Ontogeny of haemoglobin in the royal penguin Eudyptes chrysolophus schlegeli

Embryonic and foetal haemoglobins are known to occur in several vertebrate species (Manwell, 1960). Haemoglobin ontogeny has been reported for several avian species: duck (Borghese & Bertles, 1965), turkey (Manwell, Baker, Roslansky & Foght, 1963), and chicken. Its existence in the chicken was a controversial subject (reviewed by Manwell, Baker & Betz, 1966) until these authors, using a starch-gel electrophoretic technique and polypeptide chain hybridization, convincingly demonstrated the existence of three embryonic haemoglobins which were distinct from the two adult haemoglobins of this species.

The present paper describes the discovery of haemoglobins in chicks of the royal penguin Eudyptes chrysolophus schlegeli, which are electrophoretically distinct from the haemoglobins of adult birds. This penguin is endemic to Macquarie Island (latitude 54° S, longitude 159° E) where it breeds in many large colonies. It is a local form of the macaroni penguin Eudyptes chrysolophus chrysolophus which breeds on most Subantarctic islands (Carrick & Ingham, 1967).

Blood samples were collected from twenty-nine royal penguins in the summer of 1967–8 at three localities on Macquarie Island about 2–5 miles apart: Bauer Bay, Nuggets Point and the Isthmus. Four birds of known age from the Bauer Bay colony were blood-sampled; two were 2-year-olds and two were 3-year-olds. A total of sixteen chicks was bled at the Nuggets colony nearest the mouth of Nuggets Creek. Eight of these were bled in early January, when the chicks were about 3 weeks old, and another eight in early February, just before fledging, when the chicks were about 7 weeks old. Seven yearlings, which can be recognized by their small crests (Falla, 1937), were also bled at this colony. Two birds found on the Isthmus were bled; judging by their crests, both were more than a year old.

After being anaesthetized with ether, the birds were bled by cardiac puncture, using a 7-6 cm×19-gauge needle for sub-adults and a 3-8 cm×22-gauge needle for chicks. Blood was collected into an anticoagulant (citrated saline). Erythrocytes were washed in 09 % saline, and haemoglobin prepared by lysis with the gel buffer.

Vertical starch-gel electrophoresis was carried out using water-cooled gel trays with the Tris-EDTA-borate buffer system of Smithies (cited by Huehns & Shooter, 1965) at pH 8·6 and a dilution of 1 in 20. The voltage gradient was of the order of 8 V/cm.

A total of five haemoglobin zones was observed in royal penguins; they are shown in Fig. 1. One haemoglobin component migrated towards the cathode (zone V). Two components migrated towards the anode, but remained quite close to the origin (zones III and IV). Another two components (zones 1 and II), the faster of which stained more intensely, also migrated towards the anode.

Ontogeny of haemoglobin in the royal penguin is illustrated in Fig. 1. Three-week-old chicks possessed all five haemoglobin zones. Seven-week-old (i.e. fledging) chicks possessed two of the five zones, although one of eight such chicks also possessed a third zone (Fig. 1, sample 5). These two zones were the only ones detectable in yearling, 2-year-old, 3-year-old and the unknown-age birds.

The haemoglobin of adult royal penguins was not investigated. However, the haemoglobin components present in yearling and sub-adult birds (zones III and IV) are assumed to be the haemoglobins of adults of this species, and so will be referred to as the adult haemoglobins. Also, in young royal penguin chicks haemoglobins are present, though usually lost by the fledging stage, which are distinct from the adult haemoglobins: these are referred to as chick haemoglobins. It would be of interest to determine if these chick haemoglobin components are also present in embryos, for they correspond to the three embryonic haemoglobins of domestic fowl observed by Manwell et al. (1966) using a similar technique. Similarly, the two adult haemoglobins of royal penguins correspond to the situation obtaining in adult domestic fowls. However, the royal penguin chick haemoglobins are present in the chick stage for at least 3 weeks, whereas the embryonic haemoglobins of domestic fowl were not detectable in 8-day-old chicks. This suggests that there may be a set of haemoglobins in royal penguin embryos which are distinct from those present in the early chick stage. On the other hand, the haemoglobin of the embryo may be maintained in the chick for a much longer period in the royal penguin than in the domestic fowl. Haemoglobin samples taken from royal penguin eggs and chicks at short intervals would be needed to resolve this problem. Such sampling could readily be undertaken in the royal penguin as the times of egg-laying and hatching are highly synchronized (Carrick & Ingham, 1967).

The observed multiplicity of haemoglobin zones in all birds sampled may be due to polymerization. If so, this could probably be avoided by lysing the erythrocytes and performing starch-gel electrophoresis with an inorganic phosphate buffer (C. Manwell, personal communication). However, this possibility would not alter the interpretation of the existence of haemoglobin components in royal penguin chicks which are absent from adults.

Les hémoglobines du Pingouin royal (Manchot) ont été analysées par électrophorèse sur gel d’amidon, à pH 8·6, chez des poussins, des individus âgés d’un an et des sub-adultes d’âge connu.

Deux composants d’hémoglobine ont été observés chez les individus d’un an et les subadultes. On les suppose être les hémoglobines des adultes.

Trois autres composants d’hémoglobine ont été observés chez des poussins âgés de 3 semaines.

Dans 7 cas sur 8, ces hémoglobines de poussins ont disparu chez les oisillons, au stade de l’apparition des plumes.

L’existence d’autres composants d’hémoglobines dans les embryons du Pingouin royal est prédite.

The author thanks Dr M. J. Mayo and Dr R. Carrick for helpful discussions, Professor C. Manwell for reading the draft, and Mr S. R. Harris for preparing the figure.

During 1965–8 the author was a biologist with the Antarctic Division, Department of Supply, which provided the field and laboratory facilities at Macquarie Island necessary for this investigation.