Aquaculture 368–369 (2012) 36–39

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Report of significant WSSV-resistance in the Pacific white shrimp, Litopenaeusvannamei, from a Panamanian breeding program

Jorge Cuéllar-Anjel a,⁎, Brenda White-Noble b, Paul Schofield b, Roberto Chamorro a, Donald V. Lightner b

a Camaronera de Coclé S.A., Natá (Coclé), Apartado 0823-058-19, Panamáb Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 85721, USA

⁎ Corresponding author. Tel.: +507 69491976; fax: +E-mail address: jocuan@gmail.com (J. Cuéllar-Anjel)

0044-8486/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.aquaculture.2012.08.048

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 October 2011Received in revised form 29 August 2012Accepted 29 August 2012Available online 5 September 2012

Keywords:White spot diseaseWSSVWSSV resistanceShrimp breeding programLitopenaeus (Penaeus) vannamei

Three Litopenaeus vannamei families, from a breeding program in Panama and with possible WSSV resistance,were challenged per os with a reference isolate of White spot syndrome virus originally obtained from Chinain 1995 (WSSV-CN95). These F8, F9 and F12 generation families were developed from founder stocks a de-cade ago and were survivors of white spot disease. Juvenile shrimp used for WSSV challenge averaged1.5 g, and they were stocked at 50 to 96 animals per tank into nine 1000 L fiberglass tanks containing artifi-cial seawater at 30 ppt salinity and 26 °C. Three of the 1000 L tanks were used as negative control tanks, withone tank for each family. Six 1000 L tanks were used for challenging the three families with WSSV, with tworeplicate tanks for each family. A positive control consisting of 20 “Kona” SPF reference line L. vannamei (averageweight 1.5 g)was included and challengedwithWSSV in a 90 L glass aquarium. The Kona stockwas fed the samebatch ofWSSV infected tissue as the three Panamanian families to confirm infectivity and to provide a basis withwhich to compare final survival. WSSV infected minced frozen shrimp tissue was fed at a rate of 5% of averagebody weight one time on day 0. All tanks were equipped with air diffusers to provide sufficient aeration andan acclimated crushed oyster shell internal recirculating biological filter. Each tank was covered with a plasticsheet to contain aerosols and minimize water temperature fluctuations. The experimental tanks were checkeddaily andmoribund animals were collectedwhen observed and preserved in Davidson's AFA fixative. Mortalitiesin the three Panamian families ceased at 17 days post challenge. Two survivors from each tank were preservedfor histology and five shrimp per tankwere individually tested by qPCR to determine theirWSSV status and viralload. Survival at termination in the negative control families was 95%, 98% and 100%. Survival in the Kona lineWSSV positive control was 0% with all the Kona line shrimp dead by day 6 post infection. At termination onday 17, survival of Panamanian selected families in theWSSV challenged groups was 23%, 57% and 26% for fam-ilies LP-1, LP-2 and LP-3, respectively. This is the first time in the scientific literature that significant resistance ofL. vannamei against WSSV under controlled conditions is reported.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

During the past 20 years, viral diseases have been among the maincauses of economic looses in shrimp farming. White spot syndromevirus (WSSV — the causative agent of white spot disease, WSD) hasbeen one of themost prevalent and virulent disease agents in thewesternand eastern hemispheres (Chen et al., 2000; Cuéllar-Anjel et al., 2010;Huang et al., 2011; Kanchanaphum et al., 1998; Lightner, 1996, 2011;Liu et al., 2006).

Breeding programs were started about 10 years ago in severalcountries, with the intent to produce shrimp families that tolerateand/or resist WSSV natural infections in shrimp farm ponds (Huanget al., 2011; Zhao et al., 2007). In 2001, the Panamanian shrimp compa-ny Camaronera de Coclé S.A. (CAMACO, Panama) began a program forthe prevention of WSD and genetic improvement of the Pacific white

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shrimp Litopenaeus vannamei. The selection strategy consisted of firstpreventing the vertical transmission of WSSV and other viral and bac-terial shrimp pathogens (e.g. infectious hypodermal and hematopoieticnecrosis virus, IHHNV, and the bacterial agent of necrotizinghepatopancreatitis, NHP-B), using only healthy females found to befree of WSSV, IHHNV and NHP-B by nested-PCR individual analysis.

