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International Council for theExploration ofthe Sea

Cl\l 1992f.M:32, Session 0Anadromous and CatadromousFish Committee

COMPARATIVE SUSCEPTIBILITY OF NATIVE SCOTTISHAND NORWEGIAN STOCKS OF ATLANTIC SALMON,Salmo salar L., TO Gyrodactylus salaris MALMBERG:

LABoRATORY EXPERIMENTS

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T A Bakke* and K MacKenzie**

*Zoological Museum, University of Oslo, Sars gt. 1, N-0562 Oslo 5, Norway**SOAFD Marine Laboratory, Victoria Road, Aberdeen AB9 8DB, Scotland

SUMMARY

At Vikan AkvaVet, a research station for fish diseases near Namsos in Norway,150 hatchery-reared salmon parr of each of two stocks from the Rivers Shin and Cononin norlh-east Scotland, together with 150 from the River Lierelva in south-east Norway,were infected by exposing them concurrently to wild Norwegian salmon parr naturallyinfected with the monogenean Gyrodactylus salaris. After three days' exposure the fishwere transferred to six holding tanks, two tanks to each stock. Each tank held 50 fish ina pooled group and another 12 fish individua11y isolated in sma11 cages. At weeklyintervals each isolated fish and 10 ofthe pooled fish in euch tank were anaesthetised andthe numbers of G. salaris on them were counted.

No natural resistance was observed in the three stocks of salmon tested and all threewere susceptible to G. salaris. However, 3-5 weeks post-infeetion a marked heterogeneityin the course of infection was observed in a11 three stocks, with highly susceptible,moderately susceptible and responding individuals in each tank. The pooled and isolatedgroups showed different parasite population dynamies: in the pooled fish parasitepopulations increased throughout the experiment, whereas in the isolated fish they tended

. to decrease 30-36 days post-infection; The experiments ended 50 days post-infection,mainly due to fish mortality.. Possible explanations of the observed patterns of parasitepopulation growth are discussed, together with the significance ofthese results for salmonpopulations in both countries.

INTRODUCTION

The monogenean Gyrodactylus salaris Malmberg, 1957, was first reported on wild Atlanticsalmon, Salmo salar L., in Norway by Johnsen (1978). Since then heavy infeetions of G.

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salaris, in combination with secondary fungal infections, have been held responsible forsevere ieductions in the numbers of salmon parr in infected Norwegian rivers, aiid inrecent years catches of ascending mature salmon in these rivers have also dedined(Johnsen and Jensen, 1991, 1992). The distribution ofG. salaris in Norway is associatedwith stocking of fish from irifected salmonid hatchciries, and it is generally assumed thatit was introduced in the early 1970s with infected salmonid smolts from the Baltic area(see Malmberg, 1989; Halvorsen and Hartvigsen; 1989; Bakke et al., 1990; Johnsen andJensen, 1992). "

In laboratory tests, salmon from Norwe~an rivers, representirig the major East Atlanticgroup, proved to be susceptible to G. salaris, while salmon native to the River Neva, CIS,representing another major group, the Baltic, showed both innate and acquired resistanceto the parasite (Bakke et al., 1990). In subsequerit pilot experinümts with Fl progeny ofcrosses between Norwegian and Neva salmon, Jansen et al. (1991) suggested aninheritable component for resistance 1.0 G. salaris in male Neva salmon. In the UK;G.salaris was not found on more than 2,000 salmonid fish cxamined in 1989, 1990 and 1991from over 250 sites throughout the country (A.P. Shinn, pers. comm.).

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The present experiments were undertaken to test under laboratory conditions the •susceptibility of two native stocks of salmon from Scotland and one from Norway,representing two geographically distinct. sub-groups of East Atlantic salmon. Inaccordance with current theories on susceptibility and resistance offish to parasites, threehypotheses were tested: 1) thai. both Scottish and Nonvcgian stocks of salrrion aresusceptible tO infection by G. salaris; 2) that neithcr has any innate resistunce to. theparasite; and 3) that neither possesses thc ability to mount an effective response (acquiredresistance) against it.

