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Saturday, 30 May 2020

Biotechnological Advancement, Applications in Fisheries and Aquaculture

Biotechnological Advancement, Applications in Fisheries and Aquaculture


What Aquaculture offers?
Aquaculture offers the huge potential of providing nutritional as well as economic security to the ever-growing population around the globe. The beauty of aquaculture is its flexibility which allows it to be used in all the kind of water resources varying from freshwater to highly saline water. Aquaculture itself is an amalgamation of a multitude of disciplines including biology, chemistry, environment, engineering etc.  

Aquaculture and biotechnologists
Biotechnological interventions have realized it as a multi-million dollar industry. Today aquaculture is considered as an organized lucrative business sector and contributing enormously towards the GDP of several countries.

Some of the key biotechnological interventions such as the development of inducing hormone, disease diagnosis kit, probiotics, biofilter, selective breeding etc. have contributed enormously in accelerating the pace of the blue revolution. The following section will brief about the different biotechnological interventions in aquaculture along with future prospects.

What is Biotechnology?
Biotechnology is defined by several authors using various way but the simplest one is presented by the United Nations Convention on Biological Diversity as “any technological application that uses biological systems, living organisms or byproducts thereof, to make or amend products or procedures for particular use (Kafarski, P., 2012).

Wikipedia states biotechnology as functional biology that comprises the use of living organisms and bioprocess in engineering, technology, medicine and other fields requiring bio-products (source -Wikipedia). Biotechnology is used widely for human welfare such as food production, environment management, health management, etc. The integration of biotechnology with aquaculture is not a new aspect and being practiced for decades.

The versatility of Biotechnology offers many potential applications for the development of fisheries and aquaculture. The success of next-generation aquaculture will be largely guided by the technology influenced through biotechnological intervention. All the four important pillars of aquaculture such as seed, feed, disease and environment can be very well integrated with biotechnological findings which can ultimately improve the output several folds.

Interventions of Biotechnology in Aquaculture
The success of aquaculture is governed by mainly four components - quality seed, quality feed, disease management and soil water quality management. Even though the major investment in aquaculture is towards feeding management but the success of aquaculture is equally dependent on the other three components too.

Biotechnological interventions are very well prevalent in all these components and explained in detail in the following sections.

Role of Biotechnology in Seed Production
Quality and pure seed is the most important criterion to start aquaculture. Availability of quality seed with improved performance in large number is the key factor in the development of semi intensive and intensive aquaculture industry.

Development of induced breeding techniques and selective breeding are the revolutionary development in hatchery technology of aquaculture which has lightened the lamp of the blue revolution.

Induced Breeding and Biotechnology
Induced breeding based on hypophysation (Chaudhuri, H., & Alikunhi, K. H., 1957) was developed long back during the 1950s but it is the production of synthetic hormones which has helped in disseminating the technology of induced breeding to the ground level. Synthetic super-active analogues were developed using biotechnological tools such as recombinant DNA technology and protein engineering with better inducing efficacy at a lower dose.  Such analogue possesses modified amino acid in position 6, which leads to higher resistance to peptidase. It has also altered the polarity and tertiary structure of the GnRHa, which outcomes in an enhanced receptor binding affinity (Zohar and Mylonas, 2001; Thomas, P. C., 2003).

GnRH amino acid

                  Figure 2 : Different forms of GnRH amino acid sequences

The race is still on in finding species specific more efficient analogue using a bioinformatics approach to reveal the effect of amino acid substitution on receptor binding study. Currently, different types of inducing agents such as ovatide, ovaprime, ovapel etc. are commercially used widely in the hatchery (Chattopadhyay, N. R., 2016).

New candidate hormones such as Kisspeptin are also identified from different fishes as a key regulator of breeding representing upstream in the biological pathway of breeding and synthetic Kisspeptin hormone has shown to be effective in improving the reproductive performance of fishes (Rather et al., 2016).

Improved Seed Production (Selective Breeding)
Production of improved quality seed in terms of growth performance, disease resistance, and environmental adaptability is present and future of hatchery technology. Selective breeding is acting as a vital tool in the hatchery. Many successful examples of such species are GIFT, Jayanti rohu, common carp, salmon, sea bass etc. (Gjedrem et al., 2012) are available around the globe. Selective breeding is at present only viable methodology to produce specific pathogen resistant seed. Efforts are going on around the globe to produce SPR shrimp and fish for disease free aquaculture.

