19 August 2016

Aquaculture is the sustainable farming practice of raising aquatic organisms for the purpose of human consumption. Aquaculture has rapidly grown in popularity and is now poised to be a major food source for the global community over the next decade. Common species farmed by aquaculture include fish, crustaceans, molluscs, marine plants and to a lesser extent, aquatic reptiles, amphibians, and other miscellaneous invertebrates.

Aquatic animal diseases are the most significant constraint to the development and management of aquaculture worldwide. The management of animal and plant health, for both ethical and economic reasons is attracting interest from parties across the world and has inspired a range of novel therapeutic and diagnostic applications.

Challenges in the Aquaculture industry

Capture fisheries, commonly known as ‘wild fisheries’, refers to general harvesting of naturally occurring living resources in both marine and freshwater environments. Capture fisheries have become increasingly unsustainable due to overfishing and aquaculture is expected to overtake capture fisheries in supplying the world’s protein requirements (FAO 2012). Moreover, aquaculture is the fastest growing food production sector in the world, overtaking livestock production, with an average annual growth rate of 6.3% since 2000 and currently accounts for approximately 47% of the world’s fish supply (FAO 2012).

Several challenges exist which now threaten the sustainability of aquaculture. These challenges notably include (Subasinghe et al, 2003):

  1. Combating diseases and epizootics,
  2. Broodstock improvement and domestication
  3. Development of appropriate feeds and feeding mechanisms,
  4. hatchery and grow-out technology,
  5. Water-quality management.

The rapidly developing aquaculture industry has been affected by outbreaks of infectious viral, bacterial, and parasitic diseases. Waterborne pathogens can spread at faster rates than in terrestrial systems (McCallum, Harvell & Dobson 2003), and oceanographic transport processes have the potential to transmit disease across vast geographic regions. For example, pilchard herpesvirus has been shown to have spread to more than 5000 km of Australian coastline using ocean currents (Whittington et al. 1997). As a result, infectious disease is by far the biggest killer of farmed fish with outbreaks capable of wiping out entire stocks. Not only does this result in massive economic losses, but it also requires the farmer to undergo costly decontamination of the associated facilities and equipment (Pillay & Kutty 2005).  In addition, climate change and severe extreme weather events are expected to potentiate the problem with the introduction of new pathogens to due to favourable environmental conditions.

Currently, the clinical signs for infectious diseases in fish are frequently non-specific and unfortunately cannot be used for direct diagnosis. Many of the traditional diagnostic techniques used are based on observation of pathological effects of viruses in cell culture although this has the notable problem of being highly laborious and time consuming (Fernandez et al. 2008). For the detection of bacteria, molecular biology assays including PCR and microarray detection have been utilised although these techniques can lack specificity or sensitivity and be expensive (Fernandez et al. 2008). Therefore, it is clear that there is a requirement for a rapid and sensitive detection assay for pathogenic microorganisms.

Viruses, aquaculture & aptamers

Many viruses targeting commercially grown fish cause very high levels of stock mortality and are able to spread rapidly throughout a population. The most important viral diseases affecting farmed fish worldwide are caused by different genera, mostly within the families: Rhabdoviridae, Nodavirida, Birnaviridae and Iridoviridae (Leong, 2008).

A popular example is Koi herpesvirus (KHV); a highly contagious virus that causes significant morbidity and mortality in common carp varieties. KHV is considered as one of the most serious threats to carp farming in Europe and Asia, having caused $billions in losses (Walker and Winton, 2011).

Aptamers are short strands of nucleic acids that serve to bind to a target with high affinity and specificity and as such, are commonly referred to as ‘synthetic antibodies’. Aptamer targets include proteins, cells, and even small molecules which are typically problematic to antibodies. In theory, almost any target molecule can be selected, with aptamers previously having demonstrated high recognition and binding capability to low molecular mass substances (Osborne and Ellington, 1997). As a result, aptamers serve as useful tools for the identification, separation and purification of molecules and have become popular in the biomedical space (Liu and Zhang, 2015). It is also possible to further enhance the stability and improve other molecular characteristics of aptamers by developing their resistance to ion intensity, temperature, and acidity-alkalinity (Liu and Zhang, 2015).

