Nueroendocrine complex of Jamaicana flava (Caudell) (Orthoptera : Tettigoniidae)

Int. J. Insect Morphol. & Embryol. 6(1): 1-16. 1977. Pergamon Press. Printed in Great Britain.

NEUROENDOCRINE COMPLEX (CAUDELL) (ORTHOPTERA

OF JAMAICANA FLAVA • TETTIGONIIDAE)

A. PEACOCK* and J. H. ANSTEE Department of Zoology, University of Durham, Science Laboratories, South Road, Durham DH1 3LE, England

(Accepted 23 September 1976)

Abstract--The anatomy and histology of the neuroendocrine complex of adult Jamaicana tiara h~Lve been studied by means of intra-vitam injection of methylene blue followed by dissection, and by the examination of sections, cut serially, which have been stained with either paraldehyde fuchsin or chrome haematoxylin phloxine. A detailed illustration of the nerve pathways within the system is presented. The stomatogastric nervous system comprises a frontal ganglion, hypocerebral ganglion, and paired ingluvial ganglia. Nerve pathways link this system to the brain, retrocerebral glands and to the musculature of the foregut and head. Two fine nerves leave each frontal connective, FCN 1 and FCN 2. The former divides into an inner nerve, which joins with its counterpart from the opposite side, and an outer nerve which joins with a fine branch of the median nerve and supplies the dilator muscles of the cibarium and the posterior retractor muscles of the labrum. FCN 2 innervates the dorsal ,dilator muscles of the pharynx and the retractor muscles of the labrum. The median nerve leaves the anterior margin of the frontal ganglion and innervates the clypeus, epipharynx, and the anterior retractor muscles of the labrum. A single pair of pharyngeal nerves leaves the frontal ganglion and these ramify over the surface of the gut, forming a complex network. Numerous fine nerves were observed linking the recurrent nerve and the dorsal musculature of the pharynx. Three cell types were observed in the pars intercerebralis medialis of the brain, of which one was recognised as being neurosecretory. Neurosecretory 'A' material, present in these cells, was also found in the NCC 1 and the anterior storage lobe of the corpora cardiaca. Nerve fibres were observed linking the corpora cardiaca to the hypocerebral ganglion. Neurosecretory material was never observed in the hypocerebral ganglion, the recurrent nerve or the oesophageal nerves. However, stainable material was present in the large neurones of the frontal ganglion. The corpora allata are connected to the corpora cardiaca by a pair of nerves, NCA 1 and to the suboesophageal ganglion by another pair NCA 2. Neurosecretory material was nol observed in either the glands or their associated nerves. Similarities and differences between the neuroendocrine complex of Jamaicana/lava and that of other species are discussed. Index descriptors: (in addition to those in title): Stomatogastric nervous system; retrocerebral glands; anatomy; histology.

INTRODUCTION

THEREIS NOWconsiderable evidence implicating the stomatogastric nervous, neurosecretory, and retrocerebral gland systems in the control of growth and metabolism in a variety of Orthoptera. For example, interference with the stomatogastric nervous system produces a variety of effe.zts including cessation of growth and development (Clarke and Langley, 1963a, 1963b, 1963c), inhibition of egg development (Highnam, 1962; Highnam et aL, 1966; Dogra and Ewen, 1971), and reduction in protein and RNA synthesis (Clarke and Gillott, * Present address: Department of Zoology, University of Reading, Reading, England. IMAE6[1--A

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A. PEACOCKand J. H. ANSTEE

1967a, 1967b). These effects are t h o u g h t to be m e d i a t e d via the e n d o c r i n e system since interference with the integrity o f the s t o m a t o g a s t r i c nervous system reduces or prevents the release o f n e u r o s e c r e t o r y m a t e r i a l f r o m t h e p a r s i n t e r c e r e b r a l i s / c o r p o r a c a r d i a c a system (Clarke a n d Langley, 1963c; C l a r k e a n d Anstee, 1971) a n d reduces the activity o f the c o r p o r a allata ( H i g h n a m et al., 1966; Strong, 1966a; C l a r k e a n d Anstee, 1971). Whilst descriptions o f the s t o m a t o g a s t r i c nervous a n d n e u r o e n d o c r i n e systems are available for a n u m b e r o f o r t h o p t e r a n species, including Locusta migratoria (Albrecht, 1953; C l a r k e a n d Langley, 1963a, 1963c; R o o m e , 1968; C l a r k e a n d Anstee, 1971; Allure, 1973), Schistoeerca gregaria ( H i g h n a m , 1961; R o o m e , 1968; D a n d o et al., 1968), Schizodactylus monstrosus ( K h a t t e r , 1968a, 1968b) a n d Melanoplus sanguinipes ( D o g r a a n d Ewen, 1970), with the exception o f N e s b i t t (1941) a n d Cazal (1948) there is little i n f o r m a t i o n c o n c e r n i n g the m o r p h o l o g y a n d histology o f the n e u r o e n d o c r i n e c o m p l e x (i.e. the s t o m a t o g a s t r i c nervous system a n d the n e u r o s e c r e t o r y system) in tettigoniids. The present study was u n d e r t a k e n to d e t e r m i n e the a n a t o m y and histology o f the n e u r o e n d o c r i n e c o m p l e x of Jamaieanaflava (Caudell) as an essential basis for future e n d o c r i n e studies. MATERIALS AND METHODS The animals studied were sexually mature male and female J. tiara. The tettigoniids were reared in a constant-temperature room at 24°C in aluminium cages with clear Perspex fronts and sides of stiffened muslin. The background relative humidity was approximately 50%, although the daily introduction of fresh food (consisting of carrot, cabbage, banana and bran) and water into the cages may have caused some fluctuations in RH within each cage. The photoperiod was 12 hr light, 12 hr dark. However, the behaviour of the tettigoniids was such that many of the light hr were spent under sheets of corrugated cardboard on the floor of the cage. A metal tray containing moist sand and gravel was present in each cage and it was in this that the females deposited their eggs. Anatomical detail was determined by dissection aided by light microscopy; studies on the arrangement of the finer nerves were facilitated by intra vitam injection of methylene blue prior to dissection (Stark et al., 1969). Tissues used for histological examination were obtained from animals that had been killed by decapitation and immersed in Bouin's fixative. To minimise the effects that any circadian rhythms in neurosecretion might have on the appearance of the neurosecretory ceils, at various times of day, all animals were killed at the same time of day. In the case of the heads, fixation was carried out under vacuum followed by storage in fresh fixative until required. The neuroendocrine complex was then dissected out and washed in 70°g ethanol until all traces of fixative had disappeared. Tissues were embedded in paraffin wax and serial sections cut at 10 t~m. The sections were stained using either paraldehyde fuchsin (Ewen, 1962) or chrome haematoxylin phloxine (Gomori, 1941). RESULTS T h e general o r g a n i s a t i o n o f the n e u r o e n d o c r i n e c o m p l e x o f J. tiara is shown in Figs. 1 a n d 2. The s t o m a t o g a s t r i c n e r v o u s system lies on the d o r s a l surface o f the foregut a n d innervates this organ. It comprises a f r o n t a l ganglion, a h y p o c e r e b r a l ganglion, a n d p a i r e d ingluvial ganglia. N e r v e p a t h w a y s link this system to the brain. The r e t r o c e r e b r a l e n d o c r i n e system is c o m p o s e d o f the p a i r e d c o r p o r a c a r d i a c a and c o r p o r a allata.

