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Distribution of catecholamines in the cattle tick Boophilus microplus
Comp. Biochem. Physiol., 1977, Vol. 58C, pp. 21 to 28. Perffamon Press. Printed in Great Britain
D I S T R I B U T I O N O F C A T E C H O L A M I N E S IN THE CATTLE TICK BOOPHILUS M I C R O P L U S K. C. BINNINGTON AND B. F. STONE Division of Entomology, CSIRO, Long Pocket Laboratories, lndooroopilly, Australia
(Received 21 December 1976) The distribution of monoaminergic neurones in the synganglion and peripheral nervous system of the cattle tick Boophilus microplus was studied by the Falck Hillarp histofluorescence method. 2. Fluorescent cortical cell bodies were associated with the pedal and opisthosomal ganglia and with the stomodeal pons and varicosities in the neuropile, those of the pedal ganglia being the most dense. 3. Palpal, pedal and opisthosomal nerves and branches of the palpal and pedal nerves innervating the salivary glands contained fluorescent varicosities. 4. The fluorescent material in synganglion cell bodies had the spectral characteristics known to be produced by noradrenaline/dopamine. Abstract--l.
INTRODUCTION
Catecholamines have been demonstrated in the nervous system of a number of insects (Kerkut, 1973, review) and there is evidence that they may function as both central and visceral neurotransmitters (Murdock, t971, review). Although Chou et al. (1972) failed to detect biogenic amines in adults, eggs and larvae of the cattle tick Boophilus microplus we demonstrated dopamine and/or noradrenaline in cell bodies, neuropile and peripheral nerves using the Falck-Hillarp technique (Stone et al., 1973). Dopamine and noradrenaline have since been demonstrated biochemically in the synganglion and salivary gland of B. microplus (Megaw & Robertson, 1974). The present study provides the first description of the anatomical distribution of catecholamines in B. microplus, an essential step towards understanding their role in the physiology of this important parasite. MATERIALS AND METHODS
Ticks The ticks used were partially fed or engorged female
B. microplus of the Yeerongpilly strain maintained at these laboratories for many years.
H istochemistry Whole ticks or synganglia were frozen in isopentane cooled by liquid nitrogen before being freeze-dried in a Pearse tissue dryer and treated for 1 h at 80°C with formaldehyde vapour generated from paraformaldehyde equilibrated to 65 +_ 2% relative humidity (Falck & Owman, 1965). Specimens were then embedded in Paraplast, sectioned at 10pm and mounted in liquid paraffin. Whole mounts of dissected tissues were prepared by drying the tissues on cover glasses or slides for 1 hr over phosphorus pentoxide before exposing them to formaldehyde vapour. If synganglia were prepared this way both the periganglionic sheath and the neurilemma were removed before drying, this being necessary to reduce autofluorescence. As a control, engorged ticks were injected twice with a 50/tg/g solution of the catecholamine-depleting drug reserpine, the interval between injections being 24 hr. After a further 24 hr synganglia were removed and compared
for fluorescent reaction product with those of controls injected with saline.
Observation and photooraphy Preparations were examined and photographed using a Leitz Ortholux microscope fitted with a 200W mercury vapour lamp and an Orthomat camera.
