Melipona quadrifasciata quadrifasciata, Trigona spinnipes and Bombus morio ; one wasp, Polistes canadensis ; and three ants, Pseudornyrrnex phyllo- phi&s ...
Journal of
J Comp Physiol A (1989) 165:237-245
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Physiology A
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9 Springer-Verlag 1989
Comparative study of temporal summation and response form in hymenopteran photoreceptors John M. de Souza and Dora F. Ventura Departamento de Psicologia Experimental, Instituto de Psicologia, Universidade de Silo Paulo, Cx. P. 66261, Silo Paulo, SP, Brazil Accepted January 12, 1989
Summary. 1. Temporal summation was measured in green-sensitive photoreceptors of seven hymenopteran species with various life styles: three bees, Melipona quadrifasciata quadrifasciata, Trigona spinnipes and Bombus morio ; one wasp, Polistes canadensis ; and three ants, Pseudornyrrnex phyllophi&s, Camponofus rufipes, and A tta sexdens rubropilosa. In all species approximate agreement with Bloch's law was confirmed. 2. Critical durations (to), which varied from 10 ms (Pseudomyrrnex) to 46 ms (Atta), are discussed in relation to the life styles of the species and to the mechanisms causing the differences. 3. The direct measures of critical duration obtained are compared to estimates made by convolution or integration of impulse responses measured here in one species and from published data. Linear convolution of typical impulse responses is shown to result in significant departures from Bloch's law, a fact that seems to have been overlooked in the literature. 4. The method used to measure temporal summation involved recording responses to 300-ms stimuli at various intensities; the form of these responses varied greatly from species to species. Possible causes of these variations are discussed.
Introduction In visual systems, the term temporal summation refers to the fact that visual stimuli are integrated over a certain limited period of time. It has been studied psychophysically in the human visual system for over a hundred years, the first quantitative statement of a law of temporal summation being that attributed to Bloch (1885), who measured threshold stimuli of between 1.7 and 51.8 ms and
found that the product of intensity x duration was constant. The upper limit of this reciprocal relation has come to be known as the critical duration (t~) and its value in the dark-adapted human rod system is about 100 ms. Since Bloch, inumerable psychophysical and many electrophysiological studies of temporal summation have been published, most of which confirm the reciprocity law to a greater or lesser extent. It has long been established from electrophysiological experiments using the electroretinogram that temporal summation occurs in the retina (Hartline 1928 in the locust; Autrum 1950 in Calliphora, Dixippus, and Aeschna larvae; Grfisser and Kapp 1958 in the cat; Alpern and Faris 1956, Johnson and Bartlett 1956, and Biersdorf 1958 in humans), and later it was shown to occur in photoreceptors themselves (Wasserman and Kong 1975, Wulff et al. 1985 in Limulus; Daly and N o r m a n n 1985 in the turtle), with critical durations compatible with those found psychophysically. Thus, except for complex tasks (e.g., Hunter and Sigler 1940; Kaswan and Young 1963), it seems likely that the phenomenon can be explained entirely in terms of photoreceptor processes. In recent years the main underlying biochemical processes which lead to the vertebrate (Stryer 1986) and invertebrate (Devary et al. 1987) photoreceptor responses to light have been discovered. However, it is not known which stages of the cascades govern the temporal characteristics of the receptor response, nor how these can give rise to different integration times in different species and as a consequence of light adaptation. The study of such differences might be useful to the understanding of these processes. It has been shown mathematically that, assuming linearity of the visual system, integration of typical monophasic photoreceptor impulse responses results in a relation between intensity and
238
