Using iTRAQ-Based Quantitative Proteomics Analysis to Identify Differentially Expressed Proteins Related to Larval Development of Portunus trituberculatus

The swimming crab Portunus trituberculatus is an economically important marine crustacean species. Here isobaric tags for relative and absolute quantitation(iTRAQ) analysis were used to identify proteins that are differentially expressed during larval development of P. trituberculatus to elucidate the underlying mechanisms. In comparison with the first zoea stage(Z1), 3980 proteins were identified from 32789 peptides, which were matched with 115497 spectrums. A total of 241 proteins were screened with significantly differential expressions in all development stages. These 241 proteins are involved in various biological processes, such as cytoskeleton organization, protein synthesis, energy production and substance metabolism, physiological activities, and transport.Cluster analyses of the 241 differentially expressed proteins led to the generation of four protein clusters based on the overall similarity in protein expression patterns. Exactly 54, 70, 36, and 45 proteins clustered in profiles 10(0, 0, 0,-1, 0, 0), 15(0, 1, 0, 1, 0, 1), 18(0, 1, 2, 2, 1, 0), and 19(0, 1, 2, 3, 4, 5), respectively. Muscle development and exoskeleton renewal were important processes throughout the development stages. In addition, protein synthesis, degradation, and digestion actively occurred, especially at the Z4 stage. These results provide novel insights into the mechanisms underlying larval development of swimming crab and can assist in larval rearing.     

Dynamic metabolite alterations of Portunus trituberculatus during larval development

Journal of Oceanology and Limnology - A mass mortality often occurs from molting to the megalopa stage during the larval development of the swimming crab Portunus trituberculatus. Larvae with...     

Analysis of phospholipids in microalga Nitzschia closterium by UPLC-Q-TOF-MS

Precise structural identification of phospholipids in the microalga Nitzschia closterium has been established using ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS) for direct analysis of total lipid extracts. Mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in negative mode. Phospholipid molecular species identification was based on the characteristic product ions and neutral loss yielded by different phospholipids under ESI-MS/MS mode. The molecular species were confirmed by the carboxylate anions produced by phospholipids in negative mode; the regiospecificity of the two acyl chains was determined from the ratio of sn-1 to sn-2 carboxylate anion abundances. As a result, 18 lipid molecular species were identified for the first time in this microalga, comprising seven phosphatidylcholines (PC), two phosphatidylethanolamines (PE), two phosphatidylinositols (PI), and seven phosphatidylglycerols (PG). Lipid standards of PC, PE, PI, and PG were added to the total lipids as internal standards for semiquantitative analysis, revealing concentrations of phospholipids in this species between 0.09 and 3.37 nmol/mg. This method can produce a full structural profile of intact phospholipid molecular species and can be used for study of the physiological and ecological functions of lipids by monitoring their individual changes over time.     

Impacts of sampling design on the abundance estimation of Portunus trituberculatus using crab pots

Fishery-independent surveys can provide high-quality data and support fishery assessment and management.Optimization of sampling design is crucial to increase the quality of fishery surveys.Crab pots are important fishing gears used to catch crabs.We analyzed the impacts of sampling design of crab pots on the abundance of Portunus trituberculatus in the Changjiang(Yangtze) River estuary to the Hangzhou Bay and its adjacent waters in East China Sea.The crab pots were cylindrical,240 mm in height and 600 mm in diameter of the iron ring.Our sampling designs(including fixed-station sampling,simple random sampling,stratified fixed-station sampling,and stratified random sampling),three number of stations(9,16,and 24),and three numbers of crab pots(500,1 000,and 3 000) were simulated and compared with the "true" abundance that obtained from bottom trawl survey s in the study area in 2007.The scenarios with16 stations were set in stratification as a control group for comparison with unstratified designs.Results show that simple random sampling can obtain more stable results than fixed-station sampling in the abundance estimation of P.trituberculatus.In addition,stratified sampling resulted in more accurate abundance than unstratified sampling.The accuracy of the simulated results improved with the increase of the number of stations.No remarkable differences in the re sults were found among the scenarios of different number of crab pots at each station.However,resource-intensive areas exerted great impacts on simulation results.Thus,prior information or pre-survey results about resource abundance and density distribution are necessary.This study may serve as a reference for future sampling designs of crab pots of P.trituberculatus and other species.     

