May 30, 1996 - carpio L.) lines with a genetically determined ... Keywords: isogenic lines, Cyprinus carpio L., ..... gynogenetic Atlantic salmon, Salmo salar L.
~nimal~enetics, 1996,27,313-319
Characterizationof isogenic carp (Cyprinus carpio L.) lines with a genetically determined high or low antibody production G F Wiegertjes, A B J Bongers, P Voorthuis, B Zandieh Doulabi, A Groeneveld, W B Van Muiswinkel, R J M Stet
Summary
Antibody production to dinitrophenyl-keyhole limpet haemocyanin (DNP-KLH) served as the immune parameter to divergently select carp (Cyprinus carpio L.) to produce high- and lowresponder F, hybrid lines. Antibody production to trinitrophenyl-lipopolysaccharide [TNP-LPS) and to DNP-KLH were similar in magnitude. By contrast, some high-responder lines were low responders to DNP-human serum albumin, and vice versa. Low-responder carp were relatively susceptible to infection with the parasite Trypanoplasma borreli. This suggested that at least one gene with a major influence on resistance differed between the two homozygous parents (69, 85) used to generate the high- and lowresponder homozygous families, respectively. The isogenic lines showed no within-line variation in DNA fingerprints, but differed with respect to their MhcCyca-DAB genes.
Keywords: isogenic lines, Cyprinus carpio L., major histocompatibility complex (MHC), immune responsiveness Introduction
G F Wiegertjes P Voorthuis A Groeneveld W B Van Muiswinkel R J M Stet Department of Experimental Animal Morphology and Cell Biology, PO Box 338, 6700 AH Wageningen, The Netherlands A B J Bongers B Zandieh Doulabi Department of Fish Culture and Fisheries Wageninnen Agricultural University, p.0 Box 338,6700 AH Wageningen, The Netherlands
Fish have been shown to provide excellent laboratory models (Powers 1989). Inbred lines from fish species such as the zebrafish (Brachydanio rerio) (Westerfield 1993) or medaka [Oryzias latipes) (Hyodo-Taguchi ?t Egami 1985) are available, but their small size has limited their use for immunobiological studies. Ginbuna crucian carp (Carrasius gibelio 1angsdorjFii) is a larger fish species for which clonal offspring is available, and has been used to investigate major histocompatibility complex (MHC)-restricted cell transfer (Nakanishi & Onozato 1990). So far, the availability of larger genetically identical fish has remained limited, although the techniques to produce these by gynogenesis were described for several fish species (Ihssen et 01.1990).
Antibody production to a hapten-carrier complex (dinitrophenyl-keyhole limpet haemocyanin; DNP-KLH) served as the immune parameter to divergently select carp with a genetically determined high or low immune responsiveness, using gynogenesis for reproduction (Wiegertjes et 01. 1994). Here, we describe the outcome of the selection, by examination of the antibody response to DNP-KLH, in F, hybrids of homozygous carp. We examined the ubiquitous nature of the difference in immune responsiveness by immunization with other antigens, and by infection with Trypanoplasma borreli. An attempt is made to explain the immune response differences by examination of the isogenic nature of the F, hybrids using DNA fingerprint analysis, and by determination of their MHC class I1 B genes.
Materials and methods Fish A divergent selection for antibody production was initiated in a carp (Cyprinus carpio L.) base population (WAUR38/), from which two individuals were reproduced by gynogenesis according to Komen et al. (1991). The selection criteria for these two individuals were antibody production to DNP-KLH (no. 69 = high, no. 85 = low) and reproductive capacity (Wiegertjes et al. 1995a).The resulting homozygous families (69e = AbH and 85e = AbL) were tested for the presence of reproductive females and functional males with the XX genotype. The occurrence of male sex reversal in gynogenetic female carp (Komen et 01. 1992) allowed us to cross homozygous animals with a high or with a low response, and obtain isogenic F, hybrids. These were grown at 25 1°C in separate aquaria of the same recirculation system, as previously described (Wiegertjes et ul. 1995b).
Immunization with hapten-carrier complexes
. , -
Correspondence: G F Wiegertjes.
