Citarasu mku ref_2020

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
MANONMANIAM SUNDARANAR UNIVERSITY, TIRUNELVELI
CENTRE FOR MARINE SCIENCE AND TECHNOLOGY

CURRENT TRENDS IN 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
Indian Ocean (3897 m depth)
Arctic Ocean
Oceans are subdivided into 150 Seas

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

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

Invertebrates exhibit a wide range of modifications for survival
Fish have gills instead of lungs, although some species have both.
Marine mammals need to surface periodically to breathe air
Over 200,000 documented marine species
Two million marine species yet to be documented
Marine microorganisms, including protists, bacteria and viruses,
have been variously estimated as constituting about 70%

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

Marine Biotechnology Explores
Novel Pharmaceutical drugs
Cosmetics
Enzymes
Industrial Products
Bio fuels
Processes & Bioremediations
Aquaculture & sea food safety
GMO

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.

Indian scenario of Marine Biotechnology
Established in 1986
Puts 30% of its funds into public-private partnership to overall area
Involved Antarctic research since 1981 with MoES
DBT established the National Bio-resource Development Board NBDB in
1999, to help coordinate the development of India’s biodiversity towards
new products and processes.
Task Force on Aquaculture and Marine Biotechnology, set up in 1998,
which overseas individual research projects and network projects with
national and international partners.

Increasing coordination with DST,
CSIR, MoES and ICAR to avoid
duplication of research funding.
Structural and functional genomics,
cultivation of seaweeds,
bioprospecting and bioactives,
microbial extremophiles,
bioremediation and Aquaculture
DBT’s industry-focused programmes
SBIRI and BIPP are likely to be merged
and managed by BIRAC
(Biotechnology Industry Research
Assistance Council)
BIRAC promotes biotechnology across
the range from researchers to
companies
Deal with low risk and high risk
projects, IPR issues, bio-incubators,
bio-clusters and commercialization
aspects including policy matters

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
Protection of water resources
(e.g. Mangrove forests trap silt)
Nutrient Storage and Cycling
Pollution Breakdown and Absorption
Contributes to Climactic Stability
Recovery from unpredictable events
(e.g. hurricanes)
Food
Medicinal resources
Recreation

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.
Altered patterns of wind and water
circulation in the ocean environment will
influence the vertical movement of ocean
waters (i.e., upwelling and down welling).
Warming of both air and ocean
temperatures has resulted in the loss of
Arctic sea ice that affects the marine
mammals

Ocean Acidification
The oceans are normally a natural carbon sink, absorbing
carbon dioxide from the atmosphere
Because the levels of atmospheric carbon dioxide are
increasing, the oceans are becoming more acidic

Ocean Acidification [continue….]
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.

OIL POLLUTANTS
Hydrocarbon Compounds
Alkanes, naphthenes, aromatics and alkenes
Benzene, toluene, xylene and polycyclic
aromatic hydrocarbons are highly toxic.
Physical and chemical alterations &
toxication of marine habitats.
Affect photosysnthesis
Birds getting lose of water proof
Accumulate in sediments as well as fishes
and shellfishes
PAHs are potential carcinogens, mutagens
and teratogens

Heavy Metal Pollution
Toxicity and adverse health effects vary depending on the type of
metal
Dangerous metals include mercury, lead, and copper, nickel and
cadmium etc
Atmospheric contamination: Fuel combustion, electric utilities, steel
and iron manufacturing, fuel oils, fuel additives and incineration of
urban refuse
Copper is dangerous to marine organisms from anti-fouling paints

Thermal pollutants
Water released from nuclear reactors, thermal power stations: Oxygen
level changes
The altered or low level O2 irrupt the degradation level and create
suffocation
High temperature discharge from industries induce the production of
toxins
Affect the reproduction: Release of immature eggs or can prevent normal
development
Increase metabolic rate, enzyme activity & consume more food leading to
disturb the food chain

Nuclear Radiation
Radiation may found in the coastal waters
1. Natural
Cosmic rays by earths’ crust
Potassium -40
Decay products of uranium
2. Human activity
Oil exploration combustion
Phosphate production and use
Land-based mining
Discharges from nuclear power stations
Fallout from atmospheric nuclear
weapons testing and accidents
Medical diagnosis and therapy
Food conservation.

