Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Aquaculture Research Station
Non Technical Summary
C. virginica historically accounts for 86 percent of total oyster harvest in the U.S. Leading the nation in oyster production, the annual commercial landing of C. virginica in the Gulf of Mexico reached 50% of the market of the United States with a market value more than 7.6 million dollars in 2013 (National Marine Fisheries Service, 2014). However, annual statistical data clearly demonstrated that production of the Gulf oyster was significantly decreased from 25.8 million pounds in 2000 to 19.2 million pounds in 2013. The dramatic decrease of oyster production in the Gulf area had multiple causes, such as the deterioration of oyster habitat, change of water salinity caused by hurricanes, and increased pollution as a result of the BP oil spill. Many other stresses from environmental factors, such as temperature fluctuation, dissolved oxygen levels, and activities of microbial pathogens also could contribute to this decline. Exposed to complicated environmental changes, oysters adjust their metabolic and physiological responses to survive stress. Acclimation of oysters to environmental change can be accelerated by artificial intervention; however, this is difficult due to lack of information on gene regulatory mechanisms of the oyster in response to environmental stresses. The proposed study will use high throughput genomic techniques to define the gene regulatory pathway responding to stimuli caused by fluctuation of various environmental factors. Artificial modification of each gene regulatory pathway is likely to change the adaptation of C. virginica to a specific environmental change.Red swamp crawfish is one of the major local aquaculture species. Louisiana produces 95% of the crawfish in the U.S. According to the Agricultural Center of Louisiana State University, the crawfish harvest in Louisiana was more than 127 million pounds in 2014. The crawfish industry recently faced a potential risk when white spot syndrome virus (WSSV) was detected from farmed crawfish in Louisiana. Causing white spot syndrome (WSS) on the shell of most crustaceans, WSSV was first reported to wipe out the shrimp culture industry in China in 1993followed by outbreaks in East, South, and Southeast Asia. To date, WSSV has been found in all shrimp farming regions except Australia.The first appearance of WSSV in the U.S. was in late 1995. The year of introduction of WSSV into the U.S. is not clear, however, crawfish in several sites in the southeastern U.S. were found to carry WSSV between 1995 and 1997. As a type of double-stranded DNA (dsDNA) virus, WSSV can infect a wide range of captured and cultured crustaceans and other arthropods, including penaeid shrimps (Penaeus spp.), crabs, lobsters, and crawfish. The majority of WSSV studies were performed with penaeid shrimps. Three complete WSSV sequences have been identified with strains isolated from Thailand, China, and Taiwan, respectively. Slight differences have been found among the three sequences; however, WSSV strains isolated from other hosts appear to have very different pathogenicity than strains from penaeids. Comprehensive studies of WSSV in crawfish have not been performed because the crawfish industry has not yet been significantly harmed by this dangerous virus. Lack of basic knowledge of pathology and host response to WSSV in crawfish exposes the industry to risk of future WSS outbreaks. In our proposed study, we are going to identify the genomic changes of crawfish with WSSV challenge combining with the stresses from environmental factors. We are also going to compare the crawfish gene expression responses to various WSSV isolates. These results will be utilized to further investigate the innate immune responses of crustaceans to virual infections.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Goals / Objectives
I) To identify gene regulatory networks of the host responses of Eastern Oyster (Crassostrea virginica) under the stress of environmental changes.II) To understand the host defense gene regulation pathways of red swamp crawfish (Procambarus clarkii) in response to the stimulation of different strains of white spot syndrome virus (Whispovirus, WSSV).
Project Methods
1. Research on adaptive mechanisms of oysters to environmental factorsAppearance of oysters at different development stages can be very different due to effects of metamorphosis during the life cycle. In general, oysters start spawning when water temperature is above 20°C. Fertilization of the eggs will be completed in water and fertilized eggs will develop into planktonic trochophore larvae in about six hours. Within 12 to 24 hours, larvae become fully shelled veligers (D-veligers). In the next two to three weeks, free-swimming veliger larvae undergo a series of morphological changes, including change in the shape of the shell (umbo-veligers) and generation of locomotive organs (feet) to become pediveligers. Pediveligers attach to suitable underwater substrates (spat) and metamorphose to adults.Environmental conditions for zygotes and larvae thereafter will be maintained at 22°C with salinity 30‰ and pH 8.2 as an optimized control condition. During the first 24 hours post-spawning, eight samples of larvae will be collected approximately 3 hours apart. Ten samples will be collected within the following 20 days with an average interval of 2 days between collections. Spat samples will be collected at day 22 post-spawning. The majority of each sample will be snap frozen in liquid nitrogen for molecular analyses. The rest of the samples will be preserved in fixing solutions for histological and morphological studies. The control condition for both adults and larvae will be 22°C, salinity 30‰, and pH 8.2. Temperature treatments will be based on natural temperature changes of C. virginica habitats. Habitats of C. virginica are primarily distributed along the Gulf of Mexico and