Disease resistance in Atlantic salmon (Salmo salar) Coinfection of the intracellular bacterial pathogen Piscirickettsia salmonis and the sea louse Caligus rogercresseyi

Disease Resis ance in A lan ic Salmon ( Salmo sala ): Coin ec ion o he In acellula Bac e ial Pa hogen Pisci icke sia salmonis and he Sea Louse Caligus oge c esseyi Jean Paul Lho en e 1 , Jose ´A. Galla do 2 *, Bea iz Villanue a 3 , Ma ı ´a J. Ca aban ˜o 3 , Robe o Nei a 1,4 1Aquainno o S.A, Pue o Mon , Chile, 2Pon i icia Uni e sidad Ca o ´lica de Valpa aı ´so, Valpa aı ´so, Chile, 3Depa amen o de Mejo a Gene ´ ica Animal, INIA, Mad id, Spain, 4Depa amen o de P oduccio ´n Animal, Facul ad de Ciencias Ag ono ´micas, Uni e sidad de Chile, San iago, Chile Abs ac Backg ound: Na u ally occu ing coin ec ions o pa hogens ha e been epo ed in salmonids, bu hei consequences on disease esis ance a e unclea . We hypo hesized ha 1) coin ec ion o Caligus oge c esseyi educes he esis ance o A lan ic salmon o Pisci icke sia salmonis; and 2) coin ec ion esis ance is a he i able ai ha does no co ela e wi h esis ance o a single in ec ion. Me hodology: In o al, 1,634 pedig eed A lan ic salmon we e exposed o a single in ec ion (SI) o P. salmonis (p ima y pa hogen) o coin ec ion wi h C. oge c esseyi (seconda y pa hogen). Low and high le el o coin ec ion we e e alua ed (LC = 44 copepodi es pe ish; HC = 88 copepodi es pe ish). Su i al and quan i a i e gene ic analyses we e pe o med o de e mine he esis ance o he single in ec ion and coin ec ions. Main Findings: C. oge c esseyi signi ican ly inc eased he mo ali y in ish in ec ed wi h P. salmonis (SI mo ali y = 251/545; LC mo ali y = 544/544 and HC mo ali y = 545/545). He i abili y es ima es o esis ance o P. salmonis we e simila and o medium magni ude in all ea men s (h 2SI = 0.2360.07; h 2LC = 0.1760.08; h 2HC = 0.2460.07). A la ge and signi ican gene ic co ela ion wi h ega d o esis ance was obse ed be ween coin ec ion ea men s ( g LC-HC = 0.9960.01) bu no be ween he single and coin ec ion ea men s ( g SI-LC = 20.1460.33; g SI-HC = 0.3260.34). Conclusions/Signi icance: C. oge c esseyi, as a seconda y pa hogen, educes he esis ance o A lan ic salmon o he pa hogen P. salmonis. Resis ance o coin ec ion o Pisci icke sia salmonis and Caligus oge c esseyi in A lan ic salmon is a he i able ai . The absence o a gene ic co ela ion be ween esis ance o a single in ec ion and esis ance o coin ec ion indica es ha di e en genes con ol hese p ocesses. Coin ec ion o di e en pa hogens and esis ance o coin ec ion needs o be conside ed in u u e esea ch on salmon a ming, selec i e b eeding and conse a ion. Ci a ion: Lho en e JP, Galla do JA, Villanue a B, Ca aban ˜o MJ, Nei a R (2014) Disease Resis ance in A lan ic Salmon (Salmo sala ): Coin ec ion o he In acellula Bac e ial Pa hogen Pisci icke sia salmonis and he Sea Louse Caligus oge c esseyi. PLoS ONE 9(4): e95397. doi:10.1371/jou nal.pone.0095397 Edi o : J. S ephen Dumle , The Johns Hopkins Uni e si y School o Medicine, Uni ed S a es o Ame ica Recei ed Janua y 9, 2014; Accep ed Ma ch 25, 2014; Published Ap il 15, 2014 Copy igh : ß2014 Lho en e e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Funding: This s udy was inanced by INNOVA-CHILE o CORFO h ough he p ojec s: 1) Conso cio emp esa ial de gene ´ ica y desa ollo bio ecnolo ´gico pa a la indus ia salmone a (Nu206-5047), and 2) Desa ollo de una nue a me odologı ´a pa a la iden i icacio ´n y seleccio ´n de salmo ´nidos gene ´ icamen e esis en es al ec opa a ´si o Caligus oge c esseyi (07CN13PBT-61). This s udy is a collabo a i e wo k o di e en ins i u ions belonging o ‘‘Red de gene ´ ica e inmunologı ´a pa a el con ol de pa o ´genos en Acuicul u a’’ (RED CYTED Nu11RT0420). BV ecei ed unding om he Minis e io de Economı ´a y Compe i i idad, Spain (p ojec CGL2012- 39861-C02-02). The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: Jean Paul Lho en e and Robe o Nei a we e employed in AQUAINNOVO when his esea ch was pe o med. This does no al e he au ho s’ adhe ence o all he PLOS ONE policies on sha ing da a and ma e ials. * E-mail: [email p o ec ed] In oduc ion Na u ally occu ing coin ec ions o pa hogens ha e been epo ed in a ious salmonid species [1], [2]. Howe e , he consequences o o e all disease esis ance emain unclea . Mos s udies ha e shown ha p esence o a p ima y pa hogen educes he esis ance esponse o a seconda y pa hogen. Fo example, in ainbow ou , p ima y in es a ion o he pa asi e Myxobolus ce eb alis supp esses he immune sys em, inc easing mo ali y associa ed wi h a seconda y in ec ion by Ye sinia ucke i [3]. Simila ly, A lan ic salmon in ec ed wi h IPNV show signi ican ly inc eased mo ali y as smol s when exposed o Fu unculosis, Vib iosis and ISA [4], [5]. Con e sely, se e al s udies ha e shown ha coin ec ion does no necessa ily inc ease mo ali y. Fo example, some s udies on ainbow and b own ou showed ha IPNV educed he in ec ion capaci y o he hema opoie ic nec osis i us (IHNV) and ha he mo ali y o ish in ec ed wi h bo h i uses was signi ican ly lowe han ha obse ed in single challenges wi h each pa hogen [6], [7], [8], [9]. Fu he mo e, acu e coin ec ion o IPN and ISA in A lan ic salmon signi ican ly educed mo ali y compa ed wi h a single in ec ion by ISA [4]. Sea lice a e among he mos impo an sani a y p oblems in he global salmon aquacul u e indus y [10], u he hey ha e been PLOS ONE | www.plosone.o g 1 Ap il 2014 | Volume 9 | Issue 4 | e95397 linked o wild salmon and ou popula ion declines a ound he wo ld [11], [12], [13]. Salmonid in es a ion by sea lice may be associa ed wi h le hal o sub-le hal e ec s [14], [15], [16]. Sub- le hal e ec s may include s ess [17], [18], loss o appe i e, dep ession o he immune sys em and skin damage [19], [20], and he e o e, i can con ibu e o inc eased suscep ibili y o o he diseases [21], [22]. In ag eemen wi h his hypo hesis, Mus a a e al. [22] epo ed ha A lan ic salmon in ec ed wi h he sea louse Lepeoph hei us salmonis showed inc eased suscep ibili y o a mic o- spo idian pa asi e (Loma salmonae). Recen ly, Nowak e al. [23], Bus os e al. [24] and Valdes-Donoso e al. [25] sugges ed, based on ield s udies on A lan ic salmon, ha sea lice a ec disease esis ance o he amoeba Neopa amoeba pe u ans and o ISA . In Chile, caligidosis caused by Caligus oge c esseyi and pisci ick- e siosis caused by Pisci icke sia salmonis ha e his o ically been he mos impo an heal h p oblems in he salmon indus y in he g ow h-ou p oduc ion phase [26], [27], [28], [10]. C. oge c esseyi is he only sea louse ha a ec s he Chilean salmon indus y [29], and annual losses a ibu ed o his pa asi e a e es ima ed a mo e han $178 million US [27], [10]. P. salmonis, an in acellula bac e ium, was