Introduction

In shrimp farming, specifically, viral disease outbreaks in economically important species, such as Penaeus monodon, Litopenaeus vannamei, and L. stylirostris, among others, has led to significant losses in production across different geographical locations since the early 1990s. Viral disease outbreaks are one of the major constraints to aquaculture production and trade, affecting socio-economic development in this sector worldwide. The risk of disease losses is likely to increase as the shrimp sector continues to grow, with newer viral pathogens being identified every year. The economic loss due to disease outbreaks in the Asian region was estimated to be at least $1.4 billion USD in 1990. In China alone, approximately $1 billion USD in production costs was lost in 1993. The loss in 1996 due to the Yellow head virus (YHV) and White spot syndrome virus (WSSV) was estimated to be valued over $500 million USD in Thailand. Based on farm-level surveys of 16 Asian countries, the annual loss in aquaculture production due to disease and environment-related problems was estimated to be more than $3 billion USD. Major economic losses have also been estimated in other parts of the world. For example, in 1993, Ecuador lost 28,000 tons of shrimp due to an epizootic outbreak of Taura syndrome virus (TSV; Walker and Subasinghe 2000). Moreover, the incidence of viral infection in the wild brood stock, on which many shrimp aquaculture industries heavily rely, is of major concern and can impede progress in this sector. Prevalence of WSSV has been reported in shrimp captured from their natural environment in the coastal waters of Taiwan (Lo et al. 1997) and along the Atlantic coast (Chapman et al. 2004).

At present, there is no any cure for viral infections in crustaceans and the best management practice for shrimp aquaculture industry worldwide is to prevent the occurrence of viral disease to some extent. As an alternative approach to avoid incidences of viral disease, the shrimp aquaculture industry is currently placing a very high priority on obtaining specific pathogen-free (SPF) stocks of shrimp. Novel strategies, such as the use of recombinant vaccines and immunostimulants to control the occurrence viral diseases, would be highly desirable. However, invertebrates such as shrimp, which are devoid of a true adaptive immune response system, respond only by non-specific innate immune mechanisms (Chang et al. 2003). Shrimp viral defense mechanisms are not well understood. Nonetheless, recent reports indicate that studies are being aimed at the identification and characterization of immune response genes in order to have a better understanding of the mechanism involved in host–pathogen interactions and the innate immune system of shrimp. For example, an antiviral gene, PmAV (Luo et al. 2003), as well as hemocyanin (Zhang et al. 2004) and interferon-like protein (IntlP; He et al. 2005) have been identified in penaeid shrimp. More recently, the identification and up-regulation of the Rab (PjRab) gene in virus-resistant shrimp indicates a novel Rab-dependent signaling complex, triggering a phagocytic defense against viruses in crustaceans (Wu et al. 2008). However, these studies are constrained by the lack of in vitro studies in cell lines of shrimp origin and absence of shrimp genome information.

Recently, the potential of RNAi as a powerful tool for functional genomics has been realized and is now being explored as disease prevention strategies for the therapeutic treatment of viral infections (Lu et al. 2005; Tan and Yin 2004). In the present review, we discuss the application of RNAi technology to shrimp, which will elucidate an improved understanding of the immune mechanisms present in these invertebrates, as well as effective therapeutic control strategies of viral diseases in shrimp.

RNA Interference

RNAi is a cellular mechanism that is initiated by double-stranded RNA (dsRNA) and results in the sequence-specific degradation of target RNA (Fire et al. 1998). The dsRNA is cleaved by an RNase III family enzyme, the Dicer, into shorter fragments of ∼21-mer short interfering RNAs (siRNAs; Hammond et al. 2000). These siRNAs are incorporated into the RNA-induced silencing complex (RISC), which is a multi-component and multi-turnover nuclease enzyme complex (Hammond et al. 2000; Hammond et al. 2001; Martinez et al. 2002; Haley and Zamore 2004). RISC, upon incorporation of a siRNA duplex, is activated by ATP and leads to the unwinding and eventual strand separation of the siRNA duplex. The unwound antisense siRNA strand is then used by RISC to find the target mRNA to induce its endonucleolytic cleavage (Wadhwa et al. 2004). This gene-silencing mechanism, which is universally conserved, is a well-known phenomenon that exists in many eukaryotes, including invertebrates (Brown et al. 2003; Denli and Hannon 2003). The sequence-specific inhibition of viral nucleic acids can be achieved either by introducing synthetic 21–23-nucleotide duplexes of RNA (Elbashir et al. 2001a) or by transcription of an expression construct (Brummelkamp et al. 2002), resulting in the silencing of the viral pathogens, thereby inducing an antiviral response at the molecular level.

