden mandarin fish, Siniperca scherzeri – a member of the Percichthyidae family – and in another Percichthyidae species, S. chuatsi. Fish were challenged with four different doses of ISKNV‐like agent Megalocytivirus (1, 10, 100 or 1000 μg per fish) over a 30‐day period, and cumulative fish mortalities were calculated for each group. No significant mortality was observed for fish challenged with the lowest dose (1 μg per fish) relative to a control group. However, all other challenged groups showed 100% mortality over a 30‐day period in proportion to the challenge dose. Quantitative real‐time PCR was performed to measure mRNA expression levels for six immune‐related genes in golden mandarin fish following ISKNV‐like agent challenge. mRNA expression levels for IRF1, Mx, viperin and interleukin 8 significantly increased, while mRNA levels for IRF2 and IRF7 remained constant or declined during the challenge period.     

Effects of live and artificial feeds on the growth,digestion, immunity and intestinal microflora of mandarin fish hybrid (Siniperca chuatsi ♀ × Siniperca scherzeri ♂)

How to acclimate mandarin fish to eat artificial feeds has been always a challenge for researchers. The mandarin fish after hybridization (Siniperca chuatsi ♀ × Siniperca scherzeri ♂) could be fed artificial feeds which solved the problem to some extent. However, the growth performance, digestibility, immunity and intestinal microflora of mandarin fish hybrid fed artificial feeds need further study. One hundred and twenty fish of similar size (average weight, 19.5 ± 0.9 g) were randomly stocked into six 250‐L aquariums and separately fed live baits (the control) or artificial feeds in triplicate for 70 days. The weight gain and special growth rate of fish fed artificial feeds were significantly lower than those of fish fed live baits (P < 0.05). The protease activities of stomach, liver and intestine in fish fed live baits were all significantly higher than those in fish fed artificial feeds. The activities of catalase and lysozyme, the content of glutathione in serum of fish fed live baits were all significantly higher than those in fish fed artificial feeds. However, the content of malondialdehyde in liver of fish fed artificial feeds was significantly higher than that in fish fed live baits. The dominant bacteria in both groups were Proteobacteria, Bacteroidetes, Planctomycetes, Cyanobacteria, Verrucomicrobia. However, live baits greatly affected the amount of beneficial and harmful bacteria of intestine in mandarin fish hybrid and broke the balance of intestinal flora.     

A natural infection by the red sea bream iridovirus‐type Megalocytivirus in the golden mandarin fish Siniperca scherzeri

An outbreak of a Megalocytivirus infection was found in the golden mandarin fish Siniperca scherzeri during September and October 2016, in Korea. Phylogeny and genetic diversity based on the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes showed a new strain. Designated as GMIV, this strain derived from the golden mandarin fish was suggested to belong to the red sea bream iridovirus (RSIV)‐subgroup I. Additionally, this train clustered with the ehime‐1 strain from red sea bream Pagrus major in Japan and was distinguished from circulating isolates (RSIV‐type subgroup II and turbot reddish body iridovirus [TRBIV] type) in Korea. The infection level, evaluated by qPCR, ranged from 8.18 × 10² to 7.95 × 10⁶ copies/mg of tissue individually, suggesting that the infected fish were in the disease‐transmitting stage. The diseased fish showed degenerative changes associated with cytomegaly in the spleen as general sign of Megalocytivirus infection. The results confirm that the RSIV‐type Megalocytivirus might have crossed the environmental and species barriers to cause widespread infection in freshwater fish.     

Induction of heterozygous and homozygous diploid gynogenesis in Betta splendens (Regan) using hydrostatic pressure

Abstract Optimum conditions for hydrostatic pressure treatment for duplication of chromosome set in gynogenetically activated fighting fish, Betta splendens (Regan), eggs were identified. Maximum survival of heterozygous gynogens was 50%, when 2·5-min-old eggs, after insemination with UV irradiated tilapia sperm, were pressure shocked at 7000psi for 6 min. The frequency (21%) of homozygous gynogenetic fry was high, when the 34min (post-insemination) old eggs, which were inseminated with tilapia sperm, were pressure shocked for 5 min. Sex ratio of gynogenetic progeny suggested that the mechanism of sex determination in this fish is homogametic female and heterogametic male.     

