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.

Biological Characteristics of Serum Agglutinin from Portunus trituberculatus

The agglutination characteristics of serum agglutinin from Portunus trituberculatus were studied in this paper. The results showed that the serum agglutinin had no agglutination to crucian carp, Chinese soft shell turtle, grass carp, chicken or human group A, B or O blood cells, but had strong agglutination to blood cells of mice and rabbits. The activities of serum agglutinin from Portunus trituberculatus to mice blood cells reached 210, and to rabbit blood cells reached 28. Salinity had a greater effect on agglutinin activity. When the Na Cl concentration exceeded 0.6 mol/L,the serum agglutinin from Portunus trituberculatus was basically inactivated. The optimum p H for agglutinin activity was 6.0-7.4. The serum agglutinin from Portunus trituberculatus had obvious dependence to Ca2 ＋and Mg2 ＋, and EDTA could significantly inhibit its activity. The results of sugar inhibition test showed that the activity of agglutinin from Portunus trituberculatus can be specifically inhibited by N-acetylglucosamine and N-acetylmannosamine. The serum agglutinin from Portunus trituberculatus was isolated by ammonium sulfate gradient precipitation, and its activity was highest by the 25% ammonium sulfate precipitation system. The SDS polyacrylamide gel electrophoresis（SDS-PAGE） showed that the protein bands were mainly distributed within 72-95 ku.

Phylogenetic Relationships between Purple and Tea-green Individuals of the Blue Swimming Crab Portunus trituberculatus by PCR-SSCP Analysis

As an important commercial species, the blue swimming crabs with different body-colors were discovered along the coast of East China in recent years. Both purple and tea-green crabs have similar, even the same physical characteristics except for the carapace color. In order to identify the relationships between them, we amplified and compared two conserved mitochondrial gene fragments of color-different crab individuals through PCR-SSCP and DNA sequencing analyses. The results showed that there was no distinct variation of DNA bands of both the COl and 16srRNA genes detected in polyacrylamide gels through SSCP analysis between purple and tea-green crab samples, and the following sequence analysis of color-different crab individuals presented 99.81% and 99.91% nucleotide sequence identity of the COl and 16srRNA genes respectively, which indicated that there was no species or subspecies differentiation between them. In addition, the purple individuals were more conserved in DNA sequence in compadson with that of the tee-green ones.

RAPD analysis between color-different crab individuals of Portunus trituberculatus

The blue swimming crabs Portunus trituberculatus with tea-green and/or purple carapace color have been discovered on the shore of eastern part of China these days, In this paper, 40 color-different crab individuals were evaluated using 80 RAPD primers. 30 primers were selected using bulk segregation analysis （BSA） and RAPD-PCR analysis. The results showed that the ratio of polymorphic loci was 61.29%, and Nei＇s gene diversity was 0.375 6, and Shannon＇s information index was 0.554 5 among purple crab individuals; and the ratio of polymorphic loci was 62.44%, and Nei＇s gene diversity was 0.3451, and Shannon＇s information index was 0.5099 among tea-green crab individuals. It was clear that the genetic diversity between purple and tea-green crabs was similar, which indicated close phylogenetic relationships between the purple and tea-green crabs. In addition, two primers （SBS A09 and SBS G16） got specific DNA bands by comparing forty individuals of color-different crab individuals through RAPD-PCR analysis and the following electrophoresis. They indicated close phylogenetic relationships between purple and tea-green crabs.

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.

Na^+/K^+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress

Na^＋/K^＋-ATPases are membrane-associated enzymes responsible for the active transport of Na^＋ and K^＋ ions across cell membranes, generating chemical and electrical gradients. These enzymes＇ α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na^＋/K^＋-ATPase α-subunit eDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction （RT-PCR） and rapid amplification of eDNA end methods. Analysis of the nucleotide sequence revealed that the eDNA had a full-length of 3 833 base pairs （bp）, with an open reading frame of 3 120 bp, 5＇ untranslated region （UTR） of 317 bp, and 3＇ UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na^＋/K^＋-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of arnino acid sequences showed that the P. tritubereulatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit＇s transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab＇s Na^＋/K^＋-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

Effects of different dietary lipid sources on growth performance and tissue fatty acid composition of juvenile swimming crab Portunus trituberculatus

This study was conducted to evaluate the effects of dietary lipid sources on the growth performance and fatty acid composition of the swimming crab, P ortunus trituberculatus. Four isonitrogenous and isoenergetic experimental diets were formulated to contain four separate lipid sources, including fish, soybean, rapeseed, and linseed oils(FO, SO, RO, and LO, respectively). With three replicates of 18 crabs each for each diet, crabs(initial body weight, 17.00 ±0.09 g) were fed twice daily for 8 weeks. There were no significant differences among these groups in terms of weight gain, specific growth rate, and hepatosomatic index. However, the RO groups' survival rate was significantly lower than FO groups. The feed conversion and protein efficiency ratios of RO groups were poorer than other groups. The proximate compositions of whole body and hepatopancreas were significantly affected by these dietary treatments. Tissue fatty acid composition mainly reflected dietary fatty acid compositions. Crabs fed FO diets exhibited significantly higher arachidonic, eicosapentaenoic, and docosahexaenoic acid contents in muscle and hepatopancreas compared with VO crabs. Linoleic, oleic, and linolenic acids in muscle and hepatopancreas were the highest in the SO, RO, and LO groups, respectively. The present study suggested that SO and LO could substitute for FO in fishmeal-based diets for swimming crabs, without affecting growth performance and survival.

