Corpus allatum regulation during the metamorphosis of Periplaneta americana: Axon pathways

J. Insect Pllysiol.,

1977. Vol. 23. pp. 975 to 984. Peryamon Press. Printed in Great Britain

CORPUS ALLATUM REGULATION DURING THE METAMORPHOSIS OF PERIPLANETA AMERICANA: AXON PATHWAYS JACK FRASER and RUDOLPH PIPA* Division

of Entomology and Parasitology. University Berkeley, California 94720, U.S.A. (Receiwti 25 Frhrumy

of California.

1977)

Abstract--The anatomy of the retrocerebral complex was studied after supravital staining with methylene blue. and axonal tracts within the corpora allata (CA) were traced after applying the CoClz technique together with Timm’s sulfide-silver enhancement. Cobalt chloride fills of the nerves to and from the CA revealed two major sources of innervation: the brain and the subesophageal ganglion. Three cell clusters in the brain contribute axons that reach each nervus corporis allati I (NCA I) and, apparently, pass to or beyond the CA. These are: a cluster of 8 to 12 cells in the contralateral pars lateralis. a cluster of 16 to 20 cells in the ipsilateral pars lateralis. and a cluster of 50 to 60 cells in the contralateral pars intercerebralis. PAF-stained sections of other brains revealed a corresponding number of PAF-positive cells in these same regions. The medial and lateral neurons arborize in the neuropile adjacent to the pars intercerebralis. and may associate there. The lateral group also arborizes extensively in the neuropile surrounding the pedunculus of the mushroom body. At least four cell bodies located antero-ventrad in the subesophageal ganglion send axons to the CA via each nervus corporis allati II (NCA II). To determine possible inhibitory pathways to the CA, the NCA I. NCA II, and postallatal nerves of last instar larvae were severed: either singly. or in combination. Additional experiments were performed on last instar larvae to substantiate that superlarvae were a direct result of an enhanced or sustained juvenile hormone titre. These experiments included: implanting two or more CA, extirpatlng one CA. or applying 100 Llg of Ahosid topically onto allatectomized larvae. The experiments indicated that only NCA 1 is an inhibitory pathway and that superlarvae were a direct consequence of CA activation. NCA II does not seem to provide the CA with an essential excitatory innervation; when it and NCA 1 are severed a supernumerary apolysis will still result. Some of the cells in the brain

stainable by the CoCIZ method are most probably identical to those that are PAF-positive. cells may inhibit the CA in last instar larvae via neurosecretomotor junctions.

innervation of the CA motivated us to conduct the present study. We were encouraged by the possibility that now the pathways of axons that might be involved could be defined more precisely. Although there have been several anatomical studies of the retrocerebral system of Periplarlern (WILLEY. 1961: DOGRA. 1968 ; BROUSSE-GAURY,1971; ADIYODI, 1974). these were made without benefit of the powerful intracellular staining techniques recently developed for tracing individual axons. One such technique, the cobalt chloride diffusion and precipitation method (PITMAN et al., 1972) enables the investigator to trace axons within nerves to their associated cell bodies. To date, this procedure has been used to map the neural pathways to insect endocrines in only two species (MASON, 1973; NIJHOUT. 1975).

INTRODUCTION

SCHARRER(1952) made the significant discovery that severing the nervi corporis allati I (NCA I) of last instar larvae of the cockroach, Lewophaea maderae (L.), wiil produce supernumerary apolyses. She suggested that this occurred because brain inhibition had been removed from the corpora allata, thus causing the juvenile hormone (JH) titre to rise. These results were confirmed by ENCELMANNand LYJSCHER(1956a). Furthermore, ENCELMANNand L~~SCHER(1956b) and ENCELMANN(1957) were able to activate the CA of “pregnant” Leucophaea by cutting either the NCA I or the nervi corporis cardiaci I (NCCI), but not by cauterizing the nervi corporis cardiaci II (NCC II). Activation was evidenced by the onset of oiigenesis at a time when it should have been suppressed. The likelihood that the metamorphosis of another large and experimentally important cockroach, Periplanets americana. is similarly regulated by inhibitory *To whom

requests

These

MATERIALS

AND

METHODS

Periplanefa americana were taken from laboratory stock colonies reared on Purina@ dog food and water. Those cockroaches that underwent surgery were

