Deepak Agarwal1*, Ashraf Rathar2, A. Kathirvelpandian1, and S. A. Shanmugam1
1Institue of Fisheries Post Graduate Studies, TNJFU, OMR Campus, Chennai
2Sher-e- Kashmir University of Agricultural Science and Technology- Kashmir
Aquaculture is the culture of water bore plants and aquatic animals in an artificial environment mimics the natural habitat. It is an emerging science engaged in the food and nutrition security of the world. Due to the demand for fish across the globe, this science needs advancement to enhance the production and supply of nutrition to meet the increasing demand of the country. Because of that, to implement the blue revolution, a huge fund of ₹ 20,050-crore has been announced for the fisheries sector under PMMSY (Pradhan Mantri Matsya Sampada Yojana). To aid in the advancement and scalable production in aquaculture, biotechnology plays a significant role. Biotechnology is a rapidly developing field in biology through which several recent products and processes have emerged in the field of molecular biology, disease diagnosis and molecular biology. Massive reports are available on the role of biotechnology in aquaculture in term of transgenesis (AquAdvantage Salmon; Yaskowiak et al., 2006), selective breeding of fish for resistance to a specific pathogen (Das & Sahoo, 2012), species and stock identification through barcoding (Lakra et al., 2011) and molecular markers such as SSR (Sahu et al., 2013), sex control (Pandian & Sheela, 1995), hormone manipulation (Carolsfeld et al., 2000) and so on. In this article, we will discuss briefly the recent applications of biotechnology in aquaculture in terms of disease diagnosis and assisted reproductive techniques.
Introduction- SHERLOCK in Disease Diagnosis
One of the major constraints faced by aquaculture especially shrimp aquaculture is the loss due to viral diseases like WSSV, yellow head and Taura syndrome virus. The most commonly used method in disease diagnosis is Polymerase chain reaction (PCR). Several PCR based kits are available commercially to diagnose the presence of a specific pathogen in the fish sample (Yoshino et al., 2009). But it requires expensive equipment i.e. Thermocycler and skilled staff
however in some cases, the viral load is so low that even PCR is not sensitive enough. A team of scientists from McGovern Institute, Broad Institute and Harvard University recently developed a new innovative diagnostic method to detect nucleic acids using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene-editing technology. Recently, this technology has been harnessed for use as a diagnostic test.
Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), can be available to detect the specific pathogen in any aquatic species. Instead of cas9 used in CRISPER, a Cas13a enzyme is used here to binds and cleaves RNA through its “collateral cleavage” and it can be used as a diagnostic tool. SHERLOCK works by amplifying RNA using Recombinase Polymerase Amplification (RPA). RPA is a kind of polymerization which exploits recombinases enzymes to pair with the primers. Primers bind to the complementary target sequence and the resulting D-loop is stabilized through the binding of single-stranded DNA binding (SSB) protein. If the DNA target sequence is present, amplification is initiated by polymerase from the primer and progresses rapidly on an optimum temperature of 37-42°C with just a few nucleic acid molecules and the reaction finished in 3-10 minutes.
The amplified nucleotides are combined with the Cas13a nuclease, a guide RNA complementary to the target sequence, and a short nucleotide sequence coupled to a fluorescent reporter and a quencher. If the target pathogen sequence is present in the pool of amplified nucleotides, the guide RNA attaches to the target and the non-specific RNAse activity of Cas13a becomes activated.
In some circumstances, the CAs13 cuts any RNA encounters through a process called collateral cleavage. During this cleavage, Cas13 degrade the short nucleotide sequence coupled with the reporter molecule and release of quencher from the reporter resulting in activation of the fluorophore. Therefore, the fluorescent signal is used as an indicator to determine the presence of the target (Figure 1.). Furthermore, SHERLOCK reaction reagents can be lyophilized for long-term storage and be readily reconstituted on paper similar to a pregnancy test to provide a visual readout for filed application. Recently the SHERLOCK team has prepared a protocol for the detection of COVID-19 using CRISPR diagnostics (Zhang et al., 2020). Based on the same approach, the techniques can also be leveraged to detect the pathogens for the commercially important aquaculture species.
Assisted Reproductive Techniques
Induced breeding is one of the great achievements in the fisheries sector by Dr. Hiralal Chaudhary and Alikuni since 1957. It is a technique by which the commercially important fish are bred in the captive environment through artificial stimulation. One of the main prerequisites for a successfully induced breeding is the collection of gametes from the male and female fish. Hormone manipulation i.e. injection of synthetic GnRH and its analog is a common practice in induced breeding. Often, female fish produce a huge number of eggs, while the milt quantity from male fish is always a limiting factor. Milt is a collection of sperm obtained from male fish. Quality of milt is also important to achieve good fertilization rate and productivity. The concentration of live and motile sperm determines the quality of milt. Biotechnology can play a significant role to improve the milt quality by removing the non-motile or dead sperm for the milt through FACS (Fluorescent Activated Cell Sorting) by using some of the commercially available dyes. The involvement of biotechnology in breeding comes under assisted reproductive techniques (ART) which is now extensively incorporatedin the management of infertile animal species. However, this is now being used in the case of humans (Grunewald et al., 2009) and domestic animals but it can be used for the fish having a low fertilization rate. FACS is a specialized type of flow cytometer. It provides a method for sorting a heterogeneous mixture of cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. One of the earliest changes shown by apoptotic/dead spermatozoa is the externalization of phosphatidylserine (membrane phospholipid). FACS uses fluorophore-conjugated annexin V (phospholipid-binding protein) to separate non apoptotic/live spermatozoa from those with deteriorated plasma membranes and externalization of phosphatidyl serine. Thus annexin enables the identification of cells with altered membrane integrity based on annexin binding, annexin-negative (unlabeled-intact membrane; non-apoptotic) and annexin positive (labeled- altered membrane; apoptotic). The separated cells (live and motile sperms) are collected in a specific container andused furtherin induced breeding. There is an increasing demand for assisted reproductive techniques (ART) in aquaculture.
In spite, ART, the FACS can also be used in the production of monosex or male population in aquaculture. Some species of aquaculture like Tilapia is being used to produce the male population only for which hormone manipulation is a popular way. Through FACS, the sex sorting can be done by staining the sperm cells with a DNA-specific stain (Hoechst33342) which can bind to the adenine–thymine region of nucleic acids and the X chromosome-bearing sperm will adsorb more dye due to the difference in DNA content of X and Y chromosomes thus the sex shorting is done (Marzano et al., 2020).
Biotechnology is considered as one of the most promising areas to enhance fish production. Although there is a huge scope of biotechnology in aquaculture and in some areas the research is being done. Enough information is available in the public literature database. The recent techniques discussed in this article have limited research in humans and have not been used in the fisheries sector. The two methods described above are the very recent advances in biotechnology and have a great potential to bring Blue revolution in the country.
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