Genetic resistance to WSSV was developed based on mass selectionin which pure lines of L. vannamei were subjected to experimental in-fections with purified suspensions of WSSV. The positive results of the10-year breeding program have been reflected in the steady improve-ment of annual productivity (kg/ha) at the CAMACO shrimp farm,which increased from 43 kg/ha (during the 1999 epidemic of WSD);up to 1410 kg/ha in 2010 using semi-intensive farming conditions. Re-sistance to WSSV in the CAMACO shrimp lines, has increased, in the 10generations of intense selection for WSSV resistance, the average ratesof survival in Panamanian shrimp farms from5% in 2000 to 75% in 2010.

Each generation of the pure lines of shrimp was subjected to anexperimental infection with a purified preparation of WSSV. The

37J. Cuéllar-Anjel et al. / Aquaculture 368–369 (2012) 36–39

rate of survival of some of the lines in the F8 generation has been in-creased by a factor of 9000. The present article presents the method-ology and results of a challenge test performed during February andMarch 2011, at the Aquatic Pathology Laboratory of the Universityof Arizona (APL-UAZ) with the goal to evaluate, under controlled con-ditions, the resistance to WSSV virus in three lines of L. vannamei pro-duced by the breeding program of CAMACO.

2. Materials and methods

2.1. Experimental animals

The experimental animals used in this study were three L. vannameifamilies in their F8, F9 and F12 generations. These familieswere from anongoing selective breeding program initiated by the Panamanianshrimp company CAMACO in 2001. All three family lines were fromfounder stocks obtained through the selective breeding of offspringthat were survivors of experimental WSSV infections from multiplegenerations since 2001.

The family identified as “LP-1” was in its F9 generation and it wasproduced by artificial insemination from one female and one male.The second family identified as “LP-2”, was obtained by crossing fe-males from a mass selected F11 population with F8 generationmales from the “LP-1” family using natural copulation. The third fam-ily of this study is identified as “LP-3”, and it is in the F12 generationand it was obtained by crossing shrimp previously produced by massselection and natural copulation from a mixture of all individually se-lected and WSSV challenged families.

The L. vannamei stock (average weight 1.5 g) utilized in thesestudies were shipped to the APL-UAZ from CAMACO in Panama. Onarrival, the shrimp were acclimated and stocked at 50 to 96 animalsper tank into nine 1000 L fiberglass tanks containing artificial seawa-ter at 30 ppt salinity and 26 °C. The shrimp were allowed to recoverfrom shipping stress for 3 days prior to being used in the WSSV chal-lenge studies.

2.2. WSSV infected tissue preparation

SPF Kona line L. vannamei shrimp weighting 6 g average wereinjected with a semi-purified inoculum of the China WSSV isolate(WSSV-CN95), in order to produce the WSSV infected tissue used inthis experiment. This WSSV strain was chosen for this challengestudy because it is the reference isolate of WSSV most often used bythe APL-UAZ bioassay laboratory and because of the consistency invirulence that the isolate has shown since it was obtained byAPL-UAZ in 1995.

2.3. Challenge test: experiment set-up and shrimp distribution

The experimental challenge consisted of three 1000 L negative con-trol tanks, each containing representatives from each of the three fami-lies. Their siblings were challenged in separate tanks with WSSV. Atotal of six 1000 L tanks were utilized for challenging the three familieswith WSSV and included two replicates for each family. A positive con-trol consisting of 20 Kona line SPF (specific pathogen-free) referenceline L. vannamei (average weight 1.5 g) (Lightner et al., 2009; Moss andMoss, 2009) was included and challenged with WSSV in a 90 L glassaquarium. Artificial seawater (Forty Fathoms Marine Mix, Baltimore,MD) at 28.0±1.0 °C and a salinity of 30 ppt was used, all tanks wereequipped with air diffusers to provide sufficient aeration and an accli-mated crushed oyster shell in an internal recirculating biological filter(White et al., 2002). Each tank was covered with a plastic sheet to con-tain aerosols and minimize water temperature fluctuations.

The families were challenged per os with WSSV. On day 0 of thestudy, the six challenge tanks and the positive control tank, weregiven one feeding of WSSV infected minced frozen animals at a rate

of 5% of their average body weight. The WSSV content of the inocu-lum was determined to be 5.7×106 copies/μg DNA by qPCR.

