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Infrared spectral sensitivity of Melanophila acuminata
Journal of Insect Physiology 47 (2001) 1441–1450 www.elsevier.com/locate/jinsphys
Infrared spectral sensitivity of Melanophila acuminata D.X. Hammer a
a,*
, J. Seigert a, M.O. Stone b, H.G. Rylander III a, A.J. Welch
a
Department of Electrical Engineering, University of Texas, Austin, TX 78712, USA b AFRL/MLPJ, Wright-Patterson AFB, OH 45433-7702, USA Received 14 May 2001; accepted 8 August 2001
Abstract The spectral sensitivity of the pit organ of the beetle Melanophila acuminata (Coleoptera:Buprestidae) was measured using an ultrafast tunable infrared laser source and standard electrophysiological techniques. The pit organ may be classified as a broadband detector as the beetles responded to all infrared excitation wavelengths from 2 to 6 µm. There was a decrease in response threshold and latency and an increase in the magnitude of the response in the region from 2.8 to 3.5 µm, which corresponded to a region of decreased transmittance (increased absorbance) as measured by Fourier transform infrared spectroscopy. The implications of the correlation between spectral response and optical properties are discussed. 2001 Elsevier Science Ltd. All rights reserved. Keywords: M. acuminata; Sensitivity threshold; Spectral sensitivity; Action spectrum; Action potential; Generator potential; Infrared receptor
1. Introduction Buprestid beetles of the species Melanophila acuminata have shown sensitivity to infrared radiation in behavioral and physiological experiments (Evans, 1966; Schmitz and Bleckmann, 1998; Hammer et al., 2001). The beetles possess bilateral thoracic pit organs in the posteriolateral border of the coxal cavity of the second set of legs. The beetles detect infrared (IR) radiation emitted by forest fires presumably in the 2–5 µm atmospheric window, where H2O and CO2 absorption are minimal. Examination of tissue optical properties, in particular Fourier transform infrared (FTIR) spectroscopy, has indicated that the tissue which composes the IR pit organ has high absorption (low transmittance) from 2.8 to 3.5 µm (Vondran et al., 1995). The source of the absorption could be C–H and O–H groups that have vibrational stretch resonances near 3 µm (O–H: 2.70–3.23 µm, C– H: 3.28–3.57 µm (Twardowski and Anzenbacher, 1994).
* Corresponding author. Present address: Physical Sciences Inc., 20 New England Business Center, Andover, MA 01810, USA. Tel.: +1978-738-8224; fax: +1-978-687-7211. E-mail addresses: [email protected] (D.X. Hammer), [email protected] (J. Seigert), [email protected] (M.O. Stone), [email protected] (H.G. Rylander III), welch@ mail.utexas.edu (A.J. Welch).
Since these molecular groups are present in the proteins and carbohydrates that are the building blocks of all animal cells, this property of the tissue does not in itself indicate environmental adaptation for near- to mid-infrared detection. In fact, the transmission properties of the beetle pit organ are similar to those for the infraredsensitive (8–12 µm) pit membrane of Crotaline snakes of the genera Trimeresurus and Agkistrodon (Goris and Nomoto, 1967). However, the bulk tissue optical properties, as well as other characteristics of the spherule (the spherical portion of the sensillum thought to absorb the majority of infrared radiation) may contribute to the overall functional design of the organ. For example, the unique lamellar structure of the outer layers and the contents of the interior regions of the spherule may serve to enhance infrared sensitivity through IR interference and absorption respectively (Fig. 5A). Moreover, the manner in which the spherule protrudes out of the mesocuticle may attenuate lateral conduction of heat (i.e. insulate each individual sensillum by convective cooling), which thereby enhances the conduction of heat to deeper layers and isolates each sensillum. The isolation of the sensilla in the pit organ may increase the overall sensitivity of the entire pit and also enable spatial information to be detected. In order to prove that the bulk optical properties of the IR pit organ serve to enhance infrared sensitivity, it is necessary to determine the spectral sensitivity (i.e. action
0022-1910/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 1 ) 0 0 1 3 4 - 2
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spectrum) of the beetle. The action spectrum of the organ may also provide information about the transduction mechanism as it has for infrared reception in snakes (Moiseenkova et al., 2001). Therefore, the goal of the present study is to measure the spectral sensitivity of the IR pit organ of the beetle M. acuminata in the region from approximately 2 to 6 µm using proven electrophysiological techniques. To date, two previous studies have examined the spectral sensitivity of Melanophila beetles. Evans used a dispersive monochromator and globar source to irradiate the beetles in the region from 2 to 6 µm (Evans, 1966). In these behavioral experiments, the recorded response was a twitch of the antennae ipsilateral to the exposed pit organ. The magnitude of antennae movement was proportional to the stimulus intensity. Evans found that peak sensitivity was from 3 to 3.5 µm, with moderate sensitivity extending from 2.4 to 4 µm, and that the regions near 2 µm and from 5 to 6 µm required very high energy to elicit a response. Conversely, with electrophysiological techniques and a CO overtone infrared laser, Schmitz et al. (2000), recently found little difference in response magnitude (number of spikes) or latency for moderate irradiance (125.6 mW/cm2) in the region from 2.80 to 3.51 µm. However, they did find that the response was diminished at 5 µm. The present study is an attempt to supplement the data of Schmitz et al. (2000) and extend the measured spectral sensitivity region to the entire mid-infrared atmospheric window (2–5 µm). 2. Materials and methods 2.1. Electron microscopy Melanophila beetles used for scanning electron (SEM) and transmission electron microscopy (TEM) were collected from wood gathered in New Mexico. For whole beetle mounts, the beetles were sacrificed in 2.5% glutaraldehyde and fixed overnight. They were dehydrated with an ethanol solution series. Critical point drying (Samdri-790, Tousimis Research Corporation) was performed on the specimen followed by gold sputter-coating (Ladd Sputter Coater, model #30800, Ladd Research Industries Inc.). The gold coat was approximately 25 nm thick. The images were taken with a Phillips 515 SEM microscope. For transmission electron microscopy, the infrared pit organ was excised from a live beetle and fixed in 3% glutaraldehyde in 0.05 M sodium cacodylate buffer at 4°C overnight. The tissue was rinsed in buffer and postfixed with 2% osmium tetraoxide in 0.2 M sodium cacodylate buffer for 1 h. The tissue was then dehydrated in a graded series of ethanol, 15 min in each, followed by two changes of propylene oxide, 15 min each. Tissue
was then infiltrated in 1:3 Epon-Araldite–propylene oxide in a vacuum dessicator for 6 h, then 1:1 in a vacuum overnight, then pure Epon-Araldite in a vacuum for 8 h. The tissue was placed in fresh resin in flat embedding molds and hardened overnight at 60°C. Thick sections (1 µm) were obtained using a diamond knife and a Reichert Ultracut E ultramicrotome. Suitable blocks were chosen and thin sections (80–90 nm) cut and placed on formvar coated hexagonal mesh grids, stained with uranyl acetate and lead citrate and viewed and photographed with a JEOL 1200 EX II electron microscope. 2.2. Optical setup An ultrafast infrared tunable laser system (Coherent, Inc.) was used to produce infrared pulses at wavelengths from 1.2 to greater than 10 µm (Fig. 1). A complete description of the 5-stage ultrafast laser system can be found elsewhere (Hammer, 2001). Briefly, a 22-W argon ion laser pumps both a femtosecond titanium:sapphire laser and a titanium:sapphire regenerative amplifier. A seed pulse from the titanium:sapphire laser is amplified to microjoule energy in the regenerative amplifier. The pulses from the amplifier are then delivered to an optical parametric amplifier (OPA) that converts the higher energy 800-nm wavelength pulses to infrared wavelengths from 1.2 to 2.4 µm. The pulses from the OPA are converted to even longer wavelengths from 2.4 to greater than 10 µm in a difference frequency generator (DFG). The repetition rate of the infrared output radiation (250 kHz) is several orders of magnitude greater than the flicker fusion frequency of the beetle’s pit organ. Although a wide range of wavelengths can be produced with the DFG, laser output with average power greater than 1 mW is limited to the region from approximately 2.5 to 5.5 µm. Fig. 2 illustrates the tunability of the OPA and DFG. Fig. 3 shows the optical setup used to irradiate the beetles. Infrared wavelengths from both the ultrafast laser system and a helium–neon laser (Research ElectroOptics Inc., lines at 1.15 and 3.39 µm) were delivered to the beetle with gold-coated mirrors. A spectrometer (Chromex Inc.), with one of its gratings blazed at 4 µm and mercury cadmium telluride detector at the output slit, was used to characterize the wavelength of the ultrafast laser system. The power delivered to the setup was controlled with a polarizer/attenuator and/or external infrared neutral density filters. A broadband infrared beamsplitter was used to align a visible helium–neon laser collinear to the infrared lasers and to pick off a portion of the infrared beam for power measurement. The visible helium–neon laser allowed for accurate alignment of the beam on the beetle’s pit organ. It should be noted that, at comparable powers (several mW), the beetles exhibited no response to the visible helium–neon
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Fig. 1. Tunable infrared ultrafast laser system manufactured by Coherent Inc. consists of Innova 200 argon ion pump laser, Mira 900-B titanium:sapphire femtosecond laser, RegA 9000 titanium:sapphire regenerative amplifier, OPA 9800 optical parametric amplifier, and DFG difference frequency generator. Autocorrelator and Chromex Inc. 500IS/SM spectrometer are used to characterize the output of the system.
