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Serum immunoglobulin M (IgM) during early development of masu salmon (Oncorhynchus masou)
0300-9629/91$3.00+ 0.00 c 1991 Pergamon Press plc
Camp. Biochem. Physiol. Vol. 99A, No. 4, pp. 631-643, 1991 Printed in Great Britain
SERUM IMMUNOGLOBULIN M (IgM) DURING EARLY DEVELOPMENT OF MASU SALMON (ONCORHYNCHUS HIROTOSHI
FUDA,*
KIYOSHI
MASOU)
SOYANO, * FUMIO YAMAZAKI*
and
AKIHIKO
HARAt
*Department
of Biology and Aquaculture, Faculty of Fisheries, Hokkaido University, Minato, Hakodate, Hokkaido 041, Japan; tNanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University, Nanae, Kameda, Hokkaido 041-l 1, Japan (Received 30 October 1990) Abstract-l.
The development of serum immunoglobulin M (IBM) was studied by measuring IgM concentration in early developmental stages of masu salmon (Oncorhynchusmasou) using single radial immunodiffusion. 2. IgM concentration was measurable from 88 days after hatching (46.5 pg/ml) and was maintained at a relatively constant level (< 100 pg/ml) until 235 days after hatching. 3. IgM concentration increased significantly to a level of 691 + 37 pg/ml (mean + SE, N = 59) during the period from 251 to 429 days after hatching. The highest level of serum IgM was maintained from 429 days to 489 days after hatching. 4. The increase of IgM concentration was not accompanied by parallel increases in total serum protein concentration.
INTRODUCTION It is assumed that the essential function of the immunoglobulin (Ig) in vertebrates is defense against foreign antigens, for example infectious agents etc. Although five major structural classes of Ig, namely IgG, IgM, IgA, IgD and IgE, can be distinguished in
man, only one class has been identified in fish excepting cartilaginous fish belong to the order Rajiformes (Kobayashi et al., 1984, Kobayashi and Tomonaga, 1988). The fish immunoglobulin is classified as an IgM because of its similarities to mammalian IgM with respect to subunit structure, electrophoretic mobility (p-region), and the molecular weight of the intact molecule and its heavy chain (Kobayashi et al., 1982; Olesen and Jorgensen, 1986). Many studies of fish immunobiology have dealt with the phylogeny of immunoglobulin structure and function coupled with investigations into the factors affecting the immune response after antigenic stimulation (Ingram, 1980). However, there have been few studies of IgM concentration in fish serum from the point of view of physiology and fish pathology. The measurement of serum IgM concentration is a required method to obtain basic information on the immune system and for epidemiologic research. IgM concentrations have been measured in serum from the nurse shark, Ginglymostoma cirratum (Fidler et al., 1969), paddlefish, Polyodon spathula (Acton et al., 1971b, Legler et al., 1971), gar, Lepisosteus osseus (Acton ef al., 1971a), carp, Cyprinus carpio (Richter et al., 1973, Vilain et al., 1984), brown trout, Sulmo trutta (Ingram and Alexander, 1979), coho salmon, Oncorhynchus kisutch (Voss et al., 1980), chum salmon, Oncorhynchus keta (Kobayashi et al., 1982), goldfish, Curassius auratus and tenth, Tinca tinca (Vilain et al., 1984), yellowtail, Seriola quinqueradiuta (Matsubara et al., 1985), rainbow trout,
Oncorhynchus mykiss (Olesen and Jargensen, 1986) and channel catfish, Ictalurus punctatus (Ourth, 1986). Previously, we reported the purification of masu salmon (Oncorhynchus masou) IgM by a combination of salting-out, ion-exchange chromatography and gel filtration (Fuda et al., 1989). The present paper describes changes in serum IgM concentration from 46 days to 489 days after hatching and the relationship between serum IgM concentration, plasma protein levels, and body weight. MATERIALS
AND METHODS
Young masu salmon (Oncorhynchus masou) used for measurement of serum IgM were hatched and reared at the Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University. Adult fish were from Mori branch, Hokkaido Fish Hatchery. Blood was collected from hatched larvae and fry by severing their caudal peduncle. Blood was collected from adult fish by cardiac puncture. Blood samples were allowed to stand for several hours, and the serum was separated by centrifugation at 3,000 rpm for 10 min and stored at - 20°C until used for IgM immunoassay or purification. PuriJcation of serum IgM Purification of masu salmon IgM was carried out as described previously (Fuda et al., 1989). Adult masu salmon serum (100 ml) was precipitated by addition of ammonium sulfate at 50% saturation. The precipitate, collected after centrifugation at 10,OOOrpm for IOmin, was dissolved in and dialysed against 0.015 M Tris-HCl buffer, pH 8.0. The dialysate was fractionated on a DEAE cellulose (DE-52, Whatman) column (5 x 5 cm) equilibrated with Tris-HCl buffer, using a stepwise elution with the same buffer containing NaCl at 0.05, 0.1, 0.15, 0.2 and 0.4 M. The fraction eluted by 0.1 M NaCl was reprecipitated with 50% ammonium sulfate, and collected by centrifugation at 637
HIKOTOSHIFUDA
638
10,000 rpm for IOmin. The precipitate was dissolved in 0.02 M Tris-HCl buffer, pH 8.0, containing 2% NaCl and 0. I % NaN, It was then gel filtered through a Sepharose 6B (Pharmacia) column (2.2 x 56.5 cm) using the same buffer. Two distinct peaks were obtained. The first peak, collected and concentrated by lyophilization was again chromatographed on Sepharose 6B. A single and symmetrical peak was obtained and collected as the purified masu salmon IgM. Preparation of antisera Polyvalent antiserum to masu salmon serum was carried out by immunizing rabbits as described previously (Hara, 1976). Specific antiserum to masu salmon IgM was obtained from rabbits immunized with 1 ml of solution containing 25Opg of purified IgM mixed with an equal volume of Freund’s complete adjuvant. The rabbit received four injections at about 7 day intervals. Protein concentration of serum and IgM standard The total serum protein concentration of developing masu salmon was measured using a Bio-Rad Protein Assay kit using bovine serum albumin as a standard. Before assay, the serum was diluted 101 times with 0.02 M Tris-HCl buffer, pH 8.0, containing 2% NaCl and 0.1% NaN,. The protein concentration of the purified IgM was measured using the same kit but bovine IgG was used as the protein standard. Single radial immunod@iision and immunoelectrophore.vis Single radial immunodiffusion (SRID) was carried out according to the procedure of Mancini et al. (1965). Antiserum to purified masu salmon IgM was diluted at 56°C in a solution of I% (w/v) agarose (Litex, type HSA) in a 0.05 M barbital buffer, pH 8.6. Ten millilitres of the hot solution was then layered onto 7 x 1Ocm GelBond film (Pharmacia). The SRID plate was incubated in moist chamber at room temperature for 3 days. After incubation, the plate was washed with 0.9% NaCl, dried on filter paper (Tovo. tvoe 2). stained for 30 set with 1% Amido Black 10B in 7% acetic acid, and destained with 7% acetic acid. For SRID, purified masu salmon IgM standards (25. 50, 100. 200 and 400pg/ml) were used.