Aptamers have been used in virus research for the analysis of virus replication, as diagnostic biosensors, and as antiviral agents (Torres-Chavolla and Alocilja, 2009; James, 2007). A previous study has shown that aptamers inhibited viral infection at every stage of the viral replication cycle including viral entry, suggesting that aptamers might prevent infection in vivo (Binning et al., 2012)

Li et al. (2014) was able to developed DNA aptamers that bound to Singapore grouper iridovirus (SGIV), a major viral pathogen of grouper aquaculture with a high level of specificity, and reduced its replication in cultured fish cells. In fish, RNA aptamers have been also shown to provide protection against the viral hemorrhagic septicemia virus (VHSV) and the Hirame rhabdovirus (HIRRV) (Porntep et al., 2012; Hwang et al., 2012). Similarly soft-shelled turtle iridovirus (STIV), a major cause of severe systemic disease in farmed soft-shelled turtles has also been studied. Li et al. (2015) was able to develop DNA aptamers that bound STIV with a high level of specificity with aptamer QA-36 demonstrating a protective effect against STIV and inhibiting STIV infection in a dose-dependent manner.

These examples are indicative for the propensity of DNA aptamers as suitable antiviral candidates for controlling infections in marine aquaculture facilities. Aptamers can efficiently bind and inhibit many important viral enzymes, including reverse transcriptases and integrases and can also effectively bind to viral capsid proteins. Such binding inhibits the interaction between viruses and cellular receptors and prevents viral entry into the cell (Lakhin et al., 2013). Aptamers against cell-type specific protein markers can be conjugated to drugs for targeted delivery. This includes toxic and radioactive substances, drug loaded nanoparticles, liposomes, and Endogenous enzymes (Lakhin et al., 2013).

Aquatic toxins & aptamers

Aquaculture has also received substantial attention for containing different additives, contaminants, and toxins. Microcystin-LR, a class of toxins produced by certain freshwater cyanobacteria and the most toxic form of microcystins, is a major health concern due to their bioaccumulation in aquatic bodies. Eissa et al. (2014) successfully developed a highly sensitive and selective aptamer based sensor (Apta-sensor) which utilises an unlabelled DNA aptamer assembled on a graphene electrode for microcystin-LR detection. The Apta-sensor exhibited excellent selectivity for microcystin-LR with no detectable cross-reactivity to okadaic acid, microcystin-LA, and microcystin-YR. The apta-sensor also showed excellent recovery percentages once applied for the analysis of spiked water and fish samples (Eissa et al. 2014).

Similarly, toxins responsible for shellfish poisoning have also been addressed by aptamers. Brevetoxin-2 (a neurotoxin) and okadaic acid (causing diarrhoea) could both be detected in spiked shellfish using electrochemical biosensors as demonstrated by Eissa et al. (2013, 2015). Such aptamer based sensors will facilitate the routine detection of okadaic acid and Brevetoxin-2 in food samples which would reduce the chances of illness clinically described as neurological shellfish poisoning (Eissa  et al. 2015).

Monitoring of contaminants of industrial origin has also shifted from environmental samples to marine food (Kantiani et al., 2010). For example, the presence of the dye malachite green, an antifungal, antiparasitic, and antiprotozoan agent used in aquaculture since 1936, has been detected using a fluorimetric homogeneous aptamer-based assay in fish samples (Stead et al., 2010). The presence of residues of these compounds represents a risk to human health due to their toxicity with evidence suggesting carcinogenic and toxic effects on heart, kidneys and liver (Hashimoto et al., 2011). For this reason, monitoring of the presence of malachite green residues in aquaculture products is of fundamental importance to protect consumers.

A sensitive, specific method for the collection and detection of pathogenic bacteria was also demonstrated using quantum dots (QDs) with a fluorescence marker coupled along aptamers as the molecular recognition element by flow cytometry (Duan et al., 2013). The aptamers selected for Vibrio parahaemolyticus and Salmonella typhimurium were applied and Dual-color flow cytometry was developed for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium in real samples including shrimp (Duan et al., 2013).