gold-coated off-axis parabolic (OAP) mirror. The OAP was used so that the location of the minimum spot size was the same for all wavelengths. Although the location of the spot size was the same regardless of wavelength, the minimum beam radius (w0) did vary with wavelength according to w0=lf/πr, where l is the wavelength, f the focal length, and r the initial unfocused beam radius. For example, the beam radius at l=3.13 µm was 182.5 µm and at l=4.05 µm was 220.4 µm. The beetle was placed on a three-axis stage to allow accurate alignment to the infrared beam. 2.3. Electrophysiological recordings
Fig. 2. (a) OPA and (b) DFG output wavelengths. The OPA signal (peaks from 1.2 to 1.6 µm) and idler (peaks from 1.6 to 2.4 µm) wavelengths shown in (a) produce the DFG wavelengths shown in (b). The peak values for both figures are indicated in the legend.
(0.632 µm) wavelength. The power measured on detector ED1 during beetle irradiation was corrected with the ratio of power delivered to the two detectors (ED2/ED1), measured prior to the experiment with the beetle removed. The exposure duration was controlled with an electronic shutter (Vincent Associates Inc.). The beam was focused onto the beetle with a 150-mm focal length,
Generator (GP) and action potentials (AP) were recorded from the pit organ in the manner previously described (Hammer et al., 2001). The beetle was placed on its back, fixed in dental glue, and it legs were removed to allow access to the pit organ. A sharpened bent-tip tungsten electrode was advanced into the organ with a micropositioner (SD Instruments Inc.). The tip of the electrode was bent to give the infrared beam an unimpeded path to the pit organ. Signals from the neurons that innervate the pit organ were amplified (A-M Systems Inc.) and acquired with a data acquisition board (National Instruments Inc.) installed in a personal computer. With a shallow electrode penetration depth, both slowly varying generator potentials and the resulting action potentials could be recorded from the pit. Action potentials alone were recorded by setting the amplifier low-frequency cut-off to 300 Hz. Approximately ten waveforms (GPs and APs) were recorded at each power for signal averaging and histogram summing. The power was varied in the same range for each beetle (from subthreshold to supra-threshold) at each wavelength at a constant exposure duration (100 ms). Each beetle was subjected to seven different wavelengths (in random
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Fig. 3. Optical setup used to irradiate the beetles with multiple infrared wavelengths. The visible and infrared helium–neon lasers are made collinear to allow alignment of the beam on the pit organ. Beetle position is controlled with a three-axis stage. M1-7: gold mirrors (M4 was rotated to deliver the ultrafast laser pulses to the spectrometer or removed to deliver the pulses to the beetle), P/A: polarizer/attenuator, BS: BaF2 broadband infrared beam-splitter, ED1-2: power meters, A1-4: iris apertures, S: shutter, OAP: 150-mm focal length off-axis parabolic mirror.
order) so that the spectral sensitivity could be measured irrespective of inter-subject variability. In addition to beetle response, the laser power and shutter trigger signal were also acquired for accurate dosimetry calculation. Differences in the response of the beetles at different wavelengths were determined by measurement of sensitivity threshold and quantification of some of the characteristics of the response.
Table 1 Experimental parameters for measurements of spectral sensitivity. Bandwidth for each wavelength is shown in parentheses (full-width half-maximum) Parameter
Value
Number of beetles Response recorded
5 AP, GP Threshold, AP Spikes, GP Amplitude, Latency 2.64 (0.07), 3.13 (0.18), 3.39 (0.007), 3.45 (0.26), 4.05 (0.27), 4.92 (0.51), 5.28 (0.53) 100 0.3–1136 183–254
Characteristics
2.4. Fourier transform infrared spectroscopy In order to illustrate the correlation as a function of wavelength between response characteristics and the optical properties of the IR pit organ, the transmittance of dissected pits was found by FTIR. A FTIR spectrometer (Perkin–Elmer Inc.) equipped with a microscope (PE AutoIMAGE) was used to measure spectra from four different regions of the beetle: outer eye layer, wing vein, coxal capsule and pit organ. Dissected samples were placed on a BaF2 crystal for viewing under the microscope in transmission mode. Spectra were acquired between 2.5 and 14 µm. An aperture size of 100×100 µm was used. Background atmospheric absorption was subtracted from each of the acquired spectra. 2.5. Data analysis and statistical methods A summary of the experimental parameters used in the measurement of spectral sensitivity is shown in Table 1. A program was developed in LabVIEW software (vers. 6i, National Instruments Inc.) to automate the data analysis. There were three output values for each waveform: the threshold value (1: above threshold, 0: below threshold); the response magnitude (GP: amplitude, AP: spike count); and the response latency. The entire elec-
Wavelengths (µm)
Exposure duration (ms) Irradiance range (mW/cm2) Beam radius (µm)
trophysiological waveform was unbundled and each exposure separated according to the shutter trigger time. The region around each response, usually 200 ms prior and subsequent to the shutter trigger, was analyzed depending upon the response type. No digital filtering was applied to the waveforms. Generator potentials were first averaged (usually n=10) and the threshold, amplitude, and latency of the mean response found. The amplitude was calculated as the difference between the baseline (mean of all points prior to shutter trigger) and the minimum value in a userdefined window (typical duration: 100 ms) starting at the shutter trigger. If the minimum in the post-trigger window was greater than the minimum in the pre-trigger region, the response was determined to be above threshold. The latency was calculated as the time from the shutter trigger to the time the amplitude reached an absolute value equal to the maximum absolute value in the pre-trigger region.
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Summed histogram analysis was used to evaluate the action potentials (Fig. 4). First, a peak detection algorithm was applied to the waveforms. The threshold for peak detection was set equal to the maximum signal value in the region before the shutter trigger. Second, the locations of the peaks were binned and a response histogram generated. Third, each bin of the histogram was summed for the approximately ten responses that composed each waveform. The response magnitude was measured as the summed spikes in a user-defined region (typical duration: 50 ms) starting at the shutter trigger. A shorter, and hence more conservative duration window was used for the action potentials compared to the generator potentials because previous measurements (Hammer et al., 2001) have shown the beetle response to be phasic and lasting only tens of milliseconds after exposure onset. The latency was measured as the average time until the first spike after shutter trigger. Two criteria were used to determine threshold value. First, if the height of a bin in the post-trigger window was greater
Fig. 4. Example of summed histogram. (a) Single extracted waveform that indicates threshold and peaks used for the histogram. Threshold is the maximum value in a fixed period (e.g. 200 ms) prior to the onset of exposure. (b) Location of peaks in (a) are binned to create histogram. (c) Sum of ten histograms including (b). Exposure (100 ms) is indicated above each figure.
