Aquaculture 286 (2009) 1–11

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Aquaculture

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Breeding for disease resistance of Penaeid shrimps

James Cock a, Thomas Gitterle a, Marcela Salazar a, Morten Rye b,⁎a CENIACUA, Bogotá: Cra 9 B No. 113-60., Colombiab Akvaforsk Genetics Center AS, N-6600 Sunndalsøra, Norway

⁎ Corresponding author.E-mail address: morten.rye@afgc.no (M. Rye).

0044-8486/$ – see front matter © 2008 Elsevier B.V. Adoi:10.1016/j.aquaculture.2008.09.011

a b s t r a c t

a r t i c l e i n f o

Article history:

Diseases are a major constra Received 19 March 2008Received in revised form 8 September 2008Accepted 10 September 2008

Keywords:BreedingDisease resistancePenaeid shrimpsSelection

int on the intensive production of shrimps. Conditions in production ponds favourdisease development, and epidemics of several previously unreported diseases have occurred and caused severelosses. When elimination, eradication or cultural control is difficult, selective breeding for host resistance to thepathogen may be an attractive option for disease control. However, host resistance is not a panacea and shouldonly be consideredwhen (a) the disease causes severe damage (b) there are noother existing simple cost effectivecontrol measures and (c) there is demonstrable genetic variation in resistance and this is not coupled with anexcessive level of negative associations with other desirable characteristics. Shrimp have only recently beendomesticated and breeding for resistance only began in the mid 1990s; there is limited experience with shrimpbreeding in particular and crustaceans in general. Consequently, the principles and concepts behind breedingprogrammes are based largely on experiences with other species in both the plant and animal kingdoms.Commercial growers now seed ponds with shrimp populations selected for resistance to Taura Syndrome Viruswith excellent results, whilst up to now development of White Spot Syndrome Virus resistant populations hasbeen an elusive goal. The original TSV resistant populations were developed using simple mass selectiontechniques (Colombia). In later generations family based selection has been applied on populations, whichinitially had survival rates of about 30%, with care taken to reduce inbreeding and loss of genetic variation. Thissuggests that when the original populations have a reasonable level of resistance, and straightforward, effectiveselection protocols exist, it is relatively simple to breed for resistance. With catastrophic diseases, such asWSSV,which cause mortalities of 98% or more the frequency of resistance is low and it is suggested that for theoreticalreasons single gene, rather thanpolygenic, resistance is likely to develop. The low frequencyof resistance genes inbreeding populations may cause genetic bottlenecks which will greatly reduce the genetic variation in thepopulations. In order to maintain the genetic variation the genes from the small numbers of survivors should beintrogressed into populationswith broader genetic variability. Furthermore, in order tominimize the probabilityof breakdown of resistance pyramiding of resistant genes on different loci would be advantageous.Genetic variation in resistance may be encountered either in the initial base populations or may spontaneouslyarise due tomutations or new recombinants.With extremely prolific species such as shrimps,millions of animalscan readily be screened for survival and hence resistantmutants or recombinantsmay be identified. Once geneticvariation has been detected the most appropriate breeding methodology will depend on the nature of both theresistance and the disease or diseases that are of interest to the producers.

© 2008 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Diseases in intensive shrimp production systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.1. Disease avoidance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2. Host response and resistance to diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3. Disease resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.1. Evolution of genetic disease resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

4. Breeding for resistance to diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

ll rights reserved.

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4.1. Success and failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64.1.1. Taura Syndrome Virus resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64.1.2. Searching for resistance to White Spot Syndrome Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4.2. Selection procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.3. Breeding methodologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.4. Base populations and population size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1. Introduction

Diseases andpests aremajor constraints on the supplyof highqualityplant and animal products under intensive production systems.Growersand researchers have developed a whole series of crop and animalhusbandry systems geared to minimizing the harmful effects ofpathogens and pests. Recently Keen et al. (2001) observed thecommonality in microbial virulence mechanisms and the occurrenceof similar innate resistance systems in animals and plants with anancient and intertwinedhistory. Similarly, Toyodaet al. (2002) commenton the remarkable similarity of innate responses to pathogens in plants,insects and mammals. In the particular case of shrimps, cultivation isrelatively newand there is limited knowledge on response to pathogens.Hence, where there are major gaps in knowledge specific to shrimps orcrustaceans, we use experience obtained in both plants and animals asanalogous guidelines. In thefirst section of the articlewe discuss diseaseanddisease control, particularlygenetically controlledhost resistance, incultivated shrimp and other species in general terms, and then use thisinformation to provide guidelines on the circumstances when breedingfor disease resistance is an appropriate control measure and howbreeding programmes can be structured.

2. Diseases in intensive shrimp production systems

Shrimps have only recently been cultivated by man, and it is onlybeen in the last twenty to thirty years that the cultivated populations,of some species that are easily reproduced in captivity, have beenisolated from wild populations. Consequently, most populations ofcultivated shrimp have only had a relatively short period to evolve andadapt to intensive cultivated production systems.

Diseases that remain at a low level of incidence in naturalpopulations may reach epidemic levels in intensive cultivation systems.Intensive management systems in livestock production encourage theunpredictable appearance of new diseases and changes in thecharacteristics of established diseases (Biggs, 1985). Modern intensiveshrimp systems provide almost ideal conditions for the propagation ofdiseases. In the tropics shrimp are often cultivated year round with nobreak in the production cycle. Animals are confined in ponds, tanks orraceways at high densities, water exchange is limited and moribundanimals are not culled from the system. This latter is of particularimportance as cannibalism is common, disseminating pathogenseffectively. Furthermore some diseases of shrimps are common tovarious crustaceans (Wang et al., 2004, 2005; Nunan et al., 2004; Loet al., 1996; Lightner, 2005b) and it is difficult to isolate commercialproduction from other crustacean species, such as crabs, or copepods,that may be alternate hosts of shrimp diseases. All these conditionsfavour epidemics and the appearance of apparently new diseases inintensive shrimp production systems. On the Pacific coast of Central andSouth America, where a native species, Penaeus vannamei, is mostwidely cultivated, Taura Syndrome Virus (TSV) devastated the industryin the early nineties (Brock, 1997). Later White Spot Syndrome Virus(WSSV) appeared in Asia and rapidly devastated the shrimp industry inmany parts of the world. Both of these diseases were previouslyunreported. Similarly in Asia White Spot and Yellow Head epidemicshave reduced production of various Penaeid shrimp species, including

the native species P. monodon and the introduced species of P. vannamei.Furthermore, P. stylirostris, which represented close to 20% of the neo-tropical cultivated shrimp production, is now rarely cultivated due to itssusceptibility to various viruses (Lightner, 2005b).

Most of the concepts behind disease control in animal species havebeen developed for warm blooded terrestrial species. However, there aremajor differences in their environment and that of marine animals,indicating that transfer of technology from one to the other should becarriedoutwith caution.Warmblooded terrestrial landanimalsmaintaina relatively constant body temperature,whereas shrimps likemanyotheraquatic organismsare ectothermal and their body temperaturefluctuateswith that of thewater inwhich they live. Similarly, the composition of themedium in which land animals live, the air, varies little with such vitalaspects as oxygen and carbon dioxide content relatively constant on aglobal basis. On the other hand, shrimps face tremendous variability inthe environment in which they live with dramatic changes oftenoccurring abruptly. Stress, which is closely related to the manifestationof diseases (Biggs, 1985), is often induced by changes in such parametersas temperature, oxygen, salinity andammonium. Farmers on a day to daybasis attempt to control diseases by managing the environment andreducing the variability in the aquatic environment.