MATERIALS AND METHODS

The hatchery-reared salmon parr (0+ und 1+) used in these experiments came from thrcenative stocks of East Atlantic salnion und represented the progeny ofwild parerit fishcaught in their native rivers. The two Scottish stocks (0+) were from the Rivers Shin aridConon in north-east Scotland and the Norwegian stock (1+) was from thc River Lierelva,Buskerud County, iri south-east Norway. One huridred und fifty fish of each stock wereused. All of the fish were transported by air and road to Vikan AkvaVet Veterinary •Station for Contract Research in Fish Diseases, Namsos, Nord-Tioendelag County,Norway, where thc experiments were carried out.

Eggs of the two Scottish stocks were collected in March 1991, .theri hatched andmaintained until the end of September 1991 atthe hatchery of The Scottish OfficeAgriculture und Fisheries Departmerit (SOAFD) l\larine Laboratory at Tullynessle iriAberdeenshire. Ta comply with the health requiremerits of the Norwegian Ministry ofAgriculture for import of live fish to Norway, a sampie of 10 fish from each stock wastested by SOAFD inspectors for the viral diseases IPNV, VHSV, IHNV arid SVCV on14-18 June 1991. This test was repeated on five fish of each stock on 6 August 1991.Both tests were negative. (Sixty parent salmon of eaCh stock had also been tested earlierfor the same diseases und certified negative). Eggs ofthe River Lierelva stock were takenin the period 5 October-ll November 1989 to the DOFA hatchery station situated eloseto the River Glitra; a tributary of the Lierelva, where they were hatched in spring 1990and maintained until the end of September 1991.

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Before transport ta Vikan, fish of all three stocks were routinely disinfected in formalinbaths and starvcd for 24 hours before departure. They 'were transferred to largepolythene bags filled to approximately one-third with fresh water, pumped full Withoxygeri and sealed. The bags were then placed in insuluted boxes with some smallerpolythene bags filled with ice. All of the fish were alive arid active on arrival.

Fish of all threc stocks were conditioned to the experimental water temperature of 12°C(0.5°C) for three days prior to the start of the experiments under conditions of rirtificialdim illumimition from 0800 to 1600 h rind natural illumination for the rest ofthe 24-hourperiod. Although none ofthe experimental fish had ariy previous expenence of G. salaris,they were routinely disinfected in a saline hath on arrival.

The G. salaT-is strmn used for the experiment came from 60 naturally infected salmonparr, some cariying over 1.000 parasites. from the River Figgja. in Nord-TroendelagCounty. Infection of the experimental fish wasachieved by 'placing 150 fish of euch stOck

, together'with 20 of the naturally infected palT in a200 I capacity plastie tank with aconstrintly-flowing water supply. This resulted in an epidemie spreild of G. salaris amongthe exPerimental salmon; After three days exposure the experimental fish were separatedfrom thc nattirally infected ones and transferred to six 500 I capacity holding trinksmeastiring 1 m xI m. with a constantly flowing wuter supply of 5 l/min. two trinkS to euchstock. Each trink held 50 fish in a pooled group plus another 12 fish individtially isolatedin small floating cages with plastic sides and wire mesh floors meilstiring 47 x 37 cm andwith a 10 cm water column. each cagcdiVided into two cqual campartments. Thc watersupply was an untreated mixture of six parts surfacc and one part grouridwater ofpR 6.5-7.0 and salinity 0.15-0.22 ppt. Thc pooled fish werc fed an unmedicated pellet food(Ewos); the isolated fish were unfed. .

On day 0 ofthc experiment (29 September 1991), ie the day the test fish were separatedfrom the naturally infected group. and at weekly intervals thercafter, every isolated fishand 10 fish randomly selected from each pooled gfoup were anaesthetised for about 2 minin a 0.04% solution of chlorbutrinol arid thc numbers of G. scilaris on the firis arid bodysurfaces werc counted in hatchery watcr under a stereomicroscope at a magriificrition ofX120-250. Thc experiment ended on 18 November 1991 (driy 50).

Mean intensity data were compared far statistical sighificancc using analysis rifvariancc(ANOVA).

