Proteomics Research Leads to New Tests for Bitter Crab Disease, Determining When Crabs Will Molt and More

At the Northwest Atlantic Fisheries Centre in St. John's, NL, DFO senior scientist Dr. Joseph Banoub leads a Special Projects Group that is harnessing a branch of biotechnology called proteomics to develop practical applications for fisheries and aquaculture operations. By providing insight into the functions of proteins in a cell, proteomics enables fisheries researchers to study protein changes in fish and other aquatic organisms for a variety of purposes.

At the Northwest Atlantic Fisheries Centre in St. John's, NL, DFO senior scientist Dr. Joseph Banoub leads a Special Projects Group that is harnessing a branch of biotechnology called proteomics to develop practical applications for fisheries and aquaculture operations. By providing insight into the functions of proteins in a cell, proteomics enables fisheries researchers to study protein changes in fish and other aquatic organisms for a variety of purposes.

Aquatic biotechnology includes genomics, a discipline that aims to decipher and understand the entire genetic information content of plants, animals and fish, as well as micro-organisms.

The Aquatic Biotechnology and Genomics Research and Development Program of Fisheries and Oceans Canada, for example, has applied biotechnology techniques to the genetic identification of salmon populations, the diagnosis of aquatic animal diseases, the bioremediation of contaminated sites and risk assessments of genetically engineered fish.

After genomics, the next step in the study of biological systems is considered to be proteomics, a relatively new field of science that enables scientists to learn more about cellular behaviour. The "proteome" refers to all of the proteins produced by an organism, which varies with time and the requirements or stresses that a cell or organism undergoes. One gene can produce several proteins. Proteomics explores the individual functions of all the proteins in a cell to understand how the interaction of specific proteins with other parts of the cell affects the function of those proteins.

At the Northwest Atlantic Fisheries Centre in St. John's, NL, DFO senior scientist Dr. Joseph Banoub is leading a Special Projects Group to explore the application of proteomics to fisheries research in support of DFO policy and management decisions related to the ecological sustainability of wild fisheries, aquaculture and ocean ecosystems.

"By studying protein changes in fish and other aquatic organisms we can study their development over time as well as the progression of diseases," says Dr. Banoub. "Most importantly, we can use proteomics to measure the response to environmental conditions such as ocean temperature, acidity, carbon dioxide levels and pollutants including endocrine disrupting chemicals that affect the sexual maturity of fish."

A biomarker for endocrine disrupting chemicals

Ongoing research by the group is exploring a complex protein called vitellogenin, which is secreted into the bloodstream of sexually mature, female animals that lay eggs in response to circulating estrogens. Measuring levels of this protein in blood can be used for a variety of research purposes. For example, since vitellogenin appears in fish before other secondary sexual characteristics, its presence is an indicator of sexual maturity and reproductive status.

"Male fish have a gene for producing vitellogenin that is usually silenced but can be activated or 'turned on' when a fish is exposed to certain environmental conditions," says Dr. Banoub. "Unusually high levels of vitellogenin have been found in fish, including males, exposed to a variety of estrogen-mimicking compounds, and it has been proven as a biomarker for the presence of environmental endocrine disruptors in many species."

Recently, the Special Projects Group developed a simple, non-destructive method (no fish are harmed in the process) to measure vitellogenin in Rainbow Trout, Atlantic Salmon, Atlantic Cod, Haddock, Greenland Halibut and Yellowtail Flounder to identify the presence of endocrine disruptors in their environment. The test uses a very small volume of blood for analyses, enabling the fish to return to their habitat with minimal stress impact.

The team is also analyzing blood levels of vitellogenin in female fish of various species (Atlantic Salmon, Rainbow Trout, Greenland Halibut Turbot, Atlantic Cod and Yellowtail Flounder) to determine whether it can be used by fisheries and the aquaculture industry to assess the reproductive status of fish stocks.

Using mass spectrometry a scientific method for accurately measuring the weights of molecules, also called the molecular masses the team analyzed vitellogenin extracted from the blood of both control (wild) fish and fish exposed to an endocrine disrupting chemical. This enabled them to identify a small portion of the protein as unique to vitellogenin, providing a chemical 'fingerprint' for determining the amount of this protein in blood samples from the fish.

"Many studies indicate that variations in the physical ocean environment influence the production of marine organisms, making the integration of environmental information into the fishery resource assessment process a pressing issue. The ability to easily and quickly quantify vitellogenin in a wide range of these species would aid in that process," says Dr. Banoub.

Graduate students, Farid Jahou and Claudie Rene are members of the Special Projects Group that is using the science of proteomics to study a complex protein called vitellogenin. The group has developed tests to determine blood-levels of vitellogenin in several fish species, which can be used an indicator of sexual maturity and reproductive status and as a biomarker for the presence of environmental endocrine disruptors in many species.