Vaccination is a common disease control measure in warm bloodedanimals, protecting hundreds of millions of animals from disease anddeath (NOAH, 2002). Vaccines stimulate the body to produce its owndefence against infection. The defensive system “remembers” theidentity of invading organisms and combats them when a vaccinatedanimal is confronted with a specific disease organism. This protects theindividual animal and, because this animal will not develop the diseaseand will not become infective, it will also help protect the populationfrom the disease due to ‘herd immunity’ (NOAH, 2002). It is generallyaccepted that Crustaceans do not possess the capacity to acquireresistance and hence vaccination is not possible, although thisassumption is questioned by Witteveldt (2006).

2.1. Disease avoidance

Onaglobal scale simple avoidance of diseases andpests has beenoneof the most effective means of minimizing damage of cultivated orcultured species. Crop productivity is normally greater outside thecentre of origin due to less diseases and pest pressure (Jennings andCock,1977). Inplants the classic example of avoidance outside the centreof origin is the success of rubber plantations in South East Asia andAfricadue to the absence of South American Leaf Blight, which decimatesintensively cultivated populations in the Amazon basin (Davis, 1996). Inanimal populations there are also large differences in disease incidencedepending on their geographical locations with introduced speciesfacing fewer parasites (Torchin et al., 2003). The success of introducedinvasive species is often directly attributable to the lack of pathogens inthe new environment. In domesticated animal populations simpleavoidance of diseases and pests has longbeenoneof themost importantmeans of disease control, with eradication of Newcastle disease inpoultry and rinderpest and foot and mouth disease in cattle being wellknown cases (Biggs, 1985).

Disease avoidance or eradication is only possible in certaincircumstances. Exclusion of diseases has been attempted with some

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success in shrimp cultivation (McIntosh,1999;Moss,1999), with variousprogrammes emphasising the use of Specific Pathogen Free stock inbreedingprogrammes tominimize spread of diseases (Moss,1999;Mosset al., 2003; Lightner, 2005a; Hennig et al., 2005). However, it is not easyto avoid or eradicate diseases with an open air aquatic grow-outenvironment that is normally close to the sea. McCallum et al. (2004)have pointed out that, although the same basic principles apply to theepidemiology of both terrestrial and marine species, there are majorqualitative differences and these affect the management practices fortheir control. The life cycles differ considerably and the rate of spread ofepidemics appears to bemuchgreater inmarine environments and theytend to bemore devastating (McCallumet al., 2004).With the exceptionof some diseases, such as Yellow Head Virus (YHV) and MonodonBacillus Virus (MBV), most of the shrimp viruses have spread rapidlyfrom the sites where they were first recognised (Lightner 1996, 2005b;Flegel et al., 2004). The recent epidemic of White Spot Syndromeindicates how rapidly an epidemic may spread in marine species: Firstdetected in Taiwan in 1992 (Chou et al., 1995), WSSV rapidly spread tomost Asian countries (Inouye et al., 1994; Zhan et al., 1998; Flegel andAlday-Sanz, 1998; Wongteerasupaya et al., 1995) and by 1996 mostshrimp farming regions in South East Asia were affected (Flegel andAlday-Sanz,1998). In thewesternhemisphere thefirst outbreakofWSSVappeared in farmed P. vannamei and P. stylirostris in South Carolina(USA) in 1997 and it was associated with 95% cumulative losses(Lightner, 1999). By early 1999, WSSV had spread to farmed P. vannameiin CentralAmerica (JoryandDixon,1999) reaching theColombianPacificcoast in May of that year. The diseases devastated most of the majorshrimp producing areas of the world. Attempts to eradicate or excludethe disease were mostly unsuccessful. White Spot Syndrome Virusappeared to have been successfully excluded from a few shrimpproducing regions, particularly the Atlantic coast of South America;however it has recently been reported in the cooler temperature regionsof southern Brazil (Anon, 2005). It now appears that conditions on theAtlantic coast of South America were in general not conducive to thedevelopmentof full scaleWhite Spot due to thehighwater temperatures(Vidal et al., 2001). The lack of White Spot Virus epidemics in this areaappears to be related to the virus's inability to replicate at the highertemperature rather than a temperature mediated response by theshrimp (Reyes et al., 2007). Someareas in South and South East Asiamayhave escaped or have low incidence of WSSV due to higher watertemperatures. Shrimp farmers have in some cases reduced waterexchange and appear to have achieved some level of control of WSSVwith this practice, which probably both increases water temperaturesand also reduces the chances of pathogens entering the ponds.Furthermore, in Thailand the use of Specific Pathogen Free (SPF) stocksand bio-security measures have reduced WSSV incidence dramatically(anonymous reviewer, pers. comm.). On the other hand, on the PacificCoast of the neo-tropics WSSV SPFs and other bio-security measureshave not markedly reduced disease incidence. Thus, although it isgenerally difficult to avoid diseases in cultivated shrimps throughelimination of the causal agent or the use of Specific Pathogen Freestocks, it may be possible in certain circumstances to use theseapproaches to reduce incidence: in the particular case of WSSV theavoidance of disease epidemics in the neo-tropics is largely based onfinding conditions not suitable for the development of the organism,rather than through elimination of the causal agent itself.

2.2. Host response and resistance to diseases

Shrimps have evolved under conditions that are favourable forinfection and have developed a series of defence mechanisms thatprotect them from infectious diseases in their native habitat. Diseaseresistance in many animals is mediated by both innate and acquiredresistance. Innate immunity is rapid, non-specific and acts as a first lineof defence,while acquired resistance involves antigen specific responses(Bishop et al., 2002). Shrimps, as crustaceans, possess an innate immune

system that protects them from foreign organisms. It is generallyaccepted that they do not have the ability to acquire immunity andhence there is no possibility of developing vaccines for shrimps whichwill provide long term immunity to a specific disease. However, recentlyWitteveldt (2006) indicated that vaccination of shrimp against WSSVmight be possible which would open the way to the design of newstrategies to control WSSV and other invertebrate pathogens. Never-theless, this result is questioned by many immunologists. In additionthere may be possibilities to stimulate the immune system and a seriesof non-specific responses against invading organisms.

Genetically controlled behavioural characteristics may also provideresistance to disease: for example genetically controlled hygienicbehaviour in bees prevents chalk brood disease (Milne, 1983). Withcannibalism playing such an important role in infection in intensiveshrimp culture, it is possible that genetic control of cannibalisticbehaviour may be involved in providing a measure of resistance toinfection (Gitterle et al., 2005).

With diseases that are difficult to eradicate, control measures havebeen developed based on stimulation or enhancement of the naturaldefencemechanisms of the host organism, including selection for hostresistance or tolerance to diseases and modification of the environ-ment so that the disease is not favoured. In plants conscious selectivebreeding for host resistance or tolerance to diseases dates back toapproximately the middle of the twentieth century, whereasconscious selective breeding schemes for livestock disease resistanceis more recent and in the case of Penaeid shrimps selective breedingfor resistance was initiated in the 1990s.

