Marine Science and Marine Biotechnology

Dr. T. Citarasu
Associate Professor
Tele-fax: + 91-4652-253078
Mobile: +91-9994273822
Email: citarasu@gmail.com
citarasu@msuniv.ac.in
Website: www.msuniv.ac.in
www.msuniv.irins.org/profile/174278
CENTRE FOR MARINE SCIENCE AND TECHNOLOGY
MANONMANIAM SUNDARANAR UNIVERSITY, TIRUNELVELI

TRENDS IN MARINE SCIENCE AND
MARINE BIOTECHNOLOGY

OCEAN WORLD
71% of Earth’s surface and they are inter connected
The oceans are traditionally divided into 4 large basins
North and South Pacific (4028 m depth)
North and South Atlantic (3332 m depth)
Indian Ocean (3897 m depth) - 20% of water in the earth + Significant BD
Arctic Ocean (1117 m depth)
Oceans are subdivided into 150 Seas

Marine Science can be defined as the study of the ocean, its
ecosystems, and life forms; also called oceanography
Study which influences sustainable fisheries, economic growth, and
the environment
The four main branches of oceanography and grown several
centuries
The major branches are
Physical
Chemical
Geological
Biological

Physical Oceanography
Physical attributes including temperature-salinity structure, mixing,
surface waves, internal waves, surface tides, internal tides, and
currents. The topics includes
Seismic oceanography, a form of acoustic oceanography, in which
sound waves are used to study the physical properties and dynamics of
the ocean.
Ocean current: study the forces acting upon the water, including wind,
the Coriolis effect, breaking waves, cabbeling, and temperature and
salinity differences.
Ocean heat content (OHC) is a term for the energy absorbed by the
ocean

Chemical Oceanography
Understanding of seawater properties and its changes, ocean chemistry
focuses primarily on the geochemical cycles.
Marine microorganisms have a central place in the global carbon cycle
as they function as a biological pump, sequestering anthropogenic
carbon dioxide from the atmosphere in the deep ocean.
Ocean acidification describes the decrease in ocean pH that is caused
by anthropogenic carbon dioxide (CO2) emissions into the atmosphere

Many animals and some algae use carbonate ions to make calcium
carbonate shells and skeletons. Because ocean acidification decreases the
availability of carbonate ions, these organisms will have to work harder to
produce shells
Ocean acidification could make it harder for marine organisms to absorb
nitrogen, phosphorus, iron, and other elements essential for growth.

Geological Oceanography
Geological oceanography is the study of the geology of the ocean floor
including plate tectonics and paleoceanography.
Tides and energy coastal erosion and drifting of sediments, sea level
changes, depositional environments and marine deposits.
Also concerned with the occurrence of oil-traps and energy sources,
tectonic movements-underwater eruptions, mud volcanoes and impacts
of tsunamis.

Biological Oceanography
Ecology and biology of marine organisms in the context of the physical,
chemical and geological characteristics of their ocean environment.
Marine habitats: Intertidal and near shore, Estuaries, Reefs, open
Ocean, deep sea and trenches
Marine Life: Microscopic, Fungi, Plants and algae, Invertebrates and
Vertebrates
Others includes, Marine Ecosystem, Marine biology, Biotechnology,
taxonomy, conservation etc

EEZ (0.86 WC+ 0.56 EC = 2.02 mill. Sq.km) including Andaman & Nicobar
(Sea zone prescribed by the 1982 United Nations Convention on the Law of the
Sea over which a state has special rights regarding the exploration and use of
marine resources, including energy production from water and wind).
Pelagic ecosystem is the most productive & sensitive to environmental changes
Benthic ecosystem: Lesser productivity compare to pelagic
Estuarine ecosystem: Natural nurseries also help in large scale production of
fishery wealth- 2 million ha
Mangroves: Reduce coastal erosion, nursery grounds, fire wood -6740 sq.km.
Coral ecosystem: Shelter & nourishment to marine animals - 2273.8 sq. km.
Sea grass/ seaweed: Food medicine, feeding grounds for sea dugong
MARINE ECOSYSTEM IN INDIA

MARINE LIFE AND RESOURCES
Providing food, medicines, cosmetics, nutraceuticals & Tourism
Contribute to oxygen cycle & help to regulate the Earths’ climate
Shorelines are protected by marine life, some organisms even help
create new land
Marine Ecosystems: estuaries, coral reefs, marshes, lagoon, sandy
and rocky beaches, mangrove forests and sea grass beds

Defined as “ Application of scientific & engineering
principles to the processing of materials by marine
biological agents to prove goods and services”.
IMPORTANCE OF MARINE BIOTECHNOLGY

7516.6 km
News Courtesy :
Daily Thanthi 23.02.2014
Strengthen our marine biotechnology research…….
Indian Council of Agricultural Research (ICAR)
Department of Biotechnology (DBT)
Ministry of Earth Sciences (MOES)
Ministry of Environment and Forest (MOEFS)
Council of Scientific & Industrial Research (CSIR)

Untapped reservoir of biologically active compounds, which leads to the
development of new functional foods with vast diverse of novel
chemicals.

Marine Biodiversity
Defined as “Marine biodiversity therefore refers to the species
richness and abundance in the world's oceans and seas. It can also be
explored across levels of biological organization from genes, species,
functional types, habitats to ecosystems”
Biodiversity is important for
Capture and store energy
Produce organic material
Food & Medicine
Decompose organic material
Cycle water and nutrients
Recovery from Unpredictable
events
Help regulate climate and
atmospheric gases

Algae 624 sps.
Cnidarians 790 sps
Porifera 486 sps
Platyhelminthus 550 sps
Annelida 440 sps
Molluscans 3370 Sps
Crustaceans 2340 sps
Echinodermata 765 sps
Hemichordata 12 sps
Prochordata 116 sp
Pisces > 2500 sps
Amphibians 3 sps
Reptiles 26 sps
Marine birds
Mammals
BIODIVERSITY IN INDIAN COASTS

FISHERY BIODIVERSITY
India harbors >10% of global
fish biodiversity
Ranks 3rd in the world fish
production (5.78 bill. USD in
2017, 0.60 million t in 1950)
The fisheries sector
contributes 8.2 % GDP in 2018-
2019
The sector is growing at a rate
of over 10% (2% in agriculture
and animal husbandry)
Major contributors to foreign
exchange
Valuable protein, livelihood
and employment to millions of
people

Fisheries play an important role in the economic development of our
country
It is recognized as a powerful source of income and employment creation
Also seen as a good foreign exchange earner (11,34,948 met. tons)
Million tones
CAPTURE FISHERIES

THREATENING OF MARINE BIODIVERSITY

Natural Factors
Storms, cyclones, natural radiations,
volcanic lava, tsunami and freshwater
runoff are affect directly or indirectly
the marine biodiversity
Change the ocean temperature or pH
that leads to changes of nutrient
availability, biological productivity,
reproductive success, the timing of
biological processes (e.g., spawning),
biogeography, migrations, community
structure, predator-prey
relationships, and entire biomes.
Warming of both air and ocean
temperatures has resulted in the loss
of Arctic sea ice that affects the
marine mammals

POLLUTANTS TYPES &SOURCES
Oil pollutants
Persistent toxic substances (PTS)
Toxic heavy metals
Thermal pollutants
Radioactive pollutants
Nutrients and microbial contamination
Noise pollutants
Ship based threatening
Harmful algal blooms
Ocean mining
Plastics and microplastics
Ocean Acidification
Solid wastes
Coastal tourism
Ballast water
Natural calamities
Marine littering
Industrial effluents

PROBLEMS IN CAPTURE FISHERIES
Decline in catch rate due to overfishing and overdependence on
trawlers
Harmful methods and Negligent fishing – Dolphin, Turtle etc
Destructive fishing: Cyanide fishing; Dynamite fishing; long line
fishing; Ghost fishing; Purse- seine fishing and shark fining
Ecosystem destruction
Fish reduction
Rupture in food web

Attention to Genetic Diversity
Introduction of new species/strains can affect biodiversity via impacts
on the native gene pool.
New species/strains can hybridize with native stocks, and hence alter
the natural genetic architecture.
Loss of valuable genetic material such as locally adapted genes or gene
complexes or homogenization of previously structured populations via
flooding with exogenous genes.
Outcome of hybridisation between the Thai walking catfish, Clarias
macrocephalus and the African catfish C. gariepinus

CONSERVATION OF MARINE BIODIVERSITY

Role of Marine Biotechnology on Environmental Pollution
Bioremediation
“The act of adding materials to contaminated environments such as oil spill
sites, to cause an acceleration of the natural biodegradation process”.
Principles
Growth improvements of microbes by optimal nutrients/ oxygen
Reduce or neutralize the pollutants
Pycoremediation, Mycoremediation, complex strategies
Recombinant strains used for bioremediation
PET hydrolase recombinant for Polyethylene terephthalate (PET)
degradation

Antifouling
Anti-fouling paint or bottom paint is a specialized coating applied to
the hull of a ship or boat in order to slow the growth of organisms that attach
to the hull and can affect a vessel's performance and durability.
Silicone, Silicone epoxy
Siloxane
Polysiloxane
Wax
Features
Inhibitors of quorum sensing- sponge Luffariella variabilis
Neurotransmission blockers -2,5,6-tribromo-1-methtyl gramine- bryozoan
Inhibitors of adhesive production/release- Inhibit the adhesion of mussels
Enzyme inhibitors
Ion channel inhibitors- Mediterranean sponge Crambe crambe inhibits the
settlement of bryozoans

Fishing activities
Fisherman & Vessels- Follow rules
Fishing Ban period (April-May; June-
July)
Save Young ones, Populations &
Ecosystem
Endangered species
Sea Ranching
Involves introducing juveniles (either
hatchery-produced or wild-caught) into
the natural environment where they are
allowed to grow without containment
structures.

