Triploid Shrimp Research Articles

Cloning and expression analysis on a homolog of spermatogonial stem-cell renewal factor in Fenneropenaeus chinensis

The spermatogonial stem-cell renewal factor (SSRF) was named since its function in spermatogonial mitosis was reported in Japanese eel. Our previous study showed that a homolog of SSRF was highly expressed in the ovary of triploid shrimp, but not expressed in the ovary of diploid shrimp. To understand the function of SSRF in shrimp, the full-length cDNA of ssrf gene was cloned from Chinese shrimp Fenneropenaeus chinensis (Fcssrf) and its expression was analyzed. The full length of Fcssrf cDNA was 2588 bp and it contained an open reading frame encoding 450 amino acids. The predicted tertiary structure of FcSSRF was very similar to that of SSRF/eSRS34 from Anguilla japonica and TP/PD-ECGF from Homo sapiens. RT-PCR analysis showed that the Fcssrf was highly expressed in nerve, testis, hepatopancreas, gill, and stomach rather than in ovary. Expression of Fcssrf mRNA was not detected during embryonic stages and larval stages, from the nauplii to the post-larvae stage, in diploid, and triploid shrimp. However, it began to be expressed in juvenile stages (June–September) in diploid and triploid shrimp. Immunohistochemical analyses showed that FcSSRF was identified in both the diploid testis and triploid ovary. We inferred that the Fcssrf might be related to testis development.

Tetraploid inductions of Penaeus monodon using cold shock

Genetic protection of improved lines of Giant Tiger shrimp, Penaeus monodon, may be achieved through the reliable production of all triploid families, as triploid shrimp are reproductively sterile. The problem with current triploid induction methods is in producing 100 % triploid progeny reliably and doing so on a commercial scale. All triploid offspring may be achievable by mating tetraploids with diploids, overcoming current limitations. However, tetraploidy in shrimp to date has been unsuccessful, with all tetraploids dying very early in development. As the induction method of heat was found to be lethal, this study investigated an alternative method using cold water to assess induction rates and lethality. Cold-water treatments between −2 and 14 °C were applied at 18, 20, and 22 min post-spawning detection for various durations with the aim of inhibiting first mitosis. Tetraploidy was induced in treatments at or less than 1 °C; however, no nauplii hatched from these treatments. As no diploid or tetraploid nauplii hatched from any of the treatments below 3 °C, it was determined that this cold-water treatment method is not suitable for tetraploid production in P. monodon.

Triploid Penaeus monodon: Sex ratio and growth rate

This study seeks to determine the best method for preventing the second body (PBII) extrusion as a means to induce triploidy in the black tiger shrimp Penaeus monodon. Chemical (cytochalasine-B and 6-dimethylaminopurine) and temperature (heat and cold) shocks were applied to newly fertilized eggs. Cold shock that was administered at 8°C, for 10min at 8min post-spawning, was the best method for PBII triploidy induction, as evidenced by the highest percentage of forming three pronuclei in syngamy. Therefore, cold shock induction was employed to further explore the advantage of triploid over diploid shrimp. After hatching of the cold-shocked eggs, the larvae were allowed to reach juvenile and adult levels of development. The number and amount of chromosomes were determined in juveniles and adult stages, using Fluorescence Activating Cell Sorting methods, by which the shrimp were divided into diploid and triploid groups. At day 150 in culture, the average body weight of the triploid females (35.2±3.2g) and triploid males (31.5±3.5g) was significantly higher (P<0.0001) than that of their diploid counterparts (24.5±0.5g for females and 23.1±3.8g for males), having a ratio of 2 females:1 male for triploid shrimp, and 2 females:3 males for diploid shrimp. These results reveal the advantages of growing triploid over diploid P. monodon, and its feasibility for commercial production.