Nucleotide promotes feed intake and protein utilization via regulating the gene expression of feeding and nitrogen metabolism in juvenile Chinese perch (Siniperca chuatsi)

Nucleotide (NT) could enhance growth, feeding and immunity in higher vertebrate and fish. Chinese perch (Siniperca chuatsi) refuse artificial diet in natural water, whereas the NT might promote its feeding of domestication diet. The present study was designed to investigate the effect of dietary NT on growth, feed intake, feed utilization, body composition, serum biochemistry and nitrogen metabolism in juvenile Siniperca chuatsi. 0, 0.5, 1.0, 1.5, 2.0 and 4.0 g NT kg^?1 diet were supplemented in each diet, respectively. Triplicate groups of Chinese perch (36.21 ± 0.98 g) were fed twice a day to apparent satiation for 8 weeks. The results showed that fish fed with 1.5 g NT kg^?1 diet had the highest weight gain (WG), specific growth rate, feed efficiency, feed intake, protein efficiency ratio and protein retention efficiency. The crude protein content in whole body was highest in fish fed the diet containing 1.5 g NT kg^?1 diet. Dietary NT supplementation significantly increased the neuropeptide Y gene expression and significantly decreased the agouti‐related protein and pro‐opiomelanocortin gene expressions in brain. NT supplementation decreased urea nitrogen content, aspartate aminotransferase (AST) and alanine aminotransferase activities in serum. The hepatic AST activity was increased first and then decreased, and the highest activity was observed in fish fed with 1 g NT kg^?1 diet. Inversely, the activity of glutamate dehydrogenase (GDH) in liver and adenosine 5′‐monophosphate deaminase in muscle decreased first and then increased, and the lowest activity was observed in fish fed with 1.5 g NT kg^?1 diet. Similarly, the gene expression levels of GDH and arginase in liver were lowest in fish fed with 1.5 g NT kg^?1 diet. Based on the broken‐line regression analysis of WG in the present experimental condition, 1.44 g NT kg^?1 diet was the optimum supplementation level in juvenile Siniperca chuatsi.     

Induction of meiotic gynogenesis in the stinging catfish Heteropneustes fossilis (Bloch) and evidence for female homogamety

This study reports the results on induced meiotic diploid gynogenesis and female homogametic nature in the Indian catfish, Heteropneustes fossilis. The eggs of H. fossilis were inseminated with conspecific sperm. The sperm suspension was diluted to 1 × 10⁷ sperm mL⁻¹ in Hanks balanced salt solution. Sperm were irradiated under UV light, with the exposure time ranging from 15 to 360 s (7500 ergs mm⁻² for 60 s). The genetic inactivation of paternal chromosomes was confirmed by chromosome counting from the larval cells and the larvae also had a characteristic haploid syndrome. A typical ‘Hertwig effect’ in the yield of hatched larvae was observed with doses of UV exposure >75 s (9375 ergs mm²). Larvae resulting from sperm UV irradiated above 120 s (15 000 ergs mm²) were 100% haploids. Application of heat shock to the activated eggs was effective in suppressing the release of the second polar body (meiotic gynogenesis) and resulted in diploid gynogenetic larvae morphologically identical to those of the control. The best yield of diploid gynogens (49.3% with respect to the control) was found to be at 6 min after egg activation and the heat shock at 41 °C for a 1-min duration, at an ambient water temperature of 27 °C. A total of 113 diploid gynogenetic fry from seven different female fish were reared and subjected to sexing. All gynogenetic fish were female in contrast to the control, which had a mean sex ratio of 56.7% females (which was not significantly different from 50% female). From these results, the sex determination mechanism in H. fossilis was presumed to be female homogamety.     