3. Disease resistance

Genetically based host resistance is an attractive proposition fromthe point of view of the grower of improved stock. The grower, apartfrom paying for the resistant genetic stock, does not have to makefurther major outlays, although management practices that allow theresistance to be expressed may be required. A further advantage ofhost resistance is the minimal negative impact on the environment asneither antibiotics nor chemical treatments are normally needed toenhance control. On the other hand development of genetically basedhost resistance is often costly and may be impossible to achieve in theabsence of useful levels of resistance. Furthermore, each characteristicthat is added to a selective breeding program inevitably leads toslower progress in other desirable characteristics in the breeding goal.Added to this, disease resistance may be negatively associated withother desirable characteristics. These associations may be geneticallylinked to genes that are close to each other on the same chromosome,or there may be a metabolic, physiological or ecological cost of theresistance. Selective breeding for resistance is an attractive option formanaging diseases, but it is not a panacea for control of all diseases. Inorder to justify the high cost of developing genetically diseaseresistant populations this approach is only advisable when: (a) thedisease causes severe damage; (b) there are no other existing simplecost effective control measures; and (c) there is demonstrable geneticvariation in resistance and this is not coupled with an excessive levelof negative associations with other desirable characteristics.

3.1. Evolution of genetic disease resistance

Selection for disease resistance under natural conditions dependson both the advantages that ensue from being able to combatinfections and also the costs of maintaining defences in the absence ofinfection (Kraaijeveld and Godfray, 1997; Coustau et al., 2000). Thereare a small number of genetic improvement programmes with thespecies that were first bred in captivity (P. stylirostris and P. vannamei)that have passed through many generations (Goyard et al., 2002;Wyban et al., 1992). However, the Penaeid shrimp populations used inmost breeding programs are only, at the most, a few generations

4 J. Cock et al. / Aquaculture 286 (2009) 1–11

removed from the native populations from which they weredeveloped and hence levels of disease resistance will tend to reflectthe balance between the advantages and costs under naturalconditions, unless selection pressure for disease resistance undercultivated conditions has been strong.

The genetic control of disease resistance in shrimps is not wellunderstood and little research has been dedicated to the theme untilvery recently. As noted previously, shrimps appear to have no acquiredimmune response, and in this sense they are perhaps somewherebetween plants and mammals in their response, and certainly withsome interesting comparisons with invertebrates in general. Unfortu-nately the information on genetic control of disease resistance ininvertebrates is also limited. Consequently we draw inferences onvarious aspects of genetic control of disease resistance in other species,particularly plants and mammals in which there is a vast stock ofknowledge. Selective breeding for disease resistance in plants has alonger trajectory than in mammals. The early work on host plantresistance divided resistance broadly into two categories (see forexample Van der Planck, 1963, 1968; Zadoks, 2002). In simple termsvertical resistant provided effective immunity, normally throughhypersensitivity, somewhat similar to apoptosis in animals, and wascontrolled by single genes. Horizontal resistance did not provide totalimmunity but slowed the spread of the disease and was controlled bymany genes.

Recently, whilst reviewing invertebrate immunology, Rolff and Siva-Jothy (2003) indicate that when selection pressure is strong, resistancemay be monogenic and when selection pressure is lower polygenic.Similarly, Coustau et al. (2000) indicate that when there is a newenvironment inwhich the current phenotypes are not viable, theoreticalconsiderations suggest that single major mutations are likely to beinvolved in adaptation to the newenvironment. AlthoughOrr andCoyne(1992) suggest that responses to pathogens are more likely to bepolygenic with small effects, Orr (1998) indicates that long termevolution often involves the fixation of mutations of large effect. Wesuggest that in shrimps entering a new environment under intensivecultivated systems andwith the extremely high selectionpressure in thecase of diseases such asWSSV, which kill 98% or more of the populationin severe epidemics (Lightner, 1996; Vidal et al., 2001), monogenicresistance or resistance under the control of a small number of genesmay arise. Certainly resistance to antibiotics in bacteria (Mazel andDavies, 1999) and to insecticides in insect populations (Roush andMcKenzie, 1987), where selection pressure is extremely high, isfrequently conferred by single genes. This seems obvious when oneconsiders that in the case of catastrophic circumstances, in which allsusceptible organisms die, there is little chance for the slow accumula-tion of polygenic resistance: it is only the mutant or recombinant thatconfers the ability to survive that proliferates.

Although pesticide resistance in insects may not be precisely thesameas resistance to diseases causedby biological organisms, theremaybe some interesting parallels in the evolution and genetics of resistance.When chemical pesticides were first developed, insect populations inthe field were frequently faced with, from their point of view, acatastrophic situation inwhich only resistant animals could survive. Thefrequency of resistant genes was extremely low, but neverthelessresistant individuals and populations eventually emerged. In the field,where applications were sufficiently intense to cause massive mortal-ities, single gene resistance normally developed (Roush and McKenzie,1987). In studies of the development of resistance in laboratorypopulations, where the initial populations were much smaller anddosagewas often reduced so as to increase the frequency of survivors to10–20%, polygenic resistancewas the norm (Roush andMcKenzie,1987).This strongly suggests that in a selection programme, the selectionprotocol itselfmaywell affect the type of resistance that is encountered:selection procedures with a limited range of genetic variation anddosages or inoculum pressure that ensure more survivors are likely tolead to uncovering and selection for polygenic resistance, whereas

natural selection in the fieldwith larger genetic variation and extremelyhigh mortalities (well over 99%) are likely to uncover single generesistance which will normally be dominant.

Initially in plants horizontal resistance or polygenic resistance wasconsidered to be durable, and vertical (gene for gene or single gene)resistancewas subject to breakdown as the alteration of one gene in thepathogens was sufficient to overcome the one gene conferringresistance in the vertical gene-for-gene resistance. As Zadoks (2002)points out, most of the concepts related to vertical and horizontalresistance have been modified: single gene resistance can be durableand polygenic resistance can be eroded or worn down. Themost widelycited examples of durable resistance against bacterial and fungalpathogens in plants are quantitative traits (Leach, 2001). Nevertheless,there are a few cases where single gene partial resistance has beendurable in plants and it appears that this occurs when the cost to thepathogen of overcoming the resistance is high (Leach, 2001; Hulbertet al., 2001). Nevertheless the tendency in plant breeding has movedfrom breeding for vertical resistance to breeding for horizontalresistance or pyramiding of single genes as a means of making break-down of resistance less likely (see for example Pink, 2002; Pederson andLeath, 1988).

