Abstract:Chinese sturgeon (Acipenser sinensis) is a large anadromous fish listed under Category I State protectionin China and is critically endangered. This anadromous fish has historical commercial importance and waswidely dispersed in the main tributaries of the Yangtze River and the coastal regions of the Qiantang River, MinjiangRiver, and Pearl River. However, natural populations have declined severely in the last three decades as aresult of blocking of the migration route by the Gezhouba Dam. Natural reproduction of A. sinensis was interruptedduring 2013–2014 according to the monitored number of fish in the breeding population at the GezhoubaDam spawning site and catadromous juvenile fish in the estuary. Fifteen juvenile sturgeons were found in the JiangsuXupu section of the Yangtze River in June 2015. It is of great significance to evaluate whether these juvenilefish are Chinese sturgeon or hybrid sturgeon; however, few studies have tried to identify Chinese sturgeon amongother sturgeons. In this study, we successfully identified and differentiated the newly found juvenile fish fromthree other sturgeon species endemic to China, including 30 A. sinensis, 39 A. dabryanus, and 33 A. schrenckii.Morphological characters, such as bone lamella, gill rakers, and dorsal rays, of these newly found juvenile fishwere consistent with those of Chinese sturgeon. Eleven polymorphic microsatellite loci isolated and characterizedin a previous study were used to identify the three sturgeon species. The results showed that the newly foundsturgeon in the Yangtze River was closest to A. sinensis (Fst=0.039, P<0.05) and farthest from A. schrenckii(Fst=0.209, P<0.05), indicating that these fish were undoubtedly juvenile Chinese sturgeon. The level of geneticdiversity (mean number of alleles=9.455; total heterozygosity=0.796; Shannon-Wiener Diversity Index=1.998) ofthese juvenile Chinese sturgeon was lower than that of other wild Chinese sturgeon (mean number of alleles=13.455; total heterozygosity=0.810; Shannon-Wiener Diversity Index=1.886), suggesting that the size of thewild Chinese sturgeon breeding population is decreasing. No natural A. sinensis reproduction was found during2014 at the Gezhouba Dam spawning site, which is the only known spawning site for this endangered species. Wespeculate that these fish came from another spawning ground. In recent years, the Three Gorges Dam, which is theworld’s largest hydroelectric project, was put on-line, and it has had a tremendous impact on the natural reproductionof Chinese sturgeon by influencing the seasonal fluctuations in downstream water flow and temperatures,potentially resulting in loss of the eco-hydrological conditions needed by the fish for spawning. The Chinese sturgeonis an adaptable fish judging from its life history, so it is likely that other spawning sites located downstreamof the Gezhouba Dam are being used. It is necessary to identify the exact positions of these spawning grounds todescribe the hydrological conditions and better protect this endangered species. Our results will be useful formonitoring natural populations, surveying new spawning sites, and designing conservation strategies.

Abstract:Toll-like receptor (TLR) 5 plays a vital role in bacterial flagellin recognition and immune responsealerts in vertebrates. In the present study, the synergistic role of the TLR5 membrane form and the TLR5 solubleform (TLR5S) are reported in a bony fish. The full-length TLR5S cDNA was cloned using homologous cloningand rapid amplification of cDNA ends techniques to study the regulatory role of TLR5S in the innate immune responseof half-smooth tongue sole, Cynoglossus semilaevis, (designated Cs.TLR5S). Three alternative splicingvariants (Cs.TLR5S x1, Cs.TLR5S x2, and Cs.TLR5S x3) of the Cs.TLR5S cDNA sequence were found in C. semilaevis.The full-length CsTLR5S cDNA included a 308 bp 5′-untranslated region (UTR), a 1701 bp open readingframe, and 138 bp, 364 bp, and 637 bp 3′-UTRs, respectively. The cDNA encoded a polypeptide of 567 aminoacids, with a molecular mass of 64.03 kD and an isoelectric point of 8.49. Multiple sequence alignment revealedthat the Cs.TLR5S proteins are well conserved with a typical modular architecture and identical active sitesthroughout vertebrates, and shared the highest identity with Paralichthys olivaceus TLR5S (61%), suggesting aconserved function for TLR5S. A phylogenetic analysis indicated that Cs.TLR5S and homologous TLR5S sequencesfrom teleosts were clustered into a clade, and Cs.TLR5S was separated from another clade with amphibians,mammals, and other vertebrates. A tissue expression profile analysis using the quantitative real-time polymerasechain reaction (qRT-PCR) showed that Cs.TLR5S mRNA was constitutively expressed in all tested tissues,with predominant expression in liver and the lowest expression in spleen. Alternative splicing of the 3′-UTR usingqRT-PCR showed that Cs.TLR5S x3 was only expressed in liver, whereas Cs.TLR5S x1 was expressed in liver andintestine. In addition, Cs.TLR5S was expressed at different levels in liver, spleen, intestine, and head kidney after aVibrio anguillarum challenge. These results suggest that expression of the C. semilaevis Cs.TLR5S variants aredifferentially regulated in different tissues and play important roles in the immune response against bacterialpathogens.

