Effect of chronic exposure to ammonia on alterations of proteins and immunoglobulins in sea bass (Dicentrarchus labrax) serum

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Abstract

The relationship between serum protein, immunoglobulin concentrations and protein molecular weight profile (PMWP) alterations of sea bass (131.3 ± 4.3 g) reared in sea water with sublethal concentrations of ammonia was studied over two periods totalling 116 days. During the exposure period (62 days) the first group (group 1) lived in sea water with 0.204  mg.L-1unionized ammonia nitrogen equivalent to 12 % of the lethal concentration for 50 % of a population exposed for 96 h to ammonia (96-h LC50), whilst the second group (group 2) lived in sea water with 0.340  mg.L-1UIA-N equivalent to 20 % of the 96-h LC50 of ammonia. Then, the two groups were left for a recovery period (54 days) in the same water as the control group (group 0). The determination of the total immunoglobulin (Ig) concentration was carried out by enzyme-linked immunosorbant assay (ELISA). Gel filtration columns were used for the serum PMWP. Serum Ig concentration of the exposed fish (group 2: 1.76 ± 0.43 ; group 1: 1.19 ± 0.33  mg.mL-1) was lower than the control group (3.39 ± 1.01  mg.mL-1) after 21 days of exposure period but this difference was reduced at the end of this period (day 62) and treated group Ig concentrations switched to higher than the control group after the recovery period (day 116, group 2: 9.75 ± 1.84  mg.mL-1; group 1: 7.50 ± 1.22  mg.mL-1group 0: 6.38 ± 1.13  mg.mL-1). In fact, at the end of the experiment, the cumulative Ig production difference between fish exposed to ammonia and the control was less than 10 %. Although a similar evolution of the Ig serum occurred with protein concentration, the serum protein concentration deficit of group 1 was restored at the end of the exposure period (group 2: 46.49 ± 2.34  mg.mL-1; group 0: 46.74 ± 1.97  mg.mL-1) and the cumulative production during the experiment was not significantly different between group 1 and group 0. However, this remained lower for group 2. During the exposure period, the PMWP of treated fish moved towards smaller molecular weight proteins. This alteration of the PMWP showed that the 0.2-kDa fraction increased, and another fraction appeared at the end of the exposure period. However, at the end of the recovery period, no difference was found between the PMWPs of control and treated fish. The alterations of the serum protein and Ig of fish reared, for a limited period (2 months), in water with sublethal concentrations of ammonia (20 % 96-h LC50 or less) should disappear completely after a few months in normal rearing conditions.

Résumé

Modifications des protéines et des immunoglobulines sériques chez le loup (Dicentrarchus labrax) consécutives à une exposition chronique à l'ammoniaque. Les modifications des concentrations en immunoglobulines (Ig) et en protéines ainsi que celles des protéinogrammes sériques ont été suivies pendant 116 jours chez des loups méditerranéens de (131,3 ± 4,3 g) élevés en présence de doses sublétales d'ammoniaque. Pendant 62 jours, les poissons ont vécu dans de l'eau de mer additionnée d'ammoniaque à des concentrations de 0,204  mg.L-1de NH3 correspondant à 12 % de la 96-h DL50 pour le groupe 1 et 0,340  mg.L-1de NH3 correspondant à 20 % de la 96-h DL50 pour le groupe 2. Ensuite, l'ensemble des poissons a été élevé pendant 54 jours dans la même eau que celle du groupe témoin (groupe 0). L'immunoglobulinémie a été réalisée par dosage immuno-enzymatique (ELISA) ; les différents protéinogrammes sériques ont été effectués en gel filtration sur colonnes. Au cours de la période d'intoxication (21e jour), l'immunoglobulinémie reste inférieure chez les poissons traités (groupe 2 : 1,76 ± 0,43  mg.mL-1 ; groupe 1 : 1,19 ± 0,33  mg.mL-1contre 3,39 ± 1,01  mg.mL-1pour le groupe 0), mais ce déficit diminue à la fin de cette période (jour 62) pour s'inverser lors de la période de récupération (jour 116e : groupe 2 : 9.75 ± 1.84  mg.mL-1 ; groupe 1 : 7.50 ± 1.22 mg ; groupe 0 : 6.38 ± 1.13  mg.mL-1). De fait, les différences de productions cumulées des Ig entre les groupes traités et les témoins restent inférieures à 10 % en fin d'expérience. La protéinémie suit une évolution similaire à celle des Ig, mais pour le groupe 1, elle se rétablit dès la fin de la période d'intoxication (groupe 2 : 46,49 ± 2,34  mg.mL-1 ; groupe 0 : 46,74 ± 1,97  mg.mL-1). Sur la durée totale de l'expérience, les productions globales sont équivalentes entre le groupe 1 et le groupe 0 mais restent inférieures pour le groupe 2. L'étude des protéinogrammes révèle un glissement vers les protéines de faible poids moléculaire et l'émergence d'un nouveau pic en fin de période d'intoxication. Toutes ces modifications disparaissent au cours de la période de récupération. Les modifications protéiques et des Ig, consécutives à une intoxication à l'ammoniaque, de période limitée (2 mois) et pour des concentrations égales ou inférieures à 20 % de la 96-h DL50, disparaissent après quelques mois de récupération en conditions standard d'élevage.

