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Light transmission through the head capsule of an aphid, Megoura viciae
0022-1910/81/110773~4$02.00/0 0 1981 Pergamon Press Ltd.
J. Insect Physiol., Vol. 21, No. 11, pp. 113-717, 1981. Printed in Great Britain.
LIGHT TRANSMISSION THROUGH THE HEAD CAPSULE OF AN APHID, MEGOURA V1ChiE JIM HARDIE*,A. D. LEESand STEPHENYOUNG Agricultural Research Council Insect Physiology Group, Department of Pure and Applied Biology, Imperial College, London SW7 2A2, England (Received 7 April 1981)
Abstract-Spectral transmission through the dorsal head cuticle of Megoura viciae was measured. Although all wavelengths between 370and 800nm are transmitted there is more absorption of the shorter wavelengths. These data are used to demonstrate the fact that the photoperiodic receptor, which lies in the brain, is more sensitive to shorter wavelengths than has been indicated by whole animal action spectra. Key Word Index;
Light transmission, cuticle, action spectra, aphid, Megoura viciae
INTRODUCTION
ACTIONspectra for the photoperiodic response in the apterous form of Megoura viciae have been published elsewhere (LEES,1971; 1981). Such curves describe the response of the intact insect to monochromatic light at different times during the photoperiodic cycle. However, in this aphid, as in other insects, the photoreceptors are known to be extraoptic and to be located within the brain (LEES, 1964; WILLIAMSet al., 1965; CLARET,1966). ‘Deep’ photoreceptors are also associated with phase shifting response in certain circadian rhythms (TRUMAN, 1976). In Megoura the most probable site of the photoreceptive neurones is in the anterior part of the protocerebrum, just lateral to the Group I neurosecretory cells (LEES, 1964; STEEL and LEES, 1977). It is to be expected, therefore, that light reaching the receptors would be subjected to the filtering action of the cuticle and any overlying tissues. measurements of the spectral Our present transmission through the cuticle enable us to adjust the action spectra so that they reflect with more precision the spectral sensitivity of the photoreceptors. MATERIALS
AND METHODS
The dorsal head cuticle was dissected both from a newly moulted adult apterous virginopara and from older individuals where sclerotization was complete. Areas of muscle attachment do not occur in this part of the dorsum although some fibres are present near the bases of the antennae. There are no fat-body lobes overlying the protocerebrum in vivo but the epidermis and any fragments of fat-body were carefully removed during dissection. The cuticle was then rinsed and mounted in distilled water between a glass coverslip and quartz microscope slide. The y0 transmission was measured at wavelengths between 370 and 800 nm using a Shimadzu, dual beam microspectrometer *Address for correspondence: Agricultural Research Council Insect Physiology Group, Imperial College at Silwood Park, Ascot, Berks SL5 7PY. England.
(Model MPS-SOL) with a scanning spot 80 pm in diameter. The cuticular areas examined were: (A) Head/prothorax intersegmental cuticle of an old adult aptera; (B) Dorsum of an old adult; (C) Dense cuticle near antenna1 base; (D) Dorsum of a newly moulted adult aptera. (See Fig. 1). In areas A, B and C at least five sets of readings were taken of different dissections. Only a single set of data were obtained for area D between 400 and 800 nm. In addition brains were dissected from adult apterae and the presumed location of the extraoptic receptor was scanned with wavelengths between 400 and 800 nm. RESULTS
The transmission of visible light through all the cuticular areas examined increased with wavelength (Fig. 2). In older aphids the most transparent cuticle was that of the head/prothorax articulation (position A) and it is likely that the antennal/head joint cuticle has a similar transmission pattern. The dorsum region immediately above the brain area sensitive to photoperiod (position B) also shows significant transmission of all wavelengths examined ranging from ca. 19% at 370 nm to ca. 86% at 800 nm. Most of probably shows similar region the dorsum transmission values (see Fig. la). The antenna1 base regions are highly sclerotized and consequently are much more opaque with transmission values of ca. 0.5% at 370 nm to ca. 23% at 800 nm. There is no evidence for selective transmission of certain wavelengths through any of the areas of cuticle examined. Cuticle removed from the head of a newly moulted adult, before sclerotization has occurred, demonstrated high transmission values at all wavelengths examined. Scans of the anterior part of the protocerebrum, the probable location of the receptor responsive to photoperiod, showed no peaks of absorption which might be expected if a photopigment were present in appreciable concentration.