Disease resistance in most animals that have been studied iscontrolled quantitatively by many genes and in breeding programmesit is generally assumed that disease resistance is a quantitative traitunder the control of many genes (see for example Detilleux, 2001).Nevertheless there are recorded cases of single gene resistance inanimals and humans (Hill, 2001) and breeders should not ignore thepossibility of using them to confer resistance. At the same timewe notethat most animal breeding programmes and studies of the genetics ofresistance have been developed with species in which screening ofmillions of animals for disease resistance is simply not possible. Themind simply boggles at inoculation and subsequent screening of severalmillion cows to see if just one possesses a single gene for resistance tomastitis. On the other hand plant breeders regularly inoculate hundredsof thousands of plants to identify and select resistant materials.Furthermore, in the case of the development of pesticide resistance ininsects and acaroids millions of individuals were subjected to theequivalent of screening for the very few resistant genotypes every timefarmers applied insecticides to their commercial plantations. In the caseof shrimps a single hatchery can easily produce severalmillion nauplii ina day and could expose millions of larvae or subsequent stages to adisease within few days or at the most weeks and select any survivors.Hence,we suggest thatuseful single gene resistancewhich is likelyoccurfor pathogens that cause severe mortalities, but that they may exist invery low frequencies, could well be identified in shrimps and mayprovide opportunities for rapidly obtaining resistant populations. Inthese cases, in the process of selection for resistance there may be asevere genetic bottleneck, and special breeding methodologies will berequired to introgress the resistant genes into commercial populations.

Fjalestad et al. (1993) suggest that in the fish farming environment,resistance to a given pathogen will normally develop slowly. However,resistance to serious pathogens may develop through natural selectionin aquaculture populations where the animals have continuously beenexposed to the pathogen for only a fewgenerations, as in the case of TSVin P. vannamei (Gitterle, 1999), and with the QX disease in the Sydneyrock oyster Saccostrea glomerata (Nell and Hand, 2003). In shrimp,which has only recently been bred in captivity, most of the genes thatcontrol resistance will probably have come from the original nativepopulations, although their frequencymayhavebeen radicallyaltered aspopulations encountered vastly different conditions.

In most animal breeding programmes the genetic variation to beexploited is assumed to be that which exists in the base populationswhen the organisms are domesticated. Nevertheless, over severalgenerations with continuous selection pressure on populations ofprolific species, mutations probably play an important role: this viewis supported by the Iowa corn experiments (Dudley and Lambert, 2004)

5J. Cock et al. / Aquaculture 286 (2009) 1–11

andmuch of thework on fruit flies (Harshman and Hoffmann, 2000). Inthe case of shrimps, with extremely large populations and a relativelyshort reproductive cycle, mutations may play a significant role inproviding genetic variation which can be utilized. In the case of insectresistance to pesticides the estimates vary widely from one favourablegene in 102 to one in 1013 individuals (Mazel and Davies, 1999). Thefavourable mutation rate estimated for E. coli was 4 × 10− 9 pergeneration (Imhof and Schlotterer, 2001; Foster, 2004). Currentestimates suggest a surprisingly constant mutation rate for a widerange of organisms of approximately 10− 5 mutations per locus pergenerationwith less than 2% of these being favourable (Frankham et al.,2002; Drake, 1991). The percentage of favourable mutations is probableless when they have a large effect than when they have a smaller effect(Imhof and Schlotterer, 2001). However, the proportion of favourable oradvantageousmutations would seem to be high in the case of immune-system genes (Hurst and Smith,1999).We suggest a conservative figureof 1% of favourablemutations that, coupledwith amutation rate of 10− 5,gives an estimate of 10− 7 favourablemutations per locus per generation.If we then assume that resistance to a particular disease is controlled bygenes on ten loci, or could occur due to amutation on any one of ten loci,then therewill be one favourablemutation for everymillion individuals.In a pond of 10ha (105m2) stocked at 50 animals m− 2 there are 5millionanimals, which translates into five favourable mutations. In the case offavourable mutations that could possibly provide single gene resistancethere are, to our knowledge, no published estimates of the probabilitiesof encountering such genes in large populations. Nevertheless, the veryrough estimates of favourable mutation rates suggest that they mayoccur with sufficiently high frequency in shrimp cultivation for them tobe a significant source of genetic variation for disease resistance or othertraits. Certainly the opportunity should not be ignored in species whereit is a relatively simple matter to screen millions of animals.

In native or commercial populations, in order for there to be aselection advantage for disease resistance genes, the disease must bepresent in the populations. However, little is known about the incidenceandseverity of diseases of Penaeid shrimps in thewild. It is quite possiblethat diseases that are of little or no importance in the native populationsin their native habitats only becomeepidemic in the intensive conditionsof cultivated shrimp. Three of the major diseases of shrimp, TauraSyndrome Virus (TSV), Yellow Head Virus (YHV) and White SpotSyndrome Virus (WSSV) were not reported in native populations beforethe massive epidemics in cultivated shrimp and hence little was knownabout the likelihood of encountering genetic resistance. In the case ofWSSV genetic analysis indicates that it is a representative of a previouslyunknown virus group now provisionally designated as a whispovirus(Van Hulten et al., 2000). Nevertheless in both TSV and WSSV geneticdifferences in resistance have been detected (Gitterle, 1999;White et al.,2002; Zarain-Herzberg and Ascencio-Valle, 2001).

In the case of species that apparently have not previously beeninfected by a particular organism it is not unusual to find resistance.Indica rice, originally from Asia, is attacked by the Hoja Blanca virus inthe Americas, but after screening thousands of varieties, simplyinherited, dominant resistance was found at a very low frequency ingermplasm originating in Asia in Japonica type rice (Ou and Jennings,1969). Similarly, humans have only recently been exposed to HIV andyet genetic resistance has already been identified related to poly-morphism on the CCR5 allele. The resistant allele's prevalence variesby ethnicity, being as high as 4–15% in Caucasians, and virtually absentin native Africans and East Asians (O'Brien and Moore, 2000). Thisresistance differs from that of simians that do not develop AIDS(Stebbing et al., 2004) and hence does not seem to have beenmaintained at a low level after a previous pandemic. Current thinkingsuggests that the CCR5 allele might have conferred selective advan-tage in Caucasian populations under conditions where smallpox wasprevalent (Galvani and Slatkin, 2003).

These two examples, one from the plant kingdom and the otherfrom animals, indicate that resistance to new diseases or diseases that

were unimportant in the wild may occur in wild populations, but thefrequency of resistancemay be extremely low. In the case of some newdiseases, previous selection for resistance to another disease mayconfer a level of resistance. This would suggest that in shrimps, even ifnew diseases attack them in their new intensive cultivation habitats,disease resistance may well exist. This would appear to be especiallylikely in shrimps as the resistance response is not specific to aparticular causal agent. At the same time in these cases the frequencyof resistant genes is likely to be extremely low. From a breeding pointof view the HIV and Hoja Blanca cases are intriguing: If one were tosearch for HIV resistance sources in native Africans, the populationwhere the disease first infected human beings, the chances of findingit would be extremely low. Similarly, in Hoja Blanca the resistancecame from Japonica rice which was not even grown in the neo-tropics.What is certain is that in such cases it is necessary to screen largenumbers of individuals from a wide range of populations.