Diversity of bacteria isolated from crustacea larvae and their rearing water

1　Introduction　Blacktigershrimp (Penaeusmonodon)isthemajorshrimpspeciesculturedinIndonesia .Itsculturehasbecomemoreintensivebecauseofitshighdemandandeconomicvalue .Infact,shrimpexporthasyieldedthehighestIndonesianincomeinfisherysector .Itwasestimatedthattheproductionofculturedshrimpwas 80 0 0 0metrictonnesin 1995 .Consequently ,theneedforthefryforstockingpondculturesteadilyin creased .Thetotalnumberoffryrequiredwasabout 6millionperyear (FAO ,1995 )and 90 %oftherequiredanimalswereobtained…     

Monthly Variation in Crustacean Assemblage(Decapod and Stomatopod) and Its Relationships with Environmental Variables in Laizhou Bay,China

In this study,we investigated the community structure of crustaceans(decapod and stomatopod) inhabiting the sandy mud bottoms of Laizhou Bay(northeastern China) monthly from May 2011 to April 2012.Investigation was stopped from December 2011 to February 2012 because of the extreme weather and sea ice.A total of 205,057 specimens belonging to 31 species(shrimp,15;crab,15;and stomatopod,1) were collected in 148 hauls.From 2011 to 2012,Oratosquilla oratoria was the dominant biomass species(47.80%),followed by Charybdis japonica(15.49%),Alpheus japonicas(12.61%),Portunus trituberculatus(6.46%),and Crangon spp.(4.19%).Crangon spp.was the most dominant species by individual(32.55%).O.oratoria was the most-frequently encountered species(81.76%),followed by Palaemon gravieri(70.95%),C.japonica(65.54%),A.japonicas(62.16%),and P.trituberculatus(54.73%).The biomass density increased from August to September 2011 and decreased from March 2012 to April 2012.The dynamics of the ecological indices evolve in a similar manner,with high values of diversity and evenness and rich species from May to June 2011 and low values from September to October 2011.O.oratoria,C.japonica,and P.trituberculatus differed by biomass data between groups I(samples obtained from September to October 2011) and II(samples in other months).These species contributed more than 70% to the similarity of the crustacean community structure.Furthermore,the subsets of environmental variables that best matched the crustacean-assemblage structure were as follows:water depth(WD) in summer(June to August);sea surface temperature(SST),dissolved oxygen(DO),and WD in autumn(September to November);and DO,salinity,and WD in spring(March to May).The calculated correlation coefficients and significance level were higher in the period of July to August 2011 than in other months.Comparing 2011 to 2012 with 1982 to 1983,the species composition remained stable.However,the dominant species changed significantly.High value and large species,such as F.chinensis,P.trituberculatus,and T.curvirostris,have been replaced by low value and small species(i.e.,Crangon spp.,P.gravieri,and C.japonica).     

Levels of Metabolic Enzymes and Nitrogenous Compounds in the Swimming Crab Portunus trituberculatus Exposed to Elevated Ambient Ammonia-N

This study investigated the effects of ammonia-N(control and 1 and 5 mg L~(-1)) on the changes of metabolic enzymes activities and nitrogenous compounds levels in the gills, hepatopancreas, muscles, and hemolymph of the swimming crab Portunus trituberculatus. Results showed that the levels of hemolymph ammonia increased significantly at 6 h and then decreased to control level at 12 h. The activities of key enzymes involved in biosynthesis pathways of glutamine(Gln) and urea, such as glutamate dehydrogenase(GDH), glutamine synthetase(GS), and arginase(ARG), increased remarkably in different tissues of P. trituberculatus. Gln level was the highest in muscles, while urea content was the largest in hepatopancreas. The m RNA expression level of xanthine dehydrogenase(XDH) was downregulated sharply in gills but upregulated in the hepatopancreas and muscles. A small amount of uric acid was detected in the hemolymph. This result suggests that Gln in muscles may be used as an intermediary nitrogen repository, while urea may be primarily synthesized in the hepatopancreas and then transferred to other tissues via the hemolymph. Uric acid could be synthesized by the anabolism of purine nucleotides, in additon to the inhibitory activities of gills. In addition, the levels of Gln, urea and uric acid exhibited dose-and time-dependent effects with ammonia content. Therefore, P. trituberculatus could possibly convert ammonia into Gln, urea, or uric acid to detoxify ammonia during high ambient ammonia-N stress.     

