Sustainable Intensive Shrimp Farming: Development of Shrimp Farm Design and Operation Systems for Possible Control of Shrimp Diseases - Nyan Taw - International Aquaculture Consultant

In any aquaculture business, sustainability of a system improved profits. At present although biosecurity and BAqP are in place, more needed to be done. With emerging disease challenges innovated designs and operation systems are developing for sustainable production. One of the most important factors the investors, shrimp farmers and technicians need to be aware of is that whatever waste discharged into environment will come back to you in a form of disease sooner or later. Before mid1990s major threats to shrimp farming was mainly bacterial diseases. In Asia from late 1994 appearance of viral diseases such as white spot syndrome virus (WSSV) and a few others like yellow head virus (YHV), infectious myonecrosis virus (IMNV). In 2001 with availability of Specific Pathogen Free (SPF) Penaeus vannamei broodstock from Hawaii, the shrimp farming industry took off much faster.

Purpose: Although shrimp aquaculture in Sri Lanka has mainly been plagued by recurrent outbreaks of White Spot Syndrome Virus (WSSV), recent symptomatic and analytical field data of shrimps were incoherent with typical WSSV infections. As transboundary infection of new diseases is not uncommon in world shrimp aquaculture, the possibility of new diseases established in the country surfaced. Hence, the present study was aimed at evaluating the stock health of shrimps in Sri Lanka for additional six diseases commonly available in the Asian region, namely Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Infectious Myonecrosis Virus (IMNV), Taura Syndrome Virus (TSV), Yellow Head Virus (YHV), Acute Hepatopancreatic Necrosis (AHPND) and Necrotizing Hepatopancreatitis Bacterium (NHPB). Research method: Sampling was performed using broodstocks, postlarvae, cultured Penaeus monodon, wild P. semisulcatus, P. indicus and P. monodon from different locations and broodstock collecting areas in the country. Other crustacean species were also collected as possible carrier species. The presence of pathogenic organisms was identified by Polymerase Chain Reaction using respective IQ 2000TM test kits and a total of 2060 PCR tests were conducted. Findings: Of all the samples tested, no samples were positive for IMNV, TSV, YHV, AHPND and NHPB. However, confirmatory evidence was found in Sri Lanka for the presence of IHHNV with the prevalence of 31.3% in broodstock samples, 24.07% in hatchery-produced postlarvae and 18.03% in cultured shrimps. WSSV was also recorded with a prevalence of 10.34% in broodstock samples and 55.56% in cultured shrimp samples collected. None of the wild collected samples was positive for any of the tested diseases. Value: Reinstating the presence of WSSV in the Sri Lankan shrimp industry, provides confirmatory evidence of the presence of IHHNV in the country, highlighting the possibility of the emergence of new diseases in shrimp farming in Sri Lanka. Also, commonly suggested disease carriers seem to be not the major cause of disease spread during the sampling time. This study, therefore, provides much-needed information about the stock health of shrimps in Sri Lanka.

The aquaculture sector continues to be developed to support sustainable development in Indonesia. One of the efforts made to develop sustainable aquaculture in the fisheries sector is the application of sustainable technological innovations to increase aquaculture productivity. The Millennial Shrimp Farming (MSF) system for white shrimp cultivation has developed to achieve increased production that can be carried out on limited land and relatively small business capital. However, although the MSF system has been widely carried out, studies reporting on monitoring shrimp growth and viral diseases in the MSF system in Indonesia have not been widely reported. This study aims to monitor the growth of white shrimp and the presence of viral diseases during white shrimp culture using the MSF system in Indonesia. This study was conducted in 3 MSF ponds by calculating growth and identifying viral diseases, namely White Spot Syndrome Virus (WSSV), Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Infectious Myo Necrosis Virus (IMNV), and Acute hepatopancreatic necrosis disease (AHPND) with using the Polymerase Chain Reaction (PCR) method. Based on the results, the growth parameters observed on the Day of Culture (DOC) 30, 37, 44, 51, 58, 65, and 72 in the three ponds had an average weight above the standard weight compared to the Indonesian Nasional Standard (SNI 01-7246-2016) regarding the production of white shrimp in ponds with intensive technology. Meanwhile, the results of identifying viral diseases (WSSV, IHHNV, IMNV) and AHPND on DOC 1, 7, 42, 66, and 72 all showed negative results. The results above indicate that the cultivation of white shrimp with the MSF method showed optimum results for growth parameters, and no viral disease was identified during cultivation. Keywords: Litopenaeus vannamei, MSF, Virus, WSSV, IHHNV, IMNV, and AHPND

