Thermal acclimation in the perch (Perca fluviatilis L.) immunity

Journal of Thermal Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Thermal acclimation in the perch (Perca fluviatilis L.) immunity Pertti Marnila a,b,n, Esa-Matti Lilius c a b c

Natural Resources Institute Finland, FIN-31600 Jokioinen, Finland Department of Biology, Laboratory of Animal Physiology, University of Turku, FIN-20014 Turku, Finland Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland

art ic l e i nf o

a b s t r a c t

Article history: Received 28 July 2014 Received in revised form 31 December 2014 Accepted 15 January 2015

Fish immune systems must be able to cope with pathogens over a wide temperature range. Earlier research suggest that fish are more dependent on innate immune responses based on pattern recognition than acquired functions with specific recognition. If this applies to phagocytes, then opsonins (serum factors that augment phagocytosis e.g. immunoglobulins and complement proteins) attached on zymosan (Z) particles should be recognized better at higher temperatures than Z only. Z is recognized by glucan receptor representing pattern recognition. In this study perch were acclimated to 5 °C or 16 °C for 3–5 weeks. The recognition and activation of respiratory burst reaction of peripheral blood phagocytes was examined at seven different measurement temperatures (5, 10, 16, 20, 24 27, and 30 °C) when the cells were stimulated with Z and serum opsonized zymosan (OZ). Respiratory burst was measured as luminol chemiluminescence (CL) from diluted whole blood. OZ-induced CL per volume of blood was on average approximately 4.6 times higher in 16 °C acclimated fish than 5 °C acclimated perch (Po 0.0001). Z-induced CL was approximately 3 times higher at lower temperatures in 16 °C acclimated perch than 5 °C acclimated fish and 6–9 times higher at 27 °C and 30 °C (P o0.001), respectively. CL reaction kinetics were faster in perch acclimated to 5 °C than 16 °C -acclimated fish, especially at low temperatures (P o0.001). Thermal acclimation caused a 3–4 °C shift in temperature response curves of CL towards the acclimation temperature (Po 0.0001 and P o 0.053 in Z and OZ-induced CL, respectively). Serum opsonins activated perch phagocytes substantially better at higher temperatures in both acclimation groups, which is consistent with an earlier study in rainbow trout (O. mykiss). However, opsonin recognition was significantly better in 16 °C acclimated perch than 5 °C acclimated fish, which was seen as higher CLs for OZ compared to Z, especially at higher temperatures. This is opposite to previously reported results in rainbow trout. Differences between rainbow trout and perch in opsonin recognition by blood phagocytes suggest that the living habits of perch, which prefers approximately a 10 °C higher temperature than rainbow trout, may be reflected in immune cell functions. Results of the present examination suggest that also in fish phagocytes pattern recognition is the prevailing system at low temperatures, and specific recognition is more effective at high temperatures. & 2015 Elsevier Ltd. All rights reserved.

Keywords: Temperature acclimation Temperature Immunity Phagocyte Respiratory burst Opsonin recognition Perch Perca fluviatilis

1. Introduction The immune system of poikilothermic animals, such as fish, living in boreal or subarctic regions must be able to protect the host against pathogens over a wide temperature range. In general, many animals that live in boreal regions show natural seasonal acclimatization and have a capacity for thermal acclimation under laboratory conditions. Seasonal variations in water temperatures Abbreviations: CL, chemiluminescence; Z, zymosan; OZ, opsonized zymosan; Ig, immunoglobulin; IgM, M class immunoglobulin n Corresponding author at: Natural Resources Institute Finland, FIN-31600 Jokioinen, Finland. E-mail address: pertti.marnila@luke.fi (P. Marnila).

in boreal regions often exceed 20 °C. Waters in summer are substantially warmer near the surface than in deeper layers beneath the thermocline, and fish live in a temperature gradient. The fish immune system undergoes substantial seasonal changes, and factors, such as the temperature and photoperiod, affect immune functions partially via the neuroendocrine system (for reviews, see Bly and Clem, 1992; Zapata et al., 1992; Bowden et al., 2007). Therefore, the thermal history of an individual fish should be considered in the measurement of immune functions. Phagocytic leukocytes that form the first line of immune defense, play a crucial role in protection against microbial infections (Secombes and Fletcher, 1992; Dzik, 2010; Grayfer et al., 2014). Phagocytes kill microorganisms by means of lysosomal degradative enzymes and highly toxic reactive oxygen intermediates.

http://dx.doi.org/10.1016/j.jtherbio.2015.01.002 0306-4565/& 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Marnila, P., Lilius, E.-M., Thermal acclimation in the perch (Perca fluviatilis L.) immunity. J. Thermal Biol. (2015), http://dx.doi.org/10.1016/j.jtherbio.2015.01.002i

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The reactive oxygen compounds are produced, in a process called the 'respiratory burst', by the NADPH–oxidase complex in plasma membrane, and by myeloperoxidase enzyme released during degranulation from azurophilic granules into phagolysosomes and extracellular space. These processes generate electronically excited states which, on relaxation, emit photons, giving rise to phagocyte chemiluminescence (CL). The CL emission can be amplified with a chemical enhancer, luminol by a factor of
104. Luminol-amplified CL of phagocytes has been shown to be almost completely dependent on the release of myeloperoxidase from azurophilic granules. CL activity correlates well with the ingestion and killing of bacterial pathogens in salmonids (Stave et al., 1984; Ottinger et al., 1999; Nikoskelainen et al., 2005) and in human (Lilius and Nuutila, 1993; 2006). Zymosan (Z), a cell wall preparation from yeast Saccharomyces cerevisiae, is commonly used to activate phagocyte respiratory burst. Z contains glucan and mannan, which are in mammals recognized by the complement receptor 3 complex (CD11b/CD18, Mac-1). This complex recognizes also complement C3bi and fibrinogen (Lilius and Marnila, 1992; Boshra et al., 2006). Also in fish recognition of Z is mediated by glucan receptors (Ainsworth, 1994), which belong to pattern recognition receptors. The pattern recognition receptors recognize widely conserved motifs of various pathogens and initiate a rapid innate immune response. The process of opsonization is a means of identifying invading microbes by phagocytes. Binding of the serum complement components C3b and C3bi and the specific binding of the serum antibodies to the invading pathogen are normally required for a successfull recognition and destruction of the pathogen by phagocytes. Opsonization accelerates phagocytosis and killing of bacteria. Human phagocytes killed 41% of phagocytozed non-opsonized Escherichia coli K-12-bacteria in 180 min but, when the bacteria were pre-incubated in 0.4% (v/v) serum 96% were killed by phagocytes in same time (Atosuo and Lilius, 2011). Specific binding of immunoglobulins (Ig) on microbe surface accelerates killing of microbes also by the complement system (serum bacteriolytic activity). The reaction velocity rates of fish and human antibody dependent complement pathways were about two and five times higher than those of non-antibody-dependent alternative pathways, respectively (Kilpi et al., 2009). In opsonization of Z serum complement compounds and Igs are attached to Z particles. Serum opsonized Z (OZ) is recognized by human phagocytes partly via the glucal receptor (CD11b/CD18), partly by complement receptor 1 (CD35) which bind to C3b and partly by Ig-receptors (CD16 and CD64). Specific Igs and receptors recognizing the Igs and complement factors attached to Ig–antigen complexes, represent specific acquired immunity. Also in fish specific M-class Igs (IgM) and complement factors enhance phagocytosis and killing (Boshra et al., 2006; Nikoskelainen et al., 2005, 2007; Verho et al., 2005). In rainbow trout (O. mykiss) specific IgM for the bacterium Aeromonas salmonicida without an active complement was a relatively inefficient opsonin, but specific IgM with an active complement increased the magnitude of ingestion-mediated CL activity and accelerated the ingestion of target bacteria (Nikoskelainen et al., 2005). In fish the complement receptors have not yet been characterized in detail, but accumulating evidence suggests that also fish phagocytes have receptors for complement proteins and IgM (Ainsworth, 1994; Couso et al., 2001; Esteban et al., 2004; Nakao et al., 2004; Boshra et al., 2006; Stafford et al., 2006a, 2006b). Thus, we assume that also in fish OZ is recognized by IgM, complement and glucan receptors, and Z only by the glucan receptors. Low temperatures suppress humoral immunity, especially the primary antibody response, and many T-cell mediated immune functions in most fish species studied (Sypek and Burreson, 1983; Bly and Clem, 1991;1992; Ainsworth et al., 1991; Collazos et al.,