M icrospeetr ofluorometr y Excitation and emission spectra were recorded from the large fluorescent postero-dorsal cell bodies associated with the opisthosomal ganglia using smear preparations of whole synganglia mounted in liquid paraffin. A microspectrofluorometer based on a Leitz Ortholux microscope fitted with a Zeiss ultrafluar ultraviolet-transmitting objective (Stone et al., in preparation) was used. RESULTS
S yngan#lion The distribution of fluorescent cell bodies and varicosities (Fig. 1) was obtained from whole mounts (Fig. 2) and serial sections of synganglion (Fig. 3). The fluorescent cell bodies are confined to the more ventral regions of the cortex of the synganglion. Groups of fluorescent cells are associated with each of the pedal ganglia (Groups 1~,) and axons from these are directed dorsally and medially into the neuropile of the ganglion (Fig. 1). Axons from a paired group of anterior lateral cells are directed posteriorly into the stomodeal pons (Group 5) and a medial group of fluorescent cell bodies is present between the olfactory lobes adjacent to the oesophagus (Group 6). One paired group of 2-3 cell bodies (Group 7) lying posteriorly and dorsally to the 4th pedal ganglia are much ~larger (ca. 40 x 30 #m) than those of other fluorescent neurons. The axons of these cells enter the opisthosomal ganglia and can be followed into the opisthosomal nerves (Figs 4 and 5). Fluorescent varicosities are most evident in the neuropile of pedal ganglia I, II and III, less dense varicosities being present in pedal ganglia IV, in the cheliceral ganglia and in the stomodeal pons (Figs 1, 2 and 6). 21
22
K.C. B1NNINGTONAND B. F. STONE
Fig. 1. Diagrammatic lateral view of the synganglion showing the distribution of groups of fluorescent cell bodies (l 7) in the cortex and varicosities (dots) in neuropile and peripheral nerves. Abbreviations used: c. cortex; c.b. cell body; g. ch. cheliceral ganglion; g. opt. optic ganglion; 1. olf. olfactory lobe; g.p. palpal ganglion; g. pd. I-IV pedal ganglia I-IV; n. neuropile; n. op. opisthosomal nerve; n. pd IIl pedal nerve III; nu. nucleus; oes. oesophagus; p. st. stomodeal pons; s.d. salivary duct; s.a. I lII salivary acini I-III; tr. trachea; v. varicosities.
Peripheral nerves and putative effector sites Fluorescent varicosities are present in the palpal nerves, all pedal nerves, the opisthosomal nerves (Figs 1, 5 and 7) and also in branches of the palpal and pedal nerves which innervate all three acini types (I, II and III) of the salivary gland (Figs 8 and 9). Fluorescent branches of the opisthosomal nerve could be traced to the rectal region and areas of dorsal epithelium. However the final destination of these nerves could not be shown.
Effect of reserpine In the neuropile of synganglia from ticks treated with reserpine there was no discernible fluorescence
above autofluorescence (present in synganglia heated to 80°C without formaldehyde vapour) but in some synganglia weakly fluorescing cell bodies were seen (Figs 10 and 11). No varicosities were seen in peripheral nerves or organs from reserpine treated ticks.
M icrospectrofluorometr y The large opisthosomal neurons contained fluorescent material with the spectral characteristics of primary catecholamines. The uncorrected excitation and emission peaks recorded were about 418 and 480 nm respectively (Fig. 12). These were very similar to those obtained for dopamine and noradrenaline models
Fig. 2. Whole mount of synganglion with fluorescent cell bodies in the cortex and varicosities in ganglionic neuropile.
Distribution of catecholamines in the cattle tick
Fig. 3. Paraffin section of freeze-dried synganglion showing specific fluorescence in the cytoplasm of a large opisthosomal cell body (Group 7) and autoftuorescence in trachea.
Fig. 4, Whole mount of synganglion showing Group 7 cells with fluorescent axons entering the opisthosomal nerves.
23
24
K . C . BINNINGTONAND B. F. STONE
Fig. 5. Varicosities in a stretch preparation of an opisthosomal nerve.
Fig. 6. Whole mount with dense varicosities in neuropile of pedal ganglia.
Distribution of catecholamines in the cattle tick
Fig. 7. Varicosities in neuropile of pedal ganglia and in pedal nerve III.
Fig. 8. Varicosities in acinus I of salivary gland (whole mount).
25
26
K, C. BINNINGTON AND B. F. STONE
Fig. 9. Varicosities in acinus Ill of salivary gland (whole mount).
Fig. 10. Whole mount of synganglion treated in parallel with synganglia from reserpinized ticks.
Distribution of catecholamines in the cattle tick
Fig. 11. Whole mount of synganglion from a tick injected twice with reserpine and dissected 48 hr later. Compared with Fig. 10 there is a complete lack of specific fluorescence in cell bodies and neuropile.
. . . . . .
E x c i t a t i ~ Spectrum Emission Spectrum
\ I I I /
nm
Fig. 12. Excitation and emission spectra of the fluorescence from a cell body of group 7.
27
28
K. C. BINNINGTONAND B. F. STONE
using the same spectrofluorometric system and similar to those obtained by others (Ewen & Rost, 1972, review).
cal levels of catecholamines between synganglia of reserpinized and normal ticks.
DISCUSSION
Acknowled#ements--The authors thank Mr P. Atkinson for helpful discussion and Mr D. S. Fiske for assistance with illustrations.
