Metabolic depression and spleen and liver enlargement in juvenile Arctic charr Salvelinus alpinus exposed to chronic parasite infection

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1 Journal of Fish Biology (2009) 74, doi: /j x, available online at Metabolic depression and spleen and liver enlargement in juvenile Arctic charr Salvelinus alpinus exposed to chronic parasite infection E. SEPPA NEN*, H. KUUKKA, A. VOUTILAINENk, H. HUUSKONENk AND N. PEUHKURI *Finnish Game and Fisheries Research Institute (FGFRI), Laasalantie 9, FI Enonkoski, Finland, Integrative Ecology Unit, Department of Ecological and Environmental Sciences, P. O. Box 65, FI University of Helsinki, Finland, FGFRI, Sapokankatu 2, FI Kotka, Finland and kecological Research Institute, Faculty of Biosciences, University of Joensuu, P. O. Box 111, FI Joensuu, and Finland (Received 17 April 2008, Accepted 29 October 2008) The present study on the connection between standard metabolic rate (R S ) and chronic Diplostomum spp. infection resulted in a decrease in R S, and an enlargement in spleen and liver sizes in the infected juvenile Arctic charr Salvelinus alpinus compared to control fish. As splenic enlargement observed in infected fish was not due to condition-related changes in the spleen, it could most probably be explained by increased leucocyte synthesis. The higher liver masses in infected S. alpinus may have been related to disorders in energetic function, which could have had major effects on biochemical regulation by the liver. The proposed metabolic syndrome with a possible reduction in insulin sensitivity in tissues results in ineffective glucose and lipid metabolism and thus it is suggested that chronic Diplostomum infection in S. alpinus might not impose direct energetic costs, but it may weaken the efficiency of energy metabolism and thus lead to lowered R S. Journal compilation # 2009 The Fisheries Society of the British Isles Key words: Diplostomum spp.; liver; Salvelinus alpinus; spleen; standard metabolic rate. INTRODUCTION Parasites are ecologically important stressors that cause physiological and behavioural changes reducing host fitness (Møller et al., 1996; Klein et al., 2004; Östlund-Nilsson et al., 2005). Fitness costs may be due, in part, to energetic costs of maintenance and activation of the immune system (Sheldon & Verhulst, 1996; Zuk & Stoehr, 2002). Exposure to parasites (Morand & Harvey, 2000; Shinagawa et al., 2001; Khokhlova et al., 2002) or other short-term challenges to the immune system (Freitak et al., 2003; Martin et al., 2003) elevate metabolic rate, demonstrating that activation of the immune system is energetically costly. Author to whom correspondence should be addressed. Tel.: þ ; fax: þ ; eila.seppanen@rktl.fi 553 Journal compilation # 2009 The Fisheries Society of the British Isles

