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The secretory cycle of a gland involved in pheromone production in the noctuid moth, Pseudaletia separata
J. Insect Physiol., 1973, Vol. 19, pp. 19 to 28. Pergamon Press. Printed in Great Britain
THE SECRETORY CYCLE OF A GLAND INVOLVED IN PHEROMONE PRODUCTION IN THE NOCTUID MOTH, PSEUDALETIA SEPARATA J. R. CLEARWATER*
and V. SARAFIS
Department of Botany and Zoology, Massey University, Palmerston North, New Zealand (Receiwed 6 October 1971; revised 17 April 1972)
Abstract-The structure of the pheromone producing gland is briefly described. Examination of variations in gland volume, nucleus to cytoplasm ratio, and distribution and form of nucleic acids suggests that the secretory cycle of this gland consists of three phases. Two types of small vesicle are found and suggested as possible sites for pheromone metabolism.
INTRODUCTION
THE SYSTEM producing the pheromone of the noctuid appears to be extremely complex. In addition to a finely sculptured fan of hairs which serve to disperse the pheromone, a secretory mechanism consisting of at least two components is present (BIRCH, 1970). Two groups of very large cells, first described by STOBBE (1912), produce a pheromone precursor which is released into paired abdominal pouches by hairscale ducts. The scales lining these pouches are the second comBIRCH (1970) suggests that the ponent of the system (ELTRINGHAM, 1925). precursor of the pheromone in Leucania impwa is the p-glucoside of benzaldehyde, which is decomposed by the secretion of the pouch scales (presumably a ,3glucosidase). If this mechanism is correct, it shows remarkable similarities with the mechanism of synthesis of defensive secretions in Diploptera punctata (ROTH and STAY, 1958) and in Eleoaks longicollis and Tribolium castaneum (HAPP, 1968). The active pheromone is picked up when the fan is inserted into the pouches shortly after emergence (BIRCH, 1970). A histological evaluation of the physiological activity of the paired gland In insects, inactive glands are characterized by appeared a useful approach. reduced cytoplasm, a low nucleus to cytoplasm ratio, and indistinct cell boundaries. By contrast, active glands possess large swollen cells with a high nucleus to cytoplasm ratio, and cytoplasm more basophilic than that of the inactive gland. This ratio is claimed to be a reliable index of secretory activity, its major disadvantage being the time required to make the preparation (NOVAK, 1966). In order to determine this ratio, the total volume of the gland is compared with the * Present address: Alberta, Canada.
Department
of Entomology, 19
University
of Alberta,
Edmonton,
20
J. R. CLEARWATER ANDV. S-IS
number of nuclei. Many methods of estimating volume exist; LEGAY (1950) considered the average gland diameter while PFLUGFELDER (1948) prepared models of the glands and measured their water displacement. For this study, the index of NOVAK(1954) (the square root of the product of two linear dimensions) was used. Workers studying the corpus allatum have cut sections, determined the density of the nuclei and from this calculated the number of the nuclei. Because of the large size and small number of cells in the gland it was practicable to count the absolute number of the nuclei with a squash preparation. MATERIALS AND METHODS Experimental animals Male adults were obtained either from a laboratory seedlings or were captured in a light trap.
culture
fed on maize
Gland structure Glands were fixed in Bouin’s solution, dehydrated, and cleared in terpineol. After embedding in 58°C wax, serial sections were cut at 5 p, and stained with Ehrlich’s haemotoxylin and eosin. Glands were removed from males taken from stock cultures at O-5 and l-5 days following emergence and placed in EPHRUSSIand BEADLE’S(1936) saline. These were examined with a Zeiss Nomarski differential interference microscope fitted with an Olympus PM 6 camera attachment. Additional glands were transferred to a solution of O-Ol”h acridine orange in saline for 5 min before being examined under the illumination of an Olympus HLS U.V. light source. Gland physiology Male moths from stock cultures were maintained in cartons and provided with 10% sucrose until they had attained the required age. The gland was removed, placed in Ephrussi and Beadle’s saline and measured with an Olympus micrometer eyepiece at 40 x . The gland was then placed in a drop of acetocarmine, squashed, and left for 5 min before the nuclei were counted. As the nuclei stained bright red while the cytoplasm remained colourless, accurate counts were possible. RESULTS cytozogy The gland is situated deep within the fat body of the first abdominal segments with the hairscale duct leading to the hair pencil of the same side. The gland consists of an average of 68 very large cells attached to modified hairscales by scale sockets (Figs. 1, 2). The single large nucleus of each cell stains heavily with haemotoxylin. Comparison of acridine orange-stained glands of different ages revealed major differences in the distribution and fluorescence of the stain. Whereas the cytoplasm of cells 1.5 days old was translucent, that of cells O-5 days old was a deep orange.
