Appendix 2

Electroretinogram calibration


This appendix concerns the calibration of the initial response spectrum measured for butterflies using an electroretinogram (Experiment 2-4 and Experiment 3-5) to take into consideration the intensity of the mercury lamp at different wavelengths, and to create a 'response surface' with varying wavelengths and intensities.

Method & Materials

Firstly the intensity spectrum of the lamp was measured using a photodiode, RS stock number 305-462 (RS Electronics* ) in the light-level sensor circuit (see Figure A2-1). The lamp beam was passed through the monochromator, exactly as in the ERG experiment, and the photodiode was placed in the focused, monochromated beam. The response to flashes of light of different wavelengths from the photodiode circuit was recorded on the Lloyd Instruments PL3 chart recorder.

Figure A2-1 Photodiode circuit used for measurement of lamp intensity at different wavelengths.

The height of the resulting peaks was measured and normalised, and plotted as a graph of photodiode response against wavelength (see Figure A2-2).

Figure A2-2 Graph of response of photodiode against wavelength for the mercury lamp.

This graph then had to be adjusted for the wavelength-dependent sensitivity of the photodiode itself, as given by the manufacturer, and normalised to give the true lamp power at different wavelengths (see Figure A2-3).

Figure A2-3 Graph of the relative intensity of the mercury lamp at the different wavelengths used.

The photodiode measured the power of the lamp at these wavelengths, but the actual quantum flux is what affects the visual pigments of the butterfly. The energy of the photons and hence the power is a function of the wavelength of the light, and a relative quantum flux can be obtained at a given wavelength simply by multiplying the relative power by the wavelength (in nm), and then renormalising. This produces a final graph of the relative quantum flux at each wavelength (see Figure A2-4)

Figure A2-4 Graph of the relative quantum flux at each wavelength.

Having determined the intensity of the lamp at the different wavelengths, it was necessary to measure the intensity-response function of the butterfly at the wavelengths where the higher lamp intensity needed to be accounted for. The wavelengths which had the highest intensity were 375nm, 450nm and 550nm. These were close to the sensitivity peaks of the three main photoreception pigments found in insects (Chittka & Menzel, 1992, Backhaus & Menzel, 1992, Bennett et al., 1997, Bernard & Remington, 1991, Arikawa et al., 1987), at approximately 360nm, 460nm, and 520nm. Therefore measurements of the intensity-response function at the 375nm, 450nm, and 550nm done on several individuals were used to assess the similarity of the intensity function from different individuals.

A series of filters (Supergel swatch set, Rosco Filters**; Lee numerical swatch set, Lee Filters***) were used to achieve a range of intensities at these three wavelengths. The intensity of the light once passed through the filter was measured using the photodiode circuit, and a suitable series of filters selected for each wavelength to give a good spread of intensities (see Table A2-1 at end of Appendix for list of filters used).

The response of between three and five individual butterflies was then recorded as an ERG (for method, see Experiments 2-4 and 3-5). Unfortunately, only Papilio dardanus specimens could be obtained. However, the similarity of the response curves of Argynnis paphia and Papilio dardanus (shown in the results sections of Experiment 2-4 and 3-5 respectively) indicate that they may indeed have similar intensity-response functions, and therefore that using the intensity-response curves of Papilio dardanus to calibrate the spectral efficiency curve of Argynnis paphia may be a reasonable approximation.

Figure A2-5 The mean intensity-response functions of 3 to 5 individual Papilio dardanus at a) 550nm, b) 450nm, and c) 375nm.

This data indicated that the intensity response functions were smooth curves (plotted on a log scale), and that data of sufficient quality could be obtained to predict the curve with reasonable accuracy from the data of one individual. This was important, as different individuals are inevitably illuminated to different degrees due to their placement in the light beam, and thus each would give slightly shifted intensity functions. Therefore data from several individuals cannot be combined.

Therefore one individual was used, and about 6 measurements of the response were taken for both the full and filtered intensity in each case. The graphs show the ratio of the mean value of the response to the filtered intensity to the mean value of the response to the unfiltered intensity as measured immediately prior to it.

The functions obtained from a single individual were mostly relatively smooth curves (see Figure A2-6), and the function:

was fitted to each (where a and b are variable parameters controlling the scale and curvature of the function respectively).