Beginning on day 1 (post-WSSV infected carcass feeding) of thechallenge study, the WSSV challenged shrimp and the Kona stockwere fed a commercially available pelleted shrimp diet (Rangen, Buhl,Idaho). The Kona stock was fed the same batch of WSSV tissue as thethree Panamanian families to ensure that the tissue usedwas infectiousand to provide a basiswithwhich tomeasure and compare survival. Theshrimp in the three negative control tanks were fed the Rangen feedfrom day 0 of the study.

All tankswere checked daily formoribund or dead animals. To reducecannibalism in the experimental tanks, dead shrimpwere removed dailyafter virus exposure and throughout the test period until the study wasterminated on day 17. Recovery of dead and moribund animals wasrecorded daily. Dead shrimp were frozen at −70 °C. Moribund animalswhen observed, as well as and some of the survivors at termination onday 17, were preserved in Davidson's AFA fixative and processed usingroutine H&E histology (Bell and Lightner, 1988) to confirmWSSV infec-tion (Lightner, 1996) as the cause of morbidity during the study and todetermine the WSSV status of the surviving shrimp at termination onday 17. An additional five WSSV survivors from each tank were frozenand individually tested by qPCR to determine their WSSV status andviral load (Nunan and Lightner, 2011). The WSSV challenge study wasrun until mortalities ceased at 17 day post challenge. Live shrimp at ter-mination on day 17were counted as survivors. All survivors were count-ed and either frozen or preserved in Davison's AFA fixative.

2.4. Real-time PCR quantification of WSSV from infective tissue

Real-time PCR quantification (qPCR) was carried out as described byDurand and Lightner (2002), in order to determine the number ofWSSVDNA copies present in the infective tissue inoculum. Based on a samplefrom the abdominal muscle of the infected shrimp, the genomic DNAwas extracted using tissue DNA-isolation kit (Promega). The real-timePCR assay was carried out using the following primers: upstream primer(WSSV1011F): 5′-TGG TCC CGT CCT CAT CTC AG -3′ downstream primer(WSSV1079R): 5′-GCT GCC TTG CCG GAA ATT A -3′; Taqman probe:5′-AGC CAT GAAGAA TGC CGT CTA TCA CAC A -3′. The TaqMan assaywas carried out with a PerfeCTa qPCR Super Mix (Quanta BioSciencesInc., Gaithersburg, MD, USA). A sample of 10–50 ng of DNA was addedto a PCR mixture containing 0.3 μM of each primer and 0.15 μM ofTaqMan probe in a final volume of 20 μL. Real-time PCR was performedon the Mastercycler ep realplex (Eppendorf) with the following condi-tions: initial denaturation at 95 °C for 5 min, followed by 40 cycles ofamplification at 95 °C for 15 s and annealing/extension at 60 °C for1 min. Amplification data were collected and analyzedwith the Realplex2.0 software (Eppendorf). Each sample was tested in duplicate. A seriesof dilutions from a WSSV recombinant plasmid (known virus load)were made for use as standards for quantification.

2.5. Statistical analysis

Survival data was analyzed by statistical analyses according toMilton (1999). The Kaplan–Meier survival curves and cumulative sur-vival probabilities were computed by SPSS 14.0 software for Windows(Bland and Altman, 1998). Then, a 1-way ANOVA had been run to com-pare populationmeans. The significance level for the analyseswas set atPb0.05, unless noted otherwise.

3. Results

3.1. Termination survival after the WSSV challenge test

The CAMACO selected L. vannamei families showed greater surviv-al than did the unselected WSSV-susceptible (Kona) stock after chal-lenge with theWSSV-CN95 virus strain. Survival at termination in the

0

10

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30

40

50

60

70

80

90

100

% S

urv

ival

Survival at Termination

LP-1 Neg. Control

LP-1 WSSV

LP-2 Neg. Control

LP-2 WSSV

LP-3 Neg. Control

LP-3 WSSV

Kona + Control

Fig. 1. Survival by family in a WSSV challenge study of CAMACO stocks.

