MC Remany 1 *, Daly Cyriac 1, P Krishnakanth Varada Raju 1, Sruthi Prem OC 1, AK Panda 1, Jaideep Kumar 2 & YC Thampi Samraj 2

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1 Indian Journal of Experimental Biology Vol. 53, October 2015, pp Effect of preservatives, temperature and storage duration on stability of nucleic acids of pleopod tissues of Penaeus vannamei (Boone) and screening of viral infections MC Remany 1 *, Daly Cyriac 1, P Krishnakanth Varada Raju 1, Sruthi Prem OC 1, AK Panda 1, Jaideep Kumar 2 & YC Thampi Samraj 2 1 Aquatic Quarantine Facility for L. vannamei, Rajiv Gandhi Centre for Aquaculture, MPEDA (Ministry of Commerce and Industry, Govt. of India) TNFDC Hatchery Complex, Neelankarai, Chennai , Tamil Nadu, India. 2 Rajiv Gandhi Centre for Aquaculture, Technology Transfer Training & Administrative Complex, MPEDA (Ministry of Commerce and Industry, Govt. of India), Sirkali, Nagapattinam , Tamil Nadu, India. Received 16 April 2014; revised 02 July 2014 In shrimp farming, screening for economically significant viral pathogens in nucleic acids of shrimps is vital for disease surveillance programmes and further, to take necessary precautions to ensure the sustainability of the farms and thereby the shrimp industry. Different preservatives, temperature and storage durations of the pleopod tissues of Penaeus vannamei broodstock were tested to investigate its effect on the quality and quantity of the nucleic acids. The pleopods were subjected to two preservation regimes and the yield and stability of the extracted nucleic acids were monitored over a time period of 12 months. Stability of the nucleic acids was assessed with nested polymerase chain reaction, and the yield was checked spectrophotometrically. Data was analysed by performing two way ANOVA and Tukeys Paired test. Preservation treatments included storage at 20 C and 5 C in RNAlater TM and in 70 % ethanol. Significant variation (P <0.05) was observed in both DNA and RNA yield and stability from ethanol and RNAlater TM stored pleopods at 5 C. However, the yield and stability did not differ (P >0.05) in both the preservatives at 20 C. The RNA was degraded and yielded lesser quantity when pleopod tissues were stored in ethanol at 20 C than when stored in RNAlater TM during storage duration of 9 months. This study would help the shrimp farmers and researchers to adopt better preservation strategy, vital for shrimp disease surveillance programmes and for traceability studies in the event of any disease outbreak. Keywords: Broodstock, DNA yield, Litopenaeus vannamei, Prawn culture, RNAlater TM, Shrimp diseases. Shrimp nucleic acids are of paramount importance in the context of disease diagnosis, health status assessment and for disease surveillance in farms and hatcheries 1. Increasing culture of the exotic shrimp species Penaeus vannamei (syn. Litopenaeus vannamei) in India since 2009, further enhanced the need for disease diagnosis both at hatchery and farm level to overcome the risk of disease outbreak. The vannamei farmers of our country depend on the imported broodstock for seeds generally procured from Southeast Asian countries and USA. Though the stock is quarantined and screened for economically significant pathogens by the Aquatic Quarantine Facility (AQF) 2, incidences of diseases such as White Spot Syndrome (WSS), Infectious Myonecrosis (IMN), etc., are not uncommon 3. Spreading of such *Correspondence: Phone: ; Fax: rgca_sbnpc@yahoo.com diseases through infected seeds or broodstock is another challenge 4. The tissues and extracts of nucleic acids of imported vannamei are stored to serve as reference to cross check samples in the event of disease outbreak 5. However, information either on preservation method for shrimp sample storage for disease diagnosis or on shelf life of samples for disease screening is scanty. Nucleic acids, both extracted freshly from fresh or preserved target tissues viz., muscle, gills, pleopods, and haemolymph or stored extract, are vital source for disease diagnosis by molecular methods 6. However, sample preparation, preservation and storage period are critical steps for absolute nucleic acid quantification 7. Taking pleopods or the swimming appendages do not require sacrificing of the animal and hence, are the apt tissue for disease screening in brooders as it saves the valuable broodstock 6,8. Appropriate method of preservation and storage of the target tissues play a crucial role in diagnosis of