The genetic improvement of a fish stock involves selection, cross/outbreeding, and hybridization. Selectively bred stocks with superior traits such as disease resistance and rapid growth have been produced and used in aquaculture. Selective breeding could be done by two approaches one is traditional selective breeding (select high performing individuals) and another is MAS (Marker Assisted Selection) breeding. The former one could be used only for the traits which are visible like growth but for others traits like disease resistance and carcass, MAS is the best option. Molecular markers are direct reflection of the genetic diversity at the DNA level. A number of approaches have been established to obtain molecular markers, including VNTR, RAPD, SNP, AFLP, SSR, RFLP and others. These markers can be employed to tag quantitative trait loci (QTLs) and assist the breeding program. Association of markers with any trait (QTL) can lead to the identification of genes responsible for resistance or susceptibility. Selection based on marker data is called Marker Assisted Selection (MAS) and, if a gene is used, the process is called Gene Assisted Selection (GAS).

Markers like SNP which are highly abundant across the genome are very much helpful in selective breeding and with the advent of new advanced technologies like NGS (Next Generation Sequencing), identification of these markers is now very easy and economical (Agarwal et al.,2016). CIFA has released 1st batch of jayanti rohu produced through selective breeding in 1997(50 years of Indian independence so called it jayanti rohu) with a better growth rate (17% more). Further disease resistant Jayanti rohu (resistant to Aeromonas hydrophila) is also produced by CIFA (Mahapatra et al., 2017).

Assisted Reproduction Technique  
Assisted reproductive techniques (ART) have now been widely assimilated in the management of infertile couples. Although this is now being used in the case of humans but it can be very well used in the hatchery to produce quality spermatozoa. Milt collected through stripping also contains a bunch of dead spermatozoa, resulted in the failure of fertilization.

 Flow cytometry based techniques such as FACS (Fluorescent Activated Cell Sorting) can be used to segregate the inactive dead spermatozoa from the live and motile sperms using some specific dye. It provides a way for sorting a varied mixture of cells into two or more containers, one cell at a time, based upon the exact light scattering and fluorescent features of each cell. 

The separated cells (live and motile sperms) could be collected in a specific container and further used in breeding. However, this technique is still in the infancy stage but holds huge potential especially for endangered species or species with less fecundity (Agarwal et al., 2017).

Surrogate Broodstock Technology
This technology holds huge potential for aquaculture as well as in the conservation of those species which are difficult to breed in captivity. The concept of Tuna from Mackerel or Trout from Salmon (Takeuchi et al., 2004; Yoshizaki, G., & Yazawa, R., 2019) has been emerged during the recent time, spreading hope for hatchery seed production of several important species for both conservation and aquaculture point of view. 

Scientists have come up with another that offers great possible, a kind of surrogate breeding (a new aquaculture technology) in which both sperm and egg of a species are produced within a related easily cultivable species. In surrogate technology, another fish species are used to produce spermatozoa and ova. To succeed a successful surrogate technology it is compulsory to comprehend the mechanism involved in gonad development. 

Gonads progresses from a particular type of cell know as Primordial Germ Cell (PGC), giving rise to either spermatogonia or oogonia (future male testis or female ovaries). In this method, the PGC of one fish (donor) is transplanted into the embryo of another fish (receiver) and after maturation; the receiver fish produces the gametes of the donor fish. This concept offers a ray of hope to obtain big fish from small fish which are difficult to breed in captivity such as Tuna from Mackeral, which saves the time, space and input cost because small fishes need less space, less food and lower maturation time.

Transgenic fish
DNA is considered as blue print of life. The transgenic or genetically modified organism is produced by bringing out some changes in the genome using the genetic engineering approach of gene transfer. Glo fish (GMO of zebrafish) and Aquaadvantage salmon are the two GMO which are commercially available for ornamental and consumption purpose respectively. FDA defined “genetically engineered (GE) animals” as those animals altered by rDNA techniques, including all offspring that cover the modification.

Development of transgenic produced by the transfer of foreign genes into the fertilized eggs has become a powerful tool for the study of gene expression in living animals in the field of developmental biology, animal husbandry and aquaculture. By using various transgenic techniques, investigators are pursuing to progress the genetic traits of the fish used in commercial aquaculture system. 

Researchers are trying to develop fish which are larger and grow faster, more efficient in converting feed into muscle, resistant to disease, tolerant of low oxygen levels in the water, and tolerant to freezing temperatures (Agarwal et al.,2017). For example, AquAdvantage salmon, the trade label for a genetically changed Atlantic salmon developed by AquaBounty Technologies.