Induction of meiotic gynogenesis in bagrid catfish (Pseudobagrus ussuriensis) with homologous sperm and its confirmation for female homogamety

The bagrid catfish, Pseudobagrus ussuriensis, exhibits significant sexual dimorphism in growth rate and body size with males growing faster than females. Therefore, an all‐male culture can dramatically increase the output and profitability of fishery products. According to the monosex breeding route, super‐male individuals’ acquirement by XY male sex reversal and artificial gynogenesis is the key step. An effective protocol to induce meiotic gynogenesis using homologous sperms has been developed in this study. The most effective UV irradiation for sperm genetic inactivation was found to be at a distance of 20 cm with 66 μW/cm² light intensity for 25 min. Optimal treatment for cold shock was 5 min post‐fertilization at 0‐4°C for 30 min, which gave the best survival rate of 13.65 ± 2.87%. The sex ratio in the meiotic gynogens showed a significant female‐biased deviation (p < .05); thirty meiogynogens and their parents were further analysed using a male‐specific AFLP marker, of which only the male parent produced a male‐specific DNA band of 412 bp. These results indicated the female homogametic XX/XY sex determination system in P. ussuriensis. The optimization of a protocol for the successful induction of meiogynogenesis in the bagrid catfish lays the basis for all‐male production and is useful in ascertaining the genetic sex determination system in this promising aquaculture species.     

Genetic variability in meiotic gynogenetic muskellunge,Esox masquinongy (Mitchell), estimated from segregation of microsatellite alleles

Muskellunge, Esox masquinongy, is an important recreational freshwater fish native to North America. Since muskellunge populations are often maintained through stocking efforts, advances in muskellunge reproductive technologies are of direct relevance to fishery enhancement. We evaluated the efficiency of inbreeding through induced meiotic diploid gynogenesis. Eggs from six female muskellunge were manually stripped and activated using ultra violet‐irradiated yellow perch, Perca flavescens, sperm. Hydrostatic pressure shocking regimes (48 263 kPa) were then applied to the eggs to prevent second polar body expulsion producing unambiguous meiotic gynogens. Six female dams and samples of 12–20 of their gynogenetic progeny were genotyped at seven polymorphic microsatellite loci. Chromosomal recombination frequencies of microsatellite loci based on retention of heterozygosity among gynogens ranged from 0.043 to 0.839 (0.576 ± 0.237). There were no statistical differences in recombination frequency among females at any of the loci. The average inbreeding coefficient (F‐value) ranged from 0.581 to 0.979, equivalent to three to fourteen generations of full‐sibling crosses respectively. The average F‐value overall was 0.712, equivalent to between five and six generations of full‐sibling crosses. Centromere map distances of the seven microsatellite loci ranged from 2.15 to 41.95 cM and meiotic gynogenesis was useful in eliminating heterozygosity at loci proximal to the centromere, but not distal. Since the age at maturity of female muskellunge is approximately 5 years, gynogenesis may pose an expeditious alternative to traditional breeding strategies for creation of homozygous pedigrees for some loci that may be outcrossed to introduce positive heterozygosity effects in the offspring.     

Gynogenesis and sex determination in large-scale loach Paramisgurnus dabryanus (Sauvage)

Gynogenesis was induced using heterologous sperms in large-scale loach, Paramisgurnus dabryanus (Sauvage), in which a ZW/ZZ sex determination was previously proposed. Three microsatellite loci were used to monitor exclusive maternal inheritance of gynogenetic progenies. The results showed that high percentages of meiogynogens were produced at 4 min post-fertilization and mitogynogens were produced at 18 min post-fertilization by heat shocks, while meiotic gynogenesis was induced by cold shocks within a wide period and high heterozygosity was even observed in gynogens produced at 24 min post-fertilization. The sex ratios of the F₁ progenies in three gynogenetic families were significantly deviated from 1:1 expectation with a female bias in two families and a male bias in one family (P < 0.05), and the other four gynogenetic families showed approximate 1:1 sex ratios. Moreover, the self-mating between gynogenetic F₁ progenies and mating between gynogenetic F₁ progenies and normal individuals produced all-female progenies or identical proportions of females and males. The data of sex ratios generally confirmed that the sex determination in large-scale loach was determined by the putative ZW/ZZ system, and the possible reasons causing the biased sex ratios are discussed.     