21
Note the large nuclei (N) staining FIG. 1. Paraffin embedded phase II cells. heavily with Ehrlich’s haematoxylin connected to modified scales (M) by means of a socket (S). Compare the size of these cells with Figs. 2 and 3.
22
FIG. 2. Living phase I cells. The nuclear membrane (NM) separates the nucleus, which occupies much of the upper left-hand portion of the cell, from the cytoplasm. Note also the cell membrane (CM).
23
FIG. 3.
Two
populations
of vesicles.
PHEROMONE
PRODUCTION
IN
PSEUDALETIA
SEPARATA
25
The nuclei of the older cells were yellow-green. The nuclear colour of younger cells was obscured by the stain held in the cytoplasm. There were at least two types of vesicle in the cytoplasm of cells 0.5 days old (Fig. 3). These types differed in density, suggesting different chemical composition. Although the two populations were not homogenous, one type was more than twice the size of the other. The smaller type of vesicle was frequently found within the larger type. These vesicles were membrane-limited as the boundary between vesicles of the same density was sharp. Several vesicles appeared to be mobile. Physiology The gland appeared to move through a cycle of three phases (Fig. 4). From emergence to 1-O days, the gland was at the maximum observed size of between 900 and 1100 NI (NI = the square root of the product of two linear dimensions).
~OO~Phase,1
0
PhcyeIl
I
,
PtyseE
3
2
4
Initiation
Night
of
,
,
5
6
nucleus
no.
FIG. 4. The three phases of the secretory cycle of the paired gland. Each point represents 8 to 10 glands.
All of the cells were hypertrophied, some individual cells attaining diameters of over 1000 p. The nuclei of these cells were much larger than the nuclei of phase II
26
J. R. CLEARWATER ANDV. SARAFIS
cells
and in pa&in sections exhibited a lesser affinity for haematoxylin. Collapse of these giant cells signalled the beginning of the second phase. During this phase, which lasted approximately 1 day, the ratio of cytoplasm to nucleus ranges from 8.00 : 1 to 12.00 : 1 and the Novak index from 450 to 900. The third and final steep drop in volume occurred between day 2.5 and 3.5. By the end of this decline the disintegration of the nuclei and cell membranes was almost complete. The gland, now a formless mass, showed no further changes. The frequency distribution of the volume of all the glands measured revealed three distinct groups corresponding to the three phases postulated (see Fig. 5). Although all glands of the same age were not always in the same phase, the experimental population was reasonably synchronous. IO
g 2
Phase ICI
Phose U
Phase I
0 !
0
1000
800
Gland
Phase
600
size
(Novak
I
Nuclear lcytoplasmic
400
200
0
index)
Phase
ll
relationship
FIG. 5. The frequency distribution of the paired gland. DISCUSSION
The three phases of Stobbe’s gland appear to have functional significance. It is suggested that the large swollen gland of newly emerged adults (phase I) represents an active phase, possibly concerned with the elaboration of a pheromone or its precursor. After a period of decreased activity (phase II), the glands disintegrate (phase III).