southern Atlantic coasts. According to the NOAA Satellite and Information Service, the average temperature range of sea water along the coasts in these areas is from 4°C (January, coast of VA) to 32°C (June and July, Gulf of Mexico). Therefore, three temperature levels will be created for oysters; 4°C, 22°C (control), and 32°C. Water pH can be adjusted by adding CO2 (a main reason for acidification of the ocean). Natural marine water first will be filtered and sterilized. Then CO2 in the water will be depleted using microporous membrane contractors for gas gas-transfer, and CO2 will be added back to the water to set the pH value at 8.2 (control, ~200 atm pCO2), 7.7 (~1000 atm pCO2), and 7.2 (~2800 atm pCO2). Water pH will be confirmed with a pH meter . Salinities will be created as described in an earlier study. Salinities will be adjusted with artificial sea salts, sea water and freshwater. Three levels of salinity -- 15‰, 30‰ (control), and 40‰ -- will be created and confirmed with an osmometer.RNA samples will be isolated from both adult and larval C. virginica. For adults, RNA will be purified from separate tissues, such as gill, muscle, mantle, and hemolymph from each individual. For larvae and spat, RNA samples will be prepared from a pool of individuals in each condition. The number of individuals in each pool will be decided by pre-testing the amount of total RNA and the quality of RNA from different size pools. For adult samples, priority will be given to tissues with the most active stress responses including hemocytes, mantle, and gill. For larvae and spat, selection of RNA samples for sequencing will be based primarily on microscopic and histological analyses. We are particularly interested in critical morphological changes of the life cycle, such as formation of the shell, generation of the foot, and larval settlement. Therefore, larval samples that show significant differences before or after these morphological changes, under the microscope, will be selected for next-generation sequencing.2. Research on host immune response of crawfish to infection by WSSVTwo-hundred microliters of each WSSV strain, suspended in water with a concentration of 5 × 107 genomic copies per milliliter, will be injected into each crawfish at the base of the fourth walking leg. Crawfish will be maintained in tanks with recirculating systems at 22 °C with dissolved oxygen concentration above 7 mg/L. Eight samples of live crawfish will be taken at 12 hr, day 1, day 2 and each day until day 7, and survivors after 10 days of challenge. Meanwhile, the mortality of infected crawfish will be recorded every day until day 10. Total RNA will be isolated from the hemocytes and gills of all WSSV infected crawfish, and healthy individuals as controls. Preparation of crawfish hemocytes will follow protocols published previously.The virus strain originally isolated from crawfish will be used for the study of environmental effects of WSS in crawfish. To determine effects of temperature, we will hold crawfish at three temperatures; 4°C, 12°C and 22°C. Crawfish will be kept at each temperature for seven days prior to WSSV challenge. Similarly, two dissolved oxygen levels will be used; >7 mg/L (normoxic) and <3 mg/L as (hypoxic). The creation of two different levels of D.O. will be achieved by inputting mixtures of nitrogen/oxygen with different ratio. Crawfish will be maintained in each oxygen condition for seven days until WSSV challenge. Challenge, monitoring, and sampling processes for both experiments will be the same as described above.Gills of WSSV-challenged crawfish will be preserved in 10% paraformaldehyde for histological analyses. Hematoxylin and Eosin (H&E) staining will be applied to demonstrate pathological changes in the gill resulting from WSSV infection. Histological samples also will be used for in situ hybridization of WSSV in the gill of crawfish. Probes used for WSSV in situ hybridization (ISH) will be as described in a previous study. RNA samples to be used for further molecular analyses will be selected based on results of H&E staining and ISH. We will select RNA samples from individuals with double positive results in H&E staining and ISH.WSSV strains have been isolated from red swamp crawfish (Procambarus clarkii), white shrimp (Litopenaeus setiferus) and blue crab (Callinectes sapidus). The WSSV isolates will be obtained from Dr. Christopher Green at the Aquaculture Research Station (ARS), LSU AgCenter. 100 µl re-suspended WSSV will be injected intramuscularly between the carapace and abdomen into the crawfish. Different concentrations of WSSV will be applied for virulence tests. Serial dilution of a WSSV stock solution will be used to create treatments with five-fold differences between graded concentrations. WSSV with doses 0 (negative control), 102, 104, 106, 108, and 1010 genome copies per gram of shrimp will be used for injection. The maintenance of the crawfish and the WSSV administration will be performed in Dr. Green's laboratory at ARS. Samples will be collected from different tissues, including hemolymph, gill, muscle, and intestines. Samples will be preserved in Tri-Reagent for RNA extraction and 4% paraformaldehyde for histological and ISH analyses.To capture maximum alterations in gene expression of WSSV-infected crawfish, RNA samples will be screened based on qPCR analysis for molecular markers which have been previously reported. Specific primers targeting genes encoding superoxide dismutase (SOD), prophenoloxidase (proPO), and reactive oxygen species (ROS) will be used to perform the screenings because gene expressions are up-regulated by WSSV challenge according to earlier studies. RNA samples with the highest level of transcriptomes for these three genes will be selected for preparation of next-generation sequencing. RNA quality control, library construction, sequencing, and data analyses will be done in LSU BioMMED Cores following the procedures described in oyster study.