Abstract:The major histocompatibility complex (MHC) is a gene family common to vertebrates and is encoded by agroup of genes closely related to immunological functions. Many studies have indicated that MHC occurs on the surfaceof all karyocytes and that the MHC II genes of fish differ by species in structure, character, and polymorphisms.The spotted scat (Scatophagus argus) is eurythermic and euryhaline and is widely distributed in brackish water and marinehabitats of the Indo-Pacific region. Spotted scat has become an economically important species in southern Chinain recent years owing to its strong tolerance to a variety of environmental conditions. However, most spotted scat studiesin the last two decades have focused on artificial breeding, biological characteristics, gonadal development, reproductivecharacteristics, and genes related to osmotic adjustment and gonadal development, but not immune systemfunction. Furthermore, farmers have recently found that spotted scat are susceptible to infections by parasites, bacteria,and viruses, which could cause serious economic losses. Thus, we determined the complete cDNA sequence of thespotted scat MHC-IIβ gene for the first time using homology-based cloning and rapid amplification of cDNA ends polymerasechain reaction (RACE-PCR) and analyzed the introns, gene polymorphisms, and tissue expression. As results,the cDNA sequence of the spotted scat MHC-IIβ gene was 1172 bp in length, including a 34 bp 5′-untranslated region(UTR), a 388 bp 3′-UTR, and a 750 bp open reading frame (ORF). The ORF contained the signal peptide, the β1 domain,the β2 domain, a connecting peptide, a transmembrane region, and a cytoplasmic tail. The total length of the genewas 2920 bp. The MHC-IIβ gene contained six exons and five introns (85−1010 bp), and the third intron separated theβ2 domain into two parts. This structure differs from some other teleosts and provides new evidence for the lack ofconservation of MHC-IIβ gene structure. The molecular formula was C2239H3730N750O934S198, and relative molecularmass was 62.45 kD. Some classic functional sites were found in the amino acid sequence, such as a protein kinase Cphosphorylation site, an N-glycosylation site, a casein kinase II phosphorylation site, a tyrosine kinase phosphorylationsite, and an N-nutmeg acetoxylation site. Interestingly, the number of protein functional sites in the spotted scatMHC-IIβ gene was very similar to that in other fish, such as common carp, yellow croaker, and Japanese flounder, indicatingconservation of MHC in fish. We identified 48 alleles from 209 clones in 43 individuals to examine polymorphismsin the spotted scat MHC-IIβ gene. These alleles were named Scar-DXB*0101−Scar-DXB*4801, indicating highpolymorphism of the MHC-IIβ gene in S. argus. However, allelic polymorphisms of the MHC-IIβ gene are quite differentin fish, which may be an inherent characteristic of the species or it may be related to other factors, such as thefounder effect, gene flow, or gene selection during evolution. The RT-PCR analysis showed that the MHC-IIβ gene wasexpressed in all 11 tissues examined, with the highest levels in spleen, gill, intestine and skin; moderate levels in kidney,stomach, and heart; and low levels in the eyes, brain, liver, and muscle. MHC-IIβ gene mRNA expression levels variedin liver, spleen, gill, and kidney after infection with Aeromonas hydrophila, indicating that the gene has important effectson the immune response. A neighbor-joining tree showed that the S. argus MHC-IIβ gene had a fairly close geneticrelationship with those of Paralichthys olivaceus, Dicentrarchus labrax, and Salmo salar and more distant genetic relationshipswith Ginglymostoma cirratum, Gallus gallus, Mus musculus, and Homo sapiens. These results lay the foundationfor screening resistance genes and breeding S. argus for disease resistance.

Abstract:We cloned the full-length mitogen activated protein kinase kinase 3 (MKK3) cDNA sequence using therapid amplification of cDNA ends method to understand the physicochemical and functional characteristics ofMKK3 in Fenneropenaeus chinensis. F. chinensis MKK3 was analyzed using a bioinformatics method to explorethe sequence homology of MKK3 genes from different species. MKK3 gene expression levels were determined indifferent tissues by quantitative real-time polymerase chain reaction analysis before and after exposure to ammonia-N stress. The full-length cDNA sequence of the F. chinensis MKK3 gene (FcMKK3) was 1434 bp long andcontained a 33 bp 5′-untranslated region (UTR), a 390-bp 3′-UTR, and a 1011-bp open reading frame that encoded336 amino acid residues, with an isoelectric point (PI) of 6.08 and molecular mass of 37.89 kD. The homologyanalysis revealed that the FcMKK3 amino acid sequence had highly similarity with MKK3 of other species, suchas 69% identity with Nasonia vitripennis MKK3 and 68% identity with Ceratitis capitata MKK3. The phylogeneticanalysis showed that FcMKK3 was in the same class with other arthropod MKK3 genes. The FcMKK3 genewas expressed in intestine, gill, stomach, heart, lymph, hepatopancreas, muscle, and hemocytes, with significantdifferences in tissue expression levels. Relative expression of FcMKK3 in muscle was the highest, followed by thegill. FcMKK3 expression in hemocytes was the lowest. FcMKK3 expression after exposure to ammonia-N stresswas initially upregulated in intestine, gill, stomach, and muscle and then downregulated, followed by upregulation.FcMKK3 expression reached the first peak at 6 h, 6 h, 6 h, and 3 h in intestine, gill, stomach, and muscle, respectively,which was 2.33-fold (P<0.01), 1.56-fold (P<0.01), 2.99-fold (P<0.01), and 1.56-fold (P<0.01) of the fourtissues in control animals, respectively. The second peak was reached at 96 h, 72 h, 72 h, and 48 h, which was2.49-fold (P<0.01), 2.34-fold (P<0.01), 2.36-fold (P<0.01), and 5.58-fold (P<0.01) more than those in the controlgroup, respectively. Relative FcMKK3 expression levels in the experimental group were higher than those in thecontrol group at all testing points. In contrast, relative FcMKK3 expression in heart, hepatopancreas, and hemocyteswas downregulated initially and then upregulated. FcMKK3 expression was the lowest at 3 h, 3 h, and 6 h,which was 0.56-fold (P<0.01), 0.26-fold (P<0.01), and 0.72-fold (P<0.01) of the values in the three tissues fromanimals in the control group, respectively. Then, FcMKK3 expression was upregulated and reached peaks at 48 h,6 h, and 24 h, which were 2.16-fold (P<0.01), 2.53-fold (P<0.01), and 1.19-fold (P<0.05) of the control groupvalues, respectively. In conclusion, relative FcMKK3 expression was upregulated significantly in intestine, gill,stomach, heart, hepatopancreas, muscle, and hemocytes compared with those in the control group after exposure toammonia-N stress and showed different expression profiles. These results suggest that FcMKK3 might play importantroles in the F. chinensis stress response.

