De Santis, Christian (2011) Regulation of growth: understanding the myostatin functioning in two important aquaculture species, barramundi (Lates calcarifer) and black tiger shrimp (Penaeus monodon). PhD thesis, James Cook University.

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Abstract

Growth has traditionally been the most important trait amongst the many targeted to boost the productivity and profitability of aquaculture farms. Several genetic approaches have been investigated and employed in recent years to enhance the growth rate of aquatic species. Of these, the use of a single gene approach has recently gained popularity amongst aquaculture scientists looking at the improvement of productivity. The adoption of a single gene approach has spiked the characterization and study of a number of candidate genes of major effect on growth and one such gene is that of myostatin (Mstn). MSTN is a protein that inhibits muscle growth and that in beef cattle is responsible for inducing "double muscling", a phenotype with increased muscle mass. A number of studies have shown that genes similar to that of the cattle Mstn are present in the whole animal kingdom and in many species have a significant association with growth functions. Because of its central role in the regulation of growth, Mstn is therefore a very interesting candidate gene for the improvement of aquaculture productivity.

Detailed evolutionary analyses have revealed that Mstn comprises with its closest relative growth and differentiation factor-11 (Gdf-11) a small gene family that evolved from multiple events of gene or genome duplication. The structure of the Mstn/Gdf11 family resembles the "one-to-four" model seen for many genes isolated from species encompassing the diversity of the animal kingdom. In the one-to-four gene model invertebrates possess a single gene homologue (i.e. Mstn/Gdf11), higher vertebrates like mammals possess two paralog genes (i.e. Mstn and Gdf11) and at least four paralogs are present in teleost fish (i.e. Mstn-1 and Mstn-2). Gene duplication events may have engendered the origin of new functions compared to those initially exerted by the Mstn/Gdf11 ancestor. While some of these new physiological roles arisen following duplication are well studied and understood in mammalian model species, the history of functional evolution of the Mstn/Gdf11 family in species of high relevance for aquaculture production is still largely unclear. This thesis addresses key issues important for providing significant advances in the understanding of Mstn-like genes in aquaculture species using barramundi (Lates calcarifer) and the black tiger prawn (Penaeus monodon) as model species.

As at least two Mstn paralogs (Mstn-1 and Mstn-2) are present in fish like L. calcarifer, it is important for the purpose of growth improvement to understand whether subfunctionalization occurred following the event of genome duplication. In this thesis the Mstn-2 gene paralog, including its upstream region, was isolated and characterized from the L. calcarifer. Through a detailed analysis of gene expression this thesis provides preliminary evidence of differentiation of MSTN paralogs. Firstly, differential regulation as well as specific tissueresponses in the muscle, liver, gill and brain of L. calcarifer was observed after nutritional deprivation. In particular, the LcMstn-1 expression increased in liver (~4 fold) and muscle (~3 fold) and diminished in brain (~0.5 fold) and gill (~0.5 fold), while that of LcMstn-2 remained stable in brain and muscle and was up regulated in gill (~2.5 fold) and liver (~2 fold). In addition, it also suggested that Mstn genes in fish may regulate different growth and developmental processes by revealing an independent regulation of each paralog throughout the embryonic development with the Mstn-2 generally more abundant than the Mstn-1 and a diametrically opposite correlation of their expression with muscle hypertrophy in juvenile fish. Analyses of relationship between Mstn transcript abundance and muscle hypertrophy showed in fact that the expression of LcMstn-1 was only marginally associated with fiber size (r = 0.384, p = 0.064) while that of LcMstn-2 showed a highly significant negative correlation (r = -0.691, p < 0.0001). Differential regulation of Mstn paralogs was supported by in silico analyses of regulatory motifs that revealed, at least in the immediate region upstream the genes, a differentiation between Mstn-1 and Mstn-2. The Mstn-1 in particular showed a significantly higher conservation of regulatory sites among teleost species compared to its paralog indicating that this gene might have a highly conserved function in the taxon.

Conversely, invertebrates possess a single ortholog of the MSTN/GDF11 family. In this thesis the Mstn/Gdf11 gene ortholog was identified and characterized in the Penaeid shrimp, Penaeus monodon. The overall protein sequence and specific functional sites were highly conserved with other members of the MSTN/GDF11 family. Gene transcripts of pmMstn/Gdf11, assessed by real-time PCR, were detected in a variety of tissue types and were actively regulated in muscle across the moult cycle. To assess phenotypic function in shrimp, the pmMstn/Gdf11 gene expression was down-regulated by tail-muscle injection of sequence-specific double-stranded RNA. Shrimp with reduced levels of pmMstn/Gdf11 transcripts displayed a dramatic slowing in growth rate compared with that of control groups. Findings from this study place the MSTN/GDF11 gene at the centre of growth regulation in shrimp suggesting that this gene has an opposite role in invertebrates compared to higher vertebrates. In the former, levels of gene expression may positively regulate growth.

The outcomes of this thesis have provided significant advances in the understanding of Mstnlike genes in aquaculture species such as barramundi (L. calcarifer) and the black tiger prawn (P. monodon), towards the development of MSTN-based technologies for the enhancement of growth. In particular, this research emphasized that significant differences exist between Mstn-1 and Mstn-2 in fish whereby an efficient enhancement of growth may arise from specific targeting of one paralog only. It also revealed for the first time that the invertebrate Mstn/Gdf11 does not inhibit growth. This last finding in particular will cause an inversion of tendency, whereby research aiming to improve growth of invertebrates like crustaceans should investigate strategies to enhance the activity of MSTN/GDF11 and not reduce it, such as the case of vertebrate MSTN.

Item ID:	29934
Item Type:	Thesis (PhD)
Keywords:	myostatin; growth; barramundi; Lates calcarifer; black tiger shrimp; Penaeus monodon; genetic function
Copyright Information:	Copyright © 2011 Christian De Santis
Date Deposited:	25 Oct 2013 05:48
FoR Codes:	07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070401 Aquaculture @ 50% 07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070405 Fish Physiology and Genetics @ 50%
SEO Codes:	97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
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