2 666 INDIAN J EXP BIOL, OCTOBER 2015 diseases. In this study, we explored suitable preservation strategy for preservation of pleopod tissues, which are commonly used as target tissues for disease screening. Materials and Methods The exopods of the fourth pleopod were used as the target tissue for the study as per the standard operating procedures of AQF, the only Government approved facility in India which screens for known diseases in L. vannamei. These were severed from live shrimps with a pair of sterilized scissors and subjected to two preservation regimes (5 & 20 C) with two different preservatives, RNAlater TM and ethanol. The former is a commercial preservative which rapidly permeates into tissues to stabilize and protect cellular RNA and the latter is one of the widely used and recommended preservatives for tissue storage. RNAlater TM is known to preserve RNA up to 4 wk at 2-8 C, allowing transportation, storage, and shipping of samples without ice or dry ice 9. This storage reagent is also used in microarray and PCR studies 10. The two preservation regimes tested in this study were (1) RNAlater TM (Ambion) at 20 C and 5 o C; and (2) 70% ethanol at 20 C and 5 C. The tissues were stored for a maximum period of 1 year and the stability of nucleic acids were assayed at specific intervals of 3, 6, 9 and 12 months. The nucleic acids were extracted following the procedure described in IQ 2000 kit validated and certified by OIE (Office International des Epizooties). The quantitative estimation of the extracts was done spectrophotometrically by following standard procedures in a UV spectrophotometer (Unicam UV 300) and qualitative assessment for the stability of the nucleic acids was ascertained by nested PCR method. Measured DNA and RNA yield in the sample was expressed in ng per mg of the pleopod tissue. The nucleic acid extracted from fresh tissues or at day 0 served as the control. A total of 25 pleopod samples were randomly taken from both male and female broodstocks among the two treatments and the control. PCR screening was conducted for four important viral pathogens of industrial concern, which are categorized as C-1 & C The viruses screened were RNA viral groups: (1) Taura Syndrome Virus (TSV), (2) Infectious Myonecrosis Virus (IMNV); and DNA viral groups: (3) White Spot Syndrome Virus (WSSV) and (4) Infectious Hypodermal Haematopoetic Necrosis Virus (IHHNV). Of these, WSSV and TSV belong to C-1 and IHHNV fall under C-2 category. The IMNV viral group is included in both C-1 and C-2 categories. The extracts on quantification were subjected to nested PCR using specific primers designed (Farming Intelligene Tech. Corp., USA) for screening each viral disease and recognized by the OIE. The extracts were used at a concentration of 20 and 50 ng.µl -1 for DNA and RNA, respectively and subjected to nested PCR as described in the manual 12. Both the extracts and standards were diluted in yeast trna. For RNA, 2 µl of the extract was mixed with 7 µl of First PCR Premix, 0.5 µl of RT enzyme, 0.5 µl of Taq DNA polymerase (2U/µl), followed by first run of 15 cycles of amplification. The single step PCR products were further amplified by the addition of 1 µl of Taq DNA polymerase (2U/µl) and 14 µl of Nested PCR premix, which was subsequently subjected to 30 cycles of amplification. Similarly for DNA, 7.5 µl of First PCR Premix and 0.5 µl of Taq DNA polymerase (2U/µl) was added to 2 µl of the extract and amplified for 15 cycles. This was subjected to nested PCR of 30 cycles after the addition of 1 µl Taq DNA polymerase (2U/ µl) and 14 µl of nested PCR Premix. The total reaction volume used for the PCR was 25 µl. Dilutions of plasmid DNA and RNA containing 2000, 200 and 20 copies (supplied with the kit) were prepared and used as positive control and yeast trna was used as the negative control. Amplifications of the extracted viral nucleic acids were performed in a UNI IQ programmed thermal cycler (Invitrogen) specifically