R. Rajeshkannan, V. Vijayarahavan and V. Alamelu
Disease outbreak is a major impediment for the socioeconomic development of the aquaculture sector worldwide. New technologies and techniques for disease control must be developed and implemented to meet the increased demands of the expanding population. One such technology is RNA interference (RNAi).
RNAi is a recently discovered mechanism of post-transcriptional gene silencing in which double-stranded RNA (dsRNA) corresponding to a gene or coding region of interest is introduced into an organism, resulting in degradation of the corresponding mRNA.
Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to investigate the functional role of specific genes by reducing expression, without altering genotypes. The silencing effects could be used not only to study gene function, but also to identify drug targets and vaccine candidates as well as to control infectious disease by interfering with pathogen transmission, development and proliferation within the host.
In this article we summarize the current knowledge regarding the therapeutic applications of RNAi for developing alternative treatment strategies against infectious diseases in aquaculture.
RNA interference
RNA interference (RNAi) is a highly evolutionally conserved process of post-transcriptional gene silencing (PTGS) by which double-stranded RNA (dsRNA), when introduced into a cell, causes sequence-specific degradation of homologous mRNA sequences. RNAi as a mechanism of PTGS most likely evolved as a cellular defense strategy to eliminate unwanted nucleic acids (viruses and transposable elements) in plants, fungus and invertebrates, but is also widely employed in most eukaryotic cells as a mechanism to regulate the expression of endogenous genes.
Discovery of RNAi
The discovery of RNAi phenomenon was first observed by R. Jorgensen and his colleagues when plant biologists were performing experiments to enhance the hue of purple petunias. The introduction of a pigment-producing gene under the control of a promoter resulted in variegated or completely white flowers, rather than the expected deep purple colour. What was initially thought to be a peculiar effect in flowers was subsequently found to occur in fungi when scientists were attempting to boost the synthesis of orange pigment in Neurospora crassa. The phenomenon was first called co-suppression in plants and quelling in fungi.
RNAi in Ceanorhabdites elegans
The observation of RNAi in animals came accidently when Guo and Kemphues injected the antisense strand to block expression of the par-1 gene in the nematode Caenorhabditis elegans. The expression was disrupted but, upon performing their controls, they found that the sense strand also reduced the expression of that gene.
The involvement of dsRNA in gene silencing phenomena, however, was discovered by Fire et al. who found that dsRNA, but not single stranded sense or antisense RNA, mediated gene silencing in microinjected C. elegans. Subsequently, RNAi has been recognized as a highly conserved process encountered not just in unicellular protozoans and fungi but also in complex organisms such as plants and animals.Various approaches have since been developed for mammalian cells to obtain successful gene silencing. Some of the successful gene silencing approaches is listed in Table 1.
Table 1. Gene Silencing Approaches:
| Kingdom | Species | Silencing process | Induction stimulus |
| Fungi | Neurospora | Quelling | Transgene(s) |
| Saccharomyces pombe | RNAi | dsRNA | |
| Plants | Arabidopsis, Coffea canéfora, Nicotiana, Petunia | Transcriptional or Posttranscriptional gene silencing, co-suppression | Transgene(s) and viruses |
| Invertebrates | Paramecium | Homology-dependent gene silencing | Transgene(s) |
| Amblyomma americanum, Anopheles, Brugia malayi, Dugesia japonica, Hydra, Leishmania donovani, Schistosoma mansoni, Tribolium castaneum, Trypanosoma brucei, etc. | RNAi | dsRNA | |
| Ceanorhabditis elegans | RNAi, TGS | dsRNA, Transgene(s) | |
| Drosophila melanogaster | Co-suppression, RNAi, Transcriptional gene silencing | dsRNA Transgene(s) | |
| Vertebrates | Human, Mouse, Zebrafish | RNAi | dsRNA |
Applications of RNAi in aquaculture
As aquaculture is growing rapidly and poised to help in bridging the gap in the global supply and demand of aquatic animal food products, management of health and reducing losses due to disease in aquaculture is gaining high priority.
Recent advances involving the use of RNAi-based technologies promise alternative approaches for the stable silencing of genes in a variety of different animal species. Among its many applications, RNAi stands out as a powerful molecular tool to screen host genes involved in pathogenicity and other important biological processes, as well as to validate potential drug targets. In addition, silencing of viral genes stands out as a promising therapeutic approach for the development of antiviral strategies in organisms that can mount systemic antiviral RNAi response.
In this context, RNAi could help stop virus replication inside the host, reducing virus spread and, consequently, helps the control of a possible outbreak of the disease. With regard to parasitic infections, the analysis of gene function through RNAi could be used not only to investigate the interaction between host and parasite, but also to explore the parasite biology and the effects of knockdown on its survival. This could assist in the identification and validation of new anti-parasitic drug targets.