38 J. Cuéllar-Anjel et al. / Aquaculture 368–369 (2012) 36–39

CAMACO negative control families was 95%, 98% and 100%. After in-fection of the shrimp of the three selected families and the unselectedKona family, the first shrimp death appeared at day 2 in the Camacofamilies and on day 3 in Kona tank. By day 6, all of the Kona popula-tion had died from WSSV infection, resulting in 0% survival for thisfamily. Dead shrimp were observed daily in the duplicate tanks ofthe three families from CAMACO, and mortality ceased on day 17postinfection. At termination on day 17, survival in the CAMACOWSSV challenged groups was 23%, 57% and 26% for families LP-1,LP-2 and LP-3, respectively (Fig. 1; Table 1).

3.2. Clinical and histological lesions in moribund L. vannamei after WSSVinfection

Histological examination of the day 0 specimens collected prior tothe start of this study showed no signs of infection by WSSV, TSV orother significant shrimp disease agents. The severity of white spotdisease pathology was high in all of the moribund specimens collect-ed on day 4 post challenge or later in the study, but prior to termina-tion. Moribund shrimp presented an empty midgut, lethargy, whitishdiscoloration with expanded chromatophores giving a reddish color-ation of top of appendages and uropods, absence of escape response,and a lateral recumbent position on the tank bottom.

4. Discussion

PreviousWSSV challenges performed at APL-UAZ using L. vannameiduring the period of 1996 to 2010 resulted in an overall survival rate of~5%, withmost showing 0% survival and one studywith a high of 25%. Atotal of 176 families (that were not selected for resistance to WSSV)were challenged during this period. Occasionally, 1–5 survivors werenoted within a single family. The highest survival noted (25%) was atotal of 5 animals from a single family (Noble and Lightner, unpublisheddata). The results of the 2011 CAMACO challenge study are unusual inthat survival rates were 23%, 26% and 57%, the number of total survivors

Table 1Experimental design utilized in the CAMACO WSSV challenge study.

Tanknumber

Family code Tankdefinition

Terminationsurvival no./%

1A LP-1 Negative control 49 of 50/98%1B LP-2 Negative control 91 of 96/95%1C LP-3 Negative control 68 of 68/100%90L Kona WSSV positive control 0 of 20/0%B1 and B2 LP-1 WSSV challenge 24 of 104/23%B3 and B4 LP-2 WSSV challenge 74 of 129/57%B7 and B8 LP-3 WSSV challenge 34 of 130/26%

was 24, 34 and 74 animals, that these survivors were in the same tankwith shrimpwith severeWSSV infections, and thatmortalities were de-layed and did not cease until ~2 days prior to termination of the studyon day 17 post challenge.

The high severity grade of WSSV infection in most of the mori-bund specimens collected during the study (Table 2) was due to thevery high number of cells in target tissues presenting fully developedbasophilic intranuclear inclusion bodies. WSSV has an anti-apoptosisgene, which has been suggested to be the reason why species like cer-tain crab species can present severeWSSV infections while not suffer-ing high mortalities (Chen et al., 2000; Kanchanaphum et al., 1998).Perhaps, selection for WSSV resistance in the three families testedin this study is related to the upregulation of the anti-apoptosis geneofWSSV, or upregulation of a shrimp apoptosis gene, or some combina-tion of these and other possible explanations consistentwith high levelsof WSSV replication, but reduced or delayed mortality observed in thisstudy.

However, not consistent with the hypothesis of the up-regulationof the anti-apoptosis gene(s) of the virus or shrimp host, was thefinding that the shrimp that survived to termination on day 17presented no histological signs of WSSV infection nor did they con-tain a detectable level of WSSV by qPCR (Table 2). With a detectionlimit of 1 WSSV genome copy in the qPCR test used, this finding sug-gests that the day 17 survivors from all three families in this studyeither were never infected with WSSV (while others from the samefamily in the same tank died fromWSSV) or that they were sufficient-ly resistant to the virus to clear it to levels below the detection limit ofthe qPCR test (Table 2).