Disruption of bacterial balance in the gut of Portunus trituberculatus induced by Vibrio alginolyticus infection

A bstract Gut microbiota impacts the health of crustaceans. V ibrio alginolyticus is a main causative pathogen that induces the vibriosis in farmed swimming crabs, Portunus trituberculatus. However, it remains unknown whether gut bacteria perform functions during the progression of vibriosis. In this study, 16 SrRNA gene amplicon sequencing was used to investigate temporal alteration of gut bacterial community in swimming crabs in response to 72-h V. alginolyticus challenge. Our results show that V. alginolyticus infection resulted in dynamic changes of bacterial community composition in swimming crabs. Such changes were highlighted by the overwhelming overabundance of V ibrio and a significant fluctuation in the gut bacteria including the bacteria with high relative abundance and especially those with low relative abundance. These findings reveal that crab vibriosis gradually develops with the infection time of V. alginolyticus and tightly relates to the dysbiosis of gut bacterial community structure. This work contributes to our appreciation of the importance of the balance of gut bacterial community structure in maintaining the health of crustaceans.

Enhanced resistance of Portunus trituberculatus to Vibrio alginolyticus by selective breeding

We established a line (screened) of Portunus trituberculatus by selectively breeding individuals that survived from challenge with Vibrio alginolyticus, and compared the response of screened and unscreened (control) P. trituberculatus challenged with V. alginolyticus. We measured superoxide dismutase, catalase, acid phosphatase, alkaline phosphatase, and peroxidase activity and the content of hemocyanin in the plasma and phenoloxidase activity in serum. The cumulative survival rate after 24-h challenge with V. alginolyticus was significantly higher in the screened crabs than in the unscreened crabs (P<0.05). T-SOD and PO activity were significantly lower in the screened stock than in the unscreened stock (P<0.05). POD, CAT, and ACP activity and hemocyanin content were significantly higher in the screened stock than in the unscreened stock. Our results suggest that the screened stock was more resistant to infection. Furthermore, the indices we measured may be used to evaluate the health state of P. trituberculatus.

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.

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.

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.

Phylogenetic relationships between purple and tea-green individuals of the blue swimming crab Portunus trituberculatus by PCR-SSCP analysis

Given the morphological characters of purple crabs identical with that of the tea-green ones except the body color, it is expected that the purple crabs and the tea-green ones were from the same species. In order to identify the phylogenetic relationships between them at the molecular level, we amplified and compared two conserved mitochondrial gene fragments of purple and tea-green crabs through PCR-SSCP and DNA sequencing analyses. The results showed that there was no distinct variation of DNA bands of both the CO1 and 16srRNA genes detected in polyacrylamide gels through SSCP analysis between purple and tea-green crab samples, and the following sequence analysis of color-different crab individuals presented 99.81% and 99.91% nucleotide sequence identity of the CO1 and 16srRNA genes respectively. These indicated that there was no species or subspecies differentiation between purple and tea-green crabs, meaning that they belonged to the same species, Portunus trituberculatus.

Polymorphisms of clip domain serine proteinase and serine proteinase homolog in the swimming crab Portunus trituberculatus andtheir associationwith Vibrio alginolyticus

Clip domain serine proteases （cSPs） and their homologs （SPHs） play an important role in various biological processes that are essential components of extracellular signaling cascades, especially in the innate immune responses of invertebrates. Here, polymorphisms of PtcSP and PtSPH from the swimming crab Portunus tritubereulatus were investigated to explore their association with resistance/ susceptibility to Vibrio alginolyticus. Polymorphic loci were identified using Clustal X, and characterized with SPSS 16.0 software, and then the significance of genotype and allele frequencies between resistant and susceptible stocks was determined by a Zz test. A total of 109 and 77 single nucleotide polymorphisms （SNPs） were identified in the genomic fragments of PtcSP and PtSPH, respectively. Notably, nearly half of PtSPH polymorphisms were found in the non-coding exon 1. Fourteen SNPs investigated were significantly associated with susceptibility/resistance to I1. alginolyticus （P〈0.05）. Among them, eight SNPs were observed in introns, and one synonymous, four non-synonymous SNPs and one ins-del were found in coding exons. In addition, five simple sequence repeats （SSRs） were detected in intron 3 of PtcSP. Although there was no statistically significant difference of allele frequencies, the SSRs showed different polymorphic alleles on the basis of the repeat number between resistant and susceptible stocks. After fiarther validation, polymorphisms investigated here might be applied to select potential molecular markers ofP. trituberculatus with resistance to I1. alginolyticus.

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.

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.