773
JIMHARDIE,A. D. LEES AND STEPHEN YOUNG
114
80-
500
r 550
600
WAVELENGTH
650
I 700
750
I 800
(nm)
Fig. 2. Transmission of monochromatic light in the visible and near-u.v. region of the spectrum through various areas of the dorsal head capsule of adult M. viciae. A, Position A (see Fig. 1). Head/prothorax intersegmental cuticle ( + SE.); 0, Position B. Region of the dorsum ( k SE.); n , Position C. Dense cuticle near antenna1 base ( + S.E.); 0, Position D. Dorsum region of a newly moulted adult. Positions A and C are mean transmissions from 6 insects, n = 5 for Position B and n = 1 for Position D.
DISCUSSION All insect photoreceptors are activated by light that has previously passed through a layer of cuticle. In the case of eyes, with specialized dioptric apparatus, light is transmitted through cuticular lens structures whose relative transparency to visible light and near-uv. is conferred by colourless cross-linking agents in the protein (NEVILLE, 1975) but which may, however, absorb short U.V. (< 300 nm) (e.g. GOLDSMITHand FERNANDEZ,1968; CARLKIN and PHILIPSON,1972). There are few references to light transmission through less specialized cuticular areas covering extraoptic photoreceptors. WILLIAMSet al. (1965) have measured transmission through the cocoon and facial cuticle, which admits light to the pupal brain in Antheruea pernyi, thereby inducing the termination of diapause. Even allowing for the possibility that the cocoon acts as a light integrating sphere only ca. 0.5% of incident light reached the insect’s brain. BALL (1977) has reported transmission properties of the cuticle near parts of the brain believed to contain photoreceptive neurones mediating the circadian rhythm of activity in Periplaneta americana. In this insect the dorsum transmitted cu. 5-55x of light between wavelengths of 425 and 800 nm. Other regions of cuticle e.g. lightly pigmented pronotal cuticle and the 6th abdominal tergite, were more transparent. In comparison, the aphid head capsule is much less opaque with dorsum figures ranging from 19 to 86% transmission between wavelengths of 370 and 800 nm. In each of these reports transmission increased steadily from the blue to the red end of the spectrum, there was no evidence for selective transmission of certain wavelengths. On the other hand, the head capsule of many weevils acts as a filter which transmits only far red and near-i.r. wavelengths (650-900 nm; MEYER,1977).
Action spectra for the effectiveness of nearmonochromatic light interruptions in abolishing the short day (i.e. long night) photoperiodic response of M. viciae are shown in Fig. 3. Two examples have been chosen with light pulses occurring either in the early (Fig. 3a) or late (Fig. 3b) part of the scotophase. The upper curves (l), which are taken from LEES(1981) plot the intensities of spectral light that cause 50% of the aphids to develop as long-day, virginoparaproducers. The lower curves (2) represent the spectra modified for differential wavelength absorption by the area of the dorsum (area B) which overlies the putative photoperiodic receptor (LEES,1964). In addition to the cuticle, light would have to pass through the epidermis and a small haemolymph space before reaching the brain but through no other tissues. The colourless epidermal cell layer is only tenuously attached to the cuticle and it proved impossible to prepare cuticle with this layer intact. Thus in modifying the action spectra no allowance has been made for light absorption by the epidermis and haemolymph. The effect of scattered light has also been ignored. TRUMAN(1976) has concluded that in many small insects the screening action of tissue overlying the extraoptic receptors is minimal. The present results, however, indicate that this screening is far from negligible. The modified action spectra are regarded as producing a more accurate picture of the wavelength sensitivity of the photoperiodic receptor. Since I,,, remains in the blue region (430+90 nm) and the general form of the response curves is retained, the results do not affect the interpretation placed upon the action spectra, particularly in relation to pigment changes during the photoperiodic cycle (LEES, 1981). Nevertheless, the receptor mechanism is more sensitive than is indicated by the intact insect response (0.1 nW crne2 vs 0.21 PW crne2 at 450 nm for early
a
b
Fig. 1. Photomicrographs of the dorsal head cuticle examined. (a) Dissected from an old adult, fully sclerotized. (b) Dissected from a newly moulted insect. A, B, C and D refer to the specific areas where spectral transmission was recorded. ab. antenna base; e. eye. Scale bar = 100 pm.
