Modulation of the immune system of fish by their environment

Fish & Shellfish Immunology (2008) 25, 373e383

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Modulation of the immune system of fish by their environment Timothy J. Bowden* Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK Received 28 November 2007; revised 18 March 2008; accepted 23 March 2008 Available online 3 April 2008

KEYWORDS Environment; Temperature; Photoperiod; Salinity; Anoxia; Hypoxia; Particulates; Suspended solids; Oxygen capacity; Immunity; Immune system; Lysozyme

Abstract The environment impacts on the physiology and psychology of animals in a wide variety of ways. If we can develop an understanding of how different environmental factors affect different processes we may be able to predict these changes and avoid or moderate deleterious events and the resultant changes in fish health and disease resistance. In this review, advances in the understanding of environmental impacts were identified in relation to specific areas of immune function. The trends, where they can be identified, showed that increases in light, temperature, salinity, oxygen, pH or particulates results in a general increase in immune function. ª 2008 Elsevier Ltd. All rights reserved.

The Environment The environment is a generic term to describe all living and non-living things that occur on the planet or a part of it. Within this, complete landscape units function as largely discrete entities. However, we need to define the use of the term ‘environment’ more precisely. There are 2 principal environmental types; terrestrial and aquatic each with unique characteristics. The principle differences include;

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a greater nutrient load in an aquatic environment which results in more life that can be supported, the aquatic environment is more stable, aquatic organisms are seldom exposed to desiccation, oxygen and light can become limiting factors in an aquatic environment, and aquatic organisms are less influenced by gravity. The factors that will be considered include; photoperiod, temperature, pH, oxygen level, particulates, and salinity. There is a considerable body of published research looking at the effects of many environmental factors on the physiology of fish. However, most of this research focuses on husbandry (predominantly feeding) and reproduction. This review will focus on direct effects on the immune system. It will preferentially look at reports on

1050-4648/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.03.017

374

T.J. Bowden

experimental findings under controlled conditions rather than natural environmental changes. It will also look at specific environmental factors in an effort to try and detect patterns of response. The decision to focus more on experimental findings is a reflection of the difficulty in extracting useful information from results of natural change. It is extremely difficult to identify the causative factor, or factors, in these situations.

The animals The main focus of this review is on fish species of economic importance. The consequence of this means that most of those species used in the studies reviewed will be involved in commercial aquaculture or were/are being considered for commercial aquaculture. However, the paucity of data has meant that some of the species included having limited commercial appeal, such as references to mummichog and Japanese medaka, species not of commercial importance but the inclusion of which allows some analysis of immune response. Basic data regarding temperature, optimal latitudes and preferred water environment for each species included is given in Table 1. Development stages in fish can greatly influence their response to external factors. As such data presented here is for adult fish rather than larval or juveniles.

Table 1

Immune systems The immune system constitutes a highly complex defence mechanism utilising a wide range of individual systems. Because of this complexity, the immune system is often divided into subdivisions. Sometimes this is done as nonspecific/specific immunity, innate/adaptive immunity, or even mucosal/systemic immunity. These subdivisions reflect perceived discrete compartments. However, the complexity and specificity of the response often means that the boundaries are poorly defined. Much of the research presented here concentrates on innate or non-specific immune functions. This may be a reflection of the ease of study or the fact that these systems provide the first line of defence. Also, most of the studies identified here present data based on protein/enzyme studies. Finally, the influence of time on the nature of environmental changes should be considered. The difference between acute and chronic environmental impacts will be seen in the immune response where acute impacts will involve the innate immune system whereas chronic impacts will involve the adaptive immune system. For instance, short term impacts may elicit a stress response that can be measured by cortisol production. However, this response may diminish over time even though the environmental change is still present.