for reprints should be directed. 975

076

JAch FRASERAND RL’UOLPHPII’A

placed in screen-topped glass mason jars with food and water. and were kept in an incubator at 29 C under a I’D: 12L photoperiod. To facilitate tracing the nerves to the CA. 3ml of a I”,, solution of methylene blue in physiological saline IYAMASAKIand NARAHASHI,1959) were injected into each cockroach. Two hours later, the animal was immcrxcd in ;I Y’,, aqueous solution of ammonium molqbdate and dissected. When the staining was successful. nerves appeared dark blue. while muscle and fat stained less intensely. Satisfactory preparations wcrc obtainable less than 50”,, of the time. A modification of the CoC12 technique of PITMAN er (I/. (1972). employing passive diffusion rather than iontophoresih. was used to reveal the axonal tracts to and from the CA. Each cockroach was embedded in wax. \entral side down. and dissected under physiological saline. The retrocerebral complex was cxposcd by removing the overlying tracheae and fat body. and the nerve to be filled was cut far back f~om the or-~;~n which it supplied. A cup made by pushln~ ;I piccc of polyethylene tubing (id. 0.047” x o.d. 0.067”) through parafilm was filled with ;I 50OmM solution of CoC12. The saline was dr;~~nc’dof the specimen and the cut end of the nerve was immersed into the cup. To prevent leakage of the CoCI, into the surrounding tissues. the outer rim of the cup was built up with Vaseline. Exposed tissues we~-e also covered with Vaseline to minimize dehydration. To retard evaporation further. the preparation wa:, placed in a large Petri dish partly filled with water. During the ditrusion period the preparation wax refrigerated at 7 to 9 C. After 12 to I8 hr. the preparation was brought to room temperature. the insect was submerged under IO ml of saline. and the Co’ was precipitated for IO min by adding 0.2 ml of 40”,, ammonium sulfide to the \;llinc. The stained tissue was dissected out in lrcsh sal~nc. fiscd. dehydrated in ethanol. and cleat-cd and stored in methyl benzoats. All lots of clearing asent do not produce satisfactory results. Many prepat-at~un\ \WIW lost before that was realized. Only about IO”,, of the attempts to fill any nerve were successful. The routes of axons within the following nerves were determined using the CoCI, method: the nervi corporis cardiaci (NCC) I and II. the nervi corporis allati (NCA) I and II. and the posterior branch of the NCA II ( PB). Each type of fill was repeated successfully IO to I5 times. For general microscopic anatomy. brains were fixed in aqueous Bouin’s or Helly’s and stained with Mallory’s triple stain. Cobalt sulfide-stained brains were fixed in either- Carnoy’s. aqueous Bouin’s, or IO”,, formalin. Following dehydration in alcohol and clearing in methyl ben7oate. the tissues were infiltrated with Tissuemat (5X C) irl r~c~o. Serial sections were cut at 5 to 61nn. The sections of brains prepared by the CoC12 method were subsequently stained with TYRKR and Br U’S (I 974) modification of Timm’s method and

were counterstained with 0. I”,, aqueous toluidin blue. ‘10 locate the iiet~ros~crck~in cells. weal scct~ons of other brains were stained with DAWSON’S (1953) modification of HALMI’S (1952) paraldehydc fuchsin (PAF) technique. As reported by Avtvoo~ (1974). starvation results in an accumulation of PAF-positive granules in the brain of Prripltrrwrtr. so the six donors of brains to be stained with PAF were starved three weeks before they were sacrificed. Only after such treatment were the lateral neurosccretory cells of the brain revealed clearly. Neurons stained with the CoCI, method and located in the pars lateralis were counted from photographs of serial sections. The number of neurons stained with CoS in the pars intercerebralis was estimated from whole-amount preparations. The nucleolus of each PAF-positive neurosecretory cell in the pars lateralis and pars intercerebralis was counted to avoid tallying each cell more than once. All experiments were performed on last instar larvae less than one day old. Each animal was lightly anesthetized with CO?. secured to a wax dish operating stage (BODENSTEIN.1953). and covered with saline. To expose the retrocerebral complex. the cuticle from cranial vertex to cervical membrane was removed using Minuten needles as scalpels. The underlying longitudinal tracheae were pushed aside to expose the aorta. This was done carefully. for cutting just one of these major