Pacific white shrimp (Litopenaeus vannamei) is one of important aquatic animals in Thailand making a high income per year. However, outbreaks of diseases caused by yellow head virus (YHV) and white spot syndrome virus (WSSV) infection result in tremendous economic loss. Nowadays, dsRNA-mediated RNAi becomes the most promising technique to control these viruses. It has been shown that the best target genes for efficient inhibition of YHV and WSSV are protease and ribonuleotide reductase small subunit (rr2), respectively. With the sequence specificity of this RNAi technique, one dsRNA can suppress only one virus. Therefore, production of one molecule of dsRNA (multi-targeted dsRNA) to effectively inhibit both YHV and WSSV is needed. In this study, two types of recombinant plasmid (pET17b-dspro-rr2 and pET17b-dspro-dsrr2) that can produce two different forms of multi-targeted dsRNA (one-stem and two-stem) in E. coli were constructed. The multi-target dsRNA was designed specifically to both protease gene of YHV and rr2 gene of WSSV. After production of dsRNA in HT115 for 4 hours, weight ratio of dsRNA-protease and dsRNA-rr2 from the one-stem was 1:1 while that of two-stem was 1:4. The potency of each multi-targeted dsRNA on viral inhibition and shrimp mortality reduction were investigated in shrimp. Shrimp were injected with one-stem or two-stem into hemolymph before receiving YHV or WSSV. The results showed that one-stem and two-stem could inhibit both viruses (either separate or dual infection) however one-stem was more effective than two-stem when shrimp were infected by WSSV. One-stem could be maintained in shrimp hemolymph at least 5 days without loss of inhibitory effect whereas two-stem lost suppression level against YHV when dsRNA was 5 days pre-injected into shrimp. This study provides an essential information to design and achieve an effective multi-targeted dsRNA for multiple genes silencing in shrimp and reveals the potential anti-YHV and WSSV strategy for further application in the shrimp farm.

Shrimp laminin receptor binds with capsid proteins of two additional shrimp RNA viruses YHV and IMNV

White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD), has been responsible for most shrimp production losses around the world since the early 1990s. Previous research has focused mainly on the characterization of WSSV genomic variation to gain a better insight in the evolution and spread of the virus at the regional and global levels.Although WSSV genetic variation at small spatial scales has been described, the question is whether there is a correlation between this genomic variation and shrimp farming practices and disease outbreak at the local and farm level. Therefore, the present research has been carried out to describe in detail WSSV genetic variation over space and time and to estimate transmission routesof WSSV genotypes in ponds with different farming regimens. Different molecular markers, natural variation contained within the WSSV genome, were tested to quantify these relationships in semi-intensive, extensive and rice-shrimp farming systems within the Ca Mau and Bac Lieu provinces, both in the Mekong Delta, Vietnam. The outcomes of the research showed that (i) there are differences in genetic structure of WSSV populations in shrimp culture areas, with more variation found in variable number tandem repeat (VNTR) regions, short adjoining repeat sequences, than in genomic regions in which large deletions occur; (ii) a correlation betweenWSSV population structure, disease outbreak status and pond farming system was observed, providing evidence that the WSSV VNTR structure (in particular the number of repeat units in ORF94) statistically correlate with disease outbreaks and to a lesser extent to farming system; (iii) mixed-genotype WSSV infections of shrimp are correlated with fewer disease outbreaks in ponds; and (iv) it might be possible to use molecular markers (ORF94 and ORF125) to predict the outcome of WSSV infections in shrimp ponds in the future. For field applications, these findings provide important information for the development of specific management strategies to control WSD.

