metamorph brains

Experimental treatments included low and high larval density, crossed with the presence and absence of a caged predator. 100 tadpoles from each of the 12 clutches were pooled into a bucket and allocated to each tank (high density = 50 tadpoles/tank; low density = 10 tadpoles/tank). In tanks with the predator treatments, one dragonfly larva (Aeshna sp.) was placed in a cylindrical cage (diameter 8 cm; height 21 cm) made of transparent plastic film with a double net bottom (mesh size 1.5 mm) and hung 6 cm over the tank bottom. This allowed tadpoles to receive both visual and chemical cues from the predator, while the predator was unable to actually prey on tadpoles. In the no-predator treatment, the cage was left empty. During the experiment, tadpoles relied on the initial resources provided (leaves, rabbit pellets), as well as subsequent algal growth. Each treatment combination was replicated eight times, resulting in a total of 32 experimental units. 143 metamorphs (four to five per tank) were used for analyses. Formalin fixed metamorphs were weighed with a digital balance. After dissection, dorsal and right lateral views of brains were photographed with a digital camera connected to a dissecting microscope. We could only measure two dimensions for each brain part (length and width of telencephalon, diencephalon and optic tectum, and height and width of medulla oblongata) because some of the borders of the brain parts could not be identified accurately. Measurements were taken from digital photographs using tpsDig 1.37 software, and were defined as the greatest distance enclosed by the given structure in the given direction. All brains were photographed and measured three times. Repeatability of different brain measurements was high (R = 0.71 – 0.96 [mean = 0.86]).