Transactions of the American Fisheries Society ...

This article was downloaded by: [Fisheries and Oceans Canada] On: 18 August 2015, At: 09:30 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG

Transactions of the American Fisheries Society Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/utaf20

Genetic Variation in Disease Resistance and Growth of Chinook, Coho, and Chum Salmon with Respect to Vibriosis, Furunculosis, and Bacterial Kidney Disease a

T. D. Beacham & T. P. T. Evelyn

a

a

Department of Fisheries and Oceans , Biological Sciences Branch, Pacific Biological Station , Nanaimo, British Columbia, V9R 5K6, Canada Published online: 09 Jan 2011.

To cite this article: T. D. Beacham & T. P. T. Evelyn (1992) Genetic Variation in Disease Resistance and Growth of Chinook, Coho, and Chum Salmon with Respect to Vibriosis, Furunculosis, and Bacterial Kidney Disease, Transactions of the American Fisheries Society, 121:4, 456-485, DOI: 10.1577/1548-8659(1992)1212.3.CO;2 To link to this article: http:// dx.doi.org/10.1577/1548-8659(1992)1212.3.CO;2

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.

Downloaded by [Fisheries and Oceans Canada] at 09:30 18 August 2015

Terms & Conditions of access and use can be found at http://www.tandfonline.com/ page/terms-and-conditions

Transactions of the American Fisheries Society 121:456-485, 1992

Genetic Variation in Disease Resistance and Growth of Chinook, Coho, and Chum Salmon with Respect to Vibriosis, Furunculosis, and Bacterial Kidney Disease T. D. BEACHAM AND T. P. T. EVELYN


Department of Fisheries and Oceans, Biological Sciences Branch Pacific Biological Station. Nanaimo. British Columbia V9R 5K6, Canada Abstract.—We examined genetic variation in mortality, mean time to death, and (for chinook salmon) weight for one population each of chinook salmon Oncorhynchus tshawytscha. coho salmon O. kisutch, and chum salmon O. keta in British Columbia. In each of the three populations examined, 15 males were mated to 30 females in a nested breeding design. The progeny from each family were divided into groups, and each group was challenged with one of four pathogens: Vibrio anguillarum or V. ordalii, both of which cause vibriosis; Aeromonas salmonicida, which causes furunculosis; and Renibacterium salmoninarum, which causes bacterial kidney disease. When all three salmon species were considered as a group, heriiabilities of mortality (sire component, binary character) were low—less than 0.15 for the Vibrio species and A. salmonicida challenges, and less than 0.05 for the R. salmoninarum challenge. Heriiabilities of time to death were also low. Family mortality rates in the Vibrio species and A. salmonicida challenges tended to be positively correlated, but not as a result of additive genetic variation. Similar results were obtained for family mean time to death. At the end of the experiments there was no consistent genetic correlation between family weight and observed mortality rates or time to death. Our results suggest that greater improvements in disease resistance for salmon in the aquaculture industry in British Columbia can be made by judicious selection for the strain or population for brood stock rather than by selective breeding for increased resistance. Culture of Pacific salmon, particularly chinook and Amend 1977; Sawyer and Strout 1977), but salmon Oncorhynchus tshawytscha and coho control of the disease through vaccination (Evelyn salmon O. kisutch, in saltwater pens is becoming 1984, 1988) coupled with chemotherapy can sigan increasingly important industry in British Co- nificantly reduce mortality rates (Sano and Fulumbia. However, Pacific salmon reared under kuda 1987). conditions of high density during intensive culture Furunculosis, caused by infection with Aeromocan be subject to an array of diseases, and the nas salmonicida, is a systemic infection, although effect of a disease can be more severe in cultured necrotic lesions in the skin and muscles may occur populations than in wild populations because it is (McCarthy and Roberts 1980; Trust 1986). Vacaccentuated by the high rearing densities. Three cination of salmonids against furunculosis has not diseases that commonly influence survival of cul- been completely successful (Michel 1982; Munro tured salmon are vibriosis, furunculosis, and bac- 1984), and A. salmonicida is becoming more reterial kidney disease. Vibriosis is a septicemic dis- sistant to chemotherapy (Aoki et al. 1983). ease that primarily affects fish with a marine life Bacterial kidney disease, caused by Renibacterihistory stage (Anderson and Conroy 1970; Egidius um salmoninarum. is a systemic and usually 1987). In cultured salmon of the Pacific North- chronic disease that occurs in both wild (Mitchum west, vibriosis can be caused by Vibrio anguilla- et al. 1979; Banner et al. 1986) and cultured rum (Evelyn 1971) or by V. ordalii (Harrell et al. salmonids (Fryer and Sanders 1981). It is char1976). Histopathology associated with the two acterized by granulomatous lesions in the kidney, Vibrio species differs (Ransom et al. 1984); V. an- and in advanced stages the disease causes the whole guillarum is most abundant in blood-producing kidney to become enlarged and necrotic. Effective tissues and is dispersed throughout the tissues, vaccination treatments are not available to conwhereas V. ordalii is most abundant in muscle trol bacterial kidney disease (Fryer and Sanders tissue and forms microcolonies there. Vibrio an- 1981). guillarum is generally considered to be more virTechniques available to reduce disease-induced ulent (Egidius 1987) and ubiquitous (Evelyn 1988) mortality in cultured salmonids include the use of than V. ordain. Vibriosis can result in high mor- vaccines or chemotherapy, or avoidance of the talities in cultured salmon (Antipa 1976; Antipa causative agent (when such a measure is practical).