1. The stomatogastric nervous system F i g u r e 3 shows in detail the a r r a n g e m e n t o f the f r o n t a l ganglion a n d its associated nerves. The g a n g l i o n is a p p r o x i m a t e l y p e a r - s h a p e d a n d lies along the midline, d o r s a l to the p h a r y n x , in the r e g i o n where the a l i m e n t a r y c a n a l bends ventrally to f o r m the buccal cavity. It consists o f large cells a r r a n g e d d o r s a l and lateral to a ventral neuropile (Fig. 5). Typically, the c y t o p l a s m o f these cells stain light green with the P A F technique of Ewen (1962) and p i n k / p u r p l e with t h e C H P m e t h o d o f G o m o r i (1941). Occasionally, in s o m e

Neuroendocrine Complex of J. flava PC

OL ON

NCC2 NCCI HG

CC

O

N C A ~

~

DA

FCN2 LN

NW

CA

FIG. 1. Diagram of neuroendocrine complex of J. tiara as seen from right side. Suboesophageal and ingluviai ganglia are not shown. CA: corpus allatum; CC: corpus cardiacum; COC: cut end of circumoesophageal connective; D: deutocerebrum; DA: dorsal aorta; FC: frontal connective; FCN 1 and 2: nerves 1 and 2 which leave frontal connective; FG: frontal ganglion; HG: hypocerebral ganglion; LFN: labrofrontal nerve; LN: labral nerve; MN: median nerve; NC: nervus connectivus; NCA 1 : nervus corporis allati 1 ; NCA 2: cut end of nervus corporis allati 2; NCC 1 and 2: nervi corporis cardiaci 1 and 2; NW: nerve to wall of crop; OL: optic lobe; ON: optic nerve; OPN: oesophageal nerve; PC: protocerebrum; PN: pharyngeal nerve; RN: recurrent nerve; T: tritocerebrum. JG

~

'

~

""~ MGC

OPN

~

A

NCAI NCC2

DA

RN p MN

M

PV

FIG. 2. Diag,:am of stomatogastric nervous system and retrocercbral endocrine glands as seen in dorsal view following removal of brain and circumoesophageal connectives. C: crop; IG: ingluvial ganglion; M: mouth; MGC: midgut caecum; P: pharynx; other letters as in Fig. 1.

specimens, blue/purple granules are seen in the cytoplasm after P A F staining (Fig. 5). Such granules were not observed in the axons of these cells. The frontal ganglion is enveloped by an acellular neurilemma that is continuous over the whole o f the nervous system, and a cellular perineurium. The cells and nuclei o f the perineurium are oval and flattened (Fig. 5). As in other insects several nerves were observed to leave the frontal ganglion. A very fine nerve, the nervus connectivus, leaves the mid-dorsal surface of the frontal ganglion and serves to link it to the p r o t o c e r e b r u m of the brain. Figure 6 shows a portion o f this nerve leaving the frontal ganglion. A pair o f pharyngeal nerves, one on either side, leaves f r o m the postero-lateral margin of the frontal ganglion and immediately divides into 3 finer nerves (Fig. 3, PN1, PN2, PN3). O f these, the most anterior b r a n c h (PN1) passes immediately to the musculature o f the pharynx, whilst the posterior branch (PN3) divides into 3 fine nerves (Fig. 3). O f these, the middle and anterior nerve pass to the dorsal dilator muscles o f the pharynx, whilst the posterior b r a n c h innervates the surface o f the gut (Fig. 3). The third and middle branch (PN2) of the pharyngeal nerve, runs laterally over the pharynx, giving off branches to the musculature of the foregut (Figs. 1, 3). Two large nerves, the frontal connectives, emerge f r o m the antero-lateral edge o f the frontal ganglion (Figs. 3, 7) and pass back on either side of the gut to the tritocerebral

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A. PEACOCK and J. H. ANSTEE

FIG. 3.TDiagram showing)frontal ganglion and associated nerves as seen in dorsal view. A R M L : anterior retractor muscle of labrum; D D M P : dorsal muscle of pharynx; D MC: dilator muscles of cibarium; F: frons; L: labrum; M: mandible; PN 1, 2 and 3: branches 1-3 of pharyngeal nerve; PRML: posterior retractor muscles of labrum; R M A : retractor muscle of mouth angle; Nos 1, 2, 3 and 4 refer to nerves leaving median nerve; other letters as in Figs. 1, 2.