duration for constant peak amplitude which is asymptotic to two linear functions: one representing perfect summation (Bloch's law) at short durations and one representing zero summation for long durations (Daly and N o r m a n n 1985). This has been stated to be a proof that Bloch's law is a consequence of linearity (Kelly and Savoie 1978) without much attention to how large a discrepancy exists in the region of the critical duration. In the absence of any direct intracellular measurements of temporal summation in insect photoreceptors, the only comparative electrophysiological data are impulse responses from unidentified photoreceptor types (Howard et al. 1984 in eight insects; Raggenbass 1983 in drone bees) which can be integrated to give linear-range critical durations. In the present work temporal summation is measured directly in one type of photoreceptor, the green-absorbing retinular cell, in several hymenopterans which have widely different patterns of activity - diurnal vs. nocturnal, slow vs. fast, flying vs. nonflying. Critical durations are discussed in relation to the life styles of the species and compared to previous electrophysiological and behavioural data. The experimental method used, which involved recording responses to stimuli of various intensifies, revealed very significant species to species differences in the form of the response to medium and high intensities. Methods Material and preparation. The insects studied were: three bee species - the tropical stingless bees Melipona quadrifasciata quadrifaseiata and Trigona spinnipes and a bumblebee, Bombus morio; one wasp from the Polistinae subfamily, Polistes canadensis; and three ants, Pseudomyrmex phyllophilus from the Leptaleinae subfamily, Camponotus rufipes from the Formicinae subfamily, and Atta sexdens rubropilosa from the Myrmicinae subfamily. F r o m here on the names will be abbreviated to Melipona, Trigona, Bombus, Polistes, Pseudomyrmex, Camponotus, and Atta. The insect was immobilized with wax on a small brass pedestal and a glass micropipette (2.5 or 3 M KC1, 80-240 M ~ ) introduced into the eye with a Leitz micromanipulator through a small hole cut in the cornea. A silver ground wire was placed in the head capsule, or in the leg in the case of the tiny Pseudomyrmex. Stimulation and recording. Intracellular responses were received on a WPI 750 amplifier and recorded on a Hewlett Packard F M tape recorder. The eye to be recorded from was positioned at the center of a perimeter device which directed a light guide or a lightemitting diode (LED) towards it from any angle at a constant distance. Two light sources were used: (a) A 150-W xenon lamp with an all-quartz optical system and an electromechanical shutter. Intensity and wavelength could be controlled by the
J.M. de Souza and D.F. Ventura: Hymenopteran photoreceptors introduction of neutral density and interference filters. A quartz light guide led the light to the perimeter device. The exit pupil was 2.8 m m diameter at 140 m m from the eye and the unattenuated intensity at the eye was 1.3 x 104 m W / m z. (b) A green LED (4.7 m m at 62 mm, 2ma~=570 rim, halfband w i d t h = 25 nm) controlled by an Apple II microcomputer so as to produce constant-intensity square light pulses. All recording was done between 10 a.m. and 7 p.m. Temperatures varied between 20 ~ and 25 ~ The electrode was advanced through the retina in a direction roughly perpendicular to the retinular cells while the eye was stimulated with a hand lamp until an inversion of the sign of the response indicated that a cell had been penetrated. The perimeter device was then adjusted to the axis of the respective ommatidium, corresponding to the position of maximum response to stimuli from the xenon source. The spectral type of the cell was determined by comparing the responses to flashes of UV, blue, and green light; only the results of experiments on green cells with a maximum peak response of at least 25 mV are reported in this paper. Stimulation was initiated after at least 5 rain of dark adaptation; this consisted first of a series of 300-ms flashes from the xenon source, the intensities of which were varied from near threshold to close to saturation (range of 4~5 log units) and second of a series of constant-intensity flashes from the LED whose durations increased from 0.1 ms to 180ms. A n interval of 15 s was used between each flash and at least 3 rain between the two series. Whenever possible several cells were tested in each preparation. The dark-adapted impulse responses to fifty 100-~ts light pulses at - 2 . 8 log intensity were also measured and averaged in Melipona and in Musca domestica for comparison with previous work (see Discussion).
