- Email: [email protected]
Antigenicity of Pseudodactylogyrus anguillae and P. bini (Monogenea) in the European eel (Anguilla anguilla, L.) under different oxygenation conditions
Fish & Shellfish Immunology (2002) 13, 125–131 doi:10.1006/fsim.2001.0387 Available online at http://www.idealibrary.com on
Antigenicity of Pseudodactylogyrus anguillae and P. bini (Monogenea) in the European eel (Anguilla anguilla, L.) under different oxygenation conditions G. MONNI AND A. M. COGNETTI-VARRIALE Department of Animal Pathology, and Prophylaxis and Hygiene of Food, University of Pisa, Viale delle Piagge, 2—56124 Pisa, Italy (Received 11 June 2001, accepted after revision 26 October 2001, published electronically 2001) The antibody response of European eels (Anguilla anguilla, L.) to the branchial parasites Pseudodactylogyrus anguillae and P. bini under hyperoxygenation conditions was studied. The antigenic fractions of parasites were detected by means of electrophoretic techniques (SDS-PAGE) and by Western blot analysis. The results obtained demonstrate that under hyperoxygenation conditions, the eels responded to a greater number of proteins, and this was correlated with a decrease in the level of infestation. 2002 Elsevier Science Ltd. All rights reserved.
Key words: Anguilla anguilla, Pseudodactylogyrus anguillae, Pseudodactylogyrus bini, Monogenea, antibody response, antigen, oxygenation.
I. Introduction The monogenean branchial parasites Pseudodactylogyrus anguillae and P. bini produce di#use, congenerous parasitosis in both free-living and farm-bred eels, particularly in Anguilla anguilla [1]. The biological cycle of Pseudodactylogyrus is direct and greatly influenced by the temperature of the water. Temperature governs both the quantity and time of hatching of the eggs and, consequently, the entire life-cycle of the parasite [2]. Prevalence and abundance of Pseudodactylogyrus anguillae are at their highest in England in summer [3]. Environmental factors also influence the antibody response of fish. At low temperatures, fish produce lower titres of antibodies [4] and low levels of dissolved oxygen cause an increase in plasma cortisol and catecholamine, which negatively influence the immune response [5, 6]. Information regarding the influence of hyperoxygenation on infested fish, and in particular on the e#ects of high concentrations of dissolved oxygen on their immune systems, is sparse or even lacking. This investigation studied the antibody response of Anguilla anguilla to the antigens of Pseudodactylogyrus anguillae and P. bini in a comparison between hyperoxygenation and normal aeration conditions. 1050–4648/02/$-see front matter
125 2002 Elsevier Science Ltd. All rights reserved.
126
G. MONNI AND A. M. COGNETTI-VARRIALE
II. Materials and Methods FISH
Eels, A. anguilla (mean size 60·83 g), were collected from a fish farm (Novelli, Albinia, Italy) in which no anti-parasite measures had been taken. The eels were divided into three groups of 35 specimens each: the first group was examined immediately, whereas the other two groups were kept for 60 days in two 300 litre PVC tanks. The experimental conditions were: mean temperature 12 C; oxygenation (one tank had normal oxygenation—mean dissolved oxygen, DO 5·6 p.p.m., and the other had hyperoxygenation—mean DO, 14·6 p.p.m.), with pure oxygen in gaseous form measured daily by means of a microprocessor oxy-meter (OXI 320 SET, WTW). PREPARATION OF BLOOD SAMPLES
Blood was withdrawn from the caudal vein of each eel, previously anaesthetised with 0·5 ml/l of 2-Phenoxy ethanol (Sigma P-1126) and the samples were kept at room temperature for 1 h, then at 4 C over night. Thereafter, they were centrifuged first at 2500 g for 10 min then at 2000 g for a further 10 min. The serum obtained was stored at 20 C. REMOVAL AND TREATMENT OF THE PARASITES