CULTURE FISHERIES (AQUACULTURE)
Lucrative food production industry
Fresh, brackish & Marine based culture
Increasing annual growth rate
Global sea food demand-70% in the next 33 years (7 fold)
Attractive Job opportunities Million tones

Molecular Taxonomy for Conservation
Many marine organisms are poorly
described and little is known
With new molecular and analytical
techniques we can advance our
knowledge of marine biodiversity at the
species level to understand how
marine biodiversity supports
ecosystem structure, dynamics and
resilience.
Cryopreservation is extremely helpful
method to conserve rare, endangered,
threatened species
DNA banking can be considered as a
means of complimentary method for
the conservation of marine threatened
species together with conventional ex
situ approaches in preserving
biodiversity.

Protect Marine Environments
Cleaning programme
• ICC (International Coastal Clean-up )
• 2,800 peoples
• 124 tones
• 122 miles
Public awareness
Set standards for effluent discharge
Minimal use of pesticides
Strict implementation of polluter
pays principal
Strict implementation of laws
pertaining to prevention & coastal
zone management before
establishing new industrial units on
the coast.

World Environmental Day- June 5

POLICIES AND ACTS FOR PROTECTION
Marine Protection, Research, and Sanctuaries Act (1972)
Ocean Dumping Ban Act (1991)
National Estuarine Research Reserve System (1972)
Marine Mammal Protection Act (MMPA) (1972)
Endangered Species Act (1973)
Fisheries Management and Conservation Act – 1976
Oceans Act of 2000
Estuaries and Clean Waters Act of 2000

MARINE BIO-PROSPECTING AND MARINE
NATURAL PRODUCTS

Need for Marine Bio-prospecting
Repeated search in terrestrials leading to known compounds
Marine ecosystems are unique and rich reservoir of biodiversity with an
enormous potential towards improving the quality of human life.
Marine organisms have highly developed defense system in order to
survive in the hostile conditions such as extreme temperatures, varied
pressures (low or high), low energy and lack of sunlight

Multi-OMICS methodologies: Genomics, Transcriptomics, Proteomics,
Metabolomics, Metagenomics and Meta transcriptomics
Production and analysis of massive biological data
Once, the compound of interest is identified, bioactivity screening,
heterologous expression, genetic engineering and/or chemical synthesis
can follow
Characterize the activity and produce the desired bioproducts with
pharmaceutical, nutraceutical, cosmeceutical and biofuel production
applications.

Defined as “Searching for bio molecules from marine sources and the
search for new and unique bioactive components with potential
commercial applications”
Technical details & drug development includes…
Identification
Screening
Efficacy
Safety testing
Clinical trials
Large scale commercial
production

The soft bodied and unmoved organisms
are under threat with predators &
environmental factors. They escape from
the predators they have to synthesize
molecules with diverse of structures with
potent biological activities.
Even though marine bio prospecting
started the past 50 years unfortunately
only few products available in the market
and most of them under clinical trials.
Most of them treated cancers,
inflammations and antimicrobials.
Difficulties in synthesis. Structures of
some compounds are extremely
complex, the synthesis, in many case, is
not feasible.

Secondary metabolites
Produced after active growth has taken place & which perform no
vital function for the producing organisms
Interesting array of diverse and novel chemical structures with High
bioactivity potential
Heart of commercial venture & turn over - more than billion 100 $ / yr
Non Ribosomal Peptide Synthetase (NRPS) & Polyketides (PKS)
Peptide secondary metabolites produced by bacteria & Fungi
NRPS / PKS system involved diverse of bimolecular secretion
Independent of mRNA & Broad biological activities

MICROBIAL SOURCES
Microbial Metabolites
Microbes from marine sources have a
rich potential of antimicrobial active
principles for protecting themselves
from their predators
Due to the rich potential bioactive
metabolites in the marine microbes, it
may be used as drugs directly or
used as lead structures for drug
discovery
Nowadays the antibiotic resistance
and problems increased and
alternative discovery needed

Associated microbes
Secondary metabolites produced with the association between
Sponges and bacteria
Obligatory mutualism (symbionts play an essential role in the
metabolism of their host), facultative mutualism (beneficial effect on
their host, but the host will survive without the symbiont) or
commensalism (they are present without providing obvious beneficial
effects to their host).
More than 30 different phyla of Bacteria and Archaea as being
associated with sponges.
However, less than 1% of these microorganisms can be cultivated in
laboratory conditions.
Bioactive metabolites originally isolated from sponges, were in fact
synthesized or transformed by bacterial strains.

Table. Sponge associated bacterial compounds and its bioactivities
Compounds Activity Bacterial Sources Association
2-undecyl-4-
quinolone
Antimalarial
& anti-HIV
Pseudomonas sp. 1537-E7. Homophymia sp
3,6-
diisopropylpiperazin
-2,5-dione
Antimicrobial Pseudomonas sp. NJ6-3-1 Hymeniacidon perlevis
Andrimid Antibacterial Vibrio sp. M22-1 Hyatella sp.
2,4,4’-trichloro-2’-
hydroxy-
diphenylether
Antibacterial Micrococcus luteus
R-1588-10
Xestospongia sp.
Cyclo-(l-pro-l-phe) Antibacterial Alcaligenes faecalis A72 Stelletta tenuis
Iturin Antifungal Bacillus subtilis A202 Aplysina aerophoba
Manzamine A Antimalarial Micromonospora sp. Acanthostrongylophora
sp.
Surfactin, Iturin and
Fengycin
Antibacterial
antifungal
Bacillus subtilis A184 Aplysina aerophoba
Trisindoline Antibacterial Vibrio sp Hyrtios altum

Actinomycetes Origin
Actinomycetes are the intermediate group between bacteria and fungi having
high G+C (>55%) content in their DNA and abundantly present in the marine
sediment
Top most producers of antibiotics @ 70%
The important genera are Streptomyces sp, Nocardiopsis sp, Marinispora sp,
Salinispora sp and the culture- independent Dietzia sp and Rhodococcus sp
Major producers of antibacterial, antifungal, insecticidal, antitumor, anti-
inflammatory, anti-parasitic, antiviral, antifouling and anti-infective compounds

Important Secondary Metabolites
Surfactin A Synthetase C (SRFA-C)

Fungal Origin
Widely distributed in deep sea to polar ice
covers, occur in sediments, dead organic
matter, driftwood, algae, corals and
sponges etc.
Huge potential for new natural products
and an increased number of new
metabolites have become known over the
past years.
In hydrothermal sea ecosystem,
unsuspected high diversity of fungal
species identified with novel secondary
metabolites recently
Vast source of diverse antimicrobial
compounds mostly inhibit Gram negative
bacterial pathogens, HIV virus and fungus

Figure. Diversity of fungal genera obtained from the orange puffball
sponge,Tethya aurantium from the Mediterranean Sea with 29 identified genera
among 160 isolates

Antiviral compounds
Asperxanthone
Asperbiphenyl
Aspergillus sp MF-93
TMV
Anti-protozoan compounds
Varixanthone
Ascosalipyrrolidinone A
Aigialomycin D

Invertebrate Resources
Group of animals have no backbone
In halobiotic environment, they have
special adaptations, metabolic
activities, secretions helps to produce
diverse of bioactive substances,
protecting from harsh environment.
Among the invertebrates poriferans,
cnidarians, annelids, arthropods,
molluscs and echinoderms have
attracted attention due to their
antiviral, antimicrobial, antiprotozoal,
antifungal, antihelminthic and
anticancer activities