Introduction

Under intensive rearing conditions, and particularly when the water is recycled, the ammonia concentration in the water may increase. The ammonia in the water is the sum of two forms, the unionized fraction NH3 and the ionized fraction NH4+. The relative proportion of the two forms depends on pH, temperature and salinity. The former is much more toxic than the latter but toxicity is probably due to a contribution of both forms [8]. The convention is to express ammonia concentration in terms of total ammonia nitrogen (TA-N) and unionized ammonia nitrogen (UIA-N). The specific biochemical mechanism of ammonia toxicity in fish is not fully explained, but it has been shown that ammonia intoxication impairs ATP production, induces a store depletion of polysaccharide and plasma ions, alters the neuronal synaptic transmission, induces leucopenia, erythropenia, inflammation and degeneration of gills and kidneys [8].

The ammonia 96-h LC50 (lethal concentration for 50 % of a population exposed for 96 h to ammonia) ranges from 0.32  mg.L-1UIA-N in rainbow trout (Oncorhynchus mykiss) to 3.1  mg.L-1UIA-N in channel catfish (Ictalarus punctatus) [8]. The 96-h LC50 for striped bass (Morone saxatilis) is 1.01  mg.L-1UIA-N and 0.64  mg.L-1UIA-N for another freshwater dicentrarchid, the hybrid striped bass (M. saxatilis × M. chrysops) [11].

Although data in sea water are still scarce, the 96-h LC50 is 1.70  mg.L-1UIA-N for the sea bass juveniles (Dicentrarchus labrax), 2.55  mg.L-1UIA-N for the both sea bream (Sparus aurata) and turbot (Psetta maxima) [13] and 0.45  mg.L-1UIA-N for fingerling coho salmon (Oncorhynchus kisutch) [3].

An ammonia molecule is too small to act as an antigen and cannot directly modify the immune system by acting in the usual way. Moreover, it is a natural endogenous product of fish. However, during acute ammonia poisoning in fingerling coho salmon, high ammonia would be considered to act indirectly on the immune system through its toxic action or as a stressor [10].

The immune response can be modified by several stessors. Both defence mechanisms and non-specific activity may be affected [1]. However, sometimes polluant stressors may enhance certain defence parameters, e.g. an increase in circulating antibodies in the serum of striped bass exposed to water with small amounts of cadmium [15]. More recently, it has been found that antibody concentrations, after vaccination of rainbow trout against Streptococcus iniae, are higher in fish exposed to ammonia; however, there was no correlation with protection [6].

The aim of this study was to examine the correlation between chronic ammonia exposure and alteration in Ig, and total protein concentrations or in the protein molecular weight profile (PMWP), as well as evaluating, by a non-lethal procedure, the capability of sea bass to compensate for these alterations.

Section snippets

Fish

For the duration of the experiment, the fish were reared in a soundproofed room with controlled light and environmental parameters. Each tank (1 m3) received a flow of 1  m3.h-1of sea water, which was filtered (20 mm) and UV sterilized and contained the selected ammonia concentration. Temperature was kept at 22 ± 1 °C, inlet O2 was maintained between 120 and 150 % and outlet over 80 % of saturation [8].

The sea bass were provided by our Ifremer laboratory holding facilities. They were fed on a

Duplicate

Each group was followed in duplicate and non-statistical differences were found between duplicates for protein or Ig concentrations (0.94 < P < 0.09 with 28 degrees of freedom). Consequently, data from the duplicate groups were pooled and treated as one group for further analysis.

Serum concentration (table I)

On day 21 of the exposure period the protein concentrations ( figure 1, lines) of the treated groups were significantly lower than the control group concentration (group 0: 40.74 ± 1.70  mg.mL-1; group 1: 37.24 ± 1.84 

Discussion

First of all, a variation in protein and Ig concentrations versus time of the control group was observed. Protein concentration alterations over the year have been recorded in sea bass broodstock [5]. A regular increase has also been observed in protein and Ig serum concentrations in sea bass fry from 40 up to 200 g in weight, with fluctuation before stabilization around 350 g in weight.

Actually, most agents investigated as stress have other detrimental actions (infection, toxicity, etc.) [1].

Acknowledgements

We are grateful to Dr Stuart Hetherington of Mannin Seafarm, Isle of Man for critical reading of the manuscript.

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