After removal and immediate examination of the gills of all the eels, the branchial parasites were isolated, classified, counted and divided according to two stages of maturity: post-larvae and adults. Two pools of antigen were made: one containing equal quantities of adult P. anguillae and P. bini and the other containing the post-larvae. These pools were then sonicated (Vibracell sonifier, Sonics & Materials Inc.) for three 30-s cycles and the homogenised material obtained was stored at 20 C. PRODUCTION OF ANTI-EEL IGM POLYCLONAL SERUM
The anti-eel IgM polyclonal serum was obtained by using the method described by Mazzanti et al. [7] and to achieve this, 24 eels, weighing an average of 180 g were captured from the Orbetello Lagoon (Tuscany, Italy). The fish were then kept in four thermostatically controlled tanks (temperature, 15 C; salinity, 12‰, oxygen 8 p.p.m.) with a capacity of 80 l each and equipped with oxygen-pumps. After allowing the eels to adjust for approximately 20 days, they were immunised with human dinitrophenylated human serum albumin (DNP-albumin Sigma catalogue no. A-6661) according to the method indicated by Buchmann et al. [8]. The collected sera were then purified on a$nity columns, coupled to dinitrophenylated bovine serum albumin (DNP-BSA) (prepared with Bio-Rad 10 A$-Gel) with NaHCO3 (0·1 M) as bu#er. After rinsing several times with 0·1 M Tris, the IgM was collected by means of eluting with an acid bu#er (pH 2·1) of 0·1 M glycine and immediately neutralised with 0·1 M Tris. Spectrophotometry revealed the fraction that contained a total of 0·25 mg eel IgM/ml.
ANTIGENICITY OF MONOGENEA IN THE EUROPEAN EEL
127
Table 1. Chart showing the rabbit immunisation programme
1st immunisation 2nd immunisation 3rd immunisation 4th immunisation 5th immunisation
Eel IgM
Emulsifier
250 g 200 g 112.5 g 100 g 100 g
1 ml complete Freund adjuvant 0.8 ml complete Freund adjuvant 0.45 ml incomplete Freund adjuvant 0.4 ml incomplete Freund adjuvant 0.4 ml PBS
The purified eel IgM was used for immunising two female New Zealand rabbits. Each rabbit was injected subcutaneously with a total of five immunisations (Table 1); the second injection was performed 3 weeks after the first then the others were administered at 2-week intervals. Ten days after the last antigenic stimulation, the rabbits were anaesthetised and blood collected. This was kept at room temperature for 1 h, then at 4 C overnight; following this, it was centrifuged at 2000 g for 15 min and at 3000 g for a further 15 min, and the serum obtained was stored at 20 C. In order to confirm the antibody titre, the two eel anti-IgM sera obtained were assayed by means of dot-blotting: a rapid technique for immunological detection that indicates whether the serum prepared in the rabbits responds to the antigen we are interested in (IgM) and with which titre it responds. The antibody titre tested this way resulted to be positive as far as 1:25 000 dilutions.
ELECTROPHORESIS AND WESTERN BLOTTING ANALYSIS
Proteins from sonicated parasites were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method [9] using a Mini Protean II dual slab gel system (Bio-Rad). The samples were mixed with an equal volume of reducing bu#er and heated at 70 C for 10 min. Molecular weight standards (Low Range Bio Rad catalogue no. 161-0304 and Broad Range Bio-Rad catalogue no. 161-0318) and samples (10 g of parasite protein) were loaded onto a stacking gel and electrophoresed into a 12% polyacrylamide gel. All sera from all eels were tested for reaction to antigens of Pseudodactylogyrus spp. using Western Blot analysis [10]. The proteins separated by SDS-PAGE from sonicated parasites were electroblotted onto a nitro-cellulose membrane which was then cut into strips and incubated in the serum from each individual eel. The reaction between Pseudodactylogyrus antigens and eel immunoglobulins was detected with rabbit anti-eel IgM serum, diluted 1:1000 in blocking bu#er for 2 h, and then with peroxidaseconjugated goat anti-rabbit IgG (Sigma catalogue no. A 4914), diluted 1:2000 in blocking bu#er for 1 h. The strips were developed in a solution of 30 mg of 4-chloro-1-naphthol (Fluka catalogue no. 25328) in 10 ml methyl alcohol, 50 ml deionized water and 30 l 30% hydrogen peroxide.