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than all the bins in the pre-trigger region, the response was determined to be above threshold. Second, if the total number of spikes in the post-trigger window was greater than the total number of spikes in a the pre-trigger region, the response was determined to be above threshold. These two threshold criteria were applied to record a short, relatively high-frequency burst of spikes or a steady relatively low-frequency spike train, both of which are indicative of a response from the beetle. Although the data analysis was completely automated, the threshold value, response magnitude, and response latency of each waveform were independently verified. Although some aspects of the analysis algorithm are fairly simplistic, the automated technique was fairly accurate at finding the characteristics of the response. Upon further inspection, approximately 10–30% of the waveforms had one of the three output values altered from that calculated automatically. Calculation of the generator potential characteristics, although successful for high irradiances, was more problematic near threshold because of DC drift that was inevitably recorded with the amplifier low frequency cut-off set to 0.1 Hz. Probit analysis was used to find the probability curve for a stimulus response given a set of binary (YES/NO) data (Cain et al., 1996). Since 30–35 waveforms (of ten exposures each) were recorded at each wavelength for each beetle, half of which were action potentials and half of which were generator potentials, it was not possible to find the threshold response of each wavelength of each beetle via probit analysis. Probit analysis requires overlap of the above and below threshold data and thus a significant number of points in the region between ED15 and ED85. However, data from five beetles was combined and the threshold at each wavelength evaluated using probit analysis. This analysis assumes small variability within the sample. To determine statistically significant wavelengthdependent response differences, the response magnitude and latency were also evaluated. For each wavelength of each beetle, a linear fit by least-squares estimation of the response magnitude (i.e. amplitude or summed spike count) and latency as a function of irradiance (E0) on a logarithmic (base 10) plot were found: log(f(E0))=A×log E0+B, where A and B are the fit parameters (slope and intercept). Overall, the log–log fits had a slightly better coefficient of determination (R2, also the square of the correlation coefficient) than a strictly linear fit or a log-linear fit. The aim here was not to find a fit that completely described the physiological response, but merely one with reasonable accuracy to be used in further analysis. The response of each beetle at each wavelength was then compared by finding the corresponding result from the fit for a hypothetical exposure of 200 mW/cm2. This irradiance was chosen to lie roughly half-way between threshold and saturation. The two-dimensional array of these values (subject vs.
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wavelength) was finally compared by two-factor analysis of variance (ANOVA) without replication to test the hypothesis that there were differences in beetle response between wavelengths.
3. Results 3.1. Electron microscopy TEM and SEM images of the infrared pit organ are shown in Fig. 5. The bilateral organs lie on the posteriolateral border of the coxal cavity of the mesothoracic (i.e. middle) legs. Those legs are elevated during flight, which allows the receptive field of the organ to extend mainly in a lateral and ventral direction (Evans and Kuster, 1980). Within the pit, there are between 50 and 100 individual sensilla (Fig. 5C) and each sensillum has an adjoining wax gland (Fig. 5D). The pit organ is filled with wax filaments in its natural state. Each sensillum contains a spherule that is connected to the outer mesocuticle by a thin stalk (Fig. 5B) and consists of three distinguishable regions (Fig. 5A). The regions include an amorphous core (1), a middle region of irregularly shaped lacunae (2), and an outer mantle of finely layered
lamellae (3) (Schmitz and Bleckmann, 1997). Each sensillum is innervated by a single sensory neuron. The dendritic tip of the sensory neuron is embedded eccentrically into the posterior portion of spherule. (Since the section in Fig. 5B is through the center of the spherule, the dendritic tip is not seen.) The current hypothesis for transduction mechanism is that the infrared radiation is absorbed by the spherule, which compresses the dendritic tip and causes the neuron to fire. This photo-thermal-mechanical hypothesis is supported by previous examination of the fine structure of the organ (Vondran et al., 1995; Schmitz and Bleckmann, 1997). 3.2. Thresholds The generator and action potential thresholds calculated by probit analysis for the combined set of five beetles as a function of wavelength is shown in Fig. 6. The error bars indicate the 75% fiducial limits (confidence intervals). 3.3. Characteristics An example of the recorded action and generator potentials (unfiltered), as well as the response character-
Fig. 5. Electron microscopy of Melanophila beetle infrared pit organ. (A) Transmission electron micrograph through the center of a sensillum. (B) Illustrates the stalk that connects the outer (endo-)cuticle layer to the center of the sphere. (C) Scanning electron micrograph of the entire pit organ (苲70 sensilla). (D) Illustrates several sensilla and their adjoining wax glands. Scale bars: (A) 2 µm; (B) 2 µm; (C) 100 µm; (D) 10 µm.
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Fig. 6. Generator and action potential threshold as a function of wavelength. Error bars indicate 75% fiducial limits (confidence intervals).
istics of a single beetle at two different wavelengths is shown in Fig. 7. The response for the action and generator potentials was defined as summed spikes and mean amplitude (mV) respectively. For this figure, latency was measured in milliseconds and irradiance in mW/cm2. Typical single response waveforms for 3.45 µm at high irradiance (901 mW/cm2) are shown above the response characteristic curves. To illustrate receptor saturation, which is not readily apparent in the log–log plots of Fig. 7, the action potential (summed) spikes and latency for 4.05 µm are plotted on a linear scale in Fig. 8. Also shown are semi-logarithmic fits with respect to irradiance: f(E0)=A×log E0+B. The value of each response characteristic for each beetle at each wavelength at an irradiance of 200 mW/cm2 (log(E0)=2.3) was calculated and the resulting matrix (beetle vs. wavelength) analyzed with two-factor ANOVA. The results of that statistical calculation for the four characteristics (GP amplitude, GP latency, AP spikes, and AP latency) are listed in Table 2. The sample variances were compared with an F-test (confidence level a=0.05). The confidence level is the significance level related to the probability of rejecting a true hypothesis.
Fig. 7. Examples of response characteristics and fits for a single beetle at two wavelengths. Action and generator potential responses (a,b) are defined as summed spikes and mean amplitude (mV) respectively. Units for latency (c,d) are milliseconds. Fits to data are linear on a log–log scale. Typical single response waveforms (l=3.45 µm, E0=901 mW/cm2) are shown at the top of the figure (exposure denoted by bar). Note the action potentials (arrow) in the generator potential waveform.
3.4. Comparison to spectroscopy Fig. 9 is a plot of generator potential threshold, amplitude, and latency and the transmittance or absorbance (1-transmittance, reflectance is assumed to be small) found by FTIR. The thresholds are the same as those in Fig. 6. The amplitude and latency are the mean values at each wavelength calculated at 200 mW/cm2 from the fits described in Section 2.5. The FTIR spectra of all four regions of the beetle (outer eye layer, wing vein, coxal capsule and pit organ) have similar morphology.
Fig. 8. Examples of response characteristics (AP) and fits on a linear scale for a single beetle at a wavelength of 4.05 µm. Fits are semilogarithmic with irradiance.
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Table 2 ANOVA results for the spectral response characteristics SS
df
MS
F
P-value F crit
GP 8.7623×10⫺1 amplitude GP 4.6322×101 latency AP 2.5650×103 spikes AP 1.5795×102 latency
6
1.4604×10⫺1 5.0061 0.0035 2.6613
6
7.7203
4.5349 0.0057 2.6613
6
4.2749×102
5.1121 0.0032 2.6613
6
2.6324×101
3.2029 0.0255 2.6613
SS: sum of squares, df: degrees of freedom, MS: mean of square effect, F: F-test, P-value: statistical significance, F crit: F criterion.
Fig. 9. Comparison of generator potential characteristics (䉫, left axis) to Fourier transform infrared spectroscopy (—, right axis). Irradiance thresholds are determined from probit analysis. Amplitude and latency are mean values calculated at an irradiance of 200 mW/cm2 from the fits described in Section 2.5.
