1.Chromosome engineering Chromosome sex manipulation techniques to induce polyploidy (triploidy and tetraploidy) and uniparental chromosome inheritance (gynogenesis and androgenesis) have been applied extensively in cultured fish species Androgenesis Androgenesis is the process by which a progeny is produced by the male parent with no genetic contribution from female. Induction of androgenesis can produce all male population in fish which would have commercial application in aquaculture. Gynogenesis Gynogenesis is a form of all-female inheritance. In fish species, ultraviolet (UV) irradiation has been used to inactivate the sperms, and such UV-inactivated sperms are used to trigger gynogenetic development without contributing the paternal genome to the progeny Sex control:Sex control is providing to be of incraesing use in the optimization of culture systems because in most species either males, female or steriles perfrom better in culture or are more desirable in the marketplace. Development of alternative protein source:The use of fishmeal for aquaculture purposes, is not environmentally friendly because it contains a high level of phosphorous (Edun & Uka, 2011). Due to this reason, Nutritional Biotechnology are using to produce alternative plant base protein source (Adelizi, 1998). But plant-based protein contains anti- nutritional compound so this technique needs to treat those anti – nutritional factors during the processing of these plant proteins. Phytase is one example, an enzyme that breaks down the indigestible phytic acid (phytase) in cereals and oilseeds and release digestible phosphorous. which can make the best use of the phosphorous available in a plant-protein based feed Examples, processed soybean meal is a key ingredient in catfish diet . Gene Banking: Aquaculture in many countries depends on the adequate supply of quality seeds. However, problems in many hatcheries have reported stock deterioration due to poor brood stock management, inbreeding depression, genetic drift, lack of effective population size, introgressive hybridization, unconscious selection

1. Application of Biotechnology in Aquaculture
3/2/2023 1
Presented by:
Pabitra Pandey

2. Contents
1. Background
2. Challenges of Aquaculture
3. Introduction
4. Biotechnology in fish breeding
5. Chromosome engineering
6. Sex control
7. Transgenesis
8. Aquaculture nutrition
9. Fish health management
10. Gene banking
3/2/2023 2

3. Background
• Global population could grow to approximately 9.7 billion in 2050 (UNDESA,
2019)
• On a per capita basis, grew from
• An average of 9.9 kg in the 1960s
• To a record high of 20.5 kg in 2019,
• Slightly declined to 20.2 kg in 2020 (FAO, 2022)
• Around two-thirds of production is directly consumed by human and third is
processed to produce fishmeal and fish oils (Smith et al., 2011)
Source: newscientist.com
3/2/2023 3

4. Cont…
• Depend on climate driven changes
Major threat to quality and quantity of production
• 16,023 fish species are already (IUCN, 2016)
 Extinct, extinct in the wild, critically endangedred,
 endangered, vulnerable, or near threatened
Source: frontiersin.org
3/2/2023 4

5. Challenges
in
Aquaculture
Infectious and
Parasitic diseases
Poor Fecundity
and
Reproduction
Inefficient feed
conservation
Environmental
pollution
Slow growth
Reduce
viability
3/2/2023 5

6. Introduction
• In 1919, Karoly Ereky, used the term “Biotechnology”
• Bio means life and technology means
the application of knowledge for practical use
• Use of living organisms to solve problems or make useful products
• Integration of natural sciences and engineering science in order to achieve the
application of organisms, cells, parts thereof and molecular analogues for
products and services (IUPAC, 1992)
Source:
alchetron.com
3/2/2023 6

7. Application of Biotechnology in Fisheries
1.Biotechnology in fish breeding
2.Chromosome engineering
3.Sex control
4.Transgenesis
5.Aquaculture nutrition
6.Fish health management
7.Gene banking
3/2/2023 7

8. Induced breeding
• Development of GnRH technology, induced breeding successful
• Pivotal neuropeptide regulating fertility and reproduction
Fig: Hormonal control of the hypothalamus-pituitary-gonad axis of female fish and male fish
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9. Genome Editing Technique
CRISPAR/Cas
• kind of “gene scissors”
• Applied successfully in aquaculture, e.g. Atlantic salmon, rainbow trout, catfish,
tilapia and carp
• Prerequisite for improvements in production characteristics is that they are
significantly regulated by a single gene
Elimination of the dead-end gene (dnd), makes the fish sterile (Salmon)
By modifying the myostatin gene (mstn), muscle growth can be increased
Fatty acid elongase gene (evovl2)
3/2/2023 9