The inhibition of replication of human pathogenic viruses, such as polio virus (Gitlin et al. 2002), HIV-1 (Novina et al. 2002), HCV (Randall et al. 2003), and hepatitis B virus (Giladi et al. 2003) by this RNAi mechanism is well documented, proving its effectiveness to suppress these viral infections. This suggests the possibility of using RNAi as an antiviral tool for viral disease prevention in shrimp aquaculture. In the shrimp aquaculture industry, white spot syndrome caused by the white spot syndrome virus (WSSV), is one of the most serious diseases. The outbreak of this viral disease is reported to result in 100% mortality within 7–10 days in commercial shrimp farms, leading to huge economic losses worldwide (Cai et al. 1995; Lightner 1996; Flegel 1997). Presently, in the absence of any efficient therapeutic mean to control this or any other viral infections in aquaculture, the use of RNAi technology in shrimp appears very promising. Although initial studies of RNAi focused on cellular mRNA targets, the present evidence and success achieved in targeting sequence-specific viral RNAs in many organisms (Randall et al. 2003) has initiated research in targeting viruses that affect aquatic animals. In shrimp, the use of both exogenously synthetic long dsRNAs or siRNAs have been reported to induce an antiviral response (Robalino et al. 2004, 2005; Tirasophon et al. 2005; Westenberg et al. 2005; Xu et al. 2007). The various gene constructs used in shrimp RNAi technology that result in RNA interference are shown in Table 1.

Table 1 Gene constructs used in shrimp RNAi technology resulting in RNA interference
Full size table

Strategy for Target Sequence Selection

siRNAs suppress gene expression by a highly regulated enzyme-mediated process involving multiple RNA–protein interactions. These interactions can lead to biased strand selection during siRNA–RISC complex formation and activation, which may contribute to the overall efficiency of RNAi. RNAi adapts a highly sequence-specific interference of gene expression, making it convenient to design siRNA-targeting specific viral genes, thereby, preventing undesired off-target effects that commonly occur with other pharmaco-therapeutic approaches. Hence, a very important and critical factor for effective siRNA design is the appropriate and effective selection of target sequences on the gene of interest, which leads to the identification of potent siRNA. The in silico analysis for target identification is an essential step for designing the siRNA. Currently, there are quite a few of on-line software available that assist in predicting the target site for a given gene. The position of the cleavage sites in the target RNA is defined by the 5′ end of the guide siRNA, rather than its 3′ end. There are now a set of rules that are taken into consideration when using computational approaches for designing siRNA and algorithms that incorporate the findings of Elbashir et al. (2001b) and Reynolds et al. (2004). For example, low G/C content, lack of inverted repeats and other features highlight the utility and the most accepted method of rational designing to enhance the gene-silencing effect of siRNA. Based on the findings from examining siRNA in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi (Elbashir et al. 2001c), the general guidelines to be followed for effective siRNA designing are: (1) duplexes of 21 nt siRNAs with 2 nt 3′ overhangs are the most efficient triggers of sequence-specific mRNA degradation; (2) Beginning with the AUG start codon of the transcript, AA dinucleotide sequences are scanned and the 3′-adjacent 19 nucleotides are considered as potential siRNA target sites; (3) An empirical target site (5′-AAN(19)UU) in the region of mRNA with 30–50% GC content is more active than those with a higher G/C content; (iv) siRNA duplexes with an antisense siRNA 3′ overhang of AA, CC or GG are less active when compared with UU; (v) Stretches of more than 4 T’s or A’s in the target sequence when designing sequences to be expressed from an RNA pol III promoter should be avoided as a 4–6 nucleotide poly(T) tract acts as a termination signal for RNA pol III, creating RNA molecules with a short poly(U) tail; (vi) If the target mRNA does not contain 5′-AAN(19)UU, an alternative target site, 5′-AAN(21) or 5′-NAN(21), is chosen; and (vii) The potential target sites are compared to the appropriate genome database to eliminate the target sequences having homology to other coding sequences.