456


GENETIC VARIATION IN DISEASE RESISTANCE

457

Another approach is to culture populations that a seawater-farmed chinook salmon that died of show innate genetic resistance to the disease. Re- vibriosis; (2) V. ordalii isolate 74-48, obtained from searchers have reported genetic variation in mor- a seawater-farmed sockeye salmon Oncorhynchus tality among or within salmonid populations in- nerka that died of vibriosis (Evelyn and Ketchefected with the agents of vibriosis (Gjedrem and son 1980); (3) A. salmonicida isolate 76-30, obAulstad 1974), fumnculosis (Wolf 1954; Snieszko tained from a cultured coho salmon that died of et al. 1959; Ehlinger 1977), bacterial kidney dis- fumnculosis at Quinsam Hatchery, British Coease (Suzumoto et al. 1977; Winter et al. 1980; lumbia; and (4) R. salmoninarum isolate 384, obWithler and Evelyn 1990), and infectious hema- tained from a cultured chinook salmon that died topoietic necrosis (Amend and Nelson 1977; of bacterial kidney disease (Evelyn et al. 1986). Mclntyre and Amend 1978). This genetic varia- The virulence of the isolates was maintained by tion can be exploited in selective breeding pro- passage through juvenile salmon and storage at grams to produce strains that are more resistant -85°C. to specific diseases. At present, it is uncertain whether selection for Challenge Suspensions resistance to a specific disease will confer inSuspensions of the pathogens used for the chalcreased resistance specific to that disease or lenges were prepared as follows. The two Vibrio whether it will confer a more general increased species were grown for 1 d (V. anguillarum) or 2 immune response. The more general immune re- d (V. ordalii) at 21°C on plates of tryptic (tryptisponse could occur if the genetic correlations case) soy agar (TSA; Difco Laboratories) suppleamong resistance to specific diseases were posimented with 0.5% NaCl. The resulting growth was tive. However, Ehlinger (1977) reported that some suspended in cold, sterile solution of peptone strains of brook trout Salvelinus fontinalis that (0.1%, weight/volume) and saline (0.85% NaCl, were more resistant to fumnculosis were also more weight/volume), and the suspension was adjusted susceptible to gill disease; this finding indicates a to a turbidity of 1.0 optical density (OD) at 540 negative genetic correlation between resistance to nm. The A. salmonicida isolate was grown for 2 these two diseases. Winter et al. (1980) reported d on TSA at 15°C, and the resulting growth was that a population of steelhead Oncorhynchus my- suspended in peptone-saline solution as was done kiss that was less susceptible to Renibacterium with the vibrios. The R. salmoninarum isolate was species and Aeromonas hydrophila was more sus- grown on kidney disease medium-2 (KDM-2; ceptible to V. anguillarum. Negative correlations Evelyn 1977) at 15°C for 12-23 d. The resulting between growth rate and resistance to bacterial growth was suspended in the peptone-saline sokidney disease have also been reported (Winter et lution, and the suspension was adjusted to a tural. 1980). bidity of 1.25 OD at 540 nm. In the present experiment, we exposed subgroups of juvenile chinook salmon, coho salmon, and chum salmon O. keta to K anguillarum, V. Challenge Methods Challenge with one of the vibrios or A. salmonordalii, A. salmonicida, or R. salmoninarum; we measured mortality rates, mean times to death, icida was accomplished by immersing the fish (450 and growth rates. We then conducted a quanti- fish/5 L solution) for 15 min in oxygenated peptative genetic analysis of these traits and examined tone-saline solution containing a sufficient amount of the bacterial suspension to give the desired genetic correlations among them. challenge dose. To prevent foaming of the oxygenated challenge suspension, we added a few Methods drops of antifoam B (Dow Corning Corp.). ChalBacteria lenge with R. salmoninarum was accomplished We designed the experiment to measure the ge- either by the immersion method outlined for the netic variation in mortality rate, time to death, other pathogens or by intraperitoneal injection. and (for chinook salmon) weight of the salmon The injection dose consisted of 0.1 mL of the suitafter challenging subgroups of the different salmon ably diluted standardized peptone-saline suspenspecies with V. anguillarum, V. ordalii, A. sal- sion ofR. salmoninarum cells. The fish to be chalmonicida, or R. salmoninarum. The bacterial iso- lenged by injection were anesthetized with tricaine lates used in challenging the salmon were (1) K at a concentration of 50 mg/L just before the chalanguillarum isolate R20, obtained in 1989 from lenge. None of the challenges was administered

458

BEACHAM AND EVELYN


until after brands, applied to fish to denote family (details follow), had healed.

Chinook Salmon Gametes were collected from 15 male and 30 female chinook salmon on 25 October 1988 from the Robertson Creek Hatchery, located on the Somass River system on the west coast of Vancouver Island. The gametes were transported on ice to the Pacific Biological Station, Nanaimo, British Columbia, where the experiment was conducted. A nested mating design was used; each male fertilized the eggs of two females, and 30 families were produced in total for the experiment. The eggs were fertilized, water-hardened, and loaded in a vertical-stack incubator; the eggs from each family were split into two groups, and each group was placed in a separate container in the incubator. The mean temperature during development was 11.7°C, and fry emerged on 18 January 1989. At that time about 300 fry from a family were placed in a 35-L tank that was supplied with 13°C fresh water. A similar tank was set up for each family, and the photoperiod was set at 16 h light: 8 h darkness. The fish were fed Biodiet by automatic feeders (Falls 1980) during the lighted portion of the day. The experimental design required that the 30 families be distinguishable. This requirement was met by use of a hot-branding technique (Murray and Beacham 1990). The juveniles were anesthetized and branded for family identification during 14-21 March 1989. After branding, the fish were returned to the 35-L tanks immediately to allow the brands to heal. Fifteen juveniles from each family were subsequently distributed to each of ten 2,400-L tanks that were supplied with fresh water. Hence, each tank had 450 juveniles in total (15 juveniles from each of the 30 families). We allocated two 2,400-L tanks to each of the four pathogens investigated, and the final two tanks were maintained as controls. The juveniles were challenged with one of the pathogens during 1820 April 1989. When the challenges were conducted, we weighed all fish to the nearest 0.01 g. All juveniles from a 2,400-L tank were placed in an immersion bath for 15 min. Each tank group was challenged separately, but the same immersion bath was used for both replicate tank groups allocated to each disease organism investigated. Target concentrations (viable cells/mL) in the immersion baths were 1 x 104 (actual, 5.4 x 103) for V. anguillarum, 1 x 106 (actual, 1.5 x 106) for K ordalii, 5 x 103 (actual, 4.8 x 103) for A.

