217065a0Nature21751231968010665670028-0836196810.1038/217065a0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v217/n5123issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue217065a0Proteins and Haemoglobins of Salmon-Trout Hybrids
AU  - HAEN, PETER J.
AU  - O'ROURKE, FERGUS J.Department of Zoology, University College, Cork.SPECIMENS of the salmon (Salmo salar)-sea trout (S. trutta) which were developed by Dr D. J. Piggins of the Salmon Research Trust were obtained from a small lake at Galmoylestown, near Mullingar, County West-meath. The eye lens, muscle and serum proteins and haemoglobins of these F2 2 yr old (2+) specimens were compared with those of adult salmon from the River Lee, County Cork, and with those of 2+ specimens of brown trout (S. trutta) from the Fanure Fish Farm, Roscrea, County Tipperary.The proteins of the eye lens were separated electro-phoretically in agar gel on microscope slides with phosphate buffer pH 7.3 at 5 m.amp per slide for 40 min using the bath described by Koch et al2 and the technique of Scheideggar3. Muscle extracts were prepared from the middle of the body near the dorsal fin and electrophoresis was carried out in polyacrylamide gel4 using 0-05 molar veronal buffer at pH. 8.6 at 30 m.amp for 120 min. Electrophoresis of serum proteins was also carried out in polyacrylamide gel. The electrophoretograms were scanned in the automatic fully integrated 'Chromoscan' recorder using a slit width of 1/20 mm, a 3 : 1 gear ratio and cam 5-034 A. The 'Chromoscan' patterns have been reduced in the figures to one-quarter the original size.
The haemoglobins were analysed using two different supporting media; micro starch gel and micro agar gel. For starch gel studies, the haemolysates were made up in fris-hydrochloric acid buffer at pH 8.2: some fractions migrated towards the anode arid others migrated towards the cathode from the point of application. The technique used was the micro starch gel method of Koch, Bergstr m and Evans5. With the micro agar gel technique, we used the bath described by Koch et al.2 and the method of Scheideggar3. In agar gel all the haemoglobin fractions travelled towards the negative pole.
In Fig. 1 is shown the electrophoretic pattern of the lens proteins. The salmon shows at least eight components, the trout at least seven and the hybrid a minimum of ten. The hybrid pattern is not therefore like the parental patterns, nor does it seem to be a simple mixture of the two parental proteins. This is in contrast to what we have found in the case of the bream (Abramis brama)-rudd (Leuciscus erythrophihalmus) F1 hybrid whose pattern resembles the parental patterns which themselves are very similar. These results are now in preparation.
Fig. 1. Electrophoretic patterns of the eye lens proteins of salmon, trout and the salmon-trout hybrid
Fig. 2. Electrophoretic patterns of the muscle proteins of salmon, trout and the salmon-trout hybrid.
In Fig. 2 are shown the muscle protein patterns of the three fishes. There is relatively little difference between the patterns of the two parental species, and the hybrid pattern is also very much like those of the parents. Rabaey6 using agar gel electrophoresis found that closely related species had similar patterns, although Connell7 using the Tiselius method found that patterns of related species seemed to be as dissimilar as those of unrelated species. Greenberg and Kopac8 found that in the genus Xiphophorus (using paper electrophoresis with a phosphate buffer at pH 6.5) hybrid forms showed characteristics of both parental species.
The electrophoretic patterns of the serum proteins of the salmon, the sea trout and their F2 hybrid are shown in Fig. 3. It will be seen that the hybrid pattern is more like that of the trout than that of the salmon and does not seem to be a simple mixture of the parental patterns: one new hybrid serum protein fraction does, however, occur. This is similar to our finding, the literature on which is now being prepared, that in the rudd-bream hybrid, the hybrid serum protein pattern resembles that of the bream. Sanders9 in a study of three trout species and their hybrids, found that the hybrids gave serum protein electrophoretic patterns characteristic of one of the parental types. Nyman10 studied a roach (Leuciscus rutilus)-rudd hybrid, the splake, a hybrid between eastern brook trout Salvelinus fontanalis and the lake trout (S. manaycush) and the br ding, a hybrid between S. fontanalis and the char (S. alpinus). In the serum protein patterns the roach-rudd hybrid showed perfect summation, the splake pattern was indistinguishable from S. fontanalis and in the br ding "not every band system is a summation of the parental patterns".
Nyman11 reported that the F1 salmon-brown trout hybrid serum protein pattern is a simple combination of the proteins of both parents. He later12 noted that some of these proteins seem to be determined by the same genes in both parental species and only one band is present in the F1 hybrid in a concentration equal to that in the parents. He found that in the F2 generation, selection occurs against the salmon proteins of the serum and the kidney, liver and serum esterases, but others have shown that this is not true of the blood groups13.