Abstract:Exopalaemon carinicauda is the most commercially important shrimp in China based on reproduction, along growing season, and ecological flexibility. However, the frequency of disease outbreaks has increased asproduction has increased, resulting in huge economic losses. E. carinicauda relies on the innate immune responseto defend against invading pathogens. The 14-3-3 family of proteins is highly conserved and present in all eukaryoticcells. The 14-3-3 proteins are involved in many cellular pathways, including those for metabolism, thecell cycle, differentiation, signaling, apoptosis, and neoplastic transformation. Based on the 14-3-3 expressed sequencetag from a hemocyte cDNA library of E. carinicauda, the full-length cDNA of 14-3-3 from E. carinicauda(named Ec14-3-3) was cloned using the rapid amplification of cDNA ends technique. The Ec14-3-3 cDNA was2 905 bp in length and contained an open reading frame of 744 bp, encoding a mature protein of 247 amino acids,with a molecular mass of 27.95 kD and an estimated pI of 4.65. Homology analysis revealed that the Ec14-3-3amino acid sequence was highly conserved with homologs from other crustaceans, and amino acid sequence similaritywas 98% with the 14-3-3 from Penaeus monodon. Ec14-3-3 expression levels were measured in differenttissues by quantitative real-time polymerase chain reaction analysis. Ec14-3-3 expression was detected in hemocytes,gill, hepatopancreas, muscle, ovary, intestine, stomach, and the eyestalk of E. carinicauda, with the highestexpression level in hemocytes. Ec14-3-3 expression was upregulated in hemocytes and the hepatopancreas afterchallenges with Vibrio anguillarum and white spot syndrome virus. These results suggest that Ec14-3-3 may playan important role in the prawn immune response.

Abstract:Highly unsaturated fatty acids (HUFA) play a particularly important role in normal growth, immune function,and reproduction. In this study, the full-length cDNA of fatty acid elongase (ELOVL) from Chinese mitten crab (Eriocheirsinensis) was cloned for the first time using reverse transcription-polymerase chain reaction and rapid amplificationof cDNA ends analyses. The ELOVL cDNA sequence (GenBank accession number: KR005628) was 2089 bp long,with a 350-bp 5′-untranslated region (UTR), a 674-bp 3′-UTR, and an open reading frame of 1065 bp, including stopcodons that specified a protein of 354 amino acids. The presumed protein had a typical ELOVL structure with a diagnostichistidine box HVIHH motif, several conserved regions, and three putative transmembrane-spanning domains(KFTEFLDT, NTFVHIVMYVYY, and TNFQMI). The encoded protein shared 59% amino acid sequence identity withAcromyrmex echinatior (GenBank number: XP_011054336.1) and was clustered closely with Musca domestica andMarsupenaeus japonicus in a phylogenetic tree. The ELOVL gene was expressed in the hepatopancreas, intestine, muscle,stomach, heart, and gill. Higher levels were detected in the hepatopancreas and intestine (P<0.05) and lower levelswere observed in stomach, gill, cranial ganglia, muscle, thoracic ganglia, and the eyes, with trace levels expressed in theheart. We designed three different lipid-containing feeds and analyzed ELOVL mRNA expression in muscle, hepatopancreas,and gonads. The results showed the highest expression in the ovary after feeding for 98 days (P<0.05).ELOVL transcripts increased in the ovary and hepatopancreas with increased inclusion of SO in the diet, but no expressiondifferences were detected in muscle or testis. These results lay the foundation for further research into the mechanismsregulating E. sinensis HUFA biosynthesis.

Abstract:Paralichthys olivaceus is one of the most important aquaculture species in China. However, intensive breedingand many generations of inbreeding have resulted in poor disease resistance and slow growth. Therefore, it is necessaryto improve the quality and productivity of P. olivaceus through selective breeding. To study the relationship betweenmorphometric traits and body weight of P. olivaceus at different growth stages, 83 fish were selected randomly (8months old, n = 52 and 14 months old, n = 31) to measure 22 morphometric traits and body weights. The data were statisticallyprocessed using correlation, path, and stepwise regression analyses, and multivariate regression equations werecalculated. The traits were included in the equation as independent variables, and body weight was the dependent variablein six curve-fitting models to select the optimal model. The results showed that: (1)the correlation coefficients betweenbody weight and each trait varied and differed at the two developmental stages. (2)The path coefficients of X18,X4, and X11 for 8-month body weight were highly significant (P<0.01). The path coefficients of X18, X14, and X9 for14-month body weight were also significant (P<0.05). The multivariate regression equation was Y = −119.541 + 7.191X18 + 10.135 X4 + 7.197 X11 for 8 months, and Y = −484.931 + 31.959 X18 + 81.928 X14 − 17.889 X9 for 14 months. Thisresult suggests that the vital morphometric traits that affected body weight at the two growth stages were different.(3) All optimal models at 8 months were linear, including Y = −117.866 + 15.724 X18, Y = −94.579 + 24.763 X4, and Y =−100.602 + 33.184 X11. However, all optimal models at 14 months were power models, including Y = 0.036 X183.063, Y =0.095 X142.507, and Y = 62.249 X91.584. These results indicate that the growth rhythms between the two growth stageswere different. The R2 values for the multivariate regression equations at 8 and 14 months were 0.965 and 0.984, respectively,indicating that the equations can be used to predict P. olivaceus body weight. The R2 values for the X11linear model at 8 months and the X9 and X14 power models at 14 months were all < 0.85, indicating that X11, X9, and X14did not explain body weight as a single independent variable. The X18 path coefficient appeared in the multivariate regressionequations at both growth stages, indicating that X18 is a predominant morphometric trait affecting body weightand can be regarded as an assisted selection trait for body weight. These results provide a theoretical basis for selectivebreeding of P. olivaceus at different developmental stages.

