Study On The Technology Of Artificial Breeding And Expression Of Genes Involving In Reproducing Of Exopalaemon Carinicauda

1. The artifical culturing of Exopalaemon carinicauda parents(1) Morphological and histological observation on ovary development of ExopalaemoncarinicaudaThe anatomy and histology of reproductive system of female Exopalaemon carinicaudawere observed. The results showed that the reproductive system of female Exopalaemoncarinicauda was “⊥” or “Rugby” shaped and contained an oviduct. The ovary was composedof the left and right parts connected at front and end. The ovary development could be dividedinto five stages: inactive stage (I stage), minor growth stage (stage II), major growth stage(stage III), mature stage (stage IV), and spawn stage (stage V). The gonadosomatic index(GSI) increased with the ovary development, and the value of GSI was the higest at matureatage, and lowest at the spawn stage. One or two nucleolus were observed clearly and thenumber of follicle cells increased from stage I to stage III, however, the nucleus were notobserved at mature stage, the follicle cells and oocytes were fused in late ovarian stage. Theconcentration of vitellin was the highest at mature stage. Cortical rods were not observedfrom stage I to stage V.(2) The artifical culturing of Exopalaemon carinicauda parentsA technique of artifical culturing of Exopalaemon carinicauda parents was introduced inthe part, which can cutted off the spread of pathogen. Exopalaemon carinicauda can maturesynchronously under the control of manual.2. The eggs incubation of Exopalaemon carinicauda(1) Effects of temperature and salinity on the embryonic development of ExopalaemoncarinicaudaThe purpose of the present study was to determine the effects of temperature on the eggincubation period of E. carinicauda larvae reared in the laboratory. The number of days from spawning to hatching and mean rearing temperature were determined from25females. Theegg incubation period decreased exponentially from41to10days with increasing meantemperatures of15.3to28.1C, but at30C, embryonic development could not be completed.The relationship between mean temperature (T) and egg incubation period was analyzed usingthe equation T KV C, based on heat summation theory. The lower critical temperature forembryonic development represented by parameter C was12.18C, and the sum of effectivetemperature for embryonic development was3828.27C h. To conclude, water temperaturesbetween12.18C and28C were most suitable for embryonic development. There was nosignificant effect of salinity on the hatching of E. carinicauda.(2) The eggs incubation and selection of larvae of Exopalaemon carinicaudaThis study proposes a method for optimization of eggs incubation and larval selcetion ofExopalaemon carinicauda. It included the regulation of embryonic development, the bestlarval selection. And each female shrimp was reared in a container alone, so the relationshipwas very clear among different famliys.3. The rearing of Exopalaemon carinicauda larvae(1) Effects of water temperature on the survival and development period of larvae ofExopalaemon carinicaudaThe purpose of the present study was to determine the effects of temperature on thesurvival and development period of Exopalaemon carinicauda larvae reared in the laboratory.The larvae from each female were reared in1.5L beakers at six temperatures (18,20,22,24,26and28C) and fed Artemia sp. nauplii. The number of days from hatching to theattainment of each larval stage decreased with increasing temperature, the period over which90%of larvae from the T18—T28groups developed into juvenile shrimps was21,18,15,14,11and11days, respectively. The survival rates of larvae decreased with development time. Inparticular, the survival rate of the T28group decreased sharply over time. The survival rates,however, from incubation to juvenile shrimp showed no significant differences (P>0.05)between the six groups. The individual dry weight of post-larva from the T18group was thehighest of all of the six groups (P<0.05), and the individual dry weight of post-larva from theT28group was the lowest, but there was no significant difference (P>0.05) between the five groups from the T20group to the T28group. To conclude, water temperatures from22°C to26°C were most suitable for rearing E. carinicauda larvae.(2) Effects of salinity on the metamrphosis rate, survival rate and specific growth rate oflarvae and post-larvae of Exopalaemon carinicaudaThe female and male E. carinicauda were selcted from the laboratory, the ovary was atstage II of femal E. carinicauda, and then reared at different salinity water until hatching, soas to anayze the effect of salinity on metamrphosis rate, survival rate and specific growth rateof larvae and post-larvae. The results showed that there were no significant effects of salinityon metamrphosis rate and survival rate of larvae (P>0.05), but the individual dry weightsignificantly affected by salinity (P<0.05). The body length and body wet weight increasedwith salintity from5to20ppt, and the salinity of20was the optimum salinity to20-days ageE. carinicauda. When the salinity is beyond20, the growth was decreased.The expression level of Na+-K+-ATPase mRNA was investigated in gill of60-days age E.carinicauda. The result showed that the Na+-K+-ATPase mRNA level was lowest at20ofsalinity. According to correlation equations and calculation results, the optimum salinity was17.53. It indicated that the salinity17.53ppt might be equal to the hemolymph osmoticpressure of E. carinicauda.The above results suggested that the optimum salinity was from15to20ppt to larva andjuvenile growth of E. carinicauda.