et ul.
Immunoelectrophoresis was carried out according to the procedure of Grabar and Williams (1953)with I % agarose in 0.05 M barbital buffer, pH 8.6.
RESULTS AND DISCUSSION
Quantitative
measurement
of IgM
The purified IgM revealed a single precipitin line when reacted against a polyvalent antiserum to masu salmon serum proteins as well as when it was precipitated by the specific antiserum to masu salmon IgM (Fig. I). Conversely, the antiserum raised against the purified IgM preparation gave rise to a single precipitin line with the masu salmon whole serum, suggesting that the IgM preparation was immunologically pure. Different dilutions of antiserum of masu salmon IgM were incorporated into the agarose gel used for SRID. A concentration of 0.5% antiserum gave the best quantitative results (Fig. 2A). Using this dilution, a squared diameter of a precipitate ring was directly proportional to the amounts of IgM present in the sample, in the range of 25400 pg/ml sample as shown in Fig. 2B. In previous studies in which fish IgM was measured by SRID, the lower limit of IgM detection was in the range of 0.0331 mg/ml (Fidler ef al., 1969, Legler e/ al., 1971). Our lower limit of IgM detection was 0.025 mg/ml, similar to the lowest level of IgM detection in the previous reports. On the other hand, Matsubara et al. (1985) recently reported a lower limit of detection of yellowtail IgM of 0.4 +_0.17 ng/test tube by means of enzyme-linked immunosorbent assay (ELISA). The ELISA is generally a more sensitive method than SRID, and the establishment of an ELISA for masu salmon IgM is desirable for future research on IgM in the earliest developmental stages, serum IgM was not measurable in these early stages in the present study.
Fig. I. Results of immunoelectrophoresis of masu salmon serum and their purified IgM. s: masu salmon serum, M: masu salmon IgM, a-s: polyvalent antiserum to masu salmon serum proteins, a-M: specific antiserum to samu salmon IgM.
Serum
IgM during early development
IgM concentration and total serum protein during early development IgM concentration was measured in samples of serum pooled from about 100 fishes from 49 to 138 days after hatching, from about 50 fishes from 154 to 235 days after hatching and in serum samples from individual fish from 251 to 489 days after hatching. Development of IgM concentrations in the serum of masu salmon from 49 to 489 days after hatching is shown in Fig. 3. IgM concentration in the serum was already detectable (~25 pg/ml) 49 days after hatching. It was also measurable (46.5 pg/ml) using SRID from 88 days after hatching onwards. IgM concentrations were maintained at a relatively constant level
639
of masu salmon
(< lOOpg/ml) from 88 days to 235 days after hatching. On the other hand, from 251 days to 429 days after hatching, IgM concentration increased to a level of 691 + 37 pg/ml (mean + SE, N = 59). IgM concentration was highly variable in later developmental stages, but it appeared that a plateau might have been reached by 429 days of development. There was no significant difference between male and female fish IgM concentration during development. IgM concentration was measured in yearling masu salmon for 9 months before their ovulation in Mori Hatchery. The IgM concentrations appeared constant. The mean levels of IgM were 2273 _t 118p g/ml (mean f SE, N = 108) and 2049 k 97 pg/ml (N =
1
25
50
100
200 IgM
400
(pg/ml)
Fig. 2. Quantitative measurement of IgM by single radial immunodiffusion carried out in a 1.4mm thick (7 x IOcm) slab of agarose gel (lo/o), prepared in a 0.05 M barbital buffer, pH 8.6, containing 0.5% anti-IgM serum of masu salmon. (A) Increasing IgM concentration (25400 pg/ml) were added to each well and the incubated at room temperature for 3 days. (B) Linear relationship between the square diameter* of a precipitate ring (D2) and the IgM concentration in each sample.
640
HIROTOSHI FUDA et af.
1
* *
: *
. * 1.
.
= 5 F z P
:
. . *
a
:
. - *
‘
: tl D
50
49
,I
tl&*‘. 100
t50
200
#y
: -
_
250 3!w Days after%%zhing
400
450
!jcio
Fig. 3. IgM concentrations in the early developmental stages of masu salmon as measured by single radial immunodiffusion.
Closed triangle (A) represent fish of unknown sex. Closed (0) and open circles (0) represent male and female fish, respectively (see text).
125) for males and females, respectively, during the period prior to their first sexual maturation. Total serum protein concentration was measured (Fig. 4) to examine its relation to IgM concentration (Fig. 5). Total serum protein concentration increased rapidly from less than 10 mg/ml to 36.1 + 0.4 mg/ml (mean k SE, N = 185) during the period from 235 to 300 days after hatching, and reached a plateau from 300 days after hatching to the end of the measuring period (Fig. 4). The mean levels of total serum protein concentration were 37.3 + 0.9 mg/ml (mean + SE, N = IO@, and 39.7 + 0.7 mg/ml (N = 122) for yearling males and females, respectively, in the Mori Hatchery. Total serum protein concentration at this plateau stage was similar to that observed in yearling fish at the Mori Hatchery. The ratio of IgM to total serum protein concentration increased gradually from 251 days and reached a plateau from 429 days
O’
“-‘
loo
150
200
after hatching to the end of the 489 days measuring period (Fig. 5). In conclusion, both IgM concentration and total serum protein concentration increased 251 days after hatching, but we assumed that the increase of IgM concentration was not concurrent with that of total serum protein concentration. There have been numerous studies in which the maturation of humoral immunity in fish was estimated from the immune response to antigenic stimulation, but there have been few ontogenic studies in which IgM concentrations and total serum protein were measured directly. In humans, IgM concentration generally increases from 1-4 weeks after birth. The IgM concentration reaches the standard level of adults at about 9 months of age (Roitt, 1980). It appeared that maturation of IgM in serum began from 251 days after hatching in masu salmon in the present study. The increase occurred very late. and
250
3PJ
350
400
450
500
Days after hatching
Fig. 4. Development of total serum protein concentration in the early stage of masu salmon. See footnotes to Fig. 3 for each symbol.