Abstract:Culter alburnus gynogenetic diploids were created by activating eggs with ultraviolet-irradiated commoncarp sperm and preventing extrusion of the second polar body using cold shock to induce chromosome duplicationin the eggs. A three-factor five-level quadratic orthogonal rotation combined design was used to determinethe optimal combination of three experimental factors, including time after fertilization (x1), treatment duration(x2), and shock temperature (x3) to induce artificial gynogenesis in C. alburnus, using gynogenesis rate (y1)and hatching rate (y2) as indicators in the quadratic regression model. Furthermore, the response surface methodwas used to determine the effect of single factors and their interactive effect on the response value. We found thatthe optimal combination of factors to induce gynogenesis (18.00%) was cold shock for 18 min at 7℃, applied 6min after fertilization, whereas that for maximal egg hatch (33.00%) was cold shock for 23 min at 5℃, applied 6min after fertilization. The relationship between the various factors on the response values was parabolic with adownward opening. According to the factor contribution rates, cold shock start time had a highly significant effecton cold shock, followed by shock temperature and treatment duration. This is the first study to use a quadraticorthogonal rotation combined design to determine the optimal combination of factors to induce gynogenesis in C.alburnus. These results provide a basis to explore the sex-determining mechanism in C. alburnus.

Abstract:Liza haematocheila is a fish species with potential economic value; therefore, it is important to study the developmentof L. haematocheila larvae, juveniles, and young. In this study, the digestive system of Liza haematocheilafrom 1 to 39 days post-hatching (dph) was morphologically observed by light microscopy, and developmental characteristicswere described. The results showed that digestive tracts of the newly hatched larvae at 20–22℃ were composedof a single tube for digestion. At 4 dph, the upper and lower jaws appeared, and the yolk sac was absorbed. Thegastrointestinal tract was generated in coils by the digestive tract. The stomachic prototype came out of the first coils,and the following coils formed into the foregut and hindgut. At 7 dph, the yolk sac was completely absorbed, and the oilglobule were absorbed fairly well and disappeared; the post-larvae individuals obtained nutrition by exogenous feeding.At 8 dph, the digestive system was clearly differentiated into organs such as the esophagus, stomach, intestine, rectum,liver, and pancreas. At this time, nutritional requirements of the larvae gradually transitioned from endogenous to exogenous.Subsequently, with larval growth, the digestive tract changed to have increased wrinkles in the mucous layer,thickened walls, and a bigger cavity. From 15 dph to 18 dph, elongation of the digestive tract, deeper folds, and increasein the absorption surface were observed. At 18 dph, the pyloric caeca was formed, and gastric glands were observed,which indicates the beginning of digesting exogenous protein. Meanwhile, the intestinal epithelial cells further differentiated,the muscle continually thickened, and the intestines became segmented and circular. The larval feeding habitsbegan to change to herbivory. Subsequently, the digestive system gradually improved both functionally and structurally.As of 18 dph, the juveniles had rapid increase in body length by exogenous nutrition. These results showed that L.haematocheila digestive system development was consistent with functional improvement.

Abstract:The Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus) strain is one of the more successfullyintroduced farmed tilapia in China because of its strong adaptability, rapid growth, high fecundity, andability to accept a broad diet. Similar to other fish and vertebrates, tilapia cannot synthesize 18-carbon polyunsaturatedfatty acids (PUFAs) and, thus, require a dietary source of n-6 series FAs (18: 2n-6 or 20: 4n-6) for normalgrowth and reproduction. Although the optimum n-6 FA dietary requirements for tilapia < 10 g have been estimatedto be 0.5%–1.0%, few studies have investigated the linoleic acid (LA) requirement for larger juvenile tilapia.Our objective was to determine the optimal dietary LA requirement for larger juvenile GIFT. A total of 630fish (mean body weight, 60.98±3.82 g) were divided randomly into seven groups with three replicates of 30 fish ineach replicate. Seven diets with a constant dietary lipid level (8%) were formulated to contain seven levels [0.07%(control group), 0.36%, 0.61%, 1.03%, 2.00%, 3.00%, and 4.15%] of LA by supplementation with corn oil andpalmitic acid to modulate FA contents. The fish were fed three times daily (8:30, 12:30, and 16:30) to apparentsatiation for 10 weeks. At the end of the feeding trial, growth performance, body composition, serum biochemicalindices, and FA composition were measured. The results showed that weight gain rate (WGR), feed efficiency rate(FER), protein efficiency rate, and protein retention rate (PRR) of GIFT increased initially and then decreased asLA level increased. A second-order regression analysis showed that the optimal LA level for the best WGR was2.49%, and that the dietary LA level for the best FER was 2.66%. Broken-line regression analyses of PRR againstdietary LA level indicated that the dietary LA level for the best PRR was 1.02%. Increasing dietary LA level contributedto increase tissue and whole-body lipid levels. Serum total cholesterol and triglyceride (TG) levels werelowest in the group fed 1.03% LA, whereas the 1.03% LA group had the highest high-density lipoprotein cholesterollevel. Low-density lipoprotein cholesterol content declined as LA increased. Broken-line regression analysesshowed that the optimum LA requirement for the minimum TG level was 1.13%. Tissues FA composition was affectedby dietary FA composition. Muscle and liver saturated fatty acid levels declined as dietary level decreased;however, muscle and liver n-6 FA levels increased and n-3 FA levels declined with the increase in LA level. Ourcomprehensive analysis of growth performance, serum biochemical indices, and FA compositio

Abstract:Leuciscus waleckii is a cyprinid in the subfamily Leuciscinae and is distributed widely in rivers and lakes ofNortheast, Northwest, and Northern China. A unique feature of this species is its resistance to high alkalinity, whichallows it to survive and adapt to the extreme environment of Lake Dali, Inner Mongolia, with carbonate alkalinity >53.57 mmol/L (pH 9.6). Previous studies have explored the mechanisms of the extreme alkaline tolerance usingphysiological, chemical, population genetics, and transcriptomics methods. We assessed the effects of acclimating L.waleckii to different environmental alkalinity levels and exposure times on plasma free amino acids levels. L. waleckiijuveniles were acclimated to alkalinities of 18 mmol/L, 30 mmol/L, and 50 mmol/L for 22 d and 60 d, and plasma levelsof 15 free amino acids were examined and compared between the different alkalinity groups and the control. Theresults showed that total plasma free amino acid contents increased with alkalinity and exposure time, and that the levelsof nonessential amino acids changed the most. Among the nonessential amino acids, proline and valine contentsincreased significantly with increasing alkalinity (P<0.05); proline increased 23 times more than the control in the50 mmol/L alkalinity group. These results suggest that amino acids play an important role as an energy source duringacclimation of L. waleckii to high alkalinity conditions.