PORIFERA
Sponges
Most primitive metazoans (700-800 mill. yrs)
which appeared in the Cambrian period
Widely found in coastal platform to deep
waters
Sessile filter feeders with efficient defense
mechanisms against foreign attackers such
as viruses, bacteria or eukaryotic organisms.
Considered as gold mine during the past 50
years (> 10000 compounds)
Occasionally develop symbiotic relationship
with algae and microbes

Sponge derived Compounds
sesterpene
Fascaplysinopsis sp
Antibacterial
Topsentin
Spongosporites ruetzleri
Antibacterial
Anti-inflammatory
Colon cancer
2-(2’,4’-dibromophenoxy)-4,6-
dibromophenol
Dysidea granulosa
MRSA
Cyclodepsipeptides
Mirabamides A-D
Siliquariaspongia mirabilis
HIV

COELENTERATES
Cnidarians are the richest natural sources of
prostaglandins
Jelly fish, sea nettle and sea anemones mostly
secreted toxins with mixture of enzymes and
polypeptides

Hydra, Jelly fish, coral & sea anemones
1. Pseudo pterosins
Pseudopterogorgia elisabethae
Anti-inflamatory
2. Eleutherobin
Octocoral Eleutherobia sp
Anti cancer
Ascidians derived Compounds
1. Apilidine
Mediterranian tunicate Aplidium albicans
anticancer
4. Ectein ascidin 743
Ecteinascidia turbinate
Anti cancer

ANNELIDS
Of the approximately 9000 species
of annelids, more than 8000 are
polychaetes including lugworms, clam
worms, bristleworms, fire worms, palolo
worms, sea mice, featherduster worms, etc
They are found in most marine
environments from tidal zones to
hydrothermal vents
They has been found to treat several
pathophysiological conditions such as
arthritis, osteoporosis, bone cancer etc.

Arthritis
Oesteoporesis
Bone cancer
Antimicrobial
Antibacterial
Antifungal
Arenecins Hedistins
Peptides
AMP
Arenicola marina Perinereis aibuhitensis
Perinereis cultrifera

ARTHROPODS
They having an exoskeleton (external skeleton), a segmented body, and
jointed appendages (paired appendages).
Several bioactive compounds have also been isolated from marine
arthropods including horseshoe crab, crab, krill and shrimps

Krill Oil and Enzyme (Euphausia superba)
Tiny shrimp @ Whale food
Omega 3s, especially EPA and DHA
Heart disease, reduce high cholesterol, high
blood pressure, stroke, cancer,
osteoarthritis, depression

Chitin, Chitoson & Oilgomers
Shell waste with biomedical
applications
Chitin a high molecular wt polymer
N-acetyl-D-glucosamine
Chitoson-heteropolymer
Reducing LDL
Antioxidants & anticancer
Immunostimulants
DNA Vaccine delivery
Biopesticide – Agricultural
Shell waste
Wash
deprotenize- 3 % NaoH
Alkali removal by water wash
Demineralization-1.25 N Hcl
Wash
Dry and package

Lectin
Triglycerides Limulin
Limulin
Carcinoscorpin
Thiol ester protein
Fatty acids
Triglycerides
Carotenoids
Lipids
Antibacterial
Anti cancer
Antioxidant
Antiproliferative
Antimutagenic
Antiinflamatory
immune response

MOLLUSCA
Largest marine phylum, comprising about 23% of all the named
marine organisms.
Snails, octopuses, squid, clams, scallops, oysters, and chitons.
These creatures have been important to humans throughout history as a
source of food, jewelry, tools, and even pets.

Molluscan derived Compounds
1. Dolastin (Sea slug)
Dolastin 10 &15
Dollabella auricularia
Anticancer activity
2. Kahalaide F
Sea slug Elysia rufescens
Induce apoptosis
3. Spisulosine
Arctic surf clam Spisula polynyma
Anti proliferative & Anti cancer
4. Cadalmin Green mussel extract
Perna viridis –leukotrenes
Arthritis; Anti-inflamatory
Known wt of tissue + water or
methanol
Chop & homoginize/ sonicate
Centrifuge
Collect supernatant
Further purification need
Assay

commercially important marine
“archeogastropod” mollusk
Rich nutritional value, superior
taste, and various other benefits to
human health
Ginseng in the ocean- 25 vitamins
and minerals and is particularly
rich in selenium, magnesium and
B12
Abalone is favoured for its high
nutrient content that has
traditionally been used to support
eye health
Abalone

ECHINODERMATA
The phylum contains about 7000 living species including sea stars, sea
urchins, sand dollars, and sea cucumbers, as well as the sea lilies
Found at every ocean depth, from the intertidal zone to the abyssal zone
The bioactive compounds mainly are saponins

Saponins
Sterol derivatives
Arpenoids
Glycoproteins
Cerebrosides
pyrimidine nucleaosides
Thymine deoxyriboside
Uracil deoxyribose
Polysaccharides
β- carotene
Hemolytic
Antibacterial
Antifungal
Antineoplastic
Antitumor
Antiviral
Anti HIV
Antiinflamatory
anti cancer
anti allergic
Holothuria fuscocinerea
Toxopneustes pileolus Lytechinus variegates Strongylocentrotus droebachiensis

FISH AND OTHER VERTEBRATES
Metabolites extracted from fish, sea
snakes and aquatic mammals are
scanty.
Fish oil, rich in omega-3 fatty acids,
useful for various remedies including
arthritis etc.
500 species of fish are considered
toxic (Tetradotoxin (TTX))
Squalamines, a water soluble
antibiotics from dogfish shark,
Squalus acanthias
An anticancerous drug, namely “Fu-
anntai”, from sea snake had anti-
cancerous effects

Omega-3 fatty acids eicosapentaenoic
acid (EPA) and docosahexaenoic
acid (DHA)
Safety and potential nutritional and
therapeutic effects.
Heart health, Liver tonic, antioxidant &
cure cancers
Good for health, Good for Eye
Anti stress
Rich Vitamins
Fish Oil (Cod Liver oil)
Fresh Livers without Gall bladder
and veins
Wash & Chop
Steam cooking (70° C – 20 min)
Remove the floating oil
Filter & Centrifuge
Keep overnight with NaSo4
Filter & store
Thermal rupture to release oils

Steroids for fast growing !!!!! XXX

Shark Fin Cartilages and Rays
Caudal, Dorsal & pectoral fins
Known wt of dried fins
Wash
cooking with acidified water
until hydrolyze to gelatin
Remove adhering muscle
Dry in artificial dryer
Grade and pack
Shark Fin soup
Boost sexual potency
Enhance skin quality
Prevent heart disease, lower
cholesterol
Beneficial to lung & kidney
Shark Bone cartilage
Prevent variety of illness
Angiogenesis
Prevent Cancer

Fish Gelatin
Epinephelus sp, Rastrelliger sp etc
Elasticity, consistency & stability of
the products
Acid extraction
Washed skin soaked with 0.2 %
NaOH
Wash
0.2 % H2So4 + 1% citric acid
Coldwater wash
DH2o incubation @ 45° C-18hr
Filter
Dry

Fish Bone
60-70 % of inorganic substances & rich in
calcium
Essential elements for human health
Solve the problems related to bone
Collagen
Structural protein in connective tissue
Cosmetic, biomedical, pharmaceutical,
leather and film industries
Alkaline and acid extraction
Fish Bone and Collagen

ALGAL NATURAL PRODUCTS
Algae have a tremendous impact on the
sustainability of the marine ecosystem
as being the primary producers and,
therefore, a food source for other marine
organisms.
microalgae synthesize bioactive
molecules such as pigments and lipids
that exhibit health properties.
Types of Algae
Seaweed: Macro; benthic & muticellular
algae
Microalgae: Diverse group of unicellular;
occupy fresh, marine & extreme
environments

FATTY ACIDS AND HIGH VALUE OILS
Very Long chain PUFA (vlc PUFA)
Eicosapentaenoic (EPA)
Docosahexaenoic acid (DHA)
Arachidonic acid (AA)
Flexibility, fluidity and selective permeability
to cellular membranes
Brian development
Beneficial for the cardiovascular system
Nutraceutical in human and animal health

Antibiotics (fatty acids, bromophenols, tannins, terpenoids,
polysaccharides, alcohols)
Phycobiliproteins (Phycocyanin & phycoerythrin)–Natural dye for
cosmetics (lipsticks & eyeliners)
Algicides –toxin (neurotoxic and hepatotoxic)
Vitamin and vitamin precursors (ascorbic acid, riboflavin and
tocopherol)
Chlorella and Spirulina are used in skin care, sun protection and
hair care products

Carotenoids- Powerful antioxidants
Beta carotene
Algotene
Axtaxanthin
Dunaliella salina

