Journal of Invertebrate Pathology 121 (2014) 24–27
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First report on vertical transmission of a plasmid DNA in freshwater prawn, Macrobrachium rosenbergii Labrechai Mog Chowdhury a, P. Gireesh-Babu a, A. Pavan-Kumar a, P.P. Suresh Babu b, Aparna Chaudhari a,⇑ a b
Division of Fish Genetics and Biotechnology, Central Institute of Fisheries Education, Versova, Mumbai 400 061, India Central Institute of Fisheries Education, Kakinada Centre, Beach Road, Kakinada 533007, India
a r t i c l e
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Article history: Received 28 February 2014 Accepted 2 June 2014 Available online 13 June 2014 Keywords: Plasmid DNA Antisense RNA Vertical transmission Macrobrachium rosenbergii
a b s t r a c t Outbreak of WSSV disease is one of the major stumbling blocks in shrimp aquaculture. DNA vaccines have shown potential for mass scale vaccination owing to their stability, cost effectiveness and easy maintenance. Development of economically feasible delivery strategies remains to be a major challenge. This study demonstrates vertical transmission of a plasmid DNA in a decapod Macrobrachium rosenbergii for the ﬁrst time. Females at three different maturation stages (immature, matured and berried) and mature males were injected with a plasmid DNA and allowed to spawn with untreated counterparts. Using speciﬁc primers the plasmid DNA could be ampliﬁed from the offspring of all groups except that of berried females. For this conﬁrmation genomic DNA was isolated from 3 pools of 10 post larvae in each group. This presents an ideal strategy to protect young ones at zero stress. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction More than 90% of the global aquaculture production comes from Asia, where it contributes signiﬁcantly to livelihoods and national economies. Out of the total world aquaculture production of about 59.9 million tonnes, crustaceans contribute 9.6% by volume and 18% by value (FAO, 2012). Of the cultured decapod crustaceans, the marine black tiger shrimp Penaeus monodon and freshwater giant prawn Macrobrachium rosenbergii led the market share until 2010. Crippling losses due to white spot syndrome virus (WSSV) in the former and M. rosenbergii nodavirus (MrNV) in the latter have caused the aquaculturists in India and S.E. Asian countries to adopt alternative decapod species like Litopenaeus vannamei (FAO, 2011). However, there are reports of WSSV affecting the production of L. vannamei too in India (Balakrishnan et al., 2011). Effective and economically viable vaccination strategies and therapeutic approaches can recover and revive the culture of these important species. Since decapods lack an adaptive immune response like the one present in vertebrates, the standard vaccination strategies are not applicable. Nevertheless, the RNA ⇑ Corresponding author. Address: Division of Fish Genetics and Biotechnology, Central Institute of Fisheries Education, Versova, Andheri West, Mumbai 400 061, India. E-mail address: [email protected]
(A. Chaudhari). http://dx.doi.org/10.1016/j.jip.2014.06.001 0022-2011/Ó 2014 Elsevier Inc. All rights reserved.
interference pathway was recently shown to be active in shrimp, fuelling considerable effort towards developing RNAi based vaccines (Krishnan et al., 2009; Bartholomay et al., 2012). We have reported a DNA vaccine for WSSV capable of expressing long hairpin RNA (lhRNA) against vp28 envelop protein in vivo and providing 75% protection on challenge (Krishnan et al., 2009). Recently, Ahanger et al. (2014) reported successful use of antisense constructs. However, intramuscular injection of individual shrimp is not a feasible option in India where extensive farming is practiced. In this study, we have used a plasmid DNA to test the possibility of vertical transmission from the injected brooder to the offspring in the decapod M. rosenbergii at different maturation stages. Five maturation stages are recognized in M. rosenbergii females viz., immature, maturing, matured, berried and spent. In immature and maturing females, ovaries appear like ﬂabby off white or slightly orange mass of tissue and are not visible through carapace. In mature females, ovaries are well developed, orange in color and visible through carapace. Berried females carry fertilized eggs in the brood chamber between the swimming legs for incubation and subsequent hatching after 3–4 weeks. As the eggs hatch, the free-swimming larvae called zoeae are dispersed and undergo a number of microscopically distinct larval stages before metamorphosizing into post-larvae (PL). Three maturation stages of females (immature, matured and berried) were selected for the experiment.