641
Serum IgM during early development of masu salmon
0’ Av
100
150
200
300
250 Days after
350
450
400
500
hatching
Fig. 5 The ratio of IgM concentration to total serum protein concentration present in the early developmental stages of masu salmon. See footnotes to Fig. 3 for each symbol.
after 251 days after hatching have recently become a subject of considerable interest. In carp (Cyprinus carpio), lymphoid cells with surface immunoglobulin (sIg) appeared in thymus and nephros between 14 and 21 days after fertilization. On the other hand, serum Ig levels increased after 21 days as shown by immunofluorescent staining and enzyme-linked immunosorbent assay (Van Loon et al., 1981) but the quantitative level of Ig was unknown. In Atlantic salmon (Salmo salar), membrane bound immunoglobulin M was detected in fry by immunofluorescent staining at about 45 days after
the IgM concentration before 235 days after hatching was low, in comparison with the nurse shark, Ginglymostoma cirratum. In the nurse shark, concentrations of both immunoglobulins (19s and 7s) were almost half of the adult level by 48 days after hatching (Fidler et al., 1969). In masu salmon the time of increase of serum IgM might be related to when 0+ aged masu salmon (Parr) transformed into smolt in their first autumn (Hirata et al., 1986). The humoral responses and the immunohistochemistry of the lymphoid organ (thymus, kidney and spleen) and lymphocytes after antigenic stimulation before and
Table I. IgM concentration immunological stimulation
in serum of different
IgM (mg/mI)
Fish species
Age (yr)
Maw salmon (0. masou)
(Mori)S I * (Mori) 9 I + (Nanae) 0+-l + (neonatal) (adult)
_ -
2.27 f 0.12 2.05 f 0.10 0.69 + 0.04
-
(mature, 6-l 5 kg) (mature, 2-8 kg) (l-2 kg)
+
5-6(1983 68 (7s) 17.2
Nurse shark (G. cirrarum) Paddlefish (P. spat/w/a) Gar (L. osscus) Carp (C. carpio) Brown trout (S. rrurra) Coho salmon (0. kisurch) Chum salmon (0. keta) Skate (R. kenojei ) Gold fish (C. auratus) Tenth FirF) (C. carpio) Yellowtail
(S.quinqueradiotn ) Channel catfish (f. pwwcrtus) Rainbow trout (0. mvkiss)
I+ (115-3158) I+-2+ (5&200 g) (mature)3 x
+ -
1.7
-
7.3 * 0.3
-
2.1 f I
(4Oc-900 g) 3+-4+ (30&400 g) (l&llOg) (30&600 g) 4+
DrOtCin
W)
Measurement
7.09 f 0.97 5.70 + 0.39 I .89 * 0.09
SRID
Present data
SRID
Fidler el al. (1969)
SRID
40
SRID
Acton er al. (1971) Legler el al. (1971) Acton et al. (1971)
SRID
Richter
er al. (1973)
IO
SRID
Ingram
and Alexander
-
SRID
voss
Kobayashi
ef al. (1982)
Kobayashi
er al. (1984)
6.34
2.3 2.0 -
SRID
-
16.8
SRID
-
9.1
21.1
SRID
4.8
18.1
SRID
-
0.63 0.72 3.9 0.73
-
0.1-1.84 0.9l%3.04 II
-
3.3
Source of data
18 23 47
f 0.20 f 0.21 (17.9s) (8.9s) 4.7
3+-4+ (I 5&400 g)
I4
fish species
(1979)
er al. (1980)
Vilain et al. (1984)
ELISA
Matsubara
21
SRID
Ourth
6
SRID
Olesen and Jsrgensen
ef al. (1985)
(I 986) (1986)
HIROTOSHI FUDA el
642
Body Fig. 6. The relation asterisk mark
weight
ai.
(9)
between serum IgM concentration and body weight in developing (*) represent
smolting
masu
salmon.
hatching and coincident with the start of feeding (Ellis, 1977). For ontogenic research on IgM concentrations, more sensitive methods than SRID are needed. In particular, IgM concentration in serum before 49 days after hatching should be investigated. Table 1 was shown IgM concentrations in serum of masu salmon in comparison to values reported for other fish species. The values range from 0.1 to 17 mg/ml in different fishes. The IgM concentrations found in masu salmon in the present study fall within the range of IgM concentrations reported for Salmonidae as indicated in Table 1. Unfortunately, the prior exposure of fish to pathogens and their age and size were, for the most part, not reported in the studies whose results are summarized in Table 1. Olesen and Jorgensen (1986) have shown that although hypergammaglobulinaemia was found in PKD-infected rainbow trout (Salvo gairdneri) and in fish from a trout farm with VHS and ERM, the IgM concentration was low in sera from lish living under aquarium conditions with good water quality. In the present study, the serum IgM concentration of two groups of masu salmon (Nanae and Mori groups) were different. The difference of two groups might be dependent on their different living conditions as reported previously (Olesen and Jorgensen, 1986). Relation between the concentration body weight in early stage
oj” IgM
anti the
The relationship between the concentration of IgM and the body weight of our masu salmon is shown in Fig. 6. IgM concentration and the body weight appeared to be related in fish weighing up to 25 g (r = 0.65, P < O.Ol), but above 25 g the relation between IgM concentration and the body weight was insignificant (r = -0.24). On the other hand, excepting smolting masu salmon (asterisk mark), the corre-
See footnotes
masu salmon. to Fig. 3 for each symbol.
