IDENTIFICATIONS OF SHRIMP MUSCLE REGULATORY GENES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SPECIAL GRANT
• Reporting Frequency: Annual
• Accession No.: 0207701
• Grant No.: 2006-34135-17267
• Proposal No.: 2006-04751
• Project Start Date: Aug 15, 2006
• Project End Date: Aug 14, 2009
• Grant Year: 2006
• Program Code: [AH]- (N/A)

Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822

Performing Department
MOLECULAR BIOSCIENCES & BIOSYSTEMS

Non Technical Summary
Shrimp is the single most important seafood imported into the United States (3.8 billion dollars of product value). The shrimp industry is expanding rapidly with an estimated growth rate of 300 percent from 1995 to 2005 worldwide (FAO 2002). The value of Hawaii shrimp industry is approximately 40 million dollars, including sales of broodstock and fresh shrimp, and technical services for the US and Asia shrimp industry. To prevent disease problems, the industry is very interested in developing fast-growing and disease-resistant shrimp lines. The unique climate and natural resources of the Hawaiian islands provide excellent conditions for shrimp R&D programs. Research emphasis on shrimp growth not only offers the promise to enhance production efficiency, but also prevents diseases due to short growout cycles. Shrimp growth depends on the capability of muscle restoration during each molt cycle. Based on previous research of skeletal muscle and gene cloning, we believe that an integrated strategy of specific gene searching through comparative genomics is a critical and vital approach to identify the key genes that control shrimp muscle restoration. This project is designed to identify two critical muscle regulatory genes (myostatin and MyoD), and study their expressions over molt cycle and their relationships to growth performances. This research plan will not only generate significant outcomes in the genomics of shrimp muscle growth, but also offer the potential to develop molecular testing for selecting fast-growing shrimp individuals.

Animal Health Component

30%

Research Effort Categories

Basic

50%

Applied

30%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	3721	1080	50%
305	3721	1040	50%

Knowledge Area
303 - Genetic Improvement of Animals; 305 - Animal Physiological Processes;

Subject Of Investigation
3721 - Marine shrimp;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;

Keywords

marine shrimp

muscle growth

molt

cdna library

myogenesis

mrna

gene expression

genetic selection

Goals / Objectives
Shrimp is the single most important seafood imported into the United States (3.8 billion dollars of product value). The shrimp industry is expanding rapidly with an estimated growth rate of 300 percent from 1995 to 2005 worldwide (FAO 2002).In the past decade, scientific breakthroughs have revealed the detailed molecular mechanisms of muscle development in mice and humans. Critical growth factors and transcription factors that control muscle growth, development, regeneration and metabolisms have now been well characterized. Unfortunately, this knowledge has been limited to mammalian species. The genes and molecular mechanisms of shrimp growth have not yet received much scientific attention. Genomic resources from one species can be easily applied to other species through comparative genomics. Muscles in crustaceans undergo atrophy before molting, and restoration periods after molting. Like most other crustaceans, shrimp growth depends upon the capability of muscle restoration after molting. The critical genes related to shrimp muscle growth may be highly regulated by molting cycle. Based on the similar characteristics of crustacean skeletal muscle to most mammals, this research project will focus on identifying of shrimp counterparts of the known critical muscle regulatory genes from mammalian species. Two specific genes, namely myostatin and MyoD, will be initially selected. Myostatin plays a dominant role in the control of muscle mass while MyoD controls muscle cell formation. We hypothesize that these two critical genes-myostatin and MyoD, are present in shrimp muscle, and that they plays significant roles in shrimp muscle accumulations. The long-term goal of this research project is to discover the genes that control shrimp muscle growth through comparative genomics using the known DNA sequences from vertebrate species, and then to apply this knowledge to the breeding of fast-growing shrimp lines. This project has identified two specific objectives: 1) To establish shrimp muscle cDNA library and to identify shrimp counterparts of myostatin and MyoD genes; 2) To study muscle gene expression patterns across molting cycle, and their relationships to shrimp growth rates.