The catecholamines demonstrated occupy three distinct anatomical areas; those of cell bodies, neuropile and peripheral nerves. The density of fluorescent varicosities in the neuropile of pedal ganglia is suggestive of a large number of central catecholaminergic synapses which could function in integration within the central nervous system. The presence of catecholamines in nerves leading to the salivary gland and rectum suggest that they act as mediators in a stomatogastric nervous system, particularly since catecholamines have been shown in salivary gland nerves of Periplaneta americana, Schistocerca 9regaria and Manduca sexta (Robertson, 1975) and in foregut nerves of Schistocerca 9regaria (Klemm, 1971, 1972). It may be that at such sites catecholamines function as neurosecretomotor transmitters (Bern, 1966; Maddrell, 1974) that is, functioning in a way intermediate between classical rapidly acting neurotransmitters such as acetylcholine and slowly acting neurosecretory hormones. Although the axons from the large posterior cells were shown to enter the opisthosomal nerves the destination of axons from other fluorescent cell bodies is unclear. The cell bodies could either supply neurosecretomotor hormone to visceral organs or neurotransmitter to interneurons whose axons innervate the synaptic varicosities of the pedal and cheliceral ganglia. In insects, apart from their stomatogastric system, areas which apparently act as motor co-ordinator centres such as the central body and corpora pedunculata as well as the optic ganglia contain varicosities of catecholamine fluorescence (references from Murdock, 1971). However in B. microplus both the optic ganglia and glomeruli, the latter being areas of presumed integrative function (Ioffe 1963; Tsvileneva, 1964), are not rich in catecholamines, the varicosities being mainly in the pedal ganglia. F r o m the work of Tsvileneva (1964) it can be inferred that pre-synaptic terminals in the pedal ganglia could belong to peripheral sensory neurones, central association neurones or central "association-motor" neurones. Our finding that reserpine depleted the catecholamines of the synganglion as well as the peripheral nerves differs from that of Megaw & Robertson (1974) who did not find a significant difference in biochemi-
REFERENCES
BERN H. A. (1966) On the production of hormones by neurons and the role of neurosecretion in neuroendocrine mechanisms. Syrup. Soc. exp. Biol. 20. 325-344. CHou T. T., BENNETTJ. & BUEDINGE. (1972) Occurrence and concentrations of biogenic amines in trematodes. J. Parasit. 58, 1098 1102. EWEN S. W. B. & ROST F. W. D. (1972) The histochemical demonstration of catecholamines and tryptamines by acid- and aldehyde-induced fluorescence: microspectrofluorometric_ characterization of the ftuorophores in models. Histochem. J. 4, 59-69. FALCK B. & OWMAN C. (1965) A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic monoamines. Acta Univ. lund. II, 7, 1 23. IOFFE I. D. (1963) Structure of the central nervous system of Dermacentor pictus Herm. (Chelicerata, Acarina). Zool. Zh. 42, 1472 1484 (CSIRO Translation). KERKUT G. A. C. (1973) Catecholamines in invertebrates. Br. reed. Bull. 29, 100-104. KLEMM N. (1971) Monoaminh~iltige Zellelemente im stomatogastrischen Nervensystem und in den Corpora cardiaca von Schistocerca #re#aria Forsk. (Insecta, Orthoptera). Z. Naturf. 26B, 1084-1086. KLEMMN. (1972) Monoamine-containing nervous fibres in foregut and salivary gland of the desert locust, Schistocerca 9re#aria Forskal (Orthoptera, Acrididae). Comp. Biochem. Physiol. 43A, 207 211. MADDRELLS. H. P. (1974) Neurosecretion. In Insect Neurobiology (Edited by TREHERNEJ. E.), pp. 305 357. NorthHolland, Amsterdam. MEGAW M. W. J. & ROBERTSONH. A. (1974) Dopamine and noradrenaline in the salivary glands and brain of the tick, Boophilus microplus: effect of reserpine. Experientia 30, 1261-1262. MURDOCK L. L. (1971) Catecholamines in arthropods: a review. Comp. 9en. Pharmac. 2, 254-274. ROBERTSON H. A. (1975) The innervation of the salivary gland of the moth, Manduca sexta: evidence that dopamine is the transmitter. J. exp. Biol. 63, 413-419. STONE B. F., ATKINSONP. W. & BINNINGTONK. C. (1973) CSIRO Division of Entomology Annual Report, 1972-73. TSVILENEVAV. A. (1964) The nervous structure of the ixodid ganglion. Zool. Jb. 81. 579 602.