1995a; Le Morvan-Rocher et al., 1995; Alcorn et al., 2002; Magnadóttir et al., 1999; Nikoskelainen et al., 2004; Grayfer et al., 2014). It has been proposed that at low temperatures fish rely more on non-specific innate immune responses, while at higher temperatures specific immunity is used to a greater extent (Ainsworth et al., 1991; Le Morvan et al., 1998; Alcorn et al., 2002; Bowden et al., 2007). If this hypothesis is valid in phagocytic leukocytes, then at low temperatures the activation of respiratory burst via glucan receptors (which recognize Z; pattern recognition) should be functional and activation via receptors that recognize serum opsonins on Z particles (acquired specific immunity) should be relatively inactive. In that case opsonins on Z particles would not increase or accelerate phagocyte activation much but, at high temperatures opsonization of Z particles should result to substantially stronger phagocyte activation than Z alone. It is probable that thermal acclimation may alter the expression and function of different receptor types. To best of our knowledge fish phagocyte activation by Z or OZ at different temperatures has this far been examined only in rainbow trout (O. mykiss) (Nikoskelainen et al., 2004). Rainbow trout were acclimated to 5, 10, 15 and 20 °C. The recognition of opsonins was more effective at higher (15 and 20 °C) than colder temperatures (5 and 10 °C) in all acclimation groups. This is in agreement with the hypothesis above. However, phagocytes of rainbow trout that were acclimated to 5 °C or 10 °C recognized serum opsonins better at all temperatures than phagocytes of fish that were acclimated to 15 °C or 20 °C. This is somewhat controversial with the hypothesis. The authors suggested that this improved opsonin recognition due to cold acclimation could be an adaptive compensation reaction to low temperature, in which opsonin recognition is impaired (Nikoskelainen et al., 2004). Rainbow trout like other salmonids prefer cold water. It is not known if temperature or thermal acclimation has similar effects on phagocyte recognition in fish species that prefer warmer water. This information is needed in order to know if hypothesis presented above is valid in phagocyte recognition patterns. It would add on understanding on the effects of temperature on fish immunity and what sets the thermal limits in host defense. Therefore, in the present study we examined the effects of temperature and thermal acclimation of perch (Perca fluviatilis) on the ability of peripheral blood phagocytes to recognize Z and serum opsonins on Z particles and on the respiratory burst activity. Perch prefer approximately 10 °C warmer water than rainbow trout. It is a relatively eurythermic pelagial species that tolerate high water temperatures up to 30 °C.

2. Materials and methods 2.1. Animals and acclimation Perch of both sexes were captured from the Baltic Sea near the island of Seili in the archipelago of Turku (southwest coast of Finland). Fish were captured using a fishing net in winter and spring between February and early May. After capturing the perch were transported to the Archipelago Research Institute, University of Turku. The salt content of water in the capturing area is 0.6% (w/v). In winter the water temperature is þ0.2 °C under the ice and þ4 °C at deeper levels. Ice breakup occurs in April. The water temperature in May varies from þ 4 °C in deep water to þ10 °C on the surface. The mean weight of captured perch was 85 g (range 43–181). Fish were kept in 70 l brown plastic containers filled with charcoal-filtered tap water that was changed daily. A total of 5–7 perch were kept in each container, and the biomass varied approximately from 300 to 900 g per 70 l. Illumination was kept dim, and the light/dark cycle was 12/12 h. Perch were fed chironomid larvae

Please cite this article as: Marnila, P., Lilius, E.-M., Thermal acclimation in the perch (Perca fluviatilis L.) immunity. J. Thermal Biol. (2015), http://dx.doi.org/10.1016/j.jtherbio.2015.01.002i

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2–3 times a week. Temperature acclimation was initiated the day after capture, and acclimation temperatures of 5 °C or 16 °C were reached within approximately 48 h. These temperatures represent the water temperatures above the thermocline in the Northern Baltic Sea in April and the summer, respectively. Temperature acclimation lasted 3–5 weeks, which exceeded the two-week acclimation time that is generally considered sufficient for physiological changes (Lagerspetz, 1977; Talo and Tirri, 1991). 2.2. Sampling Blood samples were taken from the posterior caudal vein (vena cava posterior) using a sterile needle and syringe. The blood yield varied from 200 to 600 μl per animal. Blood samples were anticoagulated with 20 μl of 150 mM Na-EDTA in fish Ringer. Total cell numbers per volume of blood were counted using a Bürker camber and microscope. Smear preparations were drawn and stained using May-Grünwald-giemsa staining. Blood sampling and making of CL-reaction mixtures were performed at 5 °C with cold-acclimated fish and 16 °C with warm-acclimated fish because temperature changes modulate receptor expression on leukocyte cell membranes in vitro in mammals (Tennenberg et al., 1988; Glasser and Fiederlein, 1990). Lab-Animal Care & Use Committee of the University of Turku approved the study protocol. 2.3. Health status Perch were killed immediately after blood samples were taken, and a fish pathologist examined perch at the Environmental and Marine Biology laboratories, Åbo Akademi University, Turku, Finland. Results from individual fish that showed signs of injuries or infections were excluded. Only results from perch regarded as healthy were included in this study. 2.4. Reagents A stock solution of 10 mM luminol (5-amino-2,3-dihydro-1, 4-pthalazinedione, Sigma Chemicals, St. Louis, MO, USA) was prepared in 0.2 M sodium borate buffer (pH 9.0). Z (zymosan A from S. cerevisiae, Sigma) was boiled in phosphate-buffered saline (pH 7.6) and washed three times with centrifugation (10 min at 1000g) in fish Ringer (pH 7.6). A suspension of 20 mg of Z per ml was made in this buffer and stored at  70 °C. 2.5. Serum opsonization of Z To produce Z particles that have opsonins on surface (OZ) Z was incubated (20 mg/ml) in 70% fresh perch serum and 30% fish Ringer (v/v) for 30 min at 22 °C to allow serum IgM and complement factors attach on Z. Then OZ was washed three times in fish Ringer as described above. A stock solution of 20 mg/ml was prepared in fish Ringer and stored in aliquots at 70 °C. In order to examine whether sera from both groups contained sufficient amounts of opsonins, batches of Z were opsonized at 22 °C as described above with serum (70% v/v) samples obtained from 10 cold- and 10 warm-acclimated fish, and the OZ particles were washed as described above. The opsonization activity of a serum sample was defined as a percentage increase in CL maximum intensity due to the opsonization of Z compared with the CL intensity obtained with non-opsonized Z (OZ/Z-induced CL ratio). Incubation of Z particles in serum samples of perch that were acclimated to 5 and 16 °C resulted in a mean increases of 85% (S. D.736%) and 72% (S.D. 724%) in CL intensities, respectively. The difference was not statistically significant. Thus, sera from both groups can be used in opsonization. Serum pools from 5 °C acclimated fish were used to make the OZ that was used to stimulate