ANTIBACTERIAL COMPOUNDS
C-lactone malyngolide
(Lyngbya majuscula)
ANTIFUNGAL COMPOUNDS
Majuscuiamide (cyclic depsipeptide)
Gambieric acid
(Gambierdiscus toxicus –Dinoflagellate)
IMMUNOSUPPRESSIVE COMPOUNDS
Lipoproteins, microcolins A and B
ANTICANCER COMPOUNDS
Curacin A – anticancer (antimitotic
agent)
Cytotoxic pentapeptides:
majusculamide D
Deoxymajusuculamide D
Amphidinolide B
(dinoflagellate, Amphidinum sp.)
ANTIMICROBIAL & ANTICANCER COMPOUNDS

ALGAL SMALL MOLECULES
Group of secondary metabolites synthesized by biosynthetic gene
clusters (Enzyme systems producing products such as antibiotics)
Cyclic peptide (microcystin), depsipeptide ( majusculamide)
Lyngbyatoxin-A ( Lyngbya majuscule) – antifungal activity
Cryptophycin from Nostoc sp. shows a fungicidal activity
&microtubule depolymerizing agent for Cancer cells.
Heterocyclic compounds, polycyclic ethers, oxygenated polyketides
and macrolides from dinoflagellates
Dinoflagellate toxins like saxitoxin , neosaxitoxin and gonyautoxins
are sodium channel blockers

ALGAL BIOSURFACTANTS
Extracellular Amphiphilic compounds
Glycolipids, lipopeptides, fatty acids, polymeric surfactants &
particulate natures
Broad range of applications
Antibacterial, antiviral, antifungal, anti-adhesive, anti-cancer,
antioxidants, adjuvant etc
Arthrospira sp., Synechococcus nidulans, Chlorella minutissima, C.
vulgaris and C. homosphaera used for various industrial and
pharmaceutical applications

ALGAL BIO POLYMERS AND BIOPLASTICS
Typically, long chain polymers, present in the algae lipids are used for
making bioplastics
Algae is attractive for bioplastics because of its high growth rate and high
protein content.
The plastics market is worth more than $400 billion and has grown at an
average of 3.5% per year over the last two decades.
Naturally occurring polymer have many applications in medical,
pharmaceutical and food industries
Biodegradable Poly hydroxy alkanoates (PHA) are promising biopolymer
which can be alternative to petroleum based polymer
PHB poly-(hydroxy butyrate) is the most abundant PHA which is homopolymer
of hydroxybutyrate that presents in various cyanobacteria such as Chlorogloea
fritschii, Spirulina sp., Aphanothece sp. & Gloeothece sp.

ALGAL OIL PRODUCTION
Storing the Sun’s Energy
(Photosynthesis)
What is needed Storage of Energy
Sunlight Lipids and oils
CO2 Carbohydrates
Nutrients

How to produce more oils ?
The lipid content of an algae species can be increased by imposing a stress
factor, such as starving of nitrogen or silicon.
In this manner, lipid fractions up to 70-85% have been reported for nitrogen
starvation and 60% for silicon starvation

Transesterification
A reaction between an ester of one
alcohol and a second alcohol to
form an ester of the second alcohol
and an alcohol from the original
ester, as that of methyl acetate and
ethyl alcohol to form ethyl acetate
and methyl alcohol
Methods for produce biodiesel

Advantages
Rapid and faster growth rates than terrestrial crops
The per unit area yield of oil from algae is estimated to be from between
5,000 to 20,000 gallons (18,927 to 75,708 litres) per acre, per year; this is 7 to
31 times greater than the next best crop, palm oil (635 gallons or 2,404 litres)
Certain species of algae can be harvested daily
Algal biofuel contains no sulphur, non-toxic and highly bio-degradable

MICRO ALGAE IN SPACE RESEARCH
Algae produce oxygen in space
Chlorella and Euglena grown in
photobioreactor is used to produce oxygen
in the space for the Astronauts using their
waste and help them for long term
expedition in space
NASA and many other space research
station in the world open opportunities for
planktologist

SEAWEED PRODUCTS
Bioactivity Seaweeds Compounds
Antioxidant Cymopolia barbata Cymopol
Avrainvillea longicaulis Avrainvilleol
Halimeda opuntia Polyphenols
Undaria pinnatifida Fucoxanthin
Anti-inflammatory Laureafincia claviformis Pacifenol
Stypopodium flabelliforme Styfotriol
Undaria pinnatifida Fucoidon
Anticancer Gracilaria corticata Polysaccharides
Gracilaria tenuistipitata
Plocamium telfairiae
Capsosiphon fulvescens
Sargassum oligocystum

Alginate
polyuronic saccharaide
Laminaria and Fucus
D- mannuronic and L-galuronic acid
Emulsifier in food industries
Fresh seaweed washed & Dried
Pulverize to get powder
Treat with 1N HCl (pH 2-3)-ON
Wash with water & add Na2Co3
Filter & bleach with 2.5 % NaClo
Evaporate @ 60° C
Pure sodium alginate

Carrageenan
Isolated from sea weeds
Sulfated polysaccharides
Powerful antioxidants
Broad range of applications in
food and cosmetics

Fucoidon (Foo–Koy–Den)
Sulfonated polysaccharides
Antioxidants & immunostimulants
Brown sea weeds
Blood purifier
Reduced Cholesterol
Fight off viral infections
Scavenge free radicals
Liver tonic
Anticancer

Ulvan
Composed of rhamnose,
glucuronic acid, iduronic acid,
xylose and sulfate
Green seaweed, Ulva
Good gelling agent
Prevent biofilm formation
Good Nutraceuticals
Antioxidants
Anti-thrombotic activities
Treatment of gastric ulcers
Anticancer
Immunostimulants

Laminarian
Water-soluble polysaccharide
(1,3)-β-D-glucan with β (1,6) branching
and contains 20–25 glucose units
Regulator of intestinal metabolism
Tumor-inhibiting agent
Antibacterial agent
Protection against severe irradiation
Boosting the immune system
Reducing cholesterol levels in serum

TOP TEN MEDICAL APPLICATIONS
Prevents chances of stroke: Bioactive
peptides from seaweeds possess quite the same
properties as many ACE inhibitor medications.
Also helps to reduce cardiac attacks and cerebral
strokes.
Detoxifier: Alginic acid serves as a detoxifying
agent that binds up with the available toxins in the
bloodstream so that they are eliminated faster.
Remove Pb & Cd by pollution and smoking .
Rich in Vitamin B: Green seaweeds contain
many enzymes, amino acids, minerals and
Vitamin B. Chlorophyll helps to long term stamina
and over health.

Rich source of Iodine: Daily requirement ranged
between 100-200 mcg, seaweeds are right for your
health
Inflammatory diseases: Recovery people who
are suffering from cancer. Japanese consume
seaweed daily, as part of their regular diet, they are
thought to have a much lower rate of breast cancer.
Reproductive benefits: Rich in Manganese and
Vitamin B2 which contribute to sexual wellness, by
regulating the sexual hormones. Improves sperm
count & relive Pre Menstrual Syndrome and
menopausal symptoms. Helps to lengthening the
menstrual cycle, delaying menopause relieving
endometriosis as well as improving fertility.

Dental health: Preventing gingivitis & formation
of cavities enhance salivation and resists oral
tissue damage. Alginate used to make the molds
of false teeth, derives from the seaweed based
alginic acid.
Enhances skin quality: Antioxidants, vitamins
and minerals helps to relieving the aging of skin;
relieving dry skin and to straighten out wrinkles.
Energy Booster: Soups as tonics for curing
hang over, respiratory ailments, colds & fever.
Strengthen hair: Seaweed based shampoos
helps to stimulate the re-growth & quality of hairs

Cadalmin Green Algal extract
Cadalmin Green Algal extract contains a
unique blend of 100% natural marine
bioactive anti-inflammatory ingredients
extracted from selected seaweeds or marine
macro algae with a patented ecofriendly
“green” technology .
The product is effective to combat arthritic
pain and inflammatory diseases in human
beings
Patients suffering with chronic joint pain and
arthritis clinical trial-based studies revealed
that more than 98% of the respondents were
satisfied with the product with about 70-85%
relief in joint pain and arthritis

Communities which can grow at extreme environments
called extremophiles.
Some are subject to grow multiple stress conditions
Great economic value with biotechnological applications

Types of Extremophiles
Psychrophiles
-15 °C or lower for extended periods
cold soils, permafrost, polar ice, cold ocean water
etc
Polaromonas vacuolata
Thermophiles
Heat- loving microbes -115o C
Deep sea vent. Methanopyrus kandleri
Hyperthermophile
Temperatures between 80–122 °C.
Thermo acidophile
Combination of thermophile and acidophile that
prefer temperatures of 70–80 °C and pH between 2
and 3

