IDENTIFICATIONS OF SHRIMP MUSCLE REGULATORY GENES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: SPECIAL GRANT
• Reporting Frequency: Annual
• Accession No.: 0214751
• Grant No.: 2008-34135-19343
• Project No.: HAW00581-08G
• Proposal No.: 2008-03371
• Program Code: AH
• Project Start Date: Aug 1, 2008
• Project End Date: Jul 31, 2011
• Grant Year: 2008

Project Director
Ako, H.

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

Performing Department
MOLECULAR BIOSCIENCES & BIOSYSTEMS

Non Technical Summary
The shrimp industry is expanding rapidly with an estimated growth rate of 300 percent from 1995 to 2005 worldwide. The value of Hawaii shrimp industry is approximately 40 million dollars, including sales of specific pathogens-free (SPF) 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 shrimp strains using SPF broodstock. 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. This project is designed to identify two critical muscle regulatory genes, myostatin, and MyoD, through comparative genomics. Myostatin plays a dominant role in muscle mass while MyoD controls muscle cell formation in mammalian species. We will identify the shrimp counterparts of these two genes and study their mRNA expressions over the molt cycle. A quality shrimp muscle cDNA library will be prepared as a result of this research. Building 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.

Animal Health Component

(N/A)

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	3721	1040	100%

Knowledge Area
303 - Genetic Improvement of Animals;

Subject Of Investigation
3721 - Marine shrimp;

Field Of Science
1040 - Molecular biology;

Keywords

shrimp

abdominal muscle

growth

broodstock

gene

mrna

Goals / Objectives
On average, the United States imports approximately three billion dollars of shrimp per year. The shrimp industry is expanding rapidly worldwide. The unique climate and resources of Hawaii provide excellent conditions for shrimp research and aquaculture development. Along with culture technology and disease resistance, enhancement of growth rate has been emphasized as the priority area of shrimp aquaculture industry. Shrimps grow and develop through muscle restoration during the molt cycle. The rate of growth depends upon the net muscle gain during each molt. A study of muscle gene expressional changes over the molt cycle will reveal the critical genes that control shrimp muscle growth. Presently, little is known about the muscle genes, the muscle regulatory genes or factors in shrimp. Muscle growth has been well studied at the molecular and genomic levels in mammalian species. The fundamental characteristics of muscle biology are similar between mammalian and crustacean species. We hypothesize that the muscle regulatory genes present in mammalian species, or their sequence-related genes, are also involved in shrimp muscle growth. The goal of the project is to identify the shrimp genes responsible for muscle restoration and growth using genomic databases from other species. Two specific genes, namely myostatin and MyoD, are initially selected and will be our main research objectives of the project. Myostatin plays a dominant role in muscle mass while MyoD controls muscle cell formation. We will identify these two genes, and study their mRNA expressions over the molt cycle to define the critical stages of muscle atrophy and restoration in shrimp. To determine whether or not these muscle regulatory genes are associated with growth performances, we will compare their expressions between high and low-growing shrimp lines. This project is expected to generate significant outcomes through discovery of the genes that are responsible for shrimp growth. The results can be applied to shrimp broodstock industry by providing scientific insight in identifications of fast-growing individuals. Additionally, the results will also strengthen our understanding of shrimp muscle restoration during molt at the molecular level.

Project Methods
To identify specific muscle genes such as myostatin and MyoD, we will employ both cDNA library construction and reverse transcriptase-PCR amplifications of the conserved sequences of the mRNAs. Abdominal muscle tissue will be obtained from juvenile white shrimp (Litopenaeus vannamei). Poly(A)RNA will be prepared from the total RNA using two cycles of chromatography on oligo(dT)-cellulose. The muscle cDNA library has been constructed by a PCR-based library construction kit (SMART cDNA technology). We 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. 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 by 5 or 3 RACE method. In studying muscle gene expression over molt cycle, the molt cycle will be divided into four main stages. Real-time PCR methods 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 08/01/08 to 07/31/11