Abstract:This study was conducted to replace fish meal protein (FM) with plant-based protein in the tongue sole (Cynoglossussemilaevis Günther) diet without affecting growth performance, physiological and biological indices, or intestinalhistology. Six plant ingredients (wheat gluten, soy protein concentrate, soybean meal, cottonseed meal, peanutmeal, and corn gluten meal) were selected, and eight plant-based diets were formulated to replace 20% (PP20), 30%(PP30), 40% (PP40I, PP40II, PP40III, and PP40IV), 60% (PP60), and 80% (PP80) of FM. Each diet was assigned randomlyto triplicate groups of 15 fish [initial weight, (255.21 ± 0.79) g] per 500-L aquarium. The fish were maintained inflow-through aquaria and fed twice daily to apparent satiation for 9 weeks. The results showed that weight gain, specificgrowth rate, feed efficiency ratio, protein efficiency ratio, protein retention, whole-body crude protein, and plasmatriglycerides were not affected by the different plant-based protein diets compared with those of the FM-based diet (P>0.05). However, whole-body crude lipid and plasma cholesterol levels were negatively correlated with increasingplant-based protein level (P<0.05). The hepatosomatic index in fish in the PP30 treatment was significantly lower thanthat in fish in the FM treatment (P<0.05). In addition, the viscerosomatic index of the P40III diet group was significantlyhigher than that of the three other 40% FM-substituted diets. The histological results showed that intestinal villusof the hindgut were seriously damaged in the PP80 treatment, whereas they remained intact in the other treatments. Accordingto these results, 20%, 30%, 40%, 60%, and 80% FM can be replaced by plant-based protein without affectingsurvival, growth performance, or feed efficiency of C. semilaevis. However, replacing 80% of the FM with plant-basedprotein affected the physiological and histological indices, which would likely reduce growth performance and feedintake in long-term trials. Thus, our results indicate that up to 60% of FM can be replaced by plant-based protein in thetongue sole diet without affecting growth, physiological and biological indices, or intestinal histology.

Abstract:Poly-β-hydroxybutyrate (PHB) is a microbial storage compound that occurs in the presence of excess carbonsource. PHB is degraded into water-soluble β-hydroxybutyric acid in the gut of aquatic animals and slightly lowers gutpH. The acidic gut environment benefits growth of probiotic bacteria (particularly Gram-positive bacteria), which increasesenzyme secretion and improves nutrient absorption and immunity of the animal. Dietary PHB benefits thegrowth and survival of marine fish and crustaceans, such as European seabass, Dicentrarchus labrax, Siberian sturgeon,Acipenser baerii, and giant freshwater prawn, Macrobrachium rosenbergii. Our previous studies indicated thatPHB-enriched rotifers and Artemia nauplii significantly improve molting, survival, and tolerance to vibriosis challengein Chinese mitten crab, Eriocheir sinesis zoeal larvae, and that PHB supplementation in formulated feed improvesmolting of juvenile E. sinensis at the optimal dietary level of 1% PHB. However, as PHB is an organic acid-releasingcompound, little is known about its possible acidosis effect during long-term feeding or the interaction between feedingduration and dietary PHB level. In this study, the dosing and feeding period effects of dietary PHB on hepatopancreaticbiochemical composition and enzyme activities, as well as intestinal microbial diversity in juvenile E. sinensis, werestudied by feeding formulated diets containing 0%, 3%, 5%, and 10% PHB for 1, 6, 15, and 21 days, respectively. Theresults showed that dietary PHB supplementation affected crude protein content, crude lipid content, soluble proteincontent, and various hepatopancreatic digestive enzymes, as well as microbial diversity in the gastro-intestine, whichwere PHB-dose and feeding-period dependent. After 1 day of feeding, the 10% PHB supplemented group had significantlyhigher total superoxide dismutase (T-SOD) and hepatopancreatic amylase and lipase activities compared withthose in the control group fed a diet containing no PHB (P<0.05). In addition, 5% and 10% PHB resulted in significantlyhigher range-weighted richness (Rr) of the gastro-intestine microbial community (P<0.05). After 6 days of feeding,the 10% PHB treatment resulted in significantly lower amylase activity and the 3% and 5% PHB treatments resultedin significantly lower lipase activity (P<0.05). All PHB supplemented groups had significantly higher Rr values(P<0.05). On day 15, the 10% PHB treatment resulted in significantly lower T-SOD, and the 5% and 10% PHBtreatments resulted in significantly higher lipase activity (P<0.05). All PHB supplemented groups had significantlylower amylase activity but higher Rr values (P<0.05). On day 20, all PHB supplemented groups had significantly reducedT-SOD, amylase, and pepsin activities (P<0.05); the higher the PHB dose, the greater the reduction. The 10%PHB group had a significantly reduced Rr value(P<0.05), whereas 3% and 5% PHB had no effect on the Rrvalue(P>0.05). These results suggest that a longer feeding period could be applied with a lower PHB dose and viceversa to support use of dietary PHB in cultured E. sinensis.