Nutrient Inputs
Inputs of high levels of nitrogen and
phosphorus compounds, leading to
“eutrophication” leading to excessive
growth of algae (“algae bloom”)
As a consequence, oxygen
concentration decreases, while
concentrations of hydrogen sulphides
increase.
Compounding this problem, dead algae
floats on the surface and thus covers
the water, making it difficult for sunlight
to penetrate into the sea.
Fertilizer runoff: During heavy rain the
organic fertilizers run off from the
agricultural field and it affects the
marine environment and back water
bodies

Microbial Contamination
Bacteria and Viruses
Seafood contamination (Process industry + shrimp farming)
Improper methods of handling
Affect consumer levels (Cholera & typhoid)

Noise Pollution
Marine life can be susceptible
to noise or sound pollution
from sources such as passing
ships, oil exploration seismic
surveys, and naval low-
frequency active sonar.
Sound travels more rapidly
and over larger distances in
the sea than in the
atmosphere.
Between 1950 and 1975,
ambient noise in the ocean
increased by about ten
decibels (that is a ten-fold
increase)

Dead whales washedashore near Manapad coast

Ship based threatening
Pollutants are released by vessels either
operationally or accidentally.
Operational pollution:
Discharge of sewage, tank residues, bunker
oils, garbage, ballast water, emission from
vessels & antifouling paints
Accidental Pollution:
Accidentally due to collisions by contacting
with external objects, grounding, explosions,
cargo transfer failures, loss of cargos & toxic
materials discharges, oil spills etc.
Damage to habitat & Animals:
Anchoring spoil the coral beds
Propellers; ship strikes: marine mammals such
as whales

Harmful algal blooms (HABs)
Toxin producing dinoflagellates
Red tide
Sea and Fish culture Environment
Shrimp farm effluents (Algal bloom die off, large amount of dead
organic load accumulate)
Affect filter feeding animals, shrimps, lobsters

Ocean Mining
Ocean mining sites are usually large areas of polymettalic nodules or active
and extint hydrothermal vents at about 1,400-3,700 meters below the oceans’
surface (silver, gold, copper, manganese, cobalt, zinc and Diamonds)
Removing parts of the seafloor disturbs the habitat of benthic organisms,
possibly , depending on the type of mining and location causing permanent
disturbances
Leakage , spilling and corrosion would alter the mining area by chemical
contamination

Plastics
In recent years plastic pollution in the
ocean has become a significant
environmental concern for governments,
scientists, non-governmental organizations,
and members of the public worldwide.
80 % of marine debris is plastic - a
component that has been rapidly
accumulating since the end of World War II.
Discarded plastic bags, six pack rings and
other forms of plastic waste which finish up
in the ocean present dangers to wildlife and
fisheries.
Aquatic life can be threatened through
entanglement, suffocation, and ingestion
Plastic debris tends to accumulate at the
center of ocean. In particular, the Great
Pacific Garbage Patch has a very high level
of plastic particulate suspended in the
upper water column

Microplastics killing fish before they reach reproductive age……..
Fish are being killed, and prevented from reaching maturity, by the litter of plastic
particles finding their way into the world’s oceans, new research has proved
Another 30 years no fishes can’t
reproduce

Polythenes
Generated from hosehold and
industrial wastes
Recreational beaches
Mistaken for food by sea turtles
100 type of birds
31 types of sea mammals
Fishes
Release Bisphenol, phathalate &
gases
Cause cancer, heart failures

Coastal Tourism
Recreational and commercial fishing, marine vessels and marine-
industries are all sources of polluting the sea by various solid wastes.
Tourism and recreational activities account for an array of plastics,
polythenes and tin bottles being discarded along beaches and coastal
resorts. The materials carried on in-shore- and ocean currents.
Fishing gear, plastic monofilament line and nylon netting are the most
commonly noted plastic debris items with a marine source.

Marine Littering
Input of 6-10 million tones of litter into the oceans each year, 70-80 % from
land based sources, in total 100 million tones of plastics.
22 trenches within the ocean that have been identified. 18 trenches are in
the pacific ocean, 3 in the Atlantic and 1 in the Indian ocean
The deepest trench within the Mariana trench system is the Challenger
trench which is 36, 201 feet deep which is situated in the western Pacific
Ocean, to the east of the Mariana Islands

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

Important features
Extremophilc bacteria with stand high temperature, pH & salinity
Less expensive & Eco-friendly
Genetically modified organism such as super bug
Oil recovery MEOR (Minimal disruption in sites)
Remove toxic elements