PHEROMONE PRODUCTION IN PSEUDALETIA
SEPARATA
27
The giant cells are particularly interesting. STOBBE (1912), who had access only to material from wild populations, found only one specimen with giant cells. In the absence of further material, he could not discount a pathological condition. This reservation does not now seem important. As these cells are among the largest reported from insects, detailed examination of the mechanism of synthesis of secretion should prove particularly rewarding. Because of their extreme delicacy, these giant cells must be treated very gently. Examination of living phase I cells in physiological saline with interference microscopy revealed a layer of dense cytoplasm packed with vesicles. A large round body occupying a large portion of the cell is probably the nucleus since it stains red in acetocarmine, purple in Ehrlich’s haemotoxylin, and in phase II cells yellow-green in acridine orange. Preliminary electronmicrographs of conventionally prepared material reveal a very large central cavity and thin walls lined with microtubules. Electron micrographs of Phlogophora meticulosa prepared by BIRCH (1970) are very similar. Dissociation of the cells with EDTA or trypsin visibly altered their structure. Compared with living ones, paraflin embedded cells shrink to almost 10 per cent of their original volume. Acridine orange (AO) is a metachromatic dye, the fluorescence of which is dependent on the physical and chemical properties of the nucleic acids. Though evidence is not conclusive, it is thought that the metachromatic orange fluorescence is due to the presence of RNA while the orthochromatic green fluorescence is due to the presence of DNA (PEARSE,1968). If this interpretation is accepted, it can be seen that cells 0.5 days old have a considerable amount of cytoplasmic RNA while the only nucleic acid present in detectable amounts in older cells is DNA. Because of these variations in concentrations of cytoplasmic RNA, protein synthesis can be assumed to be proceeding at a much higher rate in the younger cells than in those 15 days old. The observation of much larger nuclei in phase I cells supports this interpretation. STEINBRECHT(1964) working on the pheromone secreting epithelia of female Bombyx mori demonstrated the presence of lipid vesicles with strong U.V.absorption at 240 mp. As synthetic trans-lo-cis-12-hexadecadien-l-01, the pheromone of this species, has an absorption of 230 ml*, STEINBRECHT(1964) considers that these vesicles contain a pheromone precursor. Also, GEROK(1950) has shown that lipid extracts of B. mmi have pheromone activity following reduction with LiAlH,. This led REGNIERand LAW (1968) to suggest that these vesicles contain an acid which is converted to the active pheromone by reduction of the carboxyl group. Because the noctuid P. separata is phylogenetically distant from the bombycid B. mori, any suggestion that benzaldehyde or its precursor has a similar origin can only be made with reservation. REFERENCES and function of the pheromone producing brush organs in male of Phlogophoru meticulosa (L) (Lepidoptera: Noctuidae). Trans. R. ent. Sot. Land.
BIRCH
M. C.
(1970)
22, 277-292.
Structure
28
J. R. CLEARWATER ANDV. SARAFIS
ELTRINGHAMH. (1925) On the abdominal brushes of certain noctuid moths. Trans. R. ent. Sac. Lond. 75, l-5. EPHRUSSIB. and BEADLEG. W. (1936) A technique of transplantation for Drosophila. Am. Nut. 70,218-225. GEROKW. (1950) Ph.D. Thesis, Eberhard Karls University, Tubingen, Germany. (From Regnio & Law, 1968.) HAPP G. M. (1968) Quinone and hydrocarbon production in the defensive glands of Eleodes longicollis and Tribolium castaneum. J. Insect Physiol. 14, 1821-1837. LEGAY J. M. (1950) Note sur l’evolution des corpora allata au tours de la vie larvaire de Bombyx mori. C. R. Sot. Biol., Paris 144, 512-513. NOVAK V. J. A. (1954) ‘The growth of the corpora allata during the post-embryonal development in insects’. Acta Sot. 2001. es1 18, 98-133. NOVAKV. J. A. (1966) Insect Hormones. Methuen, London. PEARsEA. G. E. (1968) Histochemistry, Theoretical and Applied, 3rd ed., Vol. 1. Churchill, London. PFLUGFELDER 0. (1948) Volumetrische Untersuchungen an den Corpora Allata der Honigbiene Apis mellifera. Biol. Zbl. 67, 223-241. REGNIERF. E. and LAW J. H. (1968) Insect pheromones. r. Lipid Res. 9, 541-551. ROTH L. M. and STAY B. (1958) The occurrence of paraquinones in some arthropods with emphasis on the quinone secreting tracheal glands of Diploptera punctutu (Blatteria). J. Insect Physiol. 1, 305-318. STEINBRECHTR. A. (1964) Feinstructur und Histochemie der Sexualduftdruse des Seidenspinners Bombyx mori. Z. Zellforsch. mikrosk. Anat. 64, 227-261. STOBBBR. (1912) Die abdominalen Duftorgane der mannlichen Sphingiden und Noctuiden. Zool. Jb. 32, 493-532.