tracheae

can result

in death

of the

animal. Directly ventral to the aorta lie the Tyndall blue cot-pora cardiaca. The scmitransluccnt corpora :III:I~;Iwere located by following the corpora cardiaca caudally. and the nerves were severed with Minuten needles. For the extirpation experiments. the CA were removed with sharpened jeweler’s forceps. Experiments were performed to confirm that superlarvae resulted from increased CA activity in the last l:ir\~al instar. Each allatectomiled lar\ a received a topical application of I /II ( IOtl ~cp)I)(‘Altn’rid (ZR 5 15). ;I compound that resemhlc\ JH both \tt uctul-alI> and by its influence on morphogenosis (H~:>;KKx PI trl., 1973). In another experiment. young last instar larvae received 2 or 3 pairs of CA. In both these experiments. the onset of a supernumerary apolysis was interpreted as evidence that an elevated titre of JH during the last larval instar was involved. The possibility that transecting a single NCA I in last instar larvae promoted the production of supcrlarvae without acting on the CA itself also was tested. This was done by extirpating the gland. If adults result at the next apolysis. then the necessity for the presence of the dencrvated CA would be demonstrated. RESULTS 7&g IIL’TL’ZIS corporis rrlluti I1 (NC/l II) c1ntl if.\ trrltfriov (AB) rend posterior (PB) hrrrrdwa. The NCA II diverges from the nervus corporis allati I (NCA I) at a point antero-lateral to the corpus allatum (Fig. I).

Axon pathways

and CA regulation

k’ig. I. Dorsal view of Pt~ipltrneru retrocerebral complex. tf’ostrrior extension of CC0 removed.) x 40. AB. anterior hrsnch of NCA 2; AC. allatal commissure: AON. aortic ncr-ve; CA. corpus allaturn: CC. corpus cardiacum; CCO. cardiaca-commissural organ; FB-CT. fat body-connective ti\buc sheath; IPN. inner postallatal nerve; MPN, middle postallatal nerve: NCA I. 2. nervi corporis allati; NCC I. 2. 3. ncrvi corporis cardiaci: OPN. outer postallatal nerve: PH. posterior branch of NCA 7; RN. recurrent nerve.

It proceeds ventrally along the tentorium to terminate on the ipsilateral mandibular nerve of the subesophageal ganglion. This differs from the description by W~LLEY (1961). who stated that NCA II passes hrtw~cn the mandibular nerves and terminates directly on the subesophageal ganglion, Two large nerves branch off the NCA II (Figs. 1, 2). The nerve PB. usually proximal to the corpus allaturn. travels caudad; the distal nerve. AB, goes cephalad. Nerve AB was described by WILLEY (1961) and AIXY~DI (1974). but neither investigator could determine where its branches ended. We found that AB becomes tripartite (Fig. 1) when it reaches the nervus corporis cardiaci III (NCC III). The medial branch joins the recurrent nerve as indicated by AIXYOI~I(1974). and the dorsal branch fuses with the NCC 111. We could not locate the terminals of the lateral. ventrally-directed branch of nerve AB. WtLt.r~ ( 1961) traced PB to the anterior lobes of the prothoracic gland in larvae, and labeled it “TO PC”. AUIYOIX (19741 could not confirm Willey’s findings. because he misidentified the outer postallatal nerve (OPN. Figs. 1. 2) as the posterior nerve (“PG”) of Willey. According to our observations, PB passes near the prothoracic gland. but apparently does not ~nnc~.v;tte it. Instead. tt joins ZBrle (not illustrated).

in Pcriplunvrtr

911

a nerve that arises from the prothoracic ganglion (PIPA and COOK, 1959). The prothoracic gland extends into the larva1 head capsule, where it envelops the foregut dilator muscles (MOSCONI-BERNARDINI. 1964). Several small nerves (not illustrated) from NCA II pass to the anterior lobes of the prothoracic gland. but it was not determined whether these nerves innervate the gland, or, instead, the underlying muscles. The al&al commissure. ADIYODI (1974) describes numerous allatal commissures forming a plexoid structure overlying the esophageal nerve between the CA. In none of our 36 animals was such a plexus encountered. Instead, the commissure consists of one to three nerves located beneath the esophageal nerve (Figs. 1. 2). When the commissure does not cross beneath the esophageal nerve it passes through it. The axons enter the perineural sheath and exit on the opposite side; they do not parallel the axons of the esophageal nerve. WILLEY (1961) and ADIYODI (1974) traced several axons of the NCA II through the CA, and into the allatal commissure. We could confirm the pathways of these axons in histological sections, and in CoCl, tills (see below). Posrallatal nerres. There are three major postallatal nerves: the inner (IPN), the middle (MPN), and the outer (OPN) (Figs. 1, 2). Their locations are fairly constant. but variations in bridges between them are common. and the anatomy can be complex (Fig. 2). These nerves were traced into a fat body-connective tissue sheath that extends caudally from the anterior lobes of the salivary glands over the esophagus. We were unable to establish the relationships amongst the postallatal nerves, the sheath, and the salivary glands. WHITEHEAD(1971), during his study of the innervation of the salivary glands of P. americunu. could demonstrate no neural connections between the CA

