Flight trials
We collected eastern monarchs (n=32; 17 male, 15 female) from migratory
stopover site St. Marks, FL in October 2016 and western monarchs (n=31;
16 male, 15 female) from an overwintering site near Oceano, CA in
December 2016 to perform flight trials. All butterflies were housed in
overwintering-like conditions in an incubator to ensure they were in the
same overwintering state during flight trials in December 2016. We used
two flight mills as described in Bradley et al 2005
(Bradley & Altizer, 2005) and an ASCO
PS-2000 datalogger (Pasco Scientific, Roseville, CA, USA) to allow
eastern and western monarchs to fly in continuous circles of 4.27 m
circumference. We recorded the time elapsed between each rotation (to
measure instantaneous speed), the cumulative flight time, and the body
mass of the monarch pre- and post- flight trial.
Flight trials were performed in a laboratory space maintained at 25°C
and controlled for light conditions. One day prior to flight trials,
monarchs were removed in groups and a steel wire attachment (32 gauge, 9
cm long) was glued to the dorsal side of the thorax using rubber cement.
Following wire attachment, monarchs were held in cylindrical mesh flight
cages (diameter= 0.38 m, height= 0.56 m) to allow acclimation to the
wire and for free feeding. We calculated five measures of flight
performance: flight duration, distance, loss of body mass relative to
total distance flown, power, and speed. During the flight trials, we
allowed monarchs to fly for 30 minutes with trials ending prior to the
30 minute maximum if monarchs suspended flight for more than 10 seconds
on 3 separate occasions. Flight trials were considered unsuccessful if
the monarch refused to fly at least one full rotation on the flight
mill. We then measured flight performance for the 29 western monarchs
(14 male, 15 female) and 27 eastern monarchs (14 male, 13 female) which
successfully completed flight trials. Distance was measured as total
distance in meters of a flight trial. Loss of body mass was calculated
as the change in body mass (massinitial -
massfinal) divided by distance flown (in m), then
log-transformed. Power was calculated as
(1/2*mass*velocity2) divided by time (in s). Speed
(m/s) was averaged across 2-minute intervals for the duration of each
flight in order to calculate the average flight speed.
We also measured morphological traits relevant to flight, including wing
size and wing shape. We measured these traits to determine if any
differences in flight behavior were due to differences in wing
morphology. Following existing protocols
(Altizer & Davis, 2010;
Li, Pierce & de Roode, 2016), forewings
were scanned on a flatbed scanner and the Fovea Pro plugin (Reindeer
Graphics, Inc., Asheville, NC) for Adobe Photoshop was used to measure
forewing area, length, breadth and perimeter. From these measurements we
calculated aspect ratio, by dividing length by breadth of the forewing,
and roundness, by using the equation 4*π*area/
(perimeter)2 (Altizer &
Davis, 2010). Using Principal Component Analysis (PCA), forewing area,
length, and width were reduced into one variable (PC1) to measure
forewing size, while forewing aspect ratio and roundness were reduced to
a second variable (PC2) to measure forewing shape.
We used analysis of variance (ANOVA) in R 3.1.3
(R Development Core Team, 2012) to test
for differences in PC1 (wing size) and PC2 (wing shape) between eastern
and western monarchs. We used analysis of covariance to test for
differences in flight duration, flight distance, loss of body mass
relative to total distance flown, flight power, and flight speed between
eastern and western monarchs. In these analyses, we included butterfly
sex as an additional explanatory variable and included PC1 (wing size)
and PC2 (wing shape) as covariates. Significance of terms in analyses of
variance and covariance was assessed by model simplification followed by
model comparison using the command “anova”
(Crawley, 2007). Models were plotted to
verify the assumptions of homogeneity of variance and normality of
errors (Crawley, 2007).