Cuticular light transmission in an aphid
111
moulted adults is complete within 2-3 hr. the relatively transparent cuticle during this period is not believed to be physiologically important to the aphid’s photoperiodic response. These comparisons represent the first attempt to compensate insect action spectra for light absorbance by structures surrounding the extraoptic receptors. The lack of absorption of specific wavelengths by the putative photopigment in the region of the brain where the receptor is located is assumed to indicate a very low concentration of photopigment. Acknowledgements-The assistance.
authors thank Tony Easty for his
REFERENCES BALLH. J. (1977) Spectral transmission through the cuticle of the American cockroach, Peripluneta americana. J. Insect Physiol. 23, 14.
CARL~ONS. D. and PH~LIP~~NB. (1972) Microspectrophotometry of the dioptric apparatus and compound rhabdom of the moth (Manduca sexta) eye. J. Insect Physiol. 18, 1721-1731. CLARETJ. (1966) Mise en evidence du rBle photortcepteur du cerveau dans l’induction de la diapause, chez Pieris brassicae (Lepido). Ann. Endoer. 27, 31 I-320. GOLDSMITH T. H. and FERNANDEZ H. R. (1968) The sensitivity of housefly photoreceptors in the midultraviolet and the limits of the visible spectrum. J. exp. Biol. 49, 669-677.
Fig. 3. (a) Action spectrum for the maternal control of virginopara production in M. vi&e. Near monochromatic light was applied for 1 hr, during the early scotophase to give a rkgime of 13.5 L:I.S D:l.O L:8.0 D. The upper curve (1) represents the incident energies at which ca. 50% of the aphids become virginopara-producers (taken from LEES, 1981). The lower curve (2) is adjusted for light absorption by the cuticle of the dorsum. (b) Action spectrum for a 0.5 hr light interruption late in the scotophase, a rtgime of 13.5 L:7.5 D:0.5 L:2.5 D. Upper curve (I) is a whole animal response as given by LEES(1981). Lower graph (2) is adjusted for cuticular absorption. light interruptions; 0.6 PW cmm2 vs 1.3 PW cmm2 at 450 nm for late night interruptions). In addition, sensitivity to shorter wavelengths is more prominent than had been suspected. As sclerotization in newly
LEESA. D. (1964) The location of the photoperiodic receptors in the aphid Megoura viciae Buckton. J. exp. Biol. 41, 119-133. LEESA. D. (1971) The relevance of action spectra in the study of insect photoperiodism. In Biochronometry (Ed. by MENAKER M.). pp. 372-379. National Academy of Sciences, Washington D.C. Lt~s A. D. (1981) Action spectra for the photoperiodic control of polymorphism in the aphid Megoura viciae. J. Insect Physiol. 27, 761-771. MEYER
J. R. (1977) Head capsule transmission of longwavelength light in the Curculionidae. Science, N. Y. 196, 524-525. NEV~LLEA. C. (1975) Biology of the Arthropod Cuticle. Springer, Berlin. STEEL C. G. H. and LEES A. D. (1977) The role of neurosecretion in the photoperiodic control of polymorphism in the aphid Megoura viciue. J. exp. Biol. 67, 117-13s. TRUMANJ. W. (1976) Extraretinal photoreception in insects. Photochem. Photobiol. 23, 215-225.
WILLIAMSC. M., ADKISKINP. L. and WALCOTTC. (1965) Physiology of insect diapause. XV. The transmission of photoperiod signals to the brain of the oak silkworm, Antheraea pernyi. Biol. BUN. mar. biol. Lab., Woods Hole u&497-507.