2 554 E. SEPPA NEN ET AL. The spleen has a central role in immune defense and haematopoiesis (Rose, 1981; John, 1994) and increased size is a possible reflection of investment in immune function (Rose, 1981; John, 1994). Spleen size has also been suggested to reflect adaptation to the level of parasite load in the host population or species (Gregory, 1991; John, 1995; Morand & Poulin, 2000). Liver enlargement occurs for various reasons such as: infection, direct toxicity, hepatic tumours, storage diseases or metabolic disorder. Changes in liver metabolism can affect the quantities of metabolites available to the peripheral tissues and consequently influence the metabolism of the whole organism (Huntington & Reynolds, 1987; Cortez-Pinto et al., 1999; Marceau et al., 1999). In fishes, liver mass indicates energy reserves and reflects overall body condition (Wootton, 1984; Lambert & Dutil, 1997). Parasitism is known to affect host metabolism in fishes (Walkey & Meakins, 1970; Lemly & Esch, 1984; Östlund-Nilsson et al., 2005). The standard metabolic rate (R S ) which is the minimal, or resting metabolic rate of an unfed fish required for maintaining critical physiological functions (Brett & Groves, 1979; Priede, 1985; Jobling, 1994), and changes in it can reflect alteration of host energy metabolism affected by parasitic infection. Metabolic rate, however, is a genetically determined, non-specific variable and has the potential to respond to natural selection (Moody et al., 1997; Khazaeli et al., 2005; Nespolo et al., 2007). Consequently, metabolic rate may be faster or slower depending on genetic factors. Diplostomum spp., including Diplostomum spathaceum and Diplostomum pseudospathaceum, are ubiquitous parasites that develop to metacercariae in the eye lens of fishes and cause cataracts due to their metabolic products and mechanical destruction of lens tissue. Cataracts reduce vision and even cause total blindness (Ersdal et al., 2001). In the present study, the effects of chronic Diplostomum spp. infection (cataracts already formed) on R S, body condition, spleen mass and liver condition in juvenile Arctic charr Salvelinus alpinus (L.) were investigated. If chronic Diplostomum spp. infection is energetically costly, infected fish were hypothesized to show increased R S and poorer body condition compared to non-infected fish. Furthermore, Diplostomum spp. infection were expected to lead to increased spleen and liver size reflecting intensified immune function and disturbances in energy metabolism, respectively. The potential influence of genetic background on the studied traits was also investigated. STUDY FISH MATERIALS AND METHODS One summer-old S. alpinus (n ¼ 100), from 10 families originating from a secondgeneration hatchery population, were used in the experiment following the principles of animal treatment and welfare for scientific experimentation approved by the ethical committee of the Finnish Game and Fisheries Research Institute (permission no. 18/05). Eggs were fertilized on 11 November A nested mating design, where two females (10 in total) were crossed with one male (n ¼ 5), enabled the study of the effects of dams and sires on the measured traits (McAdam et al., 2002; Kotiaho et al., 2003;

3 CHRONIC INFECTION AND METABOLISM 555 Roff, 2008). Infection of fish with Diplostomum spp. cercariae takes place when the water temperature is 10 C (Stables & Chappell, 1986). Hence, filtration of the rearing water was started in the beginning of April 2006, the water temperature being <9 C. The hatchery water was from two nearby lakes, Lake Pahkajarvi ( N; E) and Lake Yla-Enonvesi ( N; E). The water temperature matched the ambient temperature of the lakes during winter, but during summer the water temperature was maintained at C due to the preference of S. alpinus for cool water. Photoperiod was regulated manually matching ambient conditions (65 N). The Diplostomum spp. infected fish (n ¼ 5 fish per family) were selected from the 10 families that had been maintained in normal hatchery conditions in family-specific tanks (diameter 800 mm and water flow 18 l min 1 ). Uninfected control fish originated from the same families (n ¼ 5 fish per family) and were maintained in identical conditions but in filtered water (filter size 25 mm) from the same source. Fish were fed commercial pellet feed (Raisio Group; ad libitum. In December 2006, fish were tagged with an elastomeric colourant (VIE; Northwest Marine Technology Inc.; immediately before the respirometry experiments. Their eyes were checked for Diplostomum spp. infection and cataracts prior to tagging. The number of Diplostomum spp. in both eye lenses was counted, and the area of each lens covered by cataract opacities was evaluated by KOWA SL-15 slit-lamp microscopy (KOWA; The fish were anaesthetized with sodium bicarbonate buffered MS-222 (tricaine methane sulphonate) solution for examination and tagging. TRAIT MEASUREMENTS Oxygen consumption by S. alpinus was measured by an automated three-chamber intermittent-flow respirometer equipped with YSI 5750 polarographic oxygen sensor (YSI Inc; Forstner, 1983; Wieser et al., 1988) for the period December 2006 to February The respirometer comprised three parallel transparent acrylic chambers (volume ml), with water temperature of C(mean S.D.) and flow rate 200 ml min 1. Prior to oxygen measurement, the fish were maintained singly in aquaria, fasted for 24 h and acclimated to the experimental temperature, which was close to the rearing temperature, and the rate of change was Cperday.Three individuals at a time were chosen randomly from among all tagged fish for respirometry. Measurements continued for 24 h (photoperiod 16L:8D). Oxygen consumption in each chamber was recorded for 15 min each hour, and the average rate for this time extrapolated to an hourly value. The chambers were flushed with aerated water for 45 min after each 15 min period. Microbial oxygen consumption in empty chambers was measured at the beginning and the end of the 24 h period and subtracted from the total oxygen decline. The respirometer was cleaned and disinfected once a week. During respirometry, fish were undisturbed to minimize stress. R S was defined as the mean of the two lowest hourly oxygen consumption values during the 24 h period. After respirometry, the fish were anaesthetized, weighed (mass, M) and measured for total length (L T ). The condition factor (K) was calculated for each individual by regressing (model II regression) M on L T and using the residuals of the function (Green, 2001). The fish were killed with an overdose of MS-222 and their spleens (M S ) and livers (M L ) weighed. STATISTICAL ANALYSES To examine the effect of chronic Diplostomum spp. infection on S. alpinus general linear mixed models (GLMM) (SAS 9.1 for Windows) were constructed with R S, M L or M S as a response variable and treatment (infected or control) as an effective variable. Given that the response variables are M dependent, M was used as a covariate in the models. Dam nested under sire and sire alone were included as random factors in the analysis if they were needed in the model. In the analysis where R S was a response variable and M was used as a covariate, the total R S value (mmol O 2 h 1 ) was used instead of the mass-specific R S value (mmol O 2 g 1 h 1 ).