Acidiphiles
Sulfuric spring; Sulfolobus acidocaldarius
An organism with optimal growth at pH levels
of 3 or below
Alkaliphile
Source: Soda lake
Eg: Natronobacterium gregoryi
An organism with optimal growth at pH levels
of 9 or above

Halophiles – Salt loving microbes
Source: Salt lake
Eg. Haloferax volcanii
An organism requiring at least 0.2 M
concentrations of salt (NaCl) for growth
Solar salt works diverse of halophilic
bacteria and Actinomycetes

Active hydrothermal vents in the deep sea are chemosynthetic
ecosystems that host endemic and extraordinary organisms adapted to
life in one of the most chemically and physically extreme environments
on our planet
Hydrothermal vents are also put forward as examples of Vulnerable
Marine Ecosystem (VME)
Because vents are highly valued, there are well-known efforts to protect
them, including but not limited to the establishment of the Endeavour
Hydrothermal Vents Marine Protected Area (MPA) in 2003
Hydrothermal Vent

Applications
Molecular Biology
Taq DNA Polymerase- Thermus aquaticus
Thermostable restriction enzymes–Pyrococcus
sp-pspGIR and pspGIM
DNA and protein stabilizers - Compatible
Solutes
Osmoprotectants
Di-myo-inositol-phosphate
Di-mannosyl-di-myo-inositol-phosphate
Di-glycerol-phosphate
Mannosylglycerate
Mannosylglyceramide-

Agro, Food Industry & Environment
Thermophilic amylases and cellulases: Bakery,
glucose and fructose production
Dietary supplements, pigments -α-Linoleic acid, β-
carotene and other pigments from halophiles
Freezing protection : Antifreeze proteins from
psychrophiles
Oil recuperation - Rheologic polymers,
thermostable and halophilic enzymes that degrade
guar
Cleaning of oil spilling - Psychrophiles which
degrade oil
Phytases and phosphatases – Food application
Epoxide hydrolases- Metagenome for degradation
Xenobiotic degrading enzyme

Chemical Industry
Detergents -Lipases, pullulanases, amylases and
proteases
Thermophilic keratinases and chitinases: Wool,
feathers, chitin conversion into feed
Pharmacological Industry
Biosurfactants
Glycolipids, lipopeptides, fatty acids, polymeric
surfactants & particulate natures
Bacteriocin
Ptoteinaceous nature had antibiotic activity (Type-I,
Type II)
Liposome - Phospholipids
Vaccine Delivery
Polyhydroxybutyrate- bioplastic

IMPORTANCE OF AQUACULTURE
Lucrative food production industry
Fresh, brackish & Marine based culture
10 % annual growth rate
Global sea food demand-70% in the next
35 years (7 fold)
Capture and culture fisheries

Rearing/culture and management of aquatic organisms
Fishes
Shrimps
Seaweeds
Microalgae
Live feeds
Crabs & Lobsters
Sea cucumbers
Oysters
Mussels

SEA FARMING
Generally this marine fish farming is of 2 types
1. Setting up cages in the sea and raising fish in them. The fish is stuffed
into a cage or basket, which allows the water to move freely.
2. Coastal aquaculture (i.e. fish and shrimp farming by setting up ponds on
the beach). Cobia, Sea bass and Pompano are the most suitable species of
fin fishes.
These fish have good market potential with high cost, soft and tasty meat.
Global seafood demand will increase by 70% (7 times) in the next 35 years.

FIN FISHES IN OPEN SEA CAGE CULTURE
In the area of marine fish culture, the country is still in the experimental
phase only.
Mullets (Mugil cephalus, Liza macrolepis, V. seheli)
Groupers (Epinephelus tauvina)
Seabass (Lates calcarifer)
Milkfish (Chanos chanos)
Pearlspot (Etroplus suratensis)
Cobia (Rachycentron canadum)

Lucrative business and includes marine
and freshwater ornamental fish culture.
Coral reefs in the Indian Ocean are
home to a wide variety of ornamental
fish. Gulf of Mannar, the Palk Bay, Gulf
of Kutch, the southwest coast and
Lakshadweep and the Andaman
Islands.
We have properly utilize this resources,
our foreign exchange will increase.
Clown Fish, Damsel Fish, Moorish Idol,
Lion Fish, Parrot Fishes, Box Fishes or
Trunk Fishes, Marine Angels, Butterfly
Fish, Cleaner Wrasse, Cardinal Fishes,
Sergeant Fishes/ Unicon Fish, Rabbit
Fish, Squirrel Fish, Scorpion Fish,
Blennies and Sand-smelt Fish etc.
You can earn Rs 1 lakh per month by
investing around Rs 1 lakh in this small
business.
ORNAMENTAL FISH CULTURE

LOBSTER FATTENING
They are more profitable than shrimp and fishes & good international
market value
A good hatchery technique has not yet been standardized for lobsters
So the fishermen catch small lobsters and culture them in small cages on
the rocky seas is called as lobster fattening
It feeds on mollusks and trash fishes
About 700 tons of lobsters are harvested each year through fattening
techniques

CRAB CULTURE AND FATTENING
Tamil Nadu is one of the leading states in India with high commercial value
of shrimp and crab resources
Like lobsters, mud crabs are culturing in cages and back waters.
It has a good value for money in the international market, just like the
lobsters and its culture technology is not yet up to standardized
Live crabs are high market value

OCTOPUS CULTURE
Octopus live short lives, growing rapidly
and maturing early
Strong market demands in the
Mediterranean and in South American and
Asian countries
Octopus maya, O. bimaculoides, O.
ocellatus and O. mimus
O. vulgaris, appears strong market
potential & distributed in tropical,
subtropical and temperate waters.
Rapid growth rate of 5% body weight per
day & high FCR 30–60%
High fecundity of 100,000–500,000 eggs
per female

MUSSEL CULTURE
Mussels, Oysters and clams are important
molluscans for food.
In India, mussels are abundant on our
shores from September to January
exposed shores in the intertidal zone
In India: green mussel (Perna viridis) &
mussel (Perna indica) are found in rocky
coastal areas.
It is more profitable to grow mussels in
the raft system (in the seas), in the rack
system (in the backwaters) and in the long
line ropes.
Its meat is very tasty & oil extracted from
mussels cures arthritis.

OYSTER AND CLAM CULTURE
Indian species Crassostrea
madrasensis (Indian backwater
oyster), C. gryphoides, C. rivularis
and Saccostrea cucullata
Production of oysters in India
started during the late nineties and
increased to 2400 tonnes in 2008.
Experiments were conducted in
India to farm clam species and
results have indicated feasibility of
farming them in pen and on bottom
methods
However, there is no commercial
culture of clams in India, not
cultured
Metetrix casta, M. meretrix, Paphia
malabarica, Anadara granosa and
Villohta cyprinoides

PEARL CULTURE
A pearl is formed when the mantle tissue is injured by a parasite, an
attack of a fish or another event that damages the external fragile
rim of the shell of a mollusk shell bivalve or gastropod.
In response, the mantle tissue of the mollusk secretes nacre into the
pearl sac, a cyst that forms during the healing process
Chemically speaking, this is calcium carbonate and a fibrous protein
called conchiolin
Pinctada and Pteria species are also used for producing cultured
pearls today.

In India, pearl oysters are found in
the Gulf of Mannar and the Palk Sea,
of which six species are found in
Tamil Nadu.
Thoothukudi beach in Tamil Nadu is
very suitable for pearl oyster
farming.
This agricultural technology can be
done by raft, rack and long row
culture.
Natural pearl oysters harvested from
the Gulf of Mannar are of high
quality.
It costs about 4.5 lakhs to implement
this oyster cultivation by sowing
about 8 to 10,000 seeds in half an
acre to three quarters of an acre
After the first 1.5 years, the net profit
can be around Rs 7.5 lakhs.