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phagocytes of 5 °C acclimated perch, and serum pools from 16 °C acclimated fish were used to make the OZ to stimulate phagocytes of 16 °C acclimated fish. 2.6. Chemiluminescence assay Respiratory burst reaction of phagocytizing leukocytes in whole blood samples diluted in fish Ringer was evoked by Z or OZ and the activity was measured as luminol-enhanced CL emission. Appropriate concentration ranges for blood, luminol, Z and OZ were evaluated in preliminary experiments using similar methods than in Marnila et al. (1995), Nikoskelainen et al. (2004) and Lilius and Nuutila (2006). On the basis of these experiments the further studies were performed using 0.5 mM luminol, 0.5 mg of Z or OZ and 375 nl of blood diluted in fish Ringer (0.15% v/v) in a total reaction volume of 250 μl (1/667 dilution). Assays were performed in 2 or 3 parallel wells, and the mean of the peak CL of the wells was regarded as the CL value of that measurement. A cell background sample, which consisted of only diluted blood and luminol, was included in each assay. The magnitude of cell background was negligible compared to Z- or OZ-induced CL responses. Only the number of phagocytes affect CL emission at this and lower blood concentrations because these blood dilutions do not contain significant levels of opsonins or CL quenching erythrocytes, albumin or fibrinogen (Nikoskelainen et al., 2004, Lilius and Nuutila, 2006). Whole blood CL in perch mainly reflects granulocyte function because granulocytes are the most abundant phagocyte type in this fish (Förlin et al., 1995). The functional state of whole blood phagocytes may reflect the physiological state of the host better than isolated leukocytes because activation processes and alterations in receptor expression occur during isolation steps (Tennenberg et al., 1988; Glasser and Fiederlein, 1990). CL emission was measured at seven temperatures (5, 10, 16, 20, 24, 27 and 30 °C). Three luminometers were used at the same time because consecutive CL measurements take time. Two Wallac 1251 luminometers (Wallac-PerkinElmer Finland Ltd, Turku, Finland) were used at measurement temperatures of 20–30 °C, and one Luminoskan EL-1 luminometer (Thermo Labsystems, Helsinki, Finland) was used at 5–16 °C. Instrument signals were compared to each other at various signal levels and different temperatures using standard samples. Correction coefficients were used to fit the results onto the same scale. Reaction kinetics (CL peaktimes) were similar at the same temperatures when measurements were recorded using different luminometers. Results are presented as millivolts according to the scale of the Wallac 1251-luminometers. Samples were kept in the measuring chambers of the luminometers for 5–10 min to equalize sample temperatures before the reaction was started by the addition of Z or OZ. CL emissions were measured at 2–6 min intervals for 30–180 min depending on the temperature to obtain kinetic curves (Fig. 1A and B). The peak CL was regarded as the CL intensity value and the peaktime as the reaction velocity value of the sample. When 7 parallel samples of one blood sample were separately diluted and measured in two parallels the coefficient of variance of the 7 means was 710.1%. Blood samples of 16 °C and 5 °C acclimated perch were always measured at the same time. Subsequent CL measurements at different temperatures were performed during the first six hours after sampling. The measurement order at different temperatures was changed to avoid systematic errors due to sample storage time (see Sections 2.7 and 3.1.1.). 2.7. Measuring the effect of storage time on chemiluminescence responses The effect of blood sample storage time on leukocyte CL activity was evaluated because the measuring of CL at several temperatures

Please cite this article as: Marnila, P., Lilius, E.-M., Thermal acclimation in the perch (Perca fluviatilis L.) immunity. J. Thermal Biol. (2015), http://dx.doi.org/10.1016/j.jtherbio.2015.01.002i

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receptors we compared the Z and OZ induced CL responses of phagocytes from the same individual fish. The opsonization activity of a serum sample was defined as a percentage increase in CL maximum intensity due to the opsonization of Z compared with the CL intensity obtained with non-opsonized Z (OZ/Z-induced CL ratio). Active complement is heat sensitive whereas Igs tolerate quite high temperatures without major reduction in opsonic activity. Heating for 30 min at 45 °C is commonly used to inactivate fish complement. In order to ensure that active complement is involved in the opsonic activity which is central in this study, we tested the heat sensitivity of perch serum opsonic activity and whether it can be restored with fresh unheated serum. We pooled serum samples from 10 perch acclimated to 16 °C in order to determine the proper temperature for opsonization and the temperatures that cause heat inactivation of complement. The serum pool was divided into 20 ml aliquots in duplicate, which were preincubated for 45 min at 9 different temperatures (8, 15, 20, 25, 30, 35, 40 and 45 °C). After the preincubation, 50 ml of fresh unheated serum was added to one of the duplicates, and the sera were used to opsonize 4 mg aliquots of Z (opsonized for 30 min at 22 °C in 200 μl of pooled sera, 70% v/v in fish Ringer). OZ samples were washed three times as described above. Leukocytes of warmacclimated perch were stimulated with non-opsonized Z and OZ samples that were opsonized with differently handled serum samples, each in four parallel samples, and CL responses were measured at 24 °C as described above. 2.9. Data analyses We analyzed the overall effects of temperature and acclimation on temperature response curves (Fig. 3A and B) of CL activities from the repeated measurements at different temperatures using a covariance model in PROC MIXED of SAS/STAT (SAS1996) Statistical software for Windows release 8.01 (SAS Institute Inc., Cary, North Carolina, USA). The following model was used:

Yijk = m + ai + b j + abij + eijk

Fig. 1. Typical Z- and OZ-induced CL reaction curves in perch that were acclimated to 5 °C and 16 °C when measured at (A) 5 °C and (B) at 16 °C. The CL reaction was measured in whole blood diluted in fish Ringer (0.15% v/v) in a total reaction volume of 250 μl (1/667 dilution). The CL reaction was induced with 0.5 mg of Z or OZ. Luminol (0.5 mM) was used to enhance CL emission.

takes time, even with several luminometers. Blood samples from 5 °C acclimated fish (n¼3) were kept at 5 °C, and samples from 16 °C acclimated fish (n¼3) were maintained at 16 °C. Measurements of Z- and OZ-induced CL responses at 24 °C were initiated 45 min, 3 h, 5 h, 7 h and 9.5 h after sampling. The CL response was measured as described in Section 2.6. 2.8. Effect of temperature on serum opsonization activity To investigate the relative increase of phagocyte activation by specific receptors compared to activation by pattern recognition

Yijk is the response for the acclimation group i, temperature j and individual k; m is the overall mean; a and b are the fixed effects of acclimation and temperature, respectively; and ab is the two-factor interaction of the fixed effects. The error variables eijk are assumed to be independent and normally distributed with zero means and variances. Furthermore, the covariances between any two observations in the same subject were allowed. A logarithmic transformation was used to normalize the data in analyses of CL intensities. Contrasts at separate temperatures were performed using paired Student's T-tests for the effects of opsonins within the acclimation groups. Two-sample Student's T-test was applied when the two acclimation groups were compared (Tables 1 and 2) (the OZ/Z-induced CL ratios)

3. Results 3.1. Assessing the research methods for perch 3.1.1. Effect of storage time on chemiluminescence response Typical CL reaction curves of Z- and OZ-induced responses at 5 °C and 16 °C are presented in Fig. 1A and B. The peak CL correlates with phagocytosis and killing activity and the peaktime reflects the reaction velocity of the respiratory burst. Because the measuring of CL at several temperatures takes time it is important that the assay can be assumed to be reliable over an extended duration. As seen in Fig. 2 there was an approximately 10–20% decrease in CL activities after 5 h compared to the 45-min