Accepted 30 May 1996
0 1996 International Society for Animal Genetics
At five months of age, individuals from each line (see Tablel) were marked, bled for preimmune samples, mixed and randomly divided 313
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Il'iegcrtjes, Bongers, \:oorthuis et al.
over six tanks. At 6 months, carp were injected intramuscularly with 10 pg DNP-KLH, DNPhuman serum albumin (HSA) or with trinitrophenyl-lipopolysaccharide (TNP-LPS), as described previously (Wiegertjes et al. 1995b). DNP-specific serum antibody titres were measured by enzyme-linked immunosorbent assay (ELISA) (Wiegertjes et al. 1994) as optical density (OD) values at fixed serum dilutions (d = loo), at 1 2 , 2 1 and 28 days after immunization. Parasite infection Ten-month-old F, hybrid carp were infected with T. borreli (Steinhagen et al. 1989). To examine the influence of prior immunization with DNP-HSA at six months, non-immunized control fish were also infected (cross 7 3 x 46). Carp of each line were divided over two groups, of which one group was infected with virulent, the other with y-irradiated parasites, and placed in separate 1 2 0 1 aquaria of a flow-through system at 20 1°C. Average weight at infection was 230 k 158g. Carp were infected by intramuscular injection with 1000 virulent, or with 5 x lo4 y-irradiated T. bori-eli. Irradiation-dose was 100 Gy, at a concentration of 5 x 1O5/ml,to inactivate but not kill the parasite (G.F. Wiegertjes, unpublished observations). Parasitaemia at 3 weeks postinfection (wpi) with virulent parasites, using heparinized blood samples, was monitored with a Biirker counting chamber. Dead fish were removed daily for 6 weeks. Blood samples to
Table 1. Immunization of six-month-old F, isogenic carp (Cyprinus carpi0 L.) tzith different hapten-carrier complexes. Body weight was determined at the last +impling day (seven months)
AbII [h%)
AbH x hbl,
AhI. ( 8 5 ~ )
c 74
9h x 74
10
10
10
234
45 A 71 7 1 x 74
10
10
10
176 _t 5 7
10
10
10
101 c 34 246
3 h x 46
10
10
10
45 x 4 6
14
ND
ND
73 x 46
10
10
10
c 96 150 2 6 1 194 2 42
x 51
8
12 ~ 5 1
10
8 10
10
112 +. 63 136 t 80
76 x 5 1
12
6
7
114 t 62
2
8
DNP-KLH, d m trophenyl-keyhole limpet haemocyanin, IIKA-fiSA, dinitrophenyl-humaii serum albumin, I ILP-LPS, trinitrc%phen\1-lipopolysaccharide. ZD not don? k!
1Wh Intc~rnatiunalSocietv for Animal Genetics, Animal Genetics 2 7 , 313-319
measure serum antibody titres, elicited by irradiated T, borreli, were taken at 5 wpi. Increased specific antibody production (titre 1:320) was measured by ELISA (Jones et al. 1993). DNA fingerprinting High-molecular-weight carp DNA was obtained from liver, or from nuclei of lysed erythrocytes, from (n = 5) individuals of all crosses. DNA isolation and digestion were as described previously (Stet et al. 1993). DNA samples (50 pg) were digested in a large volume (500 p1) with HaeIII or with Hinfl (4 units pg-' DNA). Digested DNA samples (10 pg lane-') were separated on 0.8% agarose gels, run at 4OC for 960 Vh in recirculating buffer. Fingerprinting was performed as described by Johnstone & Stet (1995). The microsatellite oligonucleotide probe (GGAT), was endlabelled using polynucleotide kinase according to the manufacturer's specification. MHC class IIgenotyping Polymerase chain reaction (PCR) amplification was performed on 100-200 ng genomic DNA from the two parents of the base population (P20, P38), by combining primers based on Cyca-DAB cDNA sequences codon 4 to 4 (OL93-139 5'-CTGTCTGCTTTCACTGGAGCAG3') and codon 89 to 96 (OL93-140 5'-CTGTTTTATCACGGATCGCCGA-3'),or codon 80 to 88 (OL93-2 3 5'-CTGATAGAGTTCAGCATTATGTTTGCA-3') (Ono et al. 1993), with 200 pM of each dNTP, 