Chromosome Behavior of Heat Shock Induced Triploid in Fenneropenaeus Chinensis

INTRODUCTIONTriploidanimalsareusefulbecausetheyaresterile ,characterizedbypositivegrowthinthere productiveseason ,usefulfortesting ,forintroductionofnon nativespecies ,andforprotectionofde velopedstrains.Theinductionoftriploidyhadbeenreportedinmanyaquaculturespecies .Triploidshavebeensuccessfullyinducedinshrimpusingtemperatureshockorchemicalshock (CB ,6 DMAP)in 4species :Sicyoniaingentis (Xiangetal.,1 991 ) ,Fenneropenaeuschinensis (Xiangetal.,1 992 ;Daietal.,1 993;Baoetal.,1 993;Li…     

Artificial induction of mito-gynogenetic diploids in large yellow croaker (Pseudosciaena crocea) by hydrostatic pressure

The present study investigated conditions for inducing mito-gynogenetic (endomitosis) diploids by hydrostatic pressure in the large yellow croaker Pseudosciaena crocea. In haploid control groups, the development of eggs was activated with ultraviolet radiated semen. All fry presented typical haploid syndrome in the haploid control groups, and were verified as haploids using cytometry. After hydrostatic pressure treatment, morphologically normal fry reappeared at different frequencies according to the intensity and time of pressure shock. Fry with normal appearance in the pressure treated groups were verified as gynogenetic double haploids (GDHs), containing only one allele from the female parent at all four diagnostic microsatellite loci. For a fixed duration of 3 min, the optimal intensity of blocking the first mitosis was determined to be 40 Mpa, which was similar to that of blocking the second meiosis. There was a “window” of starting time, from 36.1 min to 38.1 min post-insemination at 25.0±1.0°C, within which the production of GDHs was not significantly different. Maximum production of morphologically normal fries, 9.36%±2.97% of developed eggs, was found when the eggs were shocked with hydrostatic pressure at 40 Mpa for 3 min, starting from 38.1 min post insemination at 25.0±1.0°C.     

Chromosome behavior of heat shock induced triploid in Fenneropenaeus chinensis

Triploidy was induced in Chinese shrimpFenneropenaeus chinensis by 30±0.5°C heat shock treatment (initiated at 20 min after fertilization) for 10 min to inhibit the release of PB2 at 18.0°C. The highest triploid rate obtained was 84.5% in nauplius stage. The effect of heat shock treatment on meiosis and cleavage of eggs was investigated in this work aimed to establish efficient procedures for triploid induction and to gain understanding of the mechanism of triploid production. Three pronuclei that could be observed in the treated eggs under fluorescence microscope developed into triploid embryos. Some abnormal chromosome behavior was observed in heat shocked eggs. Contribution No. 4407 from the Institute of Oceanology, Chinese Academy of Sciences. This work was supported by Project under Major State Basic Development Program (G1999012009) and Knowledge Innovation Program of the Chinese Academy of Sciences (KZCX 2-211)     

Individual Variation in Growth in Sea Cucumber Apostichopus japonicus （Selenck） Housed Individually

The exceptionally large individual growth variation has been previously recognized in several sea cucumber cohorts. However, there is a lack of information regarding the mechanism of such individual differences. In this study, the sea cucumber Apostichopus japonicus(Selenka)was reared individually in order to eliminate possible effects of social interaction, stocking density, etc. The results showed that there were substantial differences in growth among the sea cucumber individuals during the 100-day experiment. The special growth rate of the sea cucumber individuals differed by up to three folds(from 0. 40% to 1.01%), and the coefficient of variation in body weight increased from 12. 04% to 40. 51%. The final wet body weight, food intake and food conversion efficiency for each sea cucumber were generally positively correlated with their initial wet body weight(P<0. 05). Energy budget of the animals showed that the food energy spent on respiration was much greater(about four folds)but energy deposited for growth was much less for(initially)smaller than for larger A. Japonicus. The present result implies that there are obvious genetic differences among the sea cucumber individuals, largely accounting for the individual growth variation of the cohort sea cucumber. These results will provide some basic data for promoting selective breeding and farming of the sea cucumber.     

