三疣梭子蟹(Portunus trituberculatus)CTL基因SNP位点E4-205 C/T与抗溶藻弧菌相关性分析

C-型凝集素(C-typelectin,CTL)是甲壳类动物体液免疫中重要的免疫因子之一。但CTL基因在溶藻弧菌对三疣梭子蟹(Portunus trituberculatus)感染的过程中的表达及抗病机制尚有待进一步研究。为初步阐明CTL基因SNP E4-205 C/T位点与抗溶藻弧菌感染相关的分子机制,对不同基因型梭子蟹进行溶藻弧菌感染,通过绝对定量方法分析不同基因型梭子蟹肝胰腺、肌肉组织中溶藻弧菌的复制情况。发现溶藻弧菌感染后12 h内梭子蟹肝胰腺、肌肉组织中C/C组细菌数量显著高于T/T组,结果表明T/T组梭子蟹抗感染能力显著高于C/C组。进一步对该位点非同义突变(ACT-ATT)导致的一个氨基酸改变(Thr-Ile)的两种蛋白进行体外重组表达,并对两种重组蛋白CTL-ATT及CTL-ACT进行活性分析。发现两种重组蛋白对溶藻弧菌生长均具有一定的抑制作用,CTL-ATT的抑菌活性显著高于CTL-ACT。重组蛋白CTL-ATT与PAMPs的结合活性高于CTL-ACT与PAMPs的结合活性,同时两种蛋白与PAMPs和溶藻弧菌的结合活性具有浓度依赖性。结果表明三疣梭子蟹CTL基因参与机体感染溶藻弧菌后的免疫应答过程,其单核苷酸多态性与抗溶藻弧菌感染存在一定的关联性,三疣梭子蟹CTL基因SNPE4-205C/T位点为T/T基因型时的抗溶藻弧菌感染能力强于C/C型。

Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

Crab cell line,especially continuous crab cell line,can provide us a useful tool for studies on the virology,immunology,and molecular biology of crabs.However,no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of mitosis-arrest.In this study,long-term in vitro culture conditions for both two-(2D)and three-dimensions(3D)were successfully developed for the circulating hemocytes of swimming crab Portunus trituberculatus,designated as PTH cells.In 2D culture,a novel crab basic medium in osmolarity of 990–1100 mOsm/kg was optimized for the first time,which is different from Leibovitz's L-15 medium in mainly the components of amino acids,containing double strengths of the contents of free amino acid mixture in the crab serum.Then an optimal crab growth medium was developed by supplementing 5%fetal bovine serum,50-g/L yeast extract powder,20-μg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium,and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH cells.These 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid nitrogen.In 3D culture,using the same crab growth medium,the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60%on Day 103.This work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.

Analysis of Key Physiological Characteristics of Portunus trituberculatus in Response to Short-Term Low Salinity Stress

Salinity is a significant environmental factor that can affect the survival,metamorphosis,growth and feeding of Portunus trituberculatus.In order to analyze the key physiological characteristics of P.trituberculatus in response to short-term low salinity stress,the experiments of gradually decline and recovery as well as abrupt decline in salinity were carried out.The results showed that P.trituberculatus could survive in a certain low salinity range in the short term,and salinity 12 was the lowest tolerable salinity under the present experimental conditions.The change of the hemolymph osmotic pressure displayed significant positive correlations with water salinity,and the pressure was always higher than seawater osmotic pressure.Short-term low salinity stress changed the structure and morphology of gill tissue.The expansion of gill filament ends and epithelial cell shedding were conducive to osmotic adjustment.The activities of key ion transport enzymes such as Na^(+)-K^(+)-ATPase,carbonic anhydrase and V-ATPase also changed with the osmotic regulation,while Na^(+)-K^(+)-ATPase played a dominant role.In summary,as an osmotic adjustment species,P.trituberculatus rapidly adapt to the short-term low-salinity environment by osmotic adjustment in vivo,but salinity below salinity 12 is not conducive to its survival.Our result enriched the theoretical mechanism of osmotic regulation of P.trituberculatus,providing reference for the development of aquaculture technology of P.trituberculatus.