salmonicida. and 1 x 107 (actual, 4.2 x 106) for R. salmoninarum. An additional 30 juveniles per family were weighed to the nearest 0.01 g and their adipose fins were removed; these fish were maintained in separate tanks until the experimental juveniles were challenged with the appropriate pathogens. After the experimental fish were challenged and returned to their tanks, three of these unchallenged fish from each family were placed in each of the 2,400-L tanks. These fish were used to evaluate whether cohabitation with infected fish represented a greater pathogen challenge than the experimental challenge had been. These fish also were used to determine the extent of horizontal transmission of the pathogen among fish in the tank. We refer to them subsequently as the "crossover" juveniles. Each tank had 90 crossover fish (30 families x 3 fish/family), so the total density amounted to 540 fish per tank at this stage of the experiment. The juveniles were fed Biodiet of the appropriate size by automatic feeders, and they were exposed to a natural photoperiod. All tanks were checked daily for any dead juveniles, and water temperatures were recorded daily. The experiment involving V. anguillarum was ended 49 d after the initial challenge, because no additional mortality occurred after day 23. The mean water temperature during this experimental period was 12.2°C. At the end of this experiment, all live fish were identified to family and experimental type (challenge or crossover), and they were weighed to the nearest 0.1 g. (In this experiment and the ones subsequently reported in this article, we analyzed the weights of live fish only.) The experiment involving V. ordalii was ended 50 d after the initial challenge, and the mean water temperature during the experiment was 13.6°C. No further mortality of juveniles was observed after 27 d of rearing. All juveniles that were alive at the end of this experiment were identified and weighed as in the previous experiment. The experiment involving/!, salmonicida was ended 78 d after the initial challenge; the mean water temperature was 13.3°C. Deaths of juveniles occurred through the last day of the experiment. At the end of the experiment, all live juveniles were identified and weighed. In one of the tanks allocated to the R. salmoninarum challenge, mortality increased from 6% on day 76 after the initial challenge to 98% on day 88. Despite an intensive investigation, the cause of this sudden increase in mortality could not be



459

determined. Challenged and crossover juveniles 20 June 1989. The juveniles were subsequently died at the same time and at equal rates. As a distributed to ten 2,400-L tanks at a loading denresult of this unexplained mortality, only one tank sity of 420 fish per tank (28 families x 15 fish/ remained in which the juveniles had been chal- family). The juveniles were challenged with V. ordalii lenged with R. salmoninarum. Hence, we challenged the juveniles from one of the control tanks on 19 July 1989 by a 15-min immersion in a bath with R. salmoninarum on 27 July 1989. However, with a target concentration of 1 x 107 (actual, 3.2 this R. salmoninarum challenge was administered x 107) viable cells/mL. The challenge with A. salby a different method. Each experimental fish re- monicida occurred on 20 July 1989 by a 15-min ceived a target dose of 1 x 104 (actual, 7.3 x 103) immersion in a bath with a target concentration viable cells by intraperitoneal injection. Hence, of 5 x 105 (actual, 3.4 x 105) viable cells/mL. The for the R. salmoninarum experiment with chinook juveniles were challenged with R. salmoninarum salmon, we subsequently refer to the two different by intraperitoneal injection of a target dose of 1 challenges as the immersion challenge and the in- x 104 (actual, 7.3 x 103) viable cells on 27 July jection challenge. 1989. The challenge with V. anguillarum occurred We attempted to accelerate the effect of the R. on 18 August 1989 by a 15-min immersion in a salmoninarum challenges by transferring the ju- bath with a target concentration of 1 x 106 (actual, veniles of both challenge groups from fresh water 3.7 x 106) viable cells/mL. After the challenges (at a temperature of about 6°C) to salt water (at were completed, three crossover juveniles (marked about 10°C) during 20-26 January 1990; juveniles with adipose fin clips) from each family were addof the control tank were transferred to salt water, ed to each 2,400-L tank, resulting in a total density also. We vaccinated the juveniles against vibriosis of 504 fish per tank. The tanks were checked daily, and furunculosis before the transfer. However, and any dead individuals were removed and idenduring the vaccination, we accidently killed 15 fish tified to family. The experiments involving V. anguillarum, V. (14 challenge, 1 crossover) in the immersion tank and 47 fish (36 challenge, 11 crossover) in the ordalii, and A. salmonicida were ended 11,16, and injection tank (but none in the control tank). The 18 d, respectively, after initial challenge because fish that were killed accidently were removed from mortality of the challenged juveniles had ceased. the analysis. The R. salmoninarum experiment Mean water temperatures were 17.5°C during the was ended on 13 May 1990, 389 d after the im- V. anguillarum experiment, and 17.2°C during the mersion challenge and 290 d after the injection V. ordalii and A. salmonicida experiments. As we challenge. Mean water temperatures during the did with the chinook salmon, we attempted to experimental periods were 10.7°C for the immer- accelerate the effect of the R. salmoninarum chalsion challenge and 11.1 °C for the injection challenge by transferring the experimental groups (two lenge. tanks) and the control groups (two tanks) from fresh water to salt water during 20-26 January Coho Salmon 1990. The fish were immersion-vaccinated against Gametes were collected from 15 male and 30 vibriosis and furunculosis (with commercially female coho salmon from the Robertson Creek available vaccines) before they were transferred to Hatchery on 17 November 1988. The nested salt water, and no mortality occurred during the breeding design used and the incubation condi- vaccination and transferral process. The experitions for coho salmon were the same as those de- ment involving R. salmoninarum was ended on 6 scribed for chinook salmon. The mean water tem- September 1990, 406 d after the initial challenge; perature during development was 4.9°C, and fry the mean water temperature during this experiemerged on 14 April 1989. The initial rearing con- mental period was 11.9°C. Deaths of challenged ditions for the juvenile coho salmon were also the coho salmon occurred through the last day of the same as those described for chinook salmon. R. salmoninarum experiment. At the end of the However, before the juveniles were branded for experiments with the four pathogens, weights of family identification, the water supply to two of the surviving juveniles in the experimental tanks the 35-L tanks was interrupted, resulting in total were not recorded as they had been for the chimortality of all juvenile coho salmon in the two nook salmon. Instead, only the live fish in the tanks (two families). Each family that died origi- control tanks were identified to family and weighed nated from a different male. The remaining 28 to the nearest 0.1 g on 7 September 1990, apfamilies were branded for identification during 11- proximately 400 d after the initial challenges with

