Something at NBFGR -Lucknow

By :Pradeep Kumar Department of Fish Genetics & Biotechnology

CENTRAL INSTITUTE OF FISHERIES EDUCATION- MUMBAI EDUCATION-

What's inside..?????

Fisheries Education DNA Barcoding Genotoxicity Cell Culture Proteomics

Fisheries Education
AQUACULTURE 1. Principles of Aquaculture 2. Freshwater Aquaculture 3. Fish Nutrition & Feed Technology 4. Culture of Fish Food Organisms 5. Aquaculture Engineering 6. Ornamental Fish Production and Management 7. Coastal Aquaculture and Mariculture 8. Finfish Breeding and Hatchery Management 9. Shellfish Breeding and Hatchery Management 10. Fish Diseases and Management 11. Fish Genetics and Biotechnology
}

FRM
1. 2. 3. 4. 5. 6. 7. 8.

Taxonomy of Finfish Taxonomy of Shellfish Anatomy of Finfish and Shellfish Biology of Finfish and Shellfish Inland Capture Fisheries Physiology of Finfish and Shellfish Marine Capture Fisheries Fish Population Dynamics and Stock Assessment

AQUATIC ENVIRONMENT
1. 2. 3. 4. 5.

Meteorology and Geography Soil and Water Chemistry Limnology Oceanography Aquatic Pollution and Coastal Zone Management

HARVEST AND POSTHARVEST TECHNOLOGY

1. 2. 3. 4. 5. 6. 7. 8. 9.

Food Chemistry and Fish in Nutrition Refrigeration and Equipment Engineering Freezing Technology Fishing Craft Technology Canning and Fish Packaging Technology Navigation and Seamanship Fishing and Gear Technology Fish Products and By-Products Technology Fish Microbiology and Quality Assurance

Fisheries Extension & Economics

1. 2. 3. 4. 5. 6. 7. 8. 9.

Information and Communication Technology Statistical Methods Fisheries Economics Fisheries Extension Education Fisheries Administration and Legislation Disaster Management in Fisheries Financing and Marketing Management Entrepreneurship Development and Communication Skills Project formulation & implimantation

DNA BARCODING AND ITS APPLICATION IN

FISHERIES

Indian Biodiversity at Glance (Source-NBFGR)

Major Major Groups
No. of species . of species % of World species

Plants Animals Insects Fishes Amphibians Reptiles Birds Mammals

45,523 89,492 59,353 2,246 240 460 1,232 397

11.80 7.28 6.83 11.00 4.66 7.91 13.66 8.58

Indian Fish Diversity at Glance

Ecosystem Total Species

(Source- NBFGR)

Threatened Status

Extinct

Endang ered

Vulnerable

Rare

Indeter minate

Total

Freshwater

765

01*

03

14

02

43

63

Brackishwater Marine

113

01

03

04

1368

02

09

11

Total

Index.
} } } } } }

DNA barcoding Importance Mechanism Application Recent advancement Conclusion

The Birth of DNA Barcoding:

} The

DNA barcoding initiated at the University of

Guelp in 2003, has gathered momentum, gained extensive international participation in the form of the International Barcode of Life Project (iBOL)
} Headed

by Paul Hebert, this worldwide DNA

barcoding initiative is supported by the sequencing facilities at the Canadian Centre for DNA Barcoding (CCDB), located in the Biodiversity Institute of Ontario (BIO)

DNA barcoding

is a method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular species.

Why Barcode Fishes?

}

Due to over exploitation, conservation concerns and lists of threatened and endangered fish species have risen rapidly. There is also growing evidence of consumer fraud as low-value species are substituted for more valuable species. Highly-trained taxonomists can identify whole fish, but they cannot unambiguously identify fillets, processed product, or immature stages

How Will Barcoding Impact Fish Taxonomy?

} Preliminary

results indicate that a global barcoding

initiative will increase the fish species list for our planet by at least 10%.

Steps involved.
1.

Identification of voucher specimen by traditional taxonomy

2. 3. 4. 5.

Preservation of voucher specimen Isolation of total DNA Amplification of mitochondrial COI gene Sequencing of amplified product

Why barcode animals with mitochondrial DNA?

}

Three properties make mitochondrial genomes especially suitable for identifying species.

1.

Copy number: Each cell typically contains only 2 copies of nuclear DNA sequences, the same cell contains 100-10,000 mitochondrial genomes.

2.

Greater differences among species: Sequence differences among closely related animal species average 5- to 10-fold higher in mitochondrial than nuclear genes.

3.

Intraspecific variation in mitochondrial DNA is low in most animal species.

Primary goals of DNA barcoding

} To

focus on setting up libraries of barcode sequences

for known species in order to develop reliable, molecular tools for species identification in nature.
} This

sequences stored in BOLD (Barcode of Life

Database)

DNA barcoding, standardization helps

Speed development of economical technologies for species identification . The goal is that anyone, anywhere, anytime be able to identify quickly and accurately the species of a specimen whatever its condition.