4. Discussion It has been established that the thoracic pit organ of some species of buprestid beetles can detect infrared radiation (Evans, 1966; Schmitz and Bleckmann, 1998). Ecological observations suggest the beetles use the infrared sense to detect radiation from forest fires (Linsley, 1943). Sensitivity threshold measurements (Hammer et
al., 2001) and measurements of the beetle’s olfactory sense (Gronenberg and Schmitz, 1999) indicate the beetle may use IR radiation detection when it is in closeproximity to burning wood. This suggestion is in contrast to that reported in the literature (Van Dyke, 1926; Linsley, 1933) and may lead to further postulations of the purpose of the organ. For example, the IR pit organ may simply alert the flying beetle that it is too close to potentially fatal flames. It may also provide the beetle walking on a branch with a map of the temperature distribution in the wood from which it can choose a suitable region for oviposition. The measurement of spectral sensitivity accomplished in this study was important to determine if Melanophila beetles somehow use the bulk optical properties of their IR pit organs to accomplish IR detection. Several observations can be made about the generator and action potential thresholds (Fig. 6). As found previously (Hammer et al., 2001), the generator potential threshold was lower than the action potential threshold. This is to be expected, since the action potentials are conditionally dependent on the existence of a generator potential. The threshold at 3.39 µm (helium–neon laser) was much lower than that at 3.45 µm (tunable infrared ultrafast laser system). Although it is possible that even between these close wavelengths, there is a significant difference in threshold, the bandwidth of the tunable infrared ultrafast laser system (see Fig. 2) probably prevents the measurement of this difference with the ultrafast laser. The differences in the thresholds at 3.39 and 3.45 µm may be attributed to a difference in the spatial profile of the two lasers that could possibly lead to alignment errors. The helium–neon laser has a much ‘cleaner’ spatial profile and the fact that the near-infrared alignment line (1.15 µm) originates from the same cavity makes alignment relatively straightforward. Alignment of the ultrafast tunable infrared laser is more problematic since alignment is accomplished with the near-infrared remnant (i.e. unconverted) light that passes through the DFG crystal. Although the collinearity of the remnant light to the converted infrared light was measured with a knife-edge, the resolution of this method may not have been sufficient to detect small displacements that can lead to large differences when propagated long distances. Despite these complications, when plotted with FTIR (Fig. 9), it is apparent that there is some correlation between threshold and transmittance. The response characteristics (Fig. 7) show an increase in response magnitude (AP spike count and GP amplitude) and a decrease in latency (AP and GP) with irradiance. The log–log plots of response as a function of irradiance mask the saturation effect observed in the IR pit organ. Receptor saturation is illustrated in Fig. 8. In general, the generator potential data (b,d) was slightly less variable than the action potential data (a,c), and the latency data (c,d) was slightly more variable than the
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response magnitude data (a,b). The variability in AP characteristics when compared to GP characteristics may be attributed to the fact that the algorithm for histogram summing is more complex and more susceptible to systematic error than the simple algorithm for measurement of generator potential amplitude. The measurement of action potential latency also requires accurate histogram summing, and thus is also more prone to errors, although inherent variability in the timing of spikes elicited from the beetle upon exposure to IR radiation cannot be ruled out. Differences in the beetle response as a function of wavelength were determined to be statistically significant (Table 2). ANOVA indicated that all four characteristics measured had differences with a probability greater than 95% (pⱕ0.05). In fact, for all characteristics except AP latency, there was greater than 99% probability that a significant difference existed. The GP characteristics were slightly more significant than the AP characteristics, perhaps due to lower variability, which led to better fits (Fig. 7). The overall spectral response trends as measured by standard electrophysiological techniques can be seen in comparison to pit organ optical properties in Fig. 9. Since the measurements were made without the wax layer that covers the pit organ in its natural state, the assumption that reflectance is small or negligible is justified. The sensitivity threshold is lower in regions where there is higher absorption and vice versa. This implies either that the organ has been designed or evolved to have greater sensitivity where the tissue is highly absorbing or that the high tissue absorption dictates the sensitivity of the organ. Whether the beetle infrared sensitivity resulted from an adaptation in answer to particular tissue properties or simply that the tissue properties lead to differences in response cannot be determined from the results of this paper. With the exception of the amplitude at 3.13 µm, the GP amplitude and latency also follow similar spectral trends in optical properties. Where there is higher absorbance, the beetle responds more strongly and with a shorter latency. This outcome was expected in view of previously published results (Hammer et al., 2001), which established that with an increase in source irradiance the beetle responded with increased amplitude (spikes) and shorter latency generator (action) potentials. Fig. 9 illustrates that for a fixed irradiance, the same result applies if there is a shift to a wavelength where there is higher absorption. In Melanophila beetle infrared detection, there appears to be a strong correlation between three parameters: the atmospheric window in which the emission of radiation from forest fires is transmitted; the optical properties that make up the pit organ responsible for infrared detection; and the response characteristics of the beetle as measured by electrophysiological techniques. These results have implications for the phylogeny of
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these uniquely sensitive beetles. It is doubtful that the bulk properties of the tissue that comprises the pit organ evolved specifically for the purpose of infrared detection, since the bulk optical properties have characteristics common to many tissue types, including tissue unrelated to infrared detection found elsewhere on the beetle’s body. However, a possible descent from insect hair mechanoreceptors has been suggested from the unique structure of its sensilla (Vondran et al., 1995). In that case, the beetles may have developed the sensitivity in response to environmental considerations (i.e. atmospheric absorption), and the difference in response merely results from differences in the bulk tissue properties. This hypothesis is supported by the fact that although differences in response characteristics do exist, a response was measured from the beetle at all wavelengths in the 2.5–5.5 µm region. Therefore, the beetle infrared organ must be classified as a broad-band detector. In terms of transduction mechanism, the present results are informative but not as definitive as those of Moiseenkova et al. (2001) for the snake infrared sensitivity. They present conclusive evidence, based upon measurement over a broad range of wavelengths, that the snake sensitivity is photo-thermal. For the beetles, a dual mechanism may be involved. It seems probable that infrared radiation may be converted to thermal energy via absorption in the beetle pit organ. However, whether that thermal energy acts in some manner to produce the neural response or is converted to mechanical energy is not presently known.
5. Conclusion The spectral sensitivity of the infrared-sensitive beetle M. acuminata was measured using an ultrafast tunable infrared laser for wavelengths between 2.5 and 5.5 µm and standard electrophysiological techniques. The threshold and response characteristics of generator and action potentials recorded from the pit organ differ significantly in terms of wavelength. That difference is attributed to differences in the bulk optical properties of the organ.
Acknowledgements The authors would like to thank Drs K.-H. Apel and H. Schmitz for their help in the collection of specimens. The wood infested with beetle larvae was collected with the permission of the US Department of Agriculture-Forest Service (USDA-FS) and with considerable assistance from Terry Rogers, Entomologist, USDA-FS, Southwestern Region. The authors would also like to thank Brian Sorg, John Mendenhall, Sharon Thomsen, and Mannie Steglic for assistance with the electron
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microscopy, which was performed at the microscopy facilities of the Anatomical Pathology Services Department of MD Anderson Cancer Center and at the Institute for Cellular and Molecular Biology, University of Texas. Funding for this research was provided in part by grants from the Air Force Office of Scientific Research through the Multiple University Research Initiative from the Director of Defense Research and Engineering (F4962098-1-0480), the Defense Advanced Research Projects Agency (F49620-98-1-0489), and the Albert W. and Clemmie A. Caster Foundation.
References Cain, C.P., Noojin, G.D., Manning, L. 1996. A comparison of various probit methods for analyzing Yes/No data on a log scale. Tech. Rep. AL/OE-TR-19960102, U.S. Air Force Armstrong Laboratory. Evans, W.G., 1966. Morphology of the infrared sense organs of Melanophila acuminata (Buprestidae: Coleoptera). Annals of the Entomological Society of America 59, 873–877. Evans, W.G., Kuster, J.E., 1980. The infrared receptive fields of Melanophila acuminata (Coleoptera: Buprestidae). The Canadian Entomologist 112, 211–216. Goris, R.C., Nomoto, M., 1967. Infrared reception in oriental crotaline snakes. Comparative Biochemistry and Physiology 23, 879–892. Gronenberg, W., Schmitz, H., 1999. Afferent projections of infrared sensitive sensilla in the beetle Melanophila acuminata (Coleoptera: Buprestidae). Cell and Tissue Research 297, 311–318.
