Birds and their training
We used 30 naïve adult great tits as our model insectivorous bird
species. All birds were collected from nest boxes when 10-13 days old;
between 22nd and 24th of May 2018. All chicks from a given nest (4-6
chicks per nest), were kept together in one cage until fledging. Cages
had dimensions 0.7 × 0.4 × 0.5 m with three perches. The birds were kept
under natural winter daylight (8:15 to 17:00) augmented with fluorescent
light tubes and automatically opened/closed window shades. After
fledging, birds were moved to cages in groups of two to three birds,
irrespective of their sex. Up to that period, chicks were individually
hand fed every half‐hour with hand rearing food for all bird chicks
(Nutribird a21), a bread-like diet (made from Nutribird a21, commercial
chicken food (Country’s Best Show 1 crumble), eggs, wheat flower, sugar
and sunflower margarine, mixed and baked for ~40 minutes
at ~180°C temperature) supplemented with mealworms. This
was repeated for a period of 13.5 h (6:45 am–8:15 pm) every day. Hand
feeding was continued after fledging, but the intervals were prolonged
to 1h and later 4h, to stimulate birds to feed by themselves. At
independence, about 35 days after hatching, birds were relocated to
individual cages. From then on, the baked diet, mixed seeds, commercial
dried insects (Nobby Orlux Insect Patee Premium) and water were provided
ad libitum. Mealworms were offered only as supplementary food. During
the whole period of captivity, chicks from different nests could hear
and see each other, since they were raised in the same room.
None of the birds used in the experiment had previous direct experience
with foliage or plant volatiles except that they could have been
indirectly exposed to some plant cues associated with caterpillars and
other herbivorous insects fed to them by their parents in the first
10-13 days after hatching. As Cattley guava naturally occurs in South
America, its volatiles are evolutionary unfamiliar to the birds used in
our experiment. Moreover, the bird chicks came from nest boxes placed in
mixed oak-spruce forest, with no Scotch elm trees present in the
surroundings. As HIPVs are plant-specific, we can consider that the
birds used in our experiment are naïve to HIPVs emitted by both sapling
species used in the experiment. While Cattley guava represented a
completely novel signal to the birds, Scotch elm represented a signal to
which the birds could be evolutionarily adapted. Birds were randomly
assigned into three training groups: (1) to associate food with the
HIPVs of the Cattley guava, (2) to associate food with the HIPVs of the
Scotch elm, and (3) not trained to associate food with any green
foliage.
We performed all training, and experiments, in two Y-shaped aviaries
built with mesh screens (Figure S1). Two 2m tall dead Pedunculate oaks
in a large pot were placed in the corners of the aviary and served as a
perch. The birds whom we conditioned to find food in induced Cattley
guava were offered one induced Cattley guava with 5 mealworms pinned to
the leaves and one control cattle guava without any mealworms. The
position of the induced and control cattle guava (left or right corner)
in the aviary was selected randomly. Birds were allowed to habituate to
the aviary and search for food, thus associate the volatile compounds of
guava with the reward, for 1.5 hour every day. The birds were released
to the aviary in pairs to make the learning (from each other) faster in
first 3 trials. We conditioned birds to the volatile compounds of elm
the same way. To condition the control birds to the cage, and initiate
their interest in searching for food, we pinned 5 mealworms directly on
one of the dead Pedunculate oaks. We conducted one training trial per
bird daily. After each training trial, we checked how many meal worms
were eaten. We considered the birds to be conditioned to the volatile
compounds of the specific plant after they repeatedly ate at least 4 out
of the 5 mealworms from the induced sapling.
It took 15 trials to condition all the birds to the induced saplings
successfully. Some (40%) birds did not pay too much attention to the
plants during the first five trials but investigated mostly the aviary
netting and surroundings. After first five training trials, we started
to record some missing mealworms in most of the bird individuals, while
some (20%) bird individuals took at least 10 trials until they started
eating mealworms. After 15th trial, all birds
successfully searched for meal worms on the induced saplings. This
contrasted with previously described similar training, during which
birds were able to find the larvae on sapling after fifth
acclimatization (Amo et al. 2013a).