10. Cont…
• Japanese researchers reported in 2018
Development of a line of red sea
bream with enhanced muscle mass
(16%)
 Relatively short overall body length
• Chinese investigators reported on a
strain of yellow catfish fish from 37
percent heavier than normal with
increased muscle mass Source: thefishsite.com
3/2/2023 10

11. Cont…
• Israeli scientists recently published
Detailing the use of CRISPR to produce
true albino Nile tilapia
• Disrupt carotenoid transport genes to alter
red and white color patterns in ornamental
common carp
Gene edited albino Nile tilapia (left) beside a non-
edited version (right)
Source: thefishsite.com
3/2/2023 11

12. Chromosome Engineering
• Sexual maturation results
 Decreased growth rate
 Higher incidence of diseases
Change in organoleptic quality
• Problems can be avoided by sterile
• Chromosome manipulation techniques to induce polyploidy
• It can improve the strain for better growth
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13. Polyploidy
• Individuals with extra sets of chromosomes
• Induced triploidy is widely accepted as the most effective method
• Thermal (cold and heat), pressure or chemical shock
• Triploid Pacific oysters are sterile and grow 15% better (Eurofish Magazine, 2020)
3/2/2023 13

14. Cont…
• Cold shock were reported and observed in Common
carp in 1993
• Atlantic salmon in 1995
• Stickleback with both cold and heat shock
• Sturgeon both triploidy and tetraploidy were
induced by heat shock
Fig: Ploidy manipulation in
fish. Source: sciencedirect.com
3/2/2023 14

15. 3/2/2023 15

16. Development of Biosensors
• Basis for development of biosensors, bio- indicator, and diagnostics devices for
medicine, aquaculture and environmental monitoring
A. Lux gene:
 The enzymes responsible for toluene degradation
 For example, are synthesized only in the presence of toluene
 When lux genes are inserted into a toluene operon, the engineered bacterium
glows yellow-green in the presence of toluene
3/2/2023 16

17. Cont..
B. Gene probe:
Used identify organisms that pose health hazards
Specific gene probes can be employed, for example to detect human pathogens
in seafoods
3/2/2023 17

18. Transgenesis
• GMOs with heritable changes to the genome by integration transgene
into the host genome DNA
• Gene transfer into fish embryo (trout, salmon, carps, tilapia, medaka, goldfish,
zebrafish, loach, catfish, etc.)
• The first was demonstrated in 1985, transferred the human growth hormone
gene in goldfish
3/2/2023 18

19. Growth Enhancement
• Atlantic salmon reach market size (4-5kg) from eyed-egg stage in nearly 18
months compared to 36 months
• Also consume 25% less feed (Gomez, 2018)
• FCR: 35% higher
Source: cfs.gov.com
Source: npr.org.com
3/2/2023 19

20. Reduced Ammonia Excretion
• Growth hormone is known to increase protein retention and absorption, thought
to reduce ammonia excretion
• Over-expressed the GH gene throughout their bodies
• About 69% of that excreted by the wild-type fish over their lifetime (Kobayashi et
al., 2007)
Source: sciencedirect.com
3/2/2023 20

21. Cold Resistance
• Winter flounder, ocean pout produce Serum anti freeze protein or glycoproteins
(AFGP) to protect them from freezing
Fig: winter flounder Fig: Ocean pout
source: identifyfish.blogspot.com
3/2/2023 21

22. Cold Resistance
• Atlantic salmon :
 Where it became integrated into the germ line and then passed onto the off-
spring F3 where it was expressed specifically in the liver
• Gold fish :
Temperatures at which all the control fish died 12 h at 0˚C (Wang et al., 1995)
• Juvenile milkfish or tilapia :
led to an increase in resistance to a 26 to 13˚C drop in temperature (Wu et al.,
1998)
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23. Tomato plant with fish genes
• Arctic Flounder can create an antifreeze protein to keep its blood
liquid
• Tomatoes are very susceptible to quick freezes that can wipe out
entire crops
3/2/2023 23
Source:blogs.ubc.ca