Data measurement and analysis. The recorded responses were digitized and the peak amplitude (Vm) measured by means of a special program on a Radio Shack TRSS0 microcomputer. These were plotted as a function of log intensity, and theoretical sigmoid curves (Lipetz 1971) were fitted to the points using a program developed for the Apple II (Menzel et aI. 1986; Souza and Ventura 1987). These curves can be represented by the function Iso/I=(Vsat/Vm--1) 1Is were Vsat is the estimated saturation peak amplitude, /5o is the stimulus intensity corresponding to Vm/Vsat=50%, and S is a measure of the slope of the sigrnoid. The amplitude of the responses to the temporal stimuli were normalized in terms of V~,t and plotted as a function of log duration. The sigmoid function was then used to calculate for each duration (D) the intensity (/) which would bring each of these responses to a constant value of Vm gs~,t/2. The impulse responses were averaged on the TRS80 program. The smoothed response was convolved linearly with the stimulus for a series of durations from 1 ms to 1 s. The relative intensities (/) necessary to maintain constant peak response of the convolved function at each duration were then calculated and the results plotted as log ( I x D) vs: log D. =
Results
Figure 1 shows typical digitized receptor potentials for the intensity and duration series in Melipona. It can be seen in the intensity series that the dynamic range covers in this case 4.2 log units, and that at the lowest intensities voltage bumps of about 2 mV are present. These are generally used as an indication of dark adaptation.
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The duration series (LED source) was performed at a fixed intensity corresponding to about - 3 . 2 log attenuation of the intensity series. This resulted, in this and in most cases, in responses to the longer stimuli of 50% Vsa t o r more, i.e., well above the linear range of response. Temporal summation up to 12.5 ms is evident from the increase in amplitude of the receptor potential with increase in stimutus duration. Figure 2 presents the plots of log ( / x D) as a function of log D for each of the species studied. For these plots the values of log (Ix D) at each duration were averaged for all cells recorded in the species. Two straight lines, log (Ix D) = constant and log /--constant, corresponding respec-
tively to perfect summation (Bloch's law) and zero summation, were fitted to the data points; their intersections indicate the critical durations. The number of cells tested in each species (n) varied from 18 in Melipona to 2 in Polistes and Carnponotus, and, as expected, the scatter of the points is greater where n is small. This is especially evident at the shortest durations (more than an order of magnitude shorter than the critical duration) for which the responses were very small and subject to large relative fluctuations. For this reason these points were mostly disregarded in fitting by eye the horizontal straight lines. The critical durations varied from a minimum of 10 ms in Pseudornyrrnex to a maximum of 46 ms in Atla.
J.M. de Souza and D.F. Ventura: Hymenopteran photoreceptors
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log I50 and log Ilo are the intensities of the V/log I functions at Vm= 50% and 10% of Vs~t, respectively. S is a measure of the slope of the fitted sigmoid function: Iso/I=(V~at/Vm-1)1/5. Critical durations (to) were also estimated (Fig. 2) by averaging the data points of the temporal summation curves (see Results). The last column gives approximate rhabdomal cross-sectional areas of the ommatidia
T a b l e 1 s h o w s the a v e r a g e values o f the p a r a m eters o f the s i g m o i d f u n c t i o n s a n d o f the critical d u r a t i o n s o b t a i n e d f o r i n d i v i d u a l cells in e a c h species. It s h o u l d be n o t e d t h a t the a v e r a g e critical d u r a t i o n s p r e s e n t e d in this table were o b t a i n e d b y fitting s t r a i g h t lines to the results o f e a c h i n d i v i d u a l cell, r a t h e r t h a n to the a v e r a g e log ( I x D) values as in Fig. 2. I n s o m e cases t w o series o f t e m p o r a l stimuli were m a d e o n the s a m e cell a n d in this case they were p r o c e s s e d s e p a r a t e l y a n d the results a v e r a g e d f o r t h a t cell. A l s o given is the relative intensity c o r r e s p o n d i n g to Vm/Vsat = 10% (log l j o ) as a n a p p r o x i m a t e i n d i c a t i o n o f the a b s o l u t e sensitivity o f the r e t i n u l a r cells o f the insect (see D i s c u s sion). T h e last t w o c o l u m n s c o n t a i n the critical
d u r a t i o n s o b t a i n e d g r a p h i c a l l y f r o m Fig. 2 a n d s o m e e s t i m a t e s o f the c r o s s - s e c t i o n a l a r e a o f the o m m a t i d i a (Sim6es 1983; J o a q u i m 1985). A c o m p l e t e set o f r e s p o n s e s to the intensity series f o r one cell f r o m e a c h species is given in Fig. 3 in o r d e r to illustrate the g r e a t diversity o f forms encountered.