J. Insect Physiol., Vol. 21, No. 11, pp. 113-717, 1981. Printed in Great Britain.
LIGHT TRANSMISSION THROUGH THE HEAD CAPSULE OF AN APHID, MEGOURA V1ChiE JIM HARDIE*,A. D. LEESand STEPHENYOUNG Agricultural Research Council Insect Physiology Group, Department of Pure and Applied Biology, Imperial College, London SW7 2A2, England (Received 7 April 1981)
Abstract-Spectral transmission through the dorsal head cuticle of Megoura viciae was measured. Although all wavelengths between 370and 800nm are transmitted there is more absorption of the shorter wavelengths. These data are used to demonstrate the fact that the photoperiodic receptor, which lies in the brain, is more sensitive to shorter wavelengths than has been indicated by whole animal action spectra. Key Word Index;
Light transmission, cuticle, action spectra, aphid, Megoura viciae
INTRODUCTION
ACTIONspectra for the photoperiodic response in the apterous form of Megoura viciae have been published elsewhere (LEES,1971; 1981). Such curves describe the response of the intact insect to monochromatic light at different times during the photoperiodic cycle. However, in this aphid, as in other insects, the photoreceptors are known to be extraoptic and to be located within the brain (LEES, 1964; WILLIAMSet al., 1965; CLARET,1966). ‘Deep’ photoreceptors are also associated with phase shifting response in certain circadian rhythms (TRUMAN, 1976). In Megoura the most probable site of the photoreceptive neurones is in the anterior part of the protocerebrum, just lateral to the Group I neurosecretory cells (LEES, 1964; STEEL and LEES, 1977). It is to be expected, therefore, that light reaching the receptors would be subjected to the filtering action of the cuticle and any overlying tissues. measurements of the spectral Our present transmission through the cuticle enable us to adjust the action spectra so that they reflect with more precision the spectral sensitivity of the photoreceptors. MATERIALS
AND METHODS
The dorsal head cuticle was dissected both from a newly moulted adult apterous virginopara and from older individuals where sclerotization was complete. Areas of muscle attachment do not occur in this part of the dorsum although some fibres are present near the bases of the antennae. There are no fat-body lobes overlying the protocerebrum in vivo but the epidermis and any fragments of fat-body were carefully removed during dissection. The cuticle was then rinsed and mounted in distilled water between a glass coverslip and quartz microscope slide. The y0 transmission was measured at wavelengths between 370 and 800 nm using a Shimadzu, dual beam microspectrometer *Address for correspondence: Agricultural Research Council Insect Physiology Group, Imperial College at Silwood Park, Ascot, Berks SL5 7PY. England.
(Model MPS-SOL) with a scanning spot 80 pm in diameter. The cuticular areas examined were: (A) Head/prothorax intersegmental cuticle of an old adult aptera; (B) Dorsum of an old adult; (C) Dense cuticle near antenna1 base; (D) Dorsum of a newly moulted adult aptera. (See Fig. 1). In areas A, B and C at least five sets of readings were taken of different dissections. Only a single set of data were obtained for area D between 400 and 800 nm. In addition brains were dissected from adult apterae and the presumed location of the extraoptic receptor was scanned with wavelengths between 400 and 800 nm. RESULTS
The transmission of visible light through all the cuticular areas examined increased with wavelength (Fig. 2). In older aphids the most transparent cuticle was that of the head/prothorax articulation (position A) and it is likely that the antennal/head joint cuticle has a similar transmission pattern. The dorsum region immediately above the brain area sensitive to photoperiod (position B) also shows significant transmission of all wavelengths examined ranging from ca. 19% at 370 nm to ca. 86% at 800 nm. Most of probably shows similar region the dorsum transmission values (see Fig. la). The antenna1 base regions are highly sclerotized and consequently are much more opaque with transmission values of ca. 0.5% at 370 nm to ca. 23% at 800 nm. There is no evidence for selective transmission of certain wavelengths through any of the areas of cuticle examined. Cuticle removed from the head of a newly moulted adult, before sclerotization has occurred, demonstrated high transmission values at all wavelengths examined. Scans of the anterior part of the protocerebrum, the probable location of the receptor responsive to photoperiod, showed no peaks of absorption which might be expected if a photopigment were present in appreciable concentration.