1.5 mM MgCl,, 1 unit Taq polymerase (Eurogentec, Seraing, Belgium) and reaction buffer ('Goldstar', Eurogentec), in a total volume of 100 pl. The mixture was subjected to a thermal cycle profile (1min at 94"C, 2 min at 55"C, 1 min at 72°C) for 30 cycles. The PCR products were cloned into pGEM-TA (Promega, Madison, WI) and sequenced using the T7 DNA polymerase version 2.0 (USB, Cleveland, OH). In the parental fish, two pairs of linked genes were found: Cyca-DABl*OI, CycaDABZ*Ol, and Cyca-DAB3*01, Cyca-DAB4*01, respectively (Van Erp et al. 1996). Genomic DNA fiom n = 40 fish from the base population (including nos. 69, 85), and from the homozygous individuals used to produce the F, hybrids, was used for Cyca-DAB genotyping, using two techniques. The first technique, PCRrestriction fragment length polymorphism (RFLP) using restriction enzyme digestion of the resultant PCR product, generated differently sized fragments. Primers OL93-139 and OL93-140 were used to generate a Cyca-DABl*Ol/Cyca-DABZ*OI
315
(b) 2.0 -
2.0 -
Characterization of isogenic high- and low-responder carp Iin es
0.5
0.0
2.0
0.0 -
0.0 12
21
28
- (4
12
21
28
12
21
20
QaY
Fig. 1.Kinetics of the DNP-specific antibody respbnse. Antibody production (mean + SD) of one high-responder (0,73 x 74; AbH family) and one low-responder line (0,76 x 51; AbL family), representative for the other F, hybrids. Fish were immunized with [a) DNP-KLH, (b) DNP-HSA, or (c) with TNP-LPS. Preimmune values (OD 0.1-0.2) are not shown.
fragment, which was digested with RsaI, and separated on 1% agarose. The second technique, gene-specific PCR using primers specific for the amplification of Cyca-DAB3*Ul and CycaDAB4*01, generated fragments of 746 base pairs (bp) and 678 bp, respectively (Van Erp et al. 1996).
Results
Hapten-carrier immunization At all sample points, low-responder carp antibody production to DNP-KLH was lower than that of high-responder carp (Table 2). Antibody titre differences of maximally 0.7 OD (at 2 1 days) corresponded to lo-fold differences in serum dilutions. Frequency distributions of the antibody response to DNP-KLH were different
Table 2. Antibody response to DNP-KLH of presumed high- and low-responder isogenic F, hybrid carp Experimental design
Family AbH
AbL
Antibody response isogenic lines (mean OD * SD)
F, hybrid 8xd"
Pre-immune
12 days
2 1 days
28
36 x 74 45 x 74 73 x 74
0.1 f 0.1 0.2 f 0.1 0.1 f 0.1
0.3 0.1 0.3 f 0.1 0.5 0.3
* *
1.5 + 0.2 1.2 k 0.4 1.7 k 0.1
1.7 f 0.2 1.5 f 0.2 2.0 f 0.1
2x51 12 X 5 1 76 X 5 1
0.1 k 0.1
0.2 f 0.0 0.2 0.1 0.2 0.0
1.2 f 0.3 1.1k 0.3 1.0 0.4
1.4 f 0.3 1.5 f 0.3 0.9 0.3
0.1
* 0.1
0.1 k 0.1
* *
*
days
*
*Antibody titres are expressed as optical density (OD) values of 1 : l O O diluted serum samples. DNP-KLH; dinitrophenyl-keyhole limpet haemocyanin.
0 1996 International Society for Animal Genetics, Animal Genetics 27, 313-319
between the high- and low-responder lines, whereas the frequency distribution of the AbH x AbL lines was similar to that of high-responder carp (data not shown). The kinetics of the antibody response to the various hapten-carrier complexes were different (Fig. 1). The increase in antibody production to DNP-HSA was slower than to DNP-KLH, whereas the response to TNP-LPS was most rapid, but increased only slightly after 12 days. It was decided, based upon these results, to compare the immune responsiveness of the isogenic lines at 21 days for DNP-KLH, at 2 8 days for DNP-HSA and at 1 2 days after immunization for TNP-LPS (Fig. 2). In general, antibody production to DNP-HSA and to DNP-KLH was similar in magnitude, but, by contrast, some high responders to DNP-KLH were low responders to DNP-HSA. and vice versa.