Hammer, D.X., 2001. Infrared detection in Melanophila acuminata. Ph.D. thesis, University of Texas at Austin. Hammer, D.X., Schmitz, H., Schmitz, A., III, H.G.R., Welch, A.J., 2001. Sensitivity threshold and response characteristics of infrared detection in the beetle Melanophila acuminata (Coleoptera: Buprestidae). Comparative Biochemistry and Physiology Part A 128, 805–819. Linsley, E.G., 1933. Some observations on the swarming of Melanophila. The Pan-Pacific Entomologist 9, 138. Linsley, E.G., 1943. Attraction of Melanophila beetles by fire and smoke. Journal of Economic Entomology 36, 341–342. Moiseenkova, V., Bell, B.A., Motamedi, M., Wozniak, E., Christensen, B.N., 2001. Wide-band spectral tuning of thermal receptors. Journal of Neuroscience, submitted. Schmitz, H., Bleckmann, H., 1997. Fine structure and physiology of the infrared receptor of beetles of the genus Melanophila (Coleoptera: Buprestidae). International Journal of Insect Morphology and Embryology 26, 205–215. Schmitz, H., Bleckmann, H., 1998. The photomechanic infrared receptor for the detection of forest fires in the beetle Melanophila acuminata (Coleoptera: Buprestidae). Journal of Comparative Physiology A 182, 647–657. Schmitz, H., Mu¨ urtz, M., Bleckmann, H., 2000. Responses of the infrared sensilla of Melanophila acuminata (Coleoptera: Buprestidae) to monochromatic infrared stimulation. Journal of Comparative Physiology A 186, 543–549. Twardowski, J., Anzenbacher, P., 1994. Raman and IR Spectroscopy in Biology and Biochemistry. Ellis Horwood, New York. Van Dyke, E.C., 1926. Buprestid swarming. The Pan-Pacific Entomologist 3, 41. Vondran, T., Apel, K.H., Schmitz, H., 1995. The infrared receptor of Melanophila acuminata De Geer (Coleoptera: Buprestidae): Ultrastructural study of a unique insect thermoreceptor and its possible descent from a hair mechanoreceptor. Tissue and Cell 27, 645–658.
Infrared spectral sensitivity of Melanophila acuminata D.X. Hammer a
a,*
, J. Seigert a, M.O. Stone b, H.G. Rylander III a, A.J. Welch
a
Department of Electrical Engineering, University of Texas, Austin, TX 78712, USA b AFRL/MLPJ, Wright-Patterson AFB, OH 45433-7702, USA Received 14 May 2001; accepted 8 August 2001
Abstract The spectral sensitivity of the pit organ of the beetle Melanophila acuminata (Coleoptera:Buprestidae) was measured using an ultrafast tunable infrared laser source and standard electrophysiological techniques. The pit organ may be classified as a broadband detector as the beetles responded to all infrared excitation wavelengths from 2 to 6 µm. There was a decrease in response threshold and latency and an increase in the magnitude of the response in the region from 2.8 to 3.5 µm, which corresponded to a region of decreased transmittance (increased absorbance) as measured by Fourier transform infrared spectroscopy. The implications of the correlation between spectral response and optical properties are discussed. 2001 Elsevier Science Ltd. All rights reserved. Keywords: M. acuminata; Sensitivity threshold; Spectral sensitivity; Action spectrum; Action potential; Generator potential; Infrared receptor
1. Introduction Buprestid beetles of the species Melanophila acuminata have shown sensitivity to infrared radiation in behavioral and physiological experiments (Evans, 1966; Schmitz and Bleckmann, 1998; Hammer et al., 2001). The beetles possess bilateral thoracic pit organs in the posteriolateral border of the coxal cavity of the second set of legs. The beetles detect infrared (IR) radiation emitted by forest fires presumably in the 2–5 µm atmospheric window, where H2O and CO2 absorption are minimal. Examination of tissue optical properties, in particular Fourier transform infrared (FTIR) spectroscopy, has indicated that the tissue which composes the IR pit organ has high absorption (low transmittance) from 2.8 to 3.5 µm (Vondran et al., 1995). The source of the absorption could be C–H and O–H groups that have vibrational stretch resonances near 3 µm (O–H: 2.70–3.23 µm, C– H: 3.28–3.57 µm (Twardowski and Anzenbacher, 1994).
* Corresponding author. Present address: Physical Sciences Inc., 20 New England Business Center, Andover, MA 01810, USA. Tel.: +1978-738-8224; fax: +1-978-687-7211. E-mail addresses: [email protected] (D.X. Hammer), [email protected] (J. Seigert), [email protected] (M.O. Stone), [email protected] (H.G. Rylander III), welch@ mail.utexas.edu (A.J. Welch).
Since these molecular groups are present in the proteins and carbohydrates that are the building blocks of all animal cells, this property of the tissue does not in itself indicate environmental adaptation for near- to mid-infrared detection. In fact, the transmission properties of the beetle pit organ are similar to those for the infraredsensitive (8–12 µm) pit membrane of Crotaline snakes of the genera Trimeresurus and Agkistrodon (Goris and Nomoto, 1967). However, the bulk tissue optical properties, as well as other characteristics of the spherule (the spherical portion of the sensillum thought to absorb the majority of infrared radiation) may contribute to the overall functional design of the organ. For example, the unique lamellar structure of the outer layers and the contents of the interior regions of the spherule may serve to enhance infrared sensitivity through IR interference and absorption respectively (Fig. 5A). Moreover, the manner in which the spherule protrudes out of the mesocuticle may attenuate lateral conduction of heat (i.e. insulate each individual sensillum by convective cooling), which thereby enhances the conduction of heat to deeper layers and isolates each sensillum. The isolation of the sensilla in the pit organ may increase the overall sensitivity of the entire pit and also enable spatial information to be detected. In order to prove that the bulk optical properties of the IR pit organ serve to enhance infrared sensitivity, it is necessary to determine the spectral sensitivity (i.e. action
0022-1910/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 1 ) 0 0 1 3 4 - 2
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spectrum) of the beetle. The action spectrum of the organ may also provide information about the transduction mechanism as it has for infrared reception in snakes (Moiseenkova et al., 2001). Therefore, the goal of the present study is to measure the spectral sensitivity of the IR pit organ of the beetle M. acuminata in the region from approximately 2 to 6 µm using proven electrophysiological techniques. To date, two previous studies have examined the spectral sensitivity of Melanophila beetles. Evans used a dispersive monochromator and globar source to irradiate the beetles in the region from 2 to 6 µm (Evans, 1966). In these behavioral experiments, the recorded response was a twitch of the antennae ipsilateral to the exposed pit organ. The magnitude of antennae movement was proportional to the stimulus intensity. Evans found that peak sensitivity was from 3 to 3.5 µm, with moderate sensitivity extending from 2.4 to 4 µm, and that the regions near 2 µm and from 5 to 6 µm required very high energy to elicit a response. Conversely, with electrophysiological techniques and a CO overtone infrared laser, Schmitz et al. (2000), recently found little difference in response magnitude (number of spikes) or latency for moderate irradiance (125.6 mW/cm2) in the region from 2.80 to 3.51 µm. However, they did find that the response was diminished at 5 µm. The present study is an attempt to supplement the data of Schmitz et al. (2000) and extend the measured spectral sensitivity region to the entire mid-infrared atmospheric window (2–5 µm). 2. Materials and methods 2.1. Electron microscopy Melanophila beetles used for scanning electron (SEM) and transmission electron microscopy (TEM) were collected from wood gathered in New Mexico. For whole beetle mounts, the beetles were sacrificed in 2.5% glutaraldehyde and fixed overnight. They were dehydrated with an ethanol solution series. Critical point drying (Samdri-790, Tousimis Research Corporation) was performed on the specimen followed by gold sputter-coating (Ladd Sputter Coater, model #30800, Ladd Research Industries Inc.). The gold coat was approximately 25 nm thick. The images were taken with a Phillips 515 SEM microscope. For transmission electron microscopy, the infrared pit organ was excised from a live beetle and fixed in 3% glutaraldehyde in 0.05 M sodium cacodylate buffer at 4°C overnight. The tissue was rinsed in buffer and postfixed with 2% osmium tetraoxide in 0.2 M sodium cacodylate buffer for 1 h. The tissue was then dehydrated in a graded series of ethanol, 15 min in each, followed by two changes of propylene oxide, 15 min each. Tissue