Environmental conditions for species included in the review

Species name

Common name

Maximum length

Latitude

Temperature range

Habitat

Fundulus heteroclitus heteroclitus Cyprinus carpio

Mummichog

15 cm

52 Ne28 N

10e24  C

Common carp

120 cm

60 Ne40 N

3e32  C

Sparus aurata

Gilthead seabream Tench

70 cm

62 Ne15 N

e

70 cm

64 Ne36 N

4e24  C

103 cm

72 Ne11 N

8e24  C

Benthopelagic; non-migratory; freshwater; brackish; marine Benthopelagic; potamodromous; freshwater; brackish; pH range: 7.0e7.5; dH range: 10e15 Demersal; brackish; marine; depth range 1e150 m Demersal; potamodromous; freshwater; brackish; depth range 1e? m Demersal; oceanodromous; freshwater; brackish; marine; depth range 10e100 m Benthopelagic; oceanodromous; marine; depth range 0e250 m Demersal; oceanodromous; marine; depth range 10e200 m Demersal; oceanodromous; marine; depth range 50e2000 m Pelagic; anadromous; freshwater; brackish; marine; depth range 0e250 m Demersal; anadromous; freshwater; brackish; marine; depth range 0e250 m Benthopelagic; anadromous; freshwater; brackish; marine; depth range 0e200 m Demersal; anadromous; freshwater; brackish; marine; depth range 0e? m Benthopelagic; anadromous; freshwater; brackish; marine; depth range 0e210 m Demersal; oceanodromous; marine; depth range 20e150 m

Tinca tinca Dicentrarchus labrax Pagrus pagrus Pagrus major

European seabass Common seabream Red seabream

91 cm

57 Ne38 S

e

100 cm

34 Ne15 N

e

Hippoglossus hippoglossus Oncorhynchus nerka

Atlantic halibut

470 cm

79 Ne36 N

e

Sockeye salmon

84 cm

72 Ne42 N

0e25  C

Oncorhynchus kisutch

Coho salmon

108 cm

72 Ne22 N

0e25  C

Oncorhynchus mykiss

Rainbow trout

120 cm

63 Ne53 N

10e24  C

Salmo trutta

Brown trout

100 cm

90 Ne33 N

2e16  C

Salmo salar

Atlantic salmon

150 cm

72 Ne37 N

2e9  C

Limanda limanda

Common dab

40 cm

72 Ne43 N

e

Modulation of the immune system of fish by their environment

Photoperiod Photoperiod is one of the prime controllers of living organisms. Together with temperature it is considered a proximate cue, or zeitgeber, for circadian and seasonal influence [1]. It is clear that photoperiod changes allow an animal to track daily and seasonal rhythms and alter their physiology accordingly, including the immune system. Living systems, animals and plants, possess complex systems for monitoring light and assessing changes in light levels. Photoperiod manipulation is used as a means of resetting the body clocks of fish on a daily and seasonal basis and we have recently reviewed the impact of seasonality on fish immunity [2]. One of the principle reasons for controlling the body clock is to change the breeding cycle and allow production of eggs/fry outwith the normal breeding cycle. In salmon farming, photoperiod changes can be used to induce the animals into smolting, allowing farmers to have more than one input into seawater each year and thus stretch the production cycle.

Complement activity A study looking at short term changes in innate immunity comparing seabream (Sparus aurata) and sea bass (Dicentrarchus labrax) complement activity showed that this activity was higher during the day than at night [3]. The study exposed the animals to a constant LD 12:12 and serum was obtained from blood samples collected at 02:00, 08:00 h (light-on), 14:00, 20:00 h (light-off) and at 08:00 h again.

Haematology A recent study on rainbow trout (Oncorhynchus mykiss) looked at 3 different photoperiod regimes (control LD 12:12, experimental LD 14:10 and LD 24:0) for up to 150 days [4]. Experimental photoperiods were used for 90 days after which the animals reverted to standard LD 12:12 photoperiods. The only effect of note was a reduction in lymphocytes and thrombocytes in the LD 14:10 group at day 150. In a related study trout held under 24 h light for 2 months before being returned to an ambient photoperiod showed a significant decrease in leukocytes [5]. A study that exposed red sea bream (Pagrus major) to a constant light photoperiod showed no changes in the haemoatocrit after 8 weeks [6]. A study on Nile tilapia (Oreochromis niloticus) where the photoperiod was reduced to 6 h light:6 h dark from a LD 12:12 regime showed a significant increase in the lymphocyte count in animals exposed to the reduced light regime [7]. A further study on rainbow trout showed little change in lymphocyte populations although thrombocyte populations fell in animals exposed to adverse photoperiod regimes [8]. Experimental fish were exposed to different photoperiod regimes during 140 days: 14 acclimatising days at LD 10:14, 60 days at LD 24:0, 30 days at LD 10:14 and 30 days at LD 12:12. Control fish were kept at a ‘‘natural’’ photoperiod of 14 acclimatizing days at LD 10: 14, 92 days at LD 10: 14 and 30 days at LD 12:12. A study of Atlantic salmon (Salmo salar) pre-smolts showed animals exposed to a constant light photoperiod had reduced B-cell