desc ibed in Chile a he end o he 1980s in he X egion [30], [31], [32] and was ini ially ound o be s ongly associa ed wi h A lan ic salmon. I has now ex ended o o he salmonid species a med in Chile [33], p oducing mo ali y a es o up o 50% in ish in he sea g owing s age, wi h mone a y losses exceeding $100 million US pe yea [28]. Fu he mo e, P. salmonis has been epo ed in di e en coun ies and can in ec ainbow ou (Onco hynchus mykiss), che y salmon (Onco hynchus masou), chinook salmon (Onco hynchus shawy scha) and pink salmon (Onco hynchus go buscha) [34]. Resis ance o A lan ic salmon o C. oge c esseyi and P. salmonis has ecen ly been s udied in single-in ec ion challenges in labo a o y condi ions by Yan˜ez e al. [35] and Lho en e e al. [36]. Lho en e e al. [36] epo ed he i abili y o esis ance o A lan ic salmon o he sea louse C. oge c esseyi o low (0.03–0.06) and medium (0.22– 0.34) magni udes o he mobile and sessile s ages o he pa asi e, espec i ely. O he s udies pe o med on di e en sea lice species con i med ha A lan ic salmon ha e a he i able de ensi e mechanism agains Lepeoph hei us salmonis [37], [38], [39] and Caligus elonga us [40]. Addi ionally, i has been shown ha bo h wild and a med A lan ic salmon ha e a he i able de ensi e mechanism agains a ious bac e ia [41], [42], [43], [44], including P. salmonis [45]. Fo ins ance, Yan˜ez e al. [35] epo ed he i abili y o esis ance o A lan ic salmon o P. salmonis anging om 0.11 o 0.41. In his s udy, we hypo hesized ha coin ec ion wi h he sea louse C. oge c esseyi educes he esis ance o A lan ic salmon o P. salmonis because o he well-documen ed s ess and dep ession o he immune sys em p oduced by sea lice in ec ion in salmonids. Addi ionally, we hypo hesized ha coin ec ion esis ance is a he i able ai ha does no co ela e wi h esis ance o a single in ec ion o P. salmonis because salmonid de ense mechanisms agains bac e ia and pa asi es a e subs an ially di e en [46]. We p esen expe imen al e idence suppo ing bo h hypo heses o he in e ac ion be ween A lan ic salmon, P. salmonis and C. oge c esseyi. Ma e ials and Me hods E hics S a emen This s udy was ca ied ou in in acco dance wi h he guide o he ca e and use o expe imen al animals o he Canadian Council on Animal Ca e. The p o ocol was app o ed by he Bioe hical commi ee o he Pon i icia Uni e sidad Ca o´lica de Valpa aı ´so (Nu10/2013). The animals we e anaes he ized wi h benzocaine p io o he a ious handling p ocesses and ma king. Eu hanasia was pe o med using an o e dose o anes hesia. All e o s we e made o p o ide he bes g owing condi ions and o minimize su e ing. Fish In o al, 1,634 A lan ic salmon smol ish o 103 g a e age body weigh om 15 ull-sib amilies (co esponding o se en pa e nal hal -sib amilies) o he AquaChile Gene ic P og am we e a ailable o his s udy. The amilies o igina ed om a nes ed ma ing design (15 emales and se en males), in which one male e ilized he eggs o a leas wo emales. Eggs om each amily we e p oduced du ing he spawning season o 2009. The ish we e ed egula ly wi h a comme cial die and indi idually agged in Ap il 2010 a an a e age weigh o 5 g (SD = 8.0 g). Then, hey we e ans e ed as smol s in Feb ua y 2011 o he Aquad ice S.A. expe imen al s a ion loca ed a he Quillaipe sec o o Pue o Mon (Chile). A heal h check by PCR was pe o med p io o ans e o e i y ha he ish we e ee o i al (IPN