Mechanical Stress Induces Neuroendocrine and Immune Responses of Sea Cucumber(Apostichopus japonicus)

Grading procedure in routine sea cucumber hatchery production is thought to affect juvenile sea cucumber immunological response. The present study investigated the impact of a 3-min mechanical perturbation mimicking the grading procedure on neuroendocrine and immune parameters of the sea cucumber Apostichopus japonicus. During the application of stress, concentrations of noradrenaline and dopamine in coelomic fluid increased significantly, indicating that the mechanical perturbation resulted in a transient state of stress in sea cucumbers. Coelomocytes concentration in coelomic fluid increased transiently after the beginning of stressing, and reached the maximum in 1 h. Whereas, coelomocytes phagocytosis at 3 min, superoxide anion production from 3 min to 0.5 h, acid phosphatase activity at 0.5 h, and phenoloxidase activity from 3 min to 0.5 h were all significantly down-regulated. All of the immune parameters recovered to baseline levels after the experiment was conducted for 8 h, and an immunostimulation occurred after the stress considering the phagocytosis and acid phosphatase activity. The results suggested that, as in other marine invertebrates, neuroendocrine/immune connections exist in sea cucumber A. japonicus. Mechanical stress can elicit a profound influence on sea cucumber neuroendocrine system. Neuroendocrine messengers act in turn to modulate the immunity functions. Therefore, these effects should be considered for developing better husbandry procedures.     

Improvement of the zygote utilization and reduction of the seedling loss in the early stage of seedling production of <Emphasis Type="Italic">Sargassum thunbergii</Emphasis> (Fucales,Phaeophyta)

Artificial seedling production of Sargassum thunbergii is an effective way to relieve pressure on natural resources. In order to improve the utilization of zygotes and reduce the loss of seedlings, studies on the characteristic of the zygotes release, the development of rhizoids, the attachment of germlings, and the influence of jet washing were conducted. Results show that the percent of zygotes released was increased with time in the first 60 h. The capacity of germlings attached to the substratum was significantly increased, especially coincident with the time of the new rhizoids emerged and elongated. The detachment rate of germlings significantly decreased with the delay of starting time of jet washing or the reduction of jet washing velocity. However, the jet washing at any level applied in the experiment could cause considerable loss of germlings within the 20 days after the attachment. Our study provided some parameters to optimize the operation in the early stage of seedling production.     

Cytogenetic mechanism for the aneuploidy and mosaicism found in tetraploid Pacific oyster Crassostrea gigas (Thunberg)

Chromosome constitution was investigated in adult tetraploid Pacific oyster produced by blocking the first polar body of triploid eggs which were fertilized with haploid sperms. A high incidence of aneuploid and heteroploid mosaics were found among the offspring. Of 20 individuals identified, only 9 (45%) were eutetraploid which contained 40 chromosomes; 2 (10%) were aneuploid (hypotetraploid), which contained 39 and 38 chromosomes, respectively; and 9 (45%) were heteroploid mosaics. One mosaic was consisted of cells containing 40 and 39 chromosomes, respectiovely (1:1 in cell number), while the other 8 were consisted of cells containing chromosomes varying between tetraploid and triploid. It was also interesting to note that 3 mosaics even contained some diploid cells with 20 chromosomes. A certain number of cells of 2 tetraploids and 8 mosaics spread with 32–37 well-scattered and some clumped chromosomes at metaphase. The percentage of aneuploid cells with chromosomes varying between triploid and tetraploid correlated significantly with that of heteroploid mosaics cells with clumping chromosomes (P<0.05). Our findings suggested that reversion existed in both tetraploid and triploid oyster and chromosome clumping may underline the chromosome elimination in tetraploid oyster. It seems that the reversing cells, at least some of them, continuously eliminate their chromosomes until the most stable diploid state is established.     