460

BEACHAM AND EVELYN


V. ordalii and A. salmonicida, and about 385 d after the initial challenge with V. anguillarum.

rum experiment. A secondary infection of A. salmonicida was observed in some of the dead fish from one of the tanks in the R. salmoninarum challenge. We treated this infection in the remaining fish by applying oxolinic acid to the food and feeding this treated food to the fish for 12 d at a daily dose of 11.8 mg oxolinic acid/kg offish; oxolinic acid was used because it is inactive against R. salmoninarum (Austin 1985; Austin etal. 1985) but active against A. salmonicida. After this 12-d treatment, the secondary infection of A. salmonicida could not be detected in the fish that died. Fish that remained alive in the control tanks were identified to family and weighed to the nearest 0.1 g during 12-14 February 1990, approximately 110 d after the juveniles in the experimental tanks had been challenged with the vibrios and A. salmonicida, and about 80 d after they had been challenged with R. salmoninarum.

Chum Salmon Gametes were collected from 15 male and 30 female chum salmon on 22 November 1988 from the Chehalis River Hatchery on the lower Eraser River drainage. The nested breeding design used and the incubation conditions for chum salmon were the same as those described for chinook salmon. The mean water temperature during development was 2.8°C, and fry emerged on 18 July 1989. The initial rearing conditions for the juvenile chum salmon were also the same as those described for chinook salmon. The 30 families were branded for identification during 25-29 September 1989. The juveniles were subsequently distributed to ten 2,400-L tanks at a loading density of 450 fish per tank (30 families x 15 fish/ family). The juveniles were challenged with V. anguillarum on 26 October 1989 by a 15-min immer- Autopsy Protocol sion in a bath with a target concentration of 1 x Of the fish that died during the course of each 10s (actual, 1.7 x 105) viable cells/mL. The chal- challenge we autopsied at least one fish per family lenge with V. ordalii occurred on 26 October 1989 per tank (which amounted to 8-50% of the fish by a 15-min immersion in a bath with a target that died) to determine whether death was attribconcentration of 1 x 106 (actual, 4.4 x 105) viable utable to the organism used in the challenge. Samcells/mL. The juveniles were challenged with A. ples of kidney tissue were aseptically taken, salmonicida on 27 October 1989 by a 15-min im- cultured on TSA, and used for preparing Grammersion in a bath with a target concentration of stained smears. Fish were considered to have died 5 x 104 (actual, 3.4 x 104) viable cells/mL. The of V. anguillarum or V. ordalii (hence, vibriosis) challenge with R. salmoninarum occurred on 22 if the smears contained curved gram-negative rods November 1989 by intraperitoneal injection of a and if the rods grew within 2 d on TSA at 21°C target dose of 1 x 104 (actual, 1.5 x 10s) viable without producing pigments. To verify these clascells. After the challenges were completed, three sifications, 10% of the samples from dead fish were crossover juveniles (marked with adipose fin clips) tested for agglutination activity with rabbit antifrom each family were added to each 2,400-L tank, V. anguillarum or anti-K ordalii serum (Microresulting in a total density of 540 fish per tank. As tek). Agglutination served as further evidence that was done in the experiments with the two other death was caused by one of these vibrios. Death salmon species, the tanks were checked daily, and was considered to be due loA. salmonicida (hence, any dead individuals were removed and identified furunculosis) if the smears contained many gramto family. negative coccoid rods that grew readily on TSA at The experiments involving V. anguillarum, V. 21°C, and if these rods were nonmotile (10% of ordain, and A. salmonicida were ended 21,23, and the samples from dead fish were checked) and pro19 d, respectively, after initial challenge because duced a brown diffusing pigment. The cause of mortality of the challenged juveniles had ceased. death was determined to be R. salmoninarum Mean water temperatures were 10.3°C during the (hence, bacterial kidney disease) if the smears conV. anguillarum and A. salmonicida experiments, tained many small gram-positive rods, if the rods and 10.2°C during the V. ordalii experiment. The failed to grow on TSA at 21°C, and if the rods (in experiment involving R. salmoninarum was end- a spot check involving 10% of the smears from ed 75 d after the initial challenge; the mean water dead fish) reacted with a commercially available temperature during this experimental period was ami-/*, salmoninarum serum (Microtek) in the in7.4°C. Deaths of challenged chum salmon oc- direct fluorescent antibody technique (Bullock and curred through the last day of the R. salmonina- Stuckey 1975).