Gemmill and Read (1998) suggest that there is a major possibilitythat resistance correlates negatively with other important fitnesscomponents. Consequently, resistance genes could be subject toantagonistic selective forces, which conspire to impose an equilibriumfrequency somewhere short of complete fixation. There has been muchdebate about the cost of genetic resistance, but until recently there hasbeen little evidence to substantiate this notion (Coustau et al., 2000;Brown, 2003). Heil and Baldwin (2002) suggested, for plants, that thereare various possible trade offs between fitness and resistance. These canbe adapted to the situation in animals, with trade offs being due to(a) allocation of fitness limiting resources to resistance traits (b) consti-tutive costs of inducible resistance related to detection pathways andreserves for response (c) auto-toxicity costs in which resistance con-ferring traits are directly toxic or detrimental to the host (d) ecologicalcosts related to the interaction between the host and the environment:For example less aggressive cannibalistic feeders might avoid infectionby not consuming moribund infected animals of the same species, butwould also be less fit in situations were feed is limiting and (e) incom-patibility of resistance to onediseasewithanother. In addition theremaybe close genetic linkages between the genes that control diseaseresistance and genes that negatively effect fitness. Heil and Baldwin(2002) indicate that there is increasingevidence inplants for tradeoffs infitness related to resistance in the absence of disease pressure. Tian et al.(2003) in an elegant piece of work showed a large cost of resistance tobacterial infection in the quintessential laboratory plant, Arabadopsis(Brown, 2003). Similarly, Kraaijeveld and Godfray (1997) showed astrong trade off between the capacity for melanoid encapsulation ofparasitoids in Drosophila fruit flies and their competitiveness. Gemmilland Read (1998) give further examples of tradeoffs for moths with virusresistance andmosquitoes for resistance to protozoanparasites. Coustauet al. (2000) note that in the case of animal resistance to parasites,although the precise physiological mechanisms involved in resistanceare poorly documented, most of the evolutionary literature is based onthe central assumption of a costly investment in defence functions,leading to a trade off between resistance and other fitness related traits.Along the same lines, based on his experience, Detilleux (2001), whenreviewing genetic improvement of livestock, concluded that increasedselection pressure to improve commercially important traits is oftenaccompanied by an increase in disease problems, suggesting a trade offbetween disease tolerance and other desirable characteristics. In thecase of crustaceans our work in P. vannamei shows a negative geneticcorrelation between resistance to White Spot Syndrome Virus andgrowth (Gitterle et al., 2005) and we have repeatedly observed poorreproductive fitness of putatively resistant individuals.

Recently plant breeders have been moving to more subtleapproaches that include enhancing Induced Systemic Resistance andSystemic Acquired Resistance (SAR) in which the plant defences arepreconditioned by prior infection or treatment that results in resistance(or tolerance) against subsequent challenge by a pathogen or parasite(Vallad and Goodman, 2004). The conditioned response which only

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occurs in the presence of infection may be obtained with a lower costthan permanently maintaining a resistance mechanism.

4. Breeding for resistance to diseases

As we have suggested earlier, the high cost of developinggenetically disease resistant populations makes this approach suitablewhen the potential damage is severe, there are no other cost effectivecontrol measures and there is evidence of genetic variation for thedesired trait which is not strongly negatively correlated with otherdesirable traits.

Selection for disease resistance is directly related to its effect ongrowth and survival: the objective is not disease resistance per se butrather the impact that disease resistance will have on the desiredperformance characteristics of the selected stock. Diseases can directlyeffect both growth and survival. Diseases such as TSV andWSSV causesevere damage through mortality, although animals that survive mayhave reduced growth rates. Other diseases such as NHP and vibrio,may cause high mortality under some conditions, whilst in otherstheir main effect may be to reduce growth. Up to the present, the mainfocus in selection for disease resistance in shrimps has been toimprove survival in the face of epidemics of diseases such as TSV,which may cause mortalities of 70% or greater and WSSV withmortalities close to 100%. We suggest that this emphasis on survivalwill continue, firstly due to the importance of this trait per se andsecondly due to methodological difficulties in screening and selectingfor tolerance that allows infected animals to grow well. Furthermore,if selection for growth is carried out under commercial conditions, andchronic diseases that effect growth rate are endemic, selection forgrowth will effectively be for growth and survival (≈ yield) in thepresence of the disease. It is noteworthy that this approach isextremely difficult to implement in programmes based on SpecificPathogen Free breeding stocks.

In certain populations a sufficiently high level of disease resistancemay be reached to reduce the disease to the level of no longer beingproblematic. This has already happened in some shrimp populationsselected for Taura resistance. However, a watching brief must be kepton such diseases as the causal agent may co-evolve with the resistantstock and become a problem, or the level of resistance in the

Fig.1. Survival of shrimp in commercial ponds in the Atlantic Coast of Colombia (red squares rto colour in this figure legend, the reader is referred to the web version of this article.)

populations may decline at the low levels of disease incidence in theresistant populations.

4.1. Success and failure

In practice disease resistance breeding programs are builtempirically and two examples illustrate how a breeding program fordisease resistance can be established when knowledge is extremelylimited and consequently uncertainty is great. Furthermore, oneexample, breeding for WSSV resistance, indicates some of the pitfallsand dangers of working with very little base line information on thegenetic variation for resistance.

4.1.1. Taura Syndrome Virus resistanceIn themid 1990s the shrimp industry in Ecuador and Colombiawas

decimated by TSV. At that time most ponds were stocked with larvacaught in their native habitats or from broodstock captured in thewild. Pond survivals declined dramatically (Fig. 1) and nobody knewwhy. Therewas no doubt of the economic importance of the losses: if asolution was not found to the problem the shrimp industry wouldsimply disappear. Attempts to control the epidemic by modifyingmanagement practices were largely unsuccessful. In spite of almosttotal ignorance on the cause of the problem at that time it was notedthat 20–30% of animals survived and various leaders, technicians andresearchers (including one of us) of the sector surmised that thesurvivors could simply be escapes or, on the other hand, they could begenetically resistant animals. With little to lose and much to gain, oneof the major producers in Colombia, C.I. Oceanos S.A. initiated aprogram to select the survivors from infected ponds and use them asparents for the next generation in a simple, extremely low cost massselection scheme. Although the sector was aware of the potentialproblems of inbreeding, the scheme was deemed acceptable as itwould rapidly indicate whether there was useful genetic variance forresistance. Within two to three generations commercial pond survivalrates had once again reached their previous levels indicating thesuccess of this simple process (Fig. 1). In later years the industrychanged from a mass selection procedure to a combined family andwithin-family selection scheme, which incorporated resistant animalsinto the population and monitored levels of Taura to ensure that, with

efer to stocking density). TSV first appeared in 1995. (For interpretation of the references

7J. Cock et al. / Aquaculture 286 (2009) 1–11

the low incidence of the disease under commercial conditions,resistance was not lost.