The
lation between IgM concentration and body weight was present (P < 0.01) and the correlation coefficient was high at Y = 0.64. The date shown in Fig. 6 indicate that smolting masu salmon (asterisk) have high body weight and low IgM concentration, possibly due to suppression of their immune function. This result seems to be consistent with recent studies of relation between smoltification and the immune system in Salmonids (Maule et al., 1987). Some investigators believe that natural antibodies possibly arise without antigenic stimulation and claim that they are products of physiological development of the serum globulins. In contrast, others maintain that the gradual appearance of antibodies is due either to the increased exposure to external antigenic stimuli or to the physiological development of the immune system (Ingram, 1980). At present, it is not possible to draw any firm conclusions concerning the origins of natural antibody, but we assume that the increase of IgM concentration is related to immunological maturation. Nakanishi (1986) has shown that the size is an important factor for the determination of immunological maturation in the marine teleost (Sebastisclcs marmoratus). The results of the present study indicate that immunological maturation relates more to fish size than age since serum IgM was more closely correlated with body weight than day after hatching (time) as shown in previous reports on fishes (Nakanishi, 1986). This result is also consistent with those of a recent study of yellowtail, Seriola quinqueradiata (Matsubara et al., 198.5). Ackno~ledgemenf~-We
would like to thank to Dr Craig V. Sullivan. Department of Zoology, North Carolina State University, for critically reading the manuscript. Thanks are also due to Dr Kunihiko Kobayashi, School of Medicine, Hokkaido University, for his encouragement during this
Serum
IgM during
early development
study and Mr Shizuo Kimura, and Miss Taka Sasaki for maintenance of masu salmon. This research was also supnorted bv a Grant-in-Aid for Scientific Research from the ‘Ministry-of Education, Science and Culture, Japan.
REFERENCES Acton R. T., Weinheimer P. F., Dupree H. K., Evans E. E. and Bennett J. C. (1971a) Phylogeny of immunoglobulins. Characterization of a 14s immunoglobulin from the gar, Lepisosteus osseus. Biochemistry 10, 2028-2036. Acton R. T., Weinheimer P. F., Dupree H. K., Russell T. R., Wolcott M., Evans E. E., Schrohenloher R. E. and Bennett J. C. (1971b) Isolation and characterization of the immune macroglobulin from the paddlefish, Polyodon spathula. J. Biol. Chem. 246, 6760-6769. Ellis A. E. (1977) Ontogeny of the immune response in Salmo salur. Histogenesis of the lymphoid organs and appearance of membrane immunoglobulin and mixed leucocyte reactivity. In Dewlopmenlal Immunobiology (Edited by Solomon J. 9. and Horton J. D.), pp. 225231. Elsevier/North Holland Biomedical Press, Amsterdam. Fidler J. E., Clem L. W. and Small P. A., Jr (1969) Immunoglobulin synthesis in neonatal nurse sharks (Ginglymostoma cirrarum). Comp. Biochem. Physiol. 31, 3655371. Fuda H., Hara A. and Yamazaki F. (1989) Purification and quantification of immunoglobulin M (IgM) in serum of masu salmon (Oncorhynchus masou). Bull. Far. Fish. Hokkaido Unit. 40, 292-306. Grabar P. and Williams C. A. (1953) Mtthode permettant l’etude conjugee des proprietes tlectrophorttiques et immunochimiques d’un melange de proteins. Application au serum sanguin. Biochim. Biophys. ACIU 10,1933194. Hara A. (1976) Iron-binding activity of female-specific serum proteins of rainbow trout (Salmo guirdneri) and chum salmon (Oncorhynchus keta). Biochim. Biophys. Acta 421, 5499557. Hirata T., Goto A. and Hamada K. (1986) Bimodal length frequency distribution in 0+ aged masu salmon, Oncorhynchus masou, in a natural stream of southern Hokkaido. Japan. J. Ichthyol. 33, 2044207. Ingram G. A. (1980) Substances involved in the natural resistance of fish to infection-A review. J. Fish Biol. 16, 23360. Ingram G. A. and Alexander J. 9. (1979) The immunoglobulin of the brown trout, Salmo trutfa and its concentration in the serum of antigen-stimulated and non-stimulated fish. J. Fish Biol. 14, 249-260. Kobavashi K. and Tomonaga S. (1988) The second immunoglobulin class is commonly present in cartilaginous fish belong to the order Rajiformes. Molec. Immunol. 25, 115-120. Kobayashi K., Tomonaga S. and Kajii T. (1984) A second class of immunoglobuhn other than IgM present in the
of masu
salmon
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serum of a cartilaginous fish, the skate, Raja kenojei: Isolation and characterization. Molec. Immunol. 21, 397404. Kobayashi K., Hara A., Takano K. and Hirai H. (1982) Studies on subunit components of immunoglobulin M from a bony fish, the chum salmon (Oncorhynchus kefa). Molec. Immunol. 19, 955103. Leeler D. W.. Weinheimer P. F.. Acton R. T.. Duoree H. K. and Russell T. R. (1971) Humoral immune factors in the paddlefish, Polyodon spathula. Camp. Biochem. Physiol. 38B, 5233527. Mancini G., Carbonara A. 0. and Heremans J. F. (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2, 235-254. Matsubara A., Mihara S. and Kusuda R. (1985) Quantitation of yellowtail immunoglobulin by enzyme linked immunosorbent assay (ELISA). Bull. Japan. Sot. Sci. Fish. 51, 921-925. Maule A. G., Schreck C. 9. and Kaattari S. L. (1987) Changes in the immune system of coho salmon (Oncorhynchus kisutch) during the Parr-to-smelt transformation and after implantation of cotisol. Can. J. Fish. Aquat. Sci. 44, 161-166. Nakanishi T. (1986) Ontogenetic development of the immune response in the marine teleost Sebustiscus marmoraIUS. Bull. Japan. Sot. Sri. Fish. 52, 473477. Oleson N. J. & Jorgensen P. E. V. (1986) Quantification of serum immunoglobulin in rainbow trout Salmo gairdneri under various environmental conditions. Dis. aquaf. Org. 1,183-189. Ourth D. D. (1986) Purification and quantitation of channel catfish (Zctalurus puncfafus) immunoglobulin M. J. Appl. Ichthyol. 3, 140-143. Richter R., Frenzel E.-M., Hadge D., Kopperschlager G. and Ambrosius H. (1973) Structural and immunochemical studies on the immunoglobulin of carp (Cyprinus carpio L.) I. Analysis at the total molecule. Acfa Biol. Med. Ger. 30, 735-749. Roitt 1. M. (1980) Essential Immunology, 4th edn., pp. 110-l 15. Blackwell Scientific Publications, Oxford. Van Loon J. J. A., van Oosterom R. and van Muiswinkel W. 9. (198 I) Development of the immune system in carp (Cyprinus carpio). In Aspects of Developmental and Comparatioe Immunology I (Edited by Solomon J. B.), pp. 469470. Pergamon Press, Oxford. Vilain C., Wetzel M.-C., Du Pasquier L. and Charlemagne J. (1984) Structural and functional analysis of spontaneous anti-nitrophenyl antibodies in three cyprinid fish species: carp (Cyrinus carpio), goldfish (Carassius aurafus) and tenth (Tinca rinca). Dee. Comp. Immunol. 8, 611422. Voss E. W. Jr., Groberg W. J. Jr. and Fryer J. L. (1980) Metabolism of coho salmon Ig. Catabolic rate of coho salmon tetrameric Ig in serum. Molec. Immunol. 17, 445452.