Please cite this article as: Marnila, P., Lilius, E.-M., Thermal acclimation in the perch (Perca fluviatilis L.) immunity. J. Thermal Biol. (2015), http://dx.doi.org/10.1016/j.jtherbio.2015.01.002i

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Table 1 The relative increase (OZ/Z-induced CL ratio7 S.D.) of CL peak value due to opsonins on Z particles at different temperatures in perch acclimated to 5 °C (n¼ 13) and 16 °C (n¼17). The increase was calculated by dividing the CL peak intensity of OZinduced peak CL with Z-induced peak CL of the same individual. The OZ/Z-induced CL ratio expresses the respiratory burst activity evoked by opsonin receptors relative to glucan receptors alone because the used blood dilution of 1/667 contained insignificant levels of opsonins. The high OZ/Z ratio in the 16 °C acclimation group at 30 °C was due to very low CL activities in Z-induced CL responses. Measuring temperature (°C)

5 10 16 20 24 27 30

OZ/Z –ratio in CL Acclimated to 5 °C

Acclimated to 16 °C

1.09 7 0.11 1.197 0.20 1.167 0.23 1.23 7 0.11** 2.197 0.76** 2.82 7 1.36*** 1.56 7 0.73*

1.317 0.24**## 1.86 7 0.44**### 1.59 7 0.59***# 1.917 0.64****### 1.29 7 0.31**## 2.83 7 1.58**** 10.63 7 3.93**####

The asterisks express statistical significance between Z- and OZ-induced CL intensities within the acclimation group (paired T-test): * ¼ Po 0.10, **¼ P o 0.05, ***¼ Po 0.01, **** ¼P o 0.001. The statistical significances between the acclimation groups (OZ/Z ratio in 5 °C acclim. vs. in 16 °C acclim. perch) were calculated using two-sample T-tests, and the results are expressed with: # ¼ Po 0.10, ## ¼ Po 0.05, ### ¼ Po 0.01, #### ¼P o0.001.

time point. In warm-acclimated fish CL activity further decreased after 7 h. Therefore we carried out the CL measurements during the first six hours after sampling when comparing the effects of temperature on Z and OZ induced CL. 3.1.2. Effects of temperature on serum opsonization activity Preincubation of pooled serum for 45 min at 5–35 °C did not substantially alter opsonic activity but sera preincubation at 40 or 45 °C had 46% and 22% lower opsonic activity, respectively. This decrease was reversed by the addition of 50 ml of fresh unheated serum to the 200-ml original sample volume. The heat sensitivity of the serum opsonization activity suggests that complement factors are involved in opsonization in perch. Preincubation at 45 °C is commonly used to inactivate complement in fish serum. 3.2. Effects of temperature and thermal acclimation 3.2.1. Leukocyte numbers in blood samples The mean total numbers of leukocytes were 2.95x103/ml (S. D.71.77) and 12.17x103/ml (S.D. 7 8.16) in 5 °C acclimated and 16 °C acclimated perch, respectively (P o0.05). 3.2.2. Effects of temperature and thermal acclimation on leukocyte CL response OZ-induced (opsonin recognition) CL responses per unit volume of blood in 16 °C acclimated perch were 4.6-fold higher on

Fig. 2. Effect of blood sample storage time on leukocyte CL peak activity at 24 °C. CL responses were induced with Z and OZ 45 min, 3 h, 5 h, 7 h and 9.5 h after blood sampling. Blood samples from 5 °C acclimated fish (n ¼3) were kept at 5 °C, and samples from 16 °C acclimated fish (n¼ 3) were kept at 16 °C.

average than 5 °C acclimated perch (P o0.0001, covariance model). Z-induced (pattern recognition) CL activities were approximately 3 times higher in 16 °C acclimated perch in 5–20 °C temperature areas and 6–9 times higher at 24 and 27 °C than in 5 °C acclimated perch (Fig. 3A and B) (P o0.001, covariance model). Thermal acclimation also caused a shift in temperature dependency of CL emission towards the acclimation temperatures (Fig. 4A and B). The shift was 3–4 °C in Z- and OZ-induced CL. Statistical significances for the overall effects of acclimation on the temperature responses for CL were Po0.0001 for Z-induced CL and Po0.053 for OZ-induced CL as calculated with the covariance model for repeated measurements (see Section 2.9). Z-induced CL in 5 °C acclimated perch increased in temperature ranges from 5 to 16 °C, and OZ-induced CL increased in the 5–24 °C range (Figs. 3A and 4A and B). In 16 °C acclimated perch the CL responses to Z and OZ increased in temperature ranges 5–20 °C and 5–27 °C, respectively (Figs. 3B and 4A and B). In Fig. 3A and B fish with high CL intensity dominated the pattern of temperature response curves. Therefore, relative temperature response curves are presented in Fig. 4A and B. There the highest observed CL values for Z and OZ of each individual perch were assigned a value of 100%, and other CL activities are shown as a percent of the highest value in the same individual. The CL reaction kinetics were faster in the 5 °C acclimated group. OZ-induced CL peaktimes at low temperatures (5–10 °C)

Table 2 Phagocyte CL reaction peaktimes in minutes ( 7 S.D.) from adding of Z or OZ. Perch were acclimated to 5 °C (n ¼13) and 16 °C (n¼ 17). Measurement temperature (°C)

5 10 16 20 24 27 30

Perch acclimated to 5 °C

Perch acclimated to 16 °C

Z

OZ

Z

OZ

103.3 7 8.2## 90.17 18.2 70.0 7 5.7#### 51.6 7 24.0## 42.4 7 9.5#### 35.17 14.7### 29.9 7 13.6

98.8 7 15.6 82.7 7 14.2 73.9 7 5.3** 59.3 7 1.7 61.8 7 8.5** 53.6 7 20.3* 22.17 11.7

121.9 7 14.1 103.7 7 13.8* 91.6 7 9.2**** 71.6 7 10.5*** 71.2 7 9.0** 58.4 7 18.8*** 44.2 7 21.8

117.2 7 18.4### 96.3 7 7.4## 81.8 7 14.5 61.4 7 12.1 60.7 7 2.9 41.3 7 10.2# 54.2 7 19.6###

Asterisks (*) indicate the statistical significance between Z- and OZ-induced CL within the acclimation group at different temperatures (paired T-test): * ¼ Po 0.10, ** ¼P o0.05, *** ¼P o0.01, **** ¼P o0.001. Statistical significancies between the acclimation groups (5 °C acclim. Z vs. 16 °C acclim. Z or 5 °C acclim. OZ vs. 16 °C acclim. OZ) were calculated using two-sample T-tests, and the results are expressed with # ¼P o0.10, ## ¼P o0.05, ### ¼P o 0.01, #### ¼ Po 0.001.