RESULTS

IridividuaIly IsoIatcd Saimon (Figs. 1-6)

Overrill preva'ience of infection was 100%. initial intensities of infection on day 0 rangedfrom 29 to 236 G. salaris/fish. with means of63.9 for the Conon. 77.0 for the Lierelva. and90.6 far the Shin stock. No salmön was found to be innately resistarit to G. salaris aildnone was found to be capable of completely eliniinating thc infection in the cxperimentrilperiod of 50 days. During thc first three weeks of the experiment, fish of all threc stocksproved to be highly susceptible to G. salaris, with ri coritinuous increase in thc parasiteburden oncrich fish. Lriter in the experiment. however, there was wide variation withinall threc stocks in the ability of individual fish tri mount a response to, ör to control, theinfectian. Parasite infrapopulations on most fish reached peakS ofiritensity betwecn days

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22 and 36 und either levelled off or decreased thereafter, but in 26 fish (seven Shin, nineConon and 10 Lierclva) intensity increased continuously until cither the fish died or theexperiment cnded. In all three stocks some fish reached intensities of over1,600 parasites. The highest intensity recorded, at 37 and 43 days post-infection, wasapproximately 4,000 parasites, on a fish from Conon tank b that died between 43 und50 days post-infection (Fig. Ib). Twenty-nine other fish (seven Shin, 11 Conon und12 Lierelva) died before day 50 with maximiuu intCnsities of from 331 to 1730 parasites.

Two of the surviving fish from sitin tank b (Fig. 2b) and two from iiereiva tank a(Fig. 3a) had fewer parasites at the end thcin at the beginning of the experiment. In theShin fish the initial infeetioris of 47 and 119,parasites increased to 750 und 1,200respectively at day 22, then decreased to 27 and 49 at day 50. In the Lierelva fish theinitial infections of 47 und 184 parasites at day 0 increased to 496 at day 36 and 1,000 atday 22 respectively, then decreased to 44 and 34 at day 50.

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Although there was considerable variation in the response of individual salmon of allthree stocks to infection with G. salaris, there was no obvious difference betweeri thcstocks. Mean intensities for a11 three stocks showed initial increases tei peakS ofbetween500 und 1,000 parasites followed by decreases (Figs. 4-6)., The differencc in mean •intensity betwcen Conon tunks a and b (Fig. 4) from day 30 to day 50 was not statisticallysignificunt (P>0.05) and was mainly due to the presence in tunk b ofthe fish carrying thehighest intensity recorded in individually isolated fish (3,000-4,000). The decreases inmeun intcnsities in all six tanks towards the end of the experimerit were due partly todecreases in parasite infrapopulations und partly to selective mortalities of the mostheavily infected fish. The initial number of parasites on an individual fish at day 0 didnot seem to influerice the subsequent course of infection, the level of host response or thcparasite burden at the end of the experiment. .

Pooled Salmon (Fig. 7)

In contrast to the individually isolated fish, parasite infrapopulations on thc pooledsalmon continued to increase throughout the period ofthe experiment with no significuntdifferences between the three stocks. Due to mortalities, especially arter the fourth undfifth weeks, the initial nuniber of experimental fish in each stock was rapidly reduced.Mortalities were particularly severe in the Lierelva stock, with only 10 fish surviving today 36. Of all the experimental fish, only one, from the Conon stock, survived to day 50.For each stock, mean intensities in the two tanks closely paralleled one another, so damfrom pairs of tanks have been combined in Figure 7.

DISCUSSION

All salmon parr from the three stocks tested were initially highly susceptible to G. safaris.Of the three hypotheses tested; two proved to be correct: 1), that both Scottish aridNorwegian stocks of salmon are'susceptible to infection by G. safaris; arid 2) that neitherhas uny innate resistunce to the parasite (provided that the three stocks tested arerepresentative of Scottish and Norwegian stocks generally). Thethiid hypothesis, thatneither possesses the ability to mount an effective response against it, appeared to beincorrect. Mter aperiod of about three weeks of contiilUous parasite reproduction on allthree stocks, more than half of the iridividually isolated salnio'n appeared to resporid by

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reducing their parasiie burdens, hut none completely eliminated the infection dunng the50-day period of the experiments.

Bakke ct ai. (1990) found that the G. saiaris populations on pooled salmon pan- of twoother stocks of NOrWegian salmon increased steadily throughout the entire expenmentalperiod of five weeks, but in these experiments tho salmon were continuously exposed tothe sourco of infection for the first two weeks of the experimental penod. In anotherexperiment, individually isolated Norwegian sUlmon parr showed a continuous increaseiii parasite burden over a four-week period, but intensities of infection were nlUch lowerthari in the present cxpcnmcnts, because each salmon had been initially infected Withonly a single specimen of G. saiaris. Ir Bakke et aI. (1990) had used a larger starÜnginfeciion, and had conÜnued their experiments for longer, they may have observed aresponse by some of their fish similar to that seen in the present experiments. Kamisoand Olson (1986) found that the intensity ofinfection ofEnglish sole, Parophrys vetulusGirard, oy Gyrodactylus stellatus Crane arid Mizelle reached a peak after nine. weckSbefore beginning to decrease. This highlights the imjlOrtanCe of long-term experimentswhen testing for resistance to gyrodactYlosis.