The AquAdvantage salmon has been altered by the addition of a growth hormone regulating gene from a Pacific Chinook salmon and an antifreeze promoter from an ocean pout to the Atlantic salmon genome. Normal salmon does not grow during winters but these genes enable it to grow year-round. The purpose of the alterations is to upsurge the speed at which the fish grows, without affecting its final size or other qualities. The marketable size of this fish reaches in 16 to 18 months rather than 3 years. This technology is succeeding quickly and it is now conceivable to move genes between indistinctly related species. Glo fish is available with diverse variants and widespread between the ornamental fish keeper.

Hybrid Identification
Hybrid seeds are the major concern of recent years faced by aquaculturist, as many hatcheries are nowadays producing hybrids that they sell on the name of original species for example hybrids of IMC, hybrid of magur and gariepinus etc. It is almost impossible to distinguish the original one from hybrid seed at spawn and fry stages using external characters.

Technology for hybrid identification would really be of great use for farmers for the screening of quality seeds. This problem could be solved to a satisfactory level by using molecular approaches. Some private labs and government institutes have made some simple and reliable kits, commercially available and show good results in the identification of the hybrid with the wild one. These kits are purely PCR-based kits which can identify a hybrid in just two steps with genomic DNA as starting material. There is a particular kit manmade by CIFA, intended for the identification of Labeo rohita, Catla catla and their hybrid within a few hours in the early life stages. Three sets of primers are used to amplify three diverse microsatellite markers from the genomic DNA isolated from pectoral fins by using PCR.

The PCR products using all three primer sets differentiate the ‘hybrid–Rohu’ from wild types. The hybrid–Rohu DNA yields specific PCR products with all three primer pairs. Only two PCR products are got either from wild-type Catla DNA (by primer sets 1 and 2) or from wild-type Rohu DNA (by primer sets 1 and 3). Such PCR based hybrid identification protocol can be established for others also.     

Biotechnological interventions in Health management in aquaculture
The disease is always a nightmare and major concern in aquaculture and an estimate says that it accounts for the total loss of over 6 billion dollars per year (Leung, T. L., & Bates, A. E., 2013). Managing disease is always a challenging task but biotechnological interventions at different stages such as diagnosis, prophylaxis, therapy etc is a big help for aquaculturists. Aquaculture industries are in urgent need of fast-growing disease-resistant varieties, rapid and sensitive disease diagnostic kit, and development of economic and effective vaccines, probiotics and cell lines.

Disease diagnosis
Prevention is always better than cure and the same holds correct for aquaculture too. Monitoring pathogen at a different stage during aquaculture can help in protecting the strength of biosecurity. In this context, the development of simple, sensitive, reliable and rapid technique for the detection of the pathogens is an urgent requirement. Molecular techniques are potentially faster and more sensitive than traditionally used methods for the identification of the pathogen based on biochemical characteristics, serology and histology. PCR technology has revolutionized the science of diagnosis.

Different variants of PCR are available with the ability to detect even 1 copy number of the pathogen which is otherwise undetected in other techniques. PCR-based diagnostic means have been established for a number of pathogens disturbing aquaculture. During the last 15 years or so, molecular techniques have been progressively engaged to diagnose fish diseases. PCR based diagnosis protocols are available for almost all the OIE listed pathogens. The testing of SPF seed is carried out using PCR techniques only.  

Nowadays different variants of PCR such as LAMP, RPA etc. are available to facilitate field level sensitive and rapid diagnosis of pathogen without the requirement of any expensive machine based on isothermal amplification.

Monoclonal antibodies (MAbs) produced by hybridoma technology has contributed significantly to aquaculture. Monoclonal antibodies are being employed in disease diagnosis, pathogen classification, epidemiological analysis and development of vaccines.

Probiotics and Bioaugmentation
Applications of microbes as gut probiotics and water probiotics are gaining popularity in aquaculture to inhibit the proliferation of pathogens and maintaining healthy gut microbiota. The health of pond bottom is critical in aquaculture and water probiotics are used to reduce the organic load as well as the transformation of the toxic compounds such as ammonia into nontoxic form. Such a bioagumentation process is popularly practiced in shrimp farming nowadays.

