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The effect of seasonality on normal haematological and innate immune parameters of rainbow trout Oncorhynchus mykiss L.
Fish & Shellfish Immunology (2008) 25, 791e799
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The effect of seasonality on normal haematological and innate immune parameters of rainbow trout Oncorhynchus mykiss L. A.L. Morgan*, K.D. Thompson, N.A. Auchinachie, H. Migaud Institute of Aquaculture, University of Stirling, Stirling, Scotland FK9 4LA, United Kingdom Received 28 November 2007; revised 27 May 2008; accepted 28 May 2008 Available online 3 June 2008
KEYWORDS Season; Innate immunity; Respiratory burst; Lysozyme; Blood cell counts; Melatonin; Oncorhynchus mykiss
Abstract It is well established that seasonality dominates the life history of fish by controlling the timing of physiological events such as reproduction, food intake, locomotor activity and growth performance. Seasonal differences in immune competence and prevalence of disease have been well documented in humans. The aim of this study was to determine if season influences the immune response of rainbow trout (Oncorhynchus mykiss L.). Thus, a 12-month trial was carried out in which plasma lysozyme activity and respiratory burst of head kidney macrophages (two innate immune parameters) and total red and white blood cell counts (two haematological parameters) were monitored at monthly intervals. Since photoperiodic information is thought to be conveyed via melatonin secretion, plasma melatonin levels were also measured at four seasonal points (day and night). A general seasonal influence was observed in the parameters measured in these fish, with the exception of respiratory burst activity of head kidney macrophages, with the parameters highest in summer and lowest in winter for total white blood cell counts and lysozyme activity. ª 2008 Published by Elsevier Ltd.
Introduction During an annual cycle, the temperate aquatic environment will be affected by two primary components of season, i.e. temperature and photoperiod. These components are interlinked and follow very similar cycles. Typically during the spring, water temperatures and light periods increase while the reverse occurs in autumn. This seasonality affects the
* Corresponding author. Tel.: þ44 1786 467881; fax: þ44 1786 472133. E-mail address: [email protected] (A.L. Morgan). 1050-4648/$ - see front matter ª 2008 Published by Elsevier Ltd. doi:10.1016/j.fsi.2008.05.011
life history of fish with regard to the timing of developmental and maturational events synchronized with seasonal changes in temperature, day length and food supplies [1]. It is well documented that seasonality also affects the immune response of vertebrates [2,3]. Seasonal differences in immune competence and prevalence of disease have been well documented in humans [4,5]. Adaptive immunity in fish was shown to exhibit a seasonal cycle over a 12-month period, in particular, changes in resting antibody titre and response to antigenic challenge [6]. Other studies have shown there to be seasonal changes, in particular in the lymphoid system [7,8] and in the numbers of circulating lymphocytes [9]. Innate immunological indicators have
792 been shown to be affected by low temperatures in gilthead sea bream (Sparus aurata L.). Affected fish showed severe immunosuppression involving significant decrease of serum complement activity, decrease of plasma lysozyme activity and reduction in circulating lymphocytes [10]. It had generally been assumed that the innate immune response does not exhibit this seasonal cycle but remains constant throughout the year, always providing a defence to invading pathogens [11]. Many environmental challenges are recurrent and thus predictable [3]. Animals could enhance their survival and presumably increase their fitness, if they are able to anticipate immunologically challenging conditions in order to cope with these seasonal threats to health [12]. A potential mechanism to anticipate changes in season may be transmitted through the pineal hormone melatonin. It exhibits a strong circadian rhythm as the majority of the hormone is produced during the dark phase. Consequently, its production is affected by the seasonality of photoperiod. During the winter months, when the dark phase is at its longest, melatonin is produced for a greater length of time compared to the shorter dark phase of summer days in vertebrates [13]. In addition, melatonin production is affected by water temperature with higher levels reported in Atlantic salmon (Salmo salar L.) maintained at 12 C compared to fish maintained at 4 C [14]. The innate immune system is a fundamental defence mechanism of fish [15]. The primary aim of the current study was to determine if the innate immunity of rainbow trout is affected by seasonality. This was achieved by measuring plasma lysozyme activity and respiratory burst of head kidney macrophages (two innate immune parameters) and total red and white blood cell counts (two haematological parameters) each month over a 12-month period. The study was carried out under ambient temperature and photoperiod.
Materials and methods Fish husbandry The trial was performed at the Niall Bromage Freshwater Research Facility (NBFRF), Institute of Aquaculture (IOA), University of Stirling. Only female fish, obtained from a commercial fish farm on the Isle of Man, were used to prevent precocious males with a potentially compromised immune system skewing the results. Three tanks (B 2 m 1 m) were set up with flow through water supplied from a reservoir situated 1 km from the facility. The flow rate was approximately 2 l/s at an ambient seasonal temperature. Light was supplied by 60 W pearl, tungsten filament light bulbs (Skye Instruments, Powys, UK) housed within waterproof lamps providing an intensity of 17e19 lx at the water surface. The photoperiods were controlled by 24 h digital electronic time switches and were set to simulate the natural photoperiod by a light sensor located on the outside of the building. Fish were fed a standard commercial pelleted diet (EWOS, Livingston, UK), according to the manufacturer’s guidelines. One hundred fish (initial average weight 20.5 g) were placed in each of the three tanks at the start of the
A.L. Morgan et al. experiment (July) and five fish were sampled monthly from each tank.