In addition to thenumerous reports of resistance to TSV in L. vannamei(Argue et al., 2002; Lightner, 2011;Moss andMoss, 2009), there are otherreports of resistance to other shrimp viruses in different families ofL. vannamei. Zheng (1994) reported differences in resistance to ex-perimental infection by Baculovirus penaei among different familiesof L. vannamei. More recently reports by Zhao et al. (2007) andHuang et al. (2011) indicate that it is possible to carry out a success-ful breeding program with L. vannamei with the intent of selectingfor resistance toWSSV. Zhao et al. (2007) used suppression subtrac-tive hybridization to identify differentially expressed genes in thehepatopancreas of L. vannamei fromWSSV resistant and susceptiblefamilies. In their study, they found 40 differentiallly expressed genes intwo WSSV-resistant lines after two generations (after 4 years) of a se-lective breeding program. Following only two generations of selection,the reported survival after a WSSV challenge in a resistant line was70% (Zhao et al., 2007). Huang et al. (2011) also reported significant im-provements in WSSV resistance after three generations of selectivebreeding. In the report by Huang et al. (2011), 29 selected familieswith relatively high-resistance, moderate-resistance and susceptible

Table 2Pathology and qPCR results from samples taken during a WSSV challenge test run withthree families developed by CAMACO in Panama by selective breeding.

Day of sample Numberexm'd

Family/treatment Histologyresultsa

qPCR results

0 3, 3, 3 LP1, LP2, LP3 G0 WSSV Not done4 1 Kona/+WSSV

controlG4 WSSV Not done

4,5,12 3 LP1/WSSV G4 WSSV Not done3,4,5,11 4 LP2/WSSV G4 WSSV Not done4,5,15 5 LP3/WSSV G4 WSSV Not done17 2, 2, 2 LP1, LP2, LP3/WSSV G0 WSSV Not done17 11 LP1/WSSV Not done Not

detected17 9 LP2/WSSV Not done Not

detected17 10 LP3/WSSV Not done Not

detected

WSSV inoculum used in this challenge study: 5.7×106 copies/g DNA.a G0=no lesions due to WSSV infection; G1 to G4=lesions due to WSSV infection

were present, with G1=low severity and G4=extreme severity.

39J. Cuéllar-Anjel et al. / Aquaculture 368–369 (2012) 36–39

families were challenged with a low dose of WSSV (103 WSSV DNAcopies g−1 body weight). Average survival rates for the families withrelatively high-resistance, moderate-resistance and susceptible familiesat 10 days post challenge were 23%, 9% and 0.8% , respectively. Survi-vors from the relatively high WSSV-resistant family had significantlylower WSSV copy number (by qPCR) than did the shrimp from themoderate-resistance family. In the present study, suvivors from allthree WSSV-resistant families had undetectable levels of WSSV byqPCR, 17 days after challenge with a much larger dose of WSSV thanwas used in the Huang et al. (2011) study.

In summary, this study has demonstrated that the breeding pro-gram carried out by CAMACO since 2001, has resulted in the develop-ment of WSSV resistance in three selected lines of L. vannamei. Afternine generations of genetic selection, the three WSSV resistant fami-lies displayedmuch higher survival rates (23, 26 and 57%) than did anSPF control family (0%). Furthermore, there were no detectable WSSVviral loads in the shrimp from the three infected families that survived totermination of the study, suggesting that, in addition to being WSSV re-sistant, they are not carriers ofWSSV. These results represent an interest-ing mix of presumed genetic traits in the three lines tested.

After nine generations of selection, the three selected families de-veloped significant resistance to WSSV. Under production conditionsin earthen ponds, selected pure lines that do become WSSV infectedshow a delayed onset of WSSV infection relative to unselected stocksof L. vannamei (Cuéllar-Anjel, unpublished data). The stabilization ofthe resistance to WSSV in the homozygous pure lines may require ad-ditional generations, which have already begun at the farm level.These results, in addition to the ones obtained in this study, indicatethat significant improvements in resistance of L. vannamei shrimp toWSSV can be achieved through a systematic selective breeding program.

Acknowledgments

This studywas supported by the PanamanianAgroindustrial CALESAGroup. Thankfully acknowledged for their participation aremembers of

the CAMACO team, specially the staff from the postlarvae productioncenter “C.P.L. San Carlos”, Broodstock Department “D.R.C.”, molecularand biotechnology laboratory “UNICAM”, and the Pathology and Re-search Department. In kind support was from the United States MarineShrimp Farming Consortium under grant no. 2010-38808-21115 fromthe National Institute of Food and Agriculture, U.S. Department ofAgriculture.

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