4 556 E. SEPPA NEN ET AL. RESULTS The median number of Diplostomum spp. metacercariae and cataract intensity as a percentage of the lens area covered (eyes pooled) was 1 (range 1 3) and 30 (range 5 70), respectively. R S of Diplostomum spp. infected S. alpinus was significantly lower than that for control fish (GLMM, F 1,100, P < 0001; Fig. 1). The covariate was also highly significant (GLMM, F 35,100, P < 0001). The M S and M L of the infected fish were significantly higher than those for uninfected control fish (GLMM, F 1,100, P < 005; Fig. 2, F 1,87, P < 0001; Fig. 3, respectively). The covariates were also highly significant for both M S (GLMM, F 35,100, P < 0001) and M L (GLMM, F 35,81, P < 0001). The genetic background, dam (sire), had no effect on M L (GLMM, Z ¼ 083, P > 005). There were no differences in L T, M or K between the fish in the infected and the control groups (GLMM, all F 1,90, P > 005; Table I). The genetic background, dam (sire), had an effect on individual L T (GLMM, Z ¼ 208, P < 005), M (GLMM, Z ¼ 204, P < 005) and K (GLMM, Z ¼ 183, P < 005), suggesting that there is a maternal effect on individual size and condition. DISCUSSION Diplostomum spp. infected S. alpinus had significantly lower R S than control fish. This does not confirm earlier studies, suggesting either that parasitized fish have higher rates of oxygen consumption than the non-parasitized ones (Walkey & Meakins, 1970; Lemly & Esch, 1984; Östlund-Nilsson et al., 2005) or that parasite infection has no effect on basal metabolic rate (Meakins & Walkey, 1975). Genetic background did not explain the difference in R S between infected and control fish, indicating that the response of fish energy metabolism to chronic Diplostomum spp. infection was more or less similar in all 10 S. alpinus families examined. In addition to depressed metabolic rate, chronic Diplostomum spp. infection was associated with increased host spleen and liver size. A positive correlation FIG. 1. Mean þ S.E. standard metabolic rate (R S )ofdiplostomum spp. infected and uninfected (control) Salvelinus alpinus juveniles.