dsRNA-mediated Gene Silencing in Shrimp

dsRNA-mediated viral infections in vertebrates lead to the recognition of dsRNA by Toll-like receptor 3 and RNA-dependent protein kinases for mounting antiviral responses (Matsumoto et al. 2004; Williams 2001) by the activation of the interferon system, initiation of apoptosis, and inhibition of cellular protein synthesis. This phenomenon of innate immune activation by dsRNA was thought to be restricted to vertebrates, as the genes encoding homologues of interferons and their receptors were not found in invertebrate genomes. Recently, however, the Toll pathway has also been identified as a vital part of the Drosophila antiviral response (Zambon et al. 2005). The relatively close relationship between insects and crustaceans, in addition to the recent finding of a toll receptor in shrimp, such as Litopenaeus vannamei (Yang et al. 2007), Penaeus monodon (Arts et al. 2007), and Marsupenaeus japonicus (Mekata et al. 2008), indicates a possible role of the Toll pathway in shrimp antiviral immunity and needs further investigation. Recent studies indicate possible uses of dsRNA for effectively blocking viral disease progression in shrimp against at least three unrelated viruses: WSSV (Robalino et al. 2005), TSV (Robalino et al. 2004), and YHV (Tirasophon et al. 2005; Yodmuang et al. 2006). Several reports suggest that the dsRNA induction of antiviral immunity in shrimp is through two pathways that utilize sequence-independent and sequence-dependent dsRNAs, as described below. These two pathways may most likely be cooperatively functioned in a systemic antiviral defense in shrimp (Yodmuang et al. 2006).

One of the first reports using dsRNA in shrimp demonstrated that the exposure of a marine shrimp (Litopenaeus vannamei) to dsRNA induces a general innate antiviral immunity in a sequence-independent manner. Those results further demonstrated that the antiviral immune system of shrimp against WSSV and TSV responded to dsRNA, regardless of its sequence and base composition. For example, each of the sequences derived from the vertebrate immunoglobulin genes of ducks and pigs, fish non-coding genomic DNA, and a bacterial vector sequence (pBeloBAC11 vector), induced protection against WSSV infection (Robalino et al. 2004). An unrelated long dsRNA corresponding to the green fluorescence protein (GFP) gene is also reported to provide higher survival rates against WSSV (Kim et al. 2007). Protection using unrelated dsRNAs such as GFP and TSV-polymerase was also achieved, though partially resulting in the inhibition of YHV by 50% and 17%, respectively (Yodmuang et al. 2006). However, the innate antiviral protection offered by dsRNA in shrimp was overcome when the virus was delivered at a high dose shortly after dsRNA injection (Robalino et al. 2004). Similarly, it was observed that in the case of YHV inhibition by non-specific dsRNA of GFP, there was a greater effect at 48 h than at 72 h post-infection, indicating that non-specific inhibition diminished relatively quickly due to the degradation of long dsRNA and there was a longer effect of the specific dsRNA than the non-specific one (Tirasophon et al. 2005).