461


Data Analysis Variations in mortality rates and times to death were analyzed separately for each salmon species and each bacterial challenge with the model


Yijkl = M + Sj + DJJ + Rk + eijkl\

Yfjki is the mortality rate or time to death (days after initial challenge), M is the overall mean, 5/ is the effect of sire / (i = 1-15), D,y is the effect of the damy (j = 1 or 2) mated with sire /, R^ is the effect of tank k (k = 1 or 2), and eyu is the random error associated with fish / in subgroup ijk. A similar model without the replicate tank term was used in the analysis of each of the chinook salmon-/?, salmoninarum challenges. The mean effect of both models was considered fixed, and the other effects were considered random. To normalize values, observed times to death were log-transformed before use in the analyses of variance (ANOVA). Variance components of each random effect were determined by means of method 1 of Henderson (1953). Variances of the variance components were determined as outlined by Anderson and Bancroft (1952), and standard errors of heritability estimates were determined as outlined by Becker (1984). Mortality was analyzed as binary data (0 = alive, 1 = dead), and heritability was adjusted to a normal basis by the method of Van Vleck (1972). Heritability was estimated as four times the sire component of variance divided by the total phenotypic variance (variance components of sires, dams, tanks, and random error). Comparison of trends in fish mortality and time to death among the different pathogen challenges was conducted by simple correlation analyses and estimation of genetic correlations. Because it was not possible to determine the genetic correlation between traits when the variance component of sires was negative or zero, we also compared simple correlation coefficients to evaluate trends. Genetic covariances between observed mortality rates, times to death, and growth rates (mean family weights based on fish alive in control tank at the end of the experiment) were approximated from family means for each character by a random effects model that incorporated only sires and dams (dams were the error term). Mortality rates were transformed to arcsine square roots and the data for time to death and weight were log-transformed before genetic correlations were estimated. Genetic correlations were determined from the genetic covariances, and the genetic variances were

determined from single-trait analyses. Standard

errors of the genetic correlations were determined by the method of Mode and Robinson (1959). Results

Chinook Salmon Mortality and time to death.—Mortality of Chinook salmon challenged with V. anguillarum increased rapidly between 4 and 10 d after exposure to the pathogen, from less than 5% to about 40% mortality (Figure 1). Mortality rates declined markedly after day 10, and no additional mortality of juveniles was observed subsequent to day 23. The total mortality rate was 47%. We examined 10.5% of the fish that died, and we were able to conclude from Gram stains that V. anguillarum infection was the cause of death in 94% (47) of the fish examined. Family mortality rates ranged from 23 to 67%, and 16 of the 30 families had mortality rates of at least 50% (Table A.I). Mortality of crossover fish began 17 d after the experimental fish had been exposed to the pathogen, well after the time of substantial mortality in the experimental fish (Table 1). We examined four of the eight crossover fish that died (mortality rate, 4.4%), and V. anguillarum infection was diagnosed as the cause of death in all four fish. The total mortality rate of chinook salmon exposed to V. ordalii was 6% (Table 1), the lowest of any challenge experiment in the study. Mortality occurred between 8 and 26 d after exposure to the pathogen, but the major portion of this limited mortality occurred between 13 and 18 d after initial exposure (Figure 1). We examined 51% (28) of the fish that died and were able to confirm V. ordalii infection as the cause of death in 89% (25) of the fish examined. Family mortality rates ranged between 0 and 17%, but 9 of the 30 families had no mortality (Table A. 1). Three crossover fish died during the experiment (mortality rate, 1.7%), but we were able to confirm V. ordalii infection as the cause of death in only two of these three fish. As with the V. anguillarum challenge, mortality of the crossover fish occurred only after most of the mortality of the experimental fish had already occurred. Mortality of the chinook salmon challenged with A. salmonicida increased slowly to about 6% at day 30 after the initial exposure (Figure 1). The mortality rate then increased more rapidly, so that when the experiment was ended 78 d after the initial exposure, 30% of the challenged fish were dead (Table 1). We examined 20% (54) of the challenged fish that died and concluded that A. sal-

462

BEACHAM AND EVELYN

50-1


40-

>*

"5

o>

20-

0

10

20

30

40

50

60

70

80

200

250

30O

350

400

240

280

2O

~ R_. soimoninorum - immersion

10-

0

50

100

150

R. salmoninarum - injection

320

Days since challenge FIGURE 1 .—Cumulative percent mortality from the time of exposure for 30 families of Chinook salmon, subgroups of which were challenged with Vibrio anguillarum, V. ordalii. or Aeromonas salmonicida (by immersion exposure), or with Renibacterium salmoninarum (by immersion exposure or intraperitoneal injection). Thirty fish from each family were exposed to one of the four pathogens and were designated ''challenge" fish. For each of the four pathogen challenges, six additional unchallenged fish from each family were placed in the tanks with the challenged fish and designated "crossover" fish.

monicida infection was the cause of death in 94% (51) of the fish examined. Mortality rates of families varied widely, ranging from 13% to 80%, but only two families had mortality rates greater than 50% (Table A.I). Substantial mortality (28%) occurred in the crossover fish, and timing of their

mortality was similar to that in the experimental fish (Figure 1; Table 1). We examined 34 of the 50 crossover fish that died and concluded that death resulted from infection by A. salmonicida in 91% (31) of the fish examined. Time to death of the experimental and crossover fish was similar

463


TABLE I.—Observed mortality rates (%) and mean times to death (days after challenge) for families of chinook, coho, and chum salmon, members of which were challenged with Vibrio anguillarum, V. ordain. Aeromonas salmonicida. or Renibacterium salmoninarum. Thirty fish from each family were exposed to one of the four pathogens by immersion (imm) or intraperitoneal injection (inj). For each of the four pathogen challenges, six additional unchallenged fish from each family were placed in the tanks with the challenged fish and designated as "crossover" fish. Standard errors of the means are in parentheses.