773
JIMHARDIE,A. D. LEES AND STEPHEN YOUNG
114
80-
500
r 550
600
WAVELENGTH
650
I 700
750
I 800
(nm)
Fig. 2. Transmission of monochromatic light in the visible and near-u.v. region of the spectrum through various areas of the dorsal head capsule of adult M. viciae. A, Position A (see Fig. 1). Head/prothorax intersegmental cuticle ( + SE.); 0, Position B. Region of the dorsum ( k SE.); n , Position C. Dense cuticle near antenna1 base ( + S.E.); 0, Position D. Dorsum region of a newly moulted adult. Positions A and C are mean transmissions from 6 insects, n = 5 for Position B and n = 1 for Position D.
DISCUSSION All insect photoreceptors are activated by light that has previously passed through a layer of cuticle. In the case of eyes, with specialized dioptric apparatus, light is transmitted through cuticular lens structures whose relative transparency to visible light and near-uv. is conferred by colourless cross-linking agents in the protein (NEVILLE, 1975) but which may, however, absorb short U.V. (< 300 nm) (e.g. GOLDSMITHand FERNANDEZ,1968; CARLKIN and PHILIPSON,1972). There are few references to light transmission through less specialized cuticular areas covering extraoptic photoreceptors. WILLIAMSet al. (1965) have measured transmission through the cocoon and facial cuticle, which admits light to the pupal brain in Antheruea pernyi, thereby inducing the termination of diapause. Even allowing for the possibility that the cocoon acts as a light integrating sphere only ca. 0.5% of incident light reached the insect’s brain. BALL (1977) has reported transmission properties of the cuticle near parts of the brain believed to contain photoreceptive neurones mediating the circadian rhythm of activity in Periplaneta americana. In this insect the dorsum transmitted cu. 5-55x of light between wavelengths of 425 and 800 nm. Other regions of cuticle e.g. lightly pigmented pronotal cuticle and the 6th abdominal tergite, were more transparent. In comparison, the aphid head capsule is much less opaque with dorsum figures ranging from 19 to 86% transmission between wavelengths of 370 and 800 nm. In each of these reports transmission increased steadily from the blue to the red end of the spectrum, there was no evidence for selective transmission of certain wavelengths. On the other hand, the head capsule of many weevils acts as a filter which transmits only far red and near-i.r. wavelengths (650-900 nm; MEYER,1977).
Action spectra for the effectiveness of nearmonochromatic light interruptions in abolishing the short day (i.e. long night) photoperiodic response of M. viciae are shown in Fig. 3. Two examples have been chosen with light pulses occurring either in the early (Fig. 3a) or late (Fig. 3b) part of the scotophase. The upper curves (l), which are taken from LEES(1981) plot the intensities of spectral light that cause 50% of the aphids to develop as long-day, virginoparaproducers. The lower curves (2) represent the spectra modified for differential wavelength absorption by the area of the dorsum (area B) which overlies the putative photoperiodic receptor (LEES,1964). In addition to the cuticle, light would have to pass through the epidermis and a small haemolymph space before reaching the brain but through no other tissues. The colourless epidermal cell layer is only tenuously attached to the cuticle and it proved impossible to prepare cuticle with this layer intact. Thus in modifying the action spectra no allowance has been made for light absorption by the epidermis and haemolymph. The effect of scattered light has also been ignored. TRUMAN(1976) has concluded that in many small insects the screening action of tissue overlying the extraoptic receptors is minimal. The present results, however, indicate that this screening is far from negligible. The modified action spectra are regarded as producing a more accurate picture of the wavelength sensitivity of the photoperiodic receptor. Since I,,, remains in the blue region (430+90 nm) and the general form of the response curves is retained, the results do not affect the interpretation placed upon the action spectra, particularly in relation to pigment changes during the photoperiodic cycle (LEES, 1981). Nevertheless, the receptor mechanism is more sensitive than is indicated by the intact insect response (0.1 nW crne2 vs 0.21 PW crne2 at 450 nm for early
a
b
Fig. 1. Photomicrographs of the dorsal head cuticle examined. (a) Dissected from an old adult, fully sclerotized. (b) Dissected from a newly moulted insect. A, B, C and D refer to the specific areas where spectral transmission was recorded. ab. antenna base; e. eye. Scale bar = 100 pm.