/ Fig. 2. Dorsal view of Periplaneta retrocerebral complex, showing an extreme variation in its anatomy (CC0 removed). x 42.

97x

JACK FKASERAND RLDOLPHPIPA

and the salivary glands. That the postallatal nerkt‘s may. indeed. innervate the salivary glands of cockroaches is suggested by observations on .%rpe//u loncqipu/pcl (S.) (FRASER. unpublished). There, the intervening fat body-connective tissue sheath is absent. and an innervation of the salivary glands by the postallatal nerves is strongly indicated. C‘trriliffcLI-comnlisslrrLl/ As the corpora cardiaca

ofyrrrf trirtl the trorric

wrw5.

(CC) continue cdudally. they produce a dorsally directed structure designated by W~LLEY (1961) as the “commissurus corporis cardiac?‘. and by ADIYUDI (1974) as the “cardiacacommissural organ” (CCO). The latter designation is adopted here (Fig. I). This dorsal structure extends as far back as the distal margins of the CA. and serves as the floor of the aorta in this region. The CC0 contains PAF-positive granules similar to those found throughout the CC. ADIY~DI (1974) claimed that several nerves connect the CA with the CC0 and that these might regulate the activity of the CA. We found nerves from the NCA I (AON. Figs. 1. 2) and sometimes from the CC that terminate either on the distal limits of the CC0 or. more likely. on the aorta. The extensive CC0 plexus diagrammed by Adiyodi was not seen in any of our animals.

CoCl, ,fi/ls qf’flle wzrws corporis dltrti 1. After either of the two NCA I is filled cephalad. three clusters of neurons within the brain will stain (Fig. 3). These are: 16 to 20 cell bodies in the ipsilateral pars lateralis: 8 to 12 cell bodies in the contralateral pars lateralis; and 50 to 60 cell bodies in the contralateral pars intercerebralis. Individual differences account for the range in numbers. To elucidate further the pathways of axons within the NCA 1, the NCC 1 and NCC 11 were filled separateI). CoCl, ,fi//s of’ r/le UP~CU.S corpora c~trrtliwi 1. When NCA 1 is filled cephalad after- NCC II has been cut. thus filling NCC I, only the contralateral medial cell bodies take up the CoCI,. Their axons pass down the anterior surface of the brain and cross to the opposite hemisphere before continuing along the ventral surface of the brain to the NCC I. Proximal to these cell bodies several collaterals branch into the protocerebral neuropile surrounding the calyces, where they form a dense arborization (Fig. 5). This network extends to the lateral margins of the neuropile to a depth of about 35 pm. CoCl, ,fi/ls of’ the nercus corporis urdiuci II. When NCA I is tilled cephalad after NCC I has been cut. thus filling NCC II. nerve cell bodies in the ipsilateral and contralateral pars lateralis stain. but those in the pars intercerebralis do not. The cell bodies are located under the dorso- lateral lip of the r-lobe of the corpus pedunculatum (Figs. 8. 9). Several small branches extend ventrally from the cell bodies and arborize in the neuropile lateral to that sensory association center