It was observed that viral sequence-specific dsRNAs generated by in vitro transcription of sequences representing portions of the ribonucleotide reductase small subunit (rr2), DNA polymerase (dnapol), vp28 and vp19 from WSSV, affords significant and potent antiviral immunity in vivo when challenged with high doses of virus administered by injection and oral routes, as compared to a non-specific dsRNA. The degree of protection afforded by specific dsRNAs varies for different viral target genes, supporting the fact that target selection is an important factor for successful suppression of viral diseases by dsRNA (Robalino et al. 2005). For example, shrimp injected with dsRNAs corresponding to WSSV vp28 and protein kinase showed higher survival rates than those injected with dsRNAs corresponding to the WSSV vp281 and non-specific GFP gene (Kim et al. 2007). In case of YHV, dsRNA targeting the non-structural genes (protease, polymerase, and helicase) inhibited YHV replication in an effective manner, whereas dsRNA targeting the structural genes (gp116 and gp64) were the least effective in shrimp primary cell cultures, suggesting an important role of the copy number of the target sequences in viral inhibition (Tirasophon et al. 2005). Thus, these data indicate that not all viral-derived dsRNAs are effective in inhibiting viral replication via the RNAi pathway. Some of the non-structural genes are early genes that are essential, but are expressed at low levels during viral replication. Therefore, knocking down an early gene would be a more efficient strategy to inhibit viral infection by the RNAi mechanism (Yodmuang et al. 2006). The potency of the dsRNA on virus suppression is a dose-dependent phenomenon (Robalino et al. 2005). As reported with WSSV vp19 dsRNA, 1 μg of protease dsRNA was shown to efficiently inhibit YHV, while viral inhibition was drastically reduced in the presence of 0.2 µg (Tirasophon et al. 2005). Efficient prevention of viral propagation and protection of shrimp from mortality has been reported by the pre-administration of P. monodon with 25 µg of YHV-specific dsRNA-protease 24 h prior to YHV inoculation (Yodmuang et al. 2006). The dose-dependent effect of dsRNA has also been reported for YHV inhibition by Yodmuang et al. (2006). They showed that using 25 µg of virus-specific dsRNA (protease) was effective for complete inhibition of YHV replication. However, decreasing the dsRNA (protease) concentration to 5 or 1 µg resulted in the diminished efficiency of YHV inhibition to approximately 80% and 40%, respectively. The effect of dsRNA was observed to last for at least 5 days when shrimp were administered with YHV-specific dsRNA (protease) prior to YHV challenge, resulting in complete protection (Yodmuang et al. 2006).

The length of dsRNA also affects the RNAi efficiency, as observed in YHV suppression in shrimp cells, where a long dsRNA could generate more effective siRNAs than shorter ones (Tirasophon et al. 2005). These data imply that the involvement of RNAi-like mechanisms in the antiviral response of shrimp have stronger innate (non-specific) immune responses using a dose-dependent, sequence-specific dsRNA, rather than sequence-independent dsRNA (Robalino et al. 2005). The possible role of RNAi-like responses in L. vannamei was further tested using long dsRNA specific for two endogenous genes, hemocyanin and a novel CDP gene and checking for their expression patterns within hepatopancreas and gill cells, respectively. This revealed a substantial and specific depletion of the cognate mRNAs (Robalino et al. 2005) and suggested that the mechanism for transporting dsRNA into the cells (Feinberg and Hunter 2003) and the existence of cell surface receptors in shrimp mediate the uptake of dsRNA, as reported in Drosophila melanogaster (Ulvila et al. 2006).

For the first time, the therapeutic effect of dsRNA was reported in shrimp by curing YHV-infected shrimps. Viral replication could be prevented within 12 h, whereas shrimp mortality could be prevented using 25 µg of YHV-specific dsRNA-protease at 3 h post-YHV infection. Interestingly, non-specific dsRNA-GFP was unable to inhibit YHV propagation in a therapeutic mode (Tirasophon et al. 2007).