OPN, 3',"

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i I/ ;pv x

FIo. 4. Diagram showing position of ingluvial ganglion and associated nerves as seen in dorsolateral aspect (from the right side). M G : midgut; MT: Malpighian tubules; PV: proventriculus; Nos. 1-5: nerves leaving ingluvial ganglion and innervating crop. (1), proventriculus (2), and midgut cacea (3 and 4) and midgut (5); other letters as in Figs. 1, 2, 3.

Neuroendocrine Complex of J. tiara

FI6.5. Photomicrograph showing a transverse section through frontal ganglion. Note dorsal and lateral arrangement of cell bodies (CB) around ventral neuropile (NP), and presence of stainable (PAF positive) granules (G) in cytoplasm of cells. NL: neurilemma; P: perineurium. Scale 50/~m FIG. 6. Oblique longitudinal section through frontal ganglion (FG) showing part of nervus connectivus (NC). Scale 100 ~m FIG. 7. Slightly oblique, longitudinal section through frontal ganglion (FG) showing one of frontal connectives (FC) and median nerve (MN) leaving anterior margin of frontal ganglion as well as a fine nerve (FCN 1). Scale 100/~m Fie. 8. Transverse section through posterior region of frontal ganglion (FG) in a region where recurrent nerve (RN) lies ventral to ganglion. M : muscle of pharynx. Scale 50/~m

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A. PEACOCKand J. H. ANSTEE

lobes of the brain. Just prior to entering the tritocerebrum, each frontal connective fuses with the labral nerve on that side to form a short labrofrontal trunk (Fig. 1). At the point where each frontal connective turns posteriorly towards the tritocerebrum, a pair of nerves (FCN2), one on each side, leaves the frontal connective and runs in a dorsal direction before disappearing among the fibres of the anterior and posterior retractor muscles of the labrum, as well as the retractor muscles of the mouth angle (Fig. 3). Shortly after leaving the frontal connectives, 2 fine nerves, one on either side, leave the main nerve (FCN2) and run ventrally to innervate the dorsal dilator muscles of the pharynx (Fig. 3). The labral nerve emerges from the labrofrontal root and passes ventrally on either side of the foregut and innervates the tissues of the labrum (Fig. 1). On either side, the labral nerves give off branches that ramify extensively throughout the tissues of the lower clypeus (Fig. 3). A fine pair of nerves leaves, one on either side, from the anterior region of each frontal connective (Figs. 3, 7, (FCNI)). Each nerve travels a short distance along the dorsal surface of the pharynx before dividing into an inner and outer nerve (Fig. 3). The inner nerve joins with its counterpart from the opposite side to form a half-circle arrangement over the dorsal surface of the pharynx (Fig. 3). The outer nerve runs anteriorly over the surface of the pharynx and joins with a fine branch of the median nerve (Fig. 3). The outer nerve also supplies branches to the dilator muscles of the cibarium, as well as the posterior retractor muscles of the labrum (Fig. 3, D M C and PRML). The median nerve, referred to above, emerges from the anterior region of the frontal ganglion in the middle (Figs. 3, 7). This nerve runs along the dorsal surface of the pharynx and cibarium giving off 6 branches that innervate the tissues of these regions. The first branch, a single nerve (Fig. 3 (1)), leaves the main nerve and divides into 2 finer nerves both branches of which innervate the anterior retractor muscles. Anteriorly, in the clypeus, a very fine pair of nerves (Fig. 3(2)), next leaves the median nerve and joins up, on either side, with the fine outer branch of the frontal connective nerve F C N I . The fourth, a single nerve, leaves shortly after the latter pair, and innervates the tissues of the clypeus (Fig. 3(3)). Finally, a fine pair of nerves (Fig. 3(4)) leaves the median nerve and innervates the basal region of the anterior retractor muscles of the labrum. The median nerve continues anteriorly and eventually anastomoses with the tissues of the lower clypeus and labrum (Fig. 3). A recurrent nerve leaves the posterior ventral surface of the frontal ganglion and passes back along the mid-dorsal line of the foregut to the hypocerebral ganglion (Figs. 1, 8). Several nerves leave the recurrent nerve and pass to the tunica muscularis of the pharynx. One such nerve is shown in Fig. 9. The hypocerebral ganglion lies posterior to the brain along the midline of the oesophagus (Fig. 1). The recurrent nerve enters the ganglion at its anterior end and a single pair of oesophageal nerves emerge from the posterior margin (Fig. 1). The latter nerves run posteriorly over the surface of the crop to the proventriculus where they join the paired ingluvial ganglia (Fig. 2). The short connectives, one of which is shown in Fig. 10, leave the anterolateral regions of the ganglion and link them to the overlying corpora cardiaca. A pair of fine nerves leaves the ventral surface of the ganglion and passes to the dorsal musculature of the foregut. One of these nerves is shown in Fig. 10. In transverse section, the hypocerebral ganglion consists of a central neuropile, surrounded by large cell bodies of the neurones (Fig. 10). Neurosecretory material was never observed in the cells or the axons of the hypocerebral ganglion, nor in the axons of the recurrent and oesophageal nerves.

