RESULTS AND DISCUSSION

The hearing of H. virescens varied based on the frequency, regardless of wing position, with threshold responses of A1 approximately 20 dB SPL more sensitive than those of A2 (Fig. 2A), similar to other noctuid moths (Agee, 1967). Wing position did not play a significant role in A1 sensitivity, except at the highest frequency tested, i.e. 80 kHz (F_3,18=4.3, P=0.024; Fig. 2A); moths with unconstrained wing movement had a lower threshold for A1, but this result was not significant. There was a significant effect of wing position for A2 threshold in the 40–60 kHz range (40 kHz, F_3,18=3.8, P=0.035; 50 kHz, F_3,18=4.4, P=0.022; 60 kHz, F_3,18=3.5, P=0.044; Fig. 2A). For these frequencies, the up position always responded more sensitively than the flat wing; this trend continued for the higher frequencies but was not significantly different. However, the down position did not show a consistent trend for its A2 threshold response. Again, animals with unconstrained wing movement had a lower A2 threshold. Position also affected the maximum number of A1 action potentials measured before A2 began firing: the down position always had fewer action potentials, averaging around 6–7, while the flat and up positions averaged 8–9, and animals unconstrained from wing movement had 7–8 A1 action potentials fire before A2 started (Fig. 2B). However, this was only significantly different at 50 kHz (F_2,18=4.5, P=0.022).

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Fig. 2.

A1 and A2 threshold neural response. (A) Neurophysiological threshold response of the A1 and A2 cells according to wing position. (B) The maximum number of A1 action potentials fired just before A2 began firing, with wings in the up, flat, down and unconstrained position. Yellow regions represent significance of at least P=0.05, n=18.

Overall, these results suggest the downward wing position is significantly less sensitive to low sound levels than the flat or up position, though it may require fewer action potentials for A2 to begin firing. A similar result was found in the underwing noctuid moth, where ears were found to be less sensitive with the wings folded over in the resting position compared with an exposed up position (Faure et al., 1993). However, the results of our experiment eliminate the wing blocking the tympanal membrane as a possible explanation for this discrepancy, as the wing was surgically removed and the ears were equally exposed. Therefore, the strong result found in Faure et al. (1993) may be a factor of (1) blocking the tympanum with the wing, and (2) wing position itself altering the mechanics of the ear. In the down position, the dorsoventral muscles are relaxed and the dorsolongitudinal muscles are contracted (Macfarlane and Eaton, 1973). The muscles switch activation to get to the up position, transitioning through the flat position. This thoracic deformation could change the tension and movement of the tympanal membrane. Therefore, the next step in understanding how wing position affects hearing was measuring the mechanical response of the tympanal membrane to sound. Sound ranges that should affect A2 were the focus of the second part of the study.

The amplitude of displacement of the tympanal membrane significantly increased with higher sound levels, but not evenly across frequencies, regardless of wing position (Fig. 3A). The tympanum was most sensitive to 30–60 kHz, which corresponds to the frequency of the bat calls the moths may be avoiding. When data were divided according to wing position, fewer significant differences were seen for the sound levels below 80 dB SPL stimuli (Fig. S1). This result is notable as at 80 dB SPL the A2 neuron has already begun firing. Interestingly, from 40 to 60 kHz, the tested sound levels were not low enough to identify movement differences below 50–70 dB SPL even when wing position was not considered (Fig. 3A); despite the lack of differences, something triggers the A2 neuron to begin firing as threshold is at approximately 60–70 dB SPL. When wing position, frequency and sound level were considered together, the wing position resulted in no significant differences at any frequency/sound level combination (Fig. 3B).

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Fig. 3.

Displacement of the tympanal membrane due to sound. (A) Average displacement for all three wing positions. Color indicates significant differences according to Tukey–Kramer. For significance of each wing position, see Fig. S1. (B) Displacement of individual wing positions at each frequency for 80 dB SPL. Data were not significantly different. n=21.

Focusing on the 80 dB SPL results, as these were significantly different within each frequency level, the tympanal membrane was displaced more when the wing was in the flat rather than up position, albeit not significantly (Fig. 3B). These data oppose what could be expected based on the neural data (Fig. 2A), as more movement should amplify the deflection of the attached sensory neurons, which should in turn increase firing of the mechanosensors. Thus, it is likely that internal muscle and/or air chamber compression plays a factor in neural sensitivity. Similar to the neural data, membrane displacement in the down wing position did not follow a specific pattern across frequencies at 80 dB SPL (Fig. 3).

One explanation for the disconnection between tympanal deflection and neural response could be internal muscle tensions. The auditory nerve lies next to the flight muscles and dorsolongitudinal muscles (Fig. 1); distinct muscle groups are contracted/relaxed during the up/down strokes of flight (Macfarlane and Eaton, 1973). Tension variation in these muscles may therefore change the tension acting on the nerve, which may in turn affect its sensitivity to the same movements of the tympanal membrane.

As the auditory nerve goes directly to the pterothoracic ganglion, there should be no other afferent information influencing the auditory neurons. However, the B cell also connects to that nerve, and its role is as yet unknown (Yack and Fullard, 1993). Testing the firing rate of the B cell identified no significant difference based on wing position (averages±s.d., action potentials/0.25 s: up 3.0±1, flat 4.7±1, down: 2.3±1, F_2,8=1.25, P=0.35). Treat and Roeder (1959) also found no effect of wing position on the B cell, but did find that artificially changing the tension of the B cell changed its firing rate, and that changing the tension by thorax depressions altered the firing rate of both the B cell and the A cells. Because of the number of experimental approaches they used and the unclear results those yielded, Treat and Roeder (1959) did not draw any strong conclusions as to what the role of the B cell might be. Perhaps the firing rate is not due to a static wing position, and instead the B cell fires more dynamically based on the transitional movement of the wing. Therefore, the static mounting of the wing would miss this differing response. If the firing rate of the B cell dynamically indicates to the moth a change from down-to-up and up-to-down, this information converging with that coming from the A cells at the ganglion may facilitate the dynamic problem of hearing while flying.

Sensitivity to wing position is more obvious in the neural response than in tympanal membrane movement. While the sensory neurons are mechnoreceptors reliant on deflections of the tympanum, other factors such as muscle configuration or compression of the internal air chamber backing the tympanum may play a role. As the methods used for this study are less invasive than previous lepidopteran neural physiological analyses, this research opens possibilities to understanding responses of the animal from a more organismal approach. Future studies should examine questions of noctuid hearing sensitivity considering wing position during mounting, and could perhaps examine wing muscle activity at the same time.