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

Effluent Treatment by Probiotics
Probiotics have been defined as “live microbial
food supplements which beneficially affect the
host by improving the intestinal microflora
balance”
Gut & Water Probiotics
Competition exclusive principle
Used in Shrimp farms and polluted areas
Improved strains of Probiotics
Lactobacillus and Bifidobacterium sp

Effluent Treatment by Microalgae
Removal of nutrients
Removal excess nutrients and Co2 in
waste water systems
Solve BOD problems
Excess algal biomass may harvested
Removal of organic pollutants
Chlorella, ankistrodesmus in paper mills
& olive oil mills
Algae can produce O2 to bacteria and
bacteria degrade the nutrients
Removal of Heavy metals
Sargassum sp special affinity to heavy
metals
Waste water systems

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

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 Act
Controls dumping of waste at sea, at-sea
research, establishment of marine sanctuaries
Ocean Dumping Ban Act (1991)
Banned all sewage and industrial waste
dumping in the offshore waters
National Estuarine Research Reserve
System (1972)
Protects coastal and estuarine habitats for long
term research, water-quality monitoring,
education, and coastal stewardship

Marine Mammal Protection Act (MMPA)
(1972)
Prohibits the taking of marine mammals
 Hunting, killing, capture, harassment (tormenting,
annoying, disturbing it’s behavioral patterns )
Endangered Species Act (1973)
Administered by NOAA (National Oceanic and
atmospheric administration) and the U.S. Fish and
Wildlife Service
Designed to prevent any endangered species
from becoming extinct because of human
activities
International Whaling Commission (IWC)
An intergovernmental (international) governing
body charged with the conservation of whales
and management of whaling

Fisheries Management and
Conservation Act – 1976
Fisheries resources & Fishing
activities
Clean Water Act of 1977
Polluted water and management
Oceans Act of 2000
Hazards and pollution
Estuaries and Clean Waters Act of
2000
Restoration of estuary habitat

Molecular Taxonomy
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.
Resolving taxonomic uncertainties,
and phylogenetic relationships,
especially for those species or
populations that are endangered
and/or commercially important
Assessing genetic impacts of cultured
stocks on indigenous stocks
populations may diverge genetically
without any changes appearing in their
external morphology

Molecular Tools
Possibility to estimate biodiversity at all
levels, e.g., kingdom/class/family/species
level, in a comparatively small environmental
sample.
General assessment of comparative
biodiversity in a larger number of samples
can be achieved with fingerprinting methods
based on restriction fragment length
polymorphisms (RFLPs), RAPDs,
Microsatellites
Presence or absence of a known species can
be monitored with species specific probes
using chemiluminescent detection with dot
blot techniques or, more sophisticated, with
fluorescent in-situ hybridization (FISH) .
Distinction of individuals at the family or
even species level can be obtained using
highly variable molecular markers such as
ITS sequences (inter-transcribed spacer) or
microsatellites.

Neutral molecular markers such as
RFLP, AFLP, RAPD, single-strand
conformation polymorphism,
minisatellites, microsatellites, SNPs are
used in many conservation genetic
studies to unravel the importance of
genetic data for taxonomic distinction
and management of conservation units.
Cryopreservation is one of the
biotechnological method of ex situ
conservation and applicable for long
term storage of genetic material
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.

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

Isolation of Marine organism for Bio Prospecting
Collection techniques
Traditional snorkel Scuba diving
Self contained underwater breathing apparatus
Ship-based collection
benthic Organisms & sediments
Sediment Corers
Sediment sampling for microbes
Submersible based Collections
Act as big vacuum suckers
Burrowing organisms in benthic sediments

Remotely operated Vehicles (ROVs)
Deepwater industries for hydrocarbon extraction
Autonomous Under Water Vehicles (AUVs)
A unmanned vessels going to deeper for longer & less cost
Hybrid remotely operated Vehicle (HROVs)
Carry out targeted surveys and collections, and help carry
out detailed experiments in the deep ocean

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.
More than 5,300 different products are known from sponges and their associated
microorganisms, and more than 200 new metabolites from sponges are reported
each year.