Changes during late-stage embryonic development from egg-juvenile to free-living hatchling in Chinese freshwater crab Sinopotamon yangtsekiense (Decapoda, Brachyura, Potamidae)

This study expands on recent reports that direct development in the Chinese potamid freshwater crab Sinopotamon yangtsekiense involves the completion of all brachyuran larval stages (nauplius, zoea, and megalopa) inside the egg case during embryonic development. Detailed studies of embryonic development in this species revealed the presence of an additional larval stage (the egg-juvenile) between the megalopa and the free-living hatchling crab. We described and compared the appendages of the head, thorax, and abdomen of the egg-juvenile with those of the hatchling crab in S. yangtsekiense. Significant differences were found between most of the appendages of these two stages with a soft exoskeleton in the egg-juvenile, no joint articulation, a slimmer appearance, and a lack of setae when compared with the newly emerged free-living hatchling crab. These modifications of the appendages are related to the confinement within the egg case of the egg-megalopa and egg-juvenile during direct development, and the need for the free-living hatchling freshwater crab to move, feed, and respire. In marine crabs, the megalopa gives rise to the first crab stage whereas in freshwater crabs the egg-juvenile follows the megalopa and immediately precedes the free-living first crab stage.     

The post-larval and juvenile fish assemblage in the Sukhothai Floodplain,Thailand

This study investigated abundance, species composition and spatial and temporal distributions of fish larvae and their relationship with some environmental variables in the Sukhothai floodplain in northern Thailand. Fish larvae were collected from 33 sampling stations on 8 occasions between August 2010 and October 2013. The study collected and identified 149 296 individuals, representing 32 families and 165 taxa. The species composition of larval fish was dominated by the Cyprinidae(47.27%), Cobitidae(7.88%), Siluridae(6.67%), Bagridae(6.06%) and Mastacembelidae(3.33%) families. The mostabundant larval species were the Striped flying barb Esomus metallicus(16.90%), the Siamese mud carpHenicorhynchus siamensis(8.48%) and the Sumatran river sprat Clupeichthys goniognathus(8.31%). The greatest abundance and species diversity of larvae were found when the river flow runs onto the floodplain. PCA and nMDS analysis revealed that the samples plot is associated with temporal distribution among years. The discharge was a major factor determining fish larvae assemblage and environmental variables in the Sukhothai floodplain. Four fish larval species were positively correlated with the samples for 2013. The result of the CCA ordination plot showed that only the discharge variable was strongly correlated with fish larvae abundance, especially two cyprinid R asbora species.     

IgE Reactivity of Potential Allergens from the Swimming Crab Portunus trituberculatus and the Research of ELISA Reagent for Detecting Crab Food Anaphylaxis

Crustacean is one of the major allergic foods.It is of great significance to identify more crab allergens and research the detection methods for crab food anaphylaxis.In this study,IgE reactivity to three recombinant proteins from Portunus trituberculatus,including tropomyosin(rPtTM),myosin light chain(rPtMLC),and pancreatic lipase(rPtPL),were detected by enzyme-linked im-munosorbent assay(ELISA).The expressions of tropomyosin(TM)in various tissues of P.trituberculatus were detected by Western blot(WB).Furthermore,microplates were coated with rPtTM and the ELISA conditions were optimized.The cut-off value was deter-mined by the receiver operating characteristic(ROC)curve.Among 51 crab-allergic sera,21(41.2%)showed positive IgE to rPtTM.Other 70 crab-allergic sera more frequently recognized rPtPL(9/70;12.9%),followed by rPtMLC(1/70;1.4%).WB results showed that TM was mainly expressed in the muscle,followed by the heart and a small amount in gills and the testis.The optimal results showed that the coating condition of rPtTM was 50 ng per well with coating for 3 h at 37℃.The optimal blocking condition was 1.2%BSA with blocking for 3 h at 37℃,and the optimal dilution of the second antibody was 1:1500.The ROC curve showed that the ELISA reagent had high sensitivity(83.52%)and specificity(98.00%)when the cut-off value was 0.45.All results indicated that tropomyosin is the major allergen of P.trituberculatus,and myosin light chain and pancreatic lipase are the potential allergens.Addi-tionally,the ELISA reagent developed with the rPtTM was feasible for laboratory detection of crab anaphylaxis.     