RNAi as an antiviral tool in fish
Viruses are recognized as the most numerous organisms in the marine environment. While viral diseases can cause problems among natural fish stocks, these infectious agents are devastating and costly in aquaculture where fish are confined and intensively reared. Viruses of lower vertebrates include a large number of viral agents, belonging to different viral families and genera, with RNA and DNA genomes, displaying different host specificities. The most important viral diseases affecting farmed fish worldwide are caused by different genera, mostly within the families: Rhabdoviridae, Nodaviridae, Birnaviridae and Iridoviridae.
Some of the most significant viral pathogens of fish are members of the family Rhabdoviridae, which are enveloped negative single-stranded RNA viruses. The diseases caused by those viruses are generally characterized as acute, hemorrhagic septicaemias affecting multiple organs. Two fish rhabdoviruses, Infectious haematopoietic necrosis virus (IHNV) and Viral haemorrhagic septicaemia (VHSV), have received special attention due to rapid progression of infection and high mortality, especially in farmed salmonids. Hirame rhabdovirus (HIRRV) is also another economically significant Rhabdovirus known to cause epidemics in farmed fish.
Nodaviridae, on the other hand, are a family of small nonenveloped, isometric riboviruses, with bipartite positive-sense RNA genomes. Piscine nodaviruses belong to the genus Betanodavirus, which are the causative agents of Nervous necrosis virus (NNV), a devastating neuropathological condition that causes high mortalities in a variety of cultured marine fish.
Members of the Birnaviridae have single-shelled nonenveloped capsids and genomes comprising two segments of double-stranded RNA. This family is mostly represented by the Infectious Pancreatic Necrosis Virus (IPNV), an acute contagious systemic disease that causes gastroenteritis and destruction of the pancreas in several species of freshwater and marine fish.
Iridoviruses are large double-stranded DNA viruses with an icosahedral capsid ranging from 120 to 350 nm in diameter. Members of the family Iridoviridae are an emerging group of viral pathogens that threaten the aquaculture industry, causing great economic losses throughout the world. Rock bream and Red sea bream iridovirus (RBIV and RSIV), as well as Infectious spleen and kidney necrosis virus (ISKNV) and Rana grylio virus (RGV), are some of the viral pathogens of fish caused by iridoviruses.
Considering the substantial economic, social and environmental impact of emerging viral diseases in aquaculture, a considerable amount of research has been undertaken on viruses that cause economically important diseases. However, despite the amount of investigation undertaken, there are few vaccines available for the prevention of many piscine infectious diseases, especially those of viral origin.
Therefore, a better understanding of viral replication mechanisms, as well as the determinants of virulence, is essential to assist on the development of effective prevention methods to inhibit virus replication in fish.
RNAi as a therapeutic tool in crustaceans
The long dsRNAs are processed in cytoplasm by the Dicer-2 RNase into siRNAs. The resulting siRNAs are taken up by the RNA-induced silencing complex (RISC). The duplexed siRNA which is bound to the Ago2 protein, the central component of RISC, is unwound, and the passenger strand rapidly dissociates. Finally, this complex is coupled to the target mRNA, based on specific complementary base-pairing, to induce endonucleolytic cleavage causing the degradation of mRNA molecules.
Disease Control
Aquaculture, and more specifically the shrimp industry, is often faced with outbreaks of disease caused by bacteria and viruses. The discovery of RNAi has enabled studies on immune mechanisms and the function of genes involved in the process of fighting bacteria in shrimp species. Mechanisms that prevent bacteria from proliferation are believed to be related with prophenoloxidase. Knockdown of the inactive precursor of prophenoloxidase by RNAi resulted in a significantly increased bacterial load in M. japonicus. In another study, the QM gene from L. vannamei, encoding a tumour suppressor protein, was knocked down, resulting in a dramatic decrease in prophenoloxidase transcripts and activity in shrimp haemolymph, while the mortality was significantly increased in a bacterial challenge test with Vibrio anguillarum.
Knockdown of p38 mitogen-activated protein kinases in L. vannamei by RNAi resulted in a higher mortality of L. vannamei under Vibrio alginolyticus and Staphylococcus aureus infection, as well as a reduction in the expression of three genes, encoding antimicrobial peptides, namely PEN4, Crustin and ALF2. The expression of MjGal, encoding for a galectin of Marsupenaeus japonicus, was upregulated mostly in hemocytes and hepatopancreas tissue, and the protein was bound to both Gram-positive and Gram-negative bacteria. MjGal-silenced shrimp had significantly higher levels of bacteria in the haemolymph than the control shrimp, which confirmed that MjGal plays a key role in the shrimp defence against bacterial infections. Furthermore, the role of M. japonicus crustin such as peptide (MjCRS) was examined in vivo by RNAi, and this research suggested that MjCRS is involved in antibacterial defence and might not have a critical function against viral infections.
Viral disease outbreaks are a major concern in the development of the shrimp aquaculture industry. Investigations into host–pathogen interactions might give new insights to viral infectivity and defence mechanism. RNAi also has been widely used as a powerful tool in identifying the genes that participate in viral infection as well as defence from the host.