(Figs. 9. IO). The majol- tract extends medially. around the anterior surface of the r-lobe, and passes posteriorly under the lower ridge of the inner calyx. Several axons separate from the main tract and, just before it turns posteriorly. they arborize in the neuropile lateral to the pars intercerebralis. None of the posteriorly-directed axons appear to enter the corpus pedunculatum. though they do arborize extensively within the neuropile next to it (Fig. 6). If~~~i-trriorl of thr corpora trlkctrr r~wtrlrtl hj, CoS /“‘l”‘i/‘i/trtion. The brain and subesophageal ganglion are the two ma.ior sources of axons that pass to the (‘A. The axons from the brain reach the CA via the NCC I and NCC II: those from the subesophageal ganglion reach the CA via the NCA II. NCC I and II were filled caudally simultaneously. This revealed axons that bypass the CA to enter the NCA II. and others that arborize within the gland a&or pass through it to leave via the postallatal ner\ es. Filling the NCA II towards the CA showed 3 to 4 axons which enter the gland (Fig. 4). The largest axon traverses the midst of the gland to enter the opposite CA via the allatal commissure. This axon is most probably identical to the one that we saw in serial sections stained with Mallory’s, The remaining axons approach the center of the gland. branch there. and then turn posteriorly to exit via the postallatal nerves. We were unable to determine where the axons that ramify within the CA, or those that enter the postallatal nerves. terminate. C&I2 fills of’ tile ,suhe,sophageczl pnglion. After the NCA II of either side had been filled ventrad, CoS precipitation revealed 3 to 4 cell bodies in the antcro ~cntral aspect of the subesophageal ganglion. across from the ipsilateral labial nerve and near the mid-line. The axons enter the ganglion through the mandibular nerve. traverse the dorsal surface of the ganglion neuropile anteriorly. and pass postero-ventrad to join their cell bodies. The cell bodies seem to be located in approximately the position of the medial neurosecretory cells described by BRADY ( 1967). but whether any of them correspond to those cells remains to be determined. CoCIZ ,fil/s (!f thr posterior brunch (PB) of the NC.4 II. Axons within PB pass down the NCA II toward the subesophageal ganglion as evidenced by filling PB cephalad. We have not been able to trace the axons directly to that ganglion.

P[lrs luterulis. The pars lateralis of each protocerebral lobe contains 12 to 14 PAF-positive neuronal perikarya with diameters ranging from 17 to 25 pm and averaging 22pm (Fig. 7). These are located in approximately the same region as 8 to 20 cells that till with CoCIZ (Fig. 8). They are revealed best after the cockroaches have been starved. although KHAN and FRASER (1962) were able to demonstrate them in unstarved animals.

Fig. 3. Frontal view of brain after the right NCA cell clusters are present: the contralateral medial tions of axons from the ipsilateral NCC 11 Scale

I had been filled cephalad with CoCl,. Three distinct cluster (M), and two lateral clusters (L). The arborizaappear as a dense black area medially (arrow). = 0.3 mm.

Fig. 4. Dorsal view of corpora allata; NCA II on both sides had been filled with CoCI,. Axons (A) appear as thin black lines within each gland. and the allatal commissure (arrow) contains axons common to both CA. Scale = 0.1 mm.

980

Fig. 5. Transverse section of brain stained with Timm’s sulfdeesilver method. NCC I was filled cephalad with CoCI,. revealing dense arborizations of pars intercerebralis neurons in the neuropile (N) lateral to those cell bodies. A. alpha lobe of corpus pedunculatum. Scale = ZOpm. Fig. 6. Transverse section of brain stained with Timm’s sultide silver method. NCC II (2) was tilled cephalad with CoCI,. The arborizations of axons (unlabeled arrows) from that nerve tract occur in the neuropile medial to the pedunculus (P) of the mushroom body. C, inner calyx. Scale = 2Onm. Fig. 7. Transverse section of brain stained with paraldehyde positive nerve perikarya (arrows) are situated in the pars Scale = 20 nm.

fuchsin lateralis.

(PAF). Two clusters of PAFA. alpha lobe: N. neuropile.

Fig. X. Transverse sectton of bram stained with Timm’s suhideesilver method. NCC II (2) was filled cephalad with CoCI, to reveal nerve cell bodies (arrows) in the pars lateralis. A. alpha lobe: N. neuropile. Scale = 20 pm.

Axon pathways

and CA regulation

in Periphnetu

981

.LNC

Fig. 10. Posterior

Fig. 9. Diagrammatic anterior view of right corpus pedunculatum. depicting its relationship to the neurons associated with NCC II. x 70. A. alpha lobe; B, beta lobe; IC, inner calyx; LNC, lateral nerve cell bodies: NCC 2, nervus corporis cardiaci axon pathway; OC. outer calyx; P, pedunculus. f’ws ir7tr~c,~~rh~tr/i.~. Each half of the pars intercerebralis contains 50 to 60 PAF-positive nerve cell bodies which range from 10 to 20/1m in diameter and average I5 Itm. They are located in approximately the same place as the 50 to 60 cells that filled with CoCl,.