SEAWEED FARMING
Seaweed farming or kelp farming is the
practice of cultivating and harvesting
seaweed.
The main food species grown by
aquaculture in Japan, China and Korea
include Gelidium, Pterocladia, Porphyra
and Laminaria
Seaweed farming has frequently been
developed as an alternative to improve
economic conditions and to reduce
fishing pressure and overexploited
fisheries
Seaweeds have been harvested
throughout the world as a food source as
well as an export commodity for
production of agar and carrageenan
products
There are new long-line cultivation
methods that can be used in deeper water
approximately 7 meters in depth

They use floating cultivation lines
anchored to the bottom and are the
primary methods used in the villages
of North Sulawesi, Indonesia.
Species cultured by long-line include
those of the genera Saccharina,
Undaria, Kappaphycus, and Gracilaria
About 60 species of commercially
important sea weeds are found off the
coast of India
The Gulf of Mannar is very suitable for
sea weed farming
In south East Asian countries, it is
used as a good food, medicine, animal
and feed
On the beaches like Rameswaram and
Mandapam, women earn a few lakhs a
year through the women's self-help
group by growing algae called
Kappaphycus on bamboo rafts

SHRIMP FARMING
Asia is the largest producer- 80 %
India is the second place of Asia &
fifth place in globally
>350 shrimp hatcheries 13 billion seed
production/ annum
Top 5 Countries L. Met. tones
China 7.5
India 7.0
Vietnam 5.5
Thailand 4.5
Indonesia 1.5
Aquaculture production (2018)

CULTURE TYPES
Extensive
Semi- Intensive
Intensive
Super- Intensive
 No external diet
 Low stocking densities (0-5 m3)
 Yield: 500 kg/Ha/yr
 External diet
 Intermediate stocking (10-30 m3)
 Yield: 1-2 tonnes/Ha/yr
 Formulated complete feeds
 High density stocking (30-60 m3)
 Yield: above 2 tonnes/Ha/yr
 Closed system with raceway + aeration
 Very high density stocking (>60 m3)
 Yield: above 60 tonnes/Ha/crop

CURRENT PROBLEMS IN AQUACULTURE
Culture methods
• Extensive: 0-5 m3
• Moderate extensive : 5-15 m3
• Semi intensive : 15-30 m3
• Intensive : 30-60 m3
• Super Intensive : > 60 m3
Significant economic losses
Bacterial diseases - Vibriosis
Viral diseases – WSSV; MBV, MrNV
Stress Induced diseases

Important Shrimp Diseases in India
Vibriosis Early Mortality Syndrome
White Spot Syndrome Virus (WSSV) Enterocytozoon hepatopenaei (EHP)

CURRENT PROBLEMS IN DISEASE CONTROL
Current disease treatment
protocols – difficult
Chemicals & Synthetic drugs
Antibiotics & other chemicals
MPEDA abandoned more than
20 antibiotics

Chloramphenicol Metronidazole
Nitrofurans Ronidazole
Neomycin Ipronidazole
Nalidixic acid nitroimidazoles
Sulphamethoxazole Clenbuterol
Chlorpromazine Diethylstilbestrol
Colchicine Sulonamide
Dapsone Floroquinolones
Dimetridazole Glycopeptides
Banned Antibiotics (MPEDA)

BIOTECHNOLOGY TOOLS IN AQUATIC HEALTH

Tools can certainly revolutionize aquaculture
Genetic modification holds tremendous potential to improve the quality
and quantity of fish reared in aquaculture
Reduce the importation of foreign fish and aquaculture products and
increased foreign earnings
Tools derived from genomics can also improve our under-standing of how
aquatic organisms adapt and respond to their environments
Tools to enhanced production : Diagnostic tools; in silico approach;
Vaccine development; transgenic fish; Edible vaccine; Nanotechnology;
RNAi ; Gene editing and Phage therapy etc

BIOTECHNOLOGICAL TOOLS TO IMPROVE PRODUCTION

Herbal Medicines- Natures’ Gift
Storehouses of safer & cheaper
compounds
Low cost, effective, no side effects
& biodegradable
Various activities such as growth
promotion, immunostimulants, anti
stress, appetizing, aphrodisiac,
antibacterial, antiviral and antifungal
etc.
Diverse of active compounds
Ecliptine Tinosporone
Ocemine Aegilin

Immunostimulants
A substances that stimulate the immune system by inducing activation
or increasing activity of any of its components.
Improve immunological & haemotological parameters
Secretion of antioxidant enzymes & AMP
Resist or eliminate pathogens
CHITIN
LACTOFERIN
FUCOIDON
BETA GLUCAN
LPS
HERBALS

Herbal Immunostimulants
Phyllanthus niruri Leucus aspara Ocimum basilicum
Boost immune system
Suppress the multiplication of WSSV
Activate PO system and secretion of antioxidant enzymes
Psidium guajava
Phenol, 2,5- bis
(1,1-dimethylethyl)

MODE OF ACTION (Immunostimulation)
Possibility to activate the PRPs in SPC to activate proPO system and
immunity developed against pathogens
Immunostimulant molecules recognize PRPs (PPAE) & culminate
proteolytic cleavage of proPO to PO
Immunostimulant molecules interaction leading to PO activation
Melanin synthesis, ROI, RNI, Expression of immune genes& TLR
IMM
SPC
PRPs
PPAE
Activation
PO Activation
TLR Activation
AMP synthesis
& Phagocytosis
Immune gene Exp
& ROI, RNI

In silico Drug designing
COMPUTATIONAL DRUG SCREENING
Antiviral, immunostimulant, anti
apoptotic inhibitors and PO negative
regulator inhibitor compounds
-Terrestrial
- Marine origin – NRPS/PKS
- Solar salt works origin – NRPS/PKS
Ligand databases like PubChem
Compounds, Drug Bank, Zinc
Database etc.
3D structure of WSSV download from
PDB database or homology modeling
or threading method with I-TASSER
Active site prediction by PDBSUM
database

MOLECULAR DOCKING –Antiviral
Computational simulation of a candidate ligand binding to a
receptor and form a stable complex
Predict the suitable ligand compounds which bind the WSSV
proteins and form a complex.
The complex forming will help to arrest the multiplication of
WSSV

Inhibition of anti apoptotic protein
WSSV hijack protein AAP1 (Anti
Apoptotic Protein)
Designing drugs for inhibiting the
expression of AAP1
Help to up regulation of Caspase
expression & leading to
immunostimulation - q & semi qPCR
Inhibition of PO negative regulation
Proteinase inhibitors as negative
regulation for PO affect the
stimulation - SERPIN
By inhibit/ down regulate SERPIN PO
doesn't affect leading to good
immunostimulation
Expression of SERPIN by qRT PCR
SPC
PPAE
TLR
SERPIN
Ligand
PO positive

PREBIOTICS
Compounds in food that induce the
growth or activity of beneficial microbes
in gut, can alter the composition of
organisms in the gut microbiome.
Fructo oligosaccharides (FOS), Galacto
oligosaccharides (GOS), Inulin,
Arabinoxylo-oligosaccharides (AXOS),
short-chain Fructo oligosaccharides
(scFOS), and Mannan-oligosaccharides
(MOS)
Induce enzyme secretion, metabolic
activity, immunostimulation and fighting
against pathogens

Probiotics have been defined as “live microbial
food supplements which beneficially affect the
host by improving the intestinal microflora
balance”
Gut & Water & soil Probiotics
Competitive exclusion principle
Used in Shrimp farms and polluted areas
Improved strains of Probiotics
Lactobacillus, Enterococcus, Bacillus,
Aeromonas, Alteromonas, Arthrobacter,
Bifidobacterium, Clostridium, Paenibacillus,
Phaeobacter, Pseudoalteromonas,
Pseudomonas, Rhodosporidium,
Roseobacter and Streptomyces
PROBIOTICS

RECOMBINANT VACCINE DEVELOPMENT

Vaccine Generations
First Generation of vaccine
Live, attenuated and killed forms
killer
Cellular immune responses
Second Generation of vaccine
Subunit vaccines
T Helper cells immunity
Third Generation of vaccine
DNA vaccines
Humoral & cellular immunity
Strong & long lasting

Vaccines in Aquaculture
Whole Cell Vaccine
Bacterial Cells
Viral Particles
Inactivated forms
Virus- Grown in cell lines/ eggs
Demerit- revert
ECP Vaccine
ECP – haemolysin, virulent factors
Need adjuvant
Improved survival
Partial purified forms
52 kDa

SUBUNIT VACCINE
Proteins
Envelop / OMPs (37kDa)
Strong Immunity
Need adjuvant
Hematological & immunological improvement
BIO FILM VACCINE
Matrix of Protein, carbohydrate & DNA
90 % Bacteria secretes
Difficult to treat by antimicrobials
Modulate cytokine synthesis and
interrupt production of antibodies

Demerits of Conventional Vaccines
Gives positive effects and have some demerits
such as week and shorter immunity, reversion of
virulence, high cost, some times ineffective, heat
liable and need of high cost adjuvant etc.
Recombinant DNA vaccine technology is an
attractive alternative to traditional vaccines
because of certain advantages, which includes
straightforward design and construction, heat
stability, low production costs, long-term storage
capabilities and no risk of reversion
This approach can elicit very strong and long-
lasting immune responses, also offers economic
benefit, environmental and safety advantages,
which are particularly attractive for the farmers.