Graduate students, Farid Jahou and Claudie Rene are members of the Special Projects Group that is using the science of proteomics to study a complex protein called vitellogenin. The group has developed tests to determine blood-levels of vitellogenin in several fish species, which can be used an indicator of sexual maturity and reproductive status and as a biomarker for the presence of environmental endocrine disruptors in many species.

Rodd Hobbs of the Special Projects group analyzes purified undigested vitellogenin at the Northwest Atlantic Fisheries Centre.

Rodd Hobbs of the Special Projects group analyzes purified undigested vitellogenin at the Northwest Atlantic Fisheries Centre.

Predicting when snow crabs will molt

Snow crabs, like other crustaceans, molt or shed their shells as they mature. Dr. Banoub and his team identified a protein in snow crabs called cryptocyanin that is significantly elevated in crabs during molting. This enabled them to develop a test to analyze the amount of cryptocyanin in adolescent snow crabs to determine whether they have a high probability of molting before the fishing season.

Snow crabs, like other crustaceans, molt or shed their shells as they mature. Dr. Banoub and his team identified a protein in snow crabs called cryptocyanin that is significantly elevated in crabs during molting. This enabled them to develop a test to analyze the amount of cryptocyanin in adolescent snow crabs to determine whether they have a high probability of molting before the fishing season.

The proteomics team is also heavily involved in investigating aspects of snow crab biology. In collaboration with Dave Taylor, a DFO snow crab biology and assessment research biologist, the team began studies aimed at gaining insights into snow crab molting physiology as well as Bitter Crab Disease.

The research group has identified a protein in snow crabs, called cryptocyanin, that is significantly elevated in crabs during molting - periodic shedding of their shells. Molting is an important sign of growth as a snow crab matures and gets ready to reproduce.

"The snow crab fishing season usually takes place after the crabs have molted and have new shells," says Dr. Banoub. "But predicting the timing of that isn't always 100 percent accurate and sometimes harvesters end up catching crabs with soft shells, which are not marketable and have a poor survival rate when returned to the ocean."

To address this issue, the team developed a test to analyze the amount of cryptocyanin in blood samples from adolescent snow crabs to determine whether they have a high probability of molting before the fishing season and consequently represent a potential disruption to harvesting strategies. Future research will involve fine tuning the test to make it quicker and easier to use, since it currently requires a lot of sophisticated equipment to carry out. "Eventually the goal is to develop our method so that it can be used with any other crustacean species," says Dr. Banoub.

A new test for bitter crab disease

The group has also developed a fast, cost-effective method of detecting bitter crab disease, which is caused by the parasite Hematodinium sp. When cooked, the meat of crabs infected with the disease has an aspirin-like bitter taste, ultimately rendering the meat inedible and resulting in economic loss for the crab fishery. The new screening test uses mass spectrometry to determine the presence or absence of BCD in the blood of snow crabs.

Bitter crab disease is fatal to crabs but is not usually detectable with the naked eye during the fishing season. In many areas where the disease is prevalent, high unexpected mortalities caused by the disease can seriously disrupt recruitment to the fishery through post-season mortality. The test developed by Dr. Banoub and his team will facilitate more accurate and timely diagnosis of diseased crabs during the fishing season and enable resource managers and the industry in general to plan alternate harvesting strategies to compensate for possible shortfalls in the crab fishery.

"This method will enable us to test raw blood samples much more quickly and economically than the current detection methods," says Dr. Banoub. "In addition, once the test has been finalized for snow crab, it will be transferrable to any other species susceptible to bitter crab disease."

Ongoing research

SDS-PAGE gel of molting and non-molting crab blood samples: Lane 2 shows the stained proteins from a non-molting crab. Lane 1 shows the stained proteins from a molting crab; the intensity of colour band in Lane 1 indicates increased production of the protein cryptocyanin in the molting crab compared to the non-molting crab.

SDS-PAGE gel of molting and non-molting crab blood samples: Lane 2 shows the stained proteins from a non-molting crab. Lane 1 shows the stained proteins from a molting crab; the intensity of colour band in Lane 1 indicates increased production of the protein cryptocyanin in the molting crab compared to the non-molting crab.

Ultimately, this ongoing research will generate a series of novel practical immunochemical "dip stick" tests that can be used to exactly measure the protein concentrations in several species of fish and crustaceans.

"This cutting-edge research will improve the availability of biotechnology tools that can be used to enhance the sustainability of aquaculture production and improve the genetic and proteomic profiling of Atlantic Salmon, Rainbow Trout and Cod vitellogenins, for the control of aquaculture species," says Dr. Banoub.

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