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2. Material and methods
2.4. Breeding setup
2.1. Experimental animals
An initial dip treatment of formalin (500 ppm) for 5 min in aerated water was given to disinfect the brooders. The brooders were then transferred to FRP tanks of 300L capacity ﬁlled half with formalin treated (30 ppm) bleached seawater (7 ppt). In each tank, two to three brooders were maintained. They were fed with commercial prawn feed four times a day. Tank cleaning was done daily to remove the left over feed and 30% water exchange was done daily with fresh 7 ppt water until hatching took place. Immediately after spawning, the proto-zoea were collected and transferred to 13 ppt saline water in separate FRP tanks of 250L capacity ﬁlled half with formalin treated sea water. The temperature of the water was maintained between 28 and 30 °C.
A total of 40 male and female mature brooders of M. rosenbergii were procured from Balabadhrapuram freshwater ﬁsh farm, CIFE. The brooders were reared in FRP (Fibre-reinforced plastic) tanks (2 0.5 0.5 m) with aeration by providing commercial pelleted prawn feed (CP, India) for 2 days. Prior to use, the experimental animals were checked for the presence of any external abnormalities, disease symptoms, erratic swimming and lethargy. The animals were screened for the presence of WSSV and MrNV by PCR and only healthy animals were used for the experiment.
2.2. Antisense construct
2.5. Feeding zoea
The antisense construct against WSSV carried a 330 bp fragment of the vp28 gene of the virus in reverse orientation downstream to the Cytomegalovirus (CMV) promoter in pcDNA3.1 vector. Brieﬂy, fragment to be cloned was PCR ampliﬁed using primer set Vp28-F: AAATCTAGATGTGACCAAGACCATCGAAA and Vp28-R: AAAGATATCTGCACCATCTGCATACCAGTG containing linkers for XhoI and EcoRV restriction enzymes to facilitate directional cloning. Genomic DNA puriﬁed from WSSV-infected shrimp was used as template. The correct size of the ampliﬁed fragment was conﬁrmed using agarose gel electrophoresis, puriﬁed by Gel Extraction kit (Qiagen, Netherlands), and cloned into pcDNA3.1 vector following Sambrook et al. (2001). The positive clones were identiﬁed by colony PCR. The orientation of the cloned fragment was conﬁrmed by sequencing and the clone was named pCMVVP28AS.
The zoea were fed twice a day with artemia nauplii until 6th stage at the rate of 5 artemia nauplli per zoea. From 6th stage onwards artemia ﬂakes were also included in the diet. For 7th, 8th and 9th stages egg custard was included once in the daily diet. Conversion from one stage to the next took 2 days on average. The post larvae (PL) were obtained in 19–20 days.
2.3. Experimental design Four different treatments were designed to study the vertical transmission of DNA vaccine (Table 1). First three treatment groups include intramuscular injection of the plasmid DNA into females at three different maturation stages viz., immature, matured and berried. In the fourth treatment group, mature males were injected with plasmid DNA to test the transfer of plasmid DNA through milt. The pCMV-VP28AS construct was injected intramuscularly (i.m.) using an insulin syringe. The concentration of the pDNA was maintained at 1 lg/gm of body weight and injection volume was 100 ll. Control group received 100 ll of STE buffer (10 mM Tris Cl, 100 mM NaCl and 1 mM EDTA, pH: 8.0). The injected brooders were maintained in FRP tanks (2 0.5 0.5 m). Two mating pairs with 1:2 or 1:3 (female:male) ratio were kept in one FRP tank (Table 1). The tanks were covered with net to prevent crawling. Feeding was done twice a day with commercial prawn feed for 3 weeks until the females were berried. In 3rd week, the berried females with dark grey colored eggs were shifted to spawning tank with pre-chlorinated water with 7 ppt salinity.