Neuroendocrine Complex of J. flm'a

I I

If

7

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FIG. 9. Transverse section through recurrent nerve (RN) to show one of numerous fine nerves (N) which leave it. Scale 50 t~m FIG. 10. Transverse section through hypocerebral ganglion (HG) showing one of short connectives (C) that link it to corpora cardiaca (CC). A: aorta; M: muscle of pharynx. Scale 50 t*m FIG. 11. Section through equator of an ingluvial ganglion. Note central neuropile (NP) surrounded by cell bodies (CB). NL: neurilemma. Scale 50 t~m FIG. 12. Section through equator of a corpus allatum. N: nerve axons; CB: cell bodies. Scale 50 t~m FIG. 13. Section through a corpus allatum in a region where nervus corporis allati 1 (NCA) leaves gland. Scale 50 t*m

The ingluvial ganglia are a pair of small bodies that lie o n either side of the crop in the region of the proventriculus (Figs. 2, 4). Each g a n g l i o n consists of a small n u m b e r of large n e u r o n e s which are devoid of neurosecretory material (Fig. 11). F o u r nerves leave each g a n g l i o n a n d innervate the posterior regions of the crop, proventriculus a n d m i d g u t caeca (Fig. 4, nerves 1, 2, 3, 4 respectively). O n e of the nerves (3) supplying the m i d g u t caeca divides into 4, of which one pair (5), innervates the m i d g u t (Fig. 4).

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A. PEACOCKand J. H. ANSTEE

2. The neurosecretory and retrocerebral endocrine system Neurones that show cytological evidence of secretion are present in the pars intercerebralis of the protocerebral lobes of the brain, where they are located in 2 groups on either side of the midline (Fig. 14). Three types of cells can be identified in each group on the basis of size and staining reaction (Fig. 14). A large proportion of the cells stain deep purple with PAF and blue/black with CHP (Fig. 14, (1), (la)). Whether they are the same cells was undetermined, although Highnam (1961) reported this to be the case in Schistocerca gregaria. In J. tiara the amount of stainable material varied both in the perikarya and the axons of these cells. Where the cells are large, the inclusions are also large and completely fill the cells (Fig. 14, (1)). In others the amount of stainable material is small with the inclusions in particulate form, and evenly dispersed throughout the cytoplasm (Fig. 14 (la)). Both these types of cells are similar in their staining reactions to the 'A' type neurosecretory cells described for Schistocerca gregaria (Highnam, 1961) and Locusta migratoria (Clarke and Langley, 1963c). Another type of cell, smaller and less frequently encountered, intermingles with those above (Fig. 14 (2)). The cytoplasm of these cells stains bluish green with PAF and pink/red with CHP. Stainable material was never observed in these cells. There is also a third type of cell which is found near the periphery of each cell group. These are larger and fewer in number than either of those described above, and their cytoplasm stains pale green with PAF and pink with CHP (Fig. 14 (3)). These cells resemble the 'C' cell type of Schistocerca gregaria (Highnam, 1961) and Locusta migratoria (Clarke, 1966). In addition to the median neurosecretory cells of the pars intercerebralis, referred to above, another group of cells is distinguishable in each half of the brain. These cells lie posterior and lateral to the medial groups. Stainable material was most easily observed in the cytoplasm of these cells after PAF staining (Fig. 16). The material was in the form of small particles and although evenly distributed throughout the cytoplasm, was not observed in the axons of these cells. Hence their path through the brain remained undetermined. This is in marked contrast to the axons of the medial neurosecretory cells which can be followed within the brain owing to the presence of stainable material within them (Fig. 23). As is shown in Fig. 20, the axons from each cell group converge to form 2 nerve tracts. These run in a posterior and ventral direction keeping almost parallel with the anterior surface of the protocerebrum, before crossing one another, so that the axons from the lefthand group of neurosecretory cells, emerge from the posterior and ventral surface of the protocerebrum as the right nervus corporis cardiaci (NCC l) and vice versa (Fig. 17). The NCC 1 ensheathed by extensions of the neurilemma of the brain, proceed to the dorsal medial surface of the anterior regions of the corpora cardiaca. The stainable material within these tracts and the NCC 1, is identical in its staining properaties with the material in the neurosecretory 'A' cells of the pars intercerebralis. Within the brain, there is a region, ventral to each group of medial neurosecretory cells in which stainable material is prominent (Fig. 15). This region may correspond to the point where, after leaving the cells, the axons converge to form the tracts. The corpora cardiaca are a pair of white, transluscent structures that lie posterior to the brain and overlie the hypocerebral ganglion (Figs. 1, 2). The dorsal aorta passes between the 2 glands, such that the walls of the corpora cardiaca are effectively those of the aorta (Figs. 18, 21). The 2 corpora cardiaca are separated ventrally and dorsally except for a short region of contact mid-dorsally (Fig. 21). As with other Orthoptera which have been described (Highnam, 1961), the corpora cardiaca are composed of 2 histologically distinct regions. The major portion of the gland consists of axons of the NCC 1, cells with flattened

Neuroendocrine Complex of J. flava

FIG. 14. Section through pars intercerebralis of brain to show 2 groups of neurosecretory cells on either side of midline. Numbers 1-3 refer to types of cell observed in this region. Scale 50 t~m FIG. 15. Photomicrograph similar to Fig. 14 except that neurosecretory material (NSM) can be seen in proximal parts of nerve tracts. Scale 50 t~m FiG. 16. Section through protocerebrum of brain to show lateral group of neurosecretory cells (LC). Scale 100 ~m FI6. 17. Transverse section through ventral part of protocerebrurn (P) showing one of nervi corporis cardiaci 1 (NCC 1) emerging from brain. Scale 50 t~m

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FIG. 18. Low magnification photomicrograph showing position of medial neurosecretory cells (NSC) of protocerebrum (P) in relation to corpora cardiaca (CC) which contain neurosecretory 'A' material (NSM), A: aorta; CA: corpus allatum; G: gut; SC: cells of corpora cardiaca which do not possess neurosecretory 'A' material. Scale 100/zm FIG. 19. Oblique transverse section through oesophageal region showing nervi corporis cardiaci (NCC 2) dorsal and lateral to nervi corporis cardiaci 1 (NCC 1). RN: recurrent nerve. Scale 100 ~zm FIG. 20. Section through protocerebrum of brain to show chiasma (C) of axon tracts of the NCC 1 within brain. Scale 100 t~m Fie. 21. Transverse section through middle of corpora cardiaca (CC). A: aorta; HG: hypocerebral ganglion. Scale 100 tzm FIG. 22. Section through corpora cardiaca to show histologically distinct regions within gland. A: aorta; NSM: neurosecretory material; SC: cells in which stainable (PAF positive) material is absent. Scale 50 ~m FIG. 23. Transverse section through ventral part of protocerebrum showing neurosecretory material