Infection with Trypanoplasma borreli Between-line variation in parasitaemia was 37fold (1x lo6 - 3.7 x lo7), with the highest parasitaemia detected in the low responders (Table 3). Parasitaemia was negatively correlated with survival time and total survival. In general, high responders, and AbH x AbL carp, showed increased antibody production specific to T. borreli, whereas low-responder carp often failed to react to the parasite. Despite a slight difference in survival between previously immunized and non-immunized control fish, there were no differences in any of the other parameters measured (data not shown). No correlation could be detected between the magnitude of response to DNP-HSA and subsequent resistance to T. borreli.
Genetic effects
316
Each homozygous family consisted of animals generated from one single mother, all with a coefficient of co-ancestry f,,,. = 1 and therefore an additive genetic relation of a,,? = Zf,,, = 2. These families have no dominance variance, because of honiozygosity, but may suffer from developmental instability leading to an increase in The isogenic hybrid lines. with a genetic variance V, = 0. had equal dominance effects for all individuals within one line, and no increased environmental variation owing to homozygosity (no developmental instability). The observed varian(x ( ITp) was not reduced within the homozygous families, but a clear reduction of V , could be seen within the F, hybrids, compared with the base population (Table 4). Genomic DNA from all hvbrid crosses was subjected to fingerprint analysis. The oligonucleotide probe (GGAT), yielded multibanded fingerprints with a reasonable number of scorable fragments. The most useful enzyme for fingerprint determination was Hinfl, as digestion with I-faeIII could not detect between-line genetic: heterogeneity (data not shown). On average. Hinfr digestion yielded 10-14 scorable fragments. which varied with the isogenic line examined. Fingerprints were different between the F , hybrids, but identical within the isogenic lines (Fig. 3).
I
I
I
I
I
I
_J
0.0 (b) 2.0 r
I
I
0.0 1
I
1
T~
1
1
I
-1
I I
MHC class II genotyping
0.0
CJr.ca-LIAB1*U1 /DAB2701 linked genes were rletPcted in both parents of the base population
Ah1 I
(OOt~)
Resistance to T. borreli
Jb
i74
>42 >42 A 2 >42 >42
515
414
30323539>42 30 >42 >42 >42 >42
115 415
415
R 22
2 8 32
012
2247 1 251 81
28 31 31 32
014 013
1111L
4‘1~74 1’1224 73A74 11’317
fjll!>
(HFjt?)
2
>: 5 1
12Y51
7tj
Y
51
27 31 31
~
1
d
36 45 73 2 12 76 x46 1 x51
Hybrid crosses
Fig. 2. Antibody titres (mean SD) of high-responder (a.AbH family), low-responder (0,AbL family) and high x low-responder (0, AbH x AbL) isogenic carp. According to observed differences in kinetics arid magnitude of the response, antibody production is visualized (a) at 21 days for DNP-KLH, (b) at 28 days for DNP-HSA, and (c) at 1 2 days for immunization with TNP-LPS.
‘l’able 3 . Resistaiii c to infwtion \\-it11 TnTponoplasma borreli l~:k~,erllltt?!ltal grcii1ps
I
36 45 73 x74
115
013 013 113
’Individual x.aluc:s. tSurx-i\-cil i s n u n i k r infec:~edinumbermortalities UI), 1iot CtOill!.
P 1 O W 7 I n t c t r n a l i ~ i ~ ~Stxiidv cil for hnimal Genetics, Animal Genetics 27, 313-319
(P20, P38), whereas Cyca-DAB3*Ul/DAB4*01 linked genes were present in the female only (P38). PCR-RFLP and gene-specific PCR analyses demonstrated the presence of three Cyca-DAB genotypes, which segregated in the base population. Correlation of the response types previously described for the base population (Wiegertjes et al. 1994), with the Cyca-DAB genes, showed a significant increase in the frequence of CycaDAB1 *Ul/DAB2*01 genes within the mediumresponder group, and an absence of CycaDAB3*U1 /DAB4 *01 genes in the high-responder group (Table 5 ) .