Camp. Biochem. Physiol. Vol. 99A, No. 4, pp. 631-643, 1991 Printed in Great Britain
SERUM IMMUNOGLOBULIN M (IgM) DURING EARLY DEVELOPMENT OF MASU SALMON (ONCORHYNCHUS HIROTOSHI
FUDA,*
KIYOSHI
MASOU)
SOYANO, * FUMIO YAMAZAKI*
and
AKIHIKO
HARAt
*Department
of Biology and Aquaculture, Faculty of Fisheries, Hokkaido University, Minato, Hakodate, Hokkaido 041, Japan; tNanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University, Nanae, Kameda, Hokkaido 041-l 1, Japan (Received 30 October 1990) Abstract-l.
The development of serum immunoglobulin M (IBM) was studied by measuring IgM concentration in early developmental stages of masu salmon (Oncorhynchusmasou) using single radial immunodiffusion. 2. IgM concentration was measurable from 88 days after hatching (46.5 pg/ml) and was maintained at a relatively constant level (< 100 pg/ml) until 235 days after hatching. 3. IgM concentration increased significantly to a level of 691 + 37 pg/ml (mean + SE, N = 59) during the period from 251 to 429 days after hatching. The highest level of serum IgM was maintained from 429 days to 489 days after hatching. 4. The increase of IgM concentration was not accompanied by parallel increases in total serum protein concentration.
INTRODUCTION It is assumed that the essential function of the immunoglobulin (Ig) in vertebrates is defense against foreign antigens, for example infectious agents etc. Although five major structural classes of Ig, namely IgG, IgM, IgA, IgD and IgE, can be distinguished in
man, only one class has been identified in fish excepting cartilaginous fish belong to the order Rajiformes (Kobayashi et al., 1984, Kobayashi and Tomonaga, 1988). The fish immunoglobulin is classified as an IgM because of its similarities to mammalian IgM with respect to subunit structure, electrophoretic mobility (p-region), and the molecular weight of the intact molecule and its heavy chain (Kobayashi et al., 1982; Olesen and Jorgensen, 1986). Many studies of fish immunobiology have dealt with the phylogeny of immunoglobulin structure and function coupled with investigations into the factors affecting the immune response after antigenic stimulation (Ingram, 1980). However, there have been few studies of IgM concentration in fish serum from the point of view of physiology and fish pathology. The measurement of serum IgM concentration is a required method to obtain basic information on the immune system and for epidemiologic research. IgM concentrations have been measured in serum from the nurse shark, Ginglymostoma cirratum (Fidler et al., 1969), paddlefish, Polyodon spathula (Acton et al., 1971b, Legler et al., 1971), gar, Lepisosteus osseus (Acton ef al., 1971a), carp, Cyprinus carpio (Richter et al., 1973, Vilain et al., 1984), brown trout, Sulmo trutta (Ingram and Alexander, 1979), coho salmon, Oncorhynchus kisutch (Voss et al., 1980), chum salmon, Oncorhynchus keta (Kobayashi et al., 1982), goldfish, Curassius auratus and tenth, Tinca tinca (Vilain et al., 1984), yellowtail, Seriola quinqueradiuta (Matsubara et al., 1985), rainbow trout,
Oncorhynchus mykiss (Olesen and Jargensen, 1986) and channel catfish, Ictalurus punctatus (Ourth, 1986). Previously, we reported the purification of masu salmon (Oncorhynchus masou) IgM by a combination of salting-out, ion-exchange chromatography and gel filtration (Fuda et al., 1989). The present paper describes changes in serum IgM concentration from 46 days to 489 days after hatching and the relationship between serum IgM concentration, plasma protein levels, and body weight. MATERIALS
AND METHODS
Young masu salmon (Oncorhynchus masou) used for measurement of serum IgM were hatched and reared at the Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University. Adult fish were from Mori branch, Hokkaido Fish Hatchery. Blood was collected from hatched larvae and fry by severing their caudal peduncle. Blood was collected from adult fish by cardiac puncture. Blood samples were allowed to stand for several hours, and the serum was separated by centrifugation at 3,000 rpm for 10 min and stored at - 20°C until used for IgM immunoassay or purification. PuriJcation of serum IgM Purification of masu salmon IgM was carried out as described previously (Fuda et al., 1989). Adult masu salmon serum (100 ml) was precipitated by addition of ammonium sulfate at 50% saturation. The precipitate, collected after centrifugation at 10,OOOrpm for IOmin, was dissolved in and dialysed against 0.015 M Tris-HCl buffer, pH 8.0. The dialysate was fractionated on a DEAE cellulose (DE-52, Whatman) column (5 x 5 cm) equilibrated with Tris-HCl buffer, using a stepwise elution with the same buffer containing NaCl at 0.05, 0.1, 0.15, 0.2 and 0.4 M. The fraction eluted by 0.1 M NaCl was reprecipitated with 50% ammonium sulfate, and collected by centrifugation at 637
HIKOTOSHIFUDA
638