(3) Acute toxicity of ammonia nitrogen to different ages Exopalaemon carinicaudaAcute toxicity test of ammonia nitrogen to E. carinicauda was made by using themethod of usual biological toxicity test under the condition of seawater T=24, S=31, pH8.1.The result showed that the24,48,73,96h median lethal of total ammonia nitrogen were155.81,116.71,92.55,80.40mg/L to juvenile of E. carinicauda, respectively, and178.80,156.37,140.28,120.86mg/L to adult of E. carinicauda, respectively. The “safety level” forrearing E. carinicauda juveniles was estimated to be8.04mg/L for total ammonia nitrogen(0.26mg/L for nonionic ammonia nitrogen), and for E. carinicauda adults to be12.09mg/Lfor total ammonia nitrogen (0.50mg/L for nonionic ammonia nitrogen).4. Molecular characterization and expression analysis of vitellogenin (Vg) in Exopalaemon carinicaudaThe technique of homplgy cloning and anchored PCR were used to clone the Vg genefrom Exopalaemon carinicauda. The full length cDNA of Exopalaemon carinicauda Vg was7841bp, which contained an ORF (open reading frame) of7632bp encoding a polypeptide of2543amino acids with an estimated molecular mass of287763.7Da. The sequence of thecoding region shared33-75%identity with other known crustacean Vgs. Bioinformaticsanalysis revealed seven putative N-glycosylation sites in E. carinicauda Vg, the glycosylationof seven putative sites of E. carinicauda Vg (N151, N159, N168, N614, N660, N936and N2306) wasconfirmed. However, the Dendrobranchiata exhibited a remarkable deficiency in putativeN-glycosylation sites. E. carinicauda belonged to Pleocyemata, which were egg holdingspecies, while the Dendrobranchiata were releasing species. It seemed that there was a strongselective force against the occurrence of N-glycosylation sites in the Dendrobranchiatasuborder.The expression of the E. carinicauda Vg was observed in the most of the examinedtissues, but the expression level varied significantly among the tissues. There was a high levelin hepatopancreas, lower in ovary, while lowest in muscle and gill. In hepatopancreas, therelative level of Vg mRNA increased with the ovarian development, the highest level wasobserved at mature stage (GSI7.15%), but decreased during later stage maturation. In ovary,the relative level of Vg expression increased with the ovarian development, the highest levelwea observed at spawn stage (GSI0.67%). It indicated that the hepatopancreas was the majorsite of Vg synthesis, and the ovary might play a minor role in Vg synthesis of E. carinicauda.It might be an important reason that E. carinicauda can spawn many times in its life.5. Molecular cloning and expression analysis of vitellogenin receptor (VgR)in Exopalaemon carinicaudaA full-length cDNA encoding vitellogenin receptor (VgR) was cloned from E.carinicauda using RACE method. The full-length cDNA consist of5892bp nucleotidesincluding5661bp open reading frame, which encodes1886amino acid residues. Aphylogenetic tree was constructed by the programs of CLUSTAL and MEGA. Thecrustaceans, insect, and vertebrate were clustered together and formed a group, respectively. In the tree, E. carinicauda showed the closest relationship with Macrobrachium rosenbergii,the result was similar with the result of the BLAST. So the relationships displayed in thephylogenic tree corresponded to their classification position. Analysis of the deduced proteinsequence of VgR with SMART algorithm revealed several conserved domains similar to thelow density lipoprotein receptor family. These domains included LDL-receptor class Adomain, Low-density lipoprotein-receptor YWTD domain, EGF domain and Transmembraneregion.The expression of the E. carinicauda VgR was observed in the most of the examinedtissues by quantitative Real-time PCR, but the expression level varied significantly among thetissues(P<0.05). There was a high level in ovary, lower in hepatopancreas, while lowest inmuscle and gill. With the ovarian development, expression of the VgR in the ovary was low atthe early vitellogenic stage, and significantly higher at major growth stage (stage III),however, it slightly decreased at mature stage (stage IV), and was the highest at spawn stage(stage V)(P<0.05). The highest level of VgR mRNA expression was found in hepatopancreasat mature stage, a significant decrease of VgR mRNA expression occurred at stage I, II, IIIand V. It indicated that the main site of E. carinicauda VgR mRNA expression was the ovary,and mediated the uptake of vitellogenin (Vg) into developing oocytes. However, the excessiveVg from blood was carried into the hepatopancreas at mature stage, as the nutrients ready forovarian development next time.6. ECR cDNA characterization and its expression during ovarian andembryonic development of Exopalaemon carinicaudaEcdysteroid are steroid hormones that play an important role in development, growth,molting of larva, and reproduction in crustacean. The effect of ecdysteroids is mediated by itsbinding to ecdysteroid recptor (ECR). To investigate the role of ECR during development ofovary and embryo of E. carinicauda, we isolated and characterized cDNA of E. carinicaudaECR, and studied mRNA expression pattern. The full-length cDNA seqience was2638bp andthe open reading frame encoded570amino acids. Sequence alignment showed that the E.carinicauda ECR shared high identity with other known crustacean. There was a high level inhepatopancreas, lower in eggs, while lowest in muscle and gill, ovary and mandibular gland, not detected in blood cells.In different development stages of E. carinicauda ovary, the ECR mRNA expression wasagreed with the HSP90mRNA expression in hepatopancreas, the ECR mRNA expression wasnot agreed with the HSP90mRNA expression in ovary, and was much higher at stage III, andkeeping relative highexpression level at later development of ovary. It indicated that ECR inovary played an important role in molting before spawning, while in synthetizing vitellogeninin hepatopancreas.In differdent development stage of E. carinicauda embryo, the the relative level of ECRmRNA increased with the embryonic development, the highest level was observed at Zoea I.The HSP90mRNA increased with the embryonic development, the highest level wasobserved at Protozoea stage, and then maintained high expression levels at later developmentstage. The Vg mRNA was not observed in oosperm and sixteen cells stage, the highest levelwas observed at Metazoea stage. It indicated that ECR and HSP90were involved inembryonic development, while Vg was involved in innate immune response of embryo andlarva of E. carinicauda.