Abstract:We evaluated the effect of three different doses of Bacillus cereus as a probiotic added to rearing water on thenumber of bacteria, water quality, and disease resistant ability of Litopenaeus vannamei compared with those in a controlgroup. Shrimp were divided into four groups treated with different probiotic doses: group H (106 CFU/mL), groupM (105 CFU/mL), group L (104 CFU/mL), and the control group (0 CFU/mL). The intestinal tracts of the shrimp weresampled every 10 days to count total bacteria and Vibrio spp.. A water sample was collected at 8: 00 every 5 days. Ammonia-nitrogen and nitrite levels were measured by spectrophotometry. The shrimp were challenged with white spotsyndrome virus (WSSV) after 4 weeks, and dead shrimp were removed and recorded every day until the end of the experiment.Quantitative real-time polymerase chain reaction (PCR) analysis was performed to evaluate the expression ofthree immune-related genes after 0, 12, 24, 48, 96, and 192 h of exposure. The results showed that adding the probioticsignificantly reduced the number of bacteria in the L. vannamei intestinal tract compared with that in the control group.It also reduced the number of Vibrio spp. in culture water (P<0.05). However, no differences in ammonia-nitrogen ornitrite levels were detected, which contrasts with most previous studies. Cumulative shrimp mortality rates in group H(106 CFU/mL) and group M (105 CFU/mL) were 63.9% and 74.6%, respectively, after WSSV challenge, which weresignificantly lower than that in the control group with 100% mortality (P<0.05). Lipopolysaccharide, β-1, 3-glucanbindingprotein, and beta-1, 3-glucan-binding protein-lipoprotein mRNA levels were significantly upregulated 48 h afterthe WSSV infection, compared with those in the control group (P<0.05). Heat shock protein 70 mRNA expression wassignificantly upregulated 96 h after the WSSV infection compared with that in the control group but the difference betweenthe three experimental groups was not significant. We conclude that B. cereus as a probiotic added to rearing waterreduced the number of bacteria in the intestinal tract of L. vannamei and Vibrio spp. in culture water (P<0.05) anddid not increase ammonia-nitrogen or nitrite level. Adding B. cereus to the water improved the survival rate ofWSSV-infected L. vannamei. Moreover, B. cereus stimulated the expression of immune-related genes, which could improvedisease resistance.

Abstract:A series of demersal fish sampling surveys were conducted around the Qiansan Islets from May 2013 toApril 2014, and the captured fish were identified to 22 species, 15 families, and 6 orders. Perennial rockfish species(PRS), including Hexagrammos otakii, Sebastes schlegelii, Hexagrammos agrammus, and Conger myriasterwere dominant in the studied area. Lateolabrax maculatus, Gadus macrocephalus, and Carangidae and Scombridaespecies were seasonal migration species. Results of a canonical correspondence analysis used to analyze theeffects of environmental factors on fish community structure suggested that the distribution of fish was mainlyinfluenced by bottom-water temperature and dissolved oxygen. Bottom-water temperature had a positive effect onbiomass of H. agrammus, Pterogobius zacalles, Paralichthys olivaceus, Sebastes hubbsi, Scomber japonicus, andSeriola dumerili biomass. Biomass of both G. macrocephalus and H. otakii was closely related with bottom waterdissolved oxygen, and G. macrocephalus was only distributed in deep water with high dissolved oxygen duringseasons with low temperatures. S. schlegelii, C. myriaster, and S. pachycephalus were widely distributed in thestudy area. The trophic level results determined using the stable nitrogen isotope method suggested that PRS werein the third and the fourth trophic levels, and that they were intermediate and top carnivores, whereas L. maculatusand G. macrocephalus were apex predators above the fourth trophic level.

Abstract:A fundamental goal of ecology is to identify rules that reflect species interactions; however, empiricalexamples of assembly rules for fish species in bays are limited. Analyzing the relative roles of niche separationand habitat filtering is a good way to elucidate the species mechanism. We investigated the mechanisms of howspecies in fish assemblages within bays coexist, using Meizhou Bay as a case study with null models. C-score,number of species checkerboard pairs, and the standard effect size index based on binary data were not differentbetween the observed and simulated values. Similarly, the Pianka and Czechanowski niche overlap index also indicatedno difference between observed and simulated values, according to the abundance matrix, suggesting thatfish assemblages within this system have a random pattern that is structured simultaneously by niche-partitioningand niche-filtering effects. Therefore, preservation and conservation strategies must include habitat restoration,maintaining the connection between the bay and adjacent area, and increasing population size. These results willbe useful for developing local biodiversity management and conservation strategies.

Abstract:We performed a fixed-point survey in the southwestern sea of the Nansha Islands (SS) and the mouth ofBeibu Bay (SN) in the South China Sea from 2012 to 2013. Catch species composition, the index of relative importanceof each species, the Margalef richness index, the Shannon-Weiner diversity index, the Pielou evennessindex, and the relative density of resources were calculated. We identified 663 species in four classes, 36 orders,171 families, and 374 genera from catch resources in researched areas of the South China Sea. A total of 504 fishspecies were detected, which accounted for 76.0% of the catch. The number of dominant species in autumn washigher than that in spring, and the number of species captured in the SS was higher than that caught in the SN,with obvious seasonal fluctuations. All diversity indices were higher during the spring than those during the autumnbecause supplemental groups occurred in spring and many species moved to deep sea in autumn. However,all diversity indices in the SS were higher than those in the SN owing to greater effects of sea temperature andcurrents in the SS. Fisheries resources in the northern South China Sea were highly overfished, making the fisheryresource smaller compared with historical fishery resource data. Fisheries resources in the SS were supposed to bereasonably exploited under nationally supported policies with better relative stock density. Stability of the SouthChina Sea fishery is based on high species diversity; therefore, it is important to reasonably exploit the fisheriesresources while protecting species diversity.

Abstract:Understanding the population structure of fish species could provide the basic information for utilization andmanagement of fishery. The Red bigeye Priacanthus macracanthus was one of the economically important fish speciesthat was widely distributed in the South China Sea. However, little was known about the populations. In the presentstudy, the population genetic structure and diversity of P. macracanthus were examined in the South China Sea based ona 684 bp segment of the mitochondrial cytochrome b gene. A total of 264 individual samples were collected from sevenlocalities in the northern South China Sea and the Southwestern Nansha Islands. The bases A, T, C, and G had frequenciesof 22.7%, 28.4%, 33.4%, and 28.4%, and A + T content was similar to that of C + G (51.1% and 48.9%, respectively).Ninety-two polymorphic loci were detected and 90 haplotypes were defined. Genetic diversity was high forhaploid type (0.8130–0.9012) but nucleotide diversity was low (0.0040–0.0053). A haploid type network diagram foundadvantages for the Hap1, Hap2, Hap4, and Hap6 haploid types, but no significant geographical features were detected.A pairwise Fst analysis showed that most of the Fst value were low and non-significant (P>0.05). The overall Fst for thetotal sample was only 0.012, which showed little differentiation among populaitons. Analysis of molecular variance(AMOVA) indicated that most of the differentiation occurred within populations (98.75%) and only 1.25% occurredamong populations. Phylogenetic tree constructed using neighbor-joining method showed that the seven groups of P.macracanthus were a panmictic population, with no obvious type branches. Haplotype network using median-joiningnetwork approach also revealed no significant genealogical clades corresponding to sampling localities. Both neutralitytests and mismatch distribution analyses indicated a recent population expansion in P. macracanthus during the last glacialmaximum. These results indicate high gene flow among populations of P. macracanthus and they may belong to asingle population in the South China Sea.