Impacts of the sampling design on the abundance index estimation of Portunus trituberculatus using bottom trawl

In the survey of fishery resources,the sampling design will directly impact the accuracy of the estimation of the abundance.Therefore,it is necessary to optimize the sampling design to increase the quality of fishery surveys.The distribution and abundance of fisheries resource estimated based on the bottom trawl survey data in the Changjiang River(Yangtze River)Estuary-Hangzhou Bay and its adjacent waters in 2007 were used to simulate the"true"situation.Then the abundance index of Portunus trituberculatus were calculated and compared with its true index to evaluate the impacts of different sampling designs on the abundance estimation.Four sampling methods(including fixed-station sampling,simple random sampling,stratified fixed-station sampling,and stratified random sampling)were simulated.Three numbers of stations(9,16 and 24)were assumed for the scenarios of fixed-station sampling and simple random sampling without stratification.While 16 stations were assumed for the scenarios with stratification.Three reaction distances(1.5 m,3 m and 5 m)of P.trituberculatus to the bottom line of trawl were also assumed to adapt to the movement ability of the P.trituberculatus for different ages,seasons and substrate conditions.Generally speaking,compared with unstratified sampling design,the stratified sampling design resulted in more accurate abundance estimation of P.trituberculatus,and simple random sampling design is better than fixed-station sampling design.The accuracy of the simulated results was improved with the increase of the station number.The maximum relative estimation error(REE)was 163.43%and the minimum was 49.40%for the fixed-station sampling scenario with 9 stations,while 38.62%and 4.15%for 24 stations.With the increase of reaction distance,the relative absolute bias(RAB)and REE gradually decreased.Resource-intensive area and the seasons with high density variances have significant impacts on simulation results.Thus,it will be helpful if there are prior information or pre-survey results about density distribution.The current study can provide reference for the future sampling design of bottom trawl of P.trituberculatus and other species.

Assessment of Heavy Metals,Polycyclic Aromatic Hydrocarbons,and Perfluorinated Alkyl Substances in two Marine Crustaceans(Oratosquilla oratoria and Portunus trituberculatus)in the Zhoushan Fishing Ground of China East Sea

Heavy metals,polycyclic aromatic hydrocarbons(PAHs),and perfluorinated alkyl substances(PFASs)polluted in 155 samples of two marine crustaceans(Oratosquilla oratoria and Portunus trituberculatus)harvested in the Zhoushan fishing ground in the East China Sea were assessed.The results showed that Pb,Hg,and iAs in the whole edible parts of O.oratoria and P.trituberculatus were at trace levels,far below the limits set by the China’s national standard,whereas the exceeding standard rates of Cd in the whole edible parts of O.oratoria and P.trituberculatus were 98.6%and 75.6%,respectively.Moreover,the average Cd levels and the exceeding standard rates of Cd in the visceral tissues of O.oratoria were significantly higher than those in the muscle tissues(4.64 mg kg^(−1)vs.0.49 mg kg^(−1)and 96.7%vs 30%,respectively).PAHs with high detection frequency in the two marine crustaceans included NA,AC,FL,and PHE.Total contents of 15 PAHs(abbreviated asΣ15 PAHs)in whole edible parts of O.oratoria and P.trituberculatus were 10.75–65.9μg kg^(−1)(mean=18.7μg kg^(−1)and 13.26–181μg kg^(−1)(mean=24.2μg kg^(−1),and those in the muscle and the visceral tissues of P.trituberculatus were 9.58–160μg kg^(−1)(mean=21.1μg kg^(−1)and 18.22–201μg kg^(−1)(mean=32.7μg kg^(−1),respectively.Furthermore,PFASs that were found at high contents included PFOA,PFOS,and PFOSA,and total contents of 23 PFASs(Σ23 PFASs)ranged 0.0162–5.104μg kg^(−1)(mean=0.915μg kg^(−1).Collectively,the present work provides new data on the contamination in two marine crustaceans captured in the Zhoushan fishing ground in the China East Sea,which can be useful for making suggestions on proper consumption amounts of the two marine crustaceans.

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(500,1000,and 3000)were simulated and compared with the“true”abundance that obtained from bottom trawl surveys in the study area in 2007.The scenarios with 16 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 results 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.