In same infected rivers in Norway the salmon pair seem to sUrVive betterthan in others(Mo, in press; Jansen and Bakke, in press a,b), arid in one of tho salmon stocks tested byBakke et eil. (1990), from the River Lone in north-west Norway, the growth rate of the G.saiaris population in pooled salinon was markedly slower thari that for any of the stocksin the present expenments, from approximately the sriine initial level of intensity. Thissuggests that stockS ofsUlmon from the LOne and other rivers may havo greäter potentiaIfor controlling G. salciris infection than the Lierelva, Conon and Shiri stocks.

The difTerences between indiVidually isolated and pooled salmon in the course of G. salarisinfection mriy be explained in terms of the transmission strategy ofthe parasite. In theconstrained environment of an experimental tank, '.vith fish coming irito frequent contaetwith the tank bottcim, G. salaris can easily survive by transfer from dead fish or from thetank bottom to live fish (Bakke et al., 1991, 1992). In the present experiments dead fishwere not removed immediately ririd could He on the tank bottom for fit least one day, sotransfer ofparasites may have been a major cause ofthe higher growth rate of G. salarispopulations arid the apparent absence ofhost response in the poolrid fish. Also, parasitesdislodged from the individually isolated fish may have gone through the mesh floors ofthecages to add further to infections in the pooled groups below.

The results of our eXperiments support claims thrit G. salaris is an important cause ofmortality of pre-smolt salmon in infected Norwegian riverS (Jansen and Bakke, in pressri,b; Johnsen and Jensen, 1992; Mo, in press). They also indicate the potentiallydisastrous consequences of introducing this parasite to the UK and underline theimportance of taking all possible measures to prevent this and furt~er dispersal inNorway from happening. That the response of some fish to the infection is apparentlygenetically based (Madhavi and Anderson, 1985; Jansen et al., 1991) has importantevolutionary conscquences for both parasite and host and is of practical importUncobecause it provides a basis for genetic manipulation and selection. Selective brecdingfrom survivors of experiments like those described herein, and stocking ofinfected riverswith their progeny, could enhance the effects of natural selectiori in restoring infectedsalmon rivers to their former status.

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ACKNOWLEDGEMENTS

'We thank: Tom Isachscn, Osiomarkas Fiskcri-Administration's Fish Hatchery Station,OsIo, for valuable tcchnical assistance; Ray Johnstonc, SOAFD Marine Laboratory,Abcrdccn for maintaining thc Scottish salmon and advising on thcir packing. andtransport; Anton Rikstad, Statcns Hus, Steinkjer, for collecting the naturally infcctedsalmon parr; and RoifNordmo, Vikan AkvaVet Veterinary Station for Contract Researchin Fish Discases, Namsos, for providing research facilities at the station. This study wasa cooperative venture between thc SOAFD Marine Laboratory, Aberdeen, and TheZoological Museum, 0510, and was fundedjointly by The Scottish Office Agricultural andFisheries Department and The Directoratc for Natural Management, Trondheim, Norway.

REFERENCES

Bakke, T.A., Harris, P.D., Jansen, P.A. and Hansen, L.P. 1992. Host specificity anddispersal strategy in gyrodactylid monogeneans, with particular reference toGyrodactyZus saZaris (Platyhelminthes, Monogenea). Dis. Aquat. Org., 13,63-74.

Bakke, T.A., Jansen, P.A. and Hansen, L.P. 1990. Differences in thc host resistancc ofAtlantic salmon, SaZmo saZar L., stocks to the monogenean GyrodactyZus saZarisMalmberg, 1957. J. Fish Biol. 37, 577-588.

. .Bakke, T.A., Jansen, P.A. and Hansen, L.P. 1991. Experimental transmission of

GyrodactyZus saZaris Malmberg, 1957 (Platyhelminthes, Monogenea) from theAtlantic salmon (SaZmo saZar) to thc European eel (Anguilla anguilla). Gan. J.Zool. 69, 733-737.