was then infiltrated in 1:3 Epon-Araldite–propylene oxide in a vacuum dessicator for 6 h, then 1:1 in a vacuum overnight, then pure Epon-Araldite in a vacuum for 8 h. The tissue was placed in fresh resin in flat embedding molds and hardened overnight at 60°C. Thick sections (1 µm) were obtained using a diamond knife and a Reichert Ultracut E ultramicrotome. Suitable blocks were chosen and thin sections (80–90 nm) cut and placed on formvar coated hexagonal mesh grids, stained with uranyl acetate and lead citrate and viewed and photographed with a JEOL 1200 EX II electron microscope. 2.2. Optical setup An ultrafast infrared tunable laser system (Coherent, Inc.) was used to produce infrared pulses at wavelengths from 1.2 to greater than 10 µm (Fig. 1). A complete description of the 5-stage ultrafast laser system can be found elsewhere (Hammer, 2001). Briefly, a 22-W argon ion laser pumps both a femtosecond titanium:sapphire laser and a titanium:sapphire regenerative amplifier. A seed pulse from the titanium:sapphire laser is amplified to microjoule energy in the regenerative amplifier. The pulses from the amplifier are then delivered to an optical parametric amplifier (OPA) that converts the higher energy 800-nm wavelength pulses to infrared wavelengths from 1.2 to 2.4 µm. The pulses from the OPA are converted to even longer wavelengths from 2.4 to greater than 10 µm in a difference frequency generator (DFG). The repetition rate of the infrared output radiation (250 kHz) is several orders of magnitude greater than the flicker fusion frequency of the beetle’s pit organ. Although a wide range of wavelengths can be produced with the DFG, laser output with average power greater than 1 mW is limited to the region from approximately 2.5 to 5.5 µm. Fig. 2 illustrates the tunability of the OPA and DFG. Fig. 3 shows the optical setup used to irradiate the beetles. Infrared wavelengths from both the ultrafast laser system and a helium–neon laser (Research ElectroOptics Inc., lines at 1.15 and 3.39 µm) were delivered to the beetle with gold-coated mirrors. A spectrometer (Chromex Inc.), with one of its gratings blazed at 4 µm and mercury cadmium telluride detector at the output slit, was used to characterize the wavelength of the ultrafast laser system. The power delivered to the setup was controlled with a polarizer/attenuator and/or external infrared neutral density filters. A broadband infrared beamsplitter was used to align a visible helium–neon laser collinear to the infrared lasers and to pick off a portion of the infrared beam for power measurement. The visible helium–neon laser allowed for accurate alignment of the beam on the beetle’s pit organ. It should be noted that, at comparable powers (several mW), the beetles exhibited no response to the visible helium–neon
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Fig. 1. Tunable infrared ultrafast laser system manufactured by Coherent Inc. consists of Innova 200 argon ion pump laser, Mira 900-B titanium:sapphire femtosecond laser, RegA 9000 titanium:sapphire regenerative amplifier, OPA 9800 optical parametric amplifier, and DFG difference frequency generator. Autocorrelator and Chromex Inc. 500IS/SM spectrometer are used to characterize the output of the system.
gold-coated off-axis parabolic (OAP) mirror. The OAP was used so that the location of the minimum spot size was the same for all wavelengths. Although the location of the spot size was the same regardless of wavelength, the minimum beam radius (w0) did vary with wavelength according to w0=lf/πr, where l is the wavelength, f the focal length, and r the initial unfocused beam radius. For example, the beam radius at l=3.13 µm was 182.5 µm and at l=4.05 µm was 220.4 µm. The beetle was placed on a three-axis stage to allow accurate alignment to the infrared beam. 2.3. Electrophysiological recordings
Fig. 2. (a) OPA and (b) DFG output wavelengths. The OPA signal (peaks from 1.2 to 1.6 µm) and idler (peaks from 1.6 to 2.4 µm) wavelengths shown in (a) produce the DFG wavelengths shown in (b). The peak values for both figures are indicated in the legend.
(0.632 µm) wavelength. The power measured on detector ED1 during beetle irradiation was corrected with the ratio of power delivered to the two detectors (ED2/ED1), measured prior to the experiment with the beetle removed. The exposure duration was controlled with an electronic shutter (Vincent Associates Inc.). The beam was focused onto the beetle with a 150-mm focal length,
Generator (GP) and action potentials (AP) were recorded from the pit organ in the manner previously described (Hammer et al., 2001). The beetle was placed on its back, fixed in dental glue, and it legs were removed to allow access to the pit organ. A sharpened bent-tip tungsten electrode was advanced into the organ with a micropositioner (SD Instruments Inc.). The tip of the electrode was bent to give the infrared beam an unimpeded path to the pit organ. Signals from the neurons that innervate the pit organ were amplified (A-M Systems Inc.) and acquired with a data acquisition board (National Instruments Inc.) installed in a personal computer. With a shallow electrode penetration depth, both slowly varying generator potentials and the resulting action potentials could be recorded from the pit. Action potentials alone were recorded by setting the amplifier low-frequency cut-off to 300 Hz. Approximately ten waveforms (GPs and APs) were recorded at each power for signal averaging and histogram summing. The power was varied in the same range for each beetle (from subthreshold to supra-threshold) at each wavelength at a constant exposure duration (100 ms). Each beetle was subjected to seven different wavelengths (in random
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Fig. 3. Optical setup used to irradiate the beetles with multiple infrared wavelengths. The visible and infrared helium–neon lasers are made collinear to allow alignment of the beam on the pit organ. Beetle position is controlled with a three-axis stage. M1-7: gold mirrors (M4 was rotated to deliver the ultrafast laser pulses to the spectrometer or removed to deliver the pulses to the beetle), P/A: polarizer/attenuator, BS: BaF2 broadband infrared beam-splitter, ED1-2: power meters, A1-4: iris apertures, S: shutter, OAP: 150-mm focal length off-axis parabolic mirror.
order) so that the spectral sensitivity could be measured irrespective of inter-subject variability. In addition to beetle response, the laser power and shutter trigger signal were also acquired for accurate dosimetry calculation. Differences in the response of the beetles at different wavelengths were determined by measurement of sensitivity threshold and quantification of some of the characteristics of the response.
Table 1 Experimental parameters for measurements of spectral sensitivity. Bandwidth for each wavelength is shown in parentheses (full-width half-maximum) Parameter
Value
Number of beetles Response recorded
5 AP, GP Threshold, AP Spikes, GP Amplitude, Latency 2.64 (0.07), 3.13 (0.18), 3.39 (0.007), 3.45 (0.26), 4.05 (0.27), 4.92 (0.51), 5.28 (0.53) 100 0.3–1136 183–254
Characteristics
2.4. Fourier transform infrared spectroscopy In order to illustrate the correlation as a function of wavelength between response characteristics and the optical properties of the IR pit organ, the transmittance of dissected pits was found by FTIR. A FTIR spectrometer (Perkin–Elmer Inc.) equipped with a microscope (PE AutoIMAGE) was used to measure spectra from four different regions of the beetle: outer eye layer, wing vein, coxal capsule and pit organ. Dissected samples were placed on a BaF2 crystal for viewing under the microscope in transmission mode. Spectra were acquired between 2.5 and 14 µm. An aperture size of 100×100 µm was used. Background atmospheric absorption was subtracted from each of the acquired spectra. 2.5. Data analysis and statistical methods A summary of the experimental parameters used in the measurement of spectral sensitivity is shown in Table 1. A program was developed in LabVIEW software (vers. 6i, National Instruments Inc.) to automate the data analysis. There were three output values for each waveform: the threshold value (1: above threshold, 0: below threshold); the response magnitude (GP: amplitude, AP: spike count); and the response latency. The entire elec-
Wavelengths (µm)
Exposure duration (ms) Irradiance range (mW/cm2) Beam radius (µm)
trophysiological waveform was unbundled and each exposure separated according to the shutter trigger time. The region around each response, usually 200 ms prior and subsequent to the shutter trigger, was analyzed depending upon the response type. No digital filtering was applied to the waveforms. Generator potentials were first averaged (usually n=10) and the threshold, amplitude, and latency of the mean response found. The amplitude was calculated as the difference between the baseline (mean of all points prior to shutter trigger) and the minimum value in a userdefined window (typical duration: 100 ms) starting at the shutter trigger. If the minimum in the post-trigger window was greater than the minimum in the pre-trigger region, the response was determined to be above threshold. The latency was calculated as the time from the shutter trigger to the time the amplitude reached an absolute value equal to the maximum absolute value in the pre-trigger region.