128
G. MONNI AND A. M. COGNETTI-VARRIALE
Fig. 1. Coomassie blue stained 12% SDS-PAGE slab of sonicated adult Pseudodactylogyrus anguillae and P. bini (left lane) and molecular weight standards in kDa (right lane).
Fig. 2. Western blot of adult Pseudodactylogyrus spp. proteins incubated with sera from eels kept in hyperoxygenation conditions (left: standard molecular weights in kDa).
III. Results The abundance of P. anguillae together with P. bini in the eels on arrival at the laboratory was 8·77. Sixty day later, the abundance in the hyperoxygenated group was 2·89, and that in the normally aerated tank, 8·22. These data were statistically analysed by the Student’s t-test and by means of a non-parametric test (median test), and revealed a significant di#erence between the hyperoxygenation condition and the other two groups. Electrophoretic analysis of the sonicated Pseudodactylogyrus spp. proteins in SDS-PAGE revealed a series of bands with molecular weights ranging between 14 and 97 kDa (Fig. 1). All the sera from hyperoxygenated eels (analysed by means of Western blotting) recognised the following bands as antigenic fractions of adult Pseudodactylogyrus spp: 1 at 81 kDa, 1 between 51·2 and 81 kDa, 1 at 51·2 kDa, 1 between 33·6 and 51·2 kDa and 1 at 28·6 kDa (Fig. 2). On the other hand the sera from normally aerated eels recognised only two bands, with 81 kDa and between 51·2 and 33·6 kDa (Fig. 3). Regarding Western Blots of the Pseudodactylogyrus post-larvae, with eel serum from both types of oxygenation, recognised only one protein fraction with a molecular weight between 51·2 and 81 kDa (Fig. 4).
ANTIGENICITY OF MONOGENEA IN THE EUROPEAN EEL
129
Fig. 3. Western blot of adult Pseudodactylogyrus spp. proteins incubated with sera from eels kept in normal aeration conditions (left: standard molecular weights in kDa).
Fig. 4. Western blot of post-larvae Pseudodactylogyrus spp. proteins incubated with eel sera (right: standard molecular weights in kDa).
IV. Discussion This investigation to assess the response of Anguilla anguilla, reared in di#erent oxygenation conditions, to the proteins of Pseudodactylogyrus anguillae and P. bini, contributes to understanding the influence of oxygenation on the intensity of infestation and of the antibody repertoire produced by the eels. Infestation underwent a decrease in the number of parasites in the eels kept in hyperoxygenation conditions, which might be related to the fish adjusting to the hyperoxygenation by reducing their total respiratory surface, as Saroglia et al. [11] observed in sea bass. The higher number of antigens recognised by the sera of eels kept in hyperoxygenated water may also have contributed to the decrease in the quantity of parasites.
130
G. MONNI AND A. M. COGNETTI-VARRIALE
Some authors [12] maintain that the use of pure oxygen is capable of significantly improving production due to a better oxidation of the organic substances deriving from the catabolism of farm-bred fish, and that this improves their well-being and leads, consequently, to an increase in their immune response. Higher oxygenation may lead to greater sources of energy being available for specific immune responses against pathogens that can become virulent during stressing events [11]. This might be corroborated by the data reported by Mazzini et al. [13] in evaluation of the total immunoglobulin concentration in sea bass serum that was influenced not only by the age of the fish and by the time of year, but also by the level of oxygenation, which facilitated an increase in Ig concentration. The fact that the eel sera recognised only one protein fraction of the P. anguillae and P. bini post-larvae demonstrates that young parasites induce a low specific immune response and that adults induce antibody responses to a greater number of antigens, as reported by Abbas et al. [14] regarding infestations of homeothermic animals. These authors reported that parasites are capable of developing advanced strategies to avoid immune responses by their hosts, by either modifying the surface antigens during their life cycle or by developing protein fractions with antigenic activity only later in their life. This agrees with Zahner et al. [15], who indicated that as a parasite grows its protein fractions mature and undergo post-translational modifications which become strong protective shields against its host’s antibodies.