10,000 rpm for IOmin. The precipitate was dissolved in 0.02 M Tris-HCl buffer, pH 8.0, containing 2% NaCl and 0. I % NaN, It was then gel filtered through a Sepharose 6B (Pharmacia) column (2.2 x 56.5 cm) using the same buffer. Two distinct peaks were obtained. The first peak, collected and concentrated by lyophilization was again chromatographed on Sepharose 6B. A single and symmetrical peak was obtained and collected as the purified masu salmon IgM. Preparation of antisera Polyvalent antiserum to masu salmon serum was carried out by immunizing rabbits as described previously (Hara, 1976). Specific antiserum to masu salmon IgM was obtained from rabbits immunized with 1 ml of solution containing 25Opg of purified IgM mixed with an equal volume of Freund’s complete adjuvant. The rabbit received four injections at about 7 day intervals. Protein concentration of serum and IgM standard The total serum protein concentration of developing masu salmon was measured using a Bio-Rad Protein Assay kit using bovine serum albumin as a standard. Before assay, the serum was diluted 101 times with 0.02 M Tris-HCl buffer, pH 8.0, containing 2% NaCl and 0.1% NaN,. The protein concentration of the purified IgM was measured using the same kit but bovine IgG was used as the protein standard. Single radial immunod@iision and immunoelectrophore.vis Single radial immunodiffusion (SRID) was carried out according to the procedure of Mancini et al. (1965). Antiserum to purified masu salmon IgM was diluted at 56°C in a solution of I% (w/v) agarose (Litex, type HSA) in a 0.05 M barbital buffer, pH 8.6. Ten millilitres of the hot solution was then layered onto 7 x 1Ocm GelBond film (Pharmacia). The SRID plate was incubated in moist chamber at room temperature for 3 days. After incubation, the plate was washed with 0.9% NaCl, dried on filter paper (Tovo. tvoe 2). stained for 30 set with 1% Amido Black 10B in 7% acetic acid, and destained with 7% acetic acid. For SRID, purified masu salmon IgM standards (25. 50, 100. 200 and 400pg/ml) were used.
et ul.
Immunoelectrophoresis was carried out according to the procedure of Grabar and Williams (1953)with I % agarose in 0.05 M barbital buffer, pH 8.6.
RESULTS AND DISCUSSION
Quantitative
measurement
of IgM
The purified IgM revealed a single precipitin line when reacted against a polyvalent antiserum to masu salmon serum proteins as well as when it was precipitated by the specific antiserum to masu salmon IgM (Fig. I). Conversely, the antiserum raised against the purified IgM preparation gave rise to a single precipitin line with the masu salmon whole serum, suggesting that the IgM preparation was immunologically pure. Different dilutions of antiserum of masu salmon IgM were incorporated into the agarose gel used for SRID. A concentration of 0.5% antiserum gave the best quantitative results (Fig. 2A). Using this dilution, a squared diameter of a precipitate ring was directly proportional to the amounts of IgM present in the sample, in the range of 25400 pg/ml sample as shown in Fig. 2B. In previous studies in which fish IgM was measured by SRID, the lower limit of IgM detection was in the range of 0.0331 mg/ml (Fidler ef al., 1969, Legler e/ al., 1971). Our lower limit of IgM detection was 0.025 mg/ml, similar to the lowest level of IgM detection in the previous reports. On the other hand, Matsubara et al. (1985) recently reported a lower limit of detection of yellowtail IgM of 0.4 +_0.17 ng/test tube by means of enzyme-linked immunosorbent assay (ELISA). The ELISA is generally a more sensitive method than SRID, and the establishment of an ELISA for masu salmon IgM is desirable for future research on IgM in the earliest developmental stages, serum IgM was not measurable in these early stages in the present study.
Fig. I. Results of immunoelectrophoresis of masu salmon serum and their purified IgM. s: masu salmon serum, M: masu salmon IgM, a-s: polyvalent antiserum to masu salmon serum proteins, a-M: specific antiserum to samu salmon IgM.
Serum
IgM during early development
IgM concentration and total serum protein during early development IgM concentration was measured in samples of serum pooled from about 100 fishes from 49 to 138 days after hatching, from about 50 fishes from 154 to 235 days after hatching and in serum samples from individual fish from 251 to 489 days after hatching. Development of IgM concentrations in the serum of masu salmon from 49 to 489 days after hatching is shown in Fig. 3. IgM concentration in the serum was already detectable (~25 pg/ml) 49 days after hatching. It was also measurable (46.5 pg/ml) using SRID from 88 days after hatching onwards. IgM concentrations were maintained at a relatively constant level
639
of masu salmon
(< lOOpg/ml) from 88 days to 235 days after hatching. On the other hand, from 251 days to 429 days after hatching, IgM concentration increased to a level of 691 + 37 pg/ml (mean + SE, N = 59). IgM concentration was highly variable in later developmental stages, but it appeared that a plateau might have been reached by 429 days of development. There was no significant difference between male and female fish IgM concentration during development. IgM concentration was measured in yearling masu salmon for 9 months before their ovulation in Mori Hatchery. The IgM concentrations appeared constant. The mean levels of IgM were 2273 _t 118p g/ml (mean f SE, N = 108) and 2049 k 97 pg/ml (N =
1
25
50
100
200 IgM
400
(pg/ml)
Fig. 2. Quantitative measurement of IgM by single radial immunodiffusion carried out in a 1.4mm thick (7 x IOcm) slab of agarose gel (lo/o), prepared in a 0.05 M barbital buffer, pH 8.6, containing 0.5% anti-IgM serum of masu salmon. (A) Increasing IgM concentration (25400 pg/ml) were added to each well and the incubated at room temperature for 3 days. (B) Linear relationship between the square diameter* of a precipitate ring (D2) and the IgM concentration in each sample.
640
HIROTOSHI FUDA et af.
1
* *
: *
. * 1.
.