Please cite this article as: Marnila, P., Lilius, E.-M., Thermal acclimation in the perch (Perca fluviatilis L.) immunity. J. Thermal Biol. (2015), http://dx.doi.org/10.1016/j.jtherbio.2015.01.002i

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Fig. 3. The effects of in vitro assay temperatures on luminol-enhanced whole blood phagocyte CL peak activities. The CL response was induced with Z (filled symbol) and OZ (open symbol) in diluted (1/667) blood samples (A) from 5 °C acclimated perch (n ¼13) and (B) 16 °C acclimated perch (n¼ 17). The peak values of CL activities are plotted as the function of assay temperature. The error bars represent 7S.E.M. Asterisks indicate statistical significance (paired T-test) between Z- and OZ-induced CL peaks within the acclimation group as follows: o ¼P o0.10, n ¼ Po 0.05, nn ¼P o 0.01, nnn ¼ Po 0.001. The overall effects of temperature and acclimation on temperature response curve patterns were statistically significant in Z-induced CL (P o0.0001) and trend-setting in OZ-induced CL (Po 0.053). The covariance model used for these analyses is presented in detail in Section 2.9.

were shorter in 5 °C acclimated perch than 16 °C acclimated fish (Table 2). Peaktimes for Z-induced CL were substantially shorter in 5 °C acclimated perch (P o0.001, covariance model) at all temperatures (Table 2). 3.2.3. Effects of temperature and thermal acclimation on recognition

Fig. 4. Relative temperature response curves of (A) Z-induced CL peak activities and (B) OZ-induced CL peak activities. The highest observed CL values for Z and OZ of each individual perch were assigned value of 100%, and other CL activities were calculated as a percent of the highest value in the same individual to emphasize the effect of temperature on CL responses. In figure (A), filled circle¼Z-induced CL in perch acclimated to 5 °C (n¼ 13) and filled triangle ¼Z-induced CL in perch acclimated to 16 °C (n¼ 17). In figure (B), open circle¼ OZ-induced CL in perch acclimated to 5 °C (n¼13) and open triangle ¼ OZ-induced CL in perch acclimated to 16 °C (n ¼17). The error bars represent 7 S.E.M. Statistical analyses were not performed on this modified data.

of opsonization Temperature and thermal acclimation affected opsonin recognition. OZ was recognized better at higher temperatures in both acclimation groups (Fig. 3A and B). Serum opsonins on OZ particles did not significantly affect CL intensities in 5 °C acclimated perch at temperatures of 5–16 °C, but increased CL strongly at 24 and 27 °C (Table 1). Opsonins were recognized much weaker in 5 °C acclimated perch than 16 °C acclimated fish. The high OZ/Z

Please cite this article as: Marnila, P., Lilius, E.-M., Thermal acclimation in the perch (Perca fluviatilis L.) immunity. J. Thermal Biol. (2015), http://dx.doi.org/10.1016/j.jtherbio.2015.01.002i

P. Marnila, E.-M. Lilius / Journal of Thermal Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

-induced CL ratio in Table 1 in 5 °C acclimated fish at 24 and 27 °C is caused more by the inhibition of Z-induced CL by high temperature (Fig. 3A) than the increase in CL due to opsonins. The peaktimes of OZ-induced CL in 5 °C acclimated perch were not shorter than of Z-induced CL at any temperature. Opsonins on OZ particles increased CL responses significantly at all temperatures from 5 to 30 °C in 16 °C acclimated perch (Fig. 3B). Opsonins on OZ particles resulted in shorter peaktimes at temperatures 16–27 °C in 16 °C acclimated perch (Table 2) but, there was no statistically significant differences at 5 and 10 °C. The OZ-induced CL response was more thermotolerant than the Z-induced response in both acclimation groups.

4. Discussion CL curves measured from diluted whole perch blood are essentially similar to phagocytes of other fish species e.g., striped bass (Morone saxatilis) (Stave et al., 1983) and rainbow trout (Nikoskelainen et al., 2005, 2007; Kilpi et al., 2013). To our knowledge, perch leukocyte CL responses were not described previously in the literature. The magnitude of CL peak is considered to reflect efficacy of ingestion and killing of microbes by phagocytes and the peaktime reflect respiratory burst reaction velocity (Magrisso et al., 2000; Lilius and Nuutila, 2006; Atosuo and Lilius, 2011). Curves describing the temperature dependences of CL emission shifted 3–4 °C towards the acclimation temperatures (Fig. 4A and B). This shift may indicate a modification in respiratory burst performance over the normal temperature range of perch. We observed a same type of shift in phagocyte CL activity due to acclimation in the frog Rana temporaria (Marnila et al., 1995). In many studies adaptive responses in phagocytic cell functions due to acclimation were reported (Ainsworth et al., 1991; Hardie et al., 1994; Collazos et al., 1994, 1995b; Kurata et al., 1997; Nikoskelainen et al., 2004; Bowden et al., 2007). Phagocytes from sea bass (Dicentrarchus labrax) acclimated to 15 °C showed the highest respiratory burst response at the lowest experimental temperature of 5 °C (Angelidis et al., 1988). Collazos et al. (1994, 1995b) captured tench (Tinca tinca) in winter and summer when water temperatures were 12 °C and 30 °C, respectively, and measured phagocyte function at 12 °C, 22 °C and 33 °C. Tench phagocytes were more active in vitro in winter than summer, and phagocyte function was higher in winter at 12 °C and summer at 30 °C compared to 22 °C, which shows the strong effect of season on phagocyte function. However, in channel catfish (Ictalurus punctatus) many parameters of phagocyte function were not significantly affected by thermal acclimation, but respiratory burst in phagocytes adapted to low temperatures when the animals were acclimated to cold (Ainsworth et al., 1991; Dexiang and Ainsworth, 1991). In some studies the percentage of granulocytes and/or monocytes in blood or head kidney increased, and the proportion of lymphocytes decreased when fish were kept at low temperatures indicating the important role of phagocytes in immune defense at low temperatures (Kurata et al., 1997; Alcorn et al., 2002; Köllner and Kotterba, 2002; Engelsma et al., 2003). In present study opsonins were recognized better at high than low temperatures (Table 1, Fig. 3A and B). This result is consistent with our earlier results in rainbow trout (Nikoskelainen et al., 2004). Opsonin recognition in rainbow trout acclimated to 5 °C is substantially better at temperatures above 10 °C, but in perch acclimated to 5 °C this improvement was seen above 20 °C (Table 1, Fig. 3A). OZ is recognized better at higher temperatures than Z in both species. Efficient recognition and activation of phagocyte by opsonins require movement and clustering of receptors on cell membrane (cap formation). Our results suggest that opsonin receptors require more structural flexibility and perhaps cell