2.6. Tissue distribution To study the distribution of plasmid construct in different parts of the body after injection, both male and female brooders were dissected after mating and heart, hepatopancreas, muscle, gill, pleopod, and intestine tissues were collected. Ovaries were also collected from females. Genomic DNA was isolated and presence of construct was conﬁrmed by PCR ampliﬁcation of the CMV promoter using speciﬁc primers (CMVpF: AAAGCTAGCGAATCTGCTTAGGGTTAGG and CMVpR: AAATCTAGAAA TTTCGATAAGCCAGTAAGC) that ampliﬁed a 700 bp fragment carrying the CMV promoter from the plasmid DNA. The PCR reaction mix was prepared following Sambrook et al. (2001). The PCR cycling conditions included an initial denaturation at 94 °C for 4 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and an extension at 72 °C for 45 s and ﬁnal extension at 72 °C for 5 min. The correct size of the ampliﬁed fragment was conﬁrmed using agarose gel electrophoresis and imaging system (BioRad XR+ Gel Documentation System). 2.7. Vertical transmission Genomic DNA was isolated from 3 pools of 10 post larvae (15 days old) in each group using Qiagen DNeasy Blood and Tissue kit following manufacturer’s instructions. During sampling two washes with distilled water and one with sterile STE buffer were performed before setting up lysis for DNA isolation to prevent cross contamination with any extraneous material like feces, etc. Vertical transmission of the injected pCMV-VP28AS plasmid was conﬁrmed by PCR ampliﬁcation of CMV promoter as described in
Table 1 Experimental design for conﬁrming vertical transmission. Group
T1 T2 T3 T4 C
Intramuscular injection Intramuscular injection Intramuscular injection Intramuscular injection Un-injected control
to to to to
immature female matured female berried female matured male
2 2 2 3 (injected) 2
5 (injected) 5 (injected) 5 (injected) 7 5
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section 2.6. The correct size of the ampliﬁed fragment was conﬁrmed using agarose gel electrophoresis. 3. Results and discussion Based on its maturation stage, the female brooder spawned at some time between 3 days and one month. The offspring were separated from the brooders and housed in separate tanks immediately after spawning. This step which is a normal practice in hatcheries to avoid cannibalism also served to prevent cross contamination with the injected DNA from any parental source other than vertical transmission. The CMV promoter region of the plasmid construct could be PCR ampliﬁed from the offspring of female brooders injected at two stages – immature and matured. No ampliﬁcation was detected in the offspring of injected berried females. Interestingly, plasmid DNA was also found in offspring derived from i.m. injected male parent conﬁrming vertical transmission from male to offspring (Fig. 1). Plasmid DNA could also be ampliﬁed from various tissues of the injected male and female brooders that were examined post-spawning (Fig. 2). Lack of ampliﬁcation in one of the treatment groups (berried females) further conﬁrms absence of cross contamination from any external source. Theoretically, it is possible to detect even a single copy of the plasmid using 35 cycles of PCR ampliﬁcation with sensitivities of current gel documentation systems being as high as 0.1 ng for ethidium bromide stained gels (Biorad Bulletin). The CMV region was selected for conﬁrming presence of the plasmid because CMV is a human virus not found in shrimp. This region could not be ampliﬁed from negative controls ruling out nonspeciﬁc ampliﬁcation. The vp28 region was not used as this region could also be
Fig. 1. Vertical transmission of pCMV-VP28AS plasmid DNA from brooder to offspring conﬁrmed by PCR ampliﬁcation of CMV promoter; lane M: Generuler 100 bp plus ladder (Fermentas), lane 1: Positive control (pCMV-VP28AS plasmid), lane 2, 3 & 4: Females injected at immature, matured and berried stages, respectively, lane 5: Injected male brooder, lane 6: Negative control (un-injected).
ampliﬁed from a mild presence of WSSV that is nowadays widespread in shrimp hatcheries. The actual copy number of plasmid DNA present in the offspring could have been determined by quantitative PCR but in this short study we were only targeting the phenomenon of vertical transmission. Although speciﬁc immunoglobulins against pathogens have not been detected in shrimp, Huang and Song (1999) who studied maternal transmission of immunity in P. monodon reported that beta-1,3-1,6-glucan, derived from bakers’ yeast Saccharomyces cerevisiae, protected spawners from WSSV and that the resistance could be passed on to hatchlings. Direct introduction of plasmid into Litopenaeus vannamei zygotes through microinjection, electroporation, and transfection was reported by Sun et al. (2005) and best efﬁciency of gene transfer (40–60%) was obtained with transfection. DNA vaccines against several viral and parasitic diseases that are based on triggering the immune response have been shown to be effective when delivered through the i.m. route (Martinez-Lopez et al., 2013; Sudha et al., 2001). Moreover, they can be administered only after maturation of the immune system in that species. DNA vaccines based on RNA interference and antisense-RNA are not affected by these considerations and can be administered at early stages through dip treatment or even be vertically transmitted as reported here to protect early larval stages from viruses such as MrNV that are known to cause mortality to young ones. In addition to other known advantages of DNA vaccines like cost, stability and persistence, this method can further lower costs by requiring vaccination of only a few selected individuals. This may be a more economical option compared to even dip treatment of post-larvae. Further, DNA vaccines are known to persist for considerable durations in vivo (Evensen and Leong, 2013; Tonheim et al., 2007) and booster doses may not be required during the culture period. In future studies the possibility of administering DNA vaccine to brooders through dip treatment may be explored to even further lower stress. It can also be ascertained whether plasmid DNA is transmitted to each and every individuals as in this study pools of 10 larvae were examined. In a scenario where viral diseases represent the major bane on shrimp aquaculture this method can be veriﬁed in other species as well to deliver DNA vaccines based on antisense and RNA interference phenomena several of which have been reported to be effective on the lab scale. There is a possibility of DNA vaccine being vertically transmitted in ﬁsh too. This study is the ﬁrst report of vertical transmission of a plasmid DNA injected to parents. Acknowledgments The authors acknowledge Dr. W.S. Lakra, Director/Vice Chancellor, CIFE, Mumbai, India for providing all facilities and Indian Council of Agricultural Research, New Delhi, India for their ﬁnancial support. References
Fig. 2. Tissue distribution analysis of pCMV-VP28AS plasmid DNA in M. rosenbergii female brooder; lane M: 100 bp plus DNA ladder, lane 1: Positive control (pCMV-VP28AS plasmid), lane 2: Heart, lane 3: Gill, lane 4: Muscle, lane 5: Pleopod, lane 6: Hepatopancreas, lane 7: Ovary, lane 8: Intestine, lane 9: Negative control (un-injected).