7‘1~ ir7hibitory 7% of’ the NCA I. About 30 days after the NCA 1 on either one, or both, sides had been severed, an extra larval ecdysis occurred (Table 1). These results agree with those of SCHARRER(1952). ENGELMANNand L~SCHER (1956a). and L~WHER and ENGELMANN(1960), who demonstrated that the CA were activated similarly in L~cophaea nuderae (F.). Unlike the situation in Leucophura. in Periplnnetc7 there was no obvious change in the volume of the Table Nerves severed

1. Effects of denervation No. of replications

CA after NCA I had been transected. Activation of the CA by eliminating a source of nervous input from the brain suggests that the brain normally inhibits hormone production by this gland. R& of thr NCA II in corpora dlutcl rryultrtion. ENGELMANN(1957) suggested that in Lc~oyhurrr the NCA II is excitatory, for he found that completely denervated glands failed to become activated in “pregnant” females. and egg maturation did not occur. Our results (Table 1) discount the likelihood that this nerve is important in that regard: severing the NCA I and NCA II on one side of Periphrtu larvae is as effective in producing supernumerary apolyses as severing the NCA I alone. That the subesophageal ganglion does not inhibit the CA of Peripluneta was indicated by experiments in which we severed NCA II on both sides (Table 1). In those cases, superlarvae were not produced. Postallatal nrrws. Severing the postallatal nerves did not stimulate supernumerary apolyses (Table I), so these nerves do not seem to inhibit activity of the CA.

on CA activity

No. of larvae

view of same

in tenth

No. of superlarvae

instar

No. of adults

Prriplunrta larvae Mean no. of days (+ S.D.) from operation until next ecdysis

NCA I sham

2 2

6 2

2

25 f 5.6 24 f 1.0

posta.* sham

2 2

4 4

4 4

26 + 4.1 26 f 6.0

NCA 1. NCA II NCA 1

2 2

5 4

0 0

30 + 6.4 30 i 6.4

NCA II (bilat.)t sham

3 3

10 8

IO 8

28 i 3.0 27 + 4.2

* Postallatal nerves on one side severed; sham, animal .t Bilateral transection; sham. animal just opened up.

1

just opened

up.

JACK

9x2 Table

2. ElTects of

implanting CA or

of applying

a JH-analog

PIPA

(Altosid)

tenth instar

Pwipl~rwr~

No. of Iarb iw

No. of \uperlarcx

No. of replication5

Treatment

~KASIK ANII Rt,Lx)l.w

on incidence of supernumerary

apolyses by

larvae No. of adults

Mean no. of days () S.D.) from operation until next ecdysis

Implants 3 pr. CA

2 pi-. CA Fat body (control) JH-analog XCA*

+ Altohid Acetone (Control)

XCA* +

* Both panx of CA were removed before the compounds

indeed. the CA are responsible for the extra apolysis that occurs after one NCA I has been transected, then removing the denervated gland should prevent the rcsponse. Five of the six larvae treated that way transformed into adults within 18 f 2.8 days after the operation: only one superlarva resulted. These results \+c‘rc identical IO tho\r obtained with the six shnmoperated control larvae, and negate the possibility that some factor unrelated to CA activity may be involved. The results of experiments in which CA were implanted into last instar larvae also support the interpretation that production of superlarvae is a consequence of an increased or maintained JH titre (Table 2). It appears that three or more pairs of CA are required. To substantiate further that superlarvae are due to an increased or maintained JH titre. last instar larvae were allatectomi7ed, and I id (loO/~g) of Altosid was applied (Table 2). Such treatment simulates an increased JH titre. and resulted in superlarvae 14 to 30 days later. DISCUSSIOI\; According to the “classical scheme” (DOAN.. 1973). during the last larval instar the haemolymph JH titre declines. and the following apolysis results in an adult. There is evidence that in Lctrc~)pl~re~ (SCHAKKER. 1952: ENGFLMASXand L~?s~HEK. 195ha. 1956b) and. now. in Prrip/trr~~ttr this decline is prompted by neural inhibition of CA secretory activity. for superlarvae are produced after NCA I is transected. It ib unlikely

that

some factor

unrelated

to CA

activation

\\:I$ acting in our denervation experiments. for when the gland was removed superlarvae did not result. The cffccts of implanting CA. or of applying Altosid. a JH-analog. support further the hypothesis that an elevated or maintained JH titre was responsible for the extra apolysis. Many of the axons that pass into the NCA I arise from cell bodies located in two regions of the brain: the pars intercerebralis and the pars lateralis. Because we were unable to transect the NCC I or NCC II separately without incurring high mortality, we could not determine which of these arcas might be implicated.

