/ . Embryol. exp. Morph. Vol. 24, 2, pp. 425-428, 1970
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Printed in Great Britain
Ontogeny of haemoglobin in the royal penguin
Eudyptes chrysolophus schlegeli
By P. D. SHAUGHNESSY 1
From the Department of Genetics, and the Mawson Institute
for Antarctic Research, University of Adelaide
SUMMARY
Haemoglobins of chicks, yearlings and known-age sub-adults of the royal penguin were
investigated by starch-gel electrophoresis at pH 8-6.
Two haemoglobin components were observed in yearlings and sub-adults. These were
assumed to be the haemoglobins of adults.
Another three haemoglobin components were observed in 3-week-old chicks.
In seven out of eight fledging chicks, these chick haemoglobins had disappeared.
The existence of other haemoglobin components in royal penguin embryos is predicted.
INTRODUCTION
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).
MATERIALS AND METHODS
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
1
Author's address: Institute of Arctic Biology, University of Alaska, College, Alaska
9970J, U.S.A.
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P. D. SHAUGHNESSY
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 x 19-gauge needle for sub-adults and a 3-8 cm x 22-gauge
needle for chicks. Blood was collected into an anticoagulant (citrated saline).
Erythrocytes were washed in 0 9 % 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.
3
4
5
6
Sample number
7
8
9
Fig. 1. Ontogeny of haemoglobin of the royal penguin Eudyptes chrysolophus schlegeli.
Samples 1 and 2: 3-week-old chicks, showing all five zones. Samples 3-5: 7-weekold (fledging) chicks, showing only zones III and IV. (Sample 5 also shows zone I.)
Samples 6 and 7: yearling, showing zones III and IV. Sample 8: 2-year-old, showing
zones III and IV. Sample 9: 3-year-old, showing zones III and IV. Sodium borate
(pH 8-6) buffer system. Amido black stain.
RESULTS
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 I and I]),
the faster of which stained more intensely, also migrated towards the anode.
Ontogeny of haemoglobin
427
Ontogeny of haemoglobin in the royal penguin is illustrated in Fig. 1. Threeweek-old chicks possessed all five haemoglobin zones. Seven-week-old (i.e.
fledging) chicks possessed two of thefivezones, 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.
DISCUSSION
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.
RESUME
Ontogenese de Vhemoglobine chez le Pingouin royal
Eudyptes chrysolophus schlegeli
Les hemoglobines du Pingouin royal (Manchot) ont ete analysees par electrophorese sur
gel d'amidon, a pH 8-6, chez des poussins, des individus ages d'un an et des sub-adultes d'age
connu.
Deux composants d'hemoglobine ont ete observes chez les individus d'un an et les subadultes. On les suppose etre les hemoglobines des adultes.
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P. D. SHAUGHNESSY
Trois autres composants d'hemoglobine ont ete observes chez des poussins ages de 3
semaines.
Dans 7 cas sur 8, ces hemoglobines de poussins ont disparu chez les oisillons, au stade de
l'apparition des plumes.
L'existence d'autres composants d'hemoglobines dans les embryons du Pingouin royal est
predite.
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.
REFERENCES
T. A. & BERTLES, J. F. (1965). Haemoglobin heterogeneity: embryonic haemoglobin in the duckling and its disappearance in the adult. Science, N.Y. 148, 509-511.
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FALLA, R. A. (1937). Birds. British Australia New Zealand Antarctic Research Expedition
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BORGHESE.
(Manuscript received 10 December 1969)