Challenge Pathogen and challenge method

Mortality 0.10), and thus we analyzed the data from the two challenges separately. Mean times to death of the challenged fish and of the crossover fish were similar in the immersion challenge, but challenged fish died somewhat later than did crossover fish in the injection challenge (Table 1). We examined four of six crossover fish that died in the immersion challenge and could confirm only that one fish died of bacterial kidney disease. We examined all eight crossover fish that died in the injection challenge and confirmed that bacterial kidney disease was the cause of death in six cases. Mortality of the control fish totaled 4.0% by the end of the last challenge experiment, the R. salmoninarum experiment (Table 1). None of the control fish died during the Vibrio species experiment, and four of the control fish died during the A. salmonicida experiment. We examined all control fish that died for evidence of bacterial kidney disease, and we concluded that this disease was

464

BEACHAM AND EVELYN

TABLE 2.—Percentage of total phenotypic variation in mortality rate, time to death, and survivor weight accounted for by sire, dam, and tank effects (and random error) for 30 families of chinook salmon, members of which were challenged with one of four pathogens. Challenges were administered by immersion or intraperitoneal injection. Heritability of mortality rate was estimated as both a binary and a continuous character by the transformation of Van Vleck (1972). Standard errors of the heritability estimates are in parentheses. Heritability Percent of variation


Trait

Sire

Dam

Tank

Observed Error

Sire

Continuous Dam

Vibrio amgMUItrmm, immersion 85.6 0.08(0.05) 0.01(0.04) 89.1 0.00(0.08) 0.06(0.10) 71.9 0.40(0.21) 0.23(0.13)

Sire

Dam

0.13(0.08)

0.02(0.06)

Mortality Time to death Weight

2.1 0.0 10.0

0.2 1.4 5.9

12.1 9.5 12.2


2.4 0.0 10.7

0.4 0.0 3.8

0.0 0.0 13.6

Vibrio onUUit, immersion 97.2 0.10(0.06) 100.0 0.00(0.31) 71.9 0.43(0.20)

0.02(0.05) 0.00(0.22) 0.15(0.08)

0.40 (0.25)

0.08 (0.20)


3.5 0.0 11.1

5.6 8.3 5.5

12.5 12.7 36.4

78.4 0.14(0.11) 79.0 0.00(0.14) 47.0 0.45(0.22)

0.22(0.11) 0.33(0.19) 0.22(0.10)

0.24(0.19)

0.38(0.19)

Mortality

4.9 0.0 5.8

0.00 (0.23)

0.47 (0.35)

Time to death Weight

0.0 12.0 6.1


0.0 0.0 23.0

3.1 0.0 5.0

0.00(0.26)

0.35(0.41)

Remibaclcrimm udmomimarum, immersion 95.1 0.00(0.10) 0.20(0.15) 88.0 0.43 (0.59) 0.00 (0.62) 88.1 0.25(0.13) 0.23(0.12) Rcmibttcterimm stlmomiiuurmm, injection 96.9 0.00(0.09) 0.12(0.14) 100.0 0.00(0.09) 0.00(0.68) 72.0 0.92(0.41) 0.20(0.15)

not the cause of death for any of the fish examined. We were not able to determine a cause of death for any of the control fish. Heritability and correlations. —We investigated the effect of sires, dams, and tanks on observed variation in mortality rate and time to death for chinook salmon. We observed significant sire effects on mortality rate only in the V. anguillarum challenge (F = 2.59; df = 14, 15; P < 0.05), and significant dam effects were observed in the A. salmonicida challenge (F = 3.15; df - 15, 869; P < 0.01) and in the R. salmoninarum immersion challenge (F - 1.75; df = 15, 406; P < 0.05). Significant tank effects were observed in the V. anguillarum challenge (mortality of 60.0% in one tank versus 34.0% in the other) and in the A. salmonicida challenge (42.2 versus 18.2%). We observed no significant sire effects on time to death in any of the five challenges and dam effects were significant only in the A. salmonicida challenge (F = 1.89, P < 0.05). Significant tank effects on time to death were observed in the V. anguillarum challenge (8.3 versus 6.9 d; F= 20.59, P < 0.01) and in the A. salmonicida challenge (48.7 versus 39.5 d;F= 17.93, P < 0.01).

Additive genetic variation accounted for little of the variation in chinook salmon mortality. Sire component heritability estimates (h \in) of mortality in the five challenges ranged from 0.00 to 0.14 when mortality was considered as a binary character and from 0.00 to 0.40 when mortality was considered as a continuous character (Table 2). Maternal or nonadditive genetic effects on mortality were most substantial in the A. salmonicida and R. salmoninarum challenges. No significant additive genetic variation was observed for time to death in any of the five challenges, and sire component heritability estimates were 0.00 for four of the five challenges. Significant maternal effects on time to death were observed only in the A. salmonicida challenge. When we initially challenged the fish with the pathogens, little additive genetic variation in body weight was observed (A2«re = 0.12, SE = 0.21), but there was a substantial maternal effect (h2&m - 0.75, SE = 0.30). However, in the case of weights recorded at the end of each challenge experiment, additive genetic variation accounted for a significant portion of the weight variation, and there were no significant

nonadditive genetic or maternal effects (Table 2).


Chinook

Coho

Chum

II

= :E

IT *"

1M

1«

n

:

14

: ** •• * • *. i*".