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Summed histogram analysis was used to evaluate the action potentials (Fig. 4). First, a peak detection algorithm was applied to the waveforms. The threshold for peak detection was set equal to the maximum signal value in the region before the shutter trigger. Second, the locations of the peaks were binned and a response histogram generated. Third, each bin of the histogram was summed for the approximately ten responses that composed each waveform. The response magnitude was measured as the summed spikes in a user-defined region (typical duration: 50 ms) starting at the shutter trigger. A shorter, and hence more conservative duration window was used for the action potentials compared to the generator potentials because previous measurements (Hammer et al., 2001) have shown the beetle response to be phasic and lasting only tens of milliseconds after exposure onset. The latency was measured as the average time until the first spike after shutter trigger. Two criteria were used to determine threshold value. First, if the height of a bin in the post-trigger window was greater
Fig. 4. Example of summed histogram. (a) Single extracted waveform that indicates threshold and peaks used for the histogram. Threshold is the maximum value in a fixed period (e.g. 200 ms) prior to the onset of exposure. (b) Location of peaks in (a) are binned to create histogram. (c) Sum of ten histograms including (b). Exposure (100 ms) is indicated above each figure.
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than all the bins in the pre-trigger region, the response was determined to be above threshold. Second, if the total number of spikes in the post-trigger window was greater than the total number of spikes in a the pre-trigger region, the response was determined to be above threshold. These two threshold criteria were applied to record a short, relatively high-frequency burst of spikes or a steady relatively low-frequency spike train, both of which are indicative of a response from the beetle. Although the data analysis was completely automated, the threshold value, response magnitude, and response latency of each waveform were independently verified. Although some aspects of the analysis algorithm are fairly simplistic, the automated technique was fairly accurate at finding the characteristics of the response. Upon further inspection, approximately 10–30% of the waveforms had one of the three output values altered from that calculated automatically. Calculation of the generator potential characteristics, although successful for high irradiances, was more problematic near threshold because of DC drift that was inevitably recorded with the amplifier low frequency cut-off set to 0.1 Hz. Probit analysis was used to find the probability curve for a stimulus response given a set of binary (YES/NO) data (Cain et al., 1996). Since 30–35 waveforms (of ten exposures each) were recorded at each wavelength for each beetle, half of which were action potentials and half of which were generator potentials, it was not possible to find the threshold response of each wavelength of each beetle via probit analysis. Probit analysis requires overlap of the above and below threshold data and thus a significant number of points in the region between ED15 and ED85. However, data from five beetles was combined and the threshold at each wavelength evaluated using probit analysis. This analysis assumes small variability within the sample. To determine statistically significant wavelengthdependent response differences, the response magnitude and latency were also evaluated. For each wavelength of each beetle, a linear fit by least-squares estimation of the response magnitude (i.e. amplitude or summed spike count) and latency as a function of irradiance (E0) on a logarithmic (base 10) plot were found: log(f(E0))=A×log E0+B, where A and B are the fit parameters (slope and intercept). Overall, the log–log fits had a slightly better coefficient of determination (R2, also the square of the correlation coefficient) than a strictly linear fit or a log-linear fit. The aim here was not to find a fit that completely described the physiological response, but merely one with reasonable accuracy to be used in further analysis. The response of each beetle at each wavelength was then compared by finding the corresponding result from the fit for a hypothetical exposure of 200 mW/cm2. This irradiance was chosen to lie roughly half-way between threshold and saturation. The two-dimensional array of these values (subject vs.
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wavelength) was finally compared by two-factor analysis of variance (ANOVA) without replication to test the hypothesis that there were differences in beetle response between wavelengths.
3. Results 3.1. Electron microscopy TEM and SEM images of the infrared pit organ are shown in Fig. 5. The bilateral organs lie on the posteriolateral border of the coxal cavity of the mesothoracic (i.e. middle) legs. Those legs are elevated during flight, which allows the receptive field of the organ to extend mainly in a lateral and ventral direction (Evans and Kuster, 1980). Within the pit, there are between 50 and 100 individual sensilla (Fig. 5C) and each sensillum has an adjoining wax gland (Fig. 5D). The pit organ is filled with wax filaments in its natural state. Each sensillum contains a spherule that is connected to the outer mesocuticle by a thin stalk (Fig. 5B) and consists of three distinguishable regions (Fig. 5A). The regions include an amorphous core (1), a middle region of irregularly shaped lacunae (2), and an outer mantle of finely layered
lamellae (3) (Schmitz and Bleckmann, 1997). Each sensillum is innervated by a single sensory neuron. The dendritic tip of the sensory neuron is embedded eccentrically into the posterior portion of spherule. (Since the section in Fig. 5B is through the center of the spherule, the dendritic tip is not seen.) The current hypothesis for transduction mechanism is that the infrared radiation is absorbed by the spherule, which compresses the dendritic tip and causes the neuron to fire. This photo-thermal-mechanical hypothesis is supported by previous examination of the fine structure of the organ (Vondran et al., 1995; Schmitz and Bleckmann, 1997). 3.2. Thresholds The generator and action potential thresholds calculated by probit analysis for the combined set of five beetles as a function of wavelength is shown in Fig. 6. The error bars indicate the 75% fiducial limits (confidence intervals). 3.3. Characteristics An example of the recorded action and generator potentials (unfiltered), as well as the response character-
Fig. 5. Electron microscopy of Melanophila beetle infrared pit organ. (A) Transmission electron micrograph through the center of a sensillum. (B) Illustrates the stalk that connects the outer (endo-)cuticle layer to the center of the sphere. (C) Scanning electron micrograph of the entire pit organ (苲70 sensilla). (D) Illustrates several sensilla and their adjoining wax glands. Scale bars: (A) 2 µm; (B) 2 µm; (C) 100 µm; (D) 10 µm.
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Fig. 6. Generator and action potential threshold as a function of wavelength. Error bars indicate 75% fiducial limits (confidence intervals).
istics of a single beetle at two different wavelengths is shown in Fig. 7. The response for the action and generator potentials was defined as summed spikes and mean amplitude (mV) respectively. For this figure, latency was measured in milliseconds and irradiance in mW/cm2. Typical single response waveforms for 3.45 µm at high irradiance (901 mW/cm2) are shown above the response characteristic curves. To illustrate receptor saturation, which is not readily apparent in the log–log plots of Fig. 7, the action potential (summed) spikes and latency for 4.05 µm are plotted on a linear scale in Fig. 8. Also shown are semi-logarithmic fits with respect to irradiance: f(E0)=A×log E0+B. The value of each response characteristic for each beetle at each wavelength at an irradiance of 200 mW/cm2 (log(E0)=2.3) was calculated and the resulting matrix (beetle vs. wavelength) analyzed with two-factor ANOVA. The results of that statistical calculation for the four characteristics (GP amplitude, GP latency, AP spikes, and AP latency) are listed in Table 2. The sample variances were compared with an F-test (confidence level a=0.05). The confidence level is the significance level related to the probability of rejecting a true hypothesis.
Fig. 7. Examples of response characteristics and fits for a single beetle at two wavelengths. Action and generator potential responses (a,b) are defined as summed spikes and mean amplitude (mV) respectively. Units for latency (c,d) are milliseconds. Fits to data are linear on a log–log scale. Typical single response waveforms (l=3.45 µm, E0=901 mW/cm2) are shown at the top of the figure (exposure denoted by bar). Note the action potentials (arrow) in the generator potential waveform.
3.4. Comparison to spectroscopy Fig. 9 is a plot of generator potential threshold, amplitude, and latency and the transmittance or absorbance (1-transmittance, reflectance is assumed to be small) found by FTIR. The thresholds are the same as those in Fig. 6. The amplitude and latency are the mean values at each wavelength calculated at 200 mW/cm2 from the fits described in Section 2.5. The FTIR spectra of all four regions of the beetle (outer eye layer, wing vein, coxal capsule and pit organ) have similar morphology.
Fig. 8. Examples of response characteristics (AP) and fits on a linear scale for a single beetle at a wavelength of 4.05 µm. Fits are semilogarithmic with irradiance.