General experimental procedures Collection of plasma Blood was withdrawn monthly from fish killed by an overdose of anaesthetic (2-phenoxyethanol, SigmaeAldrich Co., UK) followed by a blow to the back of the head. Fish were bled by caudal venepuncture using 1 ml or 2 ml syringes and a 25 G or 23 G needle (Terumo Europe N.V., Belgium) depending on fish size. Prior to sampling, syringes were rinsed with heparin (4 mg ml1, SigmaeAldrich Co., UK). The blood was stored on ice and transported to the laboratory at the IOA, where blood counts were carried out prior to centrifuging the blood at 3000 g for 10 min at 4 C. Plasma was collected and stored at 70 C until analysed for lysozyme activity. Blood was also sampled at 3-month intervals for the analysis of plasma melatonin levels. Samples were taken at midday and 2 h after the onset of darkness by removing the fish from the tank under total darkness and placing them in an anaesthetic bath of a 1:20,000 concentration of 2-phenoxyethanol for 2 min. Once anaesthetised, the fish were bled under a dim red light (l Z 670e800 nm, 0.2 lx at 0.5 m). The samples were transported back to the laboratory on ice and plasma collected as above. Condition factor Whilst under anaesthesia, weight and length measurements were taken from which condition factors were calculated [Condition Factor Z (weight 100)/length3]. Total blood cell counts To determine the total white blood cell count (WBC), a 1 in 100 dilution of the blood was made in phosphate saline buffer (PBS, 0.02 M, pH 7.3). While a 1 in 1000 dilution was made to count the red blood cells (RBC). Counts were carried out using a Neubauer haemocytometer (Hawksley & Son, England) and were expressed as cells ml1. Plasma lysozyme activity Lysozyme activity in plasma was measured turbidimetrically according to Refs. [16,17]. Lyophilised Micrococcus lysodeikticus was added to a 0.04 M sodium phosphate buffer (pH 5.8) at a concentration of 0.2 mg bacteria ml1 and this was then incubated at 25 C for 20 min. Ten microlitres of plasma was added to five replicate wells of a 96 multiwell plate (Nunc, Denmark). One hundred and ninety microlitres of the bacterial suspension was added to all wells apart from the control wells, to which was added 200 ml of sodium phosphate buffer. Absorbance was read at 540 nm, 1 min after adding the buffer and then again after 5 min using a Dynex MRX II plate reader. Lysozyme activity was expressed as the amount of sample causing a decrease in absorbance of 0.001 min1. Units used were units min1 ml1. Respiratory burst activity of head kidney macrophages Macrophage suspensions were prepared from sampled fish according to Ref. [18]. Under sterile conditions the kidney
The effect of seasonality on normal haematological and innate immune parameters of Oncorhynchus mykiss was teased through sterile 100 mm mesh into 5 ml of Leibovitz (L-15, SigmaeAldrich, UK) medium containing 10 ml heparin into a 30 mm diameter Petri dish and the mesh was then rinsed with 1 ml of L-15. The macrophage suspension was briefly placed on ice prior to performing the respiratory burst assay, which was carried out as described by Secombes [19] and adapted by Burrells et al. [20]. One hundred microlitres of the macrophage suspension was aliquoted into eight replicate wells of a sterile 96-well microtitre plate (Nunc, Denmark) and left for 1 h at 21 C to allow the cells to adhere to the plate. Plates were then washed three times with L-15 medium to wash off non-adherent cells. To the first set of three replicate wells, 100 ml of 1 mg ml1 nitrobluetetrazolium (NBT, Sigmae Aldrich Co., UK) in L-15 was added and to the next three replicate wells 100 ml of NBT solution containing 1 ml ml1 of phorbol myristate acetate (PMA, SigmaeAldrich Co., UK) was added. To the remaining two wells, 100 ml of lysis buffer (citric acid, 0.1 M; Tween 20, 1.0% (v/v); crystal violet, 0.05% (w/v), SigmaeAldrich Co.; prepared in distilled water) was added. Plates were incubated at 21 C for 1 h. The contents of the first six replicate wells were removed and the plates washed three times with L-15 medium. One hundred microlitres of methanol (100% v/v) was added to the wells for 5 min to stop the reaction. The wells were then washed three times with 70% (v/v) methanol and left to air-dry (a minimum of 30 min). One hundred and twenty microlitres of dimethyl sulphoxide (DMSO, SigmaeAldrich Co., UK) and 140 ml of 2 M potassium hydroxide (KOH, BDH, UK) were added to each of the washed wells. The absorbance was then read at 610 nm using an ELISA plate reader. The average numbers of adherent cells in the wells containing lysis buffer was determined by counting the cells using a Neubauer haemocytometer. Plasma melatonin Plasma melatonin levels were measured using a melatonin radio-immunoassay (RIA) method according to Randall [21], using sheep melatonin antiserum (Stockgrand Ltd, Guilford, Surrey), and [O-methyl-3H]melatonin (Amersham International, Bucks). Plasma melatonin levels in samples (250 ml) were determined from a standard curve (3.9e500 pg/tube).