Application of Wet Waste from Shrimp (Litopenaeus vannamei) with or Without Sea Mud to Feeding Sea Cucumber (Stichopus monotuberculatus)

In the present study, the applicability of the wet waste collected from shrimp(Litopenaeus vannamei) to the culture of sea cucumber(Stichopus monotuberculatus) was determined. The effects of dietary wet shrimp waste on the survival, specific growth rate(SGR), fecal production rate(FPR), ammonia- and nitrite-nitrogen productions of sea cucumber were studied. The total organic matter(TOM) level in the feces of sea cucumber was compared with that in corresponding feeds. Diet C(50% wet shrimp waste and 50% sea mud mash) made sea cucumber grow faster than other diets. Sea cucumber fed with either diet D(25% wet shrimp waste and 75% sea mud mash) or sole sea mud exhibited negative growth. The average lowest total FPR of sea cucumber occurred in diet A(wet shrimp waste), and there was no significant difference in total FPR between diet C and diet E(sea mud mash)(P > 0.05). The average ammonia-nitrogen production of sea cucumber in different diet treatments decreased gradually with the decrease of crude protein content in different diets. The average highest nitrite-nitrogen production occurred in diet E treatment, and there was no significant difference in nitrite-nitrogen production among diet A, diet B(75% wet shrimp waste and 25% sea mud mash) and diet C treatments(P > 0.05). In each diet treatment, the total organic matter(TOM) level in feces decreased to different extent compared with that in corresponding feeds.     

Ecological effects of co-culturing sea cucumber Apostichopus japonicus (Selenka) with scallop Chlamys farreri in earthen ponds

Monthly changes in sedimentation and sediment properties were studied for three different culture treatments: sea cucumber monoculture (Mc), sea cucumber and scallop polyculture (Ps-c) and scallop monoculture (Ms). Results indicated that the survival rate of sea cucumber was significantly higher in Ps-c cultures than in Mc cultures. Sea cucumber yield was 69.6% higher in Ps-c culture than in Mc culture. No significant differences in body weight and scallop shell length were found between Ps-c and Ms cultures. The mean sedimentation rate of total particulate matter (TPM) was 72.2 g/(m2 d) in Ps-c cultures, with a maximum of 119.7 g/(m2 d), which was markedly higher than that of Mc (mean value). Sedimentation rates of organic matter (OM), total organic carbon (TOC), total nitrogen (TN) and total phosphorus (TP) in Ps-c cultures were also significantly higher than those in Mc cultures. TOC and TN contents of sediment increased rapidly in the first 5 months in Ms cultures and remained at a high level. TOC and TN contents in Mc and Ps-c cultures decreased during sea cucumber feeding seasons and increased during sea cucumber dormancy periods (summer and winter). The study demonstrates that co-culture of sea cucumber and scallop in earthen ponds is an alternative way to alleviate nutrient loads and improve water quality in coastal aquaculture systems. Moreover, it provides the additional benefit of an increased sea cucumber yield.     

糙海参(Holothuria scabra)胚胎发育观察

对糙海参的胚胎发育至幼体发生进行显微观察。结果表明:受精45 min开始出现第一次卵裂,随后经过4、8、16等裂细胞,4 h后进入不等裂多细胞期,6 h后出现可游动囊胚期,10 h后进入原肠期,24～26 h成为耳状幼体。经历10～11 d的生长与发育,变态为樽形幼体,幼体经历由小变大再由大变小的过程。樽形幼体2～6 d后再次变态为较大体形的五触手幼体,开始转入底栖,五触手期2～8 d后,幼体最终以稚参形态附着下来。整个发育过程时间为16～25 d。     

Effect of co-culture of Chinese shrimp (<Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>) and sea cucumber (<Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka) on pond environment

Monoculture of sea cucumber (pond S) and polyculture of shrimp with sea cucumber (pond SS) were established to evaluate the effect of shrimp on the environmental conditions of sea cucumber farming pond. Contributions of sediment organic matter (SOM2) resuspended from benthic sediment and the suspended particulate organic matter (SPOM) deposited from the water column to the precipitated organic matter (SOM1) collected with sediment traps were estimated with carbon stable isotope analysis. The results showed that the levels of SPOM and SOM2 in pond SS significantly decreased in comparison with those in pond S at the end of experiment (P < 0.05), indicating that co-culturing shrimp in sea cucumber farming pond could purify the farming water. Carbon stable isotope analysis showed that the proportion of SOM2 in SOM1 in pond SS (84.97% ± 0.38%) was significantly lower than that in pond S (95.20% ± 0.30%) (P < 0.05), suggesting that the resuspension of organic matter from benthic sediment into overlying water was reduced in polyculture pond. In contrast, the proportion of SPOM in SOM1 in pond SS (15.03% ± 0.38%) was significantly higher than that in pond S (4.80% ± 0.30%) (P < 0.05), indicating that the sedimentation of SPOM from water column was enhanced in pond SS owing to the biodeposition effect of shrimp.     