5 CHRONIC INFECTION AND METABOLISM 557 FIG. 2. Mean þ S.E. spleen mass (M S )ofdiplostomum spp. infected and uninfected (control) Salvelinus alpinus juveniles. between splenic enlargement and parasitism in fishes, including fish cestode and fish nematode systems, has been reported in several studies (Arnott et al., 2000; Morand & Poulin, 2000; Lefebvre et al., 2004). Ottova et al. (2005), however, found no relationship between spleen size and parasitism in common bream Abramis brama (L.) infected by Diplostomum spp. and two ectoparasites, Gyrodactylus sp. and Argulus sp. In the study by Ottova et al. (2005), splenic enlargement was associated with poor somatic condition. There were no differences in condition between the experimental groups. Consequently, splenic enlargement observed in infected fish in the present study cannot be explained by condition-related changes in the spleen. Given that spleen is a lymphoid organ having a role in defence against infections (Press & Evensen, 1999), the enlargement of spleen can most probably be explained by increased leucocyte synthesis. The spleen also has a haematological function (Press & Evensen, 1999), but there is no apparent reason to expect hyper-synthesis of erythrocytes during Diplostomum spp. infection. FIG. 3. Mean þ S.E. liver mass (M L )ofdiplostomum spp. infected and uninfected (control) Salvelinus alpinus juveniles.

6 558 E. SEPPA NEN ET AL. TABLE I. Mean S.D. total length (L T ), body mass (M) and condition factor (K) of Salvelinus alpinus juveniles Infected Control Number L T (mm) M (g) K The M L of infected S. alpinus was higher than that of uninfected fish. Although liver mass is an indication of energy reserves in fishes (Wootton, 1984; Lambert & Dutil, 1997), the higher liver masses observed in infected S. alpinus may be related to disorders in energetic function, which can have major effects on biochemical regulation by the liver. Thus, changes in the metabolism can change the proportions and the absolute quantities of metabolites that become available to the peripheral tissues and consequently can influence the metabolism of the whole organism. In humans, fatty liver is associated with the metabolic syndrome (Cortez-Pinto et al., 1999, Marceau et al., 1999) that is characterized by a remarkable reduction in insulin sensitivity in tissues (Marchesini et al., 2001), resulting in ineffective glucose and lipid metabolism. If chronic Diplostomum spp. infection in S. alpinus harms liver energetic function and decreases tissue sensitivity to insulin, depressed R S in infected fish may be a result of disturbed energy utilization. The respirometry was carried out at low temperatures in winter, and it is known that only few signs of immunological responses are found in fish eye tissues (Koppang et al., 2004), and both the humoral and cellular responses decelerate at cold temperatures (Rijkers et al., 1980; Hardie et al., 1994). The S. alpinus is, however, one of the most cold-stenotherm fish species (Sandlund et al., 1992; Lyytikäinen et al., 1997), and influence of water temperature on the immune function of S. alpinus is minor (Pylkkö et al., 2002). The possibility that splenic enlargement and impaired liver condition may result from other factors, for example, bacteria, viruses or other parasites in unfiltered water, was considered. No bacterial or viral diseases or other parasites than Diplostomum spp., however, were evident in the study fish, and therefore they were not examined more closely. Although L T, M or K did not differ between the infected and uninfected fish, they were influenced by genetic background. Dam nested under sire had a significant effect on individual L T, M or K but sire alone did not affect them, suggesting that there is a maternal effect (McAdam et al., 2002; Kotiaho et al., 2003). Maternal effects exist in salmonids (Sargent et al., 1987; Heath et al., 1999), and are found mostly early in ontogeny, diminishing as the fish age (Heath et al., 1999). To conclude, Diplostomum spp. infection may induce an immune response in S. alpinus resulting in splenic enlargement. Furthermore, chronic Diplostomum spp. infection results in liver enlargement and consequently, depressed R S.Hence, chronic Diplostomum spp. infection does not directly impose energetic costs on S. alpinus, but it reduces the efficiency of energy metabolism. It is acknowledged

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