Eff ects of climate change on the potential habitat distribution of swimming crab Portunus trituberculatus under the species distribution model

Over the last decades,the species distribution model(SDM)has become an essential tool for studying the potential eff ects of climate change on species distribution.In this study,an ensemble SDM was developed to predict the changes in species distribution of swimming crab Portunus trituberculatus across diff erent seasons in the future(2050s and 2100s)under the climate scenarios of Representative Concentration Pathway(RCP)4.5 and RCP8.5.Results of the ensemble SDM indicate that the distribution of this species will move northward and exhibit evident seasonal variations.Among the four seasons,the suitable habitat for this species will be signifi cantly reduced in summer,with loss rates ranging from 45.23%(RCP4.5)to 88.26%(RCP.8.5)by the 2100s.The loss of habitat will mostly occur in the East China Sea and the southern part of the Yellow Sea,while a slight increase in habitat will occur in the northern part of the Bohai Sea.These fi ndings provide an information forecast for this species in the future.Such forecast will be helpful in improving fi shery management under climate change.

Influences of environmental factors on the spawning stock-recruitment relationship of Portunus trituberculatus in the northern East China Sea

Based on the Ricker-type models,the spawning stock-recruitment(S-R)relationship of Portunus trituberculatus was analysed under the impacts of environmental factors(including red tide area(AORT),sea level height(SLH),sea surface salinity(SSS)and typhoon landing times(TYP))in the northern East China Sea in 2001 and 2014.Besides the traditional Ricker model,two other Ricker-type S-R models were built:Ricker model with ln-linear environmental impact(Ricker-type 2)and Ricker model with ln-quadratic polynomial environmental impact(Ricker-type 3).Results showed that AORT,SLH,SSS and TYP had great influences on the recruitment of P.trituberculatus.When SSS reached 29 and 31,recruitment decreased from 20.7×10^(3) million to 8.3×10^(3) million individuals.In this case,recruitment declined,whereas AORT and TYP increased.Analysis of the S-R model showed that the Akaike information criterion(AIC)value of the traditional Ricker model was 14.619,which remarkably decreased after addition of the environmental factors.Different numbers of environmental factors were added to the Ricker model,and the best result was obtained when four factors were added to the model together.Moreover,Ricker-type 2 model,with the AIC value of-5.307,was better than Ricker-type 3 model(add above four environmental factors at the same time).The findings indicated that the mechanisms by which various environmental factors affect the S-R relationship are different.

Music stimulus has a positive eff ect on survival and development of the larvae in swimming crab Portunus trituberculatus

The sound stimulus has a signifi cant impact on marine animals.However,many studies have focused on the negative impacts of sound on marine vertebrates.The positive impacts of sound on marine animals,especially on marine invertebrates,were rarely studied.In the present study,we evaluated the eff ects of music on the survival and development of an economically important marine invertebrate,swimming crab Portunus trituberculatus.Three pieces of classical music and a control group,i.e.,Eine Kleine Nachtmusik by Mozart(EK),Bach-Violin Concerto No.1(BV),Anonymous Romanza by Anonymous(AR),and environmental sound(EN)were used.The sound pressure received were 163-dB root mean square(RMS)re 1μPa for EK,166-dB RMS re 1μPa for BV,165-dB RMS re 1μPa for AR,44-dB RMS re 1μPa for EN,respectively.The larvae were subject to 3-h classical music transmission every day during the entire larval development period.The result showed that BV and AR signifi cantly elevated the cumulative survival rate of the larva from 3.42%±1.77%(EN)to 10.65%±1.69%(BV)and 7.12%±1.37%(AR)respectively(P<0.05).Though music did not aff ect the cumulative development time of larvae from ZoeaⅠto ZoeaⅣ,it signifi cantly increased the molting rate of megalopa from 24.00%±11.78%(EN)to 44.00%±13.06%(AR)and 46.67%±10.50%(BV)(P<0.05).Musical exposure during critical growth intervals may contribute to the survival and development of larval crustaceans.From an application point of view,music intervention is much economically feasible.The observed music eff ects imply that music could further enhance growth,welfare,and production.