鳜胚胎细胞系的建立与应用

为更好地开展鳜基因功能研究和药物筛选工作,提高基因转染效率,本研究以鳜囊胚期胚胎为材料,采用含有20%胎牛血清的DMEM培养基进行培养,建立了生长稳定的鳜胚胎细胞系MFE。在此基础上,采用绿色荧光蛋白(GFP)作为标记物,在HEK293T细胞中体外包装逆转录病毒,再感染MFE细胞系。MTT法分析表明传代后细胞培养96 h内,细胞生长率变化也经历增殖、降低到达稳定期。而且MFE细胞能稳定表达GFP基因,感染效率为20%±5%,而脂质体转染效率为3%±2%。可见包装病毒感染细胞不仅能获得稳转细胞系,效率也远高于脂质体瞬时转染。荧光定量PCR分析表明,MFE细胞系能表达Irf1、Irf3和Irf7基因,Irf1基因表达量最高。MFE细胞系受到poly I:C刺激后,Irf1、Irf3和Irf7的表达量分别升高3.5,2.3和2.1倍。因此,MFE细胞通过病毒感染可以获得较高的转染效率,该细胞可作为鳜免疫相关基因功能研究的工具。     

Artificial gynogenesis and assessment of homozygosity in meiotic gynogens of spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>)

In the present study, methodology of gynogenetic induction in spotted halibut were developed and optimized; the sex ratio of putative meiotic gynogenetic diploids was determined using AFLP-based molecular sexing technique; the homozygosity of gynogenetic population was assessed as opposed to cultivated population. The results showed that high percentage of meiotic gynogenetic diploids were generated when the eggs fertilized with irradiated heterologous sea perch frozen sperm (30–50 mJ cm⁻²) were cold shocked in sea water of −1°C for 40–75 min at 5 min after fertilization. About 15,200 diploid gynogenetic larvae were achieved and they exhibited normal morphology similar to diploid control. The gynogenetic diploids were 100% female, which first confirmed the female homogamete (XX/XY) sex determination in spotted halibut. The genetic analysis showed that the average H _O was, respectively, 0.404 and 0.724 in gynogenetic population and cultivated population, indicating an increase of homozygosity in gynogenetic population.     

Study of sex determination system in ship sturgeon, Acipenser nudiventris using meiotic gynogenesis

The sex ratios and sex determination mechanism of gynogenetic diploids of ship sturgeon, Acipenser nudiventris, have been investigated to verify the possibilities of sex control by chromosome manipulation in this species. Meiotic gynogenesis was induced in a female ship sturgeon using cold shock after egg activation with UV-irradiated sperm of a male Siberian sturgeon. Microsatellite DNA analysis was applied for verification of uniparental inheritance in the gynogenetic diploid group of fish. All the analyzed gynogenetic diploids possessed only maternal genotype in the examined experimental group of fish. In this study, a minimum of two distinctly selected diagnostic loci in the offspring was used to confirm exclusively maternal contribution. Also, these fish were analyzed for sex diagnostic. Histological analysis of gonads from gynogenetic diploids, obtained from one family, showed 73.3 % of females and 27.7 % of males. The observed sex ratio has suggested that the ship sturgeon have a female heterogametic sex determination system. Gynogenesis in this species with female heterogametic sex determination system will have important role in breeding program and reclamation of its natural population to produce both female and male progeny, while this species has been introduced in the red list of IUCN (International Union for Conservation of Nature and Natural Resources).     

Induction of sex reversal in blue drum (Nibea mitsukurii) and gynogenetic yellow drum (Nibea albiflora) by oral administration of letrozole

The objective of this study was to test the efficiency of an aromatase inhibitor, letrozole (LZ), to induce masculinization in blue drum (Nibea mitsukurii) and gynogenetic yellow drum (N. albiflora). Two experiments (Exp‐1 and Exp‐2) were performed to investigate the effect of LZ on the growth, sex ratio and gonad development in these two fishes. In Exp‐1, blue drum were treated with oral administration of letrozole at different doses (0, 1, 10, 100 mg/kg). In Exp‐2, gynogenetic yellow drum were orally administered different doses of LZ (0, 10, 100 mg/kg) and a dose of 10 mg/kg 17α‐methyltestosterone (MT). The treatments in both experiments were performed from 25 to 85 days post hatch (dph). As a result, all the LZ‐treated fish were phenotypic males despite of slower growth than the control during the treatment period. Free spermatozoa were observed from 150 dph onwards and the gonadosomatic index of the LZ‐treated fish did not differ significantly from that in the control at maturity. However, the sex‐reversed fish induced by MT exhibited a high proportion (33.4%) of testicular abnormalities. Our results showed that oral administration of LZ was efficient in inducing sex reversal in drums, and thereby facilitated their mono‐sex culture.     