Project Methods
Shrimp growth depends on the capability of muscle restoration during each molt cycle. Little is known about shrimp muscle genes and their activities during molting. Two specific genes, namely myostatin and MyoD, will be initially selected. Construction of shrimp muscle cDNA libraries is indispensable for identifying and characterizing the proposed muscle genes. Abdominal muscle tissue will be obtained from juvenile white shrimp (Litopenaeus vannamei), which will be supplied by a local certified SPF broodstock shrimp farm. Poly(A)RNA will be prepared from the total RNA using two cycles of chromatography on oligo(dT)-cellulose. The muscle cDNA library will be generated by a PCR-based library construction kit (SMART cDNA technology) of Clontech following the instruction from the manufacturer. We may also normalize the muscle cDNA library for a possible EST project in collaboration with the University Biotechnology Core Facility Laboratory. Therefore, the established cDNA libraries will have an additional use for identifications of other muscle genes through DNA sequencing. A quality shrimp muscle cDNA library will be prepared as a result of this research. Based on the sequence alignment between mammalian species and others, we will generate partial shrimp DNA sequences for myostatin and MyoD, which will then be used for identifying the full-length shrimp myostatin and MyoD from the shrimp muscle cDNA library. In studying muscle gene expression over molt cycle, the molt cycle will be divided into four main stages. Real-time PCR will be employed to quantify mRNA levels of the shrimp myostatin, MyoD, actin, and myosin heavy chain in the abdominal muscles. Further experiments will also be designed to study the expressions of these muscle genes in selected shrimp lines with slow and fast growth phenotype. The correlation between gene expression and growth rate will be analyzed to examine their possible applications to fast-growing shrimp broodstock selection. This research will not only generate significant outcomes in the genomics of shrimp muscle growth, but also have the promising potential for developing molecular testing for selecting fast-growing shrimp individuals.

Progress 08/15/06 to 08/14/09

Outputs
OUTPUTS: On average, the United States imports approximately 3.5 billion dollars of shrimp per year. The shrimp industry is expanding rapidly worldwide. Along with culture technology and disease resistances, enhancement of growth rate has been emphasized as priority areas of shrimp research and development. The goal of this project is to identify the shrimp genes that significantly influence growth performances. It is expected that the genes or mRNA identified from this project can be used for genetic selections of fast-growing shrimp broodstock. The results of the research work were disseminated to scientific communities of shrimp genomics and aquaculture through conference presentations and referenced publications. Two manuscripts were published in peer-reviewed journals. We also made two presentations in international scientific conferences. One presentation was at the international conference of Integrated Technologies to Advanced Shrimp Production (Honolulu, HI October, 2009), which has approximately 100 participants from all the world. The other presentation was at the Royal Golden Jubilee Ph.D. Congress X (Pattaya, Thailand, April 2009) with more than 400 participants from Thailand and collaborators with Thailand scientists from other countries. Some other output include collaborations and educations on shrimp genetics with local shrimp aquaculture farmers, scientists, visiting scholars and graduate student educations. PARTICIPANTS: Principal investigator Dr. Jinzeng Yang has established research collaborations with the Shrimp Department of the Oceanic Institute, and Prince of Songkla University. PARTICIPANTS: Dr. Jinzeng Yang, principal investigator, CTAHR, University of Hawaii at Manoa; Ms. Baoping Zhao, Research Associate, CTAHR, University of Hawaii at Manoa; Ms. Yanisa Laoong-u-thai, Graduate students, CTAHR, University of Hawaii at Manoa Ms. Shizu Watanabe, Graduate students, CTAHR, University of Hawaii at Manoa Mr. Dustin Moss, Graduate students, CTAHR, University of Hawaii at Manoa TARGET AUDIENCES: Animal scientists, marine biologist, shrimp aquaculture Biotechnology PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Pacific white shrimp or L. Vannamei is an important species in aquaculture. The identified genes aided the development of shrimp genome, also helped dissect genetic components that are responsible for shrimp muscle development and growth. The expression patterns of the identified genes had been planed in studying shrimp strains with different growth performance. We are targeting specific candidate genes that regulate shrimp growth. Therefore, the impact of this research project is on identifying candidate genes for economically important traits in aquaculture industry. Hopefully, the outcomes from such a technology development will become available to shrimp producers. The publications from this project have not only provided new knowledge in molecular regulation of shrimp molt cycle and muscle growth, but also are used as scientific resources for shrimp genomics and biology studies. Three Ph.D. graduate students were trained by this research project.

Publications

• 1. Laoong-u-thai Y, Zhao B, Phongdara A, Ako H, Yang J. 2009. Identifications of SUMO-1 cDNA and its expression patterns in Pacific white shrimp Litopeanaeus vannamei. Int J Biol Sci. 5:205-14.