Immunization with a circular piece of
DNA that code for an antigen
Plasmids consist of strong viral
promoter (SV-40/CMV ) to drive the
in vivo transcription and translation
of gene interest.
Intron A may sometimes be included
to improve mRNA stability and hence
increase protein expression.
Plasmids also include a strong
polyadenylation/transcriptional
termination signal, such as bovine
growth hormone or rabbit beta-
globulin polyadenylation sequences.
Multicistronic vectors are sometimes
constructed to express more than
one immunogen, or to express an
immunogen and an
immunostimulatory protein
DNA VACCINE

Recombinant plasmids enter to the host cell
Gene of interest is transcribed by the RNA polymerase II &
synthesis of messenger RNA (mRNA)
Translated into the corresponding protein in the cytoplasm
of the host cells
T- Cell as well as B-Cell immunity will developed & produced
antibody against the proteins/ keep the mammary
When pathogen (antigen) enters to the host cell, the T-Cells
as well as B cells recognize and killed.
How DNA vaccine plasmids Stimulate immune responses?

Immune system Aquatic species
Shell Fishes
Innate immune system
No clear responses
No antibody production
Pathogen persists
Infectious to others
Humoral immunity
AMP, PO & Coagulation
Cellular immunity
Encap, nodule & pago..
Adaptive immune system
Specific responses
Antibody produced
Pathogen eliminated
May or may not
Humoral immunity
B cells
Cellular immunity
T cells
Fin Fishes

Fast production of proteins with
large quantities
Short generation times, as
bacteria grow and multiply
rapidly
The expressed proteins often do
not fold properly and so are
biologically inactive
The synthesized proteins are
often toxic to bacteria
Lack of enzymes responsible for
post-translational modifications
Subunit vaccines through Bacterial Expression

Subunit vaccines through Baculovirus Expression
Viral recombinant proteins from baculovirus infected cells - Fast production
of proteins with large quantities
Advantages including improved solubility, ability to incorporate post-
translational modifications, and higher yields for secreted proteins
90 % efficiency with 500 mg of protein per liter of culture
Proper protein folding & biologically active proteins
Eukaryotic posttranslational modification

Subunit Vaccines through Yeast Expression System
The galactose induction system in Saccharomyces cerevisiae
GAL1 promoter used to conditionally over express genes
Advantages: growth speed, easy genetic manipulation, low cost media,
post translational modifications & secretory expression
Saccharomyces, Pichia, Kluyveromyces, Hansenula and Yarrowia.

Micro algal Recombinant vaccines
Antigens expressed in the chloroplast or anchored to the surface of plasma
membrane
Safe and inexpensive to immunize fishes.
Foreign antigens can be expressed in the chloroplast or the cytoplasm with
high yields
Algae are a potential food source for larval fish (10 µm)
Chlamydomonas is innocuous, nontoxic and nonpathogenic.

EDIBLE ANTIBODY OR EDIBLE VACCINES

PRODUCTION OF YOLK ANTIBODY
Principle
When chickens are faced with a foreign virus
or bacterium, they produce antibodies to
fight the invader. They pass that immunity on
to their offspring, and antibodies wind up in
the eggs.
1. Chickens are more apt than mammals to make
high-avidity antibodies
2. A single chicken can produce an enormous
amount of antibody, upto 3 grams of IgY per
month, which is 10-20 times the amount of a
rabbit
3. chickens produce antibody much quicker-high-
titre antibody is available from eggs as early as
day 25
4. Storing of eggs (antibodies) is very easy
5. It is cheaper to feed and house chickens than
rabbits
6. Effective against drug-resistant bacteria.

IgY production & purification
Yolk
DDH2O Wash
Cut Open
Isoproponanl wash 1: 3 – 2 times
Acetone wash- 1 time
Fitered & store
Complete removal of
Lipids

Anti WSSV IgY (37 k Da)
M C IgY IgY- Adj

Vaccine Delivery Methods
Injection
Small volume of DNA vaccines is
enough
0.1 or 0.2 ml volume is enough
10 µg DNA is enough (5+ 5= 2 doses
or 10= 1 dose)
Disadvantage: Cant’ apply for small
fishes
Bacterial Transformants
DNA vaccine transformant (Bacterial
mode)

Through Diets
DNA and subunit vaccine transformants /
proteins will be coated to artificial fed
Bioencapsulation
Bioencapsulation may be a preferred method
of oral delivery (rotifers, brine shrimp)
Nano delivery
Conjugate with Chitoson / sodium alginate
complex
Subunit vaccines with nanoparticles,
Poly(lactic-co-glycolic acid) (PLGA)
Micro algal delivery
Recombibnant vaccine producing micro
algae

TRANSGENIC FISH
Transgenesis
Foreign DNA is introduced into the animal, using rDNA technology, then
transmitted through the germ line so that, the animal gets the same
modified genetic material
Organisms into which heterologous DNA (transgene) has been artificially
introduced and integrated in their genomes are called transgenics
A transgenic animal is one whose genome has been changed to carry genes
from other species.
The transgenic fish may change their change their characters

Need for Transgenic fish
Growth enhancement
Adaptation for environments
Increase disease resistance
Sexual maturation
Enhance nutritional quality
Improve food utilization
Transgenic manipulation of antimicrobial
peptide genes may lead to the production
of fish strains with elevated resistance to
bacterial, viral and protozoan pathogens

TRASGENIC TELEOSTS FOR EPA AND DHA PRODUCTION

Specific Pathogen Free (SPF)
Development of SPF doubling the production in Litopenaeus
vannamei in US aqua industry during 1990s
They cross bread between two traits
CPF and Oceana Institute develop the SPF by cross bread the
better characters such as salinity, low DO, pH and disease
tolerance traits and analyze the characters at 30 generation and
commercialization

RNA Interference (RNAi) against Viral disease
“A process in which the introduction of double-stranded
RNA into a cell inhibits the expression of genes”

Post Translational Gene Silencing
(PTGS) Pathway
ds RNA in the cytoplasm triggers the multi
domain ribonuclease II enzyme DICER which
cleaves the ds RNA in to si RNA which are 21
to 23 nucleotide fragments
These si RNA is recognized by the RNA
induced Silencing complex (RISC), a multi
enzyme unit that brings about separation of
two si RNA stands.
The antisense Si RNA stand remains bound to
RISC while the sense strand is released.
Finally the antisense and RISC complex bind
the target mRNA allowing the nuclease activity
and degraded the target gene

ROLE OF RNA INTERFERENCE IN AQUATIC DISEASE CONTROL
Crustaceans such as penaeid shrimp,
which can be infected by more than twenty
different viruses.
Bunyaviridae, Herpesviridae,
Picornaviridae, Parvoviridae, Reoviridae,
Rhabdoviridae, Togaviridae, Iridoviridae or
a new virus family, the Nimaviridae
Culture practices leading to stress induced
diseases, bacterial and viral infections
leading to severe economic losses
White spot syndrome virus (WSSV), Yellow
head virus (YHV), and Taura syndrome
virus (TSV)

Phage Therapy is the therapeutic use
of lytic bacteriophages to treat
pathogenic bacterial infections.
Bacteriophages, the viruses that
infect and kill their specific hosts,
have been reported to offer scope as
an alternative to antibiotics as
therapeutic agents in controlling
bacterial infections
The purpose of phage and antibiotic
therapy are same in controlling
bacterial infections but antibiotics
are banned in many countries due to
multidrug resistant strain
development.

Phage activity is very specific, attacking
only host bacterial cells without affecting
other (normal) micro flora
The capability of phages to not only target
and destroy a specific bacterium, but also
replicate exponentially, underscores their
potential role in treating infectious
diseases
Phages also have several advantages over
antibiotics: they are ecologically safe
(i.e.harmless to humans, plants and
animals), and phage preparations are
readily producible, and easy to apply.
In aquaculture phages effectively
controlled Aeromonas salmonicida and
Vibrio harveyi

CRISPR gene editing is a method by which
the genomes of living organisms may be
edited.
It is based on a simplified version of the
bacterial CRISPR/Cas (CRISPR-Cas9)
antiviral defense system. By delivering the
Cas9 nuclease complexed with a synthetic
guide RNA (gRNA) into a cell, the cell's
genome can be cut at a desired location,
allowing existing genes to be removed
and/or new ones added
Genome editing can rapidly introduce
favorable changes to the genome, such as
fixing alleles at existing trait loci, creating
de novo alleles, or introducing alleles from
other strains or species
CRISPR-Cas technology can also be used
to control the viral and bacterial diseases
particularly in shrimps and prawns

Applications in aquaculture
Combining in vivo and in vitro screening
approaches has the potential to identify functional
disease resistance alleles for disease resistance
Salmonidae, Cyprinidae, Siluridae , Pacific oyster,
Nile tilapia, and gilthead sea bream etc
Immunity and disease resistance have already been
investigated using genome editing in Rohu carp
and Grass carp
Can also be applied to develop models for studying
fundamental immunology, such as the targeted
disruption of the TLR22 gene in carp
improved cell lines for fish species, by enabling
more efficient production of viruses for future
vaccine development by knocking out key
components of the interferon pathway

Applications in shrimp aquaculture
CRISPR-Cas technology can also be used to control the viral and bacterial
diseases particularly in shrimps and prawns
Can copy and incorporate segments of the WSSV DNA into their genome and
helps to enhance the shrimp’s immune response
RNA molecules to quickly identify and target the same DNA sequence in the
event of future viral infections
If the RNA molecules recognize an incoming sequence of foreign DNA, they
guide the CRISPR complex to that sequence.
There, the shrimp’s Cas proteins, which are specialized for cutting DNA, splice
and disable the invading gene.
This may result in protecting the shrimp from infectious diseases. Therefore, a
modern genome editing CRISPR- Cas9 technology can prove as a powerful tool
in order to prevent the infection of viral and bacterial pathogens.

