255 melatonin is crucial for the migratory orientation of pied

J. exp. Biol. 194, 255–262 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
255
MELATONIN IS CRUCIAL FOR THE MIGRATORY
ORIENTATION OF PIED FLYCATCHERS
(FICEDULA HYPOLEUCA PALLAS)
THOMAS SCHNEIDER, HANS-PETER THALAU, PETER SEMM
AND WOLFGANG WILTSCHKO
Fachbereich Biologie der Universität Frankfurt, Zoologie, Siesmayerstraße 70,
D-60054 Frankfurt am Main, Germany
Accepted 24 May 1994
Summary
After pinealectomy, young pied flycatchers tested in the geomagnetic field have been
found to be disoriented. In order to examine the possible role of the pineal hormone
melatonin, handraised flycatchers were pinealectomized (PX) at the age of 8 weeks. From
the day of operation onward, the PXMEL group received 100 mg of melatonin every
evening 1 h before darkness, the PXSOL group was injected with the solvent only, and the
PX group was untreated. Unoperated birds served as controls. During the following
autumn migration, the birds were tested for directional preference in the local
geomagnetic field, in the absence of visual cues. The controls were oriented in the
species-specific southwesterly direction; pinealectomized birds without additional
melatonin (PXSOL, PX) did not show directional preferences. The PXMEL birds that had
received daily injections of melatonin also showed significant southwesterly tendencies;
their orientation did not differ from that of the controls. This indicates that melatonin is
involved in migratory orientation, either in the processes of expressing the genetically
encoded information on the migratory course as a direction with respect to the
geomagnetic field or in the time programme controlling the specific migratory direction
at a given time.
Introduction
The amount and the direction of locomotor activity of caged migratory birds during
migratory restlessness is closely related to the actual migration of free-living conspecifics
(Berthold, 1972; Gwinner and Wiltschko, 1978; Helbig et al. 1989). Displacement
experiments and tests with handraised birds indicate that inexperienced birds possess
genetically encoded information on the direction and the distance of their migratory route
(Perdeck, 1958; Helbig, 1991). These observations led to the hypothesis that an
endogenous circannual programme controls the duration of migration and an innate
compass course its direction (e.g. Berthold, 1988). Before starting on its first migration, a
young bird must transfer the genetically coded directional information into an actual
Key words: pied flycatcher, Ficedula hypoleuca, pineal gland, melatonin, magnetic compass, migration,
innate directional information, circannual control.
256
T. SCHNEIDER AND OTHERS
flying direction, which must be maintained over the required distance. The use of the
directional information requires an external reference system, which can be provided by
the geomagnetic field (Wiltschko and Gwinner, 1974; Beck and Wiltschko, 1982).
Earlier experiments indicated a crucial role of the pineal gland in orientation during the
first migration: when pinealectomized at 8 weeks old, pied flycatchers, Ficedula
hypoleuca, were unable to orient in the earth’s magnetic field (Semm et al. 1984). This
suggested that the pineal gland was involved in the processes of establishing the
migratory direction and/or locating it with respect to the magnetic field. The specific role
of the pineal gland, however, was still unclear. Pinealectomy is known to result in
elimination of the circadian rhythms of locomotor activity and body temperature in
sparrows and other passerines (Gaston and Menaker, 1968; Binkley et al. 1971; Menaker
and Zimmerman, 1976; Zimmerman and Menaker, 1979; Gwinner, 1981). These effects
depend on the absence of the rhythmic synthesis and secretion of the pineal hormone
melatonin (Underwood and Goldman, 1987), as they can be cancelled in pinealectomized
individuals by the regular application of melatonin (Gwinner and Benzinger, 1978). In
connection with migratory orientation, however, mediation of the effects of the pineal
gland, either by its neuronal output (Korf et al. 1982; Semm and Demaine, 1984) or by its
intrinsic magnetic sensitivity (Demaine and Semm, 1985), seemed equally possible. It
was decided, therefore, to examine whether daily injections of melatonin in young,
pinealectomized flycatchers could compensate for the effects of pinealectomy on
migratory orientation.
Materials and methods
Experimental birds
Pied flycatchers are nocturnal migrants breeding in central Europe and overwintering
in western Africa at latitudes between 5 ˚ and 12 ˚ N. In autumn, the birds migrate first in a
southwesterly direction, then change to a southerly or southeasterly direction after they
reach the Iberian peninsula, to head for their African winter quarters. During spring
migration, the birds fly in a northerly direction using a more direct route via the Sahara
and the Mediterranean (Zink, 1985).
Young flycatchers were removed from their nest boxes at approximately 10 days old.
They were handraised indoors in the local geomagnetic field. After becoming
independent at approximately 4–5 weeks of age, the birds were placed in individual cages
in a room with an artificial photoperiod simulating the natural photoperiod of Frankfurt
am Main. The flycatchers were thus prevented from experiencing celestial cues.
