Acoustic Aposematism and Evasive Action in Select Chemically Defended Arctiine (Lepidoptera: Erebidae) Species: Nonchalant or Not?

Abstract

Tiger moths (Erebidae: Arctiinae) have experienced intense selective pressure from echolocating, insectivorous bats for over 65 million years. One outcome has been the evolution of acoustic signals that advertise the presence of toxins sequestered from the moths' larval host plants, i.e. acoustic aposematism. Little is known about the effectiveness of tiger moth anti-bat sounds in their natural environments. We used multiple infrared cameras to reconstruct bat-moth interactions in three-dimensional (3-D) space to examine how functional sound-producing organs called tymbals affect predation of two chemically defended tiger moth species: Pygarctia roseicapitis (Arctiini) and Cisthene martini (Lithosiini). P. roseicapitis and C. martini with intact tymbals were 1.8 and 1.6 times less likely to be captured by bats relative to those rendered silent. 3-D flight path and acoustic analyses indicated that bats actively avoided capturing sound-producing moths. Clicking behavior differed between the two tiger moth species, with P. roseicapitis responding in an earlier phase of bat attack. Evasive flight behavior in response to bat attacks was markedly different between the two tiger moth species. P. roseicapitis frequently paired evasive dives with aposematic sound production. C. martini were considerably more nonchalant and employed evasion in fewer interactions. Our results show that acoustic aposematism is effective at deterring bat predation in a natural context and that this strategy is likely to be the ancestral function of tymbal organs within the Arctiinae.

Fig 1

Morphology and acoustic emissions of Pygarctia roseicapitis (A-F) and Cisthene martini (G-L). The moths (A, G) and their corresponding tymbal organs (B, H), oscillogram (C, I), spectrogram (D, J), power spectral density plot (E, K), and the spectrogram of their response to simulated bat cries (F, L) are shown. Tymbal images are oriented with anterior on the left and ventral on the top with some scales removed. Insets show the relative position, orientation, and size of the tymbal (yellow) organ and microtymbals (red) on the thorax of each species. Insets are oriented with anterior on the left and dorsal on the top. Oscillogram, spectrogram, and power spectral density plots (C-E, I-K) show a single activation and relaxation (modulation cycle) of the tymbal organ. Moth responses to simulated bat cries (F, L) show each species’ earliest response and do not correspond to the same segment of time. Bat cries are brightest and sweep from higher to lower frequencies within a single call. Moth clicks are broadband and cluster in groups of clicks.

Fig 2. Effect of functional tymbals on the outcomes of bat-moth interactions for Pygarctia roseicapitis and Cisthene martini.

The percentages of interactions for each possible outcome recorded for each treatment group. Numbers within each bar indicate the number of interactions observed for that treatment/outcome combination.

Fig 3. Inter-pulse interval (IPI) between the two bat calls immediately preceding the first detected moth clicks for “Tymbaled” (T+ and S groups), sound-producing Pygarctia roseicapitis and Cisthene martini.

Box plot upper and lower hinges represent the 25^th and 75^th percentiles of their respective distributions. The 50^th percentile (median) is shown as a thicker black line between hinges. Tukey-style whiskers extend from each hinge to the most extreme value within 1.5*IQR (inter-quartile range). Actual data from which the box plots are constructed are displayed as points jittered along the midline of their respective box plot. Any data points beyond the whiskers are outliers. “Non-Capture” outcomes are colored black and “Capture” outcomes are colored red. Bat attack phases and their corresponding range of IPI’s are indicated as: Search Phase (white), Early Approach (light grey), Late Approach (dark grey), and Buzz (black). The right y-axis are the values of pulse repetition rate (pulse*sec^-1) corresponding to the values of IPI.

Fig 4. Number of echolocation calls bats produced between search phases for “Tymbaled” (T+ and S groups), sound-producing Pygarctia roseicapitis and Cisthene martini.

Boxplot follows plotting conventions in Fig 3. For each interaction, moth species identity has been coded as shape and the outcome of each interaction is coded by color.

Fig 5. Minimum bat-moth distances (mBMD) between “Capture, Drop” and “Non-Captured” outcomes among “Tymbaled” (T+ and S groups) and “Ablated” (T-) moths for Pygarctia roseicapitis and Cisthene martini.

Boxplot follows plotting conventions in Fig 3. Horizontal dot-dashed line demarcates 3.7 cm which was the most conservative of the smallest minimum bat-moth distances in which we could measure due to inherent limitations and error in the 3-D reconstruction process. Bat and moth should be considered to be occupying the same coordinates below this value. This plot displays the closest distance between bats and moths (T+ and S treatments) during each interaction. Interactions that resulted in “Capture, Drop” were all below 3.7 cm. All interactions that resulted in “Non-Capture” were above 3.7 cm.

Fig 6. Moth z-speed between “Capture, Drop” and “Non-Captured” outcomes among tymbaled (T+ and S groups) moths for Pygarctia roseicapitis and Cisthene martini.

Boxplot follows plotting conventions in Fig 3. The speed of the moths in the z-axis acts as a proxy for detecting diving evasive behavior. Positive values are upward flight, values near 0 m*sec^-1 are level flight, and negative values are downward flight. Only P. roseicapitis which were not captured were significantly different from 0 m*sec^-1, indicating that that species employed evasive dives. Neither outcome involving C. martini was significantly different from 0 m*sec^-1, indicating that this species did not frequently employ evasive dives. 3-D perspective plots display representative flight path data. Bats are depicted as larger points and moths as smaller points. Starting points are indicated by an arrow. Time flows from Yellow (Pre-Interaction) > Black (Interaction) > Purple (Post-Interaction). Black points are the closest distance between bat and moth and red points indicate when the moth was first detected to click. (A) “Non-Captured” P. roseicapitis diving (negative moth z-speed) in response to a bat attack. (B) “Non-Captured” C. martini taking no evasive action (moth z-speed ≈ 0) in response to a bat attack. Neither bat turned away from the clicking moth nor did they enact typical prey capture behaviors.

Fig 7. Percentage of interactions hand-scored as “Evasion” by treatment for P. roseicapitis and C. martini.

Numbers within each bar indicate the number of interactions observed for that treatment group and percentages indicate the percent of those observations that were scored as “Evasion”.

Fig 8. Ethogram showing the progression of bat attacks and possible outcomes of bat-moth interactions.

A bat approaches a moth and can either capture or not capture that moth. If the moth is not captured that encounter has ended and the moth has survived. If the bat has captured a moth it can then either drop it or consume it. In this study’s context, if a sound-producing moth is not captured it is evidence that aposematism was effective in deterring the bat attack. If a captured moth is rejected by being dropped they typically survive and this is evidence that defensive chemistry was effective in deterring consumption.