A Homolog of the Cell Apoptosis Susceptibility Gene Involved in Ovary Development of Chinese Shrimp Fenneropenaeus chinensis1

The cell apoptosis susceptibility (CAS) gene is a homolog of the yeast chromosome segregation (CSE1) gene, which functions in cell proliferation and apoptosis. In the present study, a homolog of CAS was cloned from Chinese shrimp Fenneropenaeus chinensis (FcCAS). The full-length FcCAS cDNA is 3534 bp and contains an open reading frame encoding 968 amino acids. The predicted tertiary FcCAS structure is highly similar to that of CSE1 from the yeast Saccharomyces cerevisiae. RT-PCR analysis showed that the FcCAS gene is expressed mainly in testis, ovary, stomach, lymphoid organs, gills, and hemocytes. RNA in situ hybridization showed that FcCAS transcripts were distributed mainly in the cytoplasm of oocytes. Western blot analysis showed that FcCAS could be detected only in testis and ovary, and its expression levels differed at different developmental stages of ovaries. Immunohistochemical analysis showed that FcCAS existed in both the cytoplasm and the nucleus, which suggested that FcCAS might function as a nuclear protein. No transcript was detected in the abnormally developed ovaries of triploid shrimp. Therefore, we inferred that the FcCAS gene might be one of the key genes that is closely related to ovary development in shrimp.

Triploid Black Tiger shrimp ( Penaeus monodon) performance from egg to harvest age

In this study we induced triploids in a single family of Black Tiger shrimp ( Penaeus monodon) using chemical shock induction to stop the second meiotic division. The family was reared from egg to harvest age (post-larval stage 184) in a combination of commercial hatchery and experimental controlled environment tank rearing systems. During the rearing phase ploidy, survival, weight, sex ratio and gonad morphology of sibling diploids and induced triploids were periodically assessed. This is the first published report of the culture performance of triploid P. monodon. The induction rate of triploids at the nauplii stage was 83.4 ± 0.04% but this proportion had dropped to 62.45% by the time shrimp metamorphosed to the protozeal stage. Proportions of triploids remained fairly constant throughout the remaining larval and early post-larval stages (ranging from 60.47% to 72.38%), but dropped again to 56.32% by the time shrimp reached harvest age. The reduction in triploid rate from egg to harvest age is thought to be due to a combination of factors including heightened sensitivity of triploids to continual handling and general lower survival of triploids compared to diploids. Once shrimp reached harvest age diploid females were significantly heavier (42.95 ± 2.43 g, P < 0.05) than all other categories of shrimp. There was no significant difference in weight ( P > 0.05) between triploid females, diploid males and triploid males (35.14 ± 2.26 g, 34.02 ± 0.89 g and 31.79 ± 1.56 g respectively). The final sex ratio of harvest age triploid P. monodon was 1 female:1.625 males. Histological examination of harvest age female and male gonads revealed a drastic reduction in the development of both ova and sperm in triploid shrimp compared to diploids. The limited numbers of oocytes and sperm produced in triploids failed to fully mature by harvest age. No sperm were observed within spermatophores of triploids at harvest age when a gross morphological examination was performed and no sperm bundles were seen in the vas deferens. Using the same examination technique, diploid harvest age males had sperm within the spermatophores and vas deferens, and sperm bundles. Results indicate that reproductive sterility is likely for both female and male triploid P. monodon.

Chromosomal localization of 5S rDNA in Chinese shrimp (Fenneropenaeus chinensis): a chromosome-specific marker for chromosome identification

Chinese shrimp (Fenneropenaeus chinensis) is an economically important aquaculture species in China. However, cytogenetic and genomic data is limited in the organism partly because the chromosomes are difficult to isolate and analyze. In this study, fluorescence in-situ hybridization (FISH) was used to identify the chromosomes of F. chinensis. The 5S ribosomal RNA gene (rDNA) of F. chinensis was isolated, cloned and then used as a hybridization probe. The results show that the 5S rDNA was located on one pair of homologous chromosomes in F. chinensis. In addition, triploid shrimp were used to evaluate the feasibility of chromosome identification using FISH and to validate the method. It was confirmed that 5S rDNA can be used as a chromosome-specific probe for chromosome identification in F. chinensis. The successful application of FISH in F. chinensis shows that chromosome-specific probes can be developed and this finding will facilitate further research on the chromosomes of penaeid shrimps.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp.