Experimental treatment
At 8 weeks of age, 24 of the 39 birds were pinealectomized. The operation was
conducted under Ketanest (0.4 ml kg21) and Rompun (0.5 ml kg21) anaesthesia; 0.02 ml
was injected into the pectoralis muscles. Using a pair of extra-fine forceps and a
stereomicroscope, the pineal gland and the choroid plexus were carefully removed through
a hole that had been drilled in the skull. After the orientation tests the pinealectomy was
verified histologically; no remaining pineal tissue was found in any of the birds.
Crucial role of melatonin in migratory orientation
257
Four groups of birds were used: C, 15 intact birds that served as controls; PX, 6
pinealectomized birds, left untreated; PXMEL, 14 pinealectomized birds that received
daily melatonin injections; and PXSOL, 5 pinealectomized birds that received the solvent
only.
For the injections, 50 mg of melatonin (Sigma) was dissolved in 1 ml of absolute
ethanol and 9 ml of physiological saline. After the operation, the birds in the PXMEL
group received 0.02 ml of the solution, i.e. 100 mg of melatonin daily, a dose that has been
shown to be sufficient to minimize the effects of pinealectomy in birds (Underwood and
Goldman, 1987). The solution was injected into the right pectoralis muscle, 1 h before the
lights were turned off. The PXSOL group received an injection of the solvent only at the
same time. This procedure was followed to mimic the natural circadian rhythm of the
synthesis and secretion of melatonin (Underwood and Goldman, 1987).
Orientation tests
Orientation experiments were performed from 10 August until 30 September in 1989
and 1990. They took place in small wooden huts in the garden in the local geomagnetic
field in Frankfurt (46 000 nT, 66 ˚ inclination), in the absence of visual orientation cues.
The birds were tested individually in funnel cages (Emlen and Emlen, 1966). For
recording, the funnel wall was lined with typewriter correction paper (Rabøl, 1979;
Beck and Wiltschko, 1982), upon which the bird’s hopping left scratches. The tests
began 30 min after nightfall and lasted about 90 min. All birds were tested only once per
night.
Data analysis and statistics
The lining of the funnel cages was divided into 24 sectors of 15 ˚, and the number of
scratches per sector was counted. Using vector addition, the compass heading was
calculated from these data. If the recorded activity was less than 25 scratches, the
recording was excluded from analysis.
From the headings of each individual bird, we calculated its mean vector with the
heading ab (degrees) and the length rb. Further analysis was based on the mean headings
ab. For each test group, a mean vector was calculated and tested for significant directional
preference by the Rayleigh test (Batschelet, 1981). The Mardia–Watson–Wheeler test
(Batschelet, 1981) was used to compare the distribution of the birds’ headings.
Results
The vectors of the test birds are listed in Tables 1–3. The controls (Table 1) showed
significant directional tendencies in a southwesterly direction, which correspond well
with the species-specific orientation of free-flying flycatchers, as indicated by ringing
recoveries (Zink, 1985). The PX birds, as found in earlier studies, did not show significant
preferences, nor did the PXSOL group (Table 2). The PXMEL birds (Table 3), in contrast,
were well oriented (see Fig. 1); they are significantly different from the PX and PXSOL
groups combined (P<0.01, Mardia–Watson–Wheeler test), but do not differ from the
controls (P>0.05).
258
T. SCHNEIDER AND OTHERS
Table 1. Orientation of the intact control birds
Bird
C 89/1
C 89/2
C 89/3
C 89/4
C 89/5
C 89/6
C 89/7
C 90/1
C 90/2
C 90/3
C 90/4
C 90/5
C 90/6
C 90/7
C 90/8
nb
ab
(degrees)
4
6
7
5
4
5
5
7
10
12
6
11
9
7
9
307
293
276
268
246
178
337
169
207
150
155
347
203
223
167
rb
0.65
0.17
0.17
0.37
0.35
0.14
0.17
0.68*
0.49
0.50*
0.56
0.24
0.47
0.57
0.38
For all controls, N=15; aN=229 °; rN=0.48*.
nb, number of test nights per bird; ab, rb, mean vectors of the individual bird; N, number of test birds
per group; aN, direction and rN, length of mean vector of the group, calculated from the headings ab of
the individual birds.
Asterisks indicate significant directional preference by Rayleigh test (*P<0.05; **P<0.01;
***P<0.001).
N
N
E
W
N
E
W
S
S
C
PX, PXSOL
E
W
S
PXMEL
Fig. 1. Orientation behaviour of young pied flycatchers during their first autumn migration.
The triangles at the periphery of the circles show the mean headings of the individual test
birds. The arrows represent the mean vector with the radius of the circle having a value of 1.
C, intact control birds; PX, pinealectomized birds (filled triangles); PXSOL, pinealectomized
birds that were injected daily with solvent only (open triangles); PXMEL, pinealectomized
birds that received daily injections of melatonin. The two inner circles mark the 5 % (dashed)
and the 1 % significance levels of the Rayleigh test (for numerical values, see Tables 1–3).