THE SECRETORY CYCLE OF A GLAND INVOLVED IN PHEROMONE PRODUCTION IN THE NOCTUID MOTH, PSEUDALETIA SEPARATA J. R. CLEARWATER*
and V. SARAFIS
Department of Botany and Zoology, Massey University, Palmerston North, New Zealand (Receiwed 6 October 1971; revised 17 April 1972)
Abstract-The structure of the pheromone producing gland is briefly described. Examination of variations in gland volume, nucleus to cytoplasm ratio, and distribution and form of nucleic acids suggests that the secretory cycle of this gland consists of three phases. Two types of small vesicle are found and suggested as possible sites for pheromone metabolism.
INTRODUCTION
THE SYSTEM producing the pheromone of the noctuid appears to be extremely complex. In addition to a finely sculptured fan of hairs which serve to disperse the pheromone, a secretory mechanism consisting of at least two components is present (BIRCH, 1970). Two groups of very large cells, first described by STOBBE (1912), produce a pheromone precursor which is released into paired abdominal pouches by hairscale ducts. The scales lining these pouches are the second comBIRCH (1970) suggests that the ponent of the system (ELTRINGHAM, 1925). precursor of the pheromone in Leucania impwa is the p-glucoside of benzaldehyde, which is decomposed by the secretion of the pouch scales (presumably a ,3glucosidase). If this mechanism is correct, it shows remarkable similarities with the mechanism of synthesis of defensive secretions in Diploptera punctata (ROTH and STAY, 1958) and in Eleoaks longicollis and Tribolium castaneum (HAPP, 1968). The active pheromone is picked up when the fan is inserted into the pouches shortly after emergence (BIRCH, 1970). A histological evaluation of the physiological activity of the paired gland In insects, inactive glands are characterized by appeared a useful approach. reduced cytoplasm, a low nucleus to cytoplasm ratio, and indistinct cell boundaries. By contrast, active glands possess large swollen cells with a high nucleus to cytoplasm ratio, and cytoplasm more basophilic than that of the inactive gland. This ratio is claimed to be a reliable index of secretory activity, its major disadvantage being the time required to make the preparation (NOVAK, 1966). In order to determine this ratio, the total volume of the gland is compared with the * Present address: Alberta, Canada.
Department
of Entomology, 19
University
of Alberta,
Edmonton,
20
J. R. CLEARWATER ANDV. S-IS
number of nuclei. Many methods of estimating volume exist; LEGAY (1950) considered the average gland diameter while PFLUGFELDER (1948) prepared models of the glands and measured their water displacement. For this study, the index of NOVAK(1954) (the square root of the product of two linear dimensions) was used. Workers studying the corpus allatum have cut sections, determined the density of the nuclei and from this calculated the number of the nuclei. Because of the large size and small number of cells in the gland it was practicable to count the absolute number of the nuclei with a squash preparation. MATERIALS AND METHODS Experimental animals Male adults were obtained either from a laboratory seedlings or were captured in a light trap.
culture
fed on maize
Gland structure Glands were fixed in Bouin’s solution, dehydrated, and cleared in terpineol. After embedding in 58°C wax, serial sections were cut at 5 p, and stained with Ehrlich’s haemotoxylin and eosin. Glands were removed from males taken from stock cultures at O-5 and l-5 days following emergence and placed in EPHRUSSIand BEADLE’S(1936) saline. These were examined with a Zeiss Nomarski differential interference microscope fitted with an Olympus PM 6 camera attachment. Additional glands were transferred to a solution of O-Ol”h acridine orange in saline for 5 min before being examined under the illumination of an Olympus HLS U.V. light source. Gland physiology Male moths from stock cultures were maintained in cartons and provided with 10% sucrose until they had attained the required age. The gland was removed, placed in Ephrussi and Beadle’s saline and measured with an Olympus micrometer eyepiece at 40 x . The gland was then placed in a drop of acetocarmine, squashed, and left for 5 min before the nuclei were counted. As the nuclei stained bright red while the cytoplasm remained colourless, accurate counts were possible. RESULTS cytozogy The gland is situated deep within the fat body of the first abdominal segments with the hairscale duct leading to the hair pencil of the same side. The gland consists of an average of 68 very large cells attached to modified hairscales by scale sockets (Figs. 1, 2). The single large nucleus of each cell stains heavily with haemotoxylin. Comparison of acridine orange-stained glands of different ages revealed major differences in the distribution and fluorescence of the stain. Whereas the cytoplasm of cells 1.5 days old was translucent, that of cells O-5 days old was a deep orange.
