An investigation of the genetic components controlling reproduction, germline development and sex in Marsupenaeus japonicus (Bate).

Tamera Callaghan (2011). An investigation of the genetic components controlling reproduction, germline development and sex in Marsupenaeus japonicus (Bate). PhD Thesis, School of Biological Sciences, The University of Queensland.

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Author Tamera Callaghan
Thesis Title An investigation of the genetic components controlling reproduction, germline development and sex in Marsupenaeus japonicus (Bate).
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2011-05
Thesis type PhD Thesis
Supervisor Bernard Degnan
Melony Sellars
Steve Chenoweth
Total pages 123
Total colour pages 10
Total black and white pages 113
Subjects 06 Biological Sciences
Abstract/Summary Significant advances in the domestication and genetic improvement of cultured penaeid shrimp species have been achieved globally. The domestication of species, including Litopenaeus vannamei, Penaeus monodon and Marsupenaeus japonicus, has enabled the production of stocks selectively bred for fast growth, disease resistance, improved survival and improved feed conversion efficiency (Argue et al., 2002; Preston et al., 2004; Preston et al., 2010; Glencross et al., 2010). As progress is made in selective breeding, the production of reproductively sterile stocks that are protected from unwanted or unlicensed breeding has become a research priority. Furthermore, penaeids are sexually dimorphic with females growing significantly larger than males. This is commercially important as the production of all-female populations would increase pond yields. Currently, a limited knowledge of the genetic basis of reproduction, germline development, and sex determination and differentiation restricts further progress in this research area. Several methods have been investigated to produce reproductively sterile and/or all-female populations of shrimp, including irradiation, polyploidy and hybridization (Sellars et al., 2006, 2007; Benzie et al., 2001). Although these methods have had varying degrees of success, a method that confers 100% sterility or all-female populations is yet to be achieved. This study aims to increase our understanding of the genetic basis of reproduction, sex and germline development in M. japonicus in an effort to produce reproductively sterile, all-female stocks for commercial production. The three principal components of this study are; (1) the identification of genes involved with gonad development and sexual differentiation; (2) the identification and characterization of an evolutionarily conserved germline specific gene; and (3) the assessment of RNAi as a technique to produce loss-of-function phenotypes for a germline gene. To identify genes involved with gonad development and sexual differentiation, expressed sequence tags (ESTs) were identified from reciprocal suppression subtractive hybridization (SSH) cDNA libraries from M. japonicus female and male gonads. Random sequencing of 576 clones from the two libraries revealed 296 unique ESTs. The expression profiles of 24 of these ESTs were determined in female and male gonads and developing post-larvae by qPCR. When expression was determined in gonads, six of the ESTs were expressed in ovaries only and five of the ESTs were expressed in testes only. When expression was determined in whole individuals during post-larval development, expression of the ESTs was low and inconsistent until stage PL110 (110 days since metamorphosis from mysis stage to the first post-larval stage). At PL110, seven of the ESTs were detected in females only and seven ESTs were detected in males only. Sex-specific expression at this developmental stage indicates that these ESTs act as important gonadal development markers and may have a role in gametogenesis. To further understand the genes involved with germline development and reproduction, a comparative approach was used to identify vasa, an evolutionarily conserved gene involved with germline specification. Vasa is a DEAD-box ATP-dependent RNA helicase that has a key role in germline development in many animals. Vasa orthologues were isolated from M. japonicus and P. monodon, and were named Mj-vasa and Pm-vasa respectively. The deduced amino acid sequences from Mj-vasa and Pm-vasa contained all of the conserved DEAD-box family motifs and three CCHC zinc finger repeats in their N-termini. Mj-vasa expression was examined in female and male broodstock tissues, as well as during ovarian, embryonic and larval development. Using qPCR and in situ hybridization, Mj-vasa transcripts were detected in the female and male gonads, and were localized to the cytoplasm of oocytes in female broodstock. Mj-vasa transcript abundance decreased during ovarian development. During embryogenesis and larval development, Mj-vasa transcripts were most abundant in zygotes, indicating that it is maternally expressed. Mj-vasa mRNA levels decreased between 2 hours (h) and 4 h post-spawning, and remained at low levels throughout embryogenesis and larval development. These results suggest the Mj-vasa may have a role in germ cell development and may be able to be used as a germline marker in M. japonicus. To assess whether germline and gonad genes can be knocked down to result in loss-of-function phenotypes, Mj-vasa-dsRNA was administered to female M. japonicus broodstock. The expression of Mj-vasa, six ESTs that were expressed in the ovary only and an EST that is expressed similarly in ovaries and testes were examined in control and dsRNA injected animals using qPCR. In the control animals, the expression of Mj-vasa and the six ovary specific ESTs decreased during ovarian development. In the Mj-vasa-dsRNA-injected animals, two individuals had a lower level of Mj-vasa expression than control animals with similar GSIs. This decreased level of Mj-vasa expression may have been the result of knockdown by the Mj-vasa-dsRNA injection. However, inhibition of gonad development was not observed in these individuals. Variation in gene expression and natural variation in ovarian development during a moult cycle made it difficult to assess the effect of dsRNA injection on Mj-vasa transcript abundance and ovarian development. These factors demonstrate the importance of determining the natural baseline expression level of target genes and the need to account for biological variation amongst individuals when performing gene regulation experiments. In this project a range of molecular tools were applied to identify genes involved with reproduction, sex and germline development in M. japonicus. A number of candidate genes were identified for future gene regulation studies which aim to produce reproductively sterile and/or all-female shrimp populations.
Keyword Marsupenaeus japonicus
All-female populations
Additional Notes Pages 34, 40, 60, 65, 70-71, 83-85, 87 should be printed in colour Pages 38-39, 46-47, 65 should be printed in landscape

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