The Taura case indicates several important aspects of decidingwhether to include a particular disease resistance trait into a selectivebreeding program and how it can be incorporated. In the case of Taurathere was no doubt about the economic importance of the disease, andfurthermore therewas no knownmeans of controlling or managing thedisease: when the breeding programme commenced the causal agenthad not even been identified. At the same time the simple fieldobservation of the presence of survivors under commercial conditionssuggested that useful genetic variance for resistance to whatever wascausing the disease might exist. Thus, with the undoubted economicpotential advantages of resistant populations coupled with the compel-ling, but not rigorous evidence for genetic variance in resistance, thedecision was taken to set up an extremely simple program to seewhether the supposed genetic variance in resistance could be usefullydeployed. The selection process successfully identified truly resistantindividuals; demonstrated that therewas indeed useful genetic variancefor resistance; and developed protocols for selection that identifiedcommercially useful resistance. The whole industry moved towardsclosed cycle mass selection for survival in the presence of TSV andgrowth. A hidden factor that probably contributed to the success of theTaura resistant selection program was the fact that the populations inthe sector were based on wild populations that probably had a broadgenetic base, thus increasing the probability of encountering resistance.Nowadays we know that the heritability for resistance to TSV is ratherhigh for a viral disease, with published estimates in the range 0.20–0.30(Fjalestad et al., 1997; Argue et al., 2002; CENIACUA unpublished data).Argue et al. (2002) also found a negative relationship between growthandTSV resistanceafter a single roundof selection andMosset al. (2005)reported a weak but statistically significant negative correlation (r=−0.15) betweenmean family harvestweight andmean family survival ina TSV challenge test. In other populations selected for resistance to TSV,the growth rate of animals that survived infection was equal to that ofanimals from uninfected tanks when the effects of mortality onpopulation density and its effect on growth were taken into account(CENIACUA unpublished data). Similarly, in long term studies resistanceto Taura does not appear to be strongly negatively correlated with otherdesirable traits such as general pond survival, growth and reproductivefitness (CENIACUA and AKVAFORSK unpublished data).

This latter is of particular importance in mass selection, as anyreduction of selection pressure for disease resistance, if it werenegatively correlated with growth, would then lead to increasedsusceptibility in the population as a whole. Furthermore, althoughthere is little concrete data, there is considerable circumstantial evidencethat inbreeding depression was minimal in the populations used.

4.1.2. Searching for resistance to White Spot Syndrome VirusThe Taura case contrasts with that of breeding for WSSV resistance

and indicates some of the potential pitfalls of breeding for resistancewith limited knowledge about the genetic structure of the targetresistance trait. Similar reasoning was used to attempt to rapidlyobtain resistant materials using mass selection on commercial farmsand also to incorporate resistance in nuclear stocks of a family selectionbreeding program. However, in this case successful deployment ofcommercially viable stock has been an elusive goal. Once again therewas no doubt about the economic importance of control. The first signof difficulties was the extremely low frequency of survivors, withmortalities as high as 98% in the field and in challenge tests. Thissuggested an extremely low frequency of resistance genes in the initialpopulations. Second, was the difficulty of developing a reliable meansof testing for resistance. In commercial mass selection, the selectionpressure depended on the water temperature, which varied from yearto year and in the family selection the controlled challenge tests did notappear to select truly resistant materials (Gitterle et al., 2005).Following the classical method for analyzing challenge test data,

survival was treated as a binary trait and subjected to a cross-sectionallinear model with one record per individual. The binary survival datafor individual animals were recorded when the overall survival rate inthe challenge test was approximately 50%, which is the failure rateexpected to maximize the accuracy of the linear model. We suggestthat looking at the 50% survival in quite small populations, rather thanlooking for the rare animal that could survive until maturity, will likelyuncover polygenic rather than single gene resistance. Thirdly there isnegative genetic correlation between growth and resistance and westrongly suspect that there is a strong negative association withresistance and reproductive performance. Up to the present we havenoted that it is difficult to get survivors from challenge tests toreproduce, however, others have apparently been able to obtainprogenyfrom survivors. Nevertheless, in both themass selectionprogramme runby a commercial shrimp farm and the family selection program there isconclusive evidence that resistance levels can be improvedby bothmassselection and family selection and that resistance is lost if selection isbased largely on growth which is negatively correlated with WSSV(CENIACUA unpublished data). Nevertheless, the levels of resistanceachieved so far are not sufficient to revive the shrimp industry in areaswhere WSSV is endemic and severe.

The White Spot case highlights the importance of having a broadgenetic base so as to identify sources of resistance: the frequency ofresistance genes appears to be very low and there may be sources ofresistance that are not included in the initial populations. In addition ithighlights the difficulties encountered when there is a negativecorrelation between two or more desired traits. We suspect that dueto a negative relationship between both growth and reproductivecapacity and WSSV resistance some of the most resistant material hasbeen lost from the gene pools used for breeding for resistance.Furthermore WSSV shows the necessity of selection procedures thatcan identify resistance that will manifest itself at the commercial level.

4.2. Selection procedures

Selection procedures are needed that ensure that selected stock willperform well commercially: this normally means the ability to survivean epidemic. Presently selection for disease resistance in designedbreeding schemes is normally carried out based on survival recorded incontrolled challenge tests. Moss et al. (2005) point out the difficulties ofdeveloping challenge tests that provide useful information for develop-ing populations resistant to specific pathogens under commercialconditions. Their design should be such that they (i) emulate a naturaloutbreak in the ponds and (ii) evaluate all the defencemechanismof theshrimps. In order to emulate a natural pond outbreak in a challenge test,it is necessary to identify thenatural infectionpathways of the disease soas to establish an infection protocol that closely mimics them. Selectionprocedures depend on the nature of the disease and there is no standardprotocol that canbe recommended for shrimpdiseases in general. Anewprotocol is required for each disease. The experience with Taura, inseveral breeding programmes, indicates that effective protocols can bedeveloped and usedwithin on going breeding programmes. Techniquessuch as marker assisted selection are postulated as useful; neverthelessthey can only be deployed when protocols have been established todetect resistant individuals or populations, and closely linked markersidentified. In the particular case of resistance that increases survival ofanimals in the presence of a disease in an extremely prolific species suchas shrimps, with a relatively short growth cycle, looking for markergenes may provide little advantage. It is after all very simple to infectlarge numbers of animals and simple select the survivors.

Breeders tend to look for selection protocols which are simple andthat maximize heritability and genetic variance. Whilst breeding forsurvival in the face of WSSV, the time to 50% survival in animalsinoculated withWSSV, was chosen for reasons given before. The levelsof resistance in selected populations, as measured by both 50%survival and final survival, were both increased but survival of

8 J. Cock et al. / Aquaculture 286 (2009) 1–11

resistant stock has still not reached the levels required for commercialuse of the stock.

In the particular case of breeding for survival, recently developedlongitudinal models (e.g. proportional hazards and survival scoremodels) that utilize information from the full course of the challengetest have provided greater selection accuracy than conventional cross-sectionalmodels (Gitterle et al., 2006; Ødegård et al., 2006, 2007). Theselongitudinal models facilitate analysis of time-until-death and survivalwithin sub-periods, but, for maximum accuracy, require the challengetest to proceed until all animals are dead or mortality levels off.

Fjalestad (2005) pointed out that disease resistance is a candidatefor the use of indirect selection protocols in fish due to the difficulty ofmeasuring traits such as survival on individuals. It opens the way forcombined between andwithin-family selection. Furthermore, indirectselection is particularly appropriate when the desired character isdifficult to measure precisely (Fjalestad 2005). However, for indirectselection for survival to compete with direct selection the correlationbetween the correlated trait and survival must be high. This suggeststhat when breeding for increased survival it is important to ensurethat the selection protocols, whatever the breeding methodology,truly identify the desired trait and provide the opportunity to reachlevels of resistance that are commercially useful.