Abstract:Excess ammonia-nitrogen (ammonia-N) in the aquaculture environment destroys the shrimp excretory systemand osmotic balance; thus, affecting health and growth. The behavioral responses that often accompany internalphysiological changes are a reaction to environmental stress. Therefore, behavioral observations combined withphysiological measurements provide a more complete understanding of the homeostatic perturbations of shrimp fromexternal or internal stressors. The effects of ammonia-N on growth, body composition, and immunity of shrimp havebeen studied extensively, but the effects on locomotor behavior and energy allocation are not known. We investigatedthe effects of ammonia-N on locomotor behavior and energy metabolism of Litopenaeus vannamei, a commerciallyimportant shrimp species, to provide useful information for shrimp health and regulation of the aquaculture environment.Shrimp (weight, 4.89  0.27 g) were exposed to four ammonia-N concentrations of 0.02 (control), 1.00, 2.50, and5.00 mg/L in aquaria for 2 weeks at a water temperature of (28  0.5) ℃. The locomotor behaviors of L. vannamei wererecorded by cameras, and energy budgets were determined. Video of each aquarium was recorded for 30 min at 8: 00,12: 00, and 16: 00 during the daytime and at 21: 00, 24: 00, and 3: 00 during the night and analyzed to calculate movementfrequency and locomotion distance of shrimp in each experimental group. Feeding, growth, body composition,and defecation were measured to calculate energy budgets. The results showed that frequency of movement and locomotiondistance increased initially and then decreased during the day, but decreased at night as ammonia-N concentrationincreased. Movement frequency and locomotion distance of shrimp in the 5.00 mg/L ammonia-N concentrationwere significantly lower than those of the controls, regardless of time (P < 0.05). Specific growth rate, feeding rate, andfood conversion efficiency of L. vannamei decreased significantly as ammonia-N concentration increased, and allshrimp exposed to ammonia-N had significantly lower values for these variables than those of the control (P < 0.05).Crude lipid and energy contents in shrimp exposed to 2.50 mg/L and 5.00 mg/L ammonia-N were significantly higherthan those of the controls (P < 0.05). Less energy was allocated to growth as ammonia-N concentration increased,whereas that for metabolism increased. Energy for growth in shrimp exposed to 1.00, 2.50, and 5.00 mg/L ammonia-Ndropped 2.48%, 2.19%, and 4.57%, respectively, whereas that for metabolism rose 1.32%, 1.69%, and 5.70%, respectively,compared with the controls. These findings indicate that L. vannamei reduced food intake under ammonia-Nstress, decreased locomotor behavior to adjust energy allocation to reduce energy consumption, and allocated more energyto basic physiological metabolism, resulting in decreased energy utilization efficiency and inhibited growth. Thisstudy provides a mechanism for energy allocation in L. vannamei under ammonia-N stress and shows the behavioralresponses of the shrimp to ammonia-N stress, which will contribute to producing healthy shrimp and monitoring waterquality using animal behavior.

Abstract:We isolated and screened aerobic denitrifying Bacillus stains with antagonistic effects on Streptococcusagalactiae from the intestinal contents of Nile tilapia (Oreochromis niloticus). Antagonistic ability was detectedusing morphological, physiological and biochemical characteristics, and 16S rRNA gene sequencing. Optimalgrowth conditions and the ability to hydrolyze starch and protein were tested, and drug sensitivity and safety testswere performed. We isolated a strain called NY 5 that was identified as Bacillus cereus. A total of 50 mg/L nitritenitrogenwas removed completely within 12 hours after inoculating 1% NY 5 bacterial liquid on denitrificationperformance testing medium. NY 5 had antagonistic effects on 21 strains of S. agalactiae preserved in our laboratoryfrom different sources. The mean diameter of the inhibition zone was (26.67±3.00) mm. NY 5 grew well attemperatures of 25–40℃, salinities of 0–40, and pH values of 5–9. NY 5 hydrolyzed casein and starch. NY 5 wassensitive to most of the antibiotics tested but was resistant to nine antibiotics such as Penicillin G, maddie mycin,and cefazolin, etc. Tilapia with mean body weight of (6.0 ± 1.1) g did not die or show any symptoms after exposure to2.0 × 107 CFU/mL NY 5 bacteria in the water or after injection with 200 μL of 2.0×106 CFU/mL NY 5. These resultssuggest that the NY 5 strain is B. cereus. This study provides a useful probiotic source to effectively inhibitgrowth of S. agalactiae isolated from tilapia.