designed for a better-than-gradient approach to PCR optimization with a precise control over six temperature zones. The conditions followed for amplification were: initial heating at 42 C for 30 min and at 94 C for 2 min, followed by 15 cycles of denaturation at 94 C for 20 s, annealing at 62 C for 20 s and extension at 72 C for 30 s with a final extension at 72 C for 30 s and at 20 C for 30 s. The nested PCR reaction included 30 cycles of denaturation at 94 C for 20 s, annealing at 62 C for 20 s and extension at 72 C for 30 s with a final extension of 72 C for 30 s and at 20 C for 30 s. The PCR products were then separated in 2 % agarose gel, stained in ethidium bromide (Sigma) and the results were documented using a gel documentation system (Vilber Lourmat QUANTUM ST4 imaging systems). Statistical analysis The data on total yield of the extracted nucleic acid resulting from different preservation and length of storage periods were subjected to two way ANOVA analysis with 5

3 REMANY et al.: EFFECT OF PRESERVATIVES, TEMPERATURE AND STORAGE DURATION 667 replicates (GraphPad Prism 6.00) 13 and were subsequently compared with the control to determine the long term effects of different preservation techniques. Tukey s Pairwise Comparison test was used to investigate significant differences. In all cases significance was accepted when P <0.05. Results and Discussion The total yield of bulk DNA extracted from ethanol (Et) and RNAlater TM (RL) stored pleopods at 5 C, over a storage period of 12 months ranged from ± to ± ng.mg -1 and ± 4.00 ng.mg -1 to ± ng.mg -1, respectively compared to the control DNA yield of ± 2.50 ng.mg -1. The abundance of DNA differed in the tissues when preserved in different preservatives as well as in the test runs at 5 C (Table 1). The RNA yield from Et and RL stored pleopods at 5 C, varied from ± 6.00 to ± 0.12 ng.mg -1 and ± 2.00 ng.mg -1 to ± 6.83 ng.mg -1, respectively in relation to the control RNA yield of ± 6.92 ng.mg -1. The yield of these nucleic acids too significantly differed in the tissues when preserved in different preservatives as well as in the test runs (Table 1) and was observed to be in the similar trend as that of the DNA yield when samples stored at 5 C. Pleopod stored in Et at 20 C yielded DNA in the range from ± 2.5 to ± 3.2 ng.mg -1 and those in the RL yielded ± 2.80 to ± 2.21 ng.mg -1, respectively compared to the control Table 1 Results of the analysis of variance (two-way ANOVA with interaction) of the mean yield of DNA and RNA in different preservation regimes Source df MS F P DNA yield in RL and Et at 5 C Interaction < Preservatives < Test Run < DNA yield in RL and Et at 20 C Interaction Preservatives < Test Run RNA yield in RL and Et at 5 C Interaction < Preservatives < Test Run < RNA yield in RL and Et at 20 C Interaction Preservatives < Test Run (243.75±2.50 ng.mg -1 ). At 20 C, only slight variation in yield was observed with the preservatives and the test runs (Table 1). The RNA yield from Et preserved pleopods at 20 C varied from ±2.00 to ±5.03 ng.mg -1 and those in RL yielded ±2.31 to ±6.00 ng.mg -1, respectively over a period of 12 months. The yield of RNA at 20 C varied only with the preservatives (Table 1) and not with the test runs as observed with the DNA yield. Different preservation techniques resulted in different DNA and RNA yields that varied with storage time (Fig. 1). The DNA yield from the Et preserved tissues at 5 C at specific month intervals varied significantly (P <0.05) between the control (fresh samples) and each month and also between each pair of test durations (Table 2). However, no significant variation in the yield (P >0.05) at 5 C was observed in RL stored tissues between the 6 th and 9 th month, 6 th and 12 th month and 9 th or 12 th month of storage (Fig. 1). DNA yield from samples stored at 20 C in Et and RL varied significantly (P <0.05) between 3 rd & 6 th month, 3 rd & 9 th month and 3 rd & 12 th month But no variation in the yield (P >0.05) was observed between the 6 th and 9 th month, 6 th and Fig. 1 Time-course of RNA and DNA yields (ng.mg -1 ) in pleopods stored in different conditions. Control samples obtained by analysing fresh tissues are reported as month 0.