Figure A2-6 The relative intensity functions for Papilio dardanus at a) 325nm, b) 350nm, c) 375nm, d) 400nm, e) 425nm, f) 450nm, g) 475nm, h) 500nm, i) 525nm, j) 550nm, k) 575nm, l) 600nm.

Therefore, a corrected response figure was calculated for each wavelength for which the intensity was higher than the minimum for both species, giving corrected spectral efficiency functions (shown in Figure A2-7).

Figure A2-7 The corrected spectral efficiency functions of a) Papilio dardanus and b) Argynnis paphia (using the intensity-response functions of Papilio dardanus).

Figure A2-6, however, demonstrates how the spectral efficiency function may vary depending on the intensity at which it is measured. Therefore a more useful representation of the visual spectrum of a butterfly would be represented as a surface, taking into account both wavelength and intensity. Surfaces were therefore plotted for Papilio dardanus representing both the raw data, and a surface composed of the smoothed intensity functions shown in Figure A2-6. These surfaces are shown in the results of Experiment 3-5.

FilterRelative log intensity at wavelength (nm) (uncalibrated for photodiode sensitivity):
Rosco 00-0.153-0.129          
Rosco 052-0.643           
Rosco 053-0.404           
Rosco 054-0.414           
Rosco 372-0.425           
Rosco 373-0.276           
Lee 002 -1.878-0.365  -0.172-0.253-0.416-0.823-0.368  
Lee 003 -0.378    -0.089-1.04-0.139   
Lee 004 -0.833          
Lee 007      -0.186 -0.085-0.059-0.051 
Lee 008    -0.841-1.021      
Lee 009 -0.753-0.429-0.435-0.419-0.428 -0.297   -0.073
Lee 013 -1.020-0.554-0.530-0.524-0.553-0.537  -0.170  
Lee 015         -0.206-0.081-0.074
Lee 017     -1.447    -0.708 
Lee 021          -0.380 
Lee 022           -0.352
Lee 024     -1.160    -0.939 
Lee 025         -1.040  
Lee 035       -0.271    
Lee 036         -0.556-0.411 
Lee 039-0.644-0.288     -0.189  -0.286 
Lee 048     -0.361-0.443  -1.507  
Lee 052     -0.136     -0.520
Lee 053 -0.370-0.172-0.126-0.118-0.098    -0.276 
Lee 055           -0.258
Lee 058  -0.957  -0.249-0.355     
Lee 061  -0.480  -0.059    -0.245 
Lee 063     -0.065-0.077    -0.331
Lee 068       -0.352    
Lee 071     -0.699      
Lee 075      -0.219     
Lee 079   -0.745        
Lee 088          -0.155 
Lee 089         -0.357-0.666 
Lee 100 -1.092          
Lee 102  -0.528         
Lee 104           -0.046
Lee 105          -0.147 
Lee 107  -0.576  -0.286 -0.598    
Lee 109  -0.746-0.311-0.271-0.286     -0.115
Lee 111           -0.264
Lee 116     -1.047      
Lee 130-0.563-0.214-0.115-0.071-0.068 -0.065 -0.048   
Lee 131        -0.204   
Lee 134         -0.598-0.359-0.242
Lee 135          -0.696 
Lee 136 -0.671-0.365         
Lee 137           -0.681
Lee 151         -0.273  
Lee 153  -0.432         
Lee 154 -0.542-0.284-0.224-0.209       
Lee 162  -0.256         
Lee 169           
Lee 170     -0.301      
Lee 198     -0.805      
Lee 209  -0.589         
Lee 241          -0.625 
Lee 242          -0.453 
Lee 244          -0.118 
Lee 246 -0.461-0.245     -0.082-0.076 -0.095
Lee 279  -0.126         
Table A2-1 The measured intensity of the filtered light using different filters at each wavelength (not adjusted for the sensitivity curve of the photodiode itself)

* RS Electronics, 34443 Schoolcraft, Livonia, Michigan 48150, USA
** Kangley Bridge Road, Sydenham, London SE26 5AQ
*** Central Way, Walworth Industrial Estate, Andover, Hampshire SP10 5AN, United Kingdom

Please cite this thesis as:
Freeman, ALJ; 1998; D.Phil thesis, Oxford University.
E-mail to Alexandra Freeman
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