375 numbers and increased neutrophils prior to seawater transfer [9]. Once the animals were transferred, the B-cell numbers increased and neutrophil numbers decreased. This suggested a change in the leukocyte pattern pre and post smolt.

Lysozyme Few papers have looked at changes in lysozyme levels in relation to photoperiod. One recent paper looked at differences in lysozyme levels in relation to the circadian rhythm in two fish species; seabream and sea bass. The study exposed the animals to a constant LD 12:12 and serum was obtained from blood samples collected at 02:00, 08:00 h (light-on), 14:00, 20:00 h (light-off) and at 08:00 h again [3]. The authors noted that lysozyme activity increased during light hours and decreased during dark hours in seabream, whilst in sea bass there was no significant change. Another paper looked at long term changes in lysozyme levels in Atlantic halibut (Hippoglossus hippoglossus) where one group received a constant LD 16:8 photoperiod and the other received LD 8:16 for 6 months and showed that photomanipulation could not produce the differences in lysozyme levels seen to occur in relation to season where summer levels were significantly higher than winter levels [10]. One study looked at lysozyme values for fixed photoperiod (light/dark 12:12, 24:0, and 14:10) in rainbow trout and noticed that LD 24:0 increased lysozyme after 7 days and for LD 14:10 after 30 days [11]. Another study showed no change in lysozyme of N1ile tilapia to reduced light periods [12]. The photoperiod was initially set to 12 L:12 D. Fish were allowed to acclimate for 6 days. One room was kept on the 12 L:12 D schedule. The photoperiod in the second room was altered to simulate local conditions of decreasing light (AugusteNovember).

IgM concentration Studies in Atlantic salmon have looked at antibody levels before, during and after smolting and found that they fell during the smolt window [13]. The study followed salmon for about 6 months and found that September levels were about twice those observed in March. Another study on Atlantic salmon looking at the effect of vaccination on smolting in ‘out-of-season’ animals showed that IgM levels were low during the ‘winter’ photoperiod on LD 12:12 and rose when the animals were exposed to continuous light period LD 24:0 [14].

Peroxidase The study comparing seabream and sea bass over a normal light/dark cycle suggested that seabream had a peroxidase activity peak in the morning, whilst the activity in sea bass was relatively constant [3].

Conclusion There is a trend for a reduction in leukocyte numbers with an increase in the amount of light in a 24 h cycle and an increase under reduced light conditions. There would

376 appear to be an increase in lysozyme associated with increased light and an increase in IgM levels. The effect of photoperiod on complement and peroxidase activity is unclear. The importance of photoperiods will depend on the geographical location of the species under consideration. For tropical species seasonal changes in photoperiod will be smaller than for temperate species and therefore may be less important in driving seasonal changes in physiology.

Temperature Temperature has a fundamental effect on animal physiology. It has long been known that environmental temperature effects all physiological functions of poikilothermic animals, such as fish [15]. Basic enzyme chemistry (indeed basic chemistry) teaches us the importance of temperature in governing the rate of reaction. So temperature is important in governing the rate at which body processes are carried out. As an indication of how different factors impact on an animal, there is a study on Atlantic salmon that showed how temperature could limit the impact of photoperiod during smoltification [16], suggesting a ranking of importance for these environmental cues [1]. However, a word of caution needs to be added in that it is clear that assay temperature can greatly affect the result [17,18].