Statistical analysis Before analysis, data were found to be normally distributed and homogenous without transformation. This test was performed using the appropriate function on the Minitab statistical Package (V. 10). Data were analysed (P < 0.05) using an ANOVA general linear model, Tukey pairwise comparison tests (post-hoc) and Pearson’s correlation coefficient all using Minitab (V. 10).
Results Weight and condition factor The average weight of the fish at the start of the experiment was 20.5 g and 248.7 g at the end of the trial 12 months later (Fig. 1a). The condition factor improved from 1.4 to 1.2 during this time (Fig. 1b).
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White blood cell counts There was a general increase in white blood cell counts during the spring months of the experiment peaking in July (start of the trial) and June (end of the trial) (Fig. 2a). Total white blood cell numbers decreased during the autumn and winter months to their lowest in March. Using an ANOVA (GLM) a significant effect by month was found on white blood cell counts (P Z 0.000). Using a Tukey post-hoc, pairwise comparison test, no significant differences were observed between the summer months of May, June and July (P > 0.05). There was also no significant difference in white blood cell numbers between the winter months of November, December and January. However, total white blood cell counts in the winter months were significantly lower than those measured during the summer months (P Z 0.000).
Red blood cell counts Red blood cell counts peaked during July (start of the trial) and June (end of the trial); although very little difference was observed between the other sampling months (Fig. 2b). A significant effect between month was observed (P Z 0.000). Tukey post-hoc, pairwise comparison tests established that the red blood cell counts obtained in July were not significantly different from those taken in October or April (P > 0.05), but were significantly lower than those taken in June (P Z 0.000) and significantly higher than those measured during the remaining sampling months (P > 0.05).
Lysozyme activity Plasma lysozyme activity (Fig. 3a) decreased from October to April and increased from May to July, indicating some seasonal influence on plasma lysozyme activity. The lowest levels of activity were recorded in April. A significant effect on plasma lysozyme activity was found by month (P Z 0.000). The level of plasma lysozyme activity in September was not significantly different from that of August, October or June (P > 0.05), but was significantly higher than the other months. Plasma lysozyme activity measured in March and April was significantly lower than those measured in August, September and June (P < 0.05).
Respiratory burst activity No clear seasonal pattern was observed in respiratory burst activity when measured over the 12-month period (Fig. 3b). The activity measured was greatest during February and lowest during the January. The pairwise comparisons further revealed that respiratory burst activity levels recorded in February were significantly greater than all other months (P Z 0.000) except July and August (P > 0.05). Respiratory burst activity in August was significantly greater than all months except October, February, and March (P < 0.05). There was no significant difference between March, April, May and June (P > 0.05).
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Figure 1
A.L. Morgan et al.
Variations in weight (a) and condition factor (b) of rainbow trout over a 12-month period (n Z 15; mean SE).
Plasma melatonin levels A significant difference was observed between night and daytime plasma melatonin levels (P Z 0.000) (Fig. 4). Melatonin levels were highest at night throughout the trial period. There was a significant difference between the summer melatonin levels of July and May and those measured during the winter months of November and January for both day and night samplings (P Z 0.000).
Discussion A general seasonal effect was observed for total white blood cell counts, lysozyme activity and red blood cell counts. White blood cell counts increased steadily during the spring months peaking in July and June. They then decreased during the autumn and winter months to their lowest level in March. This corroborates the work of previous studies in which white blood cell counts were
shown to exhibit a seasonal rhythm [9]. It was observed in this trial that total white blood cell counts increased during spring, peaked in the summer and then decreased in number in autumn to their lowest levels during winter. This coincides with the seasonal increase in water temperature and day length. Collazos et al. [22] reported that leucocyte counts for both male and female tench Tinca tinca L. were significantly lower in spring and winter when compared with summer and autumn, and this has since been corroborated by De Pedro et al. [23]. Although the pattern of leucocyte counts reported in these studies differed slightly to those reported here, all three studies corroborate the hypothesis of Slater and Schreck [9] ‘‘In general, all immune parameters are suppressed in winter and highest in summer’’. A slight seasonal effect was also observed for red blood cell counts, with the lowest values recorded in the winter months of November, December and January, whereas the highest red blood cell counts were recorded during July (trial start) and June (trial end). An indirect effect of lower
The effect of seasonality on normal haematological and innate immune parameters of Oncorhynchus mykiss
Figure 2
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Variations in white (a) and red (b) blood cell counts in rainbow trout over a 12-month period (n Z 15; mean SE).