Expression and function of WNT4 involved in larvae development and limb regeneration in Portunus trituberculatus

The wingless-related integration site(WNT)proteins are a family of secreted glycoproteins that are evolutionarily conserved and are believed to be involved in evolution in vertebrates and invertebrates.WNT signaling pathways may be associated with limb regeneration and development in crustaceans.However,the detail mechanisms remain unclear.Therefore,the distribution of WNT4 in the hepatopancreas,muscle,hemocyte,ganglion,heart,eyestalk,gill tissue,and diff erent larvae development stages of the swimming crab(Portunus trituberculatus)were characterized using immunofl uorescence,real-time PCR,and Western blotting.Signifi cant PtWNT4 expression was detected in heart and eyestalk.In addition,PtWNT4 was expressed in all larval stages of P.trituberculatus with a dynamic expression pattern,especially in the eyestalk and other organs in the carapace area.The injection of WNT4 dsRNA into regenerative limbs signifi cantly decreased PtWNT4 mRNA levels in the eyestalk,heart,and muscle,resulting in 1.9-fold,2.2-fold,and 2.7-fold decreases compared with those detected in the group injected with crab saline(P<0.05),respectively,indicating successful gene silencing.Overall,expression analysis on the WNT4 using RNAi provides an insight to its functional mechanism during limb regeneration in P.trituberculatus.The results not only demonstrated the requirement for WNT4 in limb regeneration of Crustaceans,but also suggested its ability to promote larval development at specifi c stages.

Growth marks in eyestalks of Portunus trituberculatus:A development of technique and evidence of molting

The analysis of growth bands in the eyestalk has been increasingly used for estimating crustacean ageing and molting.In this study,we developed an effective method to process and observe the eyestalk microstructure of the swimming crab(Portunus trituberculatus).We found that dark pigmentation as a result of boiling has an influence on the observation of the eyestalk microstructure.Choosing an unboiled eyestalk,this study compared the cross section and longitudinal section,and concluded that the cross section is suitable for the observation of growth increments with 6.1%CV(coefficient of variation),and growth bands are suitable for the observation of the longitudinal section with 5.4%CV.The width of growth increments near the edge of the endocuticle is small,and the width of growth increments of the middle part of the endocuticle is large.Relationship of number of growth bands to molting time was fitted to a linear function with the slope not significantly different from 1,indicating that growth bands are formed associated with molting.Periodicity of growth increment formation was calculated as 3.7 d,however was not verified.Our results provide a new improved technique for identification of crustacean molting and growth.

Phospholipid Compositions in Portunus trituberculatus Larvae at Different Developmental Stages

Phospholipids are used to improve the growth and survival of Portunus trituberculatus,a widely cultured crab species in China.However,only total phospholipids or several classes are applied in crab diets.In this study,we employed a targeted lipidomic method to investigate the comprehensive phospholipid composition in P.trituberculatus larvae and reveal the changing phospholipid profile over the larval development.Results showed that P.trituberculatus larvae contain 112 phospholipid species belonging to 10 phospholipid classes,in which phosphatidylcholine(PC)and phosphatidylethanolamine(PE)species are the most abundant,and PC,PE,phosphatidic acid,and phosphatidylserine(PS)are with high concentrations.The levels of all phospholipids significantly changed with larval development,which was highlighted by the downward parabolic changes in PE,phosphatidylglycerol,phosphatidylino-sitol,PS,lysophosphatidic acid,and sphingomyelin levels.In addition,nearly all phospholipid species were depleted at the M stage,which probably contributed to the mass mortality of crab larvae.These findings on the composition and alterations of phospholipids in P.trituberculatus larvae provide novel perspectives for the targeted supplementation of phospholipids in crab diets.Our work also highlights the use of targeted UHPLC-MS lipidomics in understanding the changes of phospholipids during crab development.

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.

Air Temperature and Emersion Time Can Affect the Survival Rate and Ammonium Loading of Swimming Crab Portunus trituberculatus Exposed to Air