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membrane fluidity to function properly than Z-receptors alone. The effect of thermal acclimation on opsonin recognition in perch was the opposite of rainbow trout. Opsonin recognition in rainbow trout was improved by acclimation to low temperatures (5 and 10 °C). Serum opsonic activity was also higher in rainbow trout that were acclimated to 5 and 10 °C than 15- and 20 °C-acclimated trout (Nikoskelainen et al., 2004). Perch phagocytes recognized opsonins clearly better when fish were acclimated to 16 °C than 5 °C (Table 1). This difference is likely derived from the living habits of perch. Perch prefer approximately 10 °C higher temperatures in summer than rainbow trout (Johnson and Kelsch, 1998). An analog is reported in another receptor system, namely major histocompatibility complex (MHC). Low temperature (6 °C) causes a loss of MHC-receptor (beta2-microglobulin) expression (Rodrigues et al., 1998) in the common carp (Cyprinus carpio), which prefer warm water (Johnson and Kelsch, 1998), whereas the corresponding beta2-microglobulin transcript levels were not decreased at cold in rainbow trout and Atlantic salmon (Salmo salar) even after keeping for 10 days at þ 2 °C. Rainbow trout may utilize alternative modes of immune gene regulation than common carp or perch (Kales et al., 2006). Altered opsonin recognition is seen also in CL reaction kinetics (Table 2). Opsonins on OZ particles resulted in shorter peaktimes at temperatures 16–27 °C in 16 °C acclimated perch, which is similar to mammalian phagocytes at their normal body temperatures (Lilius and Marnila, 1992). However, the differences in CL peaktimes at low temperatures of 5 and 10 °C were not statistically significant in 16 °C acclimated perch, and the peaktimes of OZinduced CL in 5 °C acclimated perch were not shorter at any temperature. Thus opsonins on OZ particles had no significant effect on respiratory burst reaction velocity. These results are probably due to weaker opsonin recognition in warm-acclimated perch at low temperatures (Table 2) and cold-acclimated fish in general. Comparisons of CL peaktimes between the acclimation groups revealed faster reaction kinetics in 5 °C acclimated perch than 16 °C acclimated perch, especially at low temperatures (Table 2). This may indicate functional and structural adaptations to cold (possibly in receptor functions, signal transduction, degranulation and cell oxidative metabolism) resulting to more rapid cell response at low temperatures. The biological relevance of impaired opsonin recognition at low temperatures is probably the need to limit harmful immune reactions. What the defense cost the host has to pay. Immunity is always balancing with the need to protect the host against microbial pathogens and with hazards of and damages caused by autoimmune reactions and overactive phagocytes infiltrating into tissues and releasing cytokines and toxic reactive oxygen intermediates. The main role of opsonins is to accelerate recognition, ingestion and killing of pathogens. In cold water pathogenic microbes grow very slowly and produce harmful metabolites and toxins at low rate. At low temperatures rapid specific immune reactions could cause more damage e.g. by autoimmune reactions than benefit the host. In summer and autumn the burden caused by microbial pathogens is higher. Microbial growth is faster and their metabolic rate is higher. Thus, effective opsonin recognition is crucial for the rapid phagocyte functions to cope with pathogens. Unfortunately, we could not reliably differentiate leukocyte types in smear preparations. Therefore, we do not know the exact numbers of neutrophils and whether the mean 4.6-fold increase in OZ-induced CL activity per volume of blood (Fig. 3A and B) is due to changes in phagocyte numbers in blood. The mean total numbers of leukocytes were 2.95x103/ml and 12.17x103/ml in 5 °C acclimated and 16 °C acclimated fish, respectively. Therefore, the increase was 4-fold. The whole blood CL response to OZ in human is a measure of neutrophil numbers (Lilius and Nuutila, 2006). Our

Please cite this article as: Marnila, P., Lilius, E.-M., Thermal acclimation in the perch (Perca fluviatilis L.) immunity. J. Thermal Biol. (2015), http://dx.doi.org/10.1016/j.jtherbio.2015.01.002i

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P. Marnila, E.-M. Lilius / Journal of Thermal Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

results suggest that the same apply to perch; the increase in the total number of leukocytes is mostly due to phagocytes because other leukocytes do not emit CL. Leukocyte numbers in many fish species increase during spring and summer (Zapata et al., 1992). For example, the increase in chub (Leuciscus cephalus) was 20% (Lamková et al., 2007) and 50– 100% in sea bass (Dientrarchus labrax) (Pascoli et al., 2011). Vaccination temporally increased phagocyte counts 2–6 times higher in rainbow trout (Lundén et al., 1998; Köllner and Kotterba, 2002), which suggests that the approximately 4 -fold increase in phagocyte numbers in perch is physiologically achievable. However, whole blood CL for OZ (measured at 24 °C) did not change in Atlantic cod (Gadus morhua) when 10 °C-acclimated juveniles were subjected to a regimen of chronically elevating temperatures (1 °C every 5 days) (Pérez-Casanova et al., 2008). These fish experienced significant stress, which was seen as elevated total blood cortisol and glucose levels. High water temperatures in the summer provide good conditions for microbial growth, and it may also support viral infectivity. The increase in phagocyte numbers per unit volume of blood and the capability to recognize opsonins (Fig. 3A and B) may be adaptive responses to the increased pathogen burden that is encountered during the warm periods of the year. A fish pathologist inspected our perch, and only individuals regarded as healthy were included in this study. However, there were individuals with infection and injuries. These individuals showed an approximately 3-fold increase in total cell numbers and CL responses to OZ compared to 16 °C acclimated healthy perch (unpublished). Therefore, in the present study with healthy fish the higher number of phagocytes in 16 °C acclimated perch was due to acclimation and not due to infections or injuries. However, we cannot exclude any stress effects. The previous discussion related OZ-induced CL to phagocyte numbers. Approximately 100% of cells react to OZ with the respiratory burst. The CL response induced by Z in humans is dependent on the number of cells that react to Z with respiratory burst in the cell population. That proportion is approximately 30% of cells in the healthy humans (Lilius and Nuutila, 2006). Notably, the increase in Z-induced CL at measuring temperatures higher than 20 °C (6-9-fold) in warm-acclimated perch was larger than the increase in cell number (4.1-fold) (Figs. 3A and B). This result suggests that the function of Z receptors improved at higher measuring temperatures in warm-acclimated perch. Perch in nature live in a wide temperature gradient in summer and autumn. The phagocyte activation enhanced progressively at temperatures from 5 to 20 °C (Fig. 4A and B), which is the ecological temperature range for perch in summer. Several poikilothermic animals respond to bacterial compounds from pathogens by behavioral fever i.e., by selecting higher environmental temperatures in a thermal gradient. This behavior was also reported in fish (Reynolds et al., 1976; Reynolds, 1977; Nagai and Iriki, 1978; Cabanac and Laberge, 1998; Gräns et al., 2012). Behavioral fever enhance survival in some species of fish, reptiles, and invertebrates during bacterial infection (Covert and Reynolds, 1977; Kluger, 1979; Kluger and Rothenburg, 1979; Louis et al., 1986; Boltaña et al., 2013). The present results suggest that in cold water opsonin recognition is impaired probably due to the need to limit adverse effects of excess immune reactions. The migration of perch to warmer water where pathogen burden may be high i.e., near the surface and waterside, would improve perch’s ability to cope with pathogenic microbes via an increase in phagocyte activity, improved opsonin recognition and glucan receptor function. These results with perch phagocytes are consistent with the hypothesis that fish are more dependent on innate immune responses than specific acquired immune functions at cold water temperatures.

Acknowledgments This work was supported by the Academy of Finland and the Foundation of Emil Aaltonen. The authors thank Dr. Hillevi Niiranen (Åbo Akademi, Turku) and biostatistician Christian Eriksson (MTT, Agrifood Research Finland), Prof. Kari Lagerspetz, Dr. Ari Tiiska, Dr. Tapani Juusti and Dr. Jari Hänninen (Univ. of Turku), and Dr. Ilpo Hakala (Lammi, Univ. of Helsinki) for their expertize and assistance.