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
VarixanthoneAscosalipyrrolidinone AAigialomycin 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

Green Fluorescent Protein (GFP)
Crystal jelly fish Aequorea victoria
238 amino acid (26.9 kDa)
Molecular biology- Reporter gene
expression
Biosensors
Clone in viral vector
protein localization in living organisms
Bacteria, Yeast and other Fungi, fish plant,
fly, and mammalian cells etc

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
PeptidesAMP
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 fuscocinereaToxopneustes 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

Carotenoids
Solvent Extraction
Astaxanthin
Isolated from Micro algae
Powerful antioxidants in aquaculture
Fucoxanthin
Brown carotenoids in certain sea weeds
Promote DHA & omega-3 fatty acids
Beta carotenoid
Found in many algae
Colored carotenoids
Antioxidants & anticancer
Immunostimulants

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

Microbial 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

Electronic & Nanotechnology
It acts as a proton pump; it captures light energy
and uses it to move protons across the membrane
out of the cell resulting production of chemical
energy
Optical “Switch” 500.000 Ghz (500x10-15 s)-
Bacteriorhodopsin from halophiles and xerophiles.
Re writeable films of high resolution (5µm) -
Bacteriorhodopsin from halophiles and xerophiles.
Photo detectors
Protein film used in artificial retinal implants etc

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

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)

Finfish Diseases
Freshwater Fishes
Motile Aeromonad septicemia
(MAS)
Hemorrhagic septicemia
A. hydrophila, A. sobria
A. caviae, A. veronii,
A. salmonicida
Marine Fishes
Vibriosis
Pathogenic Vibrios
Photobacterium sp
Micrococcus sp
Flavobacterium- Gill disease
Enterobacter- Edwardsiella
Pseduomonas
Salmonella

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

POLYMERASE CHAIN REACTION (PCR)
PCR is a technique used to make thousands of copies of a DNA used in
diagnosis including paternity testing, mutation detection for disease and
cloning genes for research
Steps including Denaturation, Annealing and extension
DNTPs, Buffer, MgCl2, Taq DNA polymerase, primers & DNA template,
In aquaculture, PCR is highly useful to detect the bacterial, viral infection at
very earlier stage of infection

TYPE OF PCR USED IN AQUATIC DISEASE DIAGNOSIS
Nested PCR
Modification of PCR that was designed to
improve sensitivity and specificity
Two sets of primers used, first set of
primers used to amplify a target
sequence and the second one used to
amplify a region within the first target
sequence
For the impossible templates where the
GC content might be high or chance of
non-specific banding is higher, nested
PCR offers the best results. It is also
useful in the amplification of genes with
the low abundance.
Highly useful to detect WSSV, EHP etc

Multiplex PCR
Simultaneous detection of multiple
targets in a single reaction well,
with a different pair of primers for
each target. This technique requires
two or more probes that can be
distinguished from each other and
detected simultaneously.
This is achieved by designing
primers, which can amplify different
regions of the same template DNA
(particular pathogen) or primers
that can be amplify two entirely
different DNA templates (two
distinct virus)

Semi quantitative PCR
Reverse transcriptase to make DNA
from the RNA in the sample, and there
for qualitatively detect gene expression
by use of the complimentary DNA.
If the target sequence to be detected is
RNA, the conventional PCR step would
precede a reverse transcription (RT)
step by which RNA is enzymatically
converted to complementary DNA
(cDNA).
An oligo deoxynucleotide primer
hybridizes to mRNA and is extended by
an RNA dependent DNA polymerase
The newly synthesized single standard
cDNA can be amplified using specific
primers in a conventional PCR.

Quantitative PCR (qRT PCR)
PCR-based technique that quantify gene expression
Amplification produces increasing amounts of double-stranded DNA, which binds
SYBR green or TaqMan probe resulting in an increase in fluorescence.
Quantification of the amount of target in unknown samples is accomplished by
measuring CT and using the standard curve to determine starting copy number.
In the diagnostic format the greatest advantage is no post-PCR manipulation involved
in the visualization of the result, cross- contamination and false positive result can be
minimized.
Increased sensitivity, reproducibility and quantitative accuracy, a part from the
decreased hands-on time and less chances of contamination is a very important tool in
aquatic health management

Loop mediated isothermal amplification (LAMP)
Nucleic acid amplification method that amplifies DNA with high specificity (109-
1010 times in 15-60 mins), efficiency and rapidity under isothermal conditions
Simple, rapid, specific and cost-effective nucleic acid amplification
DNA polymerase (Bst polymerase) and a set of four specially designed primers to
recognize six distinct regions of the target DNA
Unlike PCR, LAMP is carried out in constant temperature (60–65°C) using an auto-
cycling strand displacement DNA synthesis and does not require thermal cycler.
The amplified product can be detected as white precipitate or yellow green color
solution after addition of SYBR Green.