siRNA-mediated Gene Silencing in Shrimp

There are discrepancies in the results obtained using viral genes for siRNA constructs. This could be due to differences in the viral genes and gene fragments used in the RNAi assays. It is also emphasized that positive results are obtained only if the siRNAs are consistent with the design rules of RNAi (Xu et al. 2007). For example, WSSV vp19-siRNA delivered into shrimp was not successful in gene silencing and was a poor inducer of sequence-specific antiviral responses when compared to long dsRNA. This was attributed to improper design of siRNA, inadequate delivery into cells, limited half-life of siRNA in the extracellular environment of the shrimp, or existence of different mechanisms for uptake of siRNA (Robalino et al. 2005). It has been observed that even a one-nucleotide mutation in the siRNA gene construct results in a significant loss of the RNAi effect in shrimp (Xu et al. 2007). It is interesting to note that silencing of the non-structural viral protein genes of WSSV DNA pol, rr2 and thymidine kinase–thymidylate kinase (tk–tmk) increased the shrimp survival rate more than the silencing of the structural protein genes (vp24 and vp28; Wu et al. 2007). However, several studies on RNAi in shrimp suggested that the effective time of RNAi in vivo is limited (Wu et al. 2007; Robalino et al. 2004; Westenberg et al. 2005). This may be due to the short half-life of siRNA constructs, which prevents them from completing the process of viral replication inhibition in an infected cell, indicating the necessity for repetitive siRNA injections into the shrimp. For example, the complete eradication of virus from WSSV-infected shrimp was achieved by three injections of the vp28-siRNA. The antiviral response induced by siRNAs in shrimp is concentration dependent. The optimum concentration of vp28-siRNA was determined to be 8 µg in order to achieve an inhibitory effect of siRNA against virus replication (Xu et al. 2007). Whereas, Westenberg et al. (2005) reported that 10 µM of WSSV vp28-siRNA, when injected into shrimp, resulted in significant protection of P. monodon. However, this effect was reduced when shrimp were inoculated with an amount of siRNA ten times lower. In vitro studies showed that 5 µg of WSSV vp28 siRNA, when transfected into Sf21 cells, were able to specifically and efficiently silence the homologous gene (Westenberg et al. 2005).

Interestingly, as observed with large dsRNA molecules that can induce antiviral immunity in a sequence-independent manner (Robalino et al. 2004), similar results were obtained using sequence-independent siRNA in shrimp. For example, it was reported that shrimp injected with WSSV specific vp28 and vp15 siRNA showed a relative survival rate of 50% and 42%, respectively, which was not significantly different than the 33% relative survival rate observed in shrimp injected with non-specific GFP siRNA. This sequence-independent reaction is suggested to be due to poor intake of siRNAs by shrimp cells, resulting in their circulation in the hemolymph, thereby inducing a general defense response (Westenberg et al. 2005).

Non-specific Response Induced by dsRNA in Shrimp

siRNA and dsRNA are have been observed to induce silencing in numerous mammalian cell lines (Elbashir et al. 2001a; Paddison et al. 2002). The protein kinase R (PKR) and RNase L pathways are known to be activated by long dsRNAs in mammalian cells (Gitlin and Andino 2003). Presently, however, the mechanism of this innate immunity phenomenon in shrimp and other invertebrate taxa remains unknown. It is well known that the recognition of dsRNA by another pathway that involves RNAi is widely distributed among invertebrates as an important mechanism of the invertebrate antiviral response. Therefore, dsRNA can probably engage both innate immune pathways and an RNAi-like mechanism to stimulate potent antiviral immunity in a marine shrimp, as suggested by Robalino et al. (2005).