References Ainsworth, A.J., 1994. A beta-glucan inhibitable zymosan receptor on channel catfish neutrophils. Vet. Immunol. Immunopathol. 41 (1–2), 141–152. Ainsworth, A.J., Dexiang, C., Waterstrat, P.R., Greenway, T., 1991. Effect of temperature on the immune system of channel catfish (Ictalurus punctatus) - I. Leucocyte distribution and phagocyte function in the anterior kidney at 10 °C. Comp. Biochem. Physiol. 100A, 907–912. Alcorn, S.W., Murra, A.L., Pascho, R.J., 2002. Effects of rearing temperature on immune functions in sockeye salmon (Oncorhynchus nerka). Fish Shellfish Immunol. 12 (4), 303–334. Angelidis, P., Baudin-Laurencin, F., Youinou, P., 1988. Effects of temperature on chemiluminescence of phagocytes from sea bass, Dicentrarchus labrax L. J. Fish Dis. 11, 281–288. Atosuo, J.T., Lilius, E.-M., 2011. The real-time-based assessment of the microbial killing by the antimicrobial compounds of neutrophils. Sci. World J. 11, 2382–2390. http://dx.doi.org/10.1100/2011/376278. Bly, J.E., Clem, L.W., 1991. Temperature mediated processes in teleost immunity: in vitro immunosuppression induced by in vivo low temperature in channel catfish. Vet. Immunol. Immunopathol. 28 (3–4), 365–377. Bly, J.E., Clem, W., 1992. Temperature and teleost immunity. Fish Shellfish Immunol. 2, 159–171. Boltaña, S., Rey, S., Roher, N., Vargas, R., Huerta, M., Huntingford, F.A., Goetz, F.W., Moore, J., Garcia-Valtanen, P., Estepa, A., Mackenzie, S., 2013. Behavioural fever is a synergic signal amplifying the innate immune response. Proc. Biol. Sci. 280 (1766), 20131381. http://dx.doi.org/10.1098/rspb.2013.1381. Boshra, H., Li, J., Sunyer, J.H., 2006. Recent advances on the complement system of teleost fish. Fish Shellfish Immunol. 20 (2), 239–262. Bowden, T.J., Thompson, K.D., Morgan, A.L., Gratacap, R.M., Nikoskelainen, S., 2007. Seasonal variation and the immune response: a fish perspective. Fish Shellfish Immunol. 22 (6), 695–706. Cabanac, M., Laberge, F., 1998. Fever in goldfish is induced by pyrogens but not by handling. Physiol. Behav. 63 (3), 377–379. Collazos, M.E., Barriga, C., Ortega, E., 1994. Enhanced granulocyte phagocytosis at low winter temperature and high summer temperature in the tench (Tinca tinca L.). Comp. Biochem. Physiol. 109A, 643–648. Collazos, M.E., Ortega, E., Barriga, C., 1995a. Influence of the temperature upon the proliferative response of lymphocytes of tench (Tinca tinca) during winter and summer. Comp. Immunol. Microbiol. Infect. Dis. 18 (3), 209–214. Collazos, M.E., Barriga, C., Ortega, E., 1995b. Seasonal variations in the immune system of the cyprinid Tinca tinca. P hagocytic function. Comp. Immunol. Microbiol. Infect. Dis. 18 (2), 105–113. Covert, J.B., Reynolds, W.W., 1977. Survival value of fever in fish. Nature 267 (5606), 43–45. Couso, N., Castro, R., Noya, M., Obach, A., Lamas, J., 2001. Location of superoxide production sites in turbot neutrophils and gilthead seabream acidophilic granulocytes during phagocytosis of glucan particles. Dev. Comp. Immunol. 25 (7), 607–618. Dexiang, C., Ainsworth, A.J., 1991. Effect of temperature on the immune system of channel catfish (Ictalurus punctatus) - II. Adaptation of anterior kidney phagocytes to 10 °C. Comp. Biochem. Physiol. 100A, 913–918. Dzik, J.M., 2010. The ancestry and cumulative evolution of immune reactions. Acta Biochim. Pol. 57 (4), 443–466. Engelsma, M.Y., Hougee, S., Nap, D., Hofenk, M., Rombout, J.H., van Muiswinkel, W. B., Lidy Verburg-van Kemenade, B.M., 2003. Multiple acute temperature stress affects leucocyte populations and antibody responses in common carp, Cyprinus carpio L. Fish Shellfish Immunol. 15 (5), 397–410. Esteban, M.A., Rodriguez, A., Meseguer, J., 2004. Glucan receptor but not mannose receptor is involved in the phagocytosis of Saccharomyces cerevisiae by seabream (Sparus aurata L.) blood leucocytes. Fish Shellfish Immunol. 16 (3), 447–451. Förlin, L., Andersson, T., Balk, L., Larsson, A., 1995. Biochemical and physiological effects in fish exposed to bleached kraft mill effluents. Ecotoxicol. Environ. Saf. 30 (2), 164–170. Glasser, L., Fiederlein, R.L., 1990. The effect of various cell separation procedures on assays of neutrophil function. A critical appraisal. Am. J. Clin. Pathol. 93, 662–669. Grayfer, L., Hodgkinson, J.W., Belosevic, M., 2014. Antimicrobial responses of teleost phagocytes and innate immune evasion strategies of intracellular bacteria. Dev. Comp. Immunol. 43 (2), 223–242. http://dx.doi.org/10.1016/j.dci.2013.08.003. Gräns, A., Rosengren, M., Niklasson, L., Axelsson, M., 2012. Behavioural fever boosts

Please cite this article as: Marnila, P., Lilius, E.-M., Thermal acclimation in the perch (Perca fluviatilis L.) immunity. J. Thermal Biol. (2015), http://dx.doi.org/10.1016/j.jtherbio.2015.01.002i

P. Marnila, E.-M. Lilius / Journal of Thermal Biology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ the inflammatory response in rainbow trout Oncorhynchus mykiss. J. Fish Biol. 81 (3), 1111–1117. http://dx.doi.org/10.1111/j.1095-8649.2012.03333.x. Hardie, L.J., Fletcher, T.C., Secombes, C.J., 1994. Effect of temperature on macrophage activation and the production of macrophage activating factor by rainbow trout (Oncorhynchus mykiss) leucocytes. Dev. Comp. Immunol. 18 (1), 57–66. Johnson, J.A., Kelsch, S.W., 1998. Effects of evolutionary thermal environment on temperature–preference relationships in fishes. Environ. Biol. Fish 53, 447–458. Kales, S., Parks-Dely, J., Schulte, P., Dixon, B., 2006. Beta-2-microglobulin gene expression is maintained in rainbow trout and Atlantic salmon kept at low temperatures. Fish Shellfish Immunol. 21 (2), 176–186. Kluger, M.J., 1979. Fever in echtotherms: evolutionary implications. Am. Zool. 19, 295–304. Kluger, M.J., Rothenburg, B.,A., 1979. Fever and reduced iron: their interaction as a host defence response to bacterial infection. Science 203, 374–376. Kilpi, M.K., Atosuo, J.T., Lilius, E.-M., 2009. Bacteriolytic activity of the alternative pathway of complement differs kinetically from the classical pathway. Dev. Comp. Immunol. 33 (10), 1102–1110. http://dx.doi.org/10.1016/j. dci.2009.06.007. Kilpi, M., Nikoskelainen, S., Grannas, S., Nuutila, J., Järvisalo, O., Kause, A., Lilius, E.M., 2013. Resistance to bacterial infectious diseases in rainbow trout (Oncorhynchus mykiss). Vet. Immunol. Immunopathol. 153 (3–4), 267–278. http://dx. doi.org/10.1016/j.vetimm.2013.03.010. Kurata, O., Okamoto, N., Ikeda, Y., 1997. Adaptability of carp neutrophilic granulocytes to environmental temperatures: spontaneous cytotoxic activity and celladherent activity. Fish Shellfish Immunol. 7, 585–593. Köllner, B., Kotterba, G., 2002. Temperature dependent activation of leucocyte populations of rainbow trout, Oncorhynchus mykiss, after intraperitoneal immunisation with Aeromonas salmonicida. Fish Shellfish Immunol. 12 (1), 35–48. Lagerspetz, K.Y.H., 1977. Interactions of season and temperature acclimation in the control of metabolism in Amphibia. J. Therm. Biol. 2, 223–231. Lamková, K., Simková, A., Palíková, M., Jurajda, P., Lojek, A., 2007. Seasonal changes of immunocompetence and parasitism in chub (Leuciscus cephalus), a freshwater cyprinid fish. Parasitol. Res. 101 (3), 775–789. Le Morvan-Rocher, C., Troutaud, D., Deschaux, P., 1995. Effects of temperature on carp leukocyte mitogen-induced proliferation and nonspecific cytotoxic activity. Dev. Comp. Immunol. 19 (1), 87–95. Le Morvan, C., Troutaud, D., Deschaux, P., 1998. Differential effects of temperature on specific and nonspecific immune defences in fish. J. Exp. Biol. 201 (Pt2), 165–168. Lilius, E.-M., Marnila, P., 1992. Photon emission of phagocytes in relation to stress and disease. Experientia 48 (11–12), 1082–1091. Lilius, E.-M., Nuutila, J., 1993. Kinetics of luminol-amplified chemiluminescence in relation to phagocytosis of zymosan and its opsonized varieties in human neutrophils. In: Szalay, A., Kricka, L., Stanley, P. (Eds.), Bioluminescence and Chemiluminescence. Status Report, 1993. John Wiley & Sons, Chichester, pp. 465–469. Lilius, E.-M., Nuutila, J.T., 2006. Particle-induced myeloperoxidase release in serially diluted whole blood quantifies the number and the phagocytic activity of blood neutrophils and opsonization capacity of plasma. Luminescence 21 (3), 148–158. Louis, C., Jourdan, M., Cabanac, M., 1986. Behavioral fever and therapy in a rickettsia infected orthoptera. Am. J. Physiol. 250, R991–R995. Lundén, T., Miettinen, S., Lönnström, L.-G., Lilius, E.-M., Bylund, G., 1998. Influence of oxytetracycline and oxolinic acid on the immune response of rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 8, 217–230. Magnadóttir, B., Jonsdóttir, H., Helgason, S., Bjornsson, B., Jorgensen, T.O., Pilström, L., 1999. Humoral immune parameters in Atlantic cod (Gadus morhua L.) I. The effects of environmental temperature. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 122B (2), 173–180. Magrisso, M.Y., Alexandrova, M.L., Markova, V.I., Bechev, B.G., Bochev, P.G., 2000. Functional states of polymorphonuclear leukocytes determined by chemiluminescent kinetic analysis. Luminescence 15 (3), 143–151. Marnila, P., Tiiska, A., Lagerspetz, K., Lilius, E.-M., 1995. Phagocyte activity in the frog Rana temporaria: whole blood chemiluminescence method and the effects of temperature and thermal acclimation. Comp. Biochem. Physiol. 111A (4), 609–614. Nagai, M., Iriki, M., 1978. Autonomic response of the fish to pyrogen. Experientia 34 (9), 1177–1178. Nakao, M., Miura, C., Itoh, S., Nakahara, M., Okumura, K., Mutsuro, J., Yano, T., 2004.