Neuroendocrine Complex of J. tiara

11

or spherical nuclei and stainable neurosecretory 'A' material that has been transported from the brain (Fig. 21). In contrast stainable material is absent from the posterior/ventral regions of the gland (Fig. 22). In addition to the NCC 1, another finer pair of nerves emerges from the mid posterior surface of the protocerebrum, dorsal and lateral to the NCC 1, and joins the corpora cardiaca on their anterior surfaces (Figs. 1, 19). These nerves are the nervi corporis cardiaca 2 (NCC 2). As already described above, the corpora cardiaca are connected to the hypocerebral ganglion by 2 short, thick connectives (Fig. 10). A fine pair of nerves, the nervi corporis allati 1 (Fig. 1 (NCA 1)), connects the corpora cardiaca to the corpora allata. These nerves run parallel with the corpora cardiaca for a short distance before turning ventrally to unite with the corpora allata. The latter are a pair of small, white sub-spheres, located one on either side of the lateral line of the oesophagus and posterior to the circumoesophageal commissure (Figs. 1, 2). In addition to the NCA 1 which connects them to the corpus cardiacum they are connected by fine nerves to the suboesophageal ganglion (NCA 2) and the muscles of the crop wall. The histological appearance of a corpus allatum is shown in Fig. 12. Sections through the equator of these bodies show a small central region of axons, surrounded by the cells of the corpus allatum (Fig. 12). The nuclei of these cells are large and irregular in shape. The axons of the NCA 1 can be followed into the central regions of the corpora allata (Fig. 13). Stainable material was never observed in the axons of these nerw~s nor in the cells of the corpora allata. DISCUSSION The stomatogastric nervous system of J. _[lava is similar in general organisation to the descriptions given for other Orthoptera (Nesbitt, 1941; Willey, 1961; Clarke and Langley, 1963a, 1963b; Khatter, 1968a; Allum, 1973). Morphologically and histologically, the frontal ganglion of J.flava very closely resembles its counterpart in Periplaneta americana (Willey, 1961), Locusta migratoria (Clarke and Langley, 1963a) and Melancplus sanguinipes (Dogra and Ewen, 1970). Clarke and Langley (1963b) described neurones in the frontal ganglion of Loeusta whose cell bodies stain blueblack with chrome haematoxylin. However, they did not consider these to be neurosecretory, since discrete granules were not observed in the cytoplasm (Clarke, personal communication). On the other hand, Van der Kloot (1960) found evidence of neurosecretory material at the light microscope level in the frontal ganglion of Bombyx mori, whilst Anstee (1968) and Cazal et aL (1971) have observed neurosecretory material in the frontal ganglion of Locusta migratoria at the electron microscope level. The presence of discrete PAF-positive granules in the cells of the frontal ganglion of J. flat'a suggests that neurosecretory material is present here also. Fine nerves have been observed to leave the frontal connectives in several insect species including Naucoris cimicoides (Cazal, 1948), Periplaneta americana (Willey, 1961), Schizodactylus monstrosus (Khatter, 1968a), Blabera fusca (Brousse-Gaury, 1971) and Locusta migratoria (Roome, 1968; Allure, 1973). In Locusta, 3 fine nerves leave each frontal connective in the region where the latter nerves turn back towards the tritocerebrum (Allure, 1973). These fine nerves form a complex system whose various branches innervate the muscles of the cibarium, pharynx, and labrum. One of these fine nerves (FCN 1 of Allure, 1973) has a branch that unites with the anterior and posterior pharyngeal nerves. In contrast to Locu~ta, a single nerve (FCN 2) leaves from this region of the frontal connective of J. tiara. Indeed, the situation in J. tiara more closely resembles that found in Periplaneta