24. Disease Resistance
• One approach, bacterial pathogens is to transfer antibacterial peptide genes
 Applied to Channel catfish (Ictalarus punctatus) by the transfer of a DNA construct containing
a lytic peptide, cecropin B, driven by a cytomegalovirus (CMV) promoter
 Showed two and four-fold increases in resistance against Edwardsiella ictaluri and
Flavobacterium columnare (Dunham et al., 2002)
• B actin-human lactoferrin gene was transferred to grass carp, P1 individuals were
more resistant to Aeromonas with increased phagocytic activity (Mao et al., 2004)
3/2/2023 24

25. Ecotoxicology
• Envisioned carrying reporter genes driven by promoters that are responsive to
chemical exposure
• Transgenic zebrafish, potential use in aquatic ecotoxicology
• Cadmium and copper toxicity by
 Induction of heat-shock protein gene, oxidative stress via the induction of an
electrophile-responsive element (EpRE)
• Various organic chemicals interacting with the aryl hydrocarbon receptor-
mediated toxicity, and estrogenicity (either luciferase or GFP as reporter genes)
3/2/2023 25

26. Cont…
• Injecting blue and green biofluorescence genes from jellyfish and red
biofluorescence genes from corals into the developing eggs of zebrafish
• Fluoresced brightly under both natural white light and ultraviolet light
 Visually striking under a black light in a dark room
• Modifying the biofluorescent genes’ “on-off” switch
So GloFish would act as bio-indicators by selectively fluorescing brightly only
when exposed to environmental toxins, pollutants and estrogenic chemicals
3/2/2023 26

27. Ornamental Fish
• Color genes showed stable expression in zebrafish embryos
 Injected with GFP expression constructs under the control of the zebrafish
muscle-specific promoter of the myosin light polypeptide 2 (mylz2) gene (Ju
et al., 2003)
• Fluorescent white skirt tetra, medaka, and farmed rohu carp were successfully
produced using the zebrafish mylz2 promoter (Mohanta et al., 2014)
Source: science.org.com
Fig: Zebra fish
3/2/2023 27

28. Cont…
• Genetically engineered angelfish (Pterophyllum)
• The luminescent angel fish were created by Taiwanese bio-technologists
• World's first pink fluorescent angelfish and can view without blacklight
Fig: Angler Fish
Source: dailymail.com
3/2/2023 28

29. Surrogate technology
• Production of a germline chimera via transference of germ cells of a target
species to an appropriate host, aiming for the production of heterologous
gametes
• Approach in 1992 by transferring embryonic cells from one zebrafish embryo to
another
• Testis transplantation performed between specimens of rainbow trout
3/2/2023 29

30. Can be performed by different methodologies
Blastomere transplantation from embryo to embryo
Differentiated primordial germ cell (PGC) transferred from embryo to embryo
Differentiated PGC transplanted from larvae to embryo
Spermatogonial transplantation in larvae
Spermatogonial and Oogonia transplantation from adult to adult
3/2/2023 30

31. Sex Control by Hormonal Treatment
Source: sciencedirect.com
3/2/2023 31

32. Are you
getting bore
?
No
go on
3/2/2023 32

33. Aquaculture Nutrition
• It comprises as much as half of the operating cost of an aquaculture facilities
• This field engagaed in
1. Development of alternative protein source
• Fish meal replaced by alternative plant base protein source
• But contain anti- nutritional factor
3/2/2023 33

34. 2. Pigmentation
• Salmonids it is necessary to administer carotenoid pigments in the diet and this
currently account for 15% of the dietary cost
• Applied to pigment production
• The Yeast (Phaffia rhodozyma) produces natural astaxanthin
• Development of Phaffia strains that produce high levels of astaxanthin it is now
feasible to provide natural pigment to salmon via the diet
3/2/2023 34

35. Cont…
3. Nutraceuticals
• Application of nutraceuticals includes
Addition of feed additives, vitamins, minerals, and carotenoids etc.
Extracted including omega-3 oil, chitosan and glucosamine, originally derived
from waste products
4. Toxin binders, probiotics, prebiotics
3/2/2023 35