Outputs
OUTPUTS: Pacific white shrimp (L. vannamei) is becoming the most popular aquaculture shrimp in the world as the L. vannamei shrimp production in China and Thailand have been dramatically increased in the last several years. Specific pathogens free (SPF) shrimp were originally developed from L. vannmei in Hawaii and the local shrimp broodstock export has been developed to significant industry to the US and Asian countries. The project is designed to develop genetic tools for shrimp growth selections by identifications of muscle regulatory genes. We are targeting specific candidate genes that regulate shrimp growth. The main outputs of the project include: 1) Establishment of shrimp molt cycle identification and classification in live animals; 2) Establishment of L. vannamei abdominal muscle cDNA library and publication of the pilot sequencing results from the cDNA library; 3) Identifications of SUMO-1 protein and its expression patterns, and publication of the research results; 4) Identification of a DNA-bind protein LvDBP-23 and characterizations of its expression in L. vannamei, and publication of the research results; 5) Development of collaborations in shrimp genetics with the scientists in China and Thailand; 6) Several presentations in international scientific conferences such as shrimp technologies (2009) and aquaculture workshop in PAG meeting (2008-2011); 7) Training of four UHM graduate students and visiting graduate students from Thailand; 8) Technical support in shrimp broodstock breeding to local farms; 9) Submission of a research grant application to USDA for continued support in shrimp genetic research at UHM-CTAHR. 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: scientists, marine biologist, shrimp aquaculture Biotechnology PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results of the project are mainly at the identifications of several significant genes from L. vannamei abdominal muscle and their regulatory roles in shrimp growth. For example, LvSUMO-1 mRNA levels are high in abdominal muscle during the premolt stage, wherein it has significant activities of protein degradation, suggesting its possible role in the regulation of shrimp muscle protein degradation. A novel mRNA encoding for a putative DNA-binding protein LvDBP23 was identified. The expression of LvDBP23 mRNA is presented in abdominal muscle and swimming leg muscle. Its mRNA expression has the highest level in abdominal muscle. We are the first to report a DNA-binding protein identified from the abdominal muscle tissue of marine shrimp L. Vannamei. Its high-level specific expression during the intermot stage suggests its role in the regulation of muscle buildup during the growth phase of shrimp molt cycle.

Publications

• Laoong-u-thai Y, Zhao B, Phongdara A, Yang J. 2011. Molecular Characterizations of a Novel Putative DNA-Binding Protein LvDBP23 in Marine Shrimp L. vannamei Tissues and Molting Stages. PLoS One. 6:e19959. Epub May 20.

Progress 08/01/09 to 07/31/10

Outputs
OUTPUTS: The main output from this project in the last year is accomplishment of the thesis work of one visiting graduate student from Thailand. Also we have established collaborative research project with Prince Songka University in Thailand. One manuscript is submitted and under review. In addition, we have reached local shrimp farms about our research work in DNA-based technologies for SPF broodstock selections and hatchery operations. PARTICIPANTS: Dr. Harry Ako, UHM-CTAHR Dr. Jinzeng Yang, UHM-CTAHR Dr. Yanisa Laoong-u-thai1, Burapha University, Chonburi 20131, Thailand Dr. Amornrat Phongdara, Prince of Songkla University, Songkhla, 90112, Thailand TARGET AUDIENCES: Scientists and producers in shrimp breeding and genetic programs Researchers in muscle development PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Litopenaeus Vannamei, well known as pacific white shrimp, is the most popular shrimp in the world shrimp market. Identification and characterization of shrimp muscle regulatory genes are important for shrimp genetic improvement. A novel mRNA encoding for a putative DNA-binding protein was identified from Litopenaeus vannamei abdominal muscle cDNA library. The novel protein is named as LvDBP23. Its mRNA sequence is successfully used for producing LvDBP23 recombinant protein. In vitro nucleic acid-binding assays reveal that LvDBP23 protein can bind to both ssDNA and dsDNA, indicating its possible role of regulation of gene transcription. Its high-level specific expression during the intermot stage suggests its role in the regulation of muscle buildup during the growth phase of shrimp molt cycle. The new gene may have significant impacts on application of DNA-based selections for fast-growing shrimp strains with SPF L. Vannamei broodstock.

Publications

• No publications reported this period

Progress 08/01/08 to 07/31/09

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 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. Outcomes/Impacts PARTICIPANTS: Principal investigator Dr. Jinzeng Yang has established research collaborations with the Shrimp Departmentof 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: 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.
• 2. 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: 1377-83.