12

A. PEACOCKand J. H. ANSTEE

americana (Willey, 1961) where a single nerve leaves each frontalconnective and innervates the dilator muscles of the pharynx as well as the retractor muscles of the labrum. The fine nerves (FCN 1), arising just in front of the point of origin of the frontal connectives, have also been described for other species. Cazal (1948) described a similar pair of nerves in the Dermaptera whilst Willey (1961) observed 2 nerves leaving each frontal connective in Periplaneta americana. On the other hand, Allure (1973) reports a fine nerve leaving this region in Locusta migratoria, and noted that it either rejoins the frontal connective lower down or passes to the musculature of the pharynx. The median nerve that leaves the anterior margin of the frontal ganglion of Jamaicana tiara has also been reported for other insects (Imms, 1957; Willey, 1961; Clarke and Langley, 1963b). The present work, together with the studies of Allure (1973) confirms the earlier suggestion of Nesbitt (1941) that this nerve innervates the clypeus and epipharynx. In J. tiara it also supplies branches to the anterior retractor muscles of the labrum as well as forming part of a nerve complex with the fine nerves (FCN 1) that leave the frontal connectives. The ramifications of the median nerve with other nerves of this region have been reported for Locusta migratoria (Allure, 1973) and observed occasionally in Periplaneta americana (Willey, 1961). Fine nerves have been reported leaving the frontal ganglion between the frontal connective and recurrent nerve in Carausius morosus (Dupont-Raabe, 1957), Periplaneta americana (Willey, 1961; Davey and Treherne, 1963), Schizodactylus monstrosus (Khatter, 1968a) and Blaberafusca (Brousse-Gaury, 1971). Clarke and Langley (1963b) first described the anterior and posterior pharyngeal nerves in Loeusta migratoria, whilst Roome (1968) and later Allure (1973), observed a third pair, the median pharyngeal nerves, leaving the ganglion between the anterior and posterior pairs. Only a single pair of pharyngeal nerves was observed in J. tiara and these ramify over the surface of the gut forming a complex network. Serial sections through the foregut region revealed fine nerves linking the recurrent nerve and the dorsal musculature of the pharynx and the latter with the hypocerebral ganglion. In Periplaneta amerieana (Willey, 1961), Schizodactylus monstrosus (Katter, 1968a) and A ctias (Roome, 1968) branches of the recurrent nerve also innervate the muscle coat of the pharynx and oesophagus whilst in Dytiscus marginalis (Raabe, 1963) fine nerves leave the recurrent nerve and innervate the lateral dilator muscles of the pharynx. In Gryllus, Aeschna and Carausius (Raabe, 1963) similar branches unite with NCC 2, whereas in Loeusta migratoria (Allum, 1973) fine nerves leave the recurrent nerve and unite with branches of the posterior pharyngeal nerves before innervating the tunica muscularis of the pharynx and oesophagus. The fine nerves leaving the hypocerebral ganglion of J. tiara and passing to the surface of the oesophagus, have also been reported for Dixippus morosus (Nyst, 1942), Periplaneta (Willey, 1961), Schizodactylus monstrosus (Khatter, 1968a)and Locusta migratoria (Allum, 1973). In Grylloblata campodeiformis, Nesbitt (1956) observed nerve fibres passing from the hypocerebral ganglion and innervating the aorta. The nervus connectivus that links the frontal ganglion to the brain, was first named by Baldus (1924) in the dragon fly, Aeschna. Cazal (1948) reviews the occurrence of this nerve through the insects and finds it is present in most primitive orders, whilst Willey (1961) found it in all Orthoptera except the Saltatoria. In J. tiara, as in other insects, stainable material was most abundant in the neurones of the pars intercerebralis medialis of the protocerebrum and in the corpora cardiaca (Highnam 1961; Clarke and Langley, 1963c; Thomsen, 1965; Dogra and Ewen, 1970). Although 3

Neuroendocrine Complex of J. tiara

13

types of cells were observed in this region of the brain of J. tiara, only one type is recognised as neurosecretory and is similar to the classical Type 'A' cell of Hagadorn (1958) and Highnam (1961). The other 2 types of cell are not considered to be neurosecretory, since granules were never seen in the cytoplasm or axons of these cells. Whilst these findings are similar to th,3se reported for Calliphora erythrocephala (Thomsen, 1965) and Melanoplus sanguinipes (Dogra and Ewen, 1970), they differ from those of Oncopeltus fasciatus (Johannson, 1958), Sehistocerea gregaria (Highnam, 1961) and Locusta migratoria (Clarke, 1966). In these species, the type of cell in this region of the brain varies, being 4 in Oneopeltus (Johannson, 1958) and Schistocerca (Highnam, 1961) and 3 in Locusta (Clarke, 1966) of which the 'A' and 'B' types of cell are recognised by these authors as being neurosecretory. The 'B' cells ihave been thought to be stages in the secretory cycle of the 'A' cells (Thomsen, 1954), although Johannson (1958) and Highnam (1961) present evidence favouring the view that the 'A' and 'B' cells are distinct cell types. On the other hand, Dupont-Raabe (1956) has shown that the choice of fixative and degree of overstaining can influence the end product of neurosecretory staining methods, whilst Scharrer and Brown (1962) report that electron microscopic observations suggested that the different stainability of neurosecretory ce[{s in the earthworm Lumbricus terrestris indicated only functional states of one cell type. More recently Steel and Harmsen (1971) have provided a very significant contribution to the interpretation of the various neurosecretory cell types. From their studies on Rhodnius they propose a compromise between the two views expressed above. It is suggested that whilst interconversions are possible among the five cell types they observed they are clearly not all stages in the activity of a single cell type because two distinct cyclical processes exist in the system. However, some of the cell types are common to both processes, and ia such cells a given cytological appearance may be associated with different relative proportions of synthesis and release at different stages of the life cycle. In J./lava aggregations of stainable product were observed in the proximal regions of the NCC 1, where the axons of the neurosecretory cells converge to form the tracts. Similar deposits were reported for Oneopeltus fasciatus (Johannson, 1958), Melanoplus sanguinipes (Dogra and Ewen, 1970) and Locusta migratoria (Highnam and West, 1971). The latter authors describe this region as being a neuropilar neurosecretory reservoir. However, Mason (1973) found little evidence of stores or reservoirs of such material in Schistocerca

vaga. As has already been stated, the corpora cardiaca are structurally similar to their counterparts in Locusta migratoria and Schistocerca gregaria (Highnam, 1961) and Melanoplus sanguinipes (Dogra and Ewen, 1970) in that they are histologically divisible into two regions. Mason (1973) has shown, with Schistocerca vaga, that the fibres of the NCC 1 end mainly in the anterior region of the corpora cardiaca. It is this region of the corpora cardiaca which serves for the storage of neurosecretory material from the brain. This is most probably true of J. tiara, since material with similar staining properties was observed in the cells of the protocerebrum, NCC I and this region of the corpora cardiaca. The posterior/ventral parts of the gland, where stainable material was absent, may produce a secretion of its own, as has been suggested for other species (Highnam, 1961 ; Mordue and Goldsworthy, 1969). In the pre.,;ent work, the connection between the corpora cardiaca and the hypocerebral ganglion, whilst present, was not easily observed. The fact that such a connection does exist is of pa~:ticular interest in view of the findings of Strong (1966b) and Mason (1973) for Locusta migratoria and Schistocerca vaga respectively. Both authors observed axons of the