The disease most dangerous for the cultivation activity is virus. Viruses are organisms subseluler that contain only nucleic acid (RNA or DNA) as genetic material. Koi Herpes Virus is one type of virus that causes mortality in cultured Cyprinids. KHV disease in Indonesia started in Blitar, East Java on March 2002 because the entry of imported koi fish that carry the virus KHV, while mortality prosentase could reach 80% - 85%, which causes loss of about 5 billion rupiah. In addition of KHV, there are several types of viral diseases in shrimp is White Spot Syndrome Virus (WSSV), Taura Syndrome Virus (TSV), dan Yellow Head Virus (YHV). Disease can cause losses in farming activities, such as WSSV. WSSV is an endemic disease since 1995. disease WSSV is exotic viral disease that attacks the shrimp monodon in 1998/1999 has resulted in decreased production of very large, so the Indonesian shrimp exports down 33,000 tons. Treatment of viral diseases is difficult because the virus resistant to certain antibiotics and chemical compounds. Therefore, prevention needs to be done, one through the monitoring activities conducted on the northern coast of East Java. The method implemented is monitoring in location and identification of viruses by PCR (Polymerase Chain Reaction). Monitoring in location includes water quality measurements and sampling. Identification of virus carried by IQ 2000TM. The identification procedure includes extraction, amplification and electrophoresis. Regional monitoring conducted on the northern coast of East Java includes Gresik, Lamongan, Tuban, Bangkalan, Sampang, Pamekasan, and Sumenep. Water quality at locations quite well. Results activities of monitoring on the northern coast of East Java is disease White Spot Syndrome Virus (WSSV) was found positive in several locations: Gresik, Lamongan and Tuban, while the virus Taura Syndrome Virus (TSV) and Yellow Head Virus (YHV) was not found at all locations . In tilapia, disease Koi Herpes Virus (KHV) was found positive in Tuban.

Real-time reverse transcription polymerase chain reaction assay using TaqMan probe for detection and quantification of Infectious myonecrosis virus (IMNV)

White spot syndrome virus (WSSV) is one of the most threatening diseases in shrimp and othercrustaceans affecting global shrimp farming. Since firstly detected in Taiwan in 1992, the disease hasspread globally and followed with considerable socio-economic consequences. This research was performedto detect the WSSV infection in shrimp farming in Lombok Island’s (West Nusa Tenggara) using real-timepolymerase chain reaction. Samples of vaname (Litopenaeus vannamei) were collected from several shrimpfarming in Lombok. Results indicated that the spread of WSSV has reached shrimp farms in Lombok,especially in Lendang Jae, West Lombok. Therefore, a biosurveillance program is strongly recommendedto government to avoid and halt the spread of the disease in East Indonesia region .