Ahanger, S., Sandaka, S., Ananad, D., Mani, M.K., Kondadhasula, R., Reddy, C.S., Marappan, M., Valappil, R.K., Majumdar, K.C., Mishra, R.K., 2014. Protection of shrimp Penaeus monodon from WSSV infection using antisense constructs. Mar. Biotechnol. 16 (1), 63–73. Balakrishnan, G., Peyail, S., Kumaran, R., Theivasigamani, A., Anil, K.S., Solanki, J.B., Srinivasan, N., 2011. First report on White Spot Syndrome Virus (WSSV) infection in white leg shrimp Litopenaeus vannamei (Crustacea, Penaeidae) under semi intensive culture condition in India. Aquacult., Aquarium, Conser. Legislat. 4 (3), 301–305. Bartholomay, L.C., Loy, D.S., Dustin Loy, J., Harris, D.L., 2012. Nucleic-acid based antivirals: augmenting RNA interference to ‘vaccinate’ Litopenaeus vannamei. J. Invertebr. Pathol 110 (2), 261–266. Biorad Bulletin on Molecular Imager Ò Gel Doc™ XR+ and ChemidocTM XRS+ Systems. . Evensen, Ø., Leong, Jo-A.C., 2013. DNA vaccines against viral diseases of farmed ﬁsh. Fish Shellﬁsh Immunol. 35, 1751–1758.
L.M. Chowdhury et al. / Journal of Invertebrate Pathology 121 (2014) 24–27 FAO, 2011. The State of World Fisheries and Aquaculture. Food and Agriculture Organization, Rome, Italy. FAO, 2012. The State of World Fisheries and Aquaculture. Food and Agriculture Organization, Rome, Italy. Huang, C.C., Song, Y.L., 1999. Maternal transmission of immunity to white spot syndrome associated virus (WSSV) in shrimp (Penaeus monodon). Dev. Comp. Immunol. 23 (7–8), 545–552. Krishnan, P., Babu, P.G., Saravanan, S., Rajendran, K.V., Chaudhari, A., 2009. DNA constructs expressing long-hairpin RNA (lhRNA) protect Penaeus monodon against White Spot Syndrome Virus. Vaccine 27 (29), 3849–3855. Martinez-Lopez, A., García-Valtanen, P., Ortega-Villaizan, M., del Mar, Chico, V., Medina-Gali, R.M., Perez, L., Coll, J., Estepa, A., 2013. Increasing versatility of the DNA vaccines through modiﬁcation of the subcellular location of plasmidencoded antigen expression in the in vivo transfected cells. PLoS ONE 8, e77426.
Sambrook, S.J., Russel, D.W., Janssen, K.A., Irwuin, N.J., 2001. Molecular Cloning, A Laboratory manual, third ed. Cold Spring Harbor Laboratory Press. Sudha, P.M., Low, S., Kwang, J., Gong, Z., 2001. Multiple tissue transformation in adult zebraﬁsh by gene gun bombardment and muscular injection of naked DNA. Mar. Biotechnol. 3, 119–125. Sun, P.S., Venzon Jr., N.C., Calderon, F.R.O., Esaki, D.M., 2005. Evaluation of methods for DNA delivery into shrimp zygotes of Penaeus (Litopenaeus) vannamei. Aquaculture 243, 19–26. Tonheim, T.C., Leirvik, J., Lovoll, M., Myhr, A.I., Bogwald, J., Dalmo, R.A., 2007. Detection of supercoiled plasmid DNA and luciferase expression in Atlantic salmon (Salmo salar L.) 535 days after injection. Fish Shellﬁsh Immunol. 23, 867–876.