Complement In tilapia (Oreochromis mossabicus) acclimated to 27  C then exposed to cooler water (19 and 23 ) experienced a drop in measurable ACH50, but ACH50 was elevated when exposed to higher temperatures (31 and 35 ) [19]. A study on sockeye salmon (Oncorhynchus nerka) found no variation in ACH50 between fish cultured at 8 or 12  C [20]. A recent study in rainbow trout looking at temperature (5, 15 and 25  C) impact on immune gene expression following vaccination found up-regulation of C5a (anaphylatoxin) receptor in immunised fish at higher temperatures [21]. Complement lysis activity was measured in another study on rainbow trout maintained at a range of temperatures between 5 and 20  C which showed that lytic activity for both total complement and alternative complement only were significantly enhanced at 15 and 20  C [22].

Haematology One report on Atlantic salmon looked at changes in peripheral blood and head kidney leucocyte populations when animals were maintained at 6, 10, 14 and 18  C [23]. The greatest variations were seen in peripheral blood with the lowest temperature having the highest percentage of Igþ cells. The highest temperature had the highest number of neutrophils and the lowest Igþ cells. Head kidney populations did not seem to vary to the same extent. A study on the mitogen-induced proliferation of lymphocytes in tench (Tinca tinca) showed a marked increase in proliferation at all except winter temperatures. However, when the isolated lymphocytes were cultured at a constant 22  C instead of the equivalent ambient water temperature, the situation was reversed suggesting that the lymphocytes

T.J. Bowden need a higher temperature than ambient during winter to proliferate [24]. A study on sockeye salmon found that comparison of the percentage of lymphocytes in fish reared at 8  C and 12  C showed that 8 fish had a greater number of phagocytic kidney macrophages whilst 12 fish had a greater proportion of lymphocytes [20]. A study on phagocytosis rates in macrophages of carp maintained at three temperatures (12  C, 20  C and 28  C) found that phagocytosis was significantly reduced at 20 and 28  C compared to macrophages from fish maintained at 12  C [25]. Bly and Clem published a series of papers on temperature effects on T and B cells in channel catfish (Ictalurus punctatus) [26e29]. In these they identified a generalised immunosuppression at lower temperatures and found that virgin T cells rather than other T and B cell types were particularly susceptible to low temperature suppression [30]. A study on Japanese medaka (Oryzias latipes) looked at the effect of a 5  C rise over 1, 7 or 14 days on kidney macrophage reactive oxygen production and T-cell proliferation [31]. The authors found that reactive oxygen production was significantly reduced for a 1 day exposure but only slightly reduced after a 14 day exposure. T-cell proliferation was unaffected by any temperature change. A report from India on temperature effects on leucocyte numbers and respiratory burst activity in carp (Cyprinus carpio) showed that leucocyte number fell significantly as the temperature increased from 26  C to 36  C as did the respiratory burst activity [32]. A study of respiratory burst activity in rainbow trout found a temperature dependent increase in activity in animals maintained at temperatures between 5 and 20  C [22].

Lysozyme In tilapia acclimated to 27  C then exposed to cooler water (19 and 23 ) experienced a drop in measurable lysozyme but an elevation when exposed to higher temperatures (31 and 35 ) [19]. A study on Atlantic halibut showed that lysozyme levels increased when the culture temperature was elevated from 8  C to 18  C [33]. A study on Nile tilapia reared at a range of temperatures (18.4, 23, 28 and 33  C) found an increase in plasma lysozyme when raised at 28  C and a significant decrease in plasma lysozyme when raised at 33  C [34]. The lysozyme level at 33  C after both 2 weeks and 4 weeks was still higher than the control fish [34].

IgM concentration A study on sea bass showed that rearing these animals at higher temperatures (23  C as opposed to 17  C) significantly increased the IgM concentrations [35]. In the same study the authors varied the temperature everyday between 17  C and 23  C over a 3 month period and the IgM levels were significantly lower as a consequence. Another group reported a decrease in plasma IgM levels in cold treated (from 25 to 12  C) blue tilapia (Oreochromis aureus), whereas a cold shock of 9  C induced significant changes in leucocyte populations and antibody response in carp [36,37]. A study on Atlantic halibut looked at the effect of optimal vs. sub-optimal growth temperatures (12  C