36. Bio- remediation
• Manipulation of microorganisms in ponds to enhance mineralization of organic
matter and get rid of undesirable waste compounds (Banrie, 2013)
• Application of probiotics and enzymes
• Improve the water quality, decompose the excreta
• Reduce the level of infection and mortality by compete with pathogen
• Its excretions make the pond medium less inhabitable for bad bacteria, balance
algal growth through providing nutrients by their bio- degradation activity
•
3/2/2023 36

37. Fish Health Management
• Intensive cultural condition, high stocking density, more chance of
stress
• Results rapid spread of viral, bacterial and parasitic organisms
A. Stress evaluation and management in aquaculture
B. Disease diagnostics
3/2/2023 37

38. Cont…
• Fluorescent antibodies and enzyme linked immunoassays have been used in the
diagnosis of bacterial kidney disease (Olea et al., 1993)
• The polymerase chain reaction (PCR) has been used to detect bacterial kidney
disease DNA in single salmon eggs (Brown et al., 1994)
• Gene probes is used to detect Shrimp viruses by dot blot hybridization, in situ
hybridization and PCR (Poulos et al., 1994)
• RNAi is a promising tool for the management of viral diseases (Vijayan et al.,
2013)
3/2/2023 38

39. Gene Banking
• Ensures continuous quality seed production and genetically improved varieties
• Increases the longevity of gametes for several years without any drastic change
in the fertilizing capacity of the gametes
• Milt from over 200 species of freshwater and marine fish have been
cryopreserved (Hiemstra et al., 2006)
3/2/2023 39

40. Source:semanticscholar.org
3/2/2023 40

41. Biotechnology : Nepal
1.Monosex Culture: Sex Reversal
2.Induced Breeding
a. PG extract
b. Human Chorionic Gonadotropin (HCG)
c. Luteinizing releasing hormone-analogue (LRH-a)
d. Ovaprim and Ovulin
3. Cryopreservation
3/2/2023 41

42. Conclusions
• Play major role: expansion and intensification
• It is relatively recent practice. Nevertheless, it is a promising area to
enhance fish production
• The increased application of biotechnological tools can certainly
revolutionize fish farming
3/2/2023 42

43. References
1.McNaught, A. D., & Wilkinson, A. (1997). Compendium of chemical terminology. Blackwell Scientific publication. IUPAC recommendations.
https://doi.org/10.1351/goldbook
2.United Nations Department of Economic and Social Affairs. (2019). World Population Prospects 2019: Highlights. United Nations.
3.FAO. 2022. The State of World Fisheries and Aquaculture 2022. Towards Blue Transformation. Rome, FAO. https://doi.org/10.4060/cc0461en
4. Smith, A. D., Brown, C. J., Bulman, C. M., Fulton, E. A., Johnson, P., Kaplan, I. C., Lozano-Montes, H., Mackinson, S., Marzloff, M., Shannon, L.J., Shin,Y.J., & Tam, J.
(2011). Impacts of fishing low–trophic level species on marine ecosystems. Science, 333(6046), 1147-1150.
5. Gómez, A. (2018) Genetically modified fish in aquaculture. Frontiers in Sciences and Engineering, 8(1), 61-68.
6. Kobayashi, S. I., Morita, T., Miwa, M., Lu, J., Endo, M., Takeuchi, T., & Yoshizaki, G. (2007). Transgenic Nile tilapia (Oreochromis niloticus) over-expressing growth
hormone show reduced ammonia excretion. Aquaculture, 270(1-4), 427-435.
7.Wang, R., Zhang, P., Gong, Z., & Hew, C. L. (1995). Expression of the antifreeze protein gene in transgenic goldfish (Carassius auratus) and its implication in cold
adaptation. Molecular Marine Biology and Biotechnology, 4(1), 20-26.
8..Mohanta, R., Jayasankar, P., Mahapatra, K. D., Saha, J. N., & Barman, H. K. (2014). Molecular cloning, characterization and functional assessment of the myosin light
polypeptide chain 2 (mylz2) promoter of farmed carp, Labeo rohita. Transgenic research, 23(4), 601-607.
9.Dunham, R. (2009). Transgenic fish resistant to infectious diseases, their risk and prevention of escape into the environment and future candidate genes for disease
transgene manipulation. Comparative Immunology, Microbiology and Infectious Diseases, 32(2), 139-161
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