IMMUNODIAGNOSIS TECHNIQUES
Polyclonal Antibodies (pAbs)
Polyclonal antibodies (pAbs) are mixture of heterogeneous which are
usually produced by different B cell clones in the body.
They can recognize and bind to many different epitopes of a single antigen.
Substances foreign to the host (bacteria or virus) known as antigens are
recognized by the host immune system as invaders.
Used to develop different kits for pathogen detection

Monoclonal Antibodies (mAbs)
Introducing an antigen to a mouse and then fusing polyclonal B cells from the
mouse's spleen to myeloma cells
Hybridoma cells are cultured and continue to produce antibodies to the
antigen
Hybridoma technology allows us to produce large amounts of pure antibodies
Used to develop different kits for pathogen detection we can obtain cells that
produce antibodies naturally & can grow continually in cell culture for mAbs
production
High specificity

APPLICATIONS OF IMMUNO DIAGNOSIS
Western Blot Fluorescent Abs
ELISA
Immunoperoxidase
Lateral Flow Dot Blot

DNA BASED BLOTTING TECHNIQUES
Southern Blot
Detection of a specific DNA sequence in
DNA samples
Combines transfer of electrophoresis-
separated DNA fragments to a filter
membrane and subsequent fragment
detection by probe hybridization
Northern Blot
Detect specific RNA molecules among a
mixture of RNA
Can be used to analyze a sample of RNA
from a particular tissue or cell type in
order to measure the RNA expression of
particular genes

HYBRIDIZATION TECHNIQUES
In Situ Hybridization (ISH)
Labeled complementary DNA, RNA or modified nucleic
acids strand to localize a specific DNA or RNA
sequence in a portion or section of tissue or if the
tissue
RNA ISH is used to measure and localize RNAs
(mRNAs, lncRNAs, and miRNAs) within tissue
sections, cells, whole mounts, and circulating tumor
cells
Fluorescence In Situ Hybridization
Molecular cytogenetic technique that
uses fluorescent probes that bind to only those parts of
a nucleic acid sequence
Fluorescence microscopy can be used to find out the
signal
Detect bacterial and viral DNA in an infected cell (~ 300
bp)

METAGENOMICS
Modern genomics techniques to the study of
communities of microbial organisms directly in
their natural environments
Sampling and nucleic acids extraction, Library
construction and Analysis of metagenomics
libraries.
Nodavirus (Farfantepenaeus duorarum
nodavirus, FdNV) and a new DNA virus
possessing a circular genome designated
shrimp hepatopancreas-associated circular DNA
virus (Shrimp CDV)
Identify new latent pathogens in asymptomatic
carriers, uncharacterized pathogens causing a
new disease or multiple pathogens associated
with disease syndromes in shrimp farms

DNA MICROARRAY
Expression rate of thousands of genes and
identify wide range of pathogens from complex
samples in one single reaction
Involves hybridization of DNA with large
number of probes and can overcome the
shortcomings of multiplex PCR, which can
detect only a maximum of six pathogens at a
time
Fluorescent labeled DNA sequences that are
hybridized to the microarray slide help to
identify the pathogens
Fluorescent microarray detector and computer
program will analyze the fluorescent array for
the presence or absence of the species/strain
specific DNA sequence

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

AntibacterialAntibacterial
Piper longumMurraya koeniji
Quercus infectoria
Psoralea corylifolia
Effectively controlled the pathogens by in vitro & in vivo
Low cost & Eco-friendly
Reduce the side effects
Prevent emergence of resistant strains
Alternative to banned antibiotics
Biodegradable and non biomangnification
Block the transcription
Lyse cell wall
Interfere quorum sensing pathway
Beta Ocemine
Quercucin Psoralin
Piperine

Anti parasitic - EHP
White Feces Syndrome (WFS) is
reported to be a serious problem in L.
vannamei culture
The causative pathogen for WFS is a
microsporidian parasite, Enterocytozoon
hepatopenaei (EHP)
Target organ is hepatopancreas affects
its digestive and absorptive function
resulting poor growth and immunity
By treating the hepatoprotective and
digestives herbal active principles to the
EHP infected shrimp immunity or
hepetoprotection will developed against
the EHP infection.