were applied

It 1s probable that the inhibitory center lies within one or both of these regions and that the PAF-positive cells found there are involved. but that needs to be proved. Although it is conceivable that neurons from other parts of the brain innervate the CA. there is no evidence for that at this time. It is almost certain that some of the neurons in the brain that stained with CoS are the same as those stainable with PAF. There is a distinct possibility that these neurons inhibit the CA via junctions that are “neurosecretomotar” (BERN. 1966). In Lt~rcopl~trctr. axon terminals laden with electron-opaque granules and contiguous with CA cells are likely candidates for that function (SCHARRER.1964). The pathways of these axons, howCWT.remain to be identified. ENGELMANN and L~‘WHER(1956b) and ENGELMANN ( 1957) suggested that the CA of Le~rcophrrrtr are inhihated neurally by ;I region of the brain lentral to the pars intercercbralis, Only by thermocoagulating that portion, not the pars intercerebralis. could the CA be activated. SCHARRER (1964) writes: *‘_ it is difficult to visualize the mediation of sustained inhibitory stimuli. with pronounced effects upon cellular ultrastructure and function, by the conventional type of nervous activity”. She favors a neurohormonal control of CA activity in Lr~cophnetr. The controversy over nomenclature is resolved if one accepts a current definition of neurosecretory cells. BERN (1966) extends the concept to include neurons that terminate directly on endocrine cells: thus. those in the region described by Engelmann would be neurosecretory. The function of the subesophageal ganglion in CA rc:rulation is uncertain. In Prriplurlrttr an inhibitory ri>lc by neurons within the subesophageal ganglion is improbable: the gland is not activated by eliminating all neural input from that center. Similarly, when the NCA I is transected and superlarvae result. all neural connections to the subesophageal ganglion a~ intact. ENC;I:L.MANK (1957) suggested that in LCUc~~phw the subesophageal ganglion cxcitcs the CA, for when the gland is completely dencrvated (by removal and reimplantation) it will not become active. We have demonstrated that either one of the CA of Pcriplrrnrto larvae will be activated after the corresponding NCA 1 and NCA II are severed. If the

Axon pathways and CA regulation in Prriplanrta subesophageal ganglion did excite the CA in our experiments. it most probably did so via the allatal commissure. a tract that was not cut. Another indication that the CA are active without a neural input was shown by the implantation experiments; transplanted CA devoid of all innervation are active, though admittedly at a lower level than when they are left in situ. The evidence, though incomplete. suggests that the subesophageal ganglion does not play a major r6le in activating the CA of last instar Periplaneta larvae. With regard to the mapping of axon pathways, there are differences between our observations and those by MASON (1973), who examined Schistocerca, and NIJHOUT (1975), who studied Manduca. Mason was unable to find fibers originating from pars intercerebralis cells entering NCA I, though she was able to trace fibers from the lateral cells into this nerve. For Periplaneta, filling the NCA I reveals cells in both partes laterales plus a cluster in the contralateral pars interccrehralis. NIJHOUT (197.5) reports results similar to ours after he filled a nerve in Manduca that may be homologous to NCA I of Periplaneta. MASON (1973) and NIJHOUT (1975) were able to demonstrate a tritocerebral cluster after the NCC III was filled cephalad. Although we made many attempts, none resulted in successful fills of that nerve. The reason for these discrepancies is not clear. It is conceivable that our results. and those of Nijhout, differ from Mason’s because of dissimilarities in the proximity of the corpora cardiaca (CC) to the CA in our respective insects. Unlike those of Schistocerca. the CC and CA of Periplaneta and Manduca are almost contiguous. This makes the separation of axon pathways by the CoCl, method difficult. The nerve cell bodies located in the pars intercerebralis of Periplaneta we demonstrated by filling NCA I could belong to axons that terminate in the CC. These may have filled inadvertently, despite our precaution to dip the cut end of the CA into the CoCl, solution. Also to be kept in mind is the caveat that not all axons within a nerve necessarily fill. MASON (1973) identified 11 cell bodies in the subesophageal ganglion of Schistocerca with axons that “terminate” in each CA. We found only three to four cell bodies in the subesophageal ganglion of Periplaneta when either one of the NCA II was filled ventrad. There was evidence that axons within this nerve arborize within the CA, exit via the postallatal nerves, or enter the opposite CA via the allatal commissure. Although axons staining positively for neurosecretion have been demonstrated in the NCA II (DOGRA, 1968; AIXYODI, 1974), it would be premature to equate these with the axons we filled with CoCl,. By using a silver intensification technique modified for CoCl, fills (TYRER and BELL. 1974) we were able to identify two discrete populations of neurons that may share a common network. One group, with perikarya located in the pars intercerebralis. send axons to the contralateral NCC I and NCA I. They branch