Marine Genomics is a young scientific discipline that involves the
application of genomic techniques to investigate the phenotype of marine
organisms and the function of marine ecosystems.
Marine biology Preclinical drug safety evaluation
Genetics Controlling infectious disease
Healthcare Clinical diagnostics
Environmental biotechnology Pharmacogenomics

Need For Marine Genomics Research
Marine and coastal environments include many diverse pelagic and benthic
habitats such as open-ocean ecosystems, deep-sea communities including
thermal vent ecosystems, kelp forests, mangroves, coral reefs, etc.
Has received much less attention than its terrestrial counterpart.
Marine biosystems have been evolving for an additional 2.7 billion years
compared to terrestrial environments, and almost all the currently described
phyla are represented in the ocean while only about half have terrestrial
members.

Study of the complete set of
transcripts in a specific cell, tissue,
or organism for a given
developmental stage or
physiological condition.
This complete set of transcripts is
known as a transcriptome, including
protein-coding mRNA and non-
coding rRNA, trna and other
ncRNAs.
Unlike the relatively stable genome,
the transcriptome varies with
developmental stage, physiological
condition, and external
environment.
Powerful tool for dissecting the
relationship between genotype and
phenotype
TRANSCRIPTOMICS

Defined as “Culture independent analysis of a mixture of microbial genomes
using an approach based either on expression or sequencing” (Schloss and
Handlesmann, 2005)
METAGENOMICS
Microorganisms thrive throughout
oceans, reaching depths of 11,000
m (mean depth 3200 m), with
pressures exceeding 100 MPa and
temperatures higher than 100 °C in
deep-sea hydrothermal vents.
Typically, they associate with other
organisms: countless communities
comprise bacteria, archaea,
protists, fungi and viruses.
Marine metagenomics is, therefore,
an excellent tool for reading the
abundance of novel genetic
information and unlocking the
immensity of metabolic diversity
available from microorganisms.

Applications
Functional genes characterization
Symbiotic relationship
Microbial identification – rRNA sequencing
Pathogen identification in fish/ shrimp farm
Characterization of Viromes through Metagenomics - discover new
viruses, both in marine and freshwater environments

DNA Bar coding
Taxonomic identification
Genetic marker COI-~ 600 bp
Fast mutation rate, resulting in the generation of diversity within
and between populations
Fish Barcode of Life Initiative (FISH-BOL)- a global effort to
coordinate an assembly of a standardized DNA barcode library for
all fish species

Applications
Microbes
Sea grass & Mangroves
Marine Algae- 23 S & COI
Marine planktons
Lower chordates
Marine reptiles
Sea birds
Marine mammals

ALGAL GENOMICS
Genome sequences can be
instrumental in the reconstruction of
metabolic pathways
The metabolic pathways help to study
the production of various secondary
metabolites and increased production
etc
Sequence analyses provide information
on the evolution of different groups of
algae
Potential to develop new and
economically important products and
technologies.

Transcriptomics
Transcriptomes are sequenced among the available micro algal genomes
Transcriptome sequences have been deposited in public databases GenBank
A specific web-based database, named AlgaePath, is available for some of the
algae (http://algaepath.itps.ncku.edu.tw/)
AlgaePath integrates gene information, biological pathways and NGS datasets
Considering the great market interest of MNPs, the transcriptomic approach has
been used in order to reveal the gene pathways responsible for their synthesis.

Proteomics
Proteomics explores the mechanisms involved in many biological
processes and network functions by providing information on
proteins, including post-translational modification, sub cellular
localization and protein-protein interaction
Proteomics analysis helps to biofuel, lipid bodies, cytoplasmic lipid
droplets, β carotene-rich plastoglobuli , biosynthesis and regulation ,
nitrogen depletion, membrane protein etc (http://alga-pras.riken.jp/)

MICRO ALGAL DNA BARCODING MARKERS
Nucleic acid levels: Small sub unit (SSU), large sub unit (LSU) rRNA (18S and
5.8S +28S rRNA), internal transcribed spacers (ITS) region 1&2, Universal
Plastid Amplicon (UPA) domain including16S rRNA and 23S rRNA
Chloroplast marker genes: ribulose-1,5-bisphosphate carboxylase large
subunit (rbcL), photosystem I P700 chlorophyll a apoprotein A1(psaA),
photosystem I P700 chlorophyll a apoprotein A2 (psaB, ~1,600 bp),
photosystem II thylakoid membrane protein D1 (psbA),
Mitochondrial gene markers: cytochrome c oxidase subunit 1 DNA barcode
region (COI-5P), cytochrome c oxidase 1 (cox1), cytochrome c oxidase subunit
1 extended fragment (COI), cytochrome B (COB) and cytochrome oxidase
subunit 2-3 intergenic spacer (cox 2-3).

Genomic Application in Aquaculture
Pathogen Discovery
NGS technologies- Aquatic Viruses
(i) Discovering novel viruses associated with fish mortalities
(ii) Elucidating the mechanisms of pathogenesis
(iii) Studying the molecular epidemiology of these pathogens
Nutrition, Growth & Development
Growth, digestion, metabolism, immune function, and reproduction?
Transcriptomic profiles can be used to understand how this may alter the
physiological conditions of tissues within aquaculture species
Formulate a proper feed for better growth and development

Reproduction & Development
Transcriptomic analyses can provide some insight on fitness of
individuals and also an understanding of how reproduction at the
molecular level operates within the species of interest.
An understanding of the genetic loci responsible in determination of
sex in fishes can provide insight into reproduction, which is helpful
for aquaculture and fisheries

Immune function, Stress & Toxicology
An understanding of which genes are expressed in the presence of a
pathogen, and also the diversity of genes related to immune function is
important for aquaculture and fisheries.
Candidate tissues for transcriptomic studies would be head kidney, kidney,
and spleen for understanding immune function
Environmental stress can occur in multiple forms such as differences in
temperature, salinity, pH, spatial constraints, and pollutants

Microbiome applications and
aquaculture management
Bacterial taxa have mutualistic
relationships with fish that translate as
health of a fish
our understanding of microbial
genomes, pairing the transcriptome of
bacteria residing within the fish of
interest and aids in digestion or other
physiological functions, will provide
greater insight on genomic and
transcriptomic studies.
This approach offer many opportunities
to better understand co-evolutionary
processes that operate between the gut
microbiome and the host.
In addition, meta transcriptomics
(transcriptomes of microbiota) serves
as a useful tool for understanding how
genes within the microbe aid in
digestion.

Functional genomics in vaccine development
Multiple strains having different antigenic proteins, the challenge in
vaccine design has been to find cross protective antigens against
variant strains of the same pathogen.
NGS supported with current advances of bioinformatics tools is
expected to expedite our ability to identify the most immunogenic
proteins for vaccine production against viral diseases
WSSV- Highly immunogenic VP28

CONCLUSIONS & FUTURE DIRECTIONS

Conserve the Marine Biodiversity using novel molecular techniques
and stopping pollution to spoil marine environment and Marine
Biodiversity
Marine derived pharmaceuticals provide a novel and rich source of
chemical diversity that can contribute to design and development of
new and potentially useful pharmaceutical agents.
We must increase our use of genomics to learn more about the
oceanic environment, bio prospecting, emerging diseases,
biodiversity and extremophile research
Metagenomic approach help to find novel lead structure for drug
development. By utilizing the bioprocess technology we can produce
the compounds at large scale levels.
Improve the aquaculture activities and increase the nations’ fish/
shrimp production through advanced methods which will help to
high export
A proactive interaction between researchers, the pharmaceutical
marine biotech sector and government regulating agencies is crucial
to the incorporation of this challenging new tool in clinical medicine.

Marine Science and Marine Biotechnology

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