黄姑鱼异质雌核发育子代的遗传分析

为了解黄姑鱼（Nibea albiflora）异质雌核发育子代的基因纯合情况,利用微卫星标记（SSR）和扩增片段长度多态性标记（AFLP）对黄姑鱼异质雌核发育家系进行遗传鉴定和分析。结果显示：（1）雌核发育家系在4个SSR位点和5对AFLP引物组合扩增出的位点均未发现父本特异性等位条带,表明雌核发育体比率为100%。（2）用于遗传分析的7个SSR位点在雌核发育家系和正常交配家系中均未见完全纯合的情况,雌核发育家系7个SSR位点的平均纯合度为0.382,是正常交配家系（0.161）的2.37倍。雌核发育家系各个体的纯合位点数为0~6个,纯合位点所占比例为0~85.7%。（3）5对AFLP引物共扩增出182条清晰的扩增条带,其中有21条父本特异性条带和16条母本特异性条带。16条母本特异性条带中有7个条带在雌核发育家系中显著偏分离（P<0.05）。雌核发育家系和正常交配家系多态性条带比例分别为14.7%和20.3%。（4）雌核发育家系与母本的遗传相似度高于与正常交配家系的遗传相似度,正常交配家系同父本和母本的遗传距离大致相同。研究结果表明,黄姑鱼异质雌核发育二倍体家系的遗传纯合度显著高于正常交配家系,人工诱导雌核发育是促进基因纯合的一个有效途径,它不仅可以加速有利基因的纯合固定,还可以加速有害基因的淘汰,从而有效提高育种效率。     

Isolation and characterization of a rhabdovirus from co-infection of two viruses in mandarin fish

Co-infection of two viruses has been observed in mandarin fish (Siniperca chuatsi), but the two viruses have not been characterized. In this study, a rhabdovirus has been isolated from the co-infected two viruses extracted from the diseased mandarin fish, and its morphological structure and partial biochemical and biophysical characteristics have been observed and analyzed. The isolated rhabdovirus has a typical bullet shape, and is therefore called S. chuatsi rhabdovirus (SCRV). And, the isolated rhabdovirus produced a higher titer (10^8.5 TCID₅₀ ml^− 1) than did the co-infecting viruses (10^6.5 TCID₅₀ ml^− 1). Subsequently, the viral genome RNA was extracted, and used as template to clone the complete nucleoprotein (N) gene by RT-PCR amplification. Cloning and sequencing of the SCRV N protein revealed 42%-31% amino acid identities to that of trout rhabdovirus 903/87 and the rhabdoviruses in genus Vesiculovirus. SDS-PAGE separation of the isolated SCRV and other two rhabdoviruses also revealed obvious polypeptide profile difference. Moreover, the anti-SCRV N protein antibody was prepared, and the anti-SCRV N protein antibody only could recognize the SCRV N protein, whereas no antigenicity was detected in other two rhabdoviruses. The data suggested that the SCRV should be a rhabdovirus member related to the genus Vesiculovirus in the Rhabdoviridae.     

Ammonia nitrogen excretion in Mandarin Fish (Siniperca chuatsi) and Grass Carp (Ctenopharyngodon idellus) fed practical diets: the effects of water temperature

To understand the actual production of fish culture about the utilization of dietary protein and excreta impact on the environment between mandarin fish (Siniperca chuatsi) and grass carp (Ctenopharyngodon idellus), the study to investigate the effect of temperatures (19 ± 0.5°C, 24 ± 0.5°C and 29 ± 0.5°C) on ammonia‐N excretion in mandarin fish and grass carp under fed and fasted states was conducted. These two species were fed a practical diet containing 325.2 g kg^?1 crude protein at 3% body weight per day. The ammonia‐N excretion rate was significantly increased when temperature increased from 19 to 29°C, and a linear relationship between ammonia‐N excretion rate and temperature. The maximum ammonia‐N excretion levels of mandarin fish and grass carp were observed at 4–8 h and 2–4 h after feeding, respectively, and the minimum values for both species were observed at 24 h after feeding. Under the feeding condition, mandarin fish had a lower ammonia‐N excretion level compared to grass carp at 24°C and 29°C. The average amount of ammonia‐N excreted by mandarin fish at 24 h is significantly higher than grass carp under fasting conditions, except 19 ± 0.5°C. These results indicated that mandarin fish might make better use of protein at higher temperature than grass carp when fed practical diets in commercial production. These results of this study suggested that mandarin fish had a lower ammonia‐N excretion level compared with grass carp, making a less contribution to environmental loading in an intensive fish culture.     