Control experiments T h e m e t h o d u s e d here to calculate the t e m p o r a l s u m m a t i o n curves a n d critical d u r a t i o n s d e p e n d s f u n d a m e n t a l l y o n the p a r a l l e l i s m b e t w e e n the sigm o i d (V/log /) f u n c t i o n s f o r different d u r a t i o n s . I n o r d e r to c h e c k this, a series o f V/log I curves
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for different stimulus durations was obtained in two cells in Melipona (Fig. 4). The checking procedure consisted of finding the best fitting sigmoid to the results from the longest duration used and then fitting the data from the other durations to sigmoids of equal slope. All series could be adjusted quite well to parallel sigmoids, in this case S = 0 . 6 , and, as small variations in the value of S do not appreciably alter the resulting values of
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Melipona to verify the parallelism between V/logIcurves obtained with stimuli of different durations (D) in the same cell. Computergenerated theoretical sigmoids of constant slope (dotted curves) were fitted to the data points. Adherence to the sigmoids at all durations is within normal tolerances for single responses.
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series was provided by the fact that, although the responses at and above the critical duration varied between 20% and 90% of Vs,t, there was no correlation between these values and the critical duration. Figure 5 gives the averaged dark-adapted impulse response in Melipona and the corresponding temporal summation function derived from its convolution with the stimulus (see Data measurement and analysis). The intersection of the zeroand unit-slope asymptotes indicates a critical duration of 17 ms. Discussion
The data shown in Fig. 2 confirm that temporal summation measured in photoreceptors of seven species of hymenopterans follows the prediction of linear theory applied to typical impulse responses (Fig. 5), in spite of the fact that in most of the experiments the responses in the region of the critical duration were well above the linear range. Except for some scatter in the responses to very low-energy stimuli, intensity/duration reciprocity is approximately perfect at short durations and drops off gradually to zero summation in the region of the critical duration. Thus, Bloch's law is essentially confirmed, with the exception of the sharp break at the critical duration. Dark-adapted critical durations derived from the temporal summation data varied from a minim u m of about 10 ms to a maximum of 46 ms. Based on these values the species can be divided into three groups: fast (Melipona, Trigona, Bornbus, and Pseudomyrrnex), intermediate (Polistes) and slow (Carnponotus and Atta). It is significant
Fig. 5. Temporal summation function obtained by linear convolution of the dark-adapted impulse response in Melipona. Inset, impulse response obtained by averaging 50 responses to 100gs LED-generated light pulses at - 2 . 8 log relative intensity. The temporal summation points were obtained by comparing the peak amplitude of the convoluted responses to that of the impulse response and then calculating the stimulus energy necessary to maintain the amplitude of the impulse response at each duration. This is expressed in terms of the energy of the impulse stimulus. Straight lines, Bloch's law
that the fast species are all diurnal, fast-moving species and the slow ones are predominantly nocturnal and relatively slow-moving ants. Pseudomyrmex, the only ant among the fast species, is a small, elongated, fast-moving insect which clearly displays visual avoidance tactics when confronted with an obstacle, as opposed to the slow-moving Camponotus and Atta which seem incapable of perceiving visually objects placed in their path. Thus it would appear that the temporal characteristics of the species have evolved in some measure in accordance with their life styles, a conclusion also arrived at by Autrum (1950) and Howard et al. (1984). The fact that green-sensitive photoreceptors of relatively closely related species have such widely varying temporal characteristics poses the question of what causes these differences, since presumably the same photopigment is involved in all cases. A comparison between eyes of slow and fast insects shows morphological specializations: fast eyes must have higher spatial resolution than slow eyes, in order to avoid blurring of the image. Accordingly, fast moving, diurnal species have eyes with many small facets, and retinulae with small rhabdomal areas. Slow species have fewer larger ommatidia, with larger rhabdomal area. Our data show a positive correlation between critical duration and rhabdomal cross-sectional area (see Table 1): long critical durations are associated with large rhabdoms and vice versa. This suggests that the diffusion rate of the internal transmitter molecules might explain the differences in critical duration reported. Stieve (1986) has proposed such a mechanism, acting outside the microvilli, for the generation of bumps in Lirnulus, in view of Williams' (1983) finding that there is no relation be-