= 5 F z P
:
. . *
a
:
. - *
‘
: tl D
50
49
,I
tl&*‘. 100
t50
200
#y
: -
_
250 3!w Days after%%zhing
400
450
!jcio
Fig. 3. IgM concentrations in the early developmental stages of masu salmon as measured by single radial immunodiffusion.
Closed triangle (A) represent fish of unknown sex. Closed (0) and open circles (0) represent male and female fish, respectively (see text).
125) for males and females, respectively, during the period prior to their first sexual maturation. Total serum protein concentration was measured (Fig. 4) to examine its relation to IgM concentration (Fig. 5). Total serum protein concentration increased rapidly from less than 10 mg/ml to 36.1 + 0.4 mg/ml (mean k SE, N = 185) during the period from 235 to 300 days after hatching, and reached a plateau from 300 days after hatching to the end of the measuring period (Fig. 4). The mean levels of total serum protein concentration were 37.3 + 0.9 mg/ml (mean + SE, N = IO@, and 39.7 + 0.7 mg/ml (N = 122) for yearling males and females, respectively, in the Mori Hatchery. Total serum protein concentration at this plateau stage was similar to that observed in yearling fish at the Mori Hatchery. The ratio of IgM to total serum protein concentration increased gradually from 251 days and reached a plateau from 429 days
O’
“-‘
loo
150
200
after hatching to the end of the 489 days measuring period (Fig. 5). In conclusion, both IgM concentration and total serum protein concentration increased 251 days after hatching, but we assumed that the increase of IgM concentration was not concurrent with that of total serum protein concentration. There have been numerous studies in which the maturation of humoral immunity in fish was estimated from the immune response to antigenic stimulation, but there have been few ontogenic studies in which IgM concentrations and total serum protein were measured directly. In humans, IgM concentration generally increases from 1-4 weeks after birth. The IgM concentration reaches the standard level of adults at about 9 months of age (Roitt, 1980). It appeared that maturation of IgM in serum began from 251 days after hatching in masu salmon in the present study. The increase occurred very late. and
250
3PJ
350
400
450
500
Days after hatching
Fig. 4. Development of total serum protein concentration in the early stage of masu salmon. See footnotes to Fig. 3 for each symbol.
641
Serum IgM during early development of masu salmon
0’ Av
100
150
200
300
250 Days after
350
450
400
500
hatching
Fig. 5 The ratio of IgM concentration to total serum protein concentration present in the early developmental stages of masu salmon. See footnotes to Fig. 3 for each symbol.
after 251 days after hatching have recently become a subject of considerable interest. In carp (Cyprinus carpio), lymphoid cells with surface immunoglobulin (sIg) appeared in thymus and nephros between 14 and 21 days after fertilization. On the other hand, serum Ig levels increased after 21 days as shown by immunofluorescent staining and enzyme-linked immunosorbent assay (Van Loon et al., 1981) but the quantitative level of Ig was unknown. In Atlantic salmon (Salmo salar), membrane bound immunoglobulin M was detected in fry by immunofluorescent staining at about 45 days after
the IgM concentration before 235 days after hatching was low, in comparison with the nurse shark, Ginglymostoma cirratum. In the nurse shark, concentrations of both immunoglobulins (19s and 7s) were almost half of the adult level by 48 days after hatching (Fidler et al., 1969). In masu salmon the time of increase of serum IgM might be related to when 0+ aged masu salmon (Parr) transformed into smolt in their first autumn (Hirata et al., 1986). The humoral responses and the immunohistochemistry of the lymphoid organ (thymus, kidney and spleen) and lymphocytes after antigenic stimulation before and
Table I. IgM concentration immunological stimulation
in serum of different
IgM (mg/mI)
Fish species
Age (yr)
Maw salmon (0. masou)
(Mori)S I * (Mori) 9 I + (Nanae) 0+-l + (neonatal) (adult)
_ -
2.27 f 0.12 2.05 f 0.10 0.69 + 0.04
-
(mature, 6-l 5 kg) (mature, 2-8 kg) (l-2 kg)
+
5-6(1983 68 (7s) 17.2
Nurse shark (G. cirrarum) Paddlefish (P. spat/w/a) Gar (L. osscus) Carp (C. carpio) Brown trout (S. rrurra) Coho salmon (0. kisurch) Chum salmon (0. keta) Skate (R. kenojei ) Gold fish (C. auratus) Tenth FirF) (C. carpio) Yellowtail
(S.quinqueradiotn ) Channel catfish (f. pwwcrtus) Rainbow trout (0. mvkiss)
I+ (115-3158) I+-2+ (5&200 g) (mature)3 x
+ -
1.7
-
7.3 * 0.3
-
2.1 f I
(4Oc-900 g) 3+-4+ (30&400 g) (l&llOg) (30&600 g) 4+
DrOtCin
W)
Measurement
7.09 f 0.97 5.70 + 0.39 I .89 * 0.09
SRID
Present data
SRID
Fidler el al. (1969)
SRID
40
SRID
Acton er al. (1971) Legler el al. (1971) Acton et al. (1971)
SRID
Richter
er al. (1973)
IO
SRID
Ingram
and Alexander
-
SRID
voss
Kobayashi
ef al. (1982)
Kobayashi
er al. (1984)
6.34
2.3 2.0 -
SRID
-
16.8
SRID
-
9.1
21.1
SRID
4.8
18.1
SRID
-
0.63 0.72 3.9 0.73
-
0.1-1.84 0.9l%3.04 II
-
3.3
Source of data
18 23 47
f 0.20 f 0.21 (17.9s) (8.9s) 4.7
3+-4+ (I 5&400 g)
I4
fish species
(1979)
er al. (1980)
Vilain et al. (1984)
ELISA
Matsubara
21
SRID
Ourth
6
SRID
Olesen and Jsrgensen
ef al. (1985)
(I 986) (1986)
HIROTOSHI FUDA el
642
Body Fig. 6. The relation asterisk mark
weight
ai.