water temperature is the higher solubility of oxygen in cold water compared to warm water. Consequently, fewer red blood cells are required to carry oxygen around the body of the fish in colder weather as the oxygen is more readily available [24]. This may explain why higher levels of red blood cells were recorded in June and July. Likewise, blood becomes more viscous (thicker and harder to pump) at lower temperatures. To solve this problem, Antarctic fishes have fewer red blood cells [25] and this may also account for why fewer red blood cells were observed in the rainbow trout from this study during the winter months. However, in the warmer summer months of July and June the reduced availability of oxygen means that more red blood cells are required to absorb available oxygen. Furthermore, winter is a period of reduced activity and therefore less energy is expended, i.e. a reduced metabolism, thus less oxygen is required. This may be another reason for the observed seasonality of red blood cell counts. Plasma lysozyme exhibited a seasonal pattern of activity during the 12-month period. The pattern is very similar to that of the white blood cell counts in that activity increased
in spring peaking in late summer and then decreased over autumn to its lowest level in late winter. This has also been shown in plaice (Pleuronectes platessa L.) [26], sea trout (Salmo trutta L.) [27], dab (Limanda limanda L.) [28], Asian catfish (Clarias batrachus L.) [29] and Atlantic halibut (Hippoglossus hippoglossus L.) [30,31] in which a generally consistent seasonal trend in plasma lysozyme activity was observed, with low values being associated with winter. However, this correlation is not always observed. When lysozyme activity was measured in Nile tilapia raised at four different temperatures (18.4 C, 23 C, 28 C and 33 C), it was observed that fish cultured at 33 C for 4 weeks exhibited a decrease in lysozyme activity, although this variation was still higher than in the control fish [32]. It was consequently suggested that temperature had a limited affect on lysozyme activity in the Nile tilapia [32]. Although significant differences were recorded in respiratory burst activity (P < 0.05) according to month, these could not really be said to be seasonal. The highest levels of respiratory burst activity occurred in August and February and the lowest in January. This is corroborated by
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Figure 3 Variations in plasma lysozyme (a) and respiratory burst (b) activity in rainbow trout over a 12-month period (n Z 15; mean SE).
a seasonality study on croaker (Micropogonias furnieri) which also suggested that respiratory burst was not affected by season [33]. However, a temperature effect on respiratory burst has been reported in rainbow trout, with higher levels of activity reported with increased temperature [34]. A different method of measuring the respiratory burst, based on chemiluminescence emission, was used by the authors, and as it is a more sensitive method than the assay performed here, this may explain why they observed a seasonal difference in respiratory burst, although no effect of temperature was observed on the respiratory burst on blood leucocytes in Atlantic cod (Gadus morhua) [35]. Respiratory burst activity has been reported to increase in kidney leucocytes isolated from dab following a stress event [36]. Melatonin is known to mediate the effects of day length on both daily and seasonal behavioural and physiological events in certain vertebrates [13]. There is a cyclical daily rhythm of melatonin production, with the majority of the hormone produced during the dark phase [37,38]. This is corroborated by the results in this trial, as there was a significantly higher level of melatonin (P Z 0.000) in the night
time samples compared to daytime samples. In addition, melatonin levels appeared to change with season, with significantly higher levels measured during the summer months compared to winter in both the day and night time samples. This may have been an effect of changes in water temperature, since it has been reported that plasma melatonin levels in Atlantic salmon are significantly higher in fish maintained at 12 C compared to those at 4 C [14]. The water temperature in the present study was higher in the summer months compared to those recorded in the winter month and therefore it is not unexpected that the melatonin levels measured were higher during the warmer, summer months. Clear effects of photoperiod on the timing of reproduction and growth and the corresponding diel and seasonal patterns of melatonin provide strong circumstantial evidence that melatonin may be an intermediary in the process [39]. Similarly, melatonin may well be intermediary in the seasonality of the observed innate immune and haematological responses. The increasing age of the fish may have affected melatonin levels. The majority of studies examining the
The effect of seasonality on normal haematological and innate immune parameters of Oncorhynchus mykiss
Figure 4
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Day and night melatonin levels in rainbow trout over the 12-month trial period (n Z 15; mean SE).
effect of melatonin and age has been carried out in mammals. A study in rats concluded that pineal and plasma melatonin concentrations decline with age [40]. This pattern was not observed in the trial presented here since a strong seasonal pattern was observed with the lowest values being observed at the first sampling point which took place in winter and the highest in the early summer month of May at the end of the trial. A general seasonal pattern of the innate immune response and haematological parameters studied was observed with the exception of respiratory burst activity. Overall, immune parameters were highest in summer and lowest in winter. However, different species of fish have been shown to exhibit different seasonality in their immune response, for example in Atlantic cod non-specific immune components, especially the anti-protease activity, were more evident at low temperatures than at high temperatures whereas the parameters associated with the specific immune capacity of fish, like immunoglobulin and natural antibody level, were more prominent in cod at higher temperatures [41]. This is corroborated by a study on channel catfish which has indicated that non-specific immune components like phagocytes are more resistant to low temperatures than specific components like lymphocytes [42]. The seasonality exhibited may be in response to seasonal increases in the level of potential pathogens in the environment. The seasonal patterns in immunity may be correlated to seasonal patterns in pathogen load. For example, several diseases of the aquatic environment present a seasonal pattern, including proliferative kidney disease, one of the most economically important diseases among commercially reared rainbow trout in Europe. The disease is often seasonally dependent, occurring at water temperatures above 15 C in the summer and autumn months of the year [43]. Furunculosis (Aeromonas salmonicida) is also generally a seasonal disease, with acute outbreaks occurring when water temperatures are about 20 C and chronic infections occur when temperatures are 13 C [44]. This may help to explain the seasonal patterns
exhibited in lysozyme activity and white blood cells in the present study. The immune system may be preparing for the potential attack by seasonal pathogens and if this is the case, the change in climatic conditions must be being anticipated otherwise any preventative measures to combat the increased pathogen load would occur too late. However, the mechanisms that regulate these seasonal components have yet to be fully identified.