Cuticular light transmission in an aphid
111
moulted adults is complete within 2-3 hr. the relatively transparent cuticle during this period is not believed to be physiologically important to the aphid’s photoperiodic response. These comparisons represent the first attempt to compensate insect action spectra for light absorbance by structures surrounding the extraoptic receptors. The lack of absorption of specific wavelengths by the putative photopigment in the region of the brain where the receptor is located is assumed to indicate a very low concentration of photopigment. Acknowledgements-The assistance.
authors thank Tony Easty for his
REFERENCES BALLH. J. (1977) Spectral transmission through the cuticle of the American cockroach, Peripluneta americana. J. Insect Physiol. 23, 14.
CARL~ONS. D. and PH~LIP~~NB. (1972) Microspectrophotometry of the dioptric apparatus and compound rhabdom of the moth (Manduca sexta) eye. J. Insect Physiol. 18, 1721-1731. CLARETJ. (1966) Mise en evidence du rBle photortcepteur du cerveau dans l’induction de la diapause, chez Pieris brassicae (Lepido). Ann. Endoer. 27, 31 I-320. GOLDSMITH T. H. and FERNANDEZ H. R. (1968) The sensitivity of housefly photoreceptors in the midultraviolet and the limits of the visible spectrum. J. exp. Biol. 49, 669-677.
Fig. 3. (a) Action spectrum for the maternal control of virginopara production in M. vi&e. Near monochromatic light was applied for 1 hr, during the early scotophase to give a rkgime of 13.5 L:I.S D:l.O L:8.0 D. The upper curve (1) represents the incident energies at which ca. 50% of the aphids become virginopara-producers (taken from LEES, 1981). The lower curve (2) is adjusted for light absorption by the cuticle of the dorsum. (b) Action spectrum for a 0.5 hr light interruption late in the scotophase, a rtgime of 13.5 L:7.5 D:0.5 L:2.5 D. Upper curve (I) is a whole animal response as given by LEES(1981). Lower graph (2) is adjusted for cuticular absorption. light interruptions; 0.6 PW cmm2 vs 1.3 PW cmm2 at 450 nm for late night interruptions). In addition, sensitivity to shorter wavelengths is more prominent than had been suspected. As sclerotization in newly
LEESA. D. (1964) The location of the photoperiodic receptors in the aphid Megoura viciae Buckton. J. exp. Biol. 41, 119-133. LEESA. D. (1971) The relevance of action spectra in the study of insect photoperiodism. In Biochronometry (Ed. by MENAKER M.). pp. 372-379. National Academy of Sciences, Washington D.C. Lt~s A. D. (1981) Action spectra for the photoperiodic control of polymorphism in the aphid Megoura viciae. J. Insect Physiol. 27, 761-771. MEYER
J. R. (1977) Head capsule transmission of longwavelength light in the Curculionidae. Science, N. Y. 196, 524-525. NEV~LLEA. C. (1975) Biology of the Arthropod Cuticle. Springer, Berlin. STEEL C. G. H. and LEES A. D. (1977) The role of neurosecretion in the photoperiodic control of polymorphism in the aphid Megoura viciue. J. exp. Biol. 67, 117-13s. TRUMANJ. W. (1976) Extraretinal photoreception in insects. Photochem. Photobiol. 23, 215-225.
WILLIAMSC. M., ADKISKINP. L. and WALCOTTC. (1965) Physiology of insect diapause. XV. The transmission of photoperiod signals to the brain of the oak silkworm, Antheraea pernyi. Biol. BUN. mar. biol. Lab., Woods Hole u&497-507.