Modulation of the immune system of fish by their environment and 18  C respectively) on antibody production and found that antibody levels were higher at the higher temperature [38]. A study on sea bass investigated the antibody response at different temperatures (12, 18, 24 and 30  C) and found that elevated responses were seen only at the two higher temperatures [39]. A study on summer flounder which were maintained at a range of temperatures between 7.5 and 27  C and found the characteristic delay in antibody production although there was no effect on magnitude or duration of the response [40]. A study on Nile tilapia that were cultured at a range of temperatures (18, 23, 28 and 33  C) found a significant increase in plasma IgM levels with increasing temperature except at the highest temperature [41]. A report using Atlantic cod cultured for 12 months in water temperatures at 1, 7 and 14  C found significant and interrelated increases in plasma IgM concentration with water temperature [42]. An early study on vaccine efficacy used Atlantic salmon parr which were maintained 2, 4, 6, 8 and 10  C [43]. The animals were vaccinated against Vibrio salmonicida, the causative agent of cold water vibriosis. The authors noted that protection following challenge was better at lower temperatures (6  C or lower) although there was no significant difference in specific antibody levels between any of the groups. A study on carp noted that antibody production was inhibited at 12  C [44]. A study on rainbow trout found significant upregulation of secreted IgM encoding gene in fish maintained at 25  C although there was no effect on IgT expression [21].

MHC A report that studied MHC class I expression in common carp found that when the animals were subjected to a low temperature of 6  C expression of beta-2-microglobulin, a non-covalently associated light chain of MHC class I, was abolished in peripheral blood leucocytes [45]. A more recent report comparing rainbow trout and Atlantic salmon showed that expression of beta-2-microglobulin was not influenced by maintenance of the animals in low temperatures [46]. The authors suggested that salmonids may have a different mechanism for expressing MHC. Indeed, it has been suggested that reduced membrane fluidity, as opposed to MHC class I antigen presentation machinery, may limit T-cell activation in fish species such as cannel catfish [47]. Another study by the same group found Class II a and b gene expression was down-regulated in rainbow trout maintained at 2  C, but not at 5  C [48].

Cytokines A recent report indicated that cytokine expression (IL-1, IL-10 and IFNg) was up-regulated in rainbow trout maintained at 25 C [21]. Indeed, they found that there was a generalised up-regulation of cytokine expression at both 15  C and 25  C compared to animals at 5  C.

377 effect on haemotology with numbers of specific cell types varying both up and down with temperature changes. Lysozyme levels rose with increasing temperature and fell with lowering temperature, especially when these temperatures were from the normal/control temperature for the animal. Plasma IgM levels rose with elevated temperatures and fell with lower temperatures. Finally, MHC and cytokine appeared to have a temperature dependent expression.

Oxygen level Respiration, whether by terrestrial or aquatic animals requires oxygen. For terrestrial animals oxygen in the environment is rarely limiting. Only in enclosed spaces may oxygen be depleted. In aquatic environments however oxygen can become a limiting factor due to a wide variety of factors. One of the deciding factors for the location of fish farms is the movement of water in the surrounding area. The waters around Scotland are relatively shallow, especially compared to the deep waters found in Norwegian fjords. Therefore, Scottish fish farm sites have to ensure that tidal flows and water currents are such that there are sufficient water movements to maintain oxygen levels around the farm. Oxygen levels within the cage can be affected by the concentration of fish in any part of the cage and changes in the currents through the cage [49]. One publication linked oxygen deficiency in common dab (Limanda limanda L.) with health problems [50]. Studies on the variations in oxygen levels have looked at the effects of both hypoxia and hyperoxia. However, in the natural environment hyperoxic conditions are unlikely, whereas hypoxic conditions can and do occur, especially within fish farm cages. One study on tilapia noted that the lowest oxygen consumption was seen in animals acclimated to seawater compared to those adapted to freshwater or hypersaline water [51].

Complement A recent report on channel catfish showed that a reduction in oxygen from 6 mg/l to less than 2 mg/l resulted in a drop of complement haemolytic activity [52]. Another report using seabream induced hypoxia by air exposure found no change in alternative complement activity [53].

Haematology A study in mummichug (Fundulus heteroclitus) found that hypoxia affected the response of anterior kidney cells by reducing the production of reactive oxygen species and bactericidal activity [54]. In a study on seabream the respiratory burst activity of head-kidney leukocytes was found to be significantly depressed by hypoxia induced by air exposure [53].