One of the potential antiviral therapeutics to be considered is using RNAi to inhibit the replication of the RNA viruses as well as DNA viruses by knocking down virus-specific genes or downregulating host genes that are related to viral replication mechanisms. Rab7, a small GTPase protein of P. monodon and L. vannamei, plays a role in vesicular transport during viral infection. Silencing of the gene encoding Rab7 dramatically inhibited the replication of WSSV, yellow head virus (YHV), LSNV and TSV in infected shrimp.
The direct targeting of viral genes also allows us to inhibit viral replication in the host. This strategy has been demonstrated for targeting different kinds of viral genes, such as the genes encoding for viral coat protein in WSSV and TSV, or viral enzymes such as RNA-dependent RNA polymerase in LSNV. RNAi has also been used successfully to inhibit the replication of YHV. These results showed that dsRNA targeting nonstructural genes (proteases, polymerases, helicases) can effectively inhibit the viral replication, while the targeted structural genes were the least effective. While the studies on the applicability of RNAi to control the virus were mostly conducted on a small scale, the success of these experiments clearly showed that RNAi has enormous potential in inhibiting the replication of the virus and improve the survival of shrimp. In fact, researchers in Thailand have been succeeded in using RNAi by oral delivery against WSSV infection in P. monodon, leading to reduced percentages in cumulative mortality and delayed average time of death.
Sex Control
Using RNAi for sex control of shrimp is a remarkably practical application in aquaculture. The giant freshwater prawn (Macrobrachium rosenbergii) is considered an important freshwater shrimp in many countries, and due to their bigger size and higher economic value, male prawns are preferred over females. In this species, the androgenic gland plays a role in male sex differentiation, where a specific gene, namely insulin-like AG (Mr-IAG), is highly expressed.
In vivo silencing of Mr-IAG, by injecting the shrimp with Mr-IAG-specific dsRNA, at an early developmental stage of juvenile males induced a full and functional sex reversal of males into neo-females. Additionally, crossing neo-females with untreated males produced all male progeny. These experiments show that we can use RNAi in controlling the sex of giant freshwater prawn without changing the genetic structure of animals and therefore without creating transgenic animal species.
Limitations to RNAi in aquaculture
In general limitation of RNAi, there is some disadvantages to using RNAi in aquacultured organisms. In the case of shrimp, which are mostly reared in fresh water ponds and tanks, attempts have been made to deliver RNAi by soaking the shrimp in solutions containing in-vitro synthesized RNAi precursors. However, this method has not fully successful because the half-life of RNAi is very short when administered in-vivo.
In fish, delivery of RNAi in feed is ideal; however, fishes are reared in net pens and cages, which can result in the accumulation of uneaten feed at the bottom of the pond/lake/ocean, making it available to non-target organisms. In addition, this practice has already been shown not to be cost effective in open-water fish farms. The only effective treatment available for the disease is bathing the fish in freshwater, which involves transferring all the animals from a net pen into a freshwater-containing canvas liner and, after a period of three hours, releasing them back into the underlying net pen.
Despite its effectiveness, this procedure is not considered feasible because it is so time-consuming and laborious that it accounts for up to 20% of the production cost. Another way to deliver the RNAi is to incorporate them into additives already used in the vaccines and antibiotics commonly administrated to fish, thus avoiding new costs. However, the possibility that components of these substances could interfere with the stability and effectiveness of the RNAi precursors may prevent the use of such delivery methods.
Therefore, the delivery and stabilization of RNAi are critical hurdles to overcome the development of RNAi-based drugs. Due attention should thus be paid in the design of RNAi-based therapy.
Conclusion
RNAi acts in nature as a defense against harmful nucleic acids such as endogenous transposons and exogenous viruses. RNAi-based therapies might prove to be effective against viral infections. However, cellular delivery and stability are major hurdles to overcome the development of RNAi-based drugs.
Effective gene silencing depends on the biochemical activity as well as the charge of these nucleic acid drugs. In addition, the effectiveness of RNAi-based drugs depends on the delivery route, choice of target gene, target pathogen, and target tissue. Before RNAi based drugs find practical applications in the management of viral diseases in aquaculture, multiple issues must be addressed, including their impact on the environment, cost, and a deep understanding of RNAi at the molecular level. Despite these hurdles, RNAi provides the best opportunity for developing exciting new therapeutic approaches to the treatment of viral diseases affecting aquaculture.
Although RNAi has been successful against all major viruses known to affect aquaculture, the delivery and stability of these nucleic-acid based drugs in target cells remain important obstacles to the actual use of RNAi in the aquaculture industry. Future studies should focus on increasing the stability of siRNA in aquatic animals.
(The authors are working at Dr. MGR. Fisheries College and Research Institute, Tamil Nadu. Views expressed are personal.)
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