Ammonium overloading is a common response of aquatic organisms to air exposure during transport. This study elucidated the relationship between ammonium overloading and mortality of crab Portunus trituberculatus. Additionally, we also explored the effects of emersion time and air temperature on ammonium loading and concomitant physiological change. To test the air temperature effect, the crab was exposed to 16, 23 and 30℃ in air for 3 h, respectively, and then recovered in seawater at 23℃ for 12 h. To test the emersion time effect, crab was exposed to 23℃ in air for 0.5 and 3 h, respectively, and then recovered in seawater at 23℃ for 12 h. In the control group, crab was always immersed at 23℃. At each time interval(0.5, 1.5 and 3 h during air exposure and 0.5, 2, 4 and 12 h during recovery), ammonium excretion rate, level of total ammonium, total free amino acids and urea concentration in hemolymph and the hepatopancreas enzyme activity involved in detoxifying ammonium were analysed. Results showed that crab mortality was positively related with emersion time and temperature while ammonium loading was lower at 16 and 30℃ than at 23℃. For crab experiencing thermal inconsistence of culture media(i.e., 16 or 30℃), they were higher in ammonium excretion rate and activities of ammonium detoxification enzymes, which may be the reason that they had a lower ammonium loading. Prolonged emersion time(3.0 h vs. 0.5 h) increased the ammonium overloading and the activity of ammonium detoxification pathways in crab. Our results demonstrated that emersion-induced ammonium overloading may not be the main reason leading to P. trituberculatus death during air exposure and subsequent recovery. When the culture medium changed, thermal variation, compared with constant temperature, could reduce ammonium overloading in crab by elevating the activities of ammonium detoxification enzymes and ammonium excretion rate during recovery period.

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.

Implementing geostatistical analysis to study spatio-temporal distribution patterns of swimming crabs(Portunus trituberculatus)

This study attempted to compare the performance of local polynomial interpolation,inverse distance weighted interpolation,and ordinary kriging in studying distribution patterns of swimming crabs.Cross-validation was used to select the optimum method to get distribution results,and kriging was used for making spatial variability analysis.Data were collected from 87 sampling stations in November of 2015(autumn)and February(winter),May(spring)and August(summer)of 2016.Results indicate that swimming crabs widely distributed in autumn and summer:in the summer,they were more spatially independent,and resources in each sampling station varied a lot;in the winter and spring,the abundance of crabs was much lower,but the individual crab size was bigger,and they showed the patchy and more concentrative distribution pattern,which means they were more spatially dependent.Distribution patterns were in accordance with ecological migration features of swimming crabs,which were affected by the changing marine environment.This study could infer that it is applicable to study crab fishery or even other crustacean species using geostatistical analysis.It not only helps practitioners have a better understanding of how swimming crabs migrate from season to season,but also assists researchers in carrying out a more comprehensive assessment of the fishery.Therefore,it may facilitate advancing the implementation in the pilot quota management program of swimming crabs in northern Zhejiang fishing grounds.

Full-length transcripts facilitates Portunus trituberculatus genome structure annotation

Portunus trituberculatus is an ide al model for elucidating crustacean genetic networks.Here we combined single molecule real-time(SMRT)sequencing and Illumina RNA-seq to characterize the coding genes,non-coding RNAs and pseudogenes and further to improve the genome annotation information of P.tritub erculatus.In this study,we assembled 9694 non-redundancy full-length transcripts,and 658737307-bp repetitive sequences were identified in the P.trituberculatus full-length transcriptome.We also predicted the P.tritub erculatus genome structure based on full-length transcripts,including 18602 genes,28686 non-coding RNAs,1407 pseudogenes,740 motif,and 26434 domain.Meanwhile,14460,10211,5412,7314,and 14448 genes had significant matches with sequences in the NR,KOG,GO,KEGG,and TrEMBL database,respectively.Overall,our work firstly provided the long-read transcriptome and we believed that these data are very necessary to improve the annotation information of P.trituberculatus genome structure,and useful information for the future studies on evolution and physiological regulation of P.trituberculatus.

Effectiveness of escape vent shape in crab pots for releasing swimming crab Portunus trituberculatus in the East China sea

To stop the decline of swimming crab(Portunus trituberculatus)stocks in the East China Sea,escape vents on crab pots are urgently required to improve pot selectivity and enable release of sublegal-sized crabs.This study presents field comparative fishing experiments,which examined the effectiveness of different shapes and sizes of escape vents in crab pots for the release of swimming crabs.Two sizes each of rectangular,elliptic,and circular escape vents were tested.The results of comparative fishing experiments indicated that experimental pots with escape vents,regardless of their configuration,caught significantly fewer sublegal-sized crabs(<49 mm carapace length,or<116 mm carapace width)than unmodified control pots.Although legal-sized swimming crabs were also less caught,analysis of size selectivity showed that rectangular vents produced steeper selectivity curves than elliptic and circular escape vents.This indicates that rectangular vents may be more suitable and controllable when escape vents will become a requirement and their use will be enforced in fisheries.Underwater observations were conducted using GoPro cameras and LED lights during the first 2.5 h of the deployments(which usually lasted between 5 and 12 h).Video recordings of experimental pots equipped with rectangular or elliptic escape vents showed that swimming crabs trapped in the pots could detect the vents,and readily approached and passed through these.However,no significant difference was detected in the response behavior of swimming crabs between rectangular and elliptic vent pots when quantitative indicators,derived from video recordings,were compared.The effect of different vent shapes on the escape behavior of swimming crab gradually manifested as an increased soak time and by the onset of the escape of larger crabs.Future underwater observations should thus last the entire duration of pot soaking to study how crabs escape from the pots.