IgM Conclusions There is no conclusive indication for the effect of temperature on complement. Temperature had a varied

A study on seabream used air exposure for 2 min as a method of inducing hypoxia and found that this had no effect on serum IgM levels [55]. A different study on sea bass

378 found that mild hypoxia compared to normal and hyperoxic conditions had lower antibody responses [39]. Another study on sea bass looked at hyperoxygenation and found a 2 fold increase in serum antibody levels [56].

Conclusions Respiratory burst activity is suppressed by hypoxia. Similarly, complement can be suppressed, although there are conflicting reports for this. Finally, hypoxia can reduce antibody levels and hyperoxia can increase antibody levels. The latter result shows a 2-way response to a variable. But what the advantage may be to the animal from such a response is not clear. It may be that rather than being protective as an immune response should be, it is compensatory and allows the animal to free up resources to combat other detrimental physiological changes. This is equally true for other environmental changes where the benefit to the animal is unclear.

Particulates Also known as suspended solids, this section looks at how un-reactive material suspended in the water column can impact on the immune system of fish. The terrestrial equivalent would be air-bourne particulates such as dust storms. Much of these water-bourne suspended solids can be associated with human activities [57]. It has been shown that increases in sediment loads can disrupt the reproductive success of fish by interfering with egg and fry viability [57].

Haematology A paper by Redding et al. (1987) looked at the effect of 3 different types of suspended solid (topsoil, kaolin clay, and volcanic ash), at 2 different concentrations (high (2e3 g/L) or low (0.4e0.6 g/L) concentrations) on both coho salmon and rainbow trout [58]. They investigated the effect of different exposures of up to 7 days on haematocrits and found that both high and low concentrations in both species elevated the haematocrits. High concentration of suspended topsoil also reduced the ability of rainbow trout to resist subsequent challenge with Vibrio anguillarum. Another study on coho salmon looked at high and low concentrations of anthropogenically derived ‘‘extremely angular’’ and ‘‘round’’ silicate sediments and found decreased leukocrits were elicited by exposure to both sediment shapes when concentrations were >40 g/L and elevated haematocrit and decreased leukocrit at concentrations <41 g/L [59].

Lysozyme A study on Nile tilapia exposed to various different concentrations (0, 20, 200 and 2000 mg/l) of suspended solids (64 mm) for 2 weeks found that lysozyme levels increased with increasing sediment load [34]. The difference between the control animals and those receiving 2000 mg/l was significant [34].

T.J. Bowden

IgM concentration The study on Nile tilapia exposed to various different concentrations (0, 20, 200 and 2000 mg/l) of suspended solids (64 mm) for 2 weeks found a general increase in plasma IgM concentrations but that the increase was not significant [41].

Conclusions Particulates elevate the haematocrit and may lower the leukocrit. Particulates may also elevate the level of lysozyme and plasma IgM. This may be a response to expected elevated levels of pathogens in a particulate rich environment. The changes in haematocrit might be to compensate for reduced oxygen uptake ability if the particulates cause a reduction in the surface area of the gills.

Salinity Natural changes in salinity can occur as a consequence of rainwater diluting seawater, mixing of estuarine waters or ingress of seawater into normally freshwater areas. Many fish species are capable of withstanding large osmotic changes. In developing a method for reproducibly inducing infectious pancreatic necrosis (IPN) in Atlantic salmon post smolts we looked at the changes in susceptibility following seawater transfer and found that immediately following transfer fish experienced only low mortalities and yet had high viral titres [60]. As the time post transfer became greater the mortality levels increased, although the results were not conclusive. Freshwater dips have been used to clear parasite infections in marine aquarium fish since the fish have a greater tolerance of short term exposure to freshwater than the parasites [61].

Haematology A recent study on rainbow trout which were transferred from freshwater to seawater reported an enhanced head kidney macrophage respiratory burst in animals [62]. A study in brown trout (Salmo trutta) transferred from freshwater to seawater noted an increase in the phagocytic activity of head kidney leucocytes [63].