White spot syndrome virus (WSSV) belongs to a new virus family, Nimaviridae, genus Whispovirus and contains a large circular double-stranded DNA genome of 292,967 bp. WSSV virions are ellipsoid to bacilliform, enveloped particles with a distinctive tail-like appendage at one end. They can be found throughout the body of infected shrimp. The virions contain one nucleocapsid with a typical striated appearance and 5 major and at least 13 minor proteins. WSSV, which was first discovered in Southeast Asia around 1992, is currently the most serious viral pathogen of shrimp worldwide. It causes up to 100% mortality within 7 to 10 days in commercial shrimp farms, resulting in large economic losses amounting to billions of US dollars across different countries to the shrimp farming industry. In a natural situation, shrimp become infected through both oral and water-borne routes, and the gills are thought to be a major point of viral entry. Considering the global economic and sociological importance of shrimp farming and its continued high growth, the development of novel control measures becomes necessary against the outbreak of WSSV. A number of strategies have been used to control WSSV, each with some limitations. Conventional control strategies such as improvement of environmental conditions, stocking of pathogen-free post-larvae, and augmentation of disease resistance by oral immune-stimulants or probiotics are currently employed to control WSSV infection. Use of recombinant viral proteins as vaccines that induce a specific immune response and protection has been demonstrated to control WSSV. Other studies have shown successful vaccination of shrimp with DNA vaccines that have prolonged effects. The RNA interference (RNAi) mediated silencing of targeted viral mRNAs holds tremendous potential for controlling shrimp diseases. The silencing of viruses using RNAi has been experimentally demonstrated for WSSV in shrimp by injecting or feeding synthetic siRNA, long double-stranded RNA (dsRNA), and short/long-hairpin RNA (shRNA/lhRNA) prepared by in vitro transcription or expressed in bacteria. In addition to targeting viral proteins, protection of WSSV has also been achieved by dsRNA targeted against shrimp PmRab7, a protein important for viral entry into the host cells. Antisense constructs offered strong protection in WSSV challenged shrimp, P. monodon, with a corresponding decrease in viral load. Antisense constructs expressing VP24 and VP28 offered the best protection with a consistent reduction in WSSV copy number in both cell culture and in experimental shrimp. The advantage of using antisense constructs is their lack of toxicity and immunogenicity and their high specificity towards the desired target. The usage of edible pellet feed coated with dsRNA against WSSV has shown promising results. Overall, the present investigation clearly demonstrates that it is possible to induce strong protection in shrimp against WSSV infection using host promoter-driven antisense constructs in controlled laboratory-scale experiments. However, it is important to develop a simple and efficient delivery system for extending this study to the field level.

A multi-target dsRNA for simultaneous inhibition of yellow head virus and white spot syndrome virus in shrimp

Economic loss due to diseases in Indian shrimp farming with special reference to Enterocytozoon hepatopenaei (EHP) and white spot syndrome virus (WSSV)

Simultaneous and rapid detection of white spot syndrome virus and yellow head virus infection in shrimp with a dual immunochromatographic strip test

Biosafety evaluation and detection of shrimp viruses on field samples using dual priming oligonucleotide (DPO) system based multiplex PCR assay

Viral infections are one of the major reasons for the huge economic losses in shrimp farming. The control of viral diseases in shrimp remains a serious challenge for the shrimp aquacultural industry, with major pathogens, such as the white spot syndrome virus, yellow head virus, Taura syndrome virus, hepatopancreatic parvovirus, and baculoviruses, being geographically widespread. In the absence of a true adaptive immune response system in invertebrates such as shrimp, one of the alternative and more specific approaches to counteract viral infections in shrimp could be the use of molecular-based gene transfer technologies, such as RNA interference (RNAi). The RNAi mechanism is initiated by double-stranded RNAs (dsRNAs), which are fragmented into shorter 21-23 nucleotides of short interfering RNAs (siRNAs) by a type III endonuclease, the Dicer. RNAi, which is mediated by small interfering RNA (siRNA), results in the sequence-specific post-transcriptional silencing of a target gene. This gene-silencing mechanism is universally conserved and is a well-known phenomenon that exists in many eukaryotes, including invertebrates. It has been recently extended to shrimp as an important potential tool in viral disease prevention. RNAi technology shows considerable promise as a therapeutic approach and efficient strategy for shrimp virus control in the aquaculture industry. Further progress in understanding the mechanism of siRNAs at the molecular level, as well as strategies to achieve their tightly regulated, stable, prolonged and tissue-specific expression in an effective manner, will definitely revolutionize therapeutic approaches for counteracting viral diseases of shrimp. In the present review, the recent development and potential use of RNAi in combating shrimp viral infections is discussed.