4 668 INDIAN J EXP BIOL, OCTOBER th month and 9 th or 12 th month of storage in Et and RL preserved tissues at 20 C. Similarly variation was not significant between 3 month old and control tissue samples stored at 20 C and 5 C after preserved in Et and RL (Table 2). RNA yield showed significant variation (P <0.05) between the control and each test durations in tissues preserved in Et at 5 C. The yield also significantly (P <0.05) varied between each combination of test durations. RNA yield in tissue samples stored in RL at 5 C varied between the control and the each test duration except the third month. The yield was not significant between the 6 th & 9 th month and 9 th & 12 th month test duration. Variation in RNA yield was not observed when samples were stored at 20 C in ET between the control vs. 3 rd, control vs. 6 th, 3 rd vs. 6 th, 3 rd vs. 9 th, 6 th vs. 9 th, 6 th vs. 12 th & 9 th vs. 12 th months. The yield was significant (P <0.05) only between the control vs. 9 th, control vs. 12 th & 3 rd vs. 12 th month. Tissues stored in RL at 20 C yielded RNA with significant (P <0.05) variation only between the control vs. 12 th month. The later stored tissues yielded DNA with no significant variation (P >0.05) between the 6 th and 9 th month, 6 th and 12 th month and 9 th or 12 th month of storage (Table 2). This followed the same trend that was obtained with the RNA yield extracted from the pleopods stored in later at 20 C. The amplified extracts for each of the test viruses which were run on 2% agarose gel to compare band intensity also showed that the DNA extracted from tissues stored both in Et and RL at 20 C, did not degrade and remained in good condition for a period of 12 months in relation to the control (Figs. 2a & b). The quality of the DNA as measured by the amplification of house-keeping gene for both WSSV and IHHNV viruses at 848 bp was best preserved at 20 C in both preservatives. Storage at 5 C in ethanol showed degradation of DNA for both the viruses from 3 rd month onwards. However, degradation for both viruses was observed only on the 12 th month when tissues were stored in RL at 5 C (Figs. 2c & d). Degradation of DNA was faster at 5 C Table 2 Tukey s pairwise comparison test (α-0.05 level) on the yield of DNA and RNA on the type of preservation and storage duration Storage Period (pairwise comparison) DNA Yield RNA Yield (months) 20 C 5 C 20 C 5 C ET RL ET RL ET RL ET RL 0 vs a 2.2 a 3.4 a 14.0 a 6.4 a 2.4 a 74.4 b 12.8 a 0 vs b 20.0 b b 56.8 b 8.0 a 6.4 a b 38.4 b 0 vs b 15.0 b b 57.6 b 12.0 b 8 a b 48.8 b 0 vs b 18.0 b b 64.0 b 16.0 b 9.6 b b 64.0 b 3 vs b 17.8 b b 42.8 b 1.6 a 4.0 a 38.4 b 25.6 b 3 vs b 12.8 b b 43.6 b 5.6 a 5.6 a 70.4 b 36.0 b 3 vs b 15.8 b b 50.0 b 9.6 b 7.2 a 96.0 b 51.2 b 6 vs a 5.0 a 37.0 b 0.8 a 4.0 a 1.6 a 32.0 b 10.4 a 6 vs a 2.0 a 64.0 b 7.2 a 8.0 a 3.2 a 57.6 b 25.6 b 9 vs a 3.0 a 27.0 b 6.4 a 4.0 a 1.6 a 25.6 b 15.2 a Level of significance: P = 0.05 level.; a not significant; and b significant Fig. 2 Gel electrophoretic pattern of (a & b) WSSV and IHHNV viral DNA extracted from stored tissues in ethanol & RNAlater at 20 C; (c & d) WSSV and IHHNV viral DNA extracted from stored tissues in ethanol & RNA later at 5 C at durations of 0 month (control), 3 rd, 6 th, 9 th & 12 th months.

5 REMANY et al.: EFFECT OF PRESERVATIVES, TEMPERATURE AND STORAGE DURATION 669 Fig. 3 Gel electrophoretic pattern of (a & b) TSV and IMNV viral RNA extracted from stored tissues in ethanol & RNAlater at 20 C; and (c & d) TSV and IMNV viral RNA extracted from stored tissues in ethanol & RNA later at 5 C at durations of 0 month (control), 3 rd, 6 th, 9 th & 12 th months. in Et than the RL stored samples. RNA for both TSV and IMNV viruses degraded on the 9 th month of storage when tissues were stored in ET at 20 C. However, the quality of RNA as measured by the amplification of house-keeping gene for both TSV and IMNV viruses at 680 bp was best preserved only in RL at 20 C (Figs. 3 a & b). Quality of RNA for both the viruses reduced from 3 rd month of storage as evident in the band intensity obtained for tissues stored in ET at 5 C and from 9 th month in RL stored tissues at the same temperature (Figs. 3c & d). Degradation of RNA was observed to be faster when tissues were stored in Et at 5 C than those stored in RL. The gel visualization of the degradation of nucleic acid was in similar trend with the results of the spectrophometric measurements of the extracted DNA and RNA purity. The purity of the DNA and RNA extracted