Some of the other areas of health management in aquaculture include the development of the vaccine. Several types of research were carried out on the development of a vaccine based on Recombinant DNA technology. At this time, only one DNA vaccine for infectious hematopoietic necrosis virus (IHNV) is accessible within Canada only. RNAi based technology has shown to provide protection against pathogen but still commercial level it is not present. Even though such findings are not applied to a scale as required but future aquaculture has to adopt this for overcoming the fear of disease outbreak.

Microbial studies in aquaculture are intended to understand the benefits as well as the harmful effect of microbes on culture animals either it is fish or any other culture group such as crustacean and molluscs. In this perspective, metagenomics can present a better understanding of microbial association with the animal in different circumstances such as healthy or diseased, stunted or fast-growing, etc. by utilizing the genetic component of the sample from an organism or ecosystem. This section is intended to showcase some important potential applications of metagenomics in aquaculture.

(    A) Development of suitable probiotics
Identification of candidate probiotics is possible by utilizing the comparative microbiome study of a healthy and diseased animal. Metagenomics study reveals the in depth community composition of healthy and diseased animals and helps to find out putative potential probiotics bacteria from the healthy animal microbiome. Some of the study as present in the earlier section has shown the dominance of certain bacterial genera in the healthy animal sample and can be a candidate probiotics. A similar approach can be used to identify water probiotics by comparing the metagenome of different water bodies having good and bad quality water.

(     B)  In depth screening of antibiotic resistance gene from the aquaculture system
Antibiotic resistance is a big threat around the world and aquaculture is one of the foods producing sectors which is considered as one of the reasons for such resistance development due to indiscriminate use of antibiotics and also the aquatic system is considered as environmental hotspot of horizontal gene transfer as it receives all the kind of antimicrobials along with runoff water. The conventional method of AMR study is possible only with cultivable bacteria and hence it can generate very limited information on the prevalence of antibiotic resistance. Shotgun metagenomics offers the opportunity for in-depth screening for the presence of antibiotic resistance gene and hence it presents a better picture of AMR in aquaculture.

(     C) Bioprospecting of novel protein coding gene
The marine ecosystem supports huge microbial diversity of all the category starting from virus to bacteria and fungi and many more. All these can be an excellent source of many important lifesaving molecules such as enzymes, antibiotics, immunostimulants, etc. As the majority of the microbial population is non-cultivable hence a conventional method of microbiological study cannot be used. Here shotgun metagenomics can be used to generate in-depth genome information of the whole community which can be further used for bioprospecting of important gene coding for the novel's useful protein.

(    D) Amplicon metagenomics can be used to characterize the microbial composition of pond soil and water.

(     E)  It can be used as a part of the disease surveillance program to target all the pathogens in one go.

(     F)  Microbial composition analysis of biofloc and periphyton

Feed involves a major cost in aquaculture and the price of feed is increasing a rate much more than the growth rate of aquaculture, mainly due to reduced availability of fishes for fish meal preparation. In such a case fish meal can be substituted with other sources such as plant protein, poultry feather meal etc. The effect of these substitutions on metabolism should be studied. 

Nutrigenomics provide best approach to study these effects and hence will provide better understanding. Understanding the biochemical and metabolic pathways complicated in the use of dietary macro- or micronutrients and energy provided through feeds is beneficial for assessing the reply of organisms to nutrients obtained from different source, for optimizing dietary nutrient utilization and for diet development. Biochemical pathway or metabolism  of nutrients are regulated through one or several enzymes, a product of gene so to understand the effect of nutrients or interaction between nutrients and gene  it is necessary to study nutrition with a molecular approach. 

Nutrigenomics provides such an approach. The work of how genes and gene products interrelate with dietary signals to affect phenotype and, equally, how genes and their products metabolize nutrients is called nutritional genomics or “nutrigenomics”.  Development of molecular techniques such as quantitative PCR, DD PCR, next-generation sequencing etc. has served as a boost for nutrigenomics study. Nutrigenomics can help in precise feed formulation for the betterment of aquaculture.

Conclusion and future direction
Aquaculture is considered as a hope for growing population in terms of providing nutritional and economic security but same cannot be achieved without intensification. Biotechnology offers huge scope of integration with aquaculture for better future and accelerating the pace of blue revolution. Biotechnology can offer allot to aquaculture in bringing solution to several challenging hurdle such as quality seed, disease, balanced feed etc. Further intensive applied biotechnological research is the need of the current era for the betterment of aquaculture.

Note: Credit for writing above article goes to Dr. Sujit Kumar, Assistant Professor (Fish Biotechnology) Postgraduate Institute of Fisheries Education and Research, Kamdhenu University, Gandhinagar-India.

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