Higher survival rates of shrimp injected with long non-specific dsRNAs such as GFP (Kim et al. 2007), sequences derived from vertebrate immunoglobulin genes, fish non-coding genomic DNA, and bacterial vector sequence (Robalino et al. 2004) against viral infection indicate that long dsRNA can enhance the shrimp’s antiviral immunity in a sequence-independent manner. However, it has been reported that virus-specific dsRNAs offer higher protection than a non-specific GFP dsRNA and specific viral genes play a key role in offering higher degree of protection (Kim et al. 2007). These results indicate that dsRNA is recognized by the shrimp antiviral immune system as a molecular pattern irrespective of its sequence and base composition (Robalino et al. 2004). As mentioned earlier, it is well known that mammalian cells respond to the presence of dsRNA with a general, non-specific shutdown of translation, mediated primarily by PKR and RNase L. Presently, the pathway involved in the enhancement of antiviral immunity induced by long dsRNA is not understood and remains to be explored in shrimp. It is possible that there may be some evolutionary link between vertebrates and invertebrates leading to the recognition of dsRNA processes and innate antiviral immunity phenomena. For example, in mammals, the Toll-like receptor (TLR) is involved in the recognition of various microbial components and dsRNA (Alexopoulou et al. 2001). This leads to the regulation of gene expression of the immune system by signal transduction. The recent findings of a Toll receptor in shrimp (Yang et al. 2007; Arts et al. 2007; Mekata et al. 2008) and other shrimp genes such as ubiquitin protein, interferon-like protein, transcription factor AP-1, protein kinase raf and the peptides sharing certain similarities with the components of the complement and cytokines system (He et al. 2005), suggest the possibility of the existence of a similar antiviral immunity in response in invertebrates.

Counter Defense Mechanism to RNAi by Shrimp Viruses

Direct evidence of inhibition of the gene-silencing mechanism due to virus-induced phenomenon has been reported in shrimp. WSSV-mediated silencing of hemocyanin and homologue of the Signal Transducer and Activator of Transcription (STAT) was found to be tissue specific in hepatopancreas. This indicates that the shrimp viruses have the ability to impair the RNAi mechanism within the host (Robalino et al. 2007). The use of high doses of virus in the challenge has also been observed to result in a diminished RNAi-dependent protection mechanism offered by dsRNA in shrimp (Robalino et al. 2004). The loss of dsRNA-protease potency results in increased mortality rates and was observed at 24 h post-infection in YHV-infected shrimp (Tirasophon et al. 2007). These data suggest that the RNAi is overwhelmed by the increased virus level in vivo due to rapid replication of the virus or by accumulation of certain viral proteins that suppress the RNAi mechanism. For example, the suppression of RNA silencing by the B2 protein encoded by the flock house virus (Li et al. 2002) and Nodamura virus (Sullivan and Ganem 2005), and the core protein encoded by the hepatitis C virus (Wang et al. 2006), clearly demonstrates a conserved counter defense mechanism of RNAi by many viruses. It remains to be seen if the shrimp viruses also encode B2 proteins, as well as their role as suppressors of RNA silencing.

Limitations and Future Prospects of RNAi Technology in Shrimp

RNAi is currently considered as a very promising strategy for viral disease control in shrimp, however, the delivery of dsRNA/siRNA into target cells and tissues remains a big challenge, at present. Even though most of the reports indicate that the delivery of dsRNA into shrimp by intramuscular injection (Robalino et al. 2004; Wu et al. 2007; Xu et al. 2007) is possible, injection of dsRNA is practically impossible in shrimp farming at the field level. Hence, there is an urgent need for further investigations to develop simple and effective routes for dsRNA/siRNA delivery into shrimp. One of the alternative approaches would be to use edible dsRNA producing bacteria, as used in the case of RNAi application in C. elegans. This method made use of bacteria that were engineered to be deficient in RNase III. This, when fed continuously to C. elegans, successfully resulted in obtaining RNAi phenotypes that could be maintained over the course of several generations (Timmons et al. 2001). Similarly, ingestion of bacterially expressed double-stranded RNA was shown to inhibit gene expression involved in regenerative processes in planarians (Newmark et al. 2003). A very similar approach has been recently demonstrated in shrimp. Sixty-eight percent and 37% survival was observed in shrimp, fed with pellet feed coated with inactivated vp28-dsRNA-induced bacteria and a vp28-dsRNA–chitosan nanoparticle complex, which were used as carriers to deliver the WSSV-vp28-dsRNA, respectively (Sarathi et al. 2008a). Chitosan is a natural polysaccharide derived from crustacean shells. Previous reports demonstrated that it has successfully been used in oral delivery of a construct expressing the β-galactosidase reporter gene into fish by encapsulating the DNA and incorporating it into fish feeds (Ramos et al. 2005).