••!' 12

I.I1i

1 II

V orrfiln

-

i.i= " — '•''

u is ir

i4

•"=

o.i-

=

I.J-


*

"

."r. _•—,——. is

u

= =. S*

1J

""

5

12

-S I —:

IIS

S*

I-*'

. """! t. ".

""

'?

I2S

IS4

: •* * : •

1

8 Jtlittiiifii

8. iiliiiiiirn II

. II

IIIS

\' "

. "i 11 "* n

1J

O.I 1 II

lit

";.• * *' • i nuu

. • "•" . : :V * / 1 " • •••

II

OM

IJ

"•

"••

'

.1 M u u

0.2S ISI

1

8 silioniifiii

8. soliniioryi

R iilniiMrii

II-?

"

jj

IS

r: -«•

.

II "

• !: .

.'. ." . . . . •.

= I.I s

..: . .

". .

" ° •' "* I.I

•S

""

I.J " * i.i i i i3 is i;

IS

i.4

V ordilii

DJ

^

12

S

5

M

1

IJ

^

1.2

II

II

I.I

V. »' I'll n

i>

IS

z

i.n

. .

ri

—

."

I.I1 II

I SI

i.n

A. uliuicil'i

i.i

I.i-

StllOBildfBI

1?*

' : •• •" . t*. ir • it ——,—— . 14

i.bi

li

I JS

%

J

A. solioiicidi

n

II

.'". • \ " • • "-

II1i

Vt

I.I

1f'

*." "

A. sulieiicidc

I)

I.I

V. orlflii

i.i

§i

I.J-

Oil

• ,s!'

s* •• 0.05 for all). However, significant dam effects on mortality were observed in the V. anguillarum challenge (F = 5.17; df = 15, 869; P < 0.01), in the V. ordalii challenge (F = 2.19, P < 0.01), and in the R. salmoninarum challenge (F = 3.16, P < 0.01). Significant tank effects were observed only in the V. anguillarum challenge (F = 4.69; df = 1,869; P < 0.05), and in this case total mortality rates of challenged fish were 52.9% in one tank and 59.6% in the other. Significant sire effects on time to death were observed in the V.

ordalii challenge (F = 2.85, P < 0.05) and in the R. salmoninarum challenge (F = 2.85, P < 0.05); significant dam effects on time to death were observed only in the A. salmonicida challenge (F = 2.46, P < 0.01). Significant tank effects on time to death were observed in all pathogen challenges except the one involving V. anguillarum. Differences in mean time to death between tanks were 1.1 d (12.0 versus 13.1 d) for the V. ordalii challenge, 0.9 d (9.4 versus 10.3 d) for the A. salmonicida challenge, and 2.6 d (61.0 versus 63.6 d) for the R. salmoninarum challenge. Additive genetic variation could account for little of the observed variation in mortality rate. Sire component heritability estimates of mortality ranged between 0.00 and 0.14 when mortality was considered as a binary character, and between 0.00 and 0.28 when mortality was adjusted to a continuous scale (Table 4). Maternal effects or nonadditive genetic variation were observed for mortality rate in the V. anguillarum and R. salmoninarum challenges. Sire component heritability of time to death was generally larger than that of mortality rate. There was little evidence for any maternal or nonadditive genetic effect on time to death, except in the A. salmonicida challenge (Table 4). When we challenged the fish with the pathogens, additive genetic variation could not account for variation in body weight (h 2sjre = 0.00, SE = 0.17), but the maternal or nonadditive ge-

473


TABLE 4.—Percentage of total phenotypic variation in mortality rate and time to death accounted for by sire, dam, and tank effects (and random error) for 30 families of chum salmon, members of which were challenged with one of four pathogens. Challenges were administered by immersion or intraperitoneal injection. Heritability of mortality rate was estimated as both a binary and a continuous character by the transformation of Van Vleck (1972). Standard errors of the herilability estimates are in parentheses. Heritability


Trait

Sire

Dam

Tank

Mortality Time to death

1.5 3.5

11.9 4.2

0.7 1.2


3.4 6.0

3.8 2.4

0.5 8.0


1.6 0.7

0.3 5.3

0.0 6.5


0.0 11.2

5.9 0.6

0.0 25.2

Continuous

Observed

Percent of variation Error

Sire

Dam

Vibrio amguiUamm, immersion 85.9 0.06(0.16) 0.48(0.21) 91.1 0.14(0.10) 0.02*(0.09) Vibrio ordalii, immersion 92.3 0.14(0.11) 83.6 0.24(0.13)

0.15(0.09) 0.10(0.08)

Aeromonas salmomicMa, immersion 98.1 0.07(0.05) 0.01 (0.05) 87.5 0.03(0.09) 0.21(0.12) Renibacterium salmomimarmm, injection 94.1 0.00(0.07) 0.27(0.13) 63.0 0.45 (0.04) 0.02 (0.04)

netic effect was substantial (h 2dam = 0.56, SE = 0.23). When we weighed the fish after the last challenge experiment, the maternal or nonadditive genetic effect was small, accounting for only 1.3% of total variation (h2sire = 0.37, SE = 0.24; h 2^m = 0.43, SE = 0.17). Of the six possible paired comparisons of pathogen challenges, family mortality rates were positively correlated in four of the comparisons and negatively correlated in the other two (Figure 2). None of the correlations was statistically significant, although the correlations between family mortality rates in the V. anguillarum challenge and those in the V. ordain and the A. salmonicida challenges approached significance (0.05 < P < 0.10). When we pooled the family data by sires, three of the correlations were positive and three were negative, but all were nonsignificant. We observed a similar pattern in the genetic correlations, but this pattern was less certain because the genetic correlation based on sire variance components had a relatively large standard error. Times to death for the chum salmon were positively correlated in all six paired comparisons of the pathogen challenges, regardless of whether family data or family data pooled by sires were considered (Figure 3). Families that took longer to die when exposed to one pathogen also tended to take longer to die when exposed to a different pathogen. Correlation between family mean time to death was most marked in the two Vibrio species challenges, and the correlation between mean