Acknowledgements This research was financially supported by the National Environmental Research Council, UK and EWOS, Norway.
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journal homepage: www.elsevier.com/locate/fsi
The effect of seasonality on normal haematological and innate immune parameters of rainbow trout Oncorhynchus mykiss L. A.L. Morgan*, K.D. Thompson, N.A. Auchinachie, H. Migaud Institute of Aquaculture, University of Stirling, Stirling, Scotland FK9 4LA, United Kingdom Received 28 November 2007; revised 27 May 2008; accepted 28 May 2008 Available online 3 June 2008
KEYWORDS Season; Innate immunity; Respiratory burst; Lysozyme; Blood cell counts; Melatonin; Oncorhynchus mykiss
Abstract It is well established that seasonality dominates the life history of fish by controlling the timing of physiological events such as reproduction, food intake, locomotor activity and growth performance. Seasonal differences in immune competence and prevalence of disease have been well documented in humans. The aim of this study was to determine if season influences the immune response of rainbow trout (Oncorhynchus mykiss L.). Thus, a 12-month trial was carried out in which plasma lysozyme activity and respiratory burst of head kidney macrophages (two innate immune parameters) and total red and white blood cell counts (two haematological parameters) were monitored at monthly intervals. Since photoperiodic information is thought to be conveyed via melatonin secretion, plasma melatonin levels were also measured at four seasonal points (day and night). A general seasonal influence was observed in the parameters measured in these fish, with the exception of respiratory burst activity of head kidney macrophages, with the parameters highest in summer and lowest in winter for total white blood cell counts and lysozyme activity. ª 2008 Published by Elsevier Ltd.
Introduction During an annual cycle, the temperate aquatic environment will be affected by two primary components of season, i.e. temperature and photoperiod. These components are interlinked and follow very similar cycles. Typically during the spring, water temperatures and light periods increase while the reverse occurs in autumn. This seasonality affects the
* Corresponding author. Tel.: þ44 1786 467881; fax: þ44 1786 472133. E-mail address: [email protected] (A.L. Morgan). 1050-4648/$ - see front matter ª 2008 Published by Elsevier Ltd. doi:10.1016/j.fsi.2008.05.011
life history of fish with regard to the timing of developmental and maturational events synchronized with seasonal changes in temperature, day length and food supplies [1]. It is well documented that seasonality also affects the immune response of vertebrates [2,3]. Seasonal differences in immune competence and prevalence of disease have been well documented in humans [4,5]. Adaptive immunity in fish was shown to exhibit a seasonal cycle over a 12-month period, in particular, changes in resting antibody titre and response to antigenic challenge [6]. Other studies have shown there to be seasonal changes, in particular in the lymphoid system [7,8] and in the numbers of circulating lymphocytes [9]. Innate immunological indicators have
792 been shown to be affected by low temperatures in gilthead sea bream (Sparus aurata L.). Affected fish showed severe immunosuppression involving significant decrease of serum complement activity, decrease of plasma lysozyme activity and reduction in circulating lymphocytes [10]. It had generally been assumed that the innate immune response does not exhibit this seasonal cycle but remains constant throughout the year, always providing a defence to invading pathogens [11]. Many environmental challenges are recurrent and thus predictable [3]. Animals could enhance their survival and presumably increase their fitness, if they are able to anticipate immunologically challenging conditions in order to cope with these seasonal threats to health [12]. A potential mechanism to anticipate changes in season may be transmitted through the pineal hormone melatonin. It exhibits a strong circadian rhythm as the majority of the hormone is produced during the dark phase. Consequently, its production is affected by the seasonality of photoperiod. During the winter months, when the dark phase is at its longest, melatonin is produced for a greater length of time compared to the shorter dark phase of summer days in vertebrates [13]. In addition, melatonin production is affected by water temperature with higher levels reported in Atlantic salmon (Salmo salar L.) maintained at 12 C compared to fish maintained at 4 C [14]. The innate immune system is a fundamental defence mechanism of fish [15]. The primary aim of the current study was to determine if the innate immunity of rainbow trout is affected by seasonality. This was achieved by measuring plasma lysozyme activity and respiratory burst of head kidney macrophages (two innate immune parameters) and total red and white blood cell counts (two haematological parameters) each month over a 12-month period. The study was carried out under ambient temperature and photoperiod.
Materials and methods Fish husbandry The trial was performed at the Niall Bromage Freshwater Research Facility (NBFRF), Institute of Aquaculture (IOA), University of Stirling. Only female fish, obtained from a commercial fish farm on the Isle of Man, were used to prevent precocious males with a potentially compromised immune system skewing the results. Three tanks (B 2 m 1 m) were set up with flow through water supplied from a reservoir situated 1 km from the facility. The flow rate was approximately 2 l/s at an ambient seasonal temperature. Light was supplied by 60 W pearl, tungsten filament light bulbs (Skye Instruments, Powys, UK) housed within waterproof lamps providing an intensity of 17e19 lx at the water surface. The photoperiods were controlled by 24 h digital electronic time switches and were set to simulate the natural photoperiod by a light sensor located on the outside of the building. Fish were fed a standard commercial pelleted diet (EWOS, Livingston, UK), according to the manufacturer’s guidelines. One hundred fish (initial average weight 20.5 g) were placed in each of the three tanks at the start of the
A.L. Morgan et al. experiment (July) and five fish were sampled monthly from each tank.