from fresh tissue (day 0) was 1.8 and 2.1, respectively. Degradation of DNA was not observed in Et and RL stored samples when stored at 20 C and the values were in the range and , respectively. Storage at 5 C in Et resulted in DNA degradation from the 3 rd month, whereas RL maintained the DNA purity up to 9 months. Preservation in RNA later at 5 C, decreased the purity of RNA to 1.5 from the 6 th month onwards whereas at 20 C, the purity declined to 1.5 only from the 9 th month. The Et stored samples had a lower purity value of RNA at 5 C on the 3 rd month and remained stable when preserved in RL upto 6 months. It is often difficult for the farms and hatcheries located in remote locations, to send the samples immediately to disease diagnostic laboratories, when there is a disease outbreak. Use of liquid nitrogen or ultracold freezer is not always possible and is complicated by thawing of the materials. The different preservatives and storage duration tested in the study on degradation of the nucleic acid provides concrete information on the best preservative method that a farmer or a hatchery operator can adopt using the minimum facilities. The use of RNAlater TM (RL) in preservation of microcrustaceans without significant degradation in RNA and DNA was reported in a study conducted by Gorokhova 5. The present study revealed that preservation of pleopod tissues in RL at 5 C or in a refrigerator, can keep the nucleic acid in stable form upto a period of 12 months without much change in the yield and stability of DNA and for 6 months in the case of RNA. This also provides an alternative to storage at 20 C or 80 C without the inconvenience associated with the purchase and maintenance of costly deep freezer units by small scale farmers. The results established that the 6 month old, later stored pleopod tissues at 5 C can be used for screening both DNA and RNA viruses which were detailed in the present study. The data obtained from the study would provide baseline information on nucleic acid degradation under long term storage which is vital for genotyping and epidemiological studies 14 for tracing the movement of exotic known pathogens in vannamei culture systems of the Country. Acknowledgements The authors are grateful to National Fisheries Development Board (NFDB), Hyderabad, and MPEDA, Ministry of Commerce & Industry, Govt. of India for financial support. References 1 Angelika Niemz, Tanya Ferguson M & David Boyle S, Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol, 5 (2011) Remany MC, Daly Cyriac, Nagaraj S, Babu Rao, Panda AK, Jaideep Kumar & Thampi Samraj YC, Specific pathogen-free assurance of imported Pacific white shrimp Penaeus vannmaei (Boone, 1931) in the Aquatic Quarantine Facility, Chennai. Curr Sci, 99 (2010) 1656.

6 670 INDIAN J EXP BIOL, OCTOBER Gunalan Balakrishnan, Soundarapandian Peyail, Ramachandran Kumaran, Anand Theivasigamani, Anil SK, Jitesh BS & Nithyamary Srinivasan, First report on White Spot Syndrome Virus (WSSV) infection in white leg shrimp Litopenaeus vannamei (Crustacea, Penaeidae) under semi intensive culture condition in India. AACL Bioflux, 4 (2011) Lightner DV, Virus diseases of farmed shrimp in the Western Hemisphere: A review. Panorama Acuicola Magazine, 16 (2011) 1. 5 Department of Animal Husbandry Dairying & Fisheries order, No.35029/13/2008-Fy (T&E). Standard Operating Procedures for Aquatic Quarantine Facility for L. vannamei, 02 June (2009). 6 Office International Des Epizooties- OIE. Manual of Diagnostic Tests for Aquatic Animals, 5 th edn (OIE, Paris), 2012, Elena Gorokhova, Effects of preservation and storage of microcrustaceans in RNA later on RNA and DNA degradation. Limnol Oceanogr Methods, 3 (2005) Lightner DV, Virus diseases of farmed shrimp in the Western Hemisphere (the Americas): A review. J Invertebr Pathol, 106 (2011) Qiagen, The RNAlater principle and procedure. RNAlater Handbook, 7 (QIAGEN GmbH, Germany), 2006, Mutter GL, Zahrieh D, Liu C, Neuberg D, Finkelstein D, Baker HE & Warrington JA, Comparison of frozen and RNAlater solid tissue storage methods for use in RNA expression Microarrays. BMC Genomics, 5 (2004) World Organisation for Animal Health (OIE), chapter 1.3, Diseases listed by the OIE. Aquatic Animal Health Code, 12 th edn (OIE, Paris), 2009, IQ2000 Detection and Prevention System (nested) Manual Graphpad Pradeep B, Shekar M, Gudkoys N, Karuna Sagar I, Karunasagar I, Genotyping of white spot syndrome virus prevalent in shrimp farms of India. Dis Aquat Organ, 78 (2008) 189.