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Table 2 ANOVA results for the spectral response characteristics SS
df
MS
F
P-value F crit
GP 8.7623×10⫺1 amplitude GP 4.6322×101 latency AP 2.5650×103 spikes AP 1.5795×102 latency
6
1.4604×10⫺1 5.0061 0.0035 2.6613
6
7.7203
4.5349 0.0057 2.6613
6
4.2749×102
5.1121 0.0032 2.6613
6
2.6324×101
3.2029 0.0255 2.6613
SS: sum of squares, df: degrees of freedom, MS: mean of square effect, F: F-test, P-value: statistical significance, F crit: F criterion.
Fig. 9. Comparison of generator potential characteristics (䉫, left axis) to Fourier transform infrared spectroscopy (—, right axis). Irradiance thresholds are determined from probit analysis. Amplitude and latency are mean values calculated at an irradiance of 200 mW/cm2 from the fits described in Section 2.5.
4. Discussion It has been established that the thoracic pit organ of some species of buprestid beetles can detect infrared radiation (Evans, 1966; Schmitz and Bleckmann, 1998). Ecological observations suggest the beetles use the infrared sense to detect radiation from forest fires (Linsley, 1943). Sensitivity threshold measurements (Hammer et
al., 2001) and measurements of the beetle’s olfactory sense (Gronenberg and Schmitz, 1999) indicate the beetle may use IR radiation detection when it is in closeproximity to burning wood. This suggestion is in contrast to that reported in the literature (Van Dyke, 1926; Linsley, 1933) and may lead to further postulations of the purpose of the organ. For example, the IR pit organ may simply alert the flying beetle that it is too close to potentially fatal flames. It may also provide the beetle walking on a branch with a map of the temperature distribution in the wood from which it can choose a suitable region for oviposition. The measurement of spectral sensitivity accomplished in this study was important to determine if Melanophila beetles somehow use the bulk optical properties of their IR pit organs to accomplish IR detection. Several observations can be made about the generator and action potential thresholds (Fig. 6). As found previously (Hammer et al., 2001), the generator potential threshold was lower than the action potential threshold. This is to be expected, since the action potentials are conditionally dependent on the existence of a generator potential. The threshold at 3.39 µm (helium–neon laser) was much lower than that at 3.45 µm (tunable infrared ultrafast laser system). Although it is possible that even between these close wavelengths, there is a significant difference in threshold, the bandwidth of the tunable infrared ultrafast laser system (see Fig. 2) probably prevents the measurement of this difference with the ultrafast laser. The differences in the thresholds at 3.39 and 3.45 µm may be attributed to a difference in the spatial profile of the two lasers that could possibly lead to alignment errors. The helium–neon laser has a much ‘cleaner’ spatial profile and the fact that the near-infrared alignment line (1.15 µm) originates from the same cavity makes alignment relatively straightforward. Alignment of the ultrafast tunable infrared laser is more problematic since alignment is accomplished with the near-infrared remnant (i.e. unconverted) light that passes through the DFG crystal. Although the collinearity of the remnant light to the converted infrared light was measured with a knife-edge, the resolution of this method may not have been sufficient to detect small displacements that can lead to large differences when propagated long distances. Despite these complications, when plotted with FTIR (Fig. 9), it is apparent that there is some correlation between threshold and transmittance. The response characteristics (Fig. 7) show an increase in response magnitude (AP spike count and GP amplitude) and a decrease in latency (AP and GP) with irradiance. The log–log plots of response as a function of irradiance mask the saturation effect observed in the IR pit organ. Receptor saturation is illustrated in Fig. 8. In general, the generator potential data (b,d) was slightly less variable than the action potential data (a,c), and the latency data (c,d) was slightly more variable than the
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response magnitude data (a,b). The variability in AP characteristics when compared to GP characteristics may be attributed to the fact that the algorithm for histogram summing is more complex and more susceptible to systematic error than the simple algorithm for measurement of generator potential amplitude. The measurement of action potential latency also requires accurate histogram summing, and thus is also more prone to errors, although inherent variability in the timing of spikes elicited from the beetle upon exposure to IR radiation cannot be ruled out. Differences in the beetle response as a function of wavelength were determined to be statistically significant (Table 2). ANOVA indicated that all four characteristics measured had differences with a probability greater than 95% (pⱕ0.05). In fact, for all characteristics except AP latency, there was greater than 99% probability that a significant difference existed. The GP characteristics were slightly more significant than the AP characteristics, perhaps due to lower variability, which led to better fits (Fig. 7). The overall spectral response trends as measured by standard electrophysiological techniques can be seen in comparison to pit organ optical properties in Fig. 9. Since the measurements were made without the wax layer that covers the pit organ in its natural state, the assumption that reflectance is small or negligible is justified. The sensitivity threshold is lower in regions where there is higher absorption and vice versa. This implies either that the organ has been designed or evolved to have greater sensitivity where the tissue is highly absorbing or that the high tissue absorption dictates the sensitivity of the organ. Whether the beetle infrared sensitivity resulted from an adaptation in answer to particular tissue properties or simply that the tissue properties lead to differences in response cannot be determined from the results of this paper. With the exception of the amplitude at 3.13 µm, the GP amplitude and latency also follow similar spectral trends in optical properties. Where there is higher absorbance, the beetle responds more strongly and with a shorter latency. This outcome was expected in view of previously published results (Hammer et al., 2001), which established that with an increase in source irradiance the beetle responded with increased amplitude (spikes) and shorter latency generator (action) potentials. Fig. 9 illustrates that for a fixed irradiance, the same result applies if there is a shift to a wavelength where there is higher absorption. In Melanophila beetle infrared detection, there appears to be a strong correlation between three parameters: the atmospheric window in which the emission of radiation from forest fires is transmitted; the optical properties that make up the pit organ responsible for infrared detection; and the response characteristics of the beetle as measured by electrophysiological techniques. These results have implications for the phylogeny of
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these uniquely sensitive beetles. It is doubtful that the bulk properties of the tissue that comprises the pit organ evolved specifically for the purpose of infrared detection, since the bulk optical properties have characteristics common to many tissue types, including tissue unrelated to infrared detection found elsewhere on the beetle’s body. However, a possible descent from insect hair mechanoreceptors has been suggested from the unique structure of its sensilla (Vondran et al., 1995). In that case, the beetles may have developed the sensitivity in response to environmental considerations (i.e. atmospheric absorption), and the difference in response merely results from differences in the bulk tissue properties. This hypothesis is supported by the fact that although differences in response characteristics do exist, a response was measured from the beetle at all wavelengths in the 2.5–5.5 µm region. Therefore, the beetle infrared organ must be classified as a broad-band detector. In terms of transduction mechanism, the present results are informative but not as definitive as those of Moiseenkova et al. (2001) for the snake infrared sensitivity. They present conclusive evidence, based upon measurement over a broad range of wavelengths, that the snake sensitivity is photo-thermal. For the beetles, a dual mechanism may be involved. It seems probable that infrared radiation may be converted to thermal energy via absorption in the beetle pit organ. However, whether that thermal energy acts in some manner to produce the neural response or is converted to mechanical energy is not presently known.
5. Conclusion The spectral sensitivity of the infrared-sensitive beetle M. acuminata was measured using an ultrafast tunable infrared laser for wavelengths between 2.5 and 5.5 µm and standard electrophysiological techniques. The threshold and response characteristics of generator and action potentials recorded from the pit organ differ significantly in terms of wavelength. That difference is attributed to differences in the bulk optical properties of the organ.
Acknowledgements The authors would like to thank Drs K.-H. Apel and H. Schmitz for their help in the collection of specimens. The wood infested with beetle larvae was collected with the permission of the US Department of Agriculture-Forest Service (USDA-FS) and with considerable assistance from Terry Rogers, Entomologist, USDA-FS, Southwestern Region. The authors would also like to thank Brian Sorg, John Mendenhall, Sharon Thomsen, and Mannie Steglic for assistance with the electron
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microscopy, which was performed at the microscopy facilities of the Anatomical Pathology Services Department of MD Anderson Cancer Center and at the Institute for Cellular and Molecular Biology, University of Texas. Funding for this research was provided in part by grants from the Air Force Office of Scientific Research through the Multiple University Research Initiative from the Director of Defense Research and Engineering (F4962098-1-0480), the Defense Advanced Research Projects Agency (F49620-98-1-0489), and the Albert W. and Clemmie A. Caster Foundation.
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