14

A. PEACOCKand J. H. ANSTEE

N C C 1 passing via the c o r p o r a c a r d i a c a into the h y p o c e r e b r a l ganglion. Indeed, M a s o n (1973) was able to follow these same axons into the o u t e r o e s o p h a g e a l nerves. Stainable m a t e r i a l has been r e p o r t e d in the o e s o p h a g e a l nerves o f Melanoplus sanguinipes ( D o g r a a n d Ewen, 1970) a n d Calliphora erythrocephala (Thomsen, 1969). In b o t h cases this has been t h o u g h t to originate in the similarly stainable m e d i a l cells o f the brain. A s M a s o n (1973) p o i n t s out, this p a t h w a y is relevant to the suggestion t h a t the c o r p o r a c a r d i a c a are involved in regulating intestinal peristalsis in Locusta (Cazal, 1969) a n d in controlling intestinal p r o t e i n a s e activity in the m i d g u t o f Calliphora ( T h o m s e n a n d Moiler, 1959). The absence o f n e u r o s e c r e t o r y m a t e r i a l f r o m the o e s o p h a g e a l nerves o f J. tiara, in the present study, need n o t necessarily imply t h a t a similar p a t h w a y does not exist in this species. Clearly, the a p p l i c a t i o n o f the techniques o f a x o n a l i o n t o p h o r e s i s and c o b a l t sulphide precipitation, e m p l o y e d b y M a s o n (1973), w o u l d d e t e r m i n e whether this was the case. The c o r p o r a allata o f J . / l a v a are generally similar, histologically, to the c o r p o r a allata o f o t h e r O r t h o p t e r a (Mendes, 1948; O d h i a m b o , 1966; Joly et al., 1968; D o g r a a n d Ewen, 1970). N e u r o s e c r e t o r y material, at least, at the light m i c r o s c o p e level, was never o b s e r v e d in the cells o f the c o r p o r a allata o r along the axons o f the N C A l, a l t h o u g h o t h e r a u t h o r s have r e p o r t e d stainable m a t e r i a l in these regions o f other species ( H i g h n a m , 1961 ; Scharrer, 1964). M a s o n (1973) failed to d e m o n s t r a t e the presence o f nerve fibres, in the N C A l, originating f r o m either the N C C 1 o r the medial cells o f the b r a i n in Schistocerca vaga. She did, however, find t h a t fibres o f the lateral b r a i n cells, c a r r i e d in the N C C 2, did travel to the c o r p o r a a l l a t a ; a n o b s e r v a t i o n which is in a c c o r d with reports o f the c o n t r o l o f the c o r p o r a allata by the l a t e r a l cells (Strong, 1965a, 1965b). Finally, in J. tiara, as in Locusta (Chalaye, 1967) a n d Schistoeerca vaga ( M a s o n , 1973) the N C A 2 connects the c o r p u s a l l a t u m to the s u b o e s o p h a g e a l ganglion. I n the latter species, it is clear t h a t the axons o f the N C A 2 originate f r o m cell g r o u p s in the ganglion, a fact which presents the possibility o f an a d d i t i o n a l p a t h w a y whereby c o n t r o l m a y be exercised on the c o r p o r a allata.

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CLARKE, K. U. and C. GILLOT. 1967b. Studies on the effects of the removal of the frontal ganglion in Locusta migrataria L. II Ribonucleic acid synthesis. J. Exp. Biol. 46: 27-34. CLARKE, K. U. and P. A. LANGLEY. 1963a. Studies on the initiation of growth and moulting in Locusta migratoria migratorioides R. and F. II. The role of the stomatogastric nervous sytem. J. Insect Physio!. 9: 363-73. CLARKE, K. U. and P. A. LANGLEY. 1963b. Studies on the initiation of growth and moulting in Locusta migratoria migratorioides R. and F. III. The role of the frontal ganglion. J. Insect Physiol. 9: 411-21. CLARKE, K. U. and P. A. LANGLEY. 1963C. Studies on the initiation of growth and moulting in Locusta migratoria migratorioides R. and F. IV. The relationship between the stomatogastric nervous system and neurosecretion. J. Insect Physiol. 9: 423-30. DANDO, J., B. CHANUSSOT and M. R. DANOO. 1968. Le syst+me nerveux stomod~al post c~phalique de Schistocerca gregaria Forsk. Orthopt6re et Blabera craniifer Burro. C.R. Acad. Sci. Paris (19) 267: 1852-.';5. DAVEY, K. G. and J. E. TREHERNE. 1963. Studies on crop function in the cockroach (Periplaneta americana (L)). IL The nervous control of crop emptying. J. Exp. Biol. 40: 775-80. DOGRA, G. S, and A. B. EWEN. 1970. Histology of the neurosecretory system and the retrocerebral endocrine glands of the adult migratory grasshopper Melanoplus sanguinipes (Fab) Orthoptera Acrididae. J. MotThol. 130: 451-66. DOGRA, G. S. and A. B. EWEN. 1971. Effects of severence of stomatogastric nerves on egg laying, feeding and the neuroendocrine system in Melanoplus sanguinipes. J. Insect Physiol. 17: 483-89. DUPONT-RAABE, M. 1956. Quelques donn6es relatives aux ph6nom6nes de neuros6cr6tion chez les phasmides. Ann. Sci. Nat. (Zool). 18: 293. DtJPONT-RAABE, M. 1957. Les m6canismes de l'adaptation chromatique chez les insectes. Arch. Zool. Exp. GOn. 94" 61-294. EWEN, A. B. 1962. An improved aldehyde fuchsin staining technique for neurosecretory products in insects. Trans. Amer. Microsc. Soc. 81: 94-6. GOMORI, G. 19'll. Observations with differential stains on human islet of Langerhans. Amer. J. Pathol. 17: 395-406. HAGADORN, 1. [~. 1958. Neurosecretion and the brain of the rhynchobdellid leech Theromyzon rude (Baird 1969). J. Morphol. 102: 55-91. HtGHNAM, K. C. 1961. The histology of the neurosecretory system of the adult female desert locust Schistocerca gregaria. Quart. J. Microsc. Sci. 102: 27-38. HIGHNAM, K. C. 1962. Neurosecretory control of ovarian development in the desert locust. Mere. Soc. Endocrinol. 12:379 90. HIGHNAM, K. C., L. HILL and W. MORDUE. 1966. The endocrine system and oocyte growth in Schistocerca in relation to starvation and frontal ganglionectomy. J. Insect Physiol. 12: 977-94. HIGHNAM, K. C. and M. WEST. 1971. The neuropilar neurosecretory reservoir of Locusta migratoria migratorioides R. & F. Gen. Comp. Endocrinol. 16: 574-85. IMMS, A. D. 1957. Textbook o f Entomology (Revised Richards O. W. and Davies R. G. ) Methuen, London. JOHANNSON, A. S. 1958. Relation of nutrition to endocrine reproductive functions in the milkweed bug ( Onocgpeltus f asciatus Dallas Heteroptera: Lygaeidae). Nytt Mag. Zool. 71-132. JoLY, L., P. JOLY, A. PORTE and A. GIRARDIE. 1968. l~tude physiologique et ultra structurale des corpora allata de Locusta migratoria L. (Orthopt6re) en phase gregaria. Arch. Zool. Exp. Gen. 109: 703-28. KHATTER, N. 1968a. The stomodaeal nervous system of Schizodactylus monstrosus (Drury) (Orthoptera). J. Nat. Hist. 2: 221-30. KHATTER, N. 1968b. The neurosecretory system of Schizodactyh~s monstrosus (Drury) (Orthoptera). Bull. Soc. Zool. Fr. 91: 225-234. MASON, C. A. 1973. New features of the brain retrocerebral neuroendocrine complex of the locust Schistocerca vaga (Scudder). Z. Zellforsch. Mikrosk. Anat. 141 : 19-32. MENDES, M. V. 1948. Histology of the corpora allata of Melanoplus differentialis (Orth. Saltatoria). Biol. Bull. (Woods Hole) 94: 194-207. MORDUE, W. ~nd G. J. GOLDSWORTHY. 1969. The physiological effects of corpus cardiacum extracts in locusts. Gen. Comp. Endocrinol. 12: 360-69. INIESBITT,H. H. 1941. A comparative morphological study of the nervous system of the Orthoptera and relatecl orders. Ann. Entomol. Soc. Amer. 34: 51-81. NESBITT, H. H. J. 1956. Contributions to the anatomy of Grylloblatta campodeiformis Walker 6. The nervous system. Proc. lOth Int. Congr. Entomol. 1 : 525-29. NYST, R. H. 1942. Structure et rapports du syst6me nerveux du vaisseau dorsal et des annexes cardiaques chez. Dixippus morosus. Br Ann. Soc. Roy. 2ool. Belg. 73: 150-64. ODHIAMBO,T. R. 1966. The fine structure of the corpus allatum of the sexually mature male of the desert locust. J. Insect Physiol. 12: 819-28. RAABE, M. 1963. Existence chez difers Insectes d'une innervation trioc6r6brale des corpora cardiaca. C.R. Acad. Sci. Paris 257: 1552-55.