4.3. Breeding methodologies

The advantages and disadvantages of different breeding meth-odologies can be found in the standard texts on animal breeding. Inthis section the advantages and disadvantages of different breedingmethodologies are discussed solely with respect to the specific case ofdisease resistance. Mass selection, family selection and combinedfamily and within-family selection can all be effectively used inselective breeding for disease resistance depending on the circum-stances. Family based designs are particularly effective for those traitswhere heritability is low and hence the information on the individualbreeding candidate's own record provides little information on itsadditive genetic capacity. It seems likely that family selectionwill alsobe particularly appropriate for resistance under polygenic controlwhere the heritability is likely to be intermediate or low. On the otherhand, if disease resistance is under the control of few genes or ismonogenic, and also of extremely low frequency simple massselection may be effective but also implies a risk of greatly reducedoverall genetic variability in the population. Special breeding metho-dologies such as backcrossing may be expedient so as to introgressresistance into populations with other desirable characteristics and tomaintain genetic variability.

Family selection has several advantages over mass and individualselection when developing disease resistant populations. For manyreasons it may be advantageous to maintain the nuclear breedingstock free of diseases and several programmes are based on SpecificPathogen Free breeding nuclei. Amongst the most important is toprovide good quality stock that is not loaded with diseases fordissemination to commercial producers. With mass selection the onlypossible manner of obtaining information to select future breeders isby challenging them and this almost inevitably means exposing themto the disease followed by complex procedures to eliminate thedisease. In some cases it may be possible to select for resistance totoxins produced by pathogens or use Marker Assisted Selection andhence no exposure to the live pathogen is necessary. On the otherhand, with family selection, close relatives (usually full- and half-sibs)of the breeding candidates can be challenged with disease organismsand the information generated from these tests used to rank the non-exposed breeding candidates.

A further problemwithmass selectionwhen dealingwith extremelyvirulent diseases such as WSSV is the rapid narrowing of the geneticvariation in the population, which will restrict future genetic improve-ment and may make populations extremely vulnerable to other

diseases. Unfortunately, several of the mass selection programmes thathave been carried out by commercial operations producing their ownstock have not beenwell documented.Mass selection in the absence of aparticular diseasemay lead to low levels of resistance anddevastationbythat particular disease when it is introduced. On the other hand there issome evidence that inbreeding with mass selection for such traits asgrowth and pond survival may not always lead to serious problems inbreeding programmes. This view is supported data from the P. vannameiprogramme at the Oceanic Institute from which Moss et al. (2007)conclude that under favourable conditions inbreeding effects arestatistically significant but small for growth, and are minimal forgrow-out survival, in the absence of viral pathogens.Whilst the negativeeffects of inbreeding on survival and growth are small under favourableconditions they increase when animals grow in poor environments(Doyle et al., 2006; Moss et al., 2007). Furthermore, through selectionsubstantial gains for growth can likely be achieved at low to moderatelevels of inbreeding (Moss et al., 2007). De Donato et al. (2005) followed11 generations of mass selected lines in Venezuela and reportedincreased resistance to endemic diseases such as IHHNV and no signsof deteriorationon thefitness-related traits. In the case of shrimpsundercommercial conditions a 20%mortality in thegrow-out period and a50%mortality in the phase from spawning to stocking ponds are considerednormal and accepted. This translates into a survival rate of only 40%whichwould be unacceptable inmany species.We surmise that there islittle build up of deleterious genes in shrimp populations mass selectedfor pond survival and growth under commercial conditions; thoseanimals that carry deleterious genes will be eliminated naturally asanimals that carry them neither survive nor grow well. This type of selfelimination of poor types is readily achieved in highly prolific speciesgrown under commercial conditions where a moderate loss of unfitanimals is the norm. On the other hand inbreeding moderately toseverely affects survival in the presence of TSV and WSSV (Moss et al.,2007). Similarly the Venezuelan populations described by De Donatoet al. (2005)when confrontedwith TSV, aftermany cycles of selection inthe absence of the virus, proved to be highly susceptible. Thus control ofinbreeding is particularly importantwhen improving such traits such asdisease resistance, nevertheless, it is probably prudent for breedingprograms tomanage inbreeding irrespective of the traits under selection(Moss et al., 2007).

The experiences with TSV highlight some of dangers of loss ofheterozygositydue to inbreeding in the absence of aparticularpathogen.Themass selectedVenezuelanpopulations (DeDonato et al., 2005)werefree of TSV for many generations, but when TSV appeared the levels ofresistancewere extremely low. Similarly, the SPF Kona populations fromthe Oceanic Institute from Hawaii, that were not selected for TSVresistance proved to be extremely susceptible to TSV (Srisuvan et al.,2006). Breeding programmes that maintain their breeding nuclei asSpecific Pathogen Free populations run the risk of producing animalsthat are extremely susceptible to those specific pathogens unlessselection for resistance is incorporated into the breeding scheme: thisbecomes complex when there are several pathogens on the list.

Mass selection is not as effective as family selection whensimultaneously selecting for traits that are negatively correlated, acase which appears to be quite common for disease resistance. On theother hand, family selection for improved resistance is currently basedon sib-testing without exposing the breeding candidates to thepathogen, which only utilizes the 50% of the additive genetic varianceaccounted for by the between-family component. It is evident thatdevelopmentof appropriate testing and biosecuritymeasures thatmakeit possible to safely introduce animals surviving the challenge test (orgametes from survivors)will substantially increase the efficacy of familybased selection schemes targeting resistance to diseases. There is noindividual selection if challenge tests are carried out on separatepopulations that are not included in the breeding nucleus. In contrast,mass selection is particularly effectivewhen thedesired trait determineswhether an individual survives, when no resistance has yet been

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detected or when frequencies of the desired trait are extremely low andlarge numbers of animals fromdifferent genetic backgrounds need to bescreened.We suggest that in these situationsmass selection can be usedto screenmassive numbers of individuals, literallymillions, and to selectthose rare individuals that are able to survive a severe disease epidemicand go on to produce. In these cases it is necessary to ensure that thechallenge test used reflects commercial conditions and that the selectionpressure is maintained: if there is a cost to the resistance thenpopulations are likely to revert rapidly if the selection pressure isrelaxed. With the extremely heavy selection pressure applied in suchcases the disease resistant animals finally encountered may lack manyother desirable traits for commercial production.

Inplants it is quite common to detect disease resistance in extremelypoor plant types. In order to remedy this situation plant breedersintrogress disease resistance from unimproved or wild species intoimproved populations using backcrossing techniques. We suggest thatthis approach may be appropriate for shrimps. In those cases wherethere is not a ready source of commercially useful resistance in thecurrent commercial or nuclear breeding stock populations, it may bepossible to produce millions of larvae or juveniles and screen these forsurvival using mass selection. If resistant types are discovered, and wehave no way of knowing beforehand the probability, then the resistantanimal or animals can then be used to introgress resistance into thecommercial populations. Various techniques such as backcrossing maybe used to introgress the desired genes and eliminate the undesirablegenetic load associated with them in the initial resistant genotype.