Conclusions

Despite current progress in fish farming and aquaculture over the past decade, many outstanding needs still remain to be fulfilled. Several issues related to the interaction between viruses and their hosts must be investigated, as well as the improvement of biosecurity practices. A wide range of diagnostic tests, especially those for on-farm use, must be available to better control viral spread. Aptamers therefore offers an innovative approach to measure quantities of man-made chemical contaminants and biohazards (toxic microalgae, viruses & bacteria, biotoxins & PCBs) that can be used as an early warning system in aquaculture and as an environmental monitor to assess the good environmental status. The use of aptamers can also help prevent unwarranted outbreaks of viral diseases for enhanced regulation and better monitoring of diseases in accordance with the protocols of the OIE (Office International des Epizooties) to improve fish health management, food security, and aquaculture sustainability.

Aptamer Group

Aptamer Group takes a high-throughput approach using liquid handling robotics and dedicated researchers to identify aptamers against novel and significant targets. We are committed to finding the perfect aptamers to your target and use a proprietary selection technique to identify high affinity aptamers with specificity in as little as 3 months.

Aptamer Group is committed to the development of diagnostics/test procedures that are simple, robust, safe, and reliable enough to be performed routinely on site to improve testing and quality control throughout aquaculture value chains. Moreover, Aptamer Group’s biomarker discovery, diagnostic and therapeutic divisions aim to conduct further research by focusing on current challenges and combining with innovative approaches to drive industry standards higher.

References

Eissa, S., Ng, A., Siaj, M., Zourob, M. (2014) Label-free voltammetric aptasensor for the sensitive detection of microcystin-LR using graphene-modified electrodes. Analytical Chemistry 86(15):7551-7

Eissa, S. Siaj, M. Zourob, M. (2015) Aptamer-based competitive electrochemical biosensor for brevetoxin-2.Biosensors & Bioelectronics 15 (69): 148-54

FAO (2012) The State of World Fisheries and Aquaculture 2012. Rome,Italy.

Fernandez,L., Alvarez, B., Menendez,A., Mendez,J. Guijarro, JA., (2008). Molecular Tools for monitoring infectious diseases in aquaculture species. Dynamic Biochemistry, Process Biotechnology and Molecular Biology.

Hashimoto,JP. Paschoal, JAR., De-Queiroz,JF., Reyes,FGR. (2011) Considerations on the Use of Malachite Green in Aquaculture and Analytical Aspects of Determining the Residues in Fish: A Review. Journal of Aquatic Food Product Technology 20(3):273-294

Kantiani, L.; Llorca, M.; Sanchis, J.; Farre, M.; Barcelo, D. Emerging food contaminants:A review. Anal. Bioanal. Chem. 2010, 398, 2413–2427.

Leong JC. Fish viruses. In: Mahy BWJ, Regenmortel MHV, editors.Encyclopedia of virology. Oxford: Academic Press; 2008. p. 227-34.

Li, P., Zhou, L., Yu, Y. Yang, M., Ni, S. Wei, S. Qin, Q. (2015) Characterization of DNA aptamers generated against the soft-shelled turtle iridovirus with antiviral effects. BMC veterinary research

Liang,H., Zhou, W., Zhang,Y., Qiao,Q. (2015) Are fish fed with cyanobacteria safe, nutritious and delicious? A laboratory study. Scientific Reports. (5),15166.

Stead, S.; Ashwin, H.; Johnston, B.; Dallas, A.; Kazakov, S.; Tarbin, J.; Sharman, M.; Kay, J.;Keely, B. An RNA aptamer based assay for the detection and analysis of malachite green and leucomalachite green residues in fish tissue. Anal. Chem. 2010, 82, 2652–2660.

Subasinghe,RP., Curry,D., McGladdery,SE., Bartley,D. Recent Technological innovations in Aquaculture.(2003)

Walker, P. J., & Winton, J. R. (2010). Emerging viral diseases of fish and shrimp. Veterinary Research, 41(6), 51. http://doi.org/10.1051/vetres/2010022Lakhin, A. V., Tarantul, V. Z., & Gening, L. V. (2013). Aptamers: Problems, Solutions and Prospects. Acta Naturae, 5(4), 34–43.