and ISA ) and bac e ial pa hogens (P. salmonis,Renibac e ium salmonina um,Vib io sp and Fla obac e ia sp). A he expe imen al s a ion, he ish unde wen a h ee-week acclima ion pe iod unde seawa e condi ions (salini y o 33% and a empe a u e o 12uC). Expe imen al Design The ish we e exposed o h ee di e en in ec ion scena ios wi h h ee eplica es ( anks) o simula e single in ec ion by P. salmonis and coin ec ion by P. salmonis wi h wo di e en le els o in ec ion p essu e o he pa asi e C. oge c esseyi. In all coin ec ion’s expe imen s P. salmonis was used as p ima y pa hogen and C. oge c essyi was used as a seconda y pa hogen. Bo h le els o in ec ion p essu e we e es ablished using in o ma ion om p e ious expe imen s [47] and se up o expe imen al ish size o ensu e success ul bu di e en ial se lemen be ween ea men s and no mo ali y associa ed o single sea lice in ec ion [47,48]. Sea lice used o p oduce copepodi es we e pa hogen ee (P. salmonis and ISA ). Fish om he 15 ull-sib amilies we e equally dis ibu ed in nine anks o 0.72 m 3 such ha he same numbe o ish pe amily was used in each o he ollowing ea men s: a) Single in ec ion (SI). The ish we e in ec ed wi h P. salmonis using a cohabi a ion challenge es in an en i onmen ee o C. oge c esseyi. b) Low p essu e o coin ec ion (LC). The ish we e in ec ed wi h P. salmonis using a cohabi a ion challenge es and hen in es ed wi h C. oge c esseyi using a low in es a ion p essu e o 44 copepodi es pe ish. c) High p essu e o coin ec ion (HC). The ish we e in ec ed wi h P. salmonis using a cohabi a ion challenge es and hen in es ed wi h C. oge c esseyi using a high in es a ion p essu e o 88 copepodi es pe ish. The cohabi a ion me hod used o he p ima y in ec ion wi h P. salmonis p oduces a na u al in es a ion and educes he manipu- la ion o expe imen al ish in compa ison wi h in a-pe i oneal injec ion. A he beginning o he challenge wi h P. salmonis (day ze o), 78 ish wi h unknown pedig ee, e e ed o he e as in ec i e ish, we e in ec ed by in a-pe i oneal injec ion. A olume o 0.2 ml/ ish (a 10 6,2 TCID/ml) o a i ulen s ain o P. salmonis, isola ed om A lan ic salmon and comme cially a ailable om ADL-Diagnos ic L da., was injec ed o each in ec i e ish. The in ec i e ish we e hen placed in o cohabi a ion wi h agged ish o each an abundance o 260 ish app. pe ank and a densi y o 42 kg/m 3 . The mo ali y o he in ec i e ish was 100% a 30 days pos -in ec ion, hose ish we e no included in ou e alua ion o esis ance. Resis ance o P. salmonis was measu ed as a mo ali y Resis ance o Coin ec ion in A lan ic Salmon PLOS ONE | www.plosone.o g 2 Ap il 2014 | Volume 9 | Issue 4 | e95397 ai (dead/ali e). The sexes, ini ial weigh s and inal weigh s o all cohabi an agged ish we e eco ded. Fou days pos -in ec ion, a seconda y in ec ion was conduc ed wi h C. oge c esseyi as desc ibed by Lho en e e al. [36]. Ten in ec i e ish pe ank we e used o e alua e he e ec i e bu den o he pa asi e. We con i med he lack o sea lice (i.e., chalimus I) in he SI ea men and an inc emen al esponse o se lemen in bo h coin ec ion ea men s (LC = 2964.8 lice pe ish; HC = 6064.8 lice pe ish). S a is ical Analysis Kaplan-Meie su i al cu es we e ob ained using he so wa e Su i al ki 6 [49] o de ine he esponses o A lan ic salmon o he di e en ea men s. A chi-squa ed es was used o e alua e di e ences be ween ea men s [49]. To es ima e ixed e ec s and hei in e ac ions, an ANOVA analysis was conduc ed using a gene al lineal model (GLM) [50]. Mo ali y was de ined a 30 days a e in ec ion wi h P. salmonis and ea ed as a no mally dis ibu ed ai [43]. The GLM was: yijklm~mzCizSjzFkzTCðÞ lizCFikzSFjkzCSFijkzeijklm whe e y ijklm is he ish mo ali y condi ion, mis he popula ion mean, C i is he ea men e ec (SI, LC o HC), S j is he sex ( emale o male), F k is he ull sib amily e ec (1,2,3,…, 15), T(C) li is he l h ank wi hin he i h ea men e ec (C), CF ik is he ea men by amily in e ac ion, SF jk is he sex by amily in e ac ion, CSF ijk is he ea men by sex by amily in e ac ion and e ijklm is he andom esidual e ec . The CF ik in e ac ion de e mines whe he amilies esis an o SI a e also esis an o LC o HC coin ec ion. A quan i a i e gene ic analysis o he esis ance o P. salmonis was conduc ed o each in ec ion ea men o compa e es ima es o he addi i e gene ic a iance o he mo ali y ai e alua ed. A h eshold model (TM) was assumed o es ima e a iance componen s [51]. This model assumes a no mal unde lying liabili y a iable lde e mining he ca ego ical ou comes o he es - pe iod su i al, such ha l ijk #0 co esponds o Y ijk = 0 and l ijk .0 co esponds o y ijk = 1. The esidual a iance o lwas assumed o be 1. P (yijk~1) ~P (lijkw0) ~W(w0ih) Figu e 1. Mo ali y cu es o A lan ic salmon coin ec ed wi h P. salmonis and C. oge c esseyi .The da a show he cumula i e mo ali y o h ee eplica es ( anks) (R1–R3) o h ee di e en scena ios: single in ec ion (SI) wi h P. salmonis and co-in ec ion wi h wo le els o in es a ion p essu e o C. oge c esseyi (low p essu e o coin es a ion (LC) = 44 copepodi es pe ish; high p essu e o coin es a ion (HC) = 88 copepodi es pe ish). Each eplica e had app oxima ely 182 pedig eed ish ha we e ee o disease. The a ow indica es he day o coin ec ion. doi:10.1371/jou nal.pone.0095397.g001 Resis ance o Coin ec ion in A lan ic Salmon PLOS ONE | www.plosone.o g 3 Ap il 2014 | Volume 9 | Issue 4 | e95397 whe e W(*) co esponds o he s anda d no mal dis ibu ion, w’ i is he incidence ec o ha links he da a wi h he pa ame e s ha de ine he mean o he dis ibu ion o he liabili y indexed by he pa ame e s in hand hcon ains he popula ion mean, he addi i e gene ic alue and he signi ican (P,0.05) en i onmen al e ec s (sex, challenge ank, and ini ial weigh as co a ia e). Because he same amilies we e ep esen ed in he h ee in ec ion ea men s (SI, LC and HC), he gene ic co ela ion o P. salmonis esis ance in di e en in ec i e scena ios measu es he in e ac ion ha de e mines whe he amilies esis an o SI a e also esis an o LC o HC coin ec ion. A se o h ee bi a ia e linea model (LM) analyses was used o es ima e he gene ic co ela ion o esis ance among he di e en ea men s: y~Xb zZa ze whe e yis he obse a ions ec o o he p oposed ai s (SI, LC o HC), Xis he design ma ix, bis he ec o o signi ican Figu e 2. Kaplan–Meie su i al unc ion o A lan ic salmon coin ec ed wi h P. salmonis and C. oge c esseyi .The su i al unc ion ep esen s he esis ance o he A lan ic salmon (i.e., he p opo ion ha had no died on each day ollowing challenge) in h ee di e en ea men s: single in ec ion (SI) wi h P. salmonis and coin ec ion wi h wo le els o in es a ion p essu e o he sea louse C. oge c esseyi (low p essu e o coin ec ion (LC) = 44 copepodi es pe ish; high p essu e o co in es a