Sire

Dam

0.10(0.25)

0.76(0.33)

0.28(0.22)

0.30(0.18)

0.14(0.10)

0.02(0.10)

0.00(0.13)

0.50(0.24)

family mortality rates for these two pathogen challenges was among the highest in our experiments with chum salmon. Little of the variation in family mortality rates or times to death for the four pathogen challenges could be ascribed to additive genetic variation, yet family mortality rates and mean times to death tended to be correlated (but not significantly). The relationship between chum salmon body weight at the end of the experiments and either mortality rate or time to death was not consistent. Family mortality rate was positively correlated with mean body weight for three of the four pathogen challenges (Figure 4), and the result was similar for the genetic correlations. Mean time to death was positively correlated with mean body weight for two of the four challenges (both Vibrio species challenges) and negatively correlated with weight for the other two pathogen challenges (Figure 5), but again none of the correlations was significant. Genetic correlations based on the dam variance component were all negative, indicative of an inverse correlation between body weight and time to death. Two genetic correlations based on the sire variance component were negative and two were positive. However, the two positive genetic correlations (sire component) were much larger than the two corresponding negative correlations (dam component); this difference can account for the positive simple correlations between mean weight and time to death observed in the A. salmonicida and R. salmoninarum challenges.

474

BEACHAM AND EVELYN


Overall Comparisons When all three salmon species were considered, family mortality rates were positively correlated in 13 of the 18 paired comparisons (6 per salmon species) of pathogen challenges, and 5 were negatively correlated (0.05 < P < 0.10; sign test analysis). Four of the five negative correlations involved R. salmoninarum with another pathogen. When only the three faster-acting pathogens (the vibrios and A. salmonidda) were considered (nine correlations total), eight correlations were positive and one was negative (P < 0.05). When family data were pooled by sires, 10 of the 18 correlations were positive and 8 were negative. There seems to be some indication that mortality rate is correlated with rate of pathogen action (or perhaps inversely with fish age when the pathogen takes effect), but the effect is restricted to the dams. The correlation pattern was similar for family times to death in the 18 paired comparisons of pathogen challenges for all three salmon species. Fourteen of these pooled comparisons involved positive correlations and four involved negative correlations (P < 0.05); all negative correlations were in the R. salmoninarum comparisons. When family data were pooled by sires, 12 correlations were positive and 6 were negative. Mean times to death were correlated between families only for the challenges involving the faster-acting pathogens. There was no consistent trend in the 12 simple correlations between body weight and mortality (7 positive, 5 negative) or in those between body weight time to death (5 positive, 7 negative) for the three salmon species. Mortality rates or times to death appeared to be independent of growth rates. The relationship between family mortality rate and mean time to death was fairly consistent. Of the 12 correlations, 10 were negative and 2 were positive (Figure 8). These correlations were statistically significant (P < 0.05) for the K anguillarum. K ordain, and A. salmonidda experiments involving coho and chum salmon. Families that had lower mortality rates in a challenge also tended to have longer mean times to death; both of these traits represent increased disease resistance. Discussion

Resistance to vibriosis in salmonids has a genetic basis; variation has been observed both among and within populations (Gjedrem and Aul-

stad 1974; Winter et al. 1980; Smoker 1986). Heritability estimates of survival in Atlantic salmon Salmo salar after exposure to vibriosis range from 0.12 to 0.32 (Gjedrem and Aulstad 1974; Chevassus and Dorson 1990). In our experiment, when we considered mortality (or alternatively, survival) as a binary character, heritability estimates based on sire variance components for the three Oncorhynchus species exposed to K anguillarum ranged from 0.00 to 0.08; for mortality as a continuous character, these heritability estimates ranged from 0.00 to 0.13. After these three species were exposed to K ordalii, sire component heritability estimates for mortality (binary character) ranged from 0.10 to 0.14 (and from 0.18 to 0.40, continuous character). Transformation of mortality from a binary to a continuous character may overestimate heritability when the mean frequency of occurrence is near zero (Hill and Smith 1977), as was the case for the K ordain challenge (mortality, 6%) of chinook salmon (continuous character, /?2sirt = 0.40). The additive genetic component of characters directly associated with fitness, such as survival, has been suggested to be low (Lerner 1954; Falconer 1981). Because vibriosis more commonly is caused by infection with K anguillarum than by infection with K ordalii in British Columbia, it seems reasonable to expect that the heritabilities of mortality would be less after exposure to K anguillarum than after exposure to K ordain. The results of our experiments support this view. The sire component heritability estimates of time to death in the Vibrio species experiments were 0.00 for chinook and coho salmon, and up to 0.24 for chum salmon. Although heritability of mortality rate is low, additive genetic variation may be substantial enough in specific salmon populations to allow for selection for resistance to vibriosis. Differential susceptibility of populations or strains of salmonids to furunculosis infection has been reported by Wolf (1954), Snieszko et al. (1959), and Cipriano and Heartwell (1986), among others. Gejdrem el al. (1991) estimated heritability of survival for Atlantic salmon exposed to A. salmonidda to be 0.48 (sire component). However, the salmonid population used in their study had never been exposed to A. salmonidda previously, and thus higher heritability estimates of survival would be expected in this population than in populations where furunculosis occurs. In our experiments involving A. salmonidda, heritability estimates of mortality (sire component, binary

475


Chinook

Coho

Chum

V.ooguilloru J '

5.,

.

8.S

11. 5 |0

! ••