References 1 Ogawa, K. & Egusa, S. (1976). Studies on eel Pseudodactylogyrosis—I. Morphology and classification of three eel dactylogiridis with a proposal of a new species, Pseudodactylogyrus microrchis. Bulletin of the Japanese Society of Scientific Fisheries 42, 395–404. 2 Buchmann, K. (1988). Temperature dependent reproduction and survival of the Pseudodactylogyrus bini (Monogenea) on the European eel (Anguilla anguilla). Parasitology Research 75, 162–164. 3 Nie, P. & Kennedy, C. R. (1991). Occurrence and seasonal dynamics of Pseudodactylogyrus anguillae (Yin & Sproston) (Monogenea) in eel, Anguilla anguilla (L.), in England. Journal Fish of Biology 39, 897–900. 4 Rijkers, G. T., Wiegerinck, J. A. M., Van Oosterom, R. & Van Muiswinkel, W. B. (1981). Temperature dependence of humoral immunity in carp (Cyprinus carpio). In Aspects of Developmental and Comparative Immunology I (J. B. Solomon, eds) pp. 477–482. New York: Pergamon. 5 Kakuta, I. & Murachi, S. (1992). E#ects of hypoxia on renal function in carp. (63th Annual Meeting of the Zoological Society of Japan, Sendai, Japan, 7–8 October 1992). Zoological Science 9, 1250. 6 Sorensen, B. & Weber, R. E. (1995). E#ects of oxygenation and the stress hormones adrenaline and cortisol on the viscosity of blood from the trout Oncorhynchus mykiss. Journal of Experimental Biology 198, 953–959. 7 Mazzanti, C., Cecchini, S., Mameli, M., Cgnetti-Varriale, A. M. & Saroglia, M. (1996). Purification of seabass IgM (Dicentrarchus labrax, L.) and seabream IgM (Sparus aurata, L.) and production of polyclonal serum. Atti della Società Italiana delle Scienze Veterinarie L, 287–288. 8 Buchmann, K., Ostergaard, L. & Glamann, J. (1992). A$nity purification of antigen-specific serum immunoglobulin from the European eel (Anguilla anguilla). Scandinavian Journal of Immunology 36, 89–97.
ANTIGENICITY OF MONOGENEA IN THE EUROPEAN EEL
131
9 Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 10 Towbin, H., Stachelin, P. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76, 4350–4354. 11 Saroglia, M., Cecchini, S., Terova, G., Caricato, G. & De Stradis, A. (1998). Adaptative mechanisms and zootechnical performances of euryhaline fish species reared under hyperoxia conditions: first results of a study. Biologia Marina Mediterranea Parte seconda, Acquacoltura 5(3), 1688–1698. 12 Saroglia, M., Knight, M., Terova-Saroglia, G. & Cecchini, S. (1998). Role of water hyperoxygenation and feed ratio on the minimization of nitrogen release by intensive sea bass (Dicentrarchus labrax, L.) farming. Biologia Marina Mediterranea Parte seconda, Acquacoltura 5, 1669–1674. 13 Mazzini, M., Scapigliati, G., Abelli, L., Terribili, F. R., Scalia, D., Fanelli, M., Fausto, A. M., Mastrolia, L. & Romano, N. (1998). A study on the e#ect of hyperoxygenation on the immune system of euryaline fish in aquaculture. Biologia Marina Mediterranea Parte seconda, Acquacoltura 5, 1633–1641. 14 Abbas, A. K., Lichtman, A. H. & Pober, J. S. (1998). In Immunologia cellulare e molecolare. Ed. Piccin. 15 Zahner, H., Hobom, G. & Stim, S. (1995). The microfilarial sheath and its proteins. Parasitology Today 11, 116–120.