Abstract:Recent reports have revealed that hemocyanin in shrimp and other invertebrates is a novel multifunctionalprotein with immunological activities, such as phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities.Moreover, several studies—including our own previous findings—indicate that hemocyanin may produce some functionalfragments involved in pathogen resistance in shrimp. However, no study has comprehensively evaluated thesefragments. We conducted a comparative analysis in the proteome of the shrimp Litopenaeus vannamei after the shrimpwere infected with Vibrio parahaemolyticus for 24 h to search for novel hemocyanin fragments. Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blotting analyses of hemolymph drawn directly from the pericardialsinus revealed five peptide fragments (bands a, c, e, f, g), ranging from 6.0 to 31.0 kD, that bound to anti-shrimphemocyanin antibodies in plasma, compared with those in the control group, suggesting that they might be hemocyanindegradation products. Moreover, a 6.0 kD peptide fragment (named HMCp6) was subjected to matrix-assisted laserdesorption/ionization-tandem time-of-flight mass spectrometry analysis, and the database search results indicated thatthe HMCp6 peptide fragment was highly homologous with two subunits of L. vannamei hemocyanin (gi|325462537 andgi|854403). This result is similar to the finding that the C- and N-terminal hemocyanin fragments are significantlyaltered in Penaeus vannamei hemocytes during Taura syndrome virus infection. The HMCp6 peptide fragmentpossessed clear antibacterial activities (antibacterial rate, 98%) against V. parahaemolyticus. This result agrees with aprevious finding that a 1.9 kD antibacterial peptide from Pacifastacus leniusculus hemocyanin possesses clear antibacterialactivities against Gram-positive bacteria. In conclusion, our data suggest that the HMCp6 peptide fragment mightbe a novel fragment derived from hemocyanin with a pathogen-resistance function. These results will assist in investigationsof the mechanism of these hemocyanin fragments and help to establish strategies for controlling diseases inshrimp.

Abstract:Ultra-performance liquid chromatography tandem mass-spectrometry was used to study the environmentalbehavior of avermectin (AVM) in a simulated aquaculture pond ecosystem. AVM degradation in water, as well as theaccumulation and elimination of AVM in sediments, Elodea nuttallii, and fish and crab tissues were explored. The resultsshowed that 6 μg/L AVM had a half-life of 63.8 h in the simulated pond. At the same time, AVM was transferred tothe sediment, E. nuttallii, and aquatic animals. The peak AVM concentration, concentration time curve, and half-life insediment were 1.25 μg/kg, 469.2 μg/(kg·h), and 115.5 h, respectively. The corresponding values in E. nuttalliiwere 8.75 μg/kg, 2521.7 μg/(kg·h), and 315.0 h, respectively. These results indicate that AVM was absorbed and enrichedin E. nuttallii. In the simulated system, crucian carp (Carassius auratus gibelio) absorbed AVM from water, andpeak blood, kidney, gill, liver, and muscle concentrations were 50.9 μg/kg, 45.37 μg/kg, 21.25 μg/kg, 15.47 μg/kg, and11.9 μg/kg, respectively. AVM was only detected in the gill of Chinese mitten crab (Eriocheir sinensis) at a peak concentrationof 8.08 μg/kg but was not detected in hemolymph, muscle, or hepatopancreas. The bioconcentration factorsin descending order were crucian carp > E. nuttallii > Chinese mitten crab > sediments. These results demonstrate differentabsorption and enrichment in different environmental components and tissues in the same aquatic animal.

Abstract:We performed experiments at stocking densities of 2, 5, 8, 14, and 20 larvae/mL to determine optimallarval density for hatchery culture of bloody clam, Scapharca subcrenata. Larval developmental stages fromD-veliger to settlement and metamorphosis were included. The results indicated that total ammonia nitrogen (TAN)and nitrite increased significantly with increasing stocking density (P<0.05). TAN and nitrite levels in the 20 larvae/mL group reached maximum values of 0.089 mg/L and 0.008 mg/L, respectively. Dissolved oxygen (DO)concentrations decreased significantly with increasing stocking density (P<0.05), and DO concentrations in the14- and 20-larvae/mL groups were lower than those in the other treatments. Larval growth decreased significantlywith increasing stocking density (P<0.05). A density of 5–8 larvae/mL appeared to be optimal for normal S. subcrenatagrowth. Larval survival rate decreased with increased stocking density, and survival rate was 35% in the20 larvae/mL group(P<0.05). Larvae reared at 8 larvae/mL exhibited comparatively higher settlement density thanthat of the other stocking density groups(P<0.05). Comparatively higher settlement density was observed on thelower substrate layer than that on the upper and middle layers (P<0.05). Settlement time was prolonged and thesize of the settled larvae decreased as stocking density increased (P<0.05). Therefore, a stocking density of 5–8larvae/mL is recommended for large-scale seed production.

Abstract:We conducted this study to determine the toxicity mechanism of polycyclic aromatic hydrocarbons(PAHs) on the early development of the marine fish Centropristis striata. We analyzed individual exposure tophenanthrene (Phe), pyrene (Py), and benzo(a)pyrene (BaP) and co-exposure of each with α-naphthoflavone (ANF)to assess their toxic effects on early embryonic and larval development of C. striata. The results showed a cleardose-response relationship between Bap and Py concentration and ethoxyresorufin-O-deethylase (EROD) activitycompared with that of Phe. The effects of the PAHs on EROD activity were in the order Bap (857.52% of control)> Py (514.21%) > Phe (280.50%). Maximum EROD activity occurred at the highest concentration comparedwith that of the control group. EROD activity was inhibited (189.27%, 278.55%, and 195.40% of the control)when ANF was added to Bap, Py, and Phe, respectively. Toxicity of the PAHs to hatching rate, deformity index,and early embryo and larval mortality was in the order of Bap > Py > Phe. PAH concentration and the percentageof fertilized eggs and hatching rate were negatively correlated but PAH concentration was positively correlatedwith larval mortality and the deformity index. Bap and Py showed reduced toxicity when combined withANF. These results show that PAHs with different ring structures have obvious toxic effects, although there weredifferences in the mechanisms among them.

Abstract:Skeletal malformations are often associated with limited movement, reduced feeding capacity, depressedgrowth, and a high mortality rate. Deformed fish have a low market value and represent wasted time and effort.Skeletal malformations are observed frequently in hatchery-reared marine finfish larvae and juveniles. Such malformationscontinually hinder development of marine finfish aquaculture. This study summarizes early bone development(including jaw skeleton, vertebral column, and caudal vertebra) characteristics of commercial marinefish, such as golden pompano, yellowtail kingfish, and Asian sea bass, and compares bone malformation position,time of occurrence, and frequency in fish larvae. We also review genetic, environmental, and nutritional factorsthat regulate skeletal development in marine fish larvae to help reduced skeletal deformities during marine larvalfish culture.