Table 4. Expected variance components and observed phenotypic variance (V,) throughout the divergent selection for antibody production (OD values) to DNP-KLH Observed variance
Age Group
(months) Components V,
Base population 13 Homozygous families* 6
vA+v, +v, 2v, + v,
Homozygous familiest 12 6 F, hybrids*
~ V +AV , VE
12 days 21 days 28 days
0.19 0.02
.
0.08 0.02
0.20 0.30
ND'
0.26
0.19 0.06
0.08
0.24
DNP-KLH; dinitrophenyl-keyhole limpet haemocyanin. *Average for two families (high and low);Wiegertjes et al. (1994). tAverage for two families (high and low);Wiegertjes et al. (1995a). *Average for nine isogenic F, hybrids; this work. ND =not done.
Parent no. 69 of the AbH family was typed
Cyca-DAB1* o ~ / D ~ B 2 * 0and 1 , all carp tested within this homozygous family possessed these genes, which indicated an apparent homozygosity of the parent for Cyca-DABl*Ol/DAB2*02. Parent no. 85 of the AbL family was typed CycaDAB3*OZ/DAB4*01.Some (nos 1 2 , 46, 51), but not all (76, 2 not tested) carp tested within this family, possessed these genes, indicating the presence of a null allele. Summarizing, all highresponder hybrids were homozygous for CycaDABZ*OZ/DAB2*01,whereas all AbH x AbL crosses carried Cyca-DAB1*Ol/DABZ*OI and Cyca-DAB3*01/DAB4*Olgenes. All low-responder F, hybrids had the Cyca-DAB3*01/DAB4*01 genes.
Discussion
Fig. 3. DNA fingerprints obtained with the enzyme Hinfr and (GGAT), microsatellite probe. a TWOindividuals from each isogenic F, hybrid. The different crosses are denoted by the numbers of the two homozygous parents, as described in Table 1. b Five individuals from four representative isogenic F, hybrids.
0 1996 International Society for Animal Genetics, Animal Genetics 27, 313-319
The antibody titres to DNP-KLH differed between high- and low-responder carp, in accordance with the previously suggested genetic control of this specific antibody response (h" = 0.29-0.37) (Wiegertjes et 01. 1994, 1995a). The magnitude of antibody production in AbH x AbL lines suggested that high responsiveness to DNP-KLH was inherited in a dominant fashion. Variance analysis showed that the phenotypic variation in antibody production to DNP-KLH, within the F, hybrids (V, = VE), was reduced in comparison with the base population and homozygous families. Although, for the analysis of animal breeding experiments, an 'animal model' is the method of choice (Meyer 1989),it would be inappropriate as it does not allow for the different genetic constitution of the individuals that comprise a homozygous family. No evidence for withinline variation could be detected with DNA fingerprints, a technique previously used to establish clonal stability of natural gynogenetic Poecilia formosa (Turner et al. 1990). The antibody production to T-cell dependent DNP-HSA did not always follow the same pattern of response to DNP-KLH. Immunization with T-cell independent TNP-LPS was in agreement with the high- or low-response type. These observations are consistent with previous findings in selected homozygous families (Wiegertjes et al. 1995b), and suggest at least a carrier, or T-cell dependency of the differences in responsiveness. Immunization with a nitrophenyl-unrelated hapten coupled to KLH could possibly confirm this hypothesis. The antigendependent switch from high to low responsiveneSS in Some carp lines is typical for irmmme response (Ir) gene control (McDevitt & Sela 1965).
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Wiegertjes, Bongers, Vooorthuis et al.