21
Note the large nuclei (N) staining FIG. 1. Paraffin embedded phase II cells. heavily with Ehrlich’s haematoxylin connected to modified scales (M) by means of a socket (S). Compare the size of these cells with Figs. 2 and 3.
22
FIG. 2. Living phase I cells. The nuclear membrane (NM) separates the nucleus, which occupies much of the upper left-hand portion of the cell, from the cytoplasm. Note also the cell membrane (CM).
23
FIG. 3.
Two
populations
of vesicles.
PHEROMONE
PRODUCTION
IN
PSEUDALETIA
SEPARATA
25
The nuclei of the older cells were yellow-green. The nuclear colour of younger cells was obscured by the stain held in the cytoplasm. There were at least two types of vesicle in the cytoplasm of cells 0.5 days old (Fig. 3). These types differed in density, suggesting different chemical composition. Although the two populations were not homogenous, one type was more than twice the size of the other. The smaller type of vesicle was frequently found within the larger type. These vesicles were membrane-limited as the boundary between vesicles of the same density was sharp. Several vesicles appeared to be mobile. Physiology The gland appeared to move through a cycle of three phases (Fig. 4). From emergence to 1-O days, the gland was at the maximum observed size of between 900 and 1100 NI (NI = the square root of the product of two linear dimensions).
~OO~Phase,1
0
PhcyeIl
I
,
PtyseE
3
2
4
Initiation
Night
of
,
,
5
6
nucleus
no.
FIG. 4. The three phases of the secretory cycle of the paired gland. Each point represents 8 to 10 glands.
All of the cells were hypertrophied, some individual cells attaining diameters of over 1000 p. The nuclei of these cells were much larger than the nuclei of phase II
26
J. R. CLEARWATER ANDV. SARAFIS
cells
and in pa&in sections exhibited a lesser affinity for haematoxylin. Collapse of these giant cells signalled the beginning of the second phase. During this phase, which lasted approximately 1 day, the ratio of cytoplasm to nucleus ranges from 8.00 : 1 to 12.00 : 1 and the Novak index from 450 to 900. The third and final steep drop in volume occurred between day 2.5 and 3.5. By the end of this decline the disintegration of the nuclei and cell membranes was almost complete. The gland, now a formless mass, showed no further changes. The frequency distribution of the volume of all the glands measured revealed three distinct groups corresponding to the three phases postulated (see Fig. 5). Although all glands of the same age were not always in the same phase, the experimental population was reasonably synchronous. IO
g 2
Phase ICI
Phose U
Phase I
0 !
0
1000
800
Gland
Phase
600
size
(Novak
I
Nuclear lcytoplasmic
400
200
0
index)
Phase
ll
relationship
FIG. 5. The frequency distribution of the paired gland. DISCUSSION
The three phases of Stobbe’s gland appear to have functional significance. It is suggested that the large swollen gland of newly emerged adults (phase I) represents an active phase, possibly concerned with the elaboration of a pheromone or its precursor. After a period of decreased activity (phase II), the glands disintegrate (phase III).