Antigenicity of Pseudodactylogyrus anguillae and P. bini (Monogenea) in the European eel (Anguilla anguilla, L.) under different oxygenation conditions G. MONNI AND A. M. COGNETTI-VARRIALE Department of Animal Pathology, and Prophylaxis and Hygiene of Food, University of Pisa, Viale delle Piagge, 2—56124 Pisa, Italy (Received 11 June 2001, accepted after revision 26 October 2001, published electronically 2001) The antibody response of European eels (Anguilla anguilla, L.) to the branchial parasites Pseudodactylogyrus anguillae and P. bini under hyperoxygenation conditions was studied. The antigenic fractions of parasites were detected by means of electrophoretic techniques (SDS-PAGE) and by Western blot analysis. The results obtained demonstrate that under hyperoxygenation conditions, the eels responded to a greater number of proteins, and this was correlated with a decrease in the level of infestation. 2002 Elsevier Science Ltd. All rights reserved.
Key words: Anguilla anguilla, Pseudodactylogyrus anguillae, Pseudodactylogyrus bini, Monogenea, antibody response, antigen, oxygenation.
I. Introduction The monogenean branchial parasites Pseudodactylogyrus anguillae and P. bini produce di#use, congenerous parasitosis in both free-living and farm-bred eels, particularly in Anguilla anguilla [1]. The biological cycle of Pseudodactylogyrus is direct and greatly influenced by the temperature of the water. Temperature governs both the quantity and time of hatching of the eggs and, consequently, the entire life-cycle of the parasite [2]. Prevalence and abundance of Pseudodactylogyrus anguillae are at their highest in England in summer [3]. Environmental factors also influence the antibody response of fish. At low temperatures, fish produce lower titres of antibodies [4] and low levels of dissolved oxygen cause an increase in plasma cortisol and catecholamine, which negatively influence the immune response [5, 6]. Information regarding the influence of hyperoxygenation on infested fish, and in particular on the e#ects of high concentrations of dissolved oxygen on their immune systems, is sparse or even lacking. This investigation studied the antibody response of Anguilla anguilla to the antigens of Pseudodactylogyrus anguillae and P. bini in a comparison between hyperoxygenation and normal aeration conditions. 1050–4648/02/$-see front matter
125 2002 Elsevier Science Ltd. All rights reserved.
126
G. MONNI AND A. M. COGNETTI-VARRIALE
II. Materials and Methods FISH
Eels, A. anguilla (mean size 60·83 g), were collected from a fish farm (Novelli, Albinia, Italy) in which no anti-parasite measures had been taken. The eels were divided into three groups of 35 specimens each: the first group was examined immediately, whereas the other two groups were kept for 60 days in two 300 litre PVC tanks. The experimental conditions were: mean temperature 12 C; oxygenation (one tank had normal oxygenation—mean dissolved oxygen, DO 5·6 p.p.m., and the other had hyperoxygenation—mean DO, 14·6 p.p.m.), with pure oxygen in gaseous form measured daily by means of a microprocessor oxy-meter (OXI 320 SET, WTW). PREPARATION OF BLOOD SAMPLES
Blood was withdrawn from the caudal vein of each eel, previously anaesthetised with 0·5 ml/l of 2-Phenoxy ethanol (Sigma P-1126) and the samples were kept at room temperature for 1 h, then at 4 C over night. Thereafter, they were centrifuged first at 2500 g for 10 min then at 2000 g for a further 10 min. The serum obtained was stored at 20 C. REMOVAL AND TREATMENT OF THE PARASITES
After removal and immediate examination of the gills of all the eels, the branchial parasites were isolated, classified, counted and divided according to two stages of maturity: post-larvae and adults. Two pools of antigen were made: one containing equal quantities of adult P. anguillae and P. bini and the other containing the post-larvae. These pools were then sonicated (Vibracell sonifier, Sonics & Materials Inc.) for three 30-s cycles and the homogenised material obtained was stored at 20 C. PRODUCTION OF ANTI-EEL IGM POLYCLONAL SERUM