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis

A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm.

Microsatellite-centromere distances and microsatellite diversity in different ploidy classes of Chinese shrimp (Fenneropenaeus Chinensis)

This is the first report of microsatellite-centromere mapping in this commercial species Fenneropenaeus Chinensis, and will be important for providing fixed points in the linkage groups of genetic maps. Triploid Chinese shrimp was induced by heat shock. The fertilized eggs were treated either by retention of the first polar body or the second polar body to produce Meiosis I (MI) or Meiosis II (MII) triploid. The triploidy status in each Chinese shrimp could be confirmed by nine polymorphic microsatellite loci, in which the parents with different alleles and the female parents were each heterozygous. The nine loci were mapped in relation to their centromeres in three MII triploid families, which were induced by retention of the second polar bodies after fertilization with sperm. Microsatellite-centromere (M-C) distances ranged from 9.6 cM to 37 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in F. chinensis. Twelve polymorphic microsatellites were used to assess the heterozygosity and allelic diversity in different ploidy classes. As expected, triploids were significantly more polymorphic than diploids. The diploids had an average heterozygosity and allelic diversity value of 0.86, whereas the triploids heterozygosity averaged 0.93 and had allelic diversity value of 1.29. However, MI triploids were not significantly more polymorphic than MII in the microsatellite loci.

Evaluation of induced triploid shrimp Penaeus ( Fenneropenaeus) chinensis cultured under laboratory conditions

Triploid Penaeus ( Fenneropenaeus) chinensis was successfully produced by heat shock. Their metamorphosis, the relationship between body weight and length and difference in appearance between triploids and their diploid siblings under laboratory culture were studied. Hematological studies showed a smaller number of haemocytes, but larger cell volume, in triploids than in diploids. Triploid shrimp did not show higher growth during the immature stage, but exhibited superior growth during the maturation stage. Characteristics of reproductive organs indicated that triploid shrimp may be sterile and sex ratio can be changed through triploidization of shrimp. This paper summarizes the progress made in triploid shrimp research which would be helpful in understanding more about triploids of crustaceans.

Timing of Meiotic Maturation and First Mitosis in the Pacific White Shrimp Litopenaeus vannamei