Halvorscn, O. and Hartvigsen, R. 1989. A review of the biography and epidemiology ofGyrodactyZus saZaris. NINA Utredning, 2, 1-41.

Jansen, P.A. and Bakke,. T.A. (In press, a). Epidemiology of Gyrodactylus salarisMalmberg (Platyhelminthes: Monogenea) on Atlantic salmon (Salmo saZar L.): 1.Field studies in a south-east Norwegian river. Fish. Res.

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Jansen, P.A. and Bakke, T.A. (In press, b). Epidemiology of Gyrodactylus salaris •Malmberg (Platyhelminthes: Monogenea) on Atlantic salmon (Salmo saZar L.): 1I.Experimental studies. Fish. Res.

Jansen, P.A., Bakke, T.A. and Hansen, L.P. 1991. Resistance to GyrodactyZus saZarisMalmberg, 1957 (Monogenea) in SaZmo saZar: a genetic component (abstract). BullScand. Soc. Parasit., 1, 50.

Johnsen, RO. 1978. The efTect of an attack by the parasite GyrodactyZus saZaris on thepopulation of salmon parr in the River LakseIva, Misvaer in northern Norway.Astarte, 11, 7-9. .

Johnsen, RO. and Jensen, A.J. 1991.. The GyrodactyZus story in Norway. Aquaculture,98, 289-302.

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Johnsen, RO. and Jensen, A.J. 1992. Infection of Atlantic salmon, SaZmo saZar L., byGyrodactylus salaris Malmberg, 1957, in the River Lakselva, Misvaer in northernNorway. J. Fish Biol., 40, 433-444.

Kamiso, H.N. and Olson, RE. 1986. Host-parasite relationships between Gyrodactylusstellatus (Monogenea: Gyrodactylidae) and Parophrys vetulus (Pleuronectidae ­English sole) from coastal waters of Oregon. J. Parasit., 72, 125-129.

Madhavi, R and Anderson, RM. 1985. Variability in the susceptibility of the fish hostPoecilia reticulata to infection with Gyrodactylus bullatarudis. Parasitology, 91,531-544..

Malmberg, G. 1989. On Gyrodactylus and Pseudogyrodactylus in natural water and fishfarms. Information, 20, 46. Abo Akademi, Abo, Finland.

Mo, T.A. (In press). Seasonal variations in the prevalence and infestation intensity ofGyrodactylus salaris Malmberg, 1957 (Monogenea: Gyrodactylidae) on Atlanticsalmon parr, Salmo salar L., in the River Batnfjordelva, Norway. J. Fish Biol.

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Figure 1. Infrapopulation dynamics of G. safaris on individually isolated salmon parr ofthe Scottish Conon stock, expressed as absolute numbers of two replicate groups (tanksa and b) of 12 fish each. Each line represents a single salmon parr. Lines terminatingbefore day 50 indicate mortalities. The fish marked with an asterisk had approximately3000 parasites on day 30, approximately 4000 on days 37 and 43, and died betweendays 43 and 50.

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Figure 2. Infrapopulation dynamics of G. safaris on individually isolated salmon fry ofthe Scottish Shin stock, expressed as absolute numbers of two replicate groups(tanks a and b) of 12 fish each. Each Une represents a single salmon fry. Linesterminating before day 50 indicate mortalities.

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Figure 3. Infrapopulation dynamics of G. safaris on individually isolated salmon parr ofthe Norwegian Lierelva stock, expressed as absolute numbers of two replicate groups(tanks a and b) of 12 fish each. Each line represents a single salmon parr. Linesterminating before day 50 indicate mortalities.

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Figure 4. The course of infection of G. safaris on individually isolated salmon parr of theScottish Conon stock, expressed as mean intensities of two replicate groups (see Fig. 1).

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Figure 6. The course of infection of G. safaris on individually isolated salmon parr of theNorwegian Lierelva stock, expressed as mean intensities of two replicate groups (see Fig. 3).

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Figure 7. The course of infection of G. safaris on pooled salmon parr of the three stocks,expressed as mean intensities of two replicate groups of each stock. Mean intensities arebased on 20 fish from each sampling date up to day 36, when the mean for the Lierelvastock is based on the 10 surviving fish, and day 43, when the means for the Conon andShin stocks are based on 10 and eight survivors respectively from each stock.