The search for the rare individual that survives in the case ofcatastrophic diseases is dangerous as there is no guarantee of thedurabilityof the resistance. For several decadesplant breedershavebeenusing pyramiding of genes to lower the probability of break down ofresistance (Pink, 2002; Pederson and Leath,1988). In this process severalsingle genes that provide resistance are “pyramided” into the populationso that all individuals carry more than one gene-for-gene resistancegene. This reduces the probability of breakdown enormously. Forexample if the probability of breakdown is one in 10−8 of the pathogenovercoming either of two resistances in a given individual in a givenperiod of time it will be reduced to one in 10−16 if two genes are present.

In order to pyramid genes it is necessary to identify the sources ofresistance in resistant individuals. Until recently this was extremelydifficult to achieve: nowadays with molecular markers it may bepossible to identify different sources of single gene resistance ondifferent loci and to determine which resistant genes an individualpossesses. Hence, use of single gene resistance should preferably bebased on more than one source of single gene resistance pyramidedinto the population with marker assisted selection to ensure thatselected resistant phenotypes are also pyramided genotypes.

Breeding for resistance is a continuous dynamic process: the geneticcomposition of both pathogen and host are continually changing (Ebert,1998). Carius et al. (2001) found significant genetic variation amongclones of the freshwater crustacean Daphnia magna for susceptibility toits parasite Pasteuria ramosa and significant genetic variation amongisolates of the bacterial parasite P. ramosa for infectivity to it hostD. magna. Loss of resistance in a population can be the result of changesin the host and the genetic make up of the pathogen. After hostresistance has been developed above a certain level in a population,disease incidence may be reduced to extremely low levels. Under theseconditions selection pressure is reduced for resistance, and if there is afitness cost associated with the resistance this will be lost. We suggestthat in shrimp populations, with prolific breeders, the drift towardssusceptibility is potentially rapid. This viewpoint is supported by ourobservations of a combined family and within family selectionprogramme for resistance to TSV in which populations not subjectedto continuous selection pressure for resistance appeared to loseresistance (CENIACUA unpublished data).

Pathogen populations are continually evolving and adapting to thehost environment which is itself changing. Pathogens which are

extremely virulent and kill their host before having the opportunity toinfect a new host, as is the case for example with Ebola virus, are oftennot able to maintain themselves (Ebert, 1998)). Selection pressure inthese cases is initially for less virulence in the pathogen, as has beenshown to occur in myxomatosis although over the long term a series ofstrains of differing virulence coexist (Aparicio et al., 2004). Converselywhen host resistance levels are increased in a population the pathogenwill tend to evolve mechanisms that overcome that resistance. Recentlyvarious strains of TSV have been recognized with varying levels ofvirulence (Srisuvan et al., 2006). Fortunately it appears that shrimppopulations resistant to one particular strain are also resistant to otherstrains (Moss et al., 2005; Srisuvan et al., 2006), that is to say there is nota strain by resistant genotype interaction. In Colombia there are notabledifferences in the sequences in the CP2 section of TSV samples taken in1998 and in 2006/7, indicating evolution of the virus strains (CENIACUAunpublished data). It is not yet clear as to whether this evolution isrelated to increased virulence in the face of shrimp populations bred forresistance to TSV. Nevertheless, breeders should take into account boththe co-evolution of host resistance and pathogen virulence and thepossible lack of selection pressure for diseases by constantly monitoringlevels of resistance, even for diseases that have ceased to be a problem.Furthermore, itmay bepossible to guard against loss of resistance by thehost or evolution of more virulent pathogens by a well designeddisseminationprogramme inwhichmass selection for endemic diseasesis included as part of the process for multiplication of broodstock.

4.4. Base populations and population size

Selective breeding programmes are only appropriate when geneticvariation exists for the traits to be selected. A number of breedingprograms in fishmay have failed due to lowgenetic variation in the basepopulation (Teichert-Coddington and Smitherman, 1988; Huang andLiao, 1990). Miles and Pandey (2004) indicate that plant improvementprogram tend to make more rapid progress after the introduction ofmore diverse germplasm. Similarly, in cassava the success of modernbreeding efforts has been attributed to the great genetic variabilitywhich the modern programmes acquired from the outset (Kawano andCock, 2005). On the other hand, in the long term divergent selection foroil and protein content of maize in Illinois starting from an extremelynarrow genetic base, Dudley and Lambert (2004) state that “100generations of selection have not eliminated genetic variability and anupper limit has not been reached.” In several long term selection trialsfor body weight in mice with effective population sizes of less than 100some, but not all, populations have reached a plateau (Hill and Bunger,2004). In reviews of long term selection in laboratory and domesticanimalsmuchof the initial gains are attributed to the genetic variance inthe initial base population, and later response to mutations in thepopulation (Hill and Bunger, 2004; Weber, 2004).

Inmany animal improvement programmes the breeding designpayscareful attention to maintaining genetic variation, but less attention ispaid to ensuringmaximum diversity in the initial foundation stock. Partof the reason is undoubtedly related to the fact that most domesticatedspecies have been selected for a longperiod of time for specific traits andthe introduction of more genetic diversity to a foundation populationwould undoubtedly introducemany undesirable traits. Breeders simplycannot use native stocks to enhance their already improved stocks as thenative populations are so far behind in performance that they are notcompetitive (Hill and Bunger, 2004) and could, at least in the short terminduce negative genetic gain in the desired traits. In the case of Penaeidshrimps this is not currently a major problem as the existing improvedpopulations are mostly only a few generations from the wild popula-tions.However, aspopulations are improved itwill rapidly becomemoredifficult to incorporate genetic diversity and still maintain the geneticgains in desired traits. Furthermore, in many countries importation ofwild populations or specific populations is restricted due to biosecuritymeasures. In addition the introduction of newgermplasm into breeding

10 J. Cock et al. / Aquaculture 286 (2009) 1–11

programme with Specific Pathogen Free nuclei is onerous and timeconsuming. Thus the relatively new shrimp breeding programs shouldmake every effort to obtain genetically diverse populations before thepopulations have diverged substantially from thewild populations fromwhich they were recently derived.

Unfortunately, studies on how the base population should be formed(i.e. thenumberof individuals to be sampled fromoneor several founderstrains, their mixing, and the intensity of selection to be applied duringthe initial generations) their effects on the magnitude and variability ofthe long term selection response and inbreeding are few (Holtsmarket al., 2006).

In the case of shrimp breeding for disease resistance it would appearthat in order to detect genetic resistance in existing populations awide arange of origins of base populations should be explored and screened.Furthermore, large populations (millions of animals) should beproduced and screened to detect mutants or recombinants that conferresistance to catastrophic diseases. Any desirable traits obtained in thesemassive screening efforts should then be incorporated into the basepopulation and maintained irrespective of the breeding scheme beingused.

5. Conclusions

Selective breeding provides a useful means of controlling shrimpdiseases when these currently cause severe losses, other controlmeasures are difficult, useful genetic variation in resistance exists andresistance is not negatively and strongly associatedwith other desirabletraits. Genetic variation in resistance may be encountered either in theinitial base populations or may spontaneously arise due tomutations ornew recombinants. Effective protocols are required to detect resistance;due to the prolificacy of shrimps large populations can be screened so asto identify sources of resistance that occur at low frequencies. The mostappropriate breedingmethodology dependson thenature of thediseaseor diseases that are of interest to the producers.

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