PHEROMONE PRODUCTION IN PSEUDALETIA
SEPARATA
27
The giant cells are particularly interesting. STOBBE (1912), who had access only to material from wild populations, found only one specimen with giant cells. In the absence of further material, he could not discount a pathological condition. This reservation does not now seem important. As these cells are among the largest reported from insects, detailed examination of the mechanism of synthesis of secretion should prove particularly rewarding. Because of their extreme delicacy, these giant cells must be treated very gently. Examination of living phase I cells in physiological saline with interference microscopy revealed a layer of dense cytoplasm packed with vesicles. A large round body occupying a large portion of the cell is probably the nucleus since it stains red in acetocarmine, purple in Ehrlich’s haemotoxylin, and in phase II cells yellow-green in acridine orange. Preliminary electronmicrographs of conventionally prepared material reveal a very large central cavity and thin walls lined with microtubules. Electron micrographs of Phlogophora meticulosa prepared by BIRCH (1970) are very similar. Dissociation of the cells with EDTA or trypsin visibly altered their structure. Compared with living ones, paraflin embedded cells shrink to almost 10 per cent of their original volume. Acridine orange (AO) is a metachromatic dye, the fluorescence of which is dependent on the physical and chemical properties of the nucleic acids. Though evidence is not conclusive, it is thought that the metachromatic orange fluorescence is due to the presence of RNA while the orthochromatic green fluorescence is due to the presence of DNA (PEARSE,1968). If this interpretation is accepted, it can be seen that cells 0.5 days old have a considerable amount of cytoplasmic RNA while the only nucleic acid present in detectable amounts in older cells is DNA. Because of these variations in concentrations of cytoplasmic RNA, protein synthesis can be assumed to be proceeding at a much higher rate in the younger cells than in those 15 days old. The observation of much larger nuclei in phase I cells supports this interpretation. STEINBRECHT(1964) working on the pheromone secreting epithelia of female Bombyx mori demonstrated the presence of lipid vesicles with strong U.V.absorption at 240 mp. As synthetic trans-lo-cis-12-hexadecadien-l-01, the pheromone of this species, has an absorption of 230 ml*, STEINBRECHT(1964) considers that these vesicles contain a pheromone precursor. Also, GEROK(1950) has shown that lipid extracts of B. mmi have pheromone activity following reduction with LiAlH,. This led REGNIERand LAW (1968) to suggest that these vesicles contain an acid which is converted to the active pheromone by reduction of the carboxyl group. Because the noctuid P. separata is phylogenetically distant from the bombycid B. mori, any suggestion that benzaldehyde or its precursor has a similar origin can only be made with reservation. REFERENCES and function of the pheromone producing brush organs in male of Phlogophoru meticulosa (L) (Lepidoptera: Noctuidae). Trans. R. ent. Sot. Land.
BIRCH
M. C.
(1970)
22, 277-292.
Structure
28
J. R. CLEARWATER ANDV. SARAFIS
ELTRINGHAMH. (1925) On the abdominal brushes of certain noctuid moths. Trans. R. ent. Sac. Lond. 75, l-5. EPHRUSSIB. and BEADLEG. W. (1936) A technique of transplantation for Drosophila. Am. Nut. 70,218-225. GEROKW. (1950) Ph.D. Thesis, Eberhard Karls University, Tubingen, Germany. (From Regnio & Law, 1968.) HAPP G. M. (1968) Quinone and hydrocarbon production in the defensive glands of Eleodes longicollis and Tribolium castaneum. J. Insect Physiol. 14, 1821-1837. LEGAY J. M. (1950) Note sur l’evolution des corpora allata au tours de la vie larvaire de Bombyx mori. C. R. Sot. Biol., Paris 144, 512-513. NOVAK V. J. A. (1954) ‘The growth of the corpora allata during the post-embryonal development in insects’. Acta Sot. 2001. es1 18, 98-133. NOVAKV. J. A. (1966) Insect Hormones. Methuen, London. PEARsEA. G. E. (1968) Histochemistry, Theoretical and Applied, 3rd ed., Vol. 1. Churchill, London. PFLUGFELDER 0. (1948) Volumetrische Untersuchungen an den Corpora Allata der Honigbiene Apis mellifera. Biol. Zbl. 67, 223-241. REGNIERF. E. and LAW J. H. (1968) Insect pheromones. r. Lipid Res. 9, 541-551. ROTH L. M. and STAY B. (1958) The occurrence of paraquinones in some arthropods with emphasis on the quinone secreting tracheal glands of Diploptera punctutu (Blatteria). J. Insect Physiol. 1, 305-318. STEINBRECHTR. A. (1964) Feinstructur und Histochemie der Sexualduftdruse des Seidenspinners Bombyx mori. Z. Zellforsch. mikrosk. Anat. 64, 227-261. STOBBBR. (1912) Die abdominalen Duftorgane der mannlichen Sphingiden und Noctuiden. Zool. Jb. 32, 493-532.