罗非鱼开口饵料的初步研究

研究了配合饵料、轮虫、蛋黄、轮虫+人工配合饵料(3～12日龄投喂轮虫,13日龄起投喂配合饵料)等4种开口饵料对奥尼罗非鱼仔鱼生长、发育和存活影响。结果表明,1)投喂不同饵料对奥尼罗非鱼仔鱼生长具有显著影响(P<0.05),轮虫+配合饵料组生长较快,绝对增重率和特定增重率分别为0.011g/d和17.18%d-1;蛋黄组生长最慢,绝对增重率和特定增重率与其他各组差异显著(P<0.05)。2)轮虫+配合饵料与轮虫组的仔鱼成活率较高,分别为90.3%和89.7%,2组差异不显著(P>0.05),且仔鱼发育较好,大部分个体进入稚鱼阶段;蛋黄组成活率最低,只有40.7%,个体发育较为缓慢,与其他各组差异显著(P<0.05)。可见,投喂鲜活的天然饵料(轮虫)+配合饵料是罗非鱼苗种培育的最佳开口饵料。     

池养条件下军曹鱼仔鱼的消化道内容物

采用光镜解剖观察的方法对池塘培育的1~20日龄军曹鱼仔鱼消化道内容物的种类、数量和大小进行研究。结果显示:军曹鱼仔鱼于2日龄开口,主要摄食桡足类无节幼体和桡足幼体及枝角类;在2~20日龄阶段,桡足类在军曹鱼仔鱼食谱中占据主要成分;从13日龄开始摄食虾、蟹类幼体等大型浮游动物,两个阶段的食物选择性指数存在显著差异。     

Changes of Protein and Lipid Contents,Amino Acid and Fatty Acid Compositions in Eggs and Yolk-Sac Larvae of American Shad(Alosa sapidissima)

To investigate the changes of the biochemical composition of American shad(Alosa sapidissima) eggs and larvae at embryonic and early larval stages, samples were collected at different development stages from artificial fertilization to the end of yolk absorption including 2 h, 12 h and 30 h after fertilization and newly hatched larvae including 1 and 3 days after hatching. The composition of lipid, fatty acids, protein and amino acids were analyzed. The content of total protein exhibited a decreasing trend during embryogenesis and larval development, and a significant reduction was detected after hatching(P 0.05). The total lipid content remained relative stable. A significant reduction was detected in almost all amino acids after hatching except for glycine(P 0.05), while a significant decrease was found in the content of cysteine, proline, tyrosine, valine, isoleucine, leucine and phenylalanine during the yolk-sac phase(P 0.05). On the other hand, all the groups of fatty acids remained stable during the period of embryogenesis. But after hatching, a significant decrease was found in the content of C18:2n-6, C18:3n-6, SFA and ratio of EPA/ARA(P 0.05), while a significant increase was found in the content of C18:3n-3, C20:4n-6, C22:6n-3 and ratio of n-3/n-6(P 0.05). In conclusion, the combined data suggested that American shad utilizes the protein content as preferential energy substrates during embryonic and early larval developments with some specificity in the consumption of different amino acids.     

The potential of ocean acidification on suppressing larval development in the Pacific oyster Crassostrea gigas and blood cockle Area inflata Reeve

We evaluated the effect of pH on larval development in larval Pacific oyster(Crassostrea gigas) and blood cockle(Arca inflata Reeve).The larvae were reared at pH 8.2(control),7.9,7.6,or 7.3beginning 30 min or 24 h post fertilization.Exposure to lower pH during early embryonic development inhibited larval shell formation in both species.Compared with the control,larvae took longer to reach the D-veliger stage when reared under pH 7.6 and 7.3.Exposure to lower pH immediately after fertilization resulted in significantly delayed shell formation in the Pacific oyster larvae at pH 7.3 and blood cockle larvae at pH 7.6 and 7.3.However,when exposure was delayed until 24 h post fertilization,shell formation was only inhibited in blood cockle larvae reared at pH 7.3.Thus,the early embryonic stages were more sensitive to acidified conditions.Our results suggest that ocean acidification will have an adverse effect on embryonic development in bivalves.Although the effects appear subtle,they may accumulate and lead to subsequent issues during later larval development.     

The potential of ocean acidification on suppressing larval development in the Pacific oyster Crassostrea gigas and blood cockle Arca inflata Reeve

We evaluated the effect of pH on larval development in larval Pacific oyster （Crassostrea gigas） and blood cockle （Arca inflata Reeve）. The larvae were reared at pH 8.2 （control）, 7.9, 7.6, or 7.3 beginning 30 min or 24 h post fertilization. Exposure to lower pH during early embryonic development inhibited larval shell formation in both species. Compared with the control, larvae took longer to reach the D-veliger stage when reared under pH 7.6 and 7.3. Exposure to lower pH immediately after fertilization resulted in significantly delayed shell formation in the Pacific oyster larvae at pH 7.3 and blood cockle larvae at pH 7.6 and 7.3. However, when exposure was delayed until 24 h post fertilization, shell formation was only inhibited in blood cockle larvae reared at pH 7.3. Thus, the early embryonic stages were more sensitive to acidified conditions. Our results suggest that ocean acidification will have an adverse effect on embryonic development in bivalves. Although the effects appear subtle, they may accumulate and lead to subsequent issues during later larval development.     

Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder Platichthys stellatus

Ontogenetic changes in digestive enzyme activities and the amino acid(AA) profile of starry flounder,Platichthys stellatus,were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements.Larvae were collected at the egg stage and 0,2,4,7,12,17,24 days after hatching(DAH)for analysis.Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH.Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH.Pepsin activity increased sharply beginning 17 DAH.Lipase activity increased significantly 4 DAH and increased progressively with larval growth.Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease.High free amino acid(FAA) content was detected in starry flounder eggs(110.72mg/g dry weight).Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae.Most FAAs(except lysine and methionine) decreased 50%in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae.Changes in the protein amino acid(PAA) profile were much milder than those observed for FAAs.Most PAAs increased gradually during larval development,except lysine and phenylalanine.The percentages of free threonine,valine,isoleucine,and leucine decreased until the end of the trial,whereas the protein forms of these four AAs followed the opposite trend.A comparison of the essential AA composition of live food(rotifers,Artemia nauplii,and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA.These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food.Moreover,the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.     

Nutrient effects of broodstocks on the larvae in Patinopecten yessoensis

Patinopecten yessoensis is a commercial valuable species. This study deals with the effect of nutrient effects of the broodstock(mainly ovaries) on the larvae. Concentrations of total carbohydrate, total protein and total lipid in the gonads of P. yessoensis from three Hatcheries(Hatchery 1, Hatchery 2, and Hatchery 3) were determined before and after spawning. The relationship between the nutrient concentration in ovaries before spawning(BC) and that of larvae(LC) was assessed as well as the change in nutrient levels in ovaries after spawning(DC). Results indicate that the BC of total carbohydrate(7.66%) and total lipid(14.48%) in ovaries were significantly higher than in testes(5.20%, 5.20% respectively), whereas the BC of total protein in the ovaries was lower(61.76%) than in the testes(81.67%). The different gonadal composition suggests the different nutrient demands between male and female broodstocks in breeding season. P atinopecten yessoensis gonads contained a higher proportion of lipids, in comparison to other bivalves, which might be a response to the low ambient water temperatures. Further analysis of fatty acids showed that the concentrations of n-3PUFA, EPA and DHA in larvae(LC) were positively correlated with BC and DC, indicating the significant nutrient influence of broodstocks on the larvae. As these fatty acids are important in metabolism, and have been demonstrated to be influential to the viability of the larvae, larval growth and the settlement, spat growth, and juvenile survival in many bivalves, they could possibly be used as indexes to evaluate, and predict condition of broodstocks and larvae.     

Effects of Diet on the Volatile Flavor and Nutritional Ingredients of Common Octopus(Octopus vulgaris)

Cephalopods are important economic shellfish that have been developed extensively in the coastal water of various countries. Octopus vulgaris is a large-scaled economic cephalopod that is mainly cultured in South China. This study explored the effect of different diets on the volatile flavor and nutritional ingredients of O. vulgaris. Four diets were tested in four groups: Group A(fish(Scomberomorus niphonius)), Group B(crab(Helice tridens tientsinensis Rathbun)), Group C(clam(Mactra veneriformis)), and Group D(squid(Loligo japonica)). Octopus muscles were sampled after 36 days of feeding, and volatile flavor substances(VFSs), fatty acids(FAs), and amino acids(AAs) were detected. Results showed that the VFSs, FAs, and AAs of octopus in the four groups were obviously different. The sum of volatile ketones and aldehydes was higher in Group B than in the other groups, which could present much more flavors. All groups were abundant in unsaturated FAs, including eicosapentaenoic acid(EPA) and docosahexaenoic acid(DHA). In terms of content and variety, the FAs in Group B were more beneficial to human health than those in the other groups. The content of each AA in Group B was basically higher than those in the other groups and was significantly higher than that in Group D(P < 0.05). Comparing the VFSs, FAs, and AAs in samples fed with four kinds of diets, the results indicate that using crab to feed O. vulgaris can achieve better effects on volatile flavor and nutritional ingredients.     

Effects of calanoid copepod Schmackeria poplesia 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 culturing larval Japanese flounder, Paralichthys olivaceus. The total content of three types of HUFAs (DHA, EPA and ARA) in S. poplesia was significantly higher than that in the other two live foods (P<0.01). 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 juvenile 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 or Artemia 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.