Preferential bivalent formation in tetraploid male of Pacific oyster Crassostrea gigas Thunberg

Artificially induced tetraploid Pacific oyster, Crassostrea gigas Thunberg, produces more aneuploid gametes than normal diploid one, although they showed a comparable fecundity to diploidy. The meiotic chromosome configuration of 3 tetraploid and 1 tetraploid/triploid mosaic males were analyzed through direct chromosome observation. A majority of metaphase I spermatocytes contained both bivalents and quadrivalents. The chromosome configuration of these males was characterized by preferential formation of bivalents to quadrivalents. Bivalents appeared in all spermatocytes and consisted of 86% of all chromosome aggregates. In comparison, quadrivalents occurred in 91% spermatocytes and consisted of only 12.6% of all chromosome aggregates. The mean bivalent frequency per spermatocyte varied between 14.4 and 17.2; while that of quadrivalents varied between 2.2 and 2.7. Most quadrivalents were tandemly chained(58%) or circled(39%). The total number of chromosome aggregates per spermatocyte ranged from 13 to 20 with an average of 17.6; while 18(16 bivalents and 2 quadrivalents) was the most frequent. Univalents and trivalents appeared in very low frequency. Aneuploid(hypotetraploid) spermatocytes were observed in a low frequency. The chromosome configuration of in the mosaic individual was similar to that of tetraploid individuals. The percentage of triploid spermatocytes(2%) of the mosaic individual was significantly lower(χ2 =30, P 0.01) than that of triploid cells(46%) in its somatic tissue.     

Effects of a Potential Autochthonous Probiotic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber, <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka

The effects of Bacillus subtilis 2-1 from the intestine of healthy sea cucumber on the growth, digestive enzyme activities and intestinal microbiota of juvenile sea cucumber (Apostichopus japonicus) were determined in the present study. Sea cucumber was fed with Sargassum thunbergii powder supplemented with B. subtilis 2-1 at different concentrations varying among 0 (control), 10⁵, 10⁷, and 10⁹ CFU g^?1 for 8 weeks. Results showed that the growth performance and intestinal amylase and trypsin activities were significantly increased by dietary B. subtilis 2-1 at 10⁹ CFU g^?1 (P < 0.05). However, dietary B. subtilis 2-1 had no significant influence on the lipase activity in sea cucumber (P > 0.05). The polymerase chain reaction denaturing gradient gel electrophoresis and 16S rRNA gene sequencing analysis indicated that dietary B. subtilis 2-1 at 10⁵ and 10⁷ CFU g^?1 inhibited most of the Proteobacteria including those in genus Vibrio. Dietary B. subtilis 2-1 at 10⁹ CFU g^?1 not only decreased the abundance and species of genus Vibrio, but also increased the intensity of genera Psychrobacter and Bacillus. A specific dosage of dietary B. subtilis 2-1 could increase the growth and modulate the intestinal microbiota of sea cucumber; thus it might be a novel probiotic for keeping the health of sea cucumber.     

Identification of five sea cucumber species through PCR-RFLP analysis

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene (COI) was used to identifing five sea cucumber species (Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.     

Effects of a Potential Autochthonous Probiotic Bacillus subtilis 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber,Apostichopus japonicus Selenka

The effects of Bacillus subtilis 2-1 from the intestine of healthy sea cucumber on the growth, digestive enzyme activities and intestinal microbiota of juvenile sea cucumber(Apostichopus japonicus) were determined in the present study. Sea cucumber was fed with Sargassum thunbergii powder supplemented with B. subtilis 2-1 at different concentrations varying among 0(control), 10~5, 10~7, and 10~9 CFU g~(-1) for 8 weeks. Results showed that the growth performance and intestinal amylase and trypsin activities were significantly increased by dietary B. subtilis 2-1 at 10~9 CFU g~(-1)(P 0.05). However, dietary B. subtilis 2-1 had no significant influence on the lipase activity in sea cucumber(P 0.05). The polymerase chain reaction denaturing gradient gel electrophoresis and 16S rRNA gene sequencing analysis indicated that dietary B. subtilis 2-1 at 105 and 107 CFU g~(-1) inhibited most of the Proteobacteria including those in genus Vibrio. Dietary B. subtilis 2-1 at 10~9 CFU g~(-1) not only decreased the abundance and species of genus Vibrio, but also increased the intensity of genera Psychrobacter and Bacillus. A specific dosage of dietary B. subtilis 2-1 could increase the growth and modulate the intestinal microbiota of sea cucumber; thus it might be a novel probiotic for keeping the health of sea cucumber.     