Crucial role of melatonin in migratory orientation
259
Table 2. Orientation of the pinealectomized birds not receiving melatonin
Bird
nb
ab
(degrees)
rb
PX 89/1
PX 89/2
PX 89/3
PX 89/4
PX 90/1
PX 90/2
PXSOL 89/1
PXSOL 89/2
PXSOL 89/3
PXSOL 89/4
4
4
6
7
10
13
5
5
4
4
299
160
325
267
107
99
235
324
11
4
0.81
0.39
0.51
0.41
0.38
0.46
0.45
0.42
0.11
0.42
For all PX and PXSOL groups, N=10; aN=328 °; rN=0.25.
PX birds received no treatment; PXSOL birds received daily injections of solvent.
Symbols as in Table 1.
Table 3. Orientation of the pinealectomized birds that were receiving daily injections of
melatonin
Bird
nb
ab
(degrees)
PXMEL 89/1
PXMEL 89/2
PXMEL 89/3
PXMEL 89/4
PXMEL 89/5
PXMEL 89/6
PXMEL 89/7
PXMEL 90/1
PXMEL 90/2
PXMEL 90/3
PXMEL 90/4
PXMEL 90/5
PXMEL 90/6
PXMEL 90/7
8
5
7
7
7
5
4
7
6
8
8
11
5
9
292
166
195
285
233
228
256
208
219
137
163
193
259
154
rb
0.44
0.31
0.31
0.45
0.40
0.35
0.39
0.66*
0.45
0.63*
0.74**
0.43
0.85*
0.65*
For all PXMEL groups, N=14; aN=213 °; rN=0.70***.
Symbols as in Table 1.
Discussion
Our data clearly show that the effect of the pineal on migratory orientation is mediated
by melatonin. Melatonin could compensate for the orientational deficits normally caused
by pinealectomy; birds receiving daily injections of melatonin exhibited the directional
tendencies usually observed during that time of the year and their orientation did not
differ from that of the intact controls.
260
T. SCHNEIDER AND OTHERS
It is interesting to consider which stage of the migratory orientation process requires
melatonin for normal functioning. Migratory orientation, as we observe it in our test
cages, is the result of a complex sequence of processes, many aspects of which are not yet
fully understood. Young birds on their first journey to the yet-unfamiliar overwintering
site rely on inborn information concerning their migratory direction (Helbig, 1991). This
genetically coded directional information must be transferred into an actual direction in
space, with the help of an external reference system. Our test birds had been prevented
from seeing the sky and observing celestial rotation; hence, the only reference available
to them was the geomagnetic field (Beck and Wiltschko, 1982). When autumn migration
begins, the birds exhibit migratory restlessness and orient in the seasonally appropriate
direction. Is melatonin required for transferring the genetically coded information into a
directional angle with respect to the magnetic field, or for initiating the migratory phase
so that the directional tendencies can be expressed, or is it involved in the perception and
processing of magnetic information?
The latter seems rather unlikely. Pinealectomized homing pigeons, released in overcast
conditions, i.e. conditions where they are assumed to rely on the magnetic field, showed
normal orientation (Maffei et al. 1983; Papi et al. 1985), which suggests that they could
use their magnetic compass. Previous experiments with pied flycatchers also indicated
that magnetic orientation per se is largely independent of the melatonin levels: control
birds tested at noon, when the melatonin level is at a minimum, were as well-oriented as
those tested during the night (Thalau and Wiltschko, 1987), and pinealectomized birds
showed well-oriented nocturnal activity during spring migration (Semm et al. 1987).
Thus, an effect of melatonin on the magnetic sensor itself or on the capacity to use
magnetic information as a compass is unlikely.
Because of the general role of melatonin in the circadian system (Vakkuri et al. 1985),
its possible effects on the circadian rhythm or even on the annual cycle (Reiter, 1991;
Gwinner et al. 1993) have to be considered. All our test birds showed Zugunruhe, the
nocturnal restlessness observed in night-migrating birds during the migratory season,
although the amount of activity in the pinealectomized birds did not reach the level of
intact controls (H. P. Thalau, unpublished observations). However, the specific preferred
directions over the course of migration are also controlled by a time programme (Gwinner
and Wiltschko, 1978; Helbig et al. 1989; Munro et al. 1993), and it is still unclear whether
this programme is identical with the one controlling activity. Hence, it remains a
possibility that low melatonin levels have disruptive effects on aspects of the annual
cycle.
It is also possible that the role of melatonin is in the transfer of the genetically encoded
directional information into a direction with respect to the magnetic field. These
processes, which take place over a limited period prior to the onset of migration, are not
yet understood, nor do we know where in the brain information on the migratory direction
is processed. However, if melatonin is involved in those processes, our present findings
suggest new areas for investigation into where the important steps leading to migratory
orientation take place. For instance, the presence of melatonin-binding sites might give an
indication of the part of the brain involved.
Crucial role of melatonin in migratory orientation
261
This study was supported by the Deutsche Forschungsgemeinschaft in the Program
SFB 45 ‘Vergleichende Neurobiologie des Verhaltens’, the Stiftung Volkswagenwerk,
and a stipend from the Heisenberg Program to P.S.
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