ion (HC) = 88 copepodi es pe ish). doi:10.1371/jou nal.pone.0095397.g002 Table 1. ANOVA esul s o mo ali y 30 days a e P. salmonis in ec ion. Sou ce o a ia ion d SS SS% MS F P- alue In ec ion ea men (I) 2 87.90 28.0% 43.940 310.29 0.0001 Sex (S) 1 1.75 0.6% 1.750 12.38 0.0004 Full-sib Family (F) 14 22.30 7.1% 1.590 11.25 0.0001 Tank wi hin I (T) 6 3.72 1.2% 0.620 4.38 0.0002 I6F 28 12.94 4.1% 0.460 3.26 0.0001 S6F 2 0.53 0.2% 0.260 1.87 0.1547 S6I6F 42 6.37 2.0% 0.150 1.07 0.3517 E o 1267 179.46 57.0% To al 1362 314.98 doi:10.1371/jou nal.pone.0095397. 001 Resis ance o Coin ec ion in A lan ic Salmon PLOS ONE | www.plosone.o g 4 Ap il 2014 | Volume 9 | Issue 4 | e95397 (P,0.05) ixed e ec s wi hin each es (sex, challenge ank and ini ial weigh as a co a ia e), Zis he incidence ma ix o he andom e ec s, ais he b eeding alues ec o and eis he esidual e o ec o . The co a iance s uc u e o andom e ec s was: Va ~ai aj  ~As2aii As2aij As2aij As2ajj "# ;Va ei ej  ~Is2eii Is2eij Is2eij Is2ejj "# whe e a i/j and e i/j a e he ec o s o addi i e gene ic and esidual alues o ai s i/j, espec i ely; Ais he addi i e gene ic ela ionship ma ix; Iis he iden i y ma ix; s 2 a ii/jj and s 2 e ii/jj a e he a iances o addi i e gene ic and esidual e ec s, espec i ely, o ai s i/j and s 2 a ij and s 2 e ij a e he co a iances o addi i e gene ic and esidual e ec s, espec i ely, o he i h and j h ai s. Res ic ed Maximum Likelihood (REML) [52] and As eml so wa e [53] we e used o sol e he TM and LM models and ob ain he gene ic pa ame e s. Resul s De elopmen o Pisci icke siosis wi h and wi hou Coin ec ion wi h Sea Lice The de elopmen o pisci icke siosis was con inuously eco ded o e 53 days un il mo ali y eached 544/544 (100%) and 545/ 545 (100%) in bo h coin ec ion ea men s (Figu e 1). A ha ime, mo ali y in he g oup ha ecei ed he single in ec ion wi h P. salmonis only eached 251/545 (46%). A he beginning o he cohabi a ion challenge wi h P. salmonis, a small inc ease in mo ali y was obse ed (day 4). This inc ease was likely a consequence o he sea lice in ec ion p ocedu e and no a di ec consequence o coin ec ion. A 14–16 days pos -in ec ion, mo al- i y associa ed wi h pisci icke siosis was obse ed in cohabi an ish, and his esul was con i med by PCR a he beginning o he ou b eak in dead ish. The daily mo ali y a e was lowe in single han in coin ec ion scena ios (Figu e 1), bu no di e ences we e obse ed be ween he coin ec ion ea men s. Consis en wi h he mo ali y pa e n p e iously desc ibed, Kaplan-Meie su i al cu es (Figu e 2) con i med ha coin ec- ion o P. salmonis and C. oge c esseyi signi ican ly (p,0.05) educed he su i al o A lan ic salmon compa ed o single in ec ion wi h P. salmonis. Howe e , high pa asi e bu den did no signi ican ly educe he su i al o A lan ic salmon compa ed o low pa asi e bu den (P.0.05). Gene ic Resis ance o A lan ic Salmon o Single and Coin ec ion As shown in Table 1, all main ixed e ec s signi ican ly a ec ed he mo ali y o A lan ic salmon challenged wi h P. salmonis and coin ec ed wi h C. oge c esseyi. The in ec ion e ec (I) was highly signi ican (P,0.001) and showed he highes ela i e alue o he associa ed sum o squa es o e he o al a iabili y o mo ali y (28%). The e ec o he ull-sib amily (F) and i s in e ac ion wi h he ea men e ec (F6I) we e also highly signi ican (P,0.001). These e ec s showed a ela i e in