The anti-eel IgM polyclonal serum was obtained by using the method described by Mazzanti et al. [7] and to achieve this, 24 eels, weighing an average of 180 g were captured from the Orbetello Lagoon (Tuscany, Italy). The fish were then kept in four thermostatically controlled tanks (temperature, 15 C; salinity, 12‰, oxygen 8 p.p.m.) with a capacity of 80 l each and equipped with oxygen-pumps. After allowing the eels to adjust for approximately 20 days, they were immunised with human dinitrophenylated human serum albumin (DNP-albumin Sigma catalogue no. A-6661) according to the method indicated by Buchmann et al. [8]. The collected sera were then purified on a$nity columns, coupled to dinitrophenylated bovine serum albumin (DNP-BSA) (prepared with Bio-Rad 10 A$-Gel) with NaHCO3 (0·1 M) as bu#er. After rinsing several times with 0·1 M Tris, the IgM was collected by means of eluting with an acid bu#er (pH 2·1) of 0·1 M glycine and immediately neutralised with 0·1 M Tris. Spectrophotometry revealed the fraction that contained a total of 0·25 mg eel IgM/ml.
ANTIGENICITY OF MONOGENEA IN THE EUROPEAN EEL
127
Table 1. Chart showing the rabbit immunisation programme
1st immunisation 2nd immunisation 3rd immunisation 4th immunisation 5th immunisation
Eel IgM
Emulsifier
250 g 200 g 112.5 g 100 g 100 g
1 ml complete Freund adjuvant 0.8 ml complete Freund adjuvant 0.45 ml incomplete Freund adjuvant 0.4 ml incomplete Freund adjuvant 0.4 ml PBS
The purified eel IgM was used for immunising two female New Zealand rabbits. Each rabbit was injected subcutaneously with a total of five immunisations (Table 1); the second injection was performed 3 weeks after the first then the others were administered at 2-week intervals. Ten days after the last antigenic stimulation, the rabbits were anaesthetised and blood collected. This was kept at room temperature for 1 h, then at 4 C overnight; following this, it was centrifuged at 2000 g for 15 min and at 3000 g for a further 15 min, and the serum obtained was stored at 20 C. In order to confirm the antibody titre, the two eel anti-IgM sera obtained were assayed by means of dot-blotting: a rapid technique for immunological detection that indicates whether the serum prepared in the rabbits responds to the antigen we are interested in (IgM) and with which titre it responds. The antibody titre tested this way resulted to be positive as far as 1:25 000 dilutions.
ELECTROPHORESIS AND WESTERN BLOTTING ANALYSIS
Proteins from sonicated parasites were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method [9] using a Mini Protean II dual slab gel system (Bio-Rad). The samples were mixed with an equal volume of reducing bu#er and heated at 70 C for 10 min. Molecular weight standards (Low Range Bio Rad catalogue no. 161-0304 and Broad Range Bio-Rad catalogue no. 161-0318) and samples (10 g of parasite protein) were loaded onto a stacking gel and electrophoresed into a 12% polyacrylamide gel. All sera from all eels were tested for reaction to antigens of Pseudodactylogyrus spp. using Western Blot analysis [10]. The proteins separated by SDS-PAGE from sonicated parasites were electroblotted onto a nitro-cellulose membrane which was then cut into strips and incubated in the serum from each individual eel. The reaction between Pseudodactylogyrus antigens and eel immunoglobulins was detected with rabbit anti-eel IgM serum, diluted 1:1000 in blocking bu#er for 2 h, and then with peroxidaseconjugated goat anti-rabbit IgG (Sigma catalogue no. A 4914), diluted 1:2000 in blocking bu#er for 1 h. The strips were developed in a solution of 30 mg of 4-chloro-1-naphthol (Fluka catalogue no. 25328) in 10 ml methyl alcohol, 50 ml deionized water and 30 l 30% hydrogen peroxide.