To make siRNAs effective in inducing gene silencing, it is essential that siRNAs reach the cytoplasm of the target cell. Since the naked RNA cannot penetrate cellular lipid membranes (Li et al. 2006), it is therefore imperative that delivery of siRNA require carriers such as viral and non-viral vectors for effective transportation into the targeted cells, under in vitro and in vivo conditions. The use of viral systems result in high transfection efficiencies, however, their therapeutic applications is greatly limited due to safety concerns and high cost. In contrast, non-viral transfection agents such as cationic liposomes and polymer particles are simple, cost effective and safe. For example, polyethylenimines (PEI), which are a series of synthetic polymers, bear a high cationic charge density, providing the compactness to carry the DNA into complexes. PEI has been shown to have a high transfection efficiency in shrimp. The evaluation of gene transfer methods by microinjection, electroporation, and transfection reagent into shrimp zygotes revealed that the use of transfection reagent jetPEI had a higher efficiency of gene transfer, hatching rates, and gene expression. This transfection reagent could effectively transfect DNA into shrimp eggs, even in the presence of the shrimp jelly layer (Sun et al. 2005). This data indicates the probable use of transfection reagents having the capacity to perform large scale shrimp egg transfections with dsRNA or siRNA vectors as a means for gene delivery in the future.

A permanent shrimp cell line will greatly facilitate the work of gene transfer technology. In addition, it will lead to the understanding of the host–pathogen interaction at the molecular level, which is very critical in designing strategies, such as RNAi, for ensuring shrimp survival against viral infections. One of the earliest attempts on in vitro culture of shrimp cells was initiated in Taiwan (Chen et al. 1986). However, not much progress has been made and most of the research on shrimp cell culture is limited to the standardization of culture tissue and medium formulation (Nadala et al. 1993; Hsu et al. 1995; Tong and Miao 1996; Shimizu et al. 2001). These efforts have led to some successes, but a permanent shrimp cell line has yet to be established (Rinkevich 2005). At present, the techniques for introducing foreign genes into crustacean cells are underdeveloped and remain to be explored. Also the transfection of plasmid DNA into shrimp cells has been reported to be difficult (Shike et al. 2000). These are some of the reasons that impede the design of gene transfer strategies to create cell lines from shrimp. A modified retroviral vector has been successfully used to achieve foreign gene expression in cell cultures derived from Oka organs and the ovaries of the P. stylirostris. However, heterologous promoters of the Moloney murine leukemia virus and Rous sarcoma virus were used in this study to mediate reporter gene expression. Also, both Oka- and ovary-derived primary cell cultures were short lived to test the stability of transgene expression (Shike et al. 2000). The shrimp beta-actin promoter from P. vannamei has been used to construct an expression vector. This may find use in developing transgenic shrimps for expression of siRNA (Sun et al. 2002).

In conclusion, RNAi has been reported to be associated with viral resistance in shrimp indicating the possibility that this mechanism represents a type of sequence-directed immunity. The protection offered by specific dsRNA and siRNAs as a dose- and sequence-dependent phenomenon in shrimp indicates that the RNAi mechanism does exist in shrimp to counteract viral diseases. Therefore, it has considerable promise as a therapeutic approach and efficient strategy for shrimp virus control in the aquaculture industry. Presently, however, the use of RNAi technology in shrimp has indicated that the effective time of RNAi in vivo is limited due to the very short half-life of synthetic RNA duplexes. It is likely that siRNAs will not be present for complete elimination of virus replication in an infected tissue. In order to use RNAi technology in an effective manner to protect shrimp against viral diseases, it would be essential that future studies focus on increasing the stability of siRNA in shrimp. In addition, obtaining continuous and prolonged expression of siRNA by use of high-expression plasmids, virus-based vectors and non-viral transfection agents to introduce siRNA into shrimp remains to be explored. Moreover, a permanent shrimp cell line would be highly desirable, leading to a better understanding of the RNAi mechanism in shrimp.