The infection model, for which a genetic control of susceptibility to the disease has been suggested to be caused by an impaired humoral response to T. borreli (Wiegertjes et al. 1995c), was used to further investigate the immune responsiveness of the carp lines. In general, F, hybrids from the AbL family were more susceptible than high-responder carp, whereas AbH x AbL crosses were relatively resistant, indicating dominant inheritance of the resistant genotype. These results clearly suggest that the two parents of the AbH and AbL families differed for at least one gene, with a major influence on resistance to the parasite. Particular MHC haplotypes of the mouse, such as MHC class I1 I-Ek, have been implicated to play a major role in increased susceptibility to Trichinella spiralis, Leishmania donovani (Wassom & Kelly 1990), and Trypanosoma cruzi (Powell & Wassom 1993). Although, at this point, it is premature to link the observed differences in resistance to T. borreli to Ir gene control, the initial choice for DNPKLH as selection antigen was based upon the often observed correlation between antibody production to hapten-carrier complexes and Ir genes of the MHC (Klein 1986). Four previously described Cyca-DAB genes could be detected in the original parents (P38, P Z O ) , resulting in three different genotypes within the base population. High responsiveness could be correlated with an absence of Cyca-DAB3*DI/DAB4*1)1 genes. Owing to the low numbers of individuals (10%) typed as high or low responders, changes in frequency were often not significant. The Cyca-DAB3*Oll DAB4*Of genes are characterized by an aberrant splice site sequence for intron 1, in which GT is replaced by GC, which is highly infrequent throughout evolution (06% of the total gene sequences studied) (Jacob & Gallinaro 1989). A similar substitution has been shown to cause incomplete splicing of a high percentage of the primary transcripts of complement component
Table 5. Expecttd and observed frequencies of Cvca-DAB genotypes in highresponder ( n= !I)- low-responder ( n= 8) and medium responder [n= 23) carp of the base population MHC genotype
L:vrn-DAR 1 c01/2^01 1101/2"01 3"01,/4"01 ?-03/4*0?
Observed frequencies Expected frequencies
High responders
Medium Low responders responders
0 50
0.44 0 55 0.0
0 70' 0 09 0 21
0 25
0 25
0 50 0.25 0.25
P < 0 05 (XL-tri.) Q 1996 International Societv for Animal Genetics, Animal Genetics 27,313-319
C5 (Haviland et al. 1991). This incomplete splicing could result in lower expression levels of these MHC molecules on the cell surface, which might explain low responsiveness to DNP-KLH, but contrasts with the high responsiveness to DNP-HSA. Interestingly, recent mapping studies of the genes controlling the antibody response in Biozzi mice pointed at the MHC and immunoglobulin loci as contributing significantly to the high or low phenotypic differences (Puel et al. 1995). This is the first study that adresses an association of MHC class I1 B genes with the magnitude of immune responsiveness in fish. The large increase in information on teleost MHC genes (Dixon et al. 1995), will certainly initiate further studies.
Acknowledgements The authors wish to thank Drs J.A.M. Van Arendonk and H. Bovenhuis from the Department of Animal Breeding for helpful discussions on the genetic effects. This research was financially supported by the Netherlands Technology Foundation (STW), and was coordinated by the Life Sciences Foundation (SLW).
References Dixon B., Van Erp S.H.M., Rodrigues P.N.S., Egberts E. & Stet R.J.M. (1995) Fish major histocompatibility complex genes: an expansion. Developmental and Comparative Immunology 19,109-33. Haviland D.L., Haviland J.C., Fleisher D.T. & Wetsel R.A. (1991) Structure of the murine fifth complement component (C5) gene. A large, highly interrupted gene with a variant donor splice site and organizational homology with the third and fourth complement component genes. Journal of Biological Chemistry 18,11818-25. Hyodo-Taguchi Y. & Egami N. (1985) Establishment of inbred strains of the medaka Oryzias latipes and the usefulness of the strains for biomedical research. Zoo1ogical Science 2 , 305-16. Ihssen P.E., McKay L.R., McMillan I. & Phillips R.B. (1990) Ploidy manipulation and gynogenesis in fishes: cytogenetic and fisheries applications. Transactions ofthe American Fisheries Society 119, 698-717. Jacob M. & Gallinaro H. (1989) The 5' splice site: phylogenetic evolution and variable geometry of association with UlRNA. Nucleic Acids Research 17, 459-72. Johnstone R. & Stet R.J.M. (1995) The production of gynogenetic Atlantic salmon, Salmo salar L. Theoretical and Applied Genetics 90, 819-26. Jones S.R.M., Palmen M. & Van Muiswinkel W.B. (1993) Effects of inoculum route and dose on the immune response of common carp, Cyprinus carpio