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)

Agathi grandiflora, Aegle marmelos, Cyperus rotundus, Cynodon dactlylon,
Eclipta alba, Picrorhiza kurroa, Tinospora cordifolia, Withania somnifera and
Zingiber officinalis

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 ActivationTLR 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

MOLECULAR DYNAMICS (MD) SIMULATION
In order to the study the activity or stability of the ligand and
target protein at in vivo level MD simulation or water dynamic
analysis will be useful for the hydrogen band interaction of
particular amino acid from protein and the drug.
Physical movement of atom and molecules
Interact with water molecules in body system for more stability

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
PPAETLR
SERPIN
Ligand
PO positive

Pharmacological Important 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

OTHER BIOSURFACTANTS IN AQUACULTURE
Glycolipids from Vibrio natriegens MK3 – anti-pathogenic - V. harveyi
Brevibacterium casei MSI04 producing PHB - anti bio film - Vibrio sp
BS from Staphylococcus hominis control WSSV
Glycolipid from Staphylococcus lentus BS-SLSZ2 treat aquaculture
infections
BS from marine bacteria effectively controlled Vibrio pathogens V.
harveyi, V. alginolyticus, Vulnificus, V. fischeri , V. parahaemolyticus
and Photobacterium damselae

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.

Subunit Vaccine –WSSV VP28-pESC-URA
Benefits
Recombinant subunit vaccine developed against White Spot Syndrome
Virus (WSSV) using the immunogenic gene through yeast expression
vector pESC-URA
VP28-pESC-URA subunit vaccine administrated pacific white leg shrimp
Litopenaeus vannamei had survived more than 80 % against WSSV
infection
The vaccine helps also helped to improve the immune system by
expression of more immune genes and Toll Like Receptors (TRL) and
reduction of viral load after WSSV challenge

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
Infectious diseases are one of the primary
threats to sustainable aquaculture, with an
estimated 40% of the total potential production
lost per annum
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

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.
Although these varied environments support a rich abundance of life,
marine biodiversity 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.
Transcriptome analysis is a powerful
tool for dissecting the relationship
between genotype and phenotype,
leading to a better understanding of
the underlying pathways and
mechanisms
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.

Why Metagenomics Need?
Understanding
Metabolism
Defining the
Minimal
Gene Set
Genome
Engineering
Understanding Cell
Structure & Function
Understanding
Host Interactions
Understanding
Protein-Protein
Interactions
Understanding
Expression
(RNA/Protein)
Discover DNA
Variation, Genotyping
Forensics
Drug/Vaccine
Development

233
16S ribosomal RNA analysis of
microbial communities
16S rRNA genes have been
amplified, cloned and
sequenced from thousands of
distinct environmental niches,
but these surveys routinely
continue to identify unique new
bacterial and archaeal taxa.

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

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
Genomics and transcriptomics alone are insufficient to understand the complex
biology of microalgae, and should be complemented with a proteomic approach
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/)

Metabolomics
Metabolites are dynamic, and their properties and levels of biosynthesis depend
on genetic and/or environmental changes
Their synthesis can be triggered by physiological and/or environmental stimuli
(e.g., the presence of predators, nutrient depletion or starvation and light etc
For example metabolomic and lipidomic profiles of Nannochloropsis salina
under cold stress, and observed a total fatty acids accumulation
Metabolomics information available in Kyoto Encyclopedia of Genes and
Genomes (KEGG), PubChem and MetaCyc etc.

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

MICRO ALGAL DNA BARCODING MARKERS
DNA barcoding is a molecular technique that
uses sequence comparisons of a short region
of DNA to distinguish species.
An effective DNA barcoding marker is helpful
to poorly understood species diversity of the
micro algal species in the natural environment.
Universality and power is the important
primary criteria barcode marker
Universality is a marker’s capacity to be
amplified and sequenced routinely using as
few primer pairs as possible and for the
resulting sequences to be good quality and
require minimal editing effort.
The power of a particular marker is the ability
of that marker to distinguish between closely
related species.

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).

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

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

Growth Traits
Genetic selection in which individuals with the
best growth traits are selected as parent stock
for the next generation is one of the major
strategy
To expedite the process of identifying genetic
traits for optimal growth performance,
marker‐assisted selection (MAS) processes
such as single nucleotides polymorphism
(SNP), microsatellite, amplified fragment
length polymorphism (AFLP), random
amplified polymorphism DNA (RAPD),
restriction fragment length polymorphism
(RFLP) and quantitative trait loci (QTL) are
being used to scan chromosomal DNA of
different farmed aquatic organisms.

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.

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