128
G. MONNI AND A. M. COGNETTI-VARRIALE
Fig. 1. Coomassie blue stained 12% SDS-PAGE slab of sonicated adult Pseudodactylogyrus anguillae and P. bini (left lane) and molecular weight standards in kDa (right lane).
Fig. 2. Western blot of adult Pseudodactylogyrus spp. proteins incubated with sera from eels kept in hyperoxygenation conditions (left: standard molecular weights in kDa).
III. Results The abundance of P. anguillae together with P. bini in the eels on arrival at the laboratory was 8·77. Sixty day later, the abundance in the hyperoxygenated group was 2·89, and that in the normally aerated tank, 8·22. These data were statistically analysed by the Student’s t-test and by means of a non-parametric test (median test), and revealed a significant di#erence between the hyperoxygenation condition and the other two groups. Electrophoretic analysis of the sonicated Pseudodactylogyrus spp. proteins in SDS-PAGE revealed a series of bands with molecular weights ranging between 14 and 97 kDa (Fig. 1). All the sera from hyperoxygenated eels (analysed by means of Western blotting) recognised the following bands as antigenic fractions of adult Pseudodactylogyrus spp: 1 at 81 kDa, 1 between 51·2 and 81 kDa, 1 at 51·2 kDa, 1 between 33·6 and 51·2 kDa and 1 at 28·6 kDa (Fig. 2). On the other hand the sera from normally aerated eels recognised only two bands, with 81 kDa and between 51·2 and 33·6 kDa (Fig. 3). Regarding Western Blots of the Pseudodactylogyrus post-larvae, with eel serum from both types of oxygenation, recognised only one protein fraction with a molecular weight between 51·2 and 81 kDa (Fig. 4).
ANTIGENICITY OF MONOGENEA IN THE EUROPEAN EEL
129
Fig. 3. Western blot of adult Pseudodactylogyrus spp. proteins incubated with sera from eels kept in normal aeration conditions (left: standard molecular weights in kDa).
Fig. 4. Western blot of post-larvae Pseudodactylogyrus spp. proteins incubated with eel sera (right: standard molecular weights in kDa).
IV. Discussion This investigation to assess the response of Anguilla anguilla, reared in di#erent oxygenation conditions, to the proteins of Pseudodactylogyrus anguillae and P. bini, contributes to understanding the influence of oxygenation on the intensity of infestation and of the antibody repertoire produced by the eels. Infestation underwent a decrease in the number of parasites in the eels kept in hyperoxygenation conditions, which might be related to the fish adjusting to the hyperoxygenation by reducing their total respiratory surface, as Saroglia et al. [11] observed in sea bass. The higher number of antigens recognised by the sera of eels kept in hyperoxygenated water may also have contributed to the decrease in the quantity of parasites.
130
G. MONNI AND A. M. COGNETTI-VARRIALE
Some authors [12] maintain that the use of pure oxygen is capable of significantly improving production due to a better oxidation of the organic substances deriving from the catabolism of farm-bred fish, and that this improves their well-being and leads, consequently, to an increase in their immune response. Higher oxygenation may lead to greater sources of energy being available for specific immune responses against pathogens that can become virulent during stressing events [11]. This might be corroborated by the data reported by Mazzini et al. [13] in evaluation of the total immunoglobulin concentration in sea bass serum that was influenced not only by the age of the fish and by the time of year, but also by the level of oxygenation, which facilitated an increase in Ig concentration. The fact that the eel sera recognised only one protein fraction of the P. anguillae and P. bini post-larvae demonstrates that young parasites induce a low specific immune response and that adults induce antibody responses to a greater number of antigens, as reported by Abbas et al. [14] regarding infestations of homeothermic animals. These authors reported that parasites are capable of developing advanced strategies to avoid immune responses by their hosts, by either modifying the surface antigens during their life cycle or by developing protein fractions with antigenic activity only later in their life. This agrees with Zahner et al. [15], who indicated that as a parasite grows its protein fractions mature and undergo post-translational modifications which become strong protective shields against its host’s antibodies.