General experimental procedures Collection of plasma Blood was withdrawn monthly from fish killed by an overdose of anaesthetic (2-phenoxyethanol, SigmaeAldrich Co., UK) followed by a blow to the back of the head. Fish were bled by caudal venepuncture using 1 ml or 2 ml syringes and a 25 G or 23 G needle (Terumo Europe N.V., Belgium) depending on fish size. Prior to sampling, syringes were rinsed with heparin (4 mg ml1, SigmaeAldrich Co., UK). The blood was stored on ice and transported to the laboratory at the IOA, where blood counts were carried out prior to centrifuging the blood at 3000 g for 10 min at 4 C. Plasma was collected and stored at 70 C until analysed for lysozyme activity. Blood was also sampled at 3-month intervals for the analysis of plasma melatonin levels. Samples were taken at midday and 2 h after the onset of darkness by removing the fish from the tank under total darkness and placing them in an anaesthetic bath of a 1:20,000 concentration of 2-phenoxyethanol for 2 min. Once anaesthetised, the fish were bled under a dim red light (l Z 670e800 nm, 0.2 lx at 0.5 m). The samples were transported back to the laboratory on ice and plasma collected as above. Condition factor Whilst under anaesthesia, weight and length measurements were taken from which condition factors were calculated [Condition Factor Z (weight 100)/length3]. Total blood cell counts To determine the total white blood cell count (WBC), a 1 in 100 dilution of the blood was made in phosphate saline buffer (PBS, 0.02 M, pH 7.3). While a 1 in 1000 dilution was made to count the red blood cells (RBC). Counts were carried out using a Neubauer haemocytometer (Hawksley & Son, England) and were expressed as cells ml1. Plasma lysozyme activity Lysozyme activity in plasma was measured turbidimetrically according to Refs. [16,17]. Lyophilised Micrococcus lysodeikticus was added to a 0.04 M sodium phosphate buffer (pH 5.8) at a concentration of 0.2 mg bacteria ml1 and this was then incubated at 25 C for 20 min. Ten microlitres of plasma was added to five replicate wells of a 96 multiwell plate (Nunc, Denmark). One hundred and ninety microlitres of the bacterial suspension was added to all wells apart from the control wells, to which was added 200 ml of sodium phosphate buffer. Absorbance was read at 540 nm, 1 min after adding the buffer and then again after 5 min using a Dynex MRX II plate reader. Lysozyme activity was expressed as the amount of sample causing a decrease in absorbance of 0.001 min1. Units used were units min1 ml1. Respiratory burst activity of head kidney macrophages Macrophage suspensions were prepared from sampled fish according to Ref. [18]. Under sterile conditions the kidney
The effect of seasonality on normal haematological and innate immune parameters of Oncorhynchus mykiss was teased through sterile 100 mm mesh into 5 ml of Leibovitz (L-15, SigmaeAldrich, UK) medium containing 10 ml heparin into a 30 mm diameter Petri dish and the mesh was then rinsed with 1 ml of L-15. The macrophage suspension was briefly placed on ice prior to performing the respiratory burst assay, which was carried out as described by Secombes [19] and adapted by Burrells et al. [20]. One hundred microlitres of the macrophage suspension was aliquoted into eight replicate wells of a sterile 96-well microtitre plate (Nunc, Denmark) and left for 1 h at 21 C to allow the cells to adhere to the plate. Plates were then washed three times with L-15 medium to wash off non-adherent cells. To the first set of three replicate wells, 100 ml of 1 mg ml1 nitrobluetetrazolium (NBT, Sigmae Aldrich Co., UK) in L-15 was added and to the next three replicate wells 100 ml of NBT solution containing 1 ml ml1 of phorbol myristate acetate (PMA, SigmaeAldrich Co., UK) was added. To the remaining two wells, 100 ml of lysis buffer (citric acid, 0.1 M; Tween 20, 1.0% (v/v); crystal violet, 0.05% (w/v), SigmaeAldrich Co.; prepared in distilled water) was added. Plates were incubated at 21 C for 1 h. The contents of the first six replicate wells were removed and the plates washed three times with L-15 medium. One hundred microlitres of methanol (100% v/v) was added to the wells for 5 min to stop the reaction. The wells were then washed three times with 70% (v/v) methanol and left to air-dry (a minimum of 30 min). One hundred and twenty microlitres of dimethyl sulphoxide (DMSO, SigmaeAldrich Co., UK) and 140 ml of 2 M potassium hydroxide (KOH, BDH, UK) were added to each of the washed wells. The absorbance was then read at 610 nm using an ELISA plate reader. The average numbers of adherent cells in the wells containing lysis buffer was determined by counting the cells using a Neubauer haemocytometer. Plasma melatonin Plasma melatonin levels were measured using a melatonin radio-immunoassay (RIA) method according to Randall [21], using sheep melatonin antiserum (Stockgrand Ltd, Guilford, Surrey), and [O-methyl-3H]melatonin (Amersham International, Bucks). Plasma melatonin levels in samples (250 ml) were determined from a standard curve (3.9e500 pg/tube).