D I S T R I B U T I O N O F C A T E C H O L A M I N E S IN THE CATTLE TICK BOOPHILUS M I C R O P L U S K. C. BINNINGTON AND B. F. STONE Division of Entomology, CSIRO, Long Pocket Laboratories, lndooroopilly, Australia
(Received 21 December 1976) The distribution of monoaminergic neurones in the synganglion and peripheral nervous system of the cattle tick Boophilus microplus was studied by the Falck Hillarp histofluorescence method. 2. Fluorescent cortical cell bodies were associated with the pedal and opisthosomal ganglia and with the stomodeal pons and varicosities in the neuropile, those of the pedal ganglia being the most dense. 3. Palpal, pedal and opisthosomal nerves and branches of the palpal and pedal nerves innervating the salivary glands contained fluorescent varicosities. 4. The fluorescent material in synganglion cell bodies had the spectral characteristics known to be produced by noradrenaline/dopamine. Abstract--l.
INTRODUCTION
Catecholamines have been demonstrated in the nervous system of a number of insects (Kerkut, 1973, review) and there is evidence that they may function as both central and visceral neurotransmitters (Murdock, t971, review). Although Chou et al. (1972) failed to detect biogenic amines in adults, eggs and larvae of the cattle tick Boophilus microplus we demonstrated dopamine and/or noradrenaline in cell bodies, neuropile and peripheral nerves using the Falck-Hillarp technique (Stone et al., 1973). Dopamine and noradrenaline have since been demonstrated biochemically in the synganglion and salivary gland of B. microplus (Megaw & Robertson, 1974). The present study provides the first description of the anatomical distribution of catecholamines in B. microplus, an essential step towards understanding their role in the physiology of this important parasite. MATERIALS AND METHODS
Ticks The ticks used were partially fed or engorged female
B. microplus of the Yeerongpilly strain maintained at these laboratories for many years.
H istochemistry Whole ticks or synganglia were frozen in isopentane cooled by liquid nitrogen before being freeze-dried in a Pearse tissue dryer and treated for 1 h at 80°C with formaldehyde vapour generated from paraformaldehyde equilibrated to 65 +_ 2% relative humidity (Falck & Owman, 1965). Specimens were then embedded in Paraplast, sectioned at 10pm and mounted in liquid paraffin. Whole mounts of dissected tissues were prepared by drying the tissues on cover glasses or slides for 1 hr over phosphorus pentoxide before exposing them to formaldehyde vapour. If synganglia were prepared this way both the periganglionic sheath and the neurilemma were removed before drying, this being necessary to reduce autofluorescence. As a control, engorged ticks were injected twice with a 50/tg/g solution of the catecholamine-depleting drug reserpine, the interval between injections being 24 hr. After a further 24 hr synganglia were removed and compared
for fluorescent reaction product with those of controls injected with saline.
Observation and photooraphy Preparations were examined and photographed using a Leitz Ortholux microscope fitted with a 200W mercury vapour lamp and an Orthomat camera.
M icrospeetr ofluorometr y Excitation and emission spectra were recorded from the large fluorescent postero-dorsal cell bodies associated with the opisthosomal ganglia using smear preparations of whole synganglia mounted in liquid paraffin. A microspectrofluorometer based on a Leitz Ortholux microscope fitted with a Zeiss ultrafluar ultraviolet-transmitting objective (Stone et al., in preparation) was used. RESULTS
S yngan#lion The distribution of fluorescent cell bodies and varicosities (Fig. 1) was obtained from whole mounts (Fig. 2) and serial sections of synganglion (Fig. 3). The fluorescent cell bodies are confined to the more ventral regions of the cortex of the synganglion. Groups of fluorescent cells are associated with each of the pedal ganglia (Groups 1~,) and axons from these are directed dorsally and medially into the neuropile of the ganglion (Fig. 1). Axons from a paired group of anterior lateral cells are directed posteriorly into the stomodeal pons (Group 5) and a medial group of fluorescent cell bodies is present between the olfactory lobes adjacent to the oesophagus (Group 6). One paired group of 2-3 cell bodies (Group 7) lying posteriorly and dorsally to the 4th pedal ganglia are much ~larger (ca. 40 x 30 #m) than those of other fluorescent neurons. The axons of these cells enter the opisthosomal ganglia and can be followed into the opisthosomal nerves (Figs 4 and 5). Fluorescent varicosities are most evident in the neuropile of pedal ganglia I, II and III, less dense varicosities being present in pedal ganglia IV, in the cheliceral ganglia and in the stomodeal pons (Figs 1, 2 and 6). 21
22
K.C. B1NNINGTONAND B. F. STONE
Fig. 1. Diagrammatic lateral view of the synganglion showing the distribution of groups of fluorescent cell bodies (l 7) in the cortex and varicosities (dots) in neuropile and peripheral nerves. Abbreviations used: c. cortex; c.b. cell body; g. ch. cheliceral ganglion; g. opt. optic ganglion; 1. olf. olfactory lobe; g.p. palpal ganglion; g. pd. I-IV pedal ganglia I-IV; n. neuropile; n. op. opisthosomal nerve; n. pd IIl pedal nerve III; nu. nucleus; oes. oesophagus; p. st. stomodeal pons; s.d. salivary duct; s.a. I lII salivary acini I-III; tr. trachea; v. varicosities.