References 1 Ogawa, K. & Egusa, S. (1976). Studies on eel Pseudodactylogyrosis—I. Morphology and classification of three eel dactylogiridis with a proposal of a new species, Pseudodactylogyrus microrchis. Bulletin of the Japanese Society of Scientific Fisheries 42, 395–404. 2 Buchmann, K. (1988). Temperature dependent reproduction and survival of the Pseudodactylogyrus bini (Monogenea) on the European eel (Anguilla anguilla). Parasitology Research 75, 162–164. 3 Nie, P. & Kennedy, C. R. (1991). Occurrence and seasonal dynamics of Pseudodactylogyrus anguillae (Yin & Sproston) (Monogenea) in eel, Anguilla anguilla (L.), in England. Journal Fish of Biology 39, 897–900. 4 Rijkers, G. T., Wiegerinck, J. A. M., Van Oosterom, R. & Van Muiswinkel, W. B. (1981). Temperature dependence of humoral immunity in carp (Cyprinus carpio). In Aspects of Developmental and Comparative Immunology I (J. B. Solomon, eds) pp. 477–482. New York: Pergamon. 5 Kakuta, I. & Murachi, S. (1992). E#ects of hypoxia on renal function in carp. (63th Annual Meeting of the Zoological Society of Japan, Sendai, Japan, 7–8 October 1992). Zoological Science 9, 1250. 6 Sorensen, B. & Weber, R. E. (1995). E#ects of oxygenation and the stress hormones adrenaline and cortisol on the viscosity of blood from the trout Oncorhynchus mykiss. Journal of Experimental Biology 198, 953–959. 7 Mazzanti, C., Cecchini, S., Mameli, M., Cgnetti-Varriale, A. M. & Saroglia, M. (1996). Purification of seabass IgM (Dicentrarchus labrax, L.) and seabream IgM (Sparus aurata, L.) and production of polyclonal serum. Atti della Società Italiana delle Scienze Veterinarie L, 287–288. 8 Buchmann, K., Ostergaard, L. & Glamann, J. (1992). A$nity purification of antigen-specific serum immunoglobulin from the European eel (Anguilla anguilla). Scandinavian Journal of Immunology 36, 89–97.
ANTIGENICITY OF MONOGENEA IN THE EUROPEAN EEL
131
9 Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 10 Towbin, H., Stachelin, P. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76, 4350–4354. 11 Saroglia, M., Cecchini, S., Terova, G., Caricato, G. & De Stradis, A. (1998). Adaptative mechanisms and zootechnical performances of euryhaline fish species reared under hyperoxia conditions: first results of a study. Biologia Marina Mediterranea Parte seconda, Acquacoltura 5(3), 1688–1698. 12 Saroglia, M., Knight, M., Terova-Saroglia, G. & Cecchini, S. (1998). Role of water hyperoxygenation and feed ratio on the minimization of nitrogen release by intensive sea bass (Dicentrarchus labrax, L.) farming. Biologia Marina Mediterranea Parte seconda, Acquacoltura 5, 1669–1674. 13 Mazzini, M., Scapigliati, G., Abelli, L., Terribili, F. R., Scalia, D., Fanelli, M., Fausto, A. M., Mastrolia, L. & Romano, N. (1998). A study on the e#ect of hyperoxygenation on the immune system of euryaline fish in aquaculture. Biologia Marina Mediterranea Parte seconda, Acquacoltura 5, 1633–1641. 14 Abbas, A. K., Lichtman, A. H. & Pober, J. S. (1998). In Immunologia cellulare e molecolare. Ed. Piccin. 15 Zahner, H., Hobom, G. & Stim, S. (1995). The microfilarial sheath and its proteins. Parasitology Today 11, 116–120.