Software for DNA barcoding



Integration of FIMS (Field Information Management System) & LIMS (Laboratory Information Management System)

FISH-BOL: Current Status in India

} The

Fish Barcode of Life Initiative (FISH-BOL), is a

global effort to coordinate an assembly of a standardised reference sequence library for all fish species,
} Collected

3403 samples of 656 marine and fresh

water species.
} DNA

Barcoding of Fish initiated at NBFGR Lucknow

in July, 2005.

Barcoding Programmes in India

}

DNA Barcoding of Freshwater and Marine finfishes and shellfishes (ICAR-NBFGR, Lucknow).

} }

Barcoding Anurans of India (WII, CCMB & NoU) DNA Barcoding family Zingiberaceae: Alpinia, Zingiber & Globba (RGCB & UoC)

} } }

DNA Barcoding of Rattanas & Phyllanthus (UAS, BSI & ATREE) DNA Barcoding butterflies from Western Ghats (NCCS) DNA Barcoding Dendrobium (DU & TBGRI)

Application of barcoding
 

Identification of fish fillets fins and fragments. Identification of processed product e.g. caned fish dried fish mixture.

Identification

of

threatened

endangered

and

protected species (conservation).

 

Identification of fish eggs and larvae. Identification of pray items in stomach contents (food web and ecosystem research)

Cont
} Identification

of new species and possible fusions

insights into phylogenetic relationship. (Fish Biology Evolution)

} Possible

production of DNA microarrays from the

sequence data.
} Documentation } Identification

of range expansion

of historical archived and museum

material (taxonomy)

DNA barcode is not a..?

Supplant or invalidate existing taxonomic practice.

It is not DNA taxonomy; it does not equate species identity, formally or informally, with a particular DNA sequence.

It is not intended to duplicate or compete with efforts to resolve deep phylogeny.

Conclusion
barcoding is not a substitute to taxonomy } Reliable , cost effective, quick method to identify a species . } Can help in conservation and management of biodiversity } Can identify organism where traditional method fails
} DNA

CHROMOSOME BANDING TECHNIQUES AND GENOTOXICITY ASSAY

Chromosome preparation
 Chemicals required: I. Colchicine II. Potassium chloride(hypotonic solution) III. Methanol IV. Acetic acid V. Giemsa stain VI. Glycerol VII. Di-sodium hydrogen orthophosphate VIII. Potassium Di-hydrogen orthophosphate IX. DPX mountant X. xylene

Protocol
1. 2.

3. 4. 5. 6.

Inject with 0.05% colchicine @ 1ml/100gm of body wt. & keep it for 1-2 hrs Sacrifice the fish & collect the kidney/gill tissue, cut into small pieces and homogenize by using 6-8 ml KCl or any hypotonic solution Incubate it for 20 min. at rt. Add freshly prepared karnoys fixative. Centrifuge at 1200rpm for 15 min. Decant supernatant and add 6-8ml fixative

Cont
} Keep

it for 2-4 hrs at refrigerated temp. } Mix it well and centrifuged at 1200rpm for 10 min. } Decant supernatant and add fixative } Drop solution on slide from the height for bursting the chromosome & allow it for drying } Keep it for aging for 2-3 days. } Stain with giemsa stain in phosphate buffer for 15-20 min.

Cont

Observe metaphase under Bright field microscope Make the slide permanent by using DPX

G BANDING
o o

Chromosomes are G-banded to facilitate the identification of structural abnormalities. It is useful for identifying various genetic diseases through the photographic representation of the entire chromosome complement. Banding can be used to identify chromosomal abnormalities, such as translocations, because there is a unique pattern of light and dark bands for each chromosome.

COMET ASSAY
} Comet

assay is the method used for detection of DNA breakage & based on the migration of denatured DNA through an electrophoretic field and developed by Rydbert & Johanson in 1978 } Used to detect the genotoxic effect of Arsenic, Cu, Cd, Chromium, Methyl mercury, H2O2, etc.in fish

Steps
Individual cells or nuclei are embedded in agarose Lysed to expose DNA Alkali treatment to denature/relax DNA Electrophoresis to separate DNA DNA is visualised by EtBr, Silver stain or fluorescent dye.

Cont
} Damaged

DNA containing strands breaks migrate farther in the gel than intact DNA creating an image resembling Comet fluorescent intensity in tail is related to the number of strand breaks present with undamaged DNA will appear as intact comet heads without tail after specified electrophoresis time.

} The

} Cells

Advantages of Comet assay

1. 2. 3. 4. 5. 6. 7. 8.