Peripheral nerves and putative effector sites Fluorescent varicosities are present in the palpal nerves, all pedal nerves, the opisthosomal nerves (Figs 1, 5 and 7) and also in branches of the palpal and pedal nerves which innervate all three acini types (I, II and III) of the salivary gland (Figs 8 and 9). Fluorescent branches of the opisthosomal nerve could be traced to the rectal region and areas of dorsal epithelium. However the final destination of these nerves could not be shown.
Effect of reserpine In the neuropile of synganglia from ticks treated with reserpine there was no discernible fluorescence
above autofluorescence (present in synganglia heated to 80°C without formaldehyde vapour) but in some synganglia weakly fluorescing cell bodies were seen (Figs 10 and 11). No varicosities were seen in peripheral nerves or organs from reserpine treated ticks.
M icrospectrofluorometr y The large opisthosomal neurons contained fluorescent material with the spectral characteristics of primary catecholamines. The uncorrected excitation and emission peaks recorded were about 418 and 480 nm respectively (Fig. 12). These were very similar to those obtained for dopamine and noradrenaline models
Fig. 2. Whole mount of synganglion with fluorescent cell bodies in the cortex and varicosities in ganglionic neuropile.
Distribution of catecholamines in the cattle tick
Fig. 3. Paraffin section of freeze-dried synganglion showing specific fluorescence in the cytoplasm of a large opisthosomal cell body (Group 7) and autoftuorescence in trachea.
Fig. 4, Whole mount of synganglion showing Group 7 cells with fluorescent axons entering the opisthosomal nerves.
23
24
K . C . BINNINGTONAND B. F. STONE
Fig. 5. Varicosities in a stretch preparation of an opisthosomal nerve.
Fig. 6. Whole mount with dense varicosities in neuropile of pedal ganglia.
Distribution of catecholamines in the cattle tick
Fig. 7. Varicosities in neuropile of pedal ganglia and in pedal nerve III.
Fig. 8. Varicosities in acinus I of salivary gland (whole mount).
25
26
K, C. BINNINGTON AND B. F. STONE
Fig. 9. Varicosities in acinus Ill of salivary gland (whole mount).
Fig. 10. Whole mount of synganglion treated in parallel with synganglia from reserpinized ticks.
Distribution of catecholamines in the cattle tick
Fig. 11. Whole mount of synganglion from a tick injected twice with reserpine and dissected 48 hr later. Compared with Fig. 10 there is a complete lack of specific fluorescence in cell bodies and neuropile.
. . . . . .
E x c i t a t i ~ Spectrum Emission Spectrum
\ I I I /
nm
Fig. 12. Excitation and emission spectra of the fluorescence from a cell body of group 7.
27
28
K. C. BINNINGTONAND B. F. STONE
using the same spectrofluorometric system and similar to those obtained by others (Ewen & Rost, 1972, review).
cal levels of catecholamines between synganglia of reserpinized and normal ticks.
DISCUSSION
Acknowled#ements--The authors thank Mr P. Atkinson for helpful discussion and Mr D. S. Fiske for assistance with illustrations.