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A complement C3 fragment equivalent to mammalian C3d from the common carp (Cyprinos carpio): generation in serum after activation of the alternative pathway and detection of its receptor on the lymphocyte surface. Fish Shellfish Immunol. 16, 139–149. Nikoskelainen, S., Bylund, G., Lilius, E.-M., 2004. Effect of environmental temperature on rainbow trout (Oncorhynchus mykiss) innate immunity. Dev. Comp. Immunol. 28 (6), 581–592. Nikoskelainen, S., Verho, S., Airas, K., Lilius, E.-M., 2005. Adhesion and ingestion activities of fish phagocytes induced by bacterium Aeromonas salmonicida can be distinguished and directly measured from highly diluted whole blood of fish. Dev. Comp. Immunol. 29 (6), 525–537. Nikoskelainen, S., Verho, S., Järvinen, S., Madetoja, J., Wiklund, T., Lilius, E.-M., 2007. Multiple whole bacterial antigens in polyvalent vaccine may result in inhibition of specific responses in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 22 (3), 206–217. Ottinger, C.A., Johnson, S.C., Ewart, K.V., Brown, L.L., Ross, N.W., 1999. Enhancement of anti- Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by a mannose-binding lectin. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 123C (1), 53–59. Pascoli, F., Lanzano, G.S., Negrato, E., Poltronieri, C., Trocino, A., Radaelli, G., Bertotto, D., 2011. Seasonal effects on hematological and innate immune parameters in sea bass Dicentrarchus labrax. Fish Shellfish Immunol. 31 (6), 1081–1087. http: //dx.doi.org/10.1016/j.fsi.2011.09.014. Pérez-Casanova, J.C., Rise, M.L., Dixon, B., Afonso, L.O., Hall, J.R., Johnson, S.C., Gamperl, A.K., 2008. The immune and stress responses of Atlantic cod to longterm increases in water temperature. Fish Shellfish Immunol. 24 (5), 600–609. http://dx.doi.org/10.1016/j.fsi.2008.01.012. Reynolds, W.W., Casterlin, M.E., Covert, J.B., 1976. Behavioural fever in teleost fishes. Nature 259 (5538), 41–42. http://dx.doi.org/10.1038/259041a0. Reynolds, W.W., 1977. Fever and antipyresis in the bluegill sunfish Lepomis macrochirus. Comp. Biochem. Physiol. C 57 (2), 165–167. Rodrigues, P.N.S., Dixon, B., Roelofs, J., Rombout, J.H.M.W., Egberts, E., Pohajdak, B., Stet, R.J.M., 1998. Expression and temperature-dependent regulation of the beta2-microglobulin (Cyca-B2m) gene in a cold blooded vertebrate, the common carp (Cyprinos carpio L.). . Dev. Immunol. 5, 263–275. Secombes, C.J., Fletcher, T.C., 1992. The role of phagocytes in the protective mechanisms of fish. Annu. Rev. Fish. Dis., 53–71. Stafford, J.L., Bengten, E., Du Pasquier, L., McIntosh, R.D., Quiniou, S.M., Clem, L.W., Miller, N.W., Wilson, M., 2006a. A novel family of diversified immunomodulatory receptors in teleosts is homologous to both mammalian Fc receptors and molecules encoded within the leukocyte receptor complex. Immunogenetics 58, 758–773. Stafford, J.L., Wilson, M., Nayak, D., Quiniou, S.M., Clem, L.W., Miller, N.W., Bengten, E., 2006b. Identification and characterization of a FcR homolog in an ectothermic vertebrate, the channel catfish (Ictalurus punctatus). J. Immunol. 177 (4), 2505–2517. Stave, J.W., Roberson, B.S., Hetrick, F.M., 1983. Chemiluminescence of phagocytic cells isolated from the pronephros of striped bass. Dev. Comp. Immunol. 7 (2), 269–276. Stave, J.W., Roberson, B.S., Hetrick, F.M., 1984. Factors affecting the chemiluminscent response of fish phagocytes. J. Fish Biol. 25 (2), 197–206. Sypek, J.P., Burreson, E.M., 1983. Influence of temperature on the immune response of juvenile summer flounder, Paralichthys dentatus, and its role in the elimination of Trypanoplasma bullocki infections. Dev. Comp. Immunol. 7 (2), 277–286. Talo, A., Tirri, R., 1991. Temperature acclimation of the perch (Perca fluviatilis L.): changes in duration of cardiac action potential. J. Therm. Biol. 16 (2), 89–92. Tennenberg, S.D., Zemlan, F.P., Solomkin, J.S., 1988. Characterization of N-formylmethionyl-leucyl-phenylalanine receptors on human neutrophils. Effects of isolation and temperature on receptor expression and functional activity. J. Immunol. 141, 3937–3944. Verho, S., Järvinen, S., Nikoskelainen, S., Lilius, E.-M., 2005. Biological effect of vaccination can be assessed directly from diluted whole blood of rainbow trout using homologous blood phagocytes as immunosensors. Fish Shellfish Immunol. 19 (2), 175–183. Zapata, A.G., Varas, A., Torroba, M., 1992. Seasonal variations in the immune system of lower vertebrates. Immunol. Today 13, 142–147.

Please cite this article as: Marnila, P., Lilius, E.-M., Thermal acclimation in the perch (Perca fluviatilis L.) immunity. J. Thermal Biol. (2015), http://dx.doi.org/10.1016/j.jtherbio.2015.01.002i