(9)
between serum IgM concentration and body weight in developing (*) represent
smolting
masu
salmon.
hatching and coincident with the start of feeding (Ellis, 1977). For ontogenic research on IgM concentrations, more sensitive methods than SRID are needed. In particular, IgM concentration in serum before 49 days after hatching should be investigated. Table 1 was shown IgM concentrations in serum of masu salmon in comparison to values reported for other fish species. The values range from 0.1 to 17 mg/ml in different fishes. The IgM concentrations found in masu salmon in the present study fall within the range of IgM concentrations reported for Salmonidae as indicated in Table 1. Unfortunately, the prior exposure of fish to pathogens and their age and size were, for the most part, not reported in the studies whose results are summarized in Table 1. Olesen and Jorgensen (1986) have shown that although hypergammaglobulinaemia was found in PKD-infected rainbow trout (Salvo gairdneri) and in fish from a trout farm with VHS and ERM, the IgM concentration was low in sera from lish living under aquarium conditions with good water quality. In the present study, the serum IgM concentration of two groups of masu salmon (Nanae and Mori groups) were different. The difference of two groups might be dependent on their different living conditions as reported previously (Olesen and Jorgensen, 1986). Relation between the concentration body weight in early stage
oj” IgM
anti the
The relationship between the concentration of IgM and the body weight of our masu salmon is shown in Fig. 6. IgM concentration and the body weight appeared to be related in fish weighing up to 25 g (r = 0.65, P < O.Ol), but above 25 g the relation between IgM concentration and the body weight was insignificant (r = -0.24). On the other hand, excepting smolting masu salmon (asterisk mark), the corre-
See footnotes
masu salmon. to Fig. 3 for each symbol.
The
lation between IgM concentration and body weight was present (P < 0.01) and the correlation coefficient was high at Y = 0.64. The date shown in Fig. 6 indicate that smolting masu salmon (asterisk) have high body weight and low IgM concentration, possibly due to suppression of their immune function. This result seems to be consistent with recent studies of relation between smoltification and the immune system in Salmonids (Maule et al., 1987). Some investigators believe that natural antibodies possibly arise without antigenic stimulation and claim that they are products of physiological development of the serum globulins. In contrast, others maintain that the gradual appearance of antibodies is due either to the increased exposure to external antigenic stimuli or to the physiological development of the immune system (Ingram, 1980). At present, it is not possible to draw any firm conclusions concerning the origins of natural antibody, but we assume that the increase of IgM concentration is related to immunological maturation. Nakanishi (1986) has shown that the size is an important factor for the determination of immunological maturation in the marine teleost (Sebastisclcs marmoratus). The results of the present study indicate that immunological maturation relates more to fish size than age since serum IgM was more closely correlated with body weight than day after hatching (time) as shown in previous reports on fishes (Nakanishi, 1986). This result is also consistent with those of a recent study of yellowtail, Seriola quinqueradiata (Matsubara et al., 198.5). Ackno~ledgemenf~-We
would like to thank to Dr Craig V. Sullivan. Department of Zoology, North Carolina State University, for critically reading the manuscript. Thanks are also due to Dr Kunihiko Kobayashi, School of Medicine, Hokkaido University, for his encouragement during this
Serum
IgM during
early development
study and Mr Shizuo Kimura, and Miss Taka Sasaki for maintenance of masu salmon. This research was also supnorted bv a Grant-in-Aid for Scientific Research from the ‘Ministry-of Education, Science and Culture, Japan.
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serum of a cartilaginous fish, the skate, Raja kenojei: Isolation and characterization. Molec. Immunol. 21, 397404. Kobayashi K., Hara A., Takano K. and Hirai H. (1982) Studies on subunit components of immunoglobulin M from a bony fish, the chum salmon (Oncorhynchus kefa). Molec. Immunol. 19, 955103. Leeler D. W.. Weinheimer P. F.. Acton R. T.. Duoree H. K. and Russell T. R. (1971) Humoral immune factors in the paddlefish, Polyodon spathula. Camp. Biochem. Physiol. 38B, 5233527. Mancini G., Carbonara A. 0. and Heremans J. F. (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2, 235-254. Matsubara A., Mihara S. and Kusuda R. (1985) Quantitation of yellowtail immunoglobulin by enzyme linked immunosorbent assay (ELISA). Bull. Japan. Sot. Sci. Fish. 51, 921-925. Maule A. G., Schreck C. 9. and Kaattari S. L. (1987) Changes in the immune system of coho salmon (Oncorhynchus kisutch) during the Parr-to-smelt transformation and after implantation of cotisol. Can. J. Fish. Aquat. Sci. 44, 161-166. Nakanishi T. (1986) Ontogenetic development of the immune response in the marine teleost Sebustiscus marmoraIUS. Bull. Japan. Sot. Sri. Fish. 52, 473477. Oleson N. J. & Jorgensen P. E. V. (1986) Quantification of serum immunoglobulin in rainbow trout Salmo gairdneri under various environmental conditions. Dis. aquaf. Org. 1,183-189. Ourth D. D. (1986) Purification and quantitation of channel catfish (Zctalurus puncfafus) immunoglobulin M. J. Appl. Ichthyol. 3, 140-143. Richter R., Frenzel E.-M., Hadge D., Kopperschlager G. and Ambrosius H. (1973) Structural and immunochemical studies on the immunoglobulin of carp (Cyprinus carpio L.) I. Analysis at the total molecule. Acfa Biol. Med. Ger. 30, 735-749. Roitt 1. M. (1980) Essential Immunology, 4th edn., pp. 110-l 15. Blackwell Scientific Publications, Oxford. Van Loon J. J. A., van Oosterom R. and van Muiswinkel W. 9. (198 I) Development of the immune system in carp (Cyprinus carpio). In Aspects of Developmental and Comparatioe Immunology I (Edited by Solomon J. B.), pp. 469470. Pergamon Press, Oxford. Vilain C., Wetzel M.-C., Du Pasquier L. and Charlemagne J. (1984) Structural and functional analysis of spontaneous anti-nitrophenyl antibodies in three cyprinid fish species: carp (Cyrinus carpio), goldfish (Carassius aurafus) and tenth (Tinca rinca). Dee. Comp. Immunol. 8, 611422. Voss E. W. Jr., Groberg W. J. Jr. and Fryer J. L. (1980) Metabolism of coho salmon Ig. Catabolic rate of coho salmon tetrameric Ig in serum. Molec. Immunol. 17, 445452.