The catecholamines demonstrated occupy three distinct anatomical areas; those of cell bodies, neuropile and peripheral nerves. The density of fluorescent varicosities in the neuropile of pedal ganglia is suggestive of a large number of central catecholaminergic synapses which could function in integration within the central nervous system. The presence of catecholamines in nerves leading to the salivary gland and rectum suggest that they act as mediators in a stomatogastric nervous system, particularly since catecholamines have been shown in salivary gland nerves of Periplaneta americana, Schistocerca 9regaria and Manduca sexta (Robertson, 1975) and in foregut nerves of Schistocerca 9regaria (Klemm, 1971, 1972). It may be that at such sites catecholamines function as neurosecretomotor transmitters (Bern, 1966; Maddrell, 1974) that is, functioning in a way intermediate between classical rapidly acting neurotransmitters such as acetylcholine and slowly acting neurosecretory hormones. Although the axons from the large posterior cells were shown to enter the opisthosomal nerves the destination of axons from other fluorescent cell bodies is unclear. The cell bodies could either supply neurosecretomotor hormone to visceral organs or neurotransmitter to interneurons whose axons innervate the synaptic varicosities of the pedal and cheliceral ganglia. In insects, apart from their stomatogastric system, areas which apparently act as motor co-ordinator centres such as the central body and corpora pedunculata as well as the optic ganglia contain varicosities of catecholamine fluorescence (references from Murdock, 1971). However in B. microplus both the optic ganglia and glomeruli, the latter being areas of presumed integrative function (Ioffe 1963; Tsvileneva, 1964), are not rich in catecholamines, the varicosities being mainly in the pedal ganglia. F r o m the work of Tsvileneva (1964) it can be inferred that pre-synaptic terminals in the pedal ganglia could belong to peripheral sensory neurones, central association neurones or central "association-motor" neurones. Our finding that reserpine depleted the catecholamines of the synganglion as well as the peripheral nerves differs from that of Megaw & Robertson (1974) who did not find a significant difference in biochemi-
REFERENCES
BERN H. A. (1966) On the production of hormones by neurons and the role of neurosecretion in neuroendocrine mechanisms. Syrup. Soc. exp. Biol. 20. 325-344. CHou T. T., BENNETTJ. & BUEDINGE. (1972) Occurrence and concentrations of biogenic amines in trematodes. J. Parasit. 58, 1098 1102. EWEN S. W. B. & ROST F. W. D. (1972) The histochemical demonstration of catecholamines and tryptamines by acid- and aldehyde-induced fluorescence: microspectrofluorometric_ characterization of the ftuorophores in models. Histochem. J. 4, 59-69. FALCK B. & OWMAN C. (1965) A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic monoamines. Acta Univ. lund. II, 7, 1 23. IOFFE I. D. (1963) Structure of the central nervous system of Dermacentor pictus Herm. (Chelicerata, Acarina). Zool. Zh. 42, 1472 1484 (CSIRO Translation). KERKUT G. A. C. (1973) Catecholamines in invertebrates. Br. reed. Bull. 29, 100-104. KLEMM N. (1971) Monoaminh~iltige Zellelemente im stomatogastrischen Nervensystem und in den Corpora cardiaca von Schistocerca #re#aria Forsk. (Insecta, Orthoptera). Z. Naturf. 26B, 1084-1086. KLEMMN. (1972) Monoamine-containing nervous fibres in foregut and salivary gland of the desert locust, Schistocerca 9re#aria Forskal (Orthoptera, Acrididae). Comp. Biochem. Physiol. 43A, 207 211. MADDRELLS. H. P. (1974) Neurosecretion. In Insect Neurobiology (Edited by TREHERNEJ. E.), pp. 305 357. NorthHolland, Amsterdam. MEGAW M. W. J. & ROBERTSONH. A. (1974) Dopamine and noradrenaline in the salivary glands and brain of the tick, Boophilus microplus: effect of reserpine. Experientia 30, 1261-1262. MURDOCK L. L. (1971) Catecholamines in arthropods: a review. Comp. 9en. Pharmac. 2, 254-274. ROBERTSON H. A. (1975) The innervation of the salivary gland of the moth, Manduca sexta: evidence that dopamine is the transmitter. J. exp. Biol. 63, 413-419. STONE B. F., ATKINSONP. W. & BINNINGTONK. C. (1973) CSIRO Division of Entomology Annual Report, 1972-73. TSVILENEVAV. A. (1964) The nervous structure of the ixodid ganglion. Zool. Jb. 81. 579 602.