Improvement of the zygote utilization and reduction of the seedling loss in the early stage of seedling production of Sargassum thunbergii(Fucales,Phaeophyta)

Artificial seedling production of Sargassum thunbergii is an effective way to relieve pressure on natural resources.In order to improve the utilization of zygotes and reduce the loss of seedlings,studies on the characteristic of the zygotes release,the development of rhizoids,the attachment of germlings,and the influence of jet washing were conducted.Results show that the percent of zygotes released was increased with time in the first 60 h.The capacity of germlings attached to the substratum was significantly increased,especially coincident with the time of the new rhizoids emerged and elongated.The detachment rate of germlings significantly decreased with the delay of starting time of jet washing or the reduction of jet washing velocity.However,the jet washing at any level applied in the experiment could cause considerable loss of germlings within the 20 days after the attachment.Our study provided some parameters to optimize the operation in the early stage of seedling production.     

Study on the Free Radical Scavenging Activity of Sea Cucumber （Paracaudina chinens vat.） Gelatin Hydrolysate

Gelatin from the sea cucumber （Paracaudina chinens var.） was hydrolyzed by bromelain and the hydrolysate was found to have a high free radical scavenging activity. The hydrolysate was fractionated through an ultrafiltration membrane with 5 kDa molecular weight cutoff （MWCO）. The portion （less than 5 kDa） was further separated by Sephadex G-25. The active peak was collected and assayed for free radical scavenging activity. The scavenging rates for superoxide anion radicals （O2·^-） and hydroxyl radicals （·OH） of the fraction with the highest activity were 29.02% and 75.41%, respectively. A rabbit liver mitochondrial free radical damage model was adopted to study the free radical scavenging activity of the fraction. The results showed that the sea cucumber gelatin hydrolysate can prevent the damage of rabbit liver and mitochondria.     

Growth, metabolism and physiological response of the sea cucumber, Apostichopus japonicus Selenka during periods of inactivity

The growth, metabolism and physiological response of the sea cucumber, Apostichopus japonicus, were investigated during periods of inactivity. The body weight, oxygen consumption rate (OCR), activities of acidic phosphatase (ACP), alkaline phosphatase (AKP), catalase (CAT) and superoxide dismutase (SOD), and content of heat shock protein 70 (Hsp70) in the body wall and coelomic fluid of A. japonicus were measured during starvation, experimental aestivation and aestivation. The results showed that the body weight of sea cucumber in the three treatments decreased significantly during the experimental period (P < 0.05). The OCR of sea cucumber reduced in starvation and experimental aestivation treatments, but increased gradually in natural aestivation treatment. The activities of ACP and AKP of sea cucumber decreased gradually in all treatments, whereas those of SOD and CAT as well as Hsp70 content decreased in the starvation and experimental aestivation treatments and increased in natural aestivation treatment. The sea cucumber entered a state of aestivation at 24℃. To some extent, the animals in experimental aestivation were different from those in natural aestivation in metabolism and physiological response. These findings suggested that the aestivation mechanism of A. japonicus is complex and may not be attributed to the elevated temperature only.     

Use of species-specific PCR for the identification of 10 sea cucumber species

We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species. Ten reverse species-specific primers designed from the 16S rRNA gene, in combination with one forward universal primer, generated PCR fragments of ca. 270 bp length for each species. The specificity of the PCR assay was tested with DNA of samples of 21 sea cucumber species. Amplification was observed in specific species only. The species-specific PCR method we developed was successfully applied to authenticate species of commercial products of dehydrated sea cucumber, and was proven to be a useful, rapid, and low-cost technique to identify the origin of the sea cucumber product.