Lysozyme A study looking at transferring rainbow trout to seawater showed that moving the animals to seawater raised plasma lysozyme but reduced mucus lysozyme [62]. A study on Nile tipalia maintained at three different salinities (0, 12 and 24 ppt) found a difference between the control (0 ppt) fish and the test fish (12 and 24 ppt) with a significant increase at 24 ppt after 2 weeks exposure and significant increases at both 12 and 24 ppt after 4 weeks exposure [34]. A study in rainbow trout where animals were placed in dilute seawater (12 ppt) for 3 days after culture in freshwater showed a 3.5 fold increase in the lysozyme levels compared to control fish [64]. A further study in brown trout found a similar increase in lysozyme after seawater transfer [63]. A report that looked at three salmonid

Modulation of the immune system of fish by their environment species; rainbow trout, coho salmon and Atlantic salmon and compared data for both mucosal and plasma lysozyme from animals maintained in freshwater and animals in seawater (30  2 ppt) found significantly lower levels of mucosal lysozyme in seawater compared to freshwater in all three species [65]. They also found rainbow trout had much higher levels of plasma lysozyme compared to the other 2 species in both seawater and freshwater [65]. A study on the effect of salinity on lysozyme in tilapia observed no difference in lysozyme levels in animals acclimated to freshwater, seawater or 1.6 seawater [51].

IgM concentration A study on Nile tipalia maintained at three different salinities (0, 12 and 24 ppt) found a significant difference in the plasma IgM level between fish at 0 and fish 24 ppt with the fish at 24 ppt having a higher level [41]. A study in rainbow trout where animals were placed in dilute seawater (12 ppt) for 3 days after culture in freshwater showed no difference in plasma IgM levels [64]. A study in vaccination routes in barrimundi (Lates calcarifer) that had variously been acclimated to fresh and seawater showed that the mucosal immune system produced a higher specific immune response as measured by specific antibody levels following seawater acclimation than freshwater [66]. Whilst there was no significant different in the systemic response between fish acclimated to fresh or seawater [66].

Conclusions Phagocytosis and respiratory burst can increase with increasing salinity. Additionally, lysozyme and IgM levels can also increase with increasing salinity. Is this a response to a potential increased pathogen load? Does a higher salinity equate to an increased pathogen load since it is a more osmotically favourable environment?

pH The impact of changes in the pH of the surrounding water are really only important when considering freshwater systems as seawater has a significant buffering capacity to resist pH changes. Freshwater does not have the same level of buffering and so can experience wide changes in pH. Acid rain is the most recognisable consequence and is attributable to atmospheric pollution, such as the production of nitrates from the burning of fossil fuels, being washed into water systems [67e69]. We conducted a study on in vitro binding of salmon antibodies and showed that binding could be all but eliminated at extreme pH (pH 3 and 10) with an optimum for binding of pH 7.4 [70]. There is clear evidence that lysozyme function in vitro is affected by pH and that assays must be optimised for the pH of the species under investigation [71].

Haematology A study on lowered pH (4.5) on carp demonstrated a drop in the phagocytic rates of leucocytes after 1 week’s exposure [72]. Levels remained low after 4 weeks.

379

Lysozyme A report on rainbow trout showed that reducing the pH to 4.5 resulted in a reduction in the levels of lysozyme in the plasma [73]. A study on Nile tilapia maintained animals at pH 7.9 and pH 4 for 2 weeks and found a highly significant (P < 0.001) increase in lysozyme levels at the lower pH after 2 weeks [34].

IgM concentration The study on Nile tilapia which maintained animals at pH 7.9 and pH 4 for 2 weeks and found no difference in plasma IgM levels between the test and control groups suggesting that pH had no influence on IgM levels [41]. A study on low pH effects on carp showed that dropping to pH 4.5 lowered the levels of plasma IgM after 1 week [72]. However, levels returned to control levels after 4 weeks exposure.

Conclusion One of the studies noted that the suspended solids used in the experiment had a pH of 8, about normal for the fish species under investigation. However, the soil from which the solids were derived normally had an acid pH, which suggested that the cleaning procedure removed the acidifying material [41]. Also, there are indications that impacts may be short term in nature and that given enough time, and a progressive rather than a sudden change in pH, animals can acclimate to changes in pH [41,74]. In studying lysozyme lowering the pH appeared to lower lysozyme levels in one study and raise them in another, somewhat contradictory. Studies on IgM either showed a lowering with lower pH or no change, which is inconclusive.