Statistical analysis Before analysis, data were found to be normally distributed and homogenous without transformation. This test was performed using the appropriate function on the Minitab statistical Package (V. 10). Data were analysed (P < 0.05) using an ANOVA general linear model, Tukey pairwise comparison tests (post-hoc) and Pearson’s correlation coefficient all using Minitab (V. 10).
Results Weight and condition factor The average weight of the fish at the start of the experiment was 20.5 g and 248.7 g at the end of the trial 12 months later (Fig. 1a). The condition factor improved from 1.4 to 1.2 during this time (Fig. 1b).
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White blood cell counts There was a general increase in white blood cell counts during the spring months of the experiment peaking in July (start of the trial) and June (end of the trial) (Fig. 2a). Total white blood cell numbers decreased during the autumn and winter months to their lowest in March. Using an ANOVA (GLM) a significant effect by month was found on white blood cell counts (P Z 0.000). Using a Tukey post-hoc, pairwise comparison test, no significant differences were observed between the summer months of May, June and July (P > 0.05). There was also no significant difference in white blood cell numbers between the winter months of November, December and January. However, total white blood cell counts in the winter months were significantly lower than those measured during the summer months (P Z 0.000).
Red blood cell counts Red blood cell counts peaked during July (start of the trial) and June (end of the trial); although very little difference was observed between the other sampling months (Fig. 2b). A significant effect between month was observed (P Z 0.000). Tukey post-hoc, pairwise comparison tests established that the red blood cell counts obtained in July were not significantly different from those taken in October or April (P > 0.05), but were significantly lower than those taken in June (P Z 0.000) and significantly higher than those measured during the remaining sampling months (P > 0.05).
Lysozyme activity Plasma lysozyme activity (Fig. 3a) decreased from October to April and increased from May to July, indicating some seasonal influence on plasma lysozyme activity. The lowest levels of activity were recorded in April. A significant effect on plasma lysozyme activity was found by month (P Z 0.000). The level of plasma lysozyme activity in September was not significantly different from that of August, October or June (P > 0.05), but was significantly higher than the other months. Plasma lysozyme activity measured in March and April was significantly lower than those measured in August, September and June (P < 0.05).
Respiratory burst activity No clear seasonal pattern was observed in respiratory burst activity when measured over the 12-month period (Fig. 3b). The activity measured was greatest during February and lowest during the January. The pairwise comparisons further revealed that respiratory burst activity levels recorded in February were significantly greater than all other months (P Z 0.000) except July and August (P > 0.05). Respiratory burst activity in August was significantly greater than all months except October, February, and March (P < 0.05). There was no significant difference between March, April, May and June (P > 0.05).
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Figure 1
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Variations in weight (a) and condition factor (b) of rainbow trout over a 12-month period (n Z 15; mean SE).
Plasma melatonin levels A significant difference was observed between night and daytime plasma melatonin levels (P Z 0.000) (Fig. 4). Melatonin levels were highest at night throughout the trial period. There was a significant difference between the summer melatonin levels of July and May and those measured during the winter months of November and January for both day and night samplings (P Z 0.000).
Discussion A general seasonal effect was observed for total white blood cell counts, lysozyme activity and red blood cell counts. White blood cell counts increased steadily during the spring months peaking in July and June. They then decreased during the autumn and winter months to their lowest level in March. This corroborates the work of previous studies in which white blood cell counts were
shown to exhibit a seasonal rhythm [9]. It was observed in this trial that total white blood cell counts increased during spring, peaked in the summer and then decreased in number in autumn to their lowest levels during winter. This coincides with the seasonal increase in water temperature and day length. Collazos et al. [22] reported that leucocyte counts for both male and female tench Tinca tinca L. were significantly lower in spring and winter when compared with summer and autumn, and this has since been corroborated by De Pedro et al. [23]. Although the pattern of leucocyte counts reported in these studies differed slightly to those reported here, all three studies corroborate the hypothesis of Slater and Schreck [9] ‘‘In general, all immune parameters are suppressed in winter and highest in summer’’. A slight seasonal effect was also observed for red blood cell counts, with the lowest values recorded in the winter months of November, December and January, whereas the highest red blood cell counts were recorded during July (trial start) and June (trial end). An indirect effect of lower
The effect of seasonality on normal haematological and innate immune parameters of Oncorhynchus mykiss
Figure 2
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Variations in white (a) and red (b) blood cell counts in rainbow trout over a 12-month period (n Z 15; mean SE).