luence o 7% and 4%, espec i ely, on he a iabili y o mo ali y. Sex and ank wi hin in ec ion ea men e ec s also signi ican ly a ec ed mo ali y. Howe e , hei con ibu ions o he o e all a iabili y o mo ali y we e less han 2%. Quan i a i e gene ic pa ame e s o he esis ance o A lan ic salmon o P. salmonis o he h ee ea men s o single and coin ec ion a e shown in Table 2. The he i abili y o esis ance was e y simila be ween ea men s and o medium magni ude (0.17– 0.24). Resis ance o he single in ec ion wi h P. salmonis did no co ela e pheno ypically o gene ically wi h esis ance o P. salmonis upon coin ec ion wi h he sea louse C. oge c esseyi (Table 2). Con e sely, a high and signi ican gene ic co ela ion o esis ance o P. salmonis was obse ed be ween he wo coin ec ion ea men s ( g LC-HC = 0.9960.01), p o iding solid e idence ha hese esis ance alues a e measu emen s o he same ai . Discussion We ha e demons a ed o he i s ime ha he sea louse C. oge c esseyi, as a seconda y pa hogen, signi ican ly educes he esis ance o A lan ic salmon o he bac e ium P. salmonis. The p e alence o C. oge c esseyi in Chilean salmon a ms app oaches 100% in some seasons (i.e., sp ing and summe ) and geog aphic egions [27], [54]. The e o e, i s e ec on A lan ic salmon mo ali y may be highe han p e iously hough . In A lan ic salmon, coin ec ions o sea lice and o he pa hogens such as he amoeba Neopa amoeba pe u ans ha e also been epo ed in he USA [23] and in Chilean salmon a ms [24]. Bo h o hese s udies sugges ed ha sea lice may play an impo an ole in he epidemiology o amoebic gill disease caused by Neopa amoeba pe u ans and/o in mo ali y o A lan ic salmon in sea a ms. Simila ly, Valdes-Donoso e al. [25] epo ed ha mos o he ISAV ou b eaks be ween 2007 and 2009 in he X h egion o Chile we e associa ed wi h high sea lice bu dens. The educed su i al upon coin ec ion in A lan ic salmon migh be explained by he di ec skin damage caused by pa asi es ha allows o he pa hogens o en e he ish [55]. Al e na i ely, i may esul om he sys emic e ec s o immunosupp ession caused by sea lice [56]. Gene ic a ia ion in esis ance o disease in salmonids has been epo ed o single in ec ions o pa hogens in A lan ic salmon [40], [57], [58], [36], [42], [35], ainbow ou [59], [60], [61], [62], [63], [64], Coho salmon [41] and b ook cha [42]. Howe e , gene ic a ia ion in he esis ance o coin ec ion by wo pa hogens has no been p e iously es ima ed in salmonids. Using da a om a a med popula ion o A lan ic salmon, we demons a ed gene ic a ia ion o esis ance o P. salmonis upon coin ec ion wi h he sea louse C. oge c esseyi. Sea lice in ec ions ha e been epo ed in salmon a ms a ound he wo ld [10], bu coin ec ion wi h bac e ia, i uses o pa asi es has been minimally in es iga ed. Resis ance o coin ec ion has wo impo an implica ions o salmon b eeding. Fi s , i coin ec ion is common, selec ion o disease esis ance o Table 2. Es ima es o he i abili y (on diagonal), pheno ypic (abo e diagonal) and gene ic (below diagonal) co ela ions (6 SE) in esis ance o A lan ic salmon be ween single in ec ion (SI) wi h P. salmonis, and co-in ec ion wi h wo inc emen al le els he o sea louse C. oge c esseyi (low p essu e o coin es a ion (LC) = 44 copepodi es pe ish; high p essu e o coin es a ion (HC) = 88 copepodi es pe ish). SI LC HC SI 0.2360.07* 20.0460.09 ns 0.0660.07 ns LC 20.1460.33 ns 0.1760.08* 0.2160.01* HC 0.3260.34 ns 0.9960.01* 0.2460.07* ns: No signi ican ly di e en om ze o, p.0.05; *; signi ican ly di e en om ze o, p,0.05. doi:10.1371/jou nal.pone.0095397. 