Stress, xenobiotics and pathogens These subject areas have been avoided in this review. The reason for this is that each area presents a highly complex interaction with an animal’s physiology in general, and more specifically, the immune system. Consequently, each area would require a considerable review in its own right. However, it is important that we are aware that these factors can impact through the environment, even though they may be present mainly through anthropogenic activities. In the case of farmed animals stress usually presents through normal husbandry procedures. Reviews of the effects of stress on the immune system of fish showed how complex these interactions could become [75e78]. A study looking at husbandry stress in sea bass used tank scrubbing as the stressor and found that this significantly reduced serum IgM levels [35]. Further evidence shows how the environment impacts on the immune systems through the endocrine system [79]. A study on stress in rainbow trout showed how complicated the measurement of cortisol could be and the consequences on accurately measuring stress response [80]. A recent review of the effects of polycyclic aromatic hydrocarbons, or PAH’s, on fish made the point that whilst it is clear that PAH’s are immunotoxic, with effects on both innate and acquired immune

380 systems, in fish these effects can be contradictory and dependent on the mode of exposure and dose, whereas in mammals the effects were more predictable [81]. Xenobiotics are by definition alien elements of the environment. Natural pollution incidents are rare but can occur. These may involve runoff from land such as acid leaching from coniferous forests or algal blooms causing rapid changes in local water chemistry but mainly such incidents can usually be shown to be exacerbated by human activities.

Discussion One of the greatest problems in identifying the causes of alterations in immune function is the wide variety of possible causes. Also, there is difficulty in interpreting results when the true baseline is unknown. Even for the common research relevant species such as rainbow trout and Atlantic salmon the availability of good baseline data is patchy at best and in many species lacking entirely. Additionally, the data that is published is usually a snapshot in time, with little consideration for natural variations that can, and will, occur over time as seasons change and the animal ages. In this review we have centered on the effects of some obvious environmental factors such as temperature and oxygen level. However, other factors can influence immune functioning. Complement levels can be influenced by diet composition and starvation [82e85]. One factor that should be identified is time. The difference between an acute or chronic factor can be considerable. Reviews of acute and chronic stress on fish immunology show how the responses vary with time [86,87]. Evidence for changes in response to repeated exposure to stressors is plentiful [88]. One of the papers reviewed here showed how one measure, reactive oxygen production by macrophages, altered with changing exposure time to temperature elevation with a big response to an acute exposure and a small response to a chronic exposure [31]. This is seen in many other papers with attenuation of the response occurring as the time exposure increases from an acute exposure to a chronic exposure. A report on stress in rainbow trout looked at lysozyme levels following acute and chronic stress and showed that acute stress elevated lysozyme levels and chronic stress suppressed lysozyme [80]. It is clear that technical developments will help expand our knowledge. The use of techniques such as microarrays, proteomics and bioinformatics will allow the development of a shotgun approach to screening of the immune response so that instead in individual immune parameters, or a small number of parameters, being investigated, a wide range on immune factors can be screened for changes in translation/ transcription/expression levels [89e91]. As yet there has been very little information published on immune parameters but given the number of projects currently using these tools this is likely to change very quickly. In summarising the findings it might be considered obtuse to point out the obvious, but the conclusions of each section would suggest that, in general, elevations in a given environmental factor leads to an elevation in the corresponding measured immune parameter. Correspondingly, reductions in any environmental factor lead to a reduction in the immune parameter. When viewed in detail it

T.J. Bowden might seem reasonable that a reduction in environmental temperature would lead to a reduction in immune function. It might be expected that the lower temperature would limit pathogen events and so reducing the immune response may reduce the physiological pressure on an animal to defend itself. Changes in other environmental factors might be more difficult to explain. The development of bioinformatics techniques, which can look at physiological, biochemical and genetic changes on a much bigger scale, may allow a better understanding of the whole animal response. And so it might be premature to advise a fish farm manager that husbandry systems can be relaxed when the temperature falls or light levels are reduced.

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