J.M. de Souza and D.F. Ventura: Hymenopteran photoreceptors
tween bump latencies and microvillar length in the day and night states in locust. The temporal and gain characteristics of the three bee species are very similar in all respects. This corroborates earlier evidence that bees all have similar photoreceptors (Hertel and Ventura 1985; Menzel et al. 1988). Although only two cells in one specimen of Polistes were tested, they both gave very similar results: it is not clear why a wasp should differ from the bees, but the fact that its nest is completely open might explain the lower absolute sensitivity (log Ilo). It is perhaps surprising that a nonflying insect, Pseudomyrmex, has the fastest photoreceptor among the species tested here; the speed of insect visual systems has usually been associated with the necessity of fast reactions during flight (Howard et al. 1984; Srinivasan and Lehrer 1984). An interesting aspect of the data presented in Table 1 is that the slopes (S) of the V/log I functions are all close to 0.7 except that of Atta (0.97); one would expect that a nocturnal insect, which is, however, also active during the day, should benefit from a larger dynamic range than diurnal species. Atta has fewer ommatidia (lower spatial resolution) with a smaller dynamic range, slower temporal resolution, and lower absolute sensitivity (log llo) than the bee species. The unexpected lower absolute sensitivity is probably due to the fact that the V/log I data were measured (a) with a narrow stimulus source ( ~ 1~ which would be insufficient to stimulate fully Atta's widely spaced ommatidia, and (b) during the day. In the same species Felisberti and Ventura (1989) found considerable reduction in the diameter of the cones behind the corneal lens and pigment migration towards the rhabdom in the day state and Ventura et al. (1976) measured an electroretinogram sensitivity reduction of about 1 log unit during the day. Direct intracellular determination of critical duration in invertebrate photoreceptors has only been reported by Wasserman and Kong (Wasserman and Kong 1975; Kong and Wasserman 1978) who found values from 65 to 170 ms in Lirnulus depending on the peak potential used as a criterion for constancy. Their data showed partial summation below the critical duration. Had they used the total summation asymptote for short durations rather than a straight line representing partial summation, they would have found much less variation (about 60-100 ms). As explained below, perceptible partial summation is to be expected over a range of durations of about 1 log unit around the critical duration. Daly and Normann (1985) obtained critical du-
243
rations in turtle photoreceptors (about 150 ms, dark adapted) by measuring responses to what appear to have been equal-energy stimuli of varying duration giving responses in the linear range and then calculating their respective sensitivities. They also showed that, assuming linearity, critical duration for peak amplitude can be calculated by integrating the impulse response and dividing by its peak amplitude, but their assertion that integration time is equal to critical duration is a gross approximation. Integration time for peak response is equal to the width of the impulse response at its base and the critical duration would only be equal to this if the impulse response were rectangular, which is definitely not the case in any known animal. Similarly, linear integration of typical bellshaped impulse responses, such as those Daly and Normann measured in dark-adapted turtle receptors, results in appreciable partial summation over a range of durations of about 1 log unit around the critical duration (see Fig. 5). It is thus surprising that so many psychophysical studies of temporal summation have shown such close adherence to Bloch's law in this region. Daly and Normann also present temporal modulation functions calculated by taking Fourier tranforms of the impulse responses. Howard et al. (1984) recorded dark- and lightadapted impulse responses in unidentified photoreceptor types of eight flying insects; these can be transformed into critical durations by convolution or integration and give dark-adapted values of about 24 ms (dronefly) to 49 ms (dragonfly) and peak integration times of up to 120 ms, i.e., in the intermediate to long range in comparison with the results presented here. The authors used time to peak of the impulse response as a measure of the speed of the receptor process. We consider that critical duration, which is directly related to integration time, is a more adequate measure, because time to peak refers only to the first stimulus, whereas perception of a sequence of events requires that the photoreceptors have time to recover from each previous stimulus before processing the next. The latency from stimulus onset to response onset, which is part of the time to peak, is thus not a relevant feature. Raggenbass (1983) gives impulse responses of photoreceptors of Apis rnellifera drones recorded from superfused slices of retina which correspond to critical durations in the region of 30 ms, also considerably longer than those reported here for three species of bee. In view of these differences and as a further check against the possibility that our method may have introduced an adaptational effect, we re-