002 Resis ance o Coin ec ion in A lan ic Salmon PLOS ONE | www.plosone.o g 5 Ap il 2014 | Volume 9 | Issue 4 | e95397 wo o mo e pa hogens, e alua ed independen ly as p oposed by Ødega˚ d e al. [65], could be an ine icien me hod unless esis ance o single and coin ec ion is posi i ely co ela ed. Second, e alua ion o esis ance o wo di e en pa hogens could be pe o med in a simple assay, educing cos s associa ed wi h labo a o y es ing. Fu he s udies a e necessa y o de e mine whe he esis ance o coin ec ion by P. salmonis and C. oge c esseyi o o coin ec ion by o he pa hogens ele an o salmon a ming such as ISAV, Ae omonas salmonicida o Neopa amoeba pe u ans occu s in o he popula ions o salmonids. The gene ic co ela ion o esis ance among a ious A lan ic salmon pa hogens has been desc ibed o some bac e ia [66], [67], [68], [69], [70], [71] and i uses [69], [71], [72]. Howe e , he gene ic co ela ion o esis ance be ween single and coin ec ion o wo pa hogens has no been p e iously es ima ed in salmonids. Ou esul s s ongly sugges ha he esis ance o A lan ic salmon o a single in ec ion o P. salmonis and ha o coin ec ion wi h he sea louse C. oge c esseyi a e no gene ically ela ed. The e o e, we can in e ha he bes s a egy o de eloping esis ance o P. salmonis should conside coin ec ion wi h sea lice. Howe e , u he s udies a e necessa y o es ablish whe he he esis ance o coin ec ion obse ed in expe imen al condi ions co ela es wi h highe su i al a es in he ield. A high gene ic co ela ion o esis ance be ween esh and sea wa e has been desc ibed o o he diseases such as u unculosis, sea lice and IPN [67], [38], [73]. Conclusion In ec ion wi h he sea louse C. oge c esseyi, as a seconda y pa hogen, educes he esis ance o A lan ic salmon o he pa hogen P. salmonis. Resis ance o coin ec ion o Pisci icke sia salmonis and Caligus oge c esseyi in A lan ic salmon is a he i able ai . The absence o a gene ic co ela ion be ween he esis ance o single in ec ion and ha o coin ec ion indica es ha di e en genes con ol hese p ocesses. Fu he s udies a e necessa y o in es iga e he e ec s o coin ec ion when he sea louse is he p ima y pa hogen. I is clea ha coin ec ion o di e en pa hogens and esis ance o coin ec ion needs o be conside ed in u u e esea ch on salmon a ming, selec i e b eeding and conse a ion. Acknowledgmen s The au ho s would like o hank Ma ı ´n He ia o his aluable con ibu ion o he de elopmen o he expe imen s in Aquad ice S.A. Au ho Con ibu ions Concei ed and designed he expe imen s: JPL JAG RN. Pe o med he expe imen s: JPL JAG. Analyzed he da a: JPL JAG. Con ibu ed eagen s/ma e ials/analysis ools: BV MJC. W o e he pape : JPL JAG. Con ibu ed o designing he expe imen s and analyzing he da a: BV MJC. Re e ences 1. Mulcahy D, F ye JL (1976). Double in ec ion o ainbow ou y wi h IHN and IPN i uses. Am Fish Soc/Fish Heal h Sec Newsl 5: 5–6. 2. Vilas MP, Rod ı ´guez S, Pe ez S (1994) A case o coin ec ion o IPN and IHN i us in a med ainbow ou in Spain. Bull Eu Assoc Fish Pa hol 14 : 1–4. 3. 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Resis ance o Coin ec ion in A lan ic Salmon PLOS ONE | www.plosone.o g 7 Ap il 2014 | Volume 9 | Issue 4 | e95397