water temperature is the higher solubility of oxygen in cold water compared to warm water. Consequently, fewer red blood cells are required to carry oxygen around the body of the fish in colder weather as the oxygen is more readily available [24]. This may explain why higher levels of red blood cells were recorded in June and July. Likewise, blood becomes more viscous (thicker and harder to pump) at lower temperatures. To solve this problem, Antarctic fishes have fewer red blood cells [25] and this may also account for why fewer red blood cells were observed in the rainbow trout from this study during the winter months. However, in the warmer summer months of July and June the reduced availability of oxygen means that more red blood cells are required to absorb available oxygen. Furthermore, winter is a period of reduced activity and therefore less energy is expended, i.e. a reduced metabolism, thus less oxygen is required. This may be another reason for the observed seasonality of red blood cell counts. Plasma lysozyme exhibited a seasonal pattern of activity during the 12-month period. The pattern is very similar to that of the white blood cell counts in that activity increased
in spring peaking in late summer and then decreased over autumn to its lowest level in late winter. This has also been shown in plaice (Pleuronectes platessa L.) [26], sea trout (Salmo trutta L.) [27], dab (Limanda limanda L.) [28], Asian catfish (Clarias batrachus L.) [29] and Atlantic halibut (Hippoglossus hippoglossus L.) [30,31] in which a generally consistent seasonal trend in plasma lysozyme activity was observed, with low values being associated with winter. However, this correlation is not always observed. When lysozyme activity was measured in Nile tilapia raised at four different temperatures (18.4 C, 23 C, 28 C and 33 C), it was observed that fish cultured at 33 C for 4 weeks exhibited a decrease in lysozyme activity, although this variation was still higher than in the control fish [32]. It was consequently suggested that temperature had a limited affect on lysozyme activity in the Nile tilapia [32]. Although significant differences were recorded in respiratory burst activity (P < 0.05) according to month, these could not really be said to be seasonal. The highest levels of respiratory burst activity occurred in August and February and the lowest in January. This is corroborated by
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Figure 3 Variations in plasma lysozyme (a) and respiratory burst (b) activity in rainbow trout over a 12-month period (n Z 15; mean SE).
a seasonality study on croaker (Micropogonias furnieri) which also suggested that respiratory burst was not affected by season [33]. However, a temperature effect on respiratory burst has been reported in rainbow trout, with higher levels of activity reported with increased temperature [34]. A different method of measuring the respiratory burst, based on chemiluminescence emission, was used by the authors, and as it is a more sensitive method than the assay performed here, this may explain why they observed a seasonal difference in respiratory burst, although no effect of temperature was observed on the respiratory burst on blood leucocytes in Atlantic cod (Gadus morhua) [35]. Respiratory burst activity has been reported to increase in kidney leucocytes isolated from dab following a stress event [36]. Melatonin is known to mediate the effects of day length on both daily and seasonal behavioural and physiological events in certain vertebrates [13]. There is a cyclical daily rhythm of melatonin production, with the majority of the hormone produced during the dark phase [37,38]. This is corroborated by the results in this trial, as there was a significantly higher level of melatonin (P Z 0.000) in the night
time samples compared to daytime samples. In addition, melatonin levels appeared to change with season, with significantly higher levels measured during the summer months compared to winter in both the day and night time samples. This may have been an effect of changes in water temperature, since it has been reported that plasma melatonin levels in Atlantic salmon are significantly higher in fish maintained at 12 C compared to those at 4 C [14]. The water temperature in the present study was higher in the summer months compared to those recorded in the winter month and therefore it is not unexpected that the melatonin levels measured were higher during the warmer, summer months. Clear effects of photoperiod on the timing of reproduction and growth and the corresponding diel and seasonal patterns of melatonin provide strong circumstantial evidence that melatonin may be an intermediary in the process [39]. Similarly, melatonin may well be intermediary in the seasonality of the observed innate immune and haematological responses. The increasing age of the fish may have affected melatonin levels. The majority of studies examining the
The effect of seasonality on normal haematological and innate immune parameters of Oncorhynchus mykiss
Figure 4
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Day and night melatonin levels in rainbow trout over the 12-month trial period (n Z 15; mean SE).
effect of melatonin and age has been carried out in mammals. A study in rats concluded that pineal and plasma melatonin concentrations decline with age [40]. This pattern was not observed in the trial presented here since a strong seasonal pattern was observed with the lowest values being observed at the first sampling point which took place in winter and the highest in the early summer month of May at the end of the trial. A general seasonal pattern of the innate immune response and haematological parameters studied was observed with the exception of respiratory burst activity. Overall, immune parameters were highest in summer and lowest in winter. However, different species of fish have been shown to exhibit different seasonality in their immune response, for example in Atlantic cod non-specific immune components, especially the anti-protease activity, were more evident at low temperatures than at high temperatures whereas the parameters associated with the specific immune capacity of fish, like immunoglobulin and natural antibody level, were more prominent in cod at higher temperatures [41]. This is corroborated by a study on channel catfish which has indicated that non-specific immune components like phagocytes are more resistant to low temperatures than specific components like lymphocytes [42]. The seasonality exhibited may be in response to seasonal increases in the level of potential pathogens in the environment. The seasonal patterns in immunity may be correlated to seasonal patterns in pathogen load. For example, several diseases of the aquatic environment present a seasonal pattern, including proliferative kidney disease, one of the most economically important diseases among commercially reared rainbow trout in Europe. The disease is often seasonally dependent, occurring at water temperatures above 15 C in the summer and autumn months of the year [43]. Furunculosis (Aeromonas salmonicida) is also generally a seasonal disease, with acute outbreaks occurring when water temperatures are about 20 C and chronic infections occur when temperatures are 13 C [44]. This may help to explain the seasonal patterns
exhibited in lysozyme activity and white blood cells in the present study. The immune system may be preparing for the potential attack by seasonal pathogens and if this is the case, the change in climatic conditions must be being anticipated otherwise any preventative measures to combat the increased pathogen load would occur too late. However, the mechanisms that regulate these seasonal components have yet to be fully identified.
Acknowledgements This research was financially supported by the National Environmental Research Council, UK and EWOS, Norway.
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