Progress 08/15/07 to 08/14/08

Outputs
OUTPUTS: The primary activities of the research project are 1) establishment of the muscle cDNA libraries of Pacific white shrimp; 2) identifications of significant muscle regulatory genes. Research results were presented to the annual meeting of Plant and Animal Genomics Conference in 2008. One manuscript was recently published in the journal of Animal. We have also developed collaborations with Oceanic Institute, Prince of Songka University (Thailand). Scientists from both institutes are also interested in basic genomic studies of shrimp growth. Initial data analysis from shrimp muscle EST sequencing showed that a large of number of shrimp muscle mRNA sequences was identified from this project. Some of EST sequences were submitted to the NCBI database for public use. Two important genes related to shrimp muscle molting and development has been studies in details for their specific functions. A manuscript was recently submitted to the journal of Gene. PARTICIPANTS: Harry Ako, Ph.D. Principal Investigator, CTAHR; Jinzeng Yang, Ph.D., Co-principal Investigator CTAHR; Jose Renato Cesar, Ph.D. Student, CTAHR; Baoping "Beth" Zhao, Research Associate, CTAHR; Yanisa Laoong-u-thai, Ph.D. Student, Prince of Songkla University, (Thailand) TARGET AUDIENCES: Animal geneticist; Aquaculture PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Gene identifications will have application for effective animal selection for shrimp breeding program. Our results will be facilitating DNA and/or genetic testing for selection of fast-grower of broodstock shrimp. At this time there are no immediate impacts on shrimp aquaculture from this research project.

Publications

• Cesar, JR; Zhao, B; Yang, J. 2008. Analysis of expressed sequence tags from abdominal muscle cDNA library of the pacific white shrimp Litopenaeus vannamei. ANIMAL 2: 1377-1383

Progress 08/15/06 to 08/14/07

Outputs
OUTPUTS: Research results were presented to the annual meeting of Plant and Animal Genomics Conference in 2007. Fifty expressed sequence tags from shrimp muscle cDNA library were submitted to the Genbank or NCBI database. PARTICIPANTS: Harrry Ako, Ph.D. Principal Investigator, CTAHR, Jinzeng Yang, Ph.D., Co-principal Investigator CTAHR, Jose Renato Cesar, Ph.D. Student, CTAHR Baoping "Beth" Zhao, Research Associate, CTAHR, Yanisa Laoong-u-thai, Ph.D. Student, Prince of Songkla University, (Thailand) TARGET AUDIENCES: Aquaculture and animal genomics

Impacts
The pacific white shrimp, Litopenaeus vannamei, is a popular species in aquaculture. A cDNA library based on juvenile abdominal muscle was established by a PCR-based SMARTTM cDNA technology. Library size was 5.0 x 106 pfu independent clones per microgram of starting RNA with the percentage of recombinant clones >95%. Sequence analysis of 311 randomly picked positive clones revealed 197 expressed sequence tags with average insert size of 745 nucleotides, 56% (110 of 197) clones having 5'-end sequence and 44% (87 of 197) clones having 3'-end sequence. By Blast queries of the sequences we identified 160 unique clones, including 67 sequences of 100% identity matches, 28 high homologies (80-90% sequence match, >100 bits hit score in Blastn), 65 medium homologies (>100 bits hit score in Blastp) to the known genes in the database. We identified 37 unknown sequences, including 1 sequence matching a hypothetical protein and 2 sequences matching DNA microsatellite markers. Among the 100% to high identity-matched ESTs, 12S ribosomal RNA, actin 1, actin 2 and arginine kinase and beta-actin were most abundant transcripts with 5 to 20 times of hit. Primary hit sequences originate from shrimp, insects, lobsters, crabs, and crayfish. According to the function of gene products, genes were categorized as muscle structural proteins (24%), protein synthesis (24%), followed by mitochondrial functions (22%), exoskeleton (14%), enzymes (6%), and RNA splicing (2%). Preliminary analysis of sequence data suggests that the established cDNA library supports a full-scale EST strategy for discovery of the transcripts responsible for shrimp muscle biology. A further analysis and identifications of the full-length cDNA sequences will significantly facilitate shrimp genomic program.

Publications

• Yang J, Cesar JR, Zhao B, Ako H. Identifications Of Shrimp Muscle Regulatory Genes By Comparative Genome Analysis. Plant & Animal Genomes XV Conference (January, 2007, San Diego, CA), P633.