Effect of escape vents on retention and size selectivity of crab pots for swimming crab Portunus trituberculatus in the East China Sea

An increase in the crab pot fishery in the East China Sea has caused great pressure on swimming crab Portunus trituberculatus resources.Thus,it is essential to implement suitable measures to release sublegal-sized crabs to increase the number of recruits for legal-sized crabs.One of the measures considered is the installation of escape vents on crab pots.We tested crab pots with one and two escape vents and compared the catchability and size selectivity of these pots with control pots without an escape vent.Pots with one escape vent located on single side of the pot,top or bottom(SS),and another on both sides at top and bottom edges(BS)were tested in sea trial experiments following typical commercial fishing practice.The results show that both SS pots and BS pots caught significantly smaller number of sublegal-sized crabs(<116 mm carapace width,CW),but the difference in catch between the two types of crab pots is not significant.The 50%selective CWs(CW_(50))and the selective ranges(SRs)of both BS pots are slightly larger than those of SS pots.However,95%confidence intervals of CW_(50)s and SRs are overlapped,indicating that the discrepancy between them is small.Then,in a mixed effect model,the effect of locations of escape vents,taken as the fixed effect,on selectivity parameters and indexes was analyzed by hypothesis testing.The results show that null hypothesis of no effect of location of vents on size selectivity cannot be rejected,indicating that there is no significant difference in size selectivity between the two types of pots for P.trituberculatus.

Dietary cholesterol promotes growth and ecdysone signalling pathway by modulating cholesterol transport in swimming crabs(Portunus trituberculatus)

Cholesterol,as an indispensable nutrient,regulates molting and growth in crustacean.As crustaceans are unable to biosynthesize cholesterol de novo,it is central to understand how dietary cholesterol affects molting in crustaceans.An 8-week feeding trial was conducted to evaluate the effects of dietary cholesterol level(0.12%,0.43%,0.79%,1.00%,1.30%and 2.50%)on growth,cholesterol metabolism and expression of genes related to lipid and ecdysone metabolism in female swimming crabs(Portunus trituberculatus).A total of 192 crabs(1.41±0.05 g)were randomly distributed into 192 aquaria.Each treatment had 4 replicates with each replicate containing 8 crabs.Crabs fed the 1.00%cholesterol diet showed best growth performance,and thus based on percent weight gain,the optimal dietary cholesterol requirement was calculated at 1.01%.Tissue cholesterol concentrations were positively correlated with dietary cholesterol level.The contents of functional fatty acids in hepatopancreas significantly increased as dietary cholesterol increased from 0.12%to 2.50%(P<0.05).The expression levels of genes related to lipogenesis pathway,lipid catabolism and fatty acid oxidation were significantly downregulated with increased dietary cholesterol level(P<0.05).The highest expression levels of cholesterol transport genes,low-density lipoprotein receptor(ldlr)and low-density lipoprotein receptorrelated protein 2(lrp2)occurred in crabs fed the 1.30%cholesterol diet.Moreover,hormones related to molting such as crustacean hyperglycemic hormone(CHH),methyl farnesoate(MF),molt-inhibiting hormone(MIH),and ecdysone in hemolymph were significantly influenced by dietary cholesterol level(P<0.05).The highest expression levels of ecdysone receptor(ecr)and chitinase 1(chi1)in eyestalk and hepatopancreas were found in crabs fed the diet containing 1.00%cholesterol(P<0.05).In conclusion,the optimal dietary level was beneficial to functional fatty acid accumulation,regulated lipid metabolism,promoted the ecdysone signalling pathway by improving the cholesterol transport,and improved the molting rate and growth of swimming crabs.