Sensitive Can detect SSB, DSB, etc Require small no. of cells/sample Flexible Easy for application Require less time for the study Rapid Economic

CellCulture
} Cell

culture is the process by which cells are grown under controlled conditions. Or Cultivation of cells in vitro at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as cell culture. } Cell culture was first successfully undertaken by Ross Harrison in 1907 } Roux in 1885 for the first time maintained embryonic chick cells in a cell culture

Applications of cell culture technology

} Model

systems for:- Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells } Toxicity testing and toxicity assay:- Study the effects of new drugs } Production of monoclonal antibodies } Cancer research:- Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells

Conti
} Virology

:- Cultivation of virus for vaccine production, also used to study there infectious cycle } Genetic Engineering:- Production of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles. } Gene therapy:- Cells having a functional gene can be replaced to cells which are having nonfunctional gene

Design and Equipment for the Cell Culture Laboratory

}

Equipment :Laminar flow cabinet(vertical),Incubator (BOD and CO2) Storage space for sterile equipment Sterilizer / Autoclave Refrigerators and freezers, Hot air oven, Inverted microscope, Centrifuge, Electronic Balance Hemocytometer, Water Bath Aspiration Pump, pH meter Osmometer, Magnetic stirrer Pipettes

Materials: 70% (v/v) ethanol for swabbing, Sterile scissors, forceps and probes, Sterile petri dishes, Phosphate buffered saline (PBS), Trypsin, cold sterilized in a 125 ml sterile erlenmeyer containing a magnetic stirring bar, Minimum Essential Medium / L-15 medium, Fetal Bovine Serum / Fetal Calf Serum,Culture Flasks

Laboratory Design
work should be conducted in a single use facility which, if at all possible, should be separated into an area reserved for handling newly received material (quarantine area) and an area for material which is known to be free of contaminants main tissue culture facility. } All new material should be handled as quarantine material until it has been shown to be free of contaminants such as bacteria, fungi and particularly mycoplasma.
} Ideally

Conti..
Care and Maintenance of Laboratory Areas  Safety Considerations I. Sterile Techniques II. Contamination Control


Protocol
Media and supplements:Leibovitzs-15 (L-15, Sigma chemicals, Sigma Aldrich,St. Louis, MO, USA) supplemented with 20% fetal bovineserum (FBS, Sigma chemicals). After the initiation of primary culture, fish serum was added at 1% final concentration


Explant Preparation
I. II.

Preparation of donor fish:- depurinate the fish & then sacrifice it after plunging in ice for 10-15 min. Decontamination:- The tissue of interest was aseptically picked up and washed three to five times with the antibiotic solution {penicillin (400 IU/ml) and streptomycin (400 g / ml) with an antifungal amphotericin B (10 g/ml)}.

Cont..

III.Dissection or Disagregation:} Two A.

B.

major methods for initiating a primary culture:Tissues were disaggregated into its component cells. This was achieved by enzymatic digestion using trypsin or collagenase supplemented with EDTA. The required tissue was picked up and cut in small pieces to prepare explants of 1 mm3 size and planted in culture dishes.

Culture of cells following seeding

}

The explants were seeded in 25 cm2 tissue culture flask and kept semi dry for a few minutes. The adherence of explants was accomplished by incubation with 0.5 mL of FBS at 28o C. After 8-10 hrs, the growth medium, L-15 (Leibovitz) was added gently. Fifty percent of the media was exchanged once in every 3 days. The dispersed cells adhere on the culture substrate and start to proliferate. Dead cells can not secrete substrate adhesion molecules and hence float. They were removed by subsequent medium exchange. The optimum pH and incubation temperature maintained were 7.4 and 22-280 C respectively for culture of fish cells. Except L-15, other media require a 5-10% CO2 in the culture medium in order to stabilize the pH at required level.

Subculture and maintenance

} When

the primary culture attains confluency, it is subcultured using a TPVG solution as detachment agents. Cells intolerant to trypsin can be scraped using a cell scraper or dispersed by rocking followed by gentle pipetting. Detached cells can then be distributed to 2 to 4 flasks containing fresh medium depending on the split ratio required.

} Maintenance

PROTEOMICS
The word "proteome" is a blend of "protein" and "genome", and was coined by Marc Wilkins in 1994.

&
is the large-scale study of proteins, particularly their structures and functions.

STEPS
I. II. III. IV. V. VI. VII. VIII. IX.

Sample preparation:- take care of salt contamination & check the purity of sample through SDS PAGE Rehydration:- peptide should get absorbed in gel IEF:- placing IPG strip on protean IEF cell(native protein run basis of separation is charge i.e its isoelectric point) SDS PAGE:- 2nd dimension run of protein on the basis of its mol. Wt. Gel image analysis Spot picking In gel trypsin digestion Mass spectrometry analysis Protein identification through BLAST

Open for discussion & Thank YOU