Intense efforts are currently underway to develop polyploid strains of cultured shrimp species, both for enhanced culture performance and protection of intellectual property (Fast and Wyban 2001). Triploidy induction has been reported for several species of Superfamily Penaeoidea, including the Chinese shrimp Penaeus chinensis = Fenneropenaeus chinensis (Bao et al. 1994), the Pacific white shrimp Litopenaeus vannamei (Dumas and Ramos 1999), and the Indian white shrimp Penaeus indicus = Fenneropenaeus indicus (Morelli and Aquacop 2003), all members of Family Penaeidae, and Sicyonia ingentis, a member of Family Sicyoniidae (Hertzler 2002; for new genera see PCrez Farfante and Kensley 1997). Gonadal development has been shown to be impaired in triploid shrimp, and a higher ratio of females to males develops (Li et al. 2003b). Current efforts are focused on producing tetraploid strains carrying desirable traits such as disease resistance or enhanced growth, which can then be mated to diploids to produce 100% triploid (presumably sterile) offspring, thereby protecting the financial investment of those who developed the strains. Tetraploidy induction has been reported for E chinensis (Xiang et al. 1991; Li et al. 2003a). Polyploidy induction typically uses either temperature shock (heat or cold) or drugs such as cytochalasins or 6-dimethylaminopufine (6-DMAP) to suppress cell division while allowing extra chromosome sets to be retained. The optimal procedures are determined empirically and depend on a critical synchronization with the events of meiotic maturation, DNA synthesis, and first mitosis that accompany fertilization. However, the details of these processes in penaeoidean shrimp have only been described for the ridgeback prawn S. ingentis (Lindsay et al. 1992; Hertzler and Clark 1993) and recently for E indicus (Morelli and Aquacop 2003). In S. ingentis (Superfamily Penaeoidea, Family Sicyoniidae), egg jelly release and meiotic maturation are triggered by exposure to seawater at spawning, and occur in both fertilized and non-fertilized oocytes (Pillai and Clark 1987; Lindsay et al. 1992). The hatching envelope develops after first polar body formation but before second polar body formation, so that the first polar body lies outside of the hatching envelope and the second polar body lies inside of it (Lynn et al. 1991; Hertzler 2002). Sperm are first detected in eggs after about 35 min post-spawning, following an elaborate acrosomal reaction which includes the formation of an acrosomal filament. Pronuclear migration requires microtubules, and the first cleavage occurs at about 90-min postspawning at 22 C (Hertzler and Clark 1993). In contrast, in many species of Family Penaeidae, the time to first cleavage is much faster since most live in warm water. For example, in E indicus first cleavage occurs by 35 min at 29.5 C (Morelli and Aquacop 2003), while in L. vannamei first cleavage occurs within 50 min of spawning at 27 C (Wyban and Sweeney 1991). Furthermore, sperm of the Penaeidae do not form an acrosomal filament (Alfaro et al. 1993; Pongtippatee-Taweepreda et al. 2004). This report describes the events of fertilization and first mitosis in the Pacific white shrimp L. vannamei, using fluorescent probes for DNA and tubulin and confocal microscopy. While S. ingentis and L. vannamei are in different families and have different thylecum types, meiotic maturation, pronuclear migration, and first mitosis proceed in a similar way but with different timing. Knowledge of the timing of these events may be useful for efforts aimed at

Gonad Development Characteristics and Sex Ratio in Triploid Chinese Shrimp ( Fenneropenaeus chinensis )

This paper details for the first time the gonad development characteristics and sex ratio of triploid shrimp ( Fenneropenaeus chinensis). In triploid shrimp the development of gonad is apparently impaired, especially in females. In the ovary of triploids, germ cells mainly remain at oogonia stage during September through December. From January to February of the next year, partial primary oocytes developed in the ovary lobes. Spermatocytes and spermatids could be observed in the testes of triploids, and a few sperm were observed in the vas deferens and spermatophores. The morphology of sperms in triploid shrimp was abnormal. Flow cytometry was used to detect the ploidy of sperm in the vas deferens. The data showed that triploidy could affect the sex ratio in Chinese shrimp. The female-to-male ratio in triploids of about 4:1 will favor triploid shrimp aquaculture.

Optimization of triploid induction by heat shock in Chinese shrimp Fenneropenaeus chinensis

Chromosome manipulation for commercially valuable marine animals plays an important role in aquaculture. The special reproductive characteristics of shrimp make it difficult to control fertilization and synchronize egg development, so research on chromosome manipulation in shrimp has proceeded very slowly. In the present study, triploid shrimp Fenneropenaeus chinensis were induced by heat shocks and the optimal-inducing condition was screened at different spawning temperatures. Level of triploid induction for each treatment was evaluated by flow cytometry at nauplius stage. The highest level of triploid induction reached to more than 90%. Starting time for each treatment was very crucial for triploid induction in shrimp. One optimal treatment condition for triploid induction was heat shock (29–32 °C), starting at 18–20 min for duration of 10 min. These conditions varied depending on the temperature at spawning. Triploid level at embryo stage and nauplius stage was not different, suggesting the same hatching rate between diploids and triploids. Heat shock is a very effective way to induce triploids in this species, and can be easily used on large scale without any harmful effect on the environment as compared with chemical treatment.