244
corded dark-adapted impulse responses in Melipona and Musca domestica. Figure 5 shows the average of 50 responses in Melpona and the resulting temporal summation curve. It can be seen that partial summation is discernible from durations of a few milliseconds up to about 40 ms, or roughly i log unit, and the critical duration of 17 ms is compatible with the values obtained with the main method used in this paper. The same value was obtained by integrating the response and dividing by its peak amplitude. A further series of responses to 180 light pulses over a period of 30 min, after 2 min adaptation to - 3 . 4 log intensity, gave a terminal critical duration of 21.8 ms but very little adaptation occurred after 10 rain; this result is still within the range of values obtained in the main experiments. In Musca the critical duration derived from the thoroughly dark-adapted impulse response was 24 ms as compared to 37 ms from the data of Howard et al. (1984), although time to peak was close at 39 ms against 43. Only a few behavioral studies of the temporal characteristics of insect visual systems have been made: flicker cutoff frequencies in Apis mellifera were found at 50 Hz for negative geotaxis (Wolf 1933), and at 100 Hz (Kunze 1961) and 220 Hz (Autrum and St6cker 1950) for optomotor responses. Srinivasan and Lehrer (1984) used bees' movement avoidance response to find a cutoff frequency of 200 Hz and a peak at about 60 Hz. They also report (Srinivasan and Lehrer 1985) cutoff frequencies of 80-120 Hz for mixtures of colors. It is not easy to compare these values to darkadapted critical durations, but Matin (1968) compared human psychophysical flash and flicker data and concluded that critical flicker frequency can be equated to the reciprocal of the critical duration. Thus a cutoff frequency of 200 Hz would correspond to a strongly light-adapted critical duration of 5 ms. Using double light pulses and moderate light adaptation, we have obtained critical durations of down to 5 ms in Melipona (Souza 1986). The sets of typical responses to the intensity series in each of the species given in Fig. 3 show that very large differences in response form exist in these closely related insects. These are evident at lower intensities in the greater or lesser incidence of voltage bumps : although considerable variation occurred between individual cells of each species, the largest bumps were found in Camponotus (up to 10 mV) and the smallest in Atta (1 mV). These differences in bump size do not seem to be artifactual, but rather the result of real differences in the photoreceptors' dark-adapted voltage gain, as described by Laughlin (1981) for other species of in-
J.M. de Souza and D.F. Ventura: Hymenopteran photoreceptors
sects. Apart from this, all species have approximately rectangular responses to the 300-ms stimuli at low intensities. As the intensity is increased, however, the responses of the faster species become characterized by a phasic on-peak followed by a sustained plateau, whereas the slow species continue to give almost rectangular responses with little or no peak. It would seem that the mechanism of adaptation which reduces the potential from the peak to the plateau, generally attributed to a lightdependent Ca 2+ influx (Muijser 1979; Brown 1986), is more active in some species than in others. As the intensity is increased further, in the fast species a trough separates the peak from the plateau and finally, at intensities close to saturation, the trough disappears again. At high intensities the peaks are sharpened by what appears to be a second, faster adaptation process as evidenced by a discontinuity in the declining phase of the peak potential. In all species, return to baseline at stimulus offset becomes slower at the highest intensities and, in the case of Bombus, the potential actually increases at this point and a slow depolarizing afterpotential ensues. Whether these variations in response form to relatively long and intense stimuli can throw any light on the overall transduction characteristics of the photoreceptors remains to be seen. It could be speculated that the species might differ in lightinduced intracellular Ca 2+ levels. Intracellular Ca 2+ may be regulated either by extracellular C a 2+ o r by release from intracellular stores through the action of inositol polyphosphates (Brown 1986). By this line of reasoning, species such as Camponotus and Atta, which show little or no peak to plateau adaptation, should have lower intracellular C a 2+ than the other species. The large bumps encountered in Camponotus might also be related to low Ca2+; Stieve and Bruns (1980) reported increased bump size with l o w e r C a 2 + in Limulus. However, this would not explain the small bump size in Atta. Interestingly, another insect tested, an orthopteran from the Tettigoniidae family, also had very small adaptation and large bumps. Acknowledgements. This work was supported by research grants from CNPq and FINEP.
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