It may be seen from Fig. 4 that no new hybrid haemoglobin was found using the starch gel technique; the hybrid pattern is not, however, just a mixture of the two parental patterns. On both the anode and the cathode side, only four of the five salmon fractions are present in the hybrid and the C1, C2 and C4 fractions of the trout do not occur in the hybrid. The micro agar gel technique also shows that there is an overall reduction in the number effractions in the hybrid, as seen in Fig. 5. Koch et al. have shown that the relative quantities of the components, which at pH 8.2 migrate to the anode, decrease as the salmon and sea trout grow.
Sick, Frydenberg and Nielsen14 studied the haemoglobin patterns of the plaice (Pleuronectes platessa) and the flounder (Platichthys flesus) and their natural hybrids. Nearly all the hybrids (thirty-three out of thirty-four) showed the same haemoglobin pattern which clearly differed from that obtained by running an in vitro mixture of haemoglobins from the two parent species. The authors explained that the presence of new haemoglobins in the hybrid was evidence for the occurrence of at least two different sub-units in the tetrameric haemoglobin molecules of the parental species. The single deviant pattern found among the 327 individuals which were studied was recognizable morphologically as a hybrid which could have been the result of a mating between two hybrids or between a hybrid and one of the parent species.
Manwell et al.15 noted that there was little evidence for the formation of any new hybrid protein in various animals. These authors studied the haemoglobins of some hybrid birds and fishes to investigate the possibility that new haemoglobins might arise in hybrids from recombinations of the different polypeptide chains and be significant in explaining some aspects of hybrid vigour.
Fig. 3. Electrophoretic patterns of the serum proteins of salmon, trout and the salmon-trout hybrid.
In a study of four species of centrarchid fishes, it was found12 that in most cases the F1 hybrids yielded a haemoglobin pattern which was identical to that obtained by simply mixing the haemoglobins of the two parents. In some of these crosses, however, the electrophoretic properties of 25-40 per cent of F1 hybrids were different from those of the parental species.
Fig. 4. Haemoglobin patterns of salmon, trout, salmon-trout hybrid and an in vitro mixture of salmon and trout blood in starch gels.
Fig. 5. Haemoglobin patterns of salmon (1), trout (3) and salmon-trout hybrid (2) in agar gels, C1 and C2 of the hybrid pattern correspond to the C2 and C3 of the salmon and trout patterns. The C3 component of the hybrid has a migration rate similar to the C4 of the trout, while the C4 fraction of the hybrid has comparable components in C5 of the salmon and C5 of the trout.
"Artificial hybridization" of haemoglobins from the small mouth bass Micropterus dolomieu and the large mouth bass M. salmoides was achieved by mixing haemolysates and leaving the mixture in pure carbon monoxide for several days. The new haemoglobins which were produced seem to be identical with those of the red-eyed bass M. coosae and the spotted bass M. punctulatus which, it is suggested, may have evolved from hybrids of the large-mouth and small-mouth bass. Hybrid fishes do not seem to have any unique haemoglobin peptides, which again suggests that polypeptide chain recombination rather than biosynthetic interaction is responsible for the appearance of new hybrid haemoglobins. Manwell et al.15 have shown that in some hybrid fishes there is better blood gas transport by the new haemoglobin, thus providing an understanding of the nature of hybrid vigour at the molecular level15.Piggins, , D. J., Ann. Rep. Salm. Res. Trust, Ire., 1964, 27 (1965).Koch, , H. J., Bergstrom, , E., and Evans, , J., Medel. Vlaam. Acad. Kl. Wet, 26, 1 (1964).Scheideggar, , J. J., Intern. Arch. Allergy appl. Immunol., 7, 103 (1955).Raymond, , S., and Weintraub, , L., Science, 130, 711 (1959).PubMedISIChemPortKoch, , H. J., Bergstrom, , E., and Evans, , J., Laxforskningsinstitutet Meddelande, 6, 1 (1964).Rabaey, Protides Biol. Fluids, 12, 273 (1964).Connell, , J. J., Biochem. J., 55, 378 (1953).PubMedISIChemPortGreenberg, , S. S., and Kopac, , M. J., Physiol. Zool., 38, 149 (1965).ISISanders, , B. G., and Leone, , C. A., Taxonomic Biochemistry and Serology (New York, 1964).Nyman, , L., Laxforskningsinstitutet Meddelande, 13, 1 (1965).Nyman, , L., Swed. Salmon Res. Inst. Rep. LFI Medd., 3, 1 (1966).Nyman, , L., Rep. Inst. Freshwat. Res. Drottningholm, 47, 5 (1967).Alabaster, , J. S., and Durbin, , F. J., Rep. Salm. Res. Trust Ire., 38 (1964).Sick, , K., Frydenberg, , O., and Nielsen, , J. T., Nature, 198, 712 (1963).Manwell, , C., Baker, , A., and Childers, , W., Comp. Biochem. Physiol., 10, 103 (1963).PubMedISIChemPort