Cloning,characterization and expression of the pepsinogen C from the golden mandarin fish <Emphasis Type="Italic">Siniperca scherzeri</Emphasis> (Teleostei: Perciformes)

Pepsinogens are precursors of pepsins, which are gastric digestive proteinases that degrade food proteins into peptides. In the study reported here, the cDNA and its corresponding genomic DNA of the golden mandarin fish (Siniperca scherzeri, Perciformes) pepsinogen C (PGC) were cloned and sequenced. The golden mandarin fish PGC gene was deduced to have nine exons and eight introns, a structure similar to the PGCs of other vertebrates. The full-length cDNA was found to contain a 37-bp 5′-untranslated region, a 1,164-bp open reading frame, and a 304-bp 3′-untranslated region and the PGC protein to consist of a signal peptide, an activation segment, and a pepsin moiety. A sequence analysis revealed that pairwise sequence similarities of PGC proteins are around 70% between golden mandarin fish and other vertebrate groups, and around 90% within the fish group. A comparison of vertebrate PGC protein sequences revealed two motifs. One was in the activation segment that occurred only in the mammal and avian PGCs, suggesting that PGCs active in homeotherms (mammal and avian) have different activation mechanisms than those in poikilotherms (amphibian and fish). The second was in the pepsin moiety that occurred only in fish, suggesting the primitive position of fish among vertebrates. PGC mRNA is mainly expressed in the stomach and esophagus and at much lower levels in the skin and muscle. Taken together with results reported from other studies, the results reported here will lead to a better understanding of the molecular mechanisms of fish digestive physiology and the evolution of fish pepsinogen genes.     

Genetic contribution of parents in sex determination of honmoroko Gnathopogon caerulescens

The honmoroko has been inferred to have an XX/XY sex determination system, but the parental genome can also affect the sex ratio of the offspring. The extent of parental effects on sex determination was examined by checking the sex ratios of F1 and F2 gynogenetic diploids and control diploids. Eleven gynogenetic broods from different females consisted of all or nearly all females, but eight broods showed a variable proportion of males (<50 %). One second-generation brood of gynogenetic diploids consisted wholly of females, but others produced some males. In crosses with a control diploid female, four males from a high-percentage male brood of gynogenetic diploids produced offspring with a balanced sex ratio. Sib-mating between a gynogenetic female and three gynogenetic males from the brood produced predominantly male progeny. These results suggest that there are at least four possible genotypes: genotypic female (XX), phenotypic female carrying a silent Y chromosome, genotypic male (XY), and genotypic supermale (YY). These inferences suggest that this fish has an XY system but a relatively high proportion of females possess a mutated, silent Y chromosome which does not lead to testis formation.     

Dietary protein requirement for juvenile mandarin fish,Siniperca scherzeri

An 8‐week feeding trial was conducted to investigate the dietary protein requirement of juvenile mandarin fish, Siniperca scherzeri, a demersal freshwater piscivore with high commercial value in East Asia. Five isolipidic (100 g/kg) and isoenergetic (20 MJ/kg) practical diets were formulated to contain graded levels of 450, 500, 550, 600, and 650 g/kg crude protein, using mackerel fishmeal as the sole source of protein. Each of the five test diets was fed to visual satiety to triplicate groups of fish (initial mean body weight ± SE; 20.1 ± 0.2 g) reared in a recirculating freshwater system over the experimental period. Fish growth performance in terms of weight gain tended to improve with increasing dietary protein level, and the highest values were observed in those fish fed the 600–650 g/kg diets. Feed intake significantly declined as the protein content of the diet increased. Feed efficiency and protein retention tended to increase with increasing dietary protein levels, from 450 to 600 g/kg, and then declined when dietary protein content further increased to 650 g/kg. A similar trend was also found for the protein efficiency ratio, although the observed changes did not reach statistical significance. Whole‐body composition and plasma biochemical parameters were not significantly affected by the dietary protein content. Based on these findings and a broken‐line model of growth, 614.4 g/kg seems to be the proper amount of protein in a practical diet for >20 g mandarin fish.