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ROOME, R. E. 1968. The function of the stomatogastric nervous system as a link between feeding, endocrine secretion and growth in insects. Ph.D. Thesis, University of Nottingham. SCHARRER, B. 1964. Histophysiological studies on the corpus allatum of Leucophaea maderae. IV. Ultrastructure during normal activity cycle. Z. Zellforsch Mikrosk. Anat. 62: 125-48. SCHARRER, E. and S. BROWN. 1962. Electron microscope studies on secretory cells in Lumbricus terrestris. Mam. Soc. Endocrinol. 12: 103-08. STARK, M. J., K. N. SMALLEYand E. C. ROWE. 1969. Methylene blue staining of axons in the ventral nerve cord of insects. Stain Technol. 44: 97-102. STEELE, C. G. and R. HARMSEN. 1971. Dynamics of the neurosecretory system of the brain of an insect, Rhodnius prolixus, during growth and moulting. Gen. Comp. Endocrinol. 17: 125-41. STRONG, L. 1965a. The relationship between the brain, corpora allata, and oocyte growth in the central american locust Schistocerca sp. I. The cerebral neurosecretory system, the corpora allata, and oocyte growth. J. Insect Physiol. 11 : 135-46. STRONG, L. 1965b. The relationship between the brain, corpora allata, and occyte growth in the central american locust, Schistocerca sp II. The innervation of the corpora allata, the lateral neurosecretory complex, and oocyte growth. J. Insect Physiol. 11: 271-80. STRONG, L. 1966a. Effect of removal of frontal ganglion on corpus allatum function in Locusta migratoria migratorioides R. and F. Nature (Lond.) 210: 330-31. STRONG, L. 1966b. On the occurrence of neuroglandular axons within the sympathetic nervous system of a locust Loeusta migratoria migratorioides. J.R. Microsc. Soc. 86: 141-49. THOMSEN, E. and I. MOLLER. 1959. Neurosecretion and intestinal proteinase activity in an insect Calliphora erythrocephala Meig. Nature (Lond.) 183: 1401q)2. THOMSEN, M. 1954. Neurosecretion in some Hymenoptera. Dan. Biol. Skr. 7: 1-24. THOMSEN, M. 1965. The neurosecretory system of the adult Calliphora erythrocephala. II. Histology of the neurosecretory cells of the brain and some related structures. Z. Zellforsch. 67: 693-717. THOMSEN, M. 1969. The neurosecretory system of the adult Calliphora erythrocephala. IV. A histological study of the corpus cardiacum and its connections with the nervous system. Z. Zellforsch. Mikrostk. Anat. 94: 205-19. VAN DER KLOOT, W. G. 1960. Neurosecretion in insects. Annu. Rev. Entomol. 5: 35-52. WILLEY, B. 1961. The morphology of the stomodeal nervous system in Periplaneta americana (L) and other Blattaria. J. Morphol. 108: 219-61.