9X3

extensively in the neuropile adjacent to the pars intercerebralis. and are very likely equivalent to the arborizing neurosecretory cells described by ADIYODI and BERN (1968). The second population, with axons in NCC II and NCA 1. have perikarya located in the pars lateralis. These neurons arborize in the same region as the first population. and extensively in the neuropile surrounding the pedunculus of the mushroom body. This latter group, too. may consist of neurosecretory cells. The possibility that these separate populations of neurons effectuate neuroendocrine reflexes by communicating directly, or through mutual interneurons. should be explored further, Ackno~4edyrment~+ We thank Dr. CORY GOODMAN for his helpful advice regarding the CoClz diffusion and percipitation technique. and Dr. MARK SCHROEIXR. Shell Development Laboratories. Modesto. CA. for supplying cockroaches. REFERENCES ADIYODI K. G. (1974) Extracerebral cephahc neuroendocrine complex of the blattids. Peripkmetu americunu (L.) and Neost)jlopygu rhomhifolia (Stall.): An in sirlr study. J. Morph. 144, 469-484. ADIYODI K. G. and BERN H. A. (1968) Neuronal appearance of neurosecretory cells in the pars intercerebralis of Periplaneta americana (L.) Gen. Camp. Emloc,r. 1 I, 88-91. BERN H. A. (1966) On the production of hormones by neurones and the role of neurosecretion in neuroendocrine mechanisms. Symp. SW. ezp. Biol. 20, 325 -344. BODENSTEIND. (1953) Studies on the humoral mechanisms in growth and metamorphosis of the cockroach, Pcriplaneta americana II. The function of the prothoracic gland and the corpus cardiacum. J. u.~p. Zool. 123, 4 I 3-434. BRADY .I. (1967) Histological observations on circadian changes in the neurosecretory cells of cockroach suboesophageal ganglia. J. Insect Physiol. 13, 201-113. BROUSSE-GALJRYP. (1971) Influence de stimuli externes sur le comportement neuro-endocrinien de blattes. 1. Les organes sensoriels ckphaliques, point de depart de rCflexes neuroendocriniens. Ann. SC?. tlut. Zoo/. Rio/. Anim. 13. 181-332. DAWSON A. B. (1953) Evidence for the termination of neurosecretory fibers within the pars intermedia of the hypophysis of the frog, Rana pipirns. Anut. Rec. 115, 63-69. DOANEW. W. (1973) Role of hormones in Insect development. In Developmental Sysrrms: Insects (Ed. by COI~NCE S. J. and WADDINGTON C. H.). Pp. 291-497. Academic Press. London. DOGRAG.. S. (1968) The study of the neurosecretory system of Periplaneta americana (L.) in situ using a technique specific for cystine and/or cysteine. Acrrr .4rut. 70, X-303. ENGELMANN F. (1957) Die Steuerung der Ovarfunktion bei der ovoviviparen Schabe Leucopkaect mtrderur (Fabr.). .1. Insect Physiol. 1, 257-278. ENGELMANI\: F. and LDSCHER M. (1956a) Zur Frage der AuslGsung der Metamorphosen bei Insekten. Ntrtltrwissenscliqftfvt43, 43-44.

9x4

JAC.I
F. and L~~s(.HBK M. (IY56b) Die hemmende Wirkung des Gehirns auf die Corpora allata bei Lrlrc+ /‘htrrcc rnudurtrr (Orthoptera). l’erh. dt,sch.Zoo/. C;r,\. (1956), 215~~Z’O. HALMI N. S. (1952) Differentiation of two types of basophilia in the adenohypophysis of the rat and the mouse. Stair] TCK/L 27, 61-64. HFNRKK C. A.. STAAL G. B.. and SIDIMLL J. 13. (1973) Alkyl 3,7.l I -trimethyl-2.4-dodecadienoates. a new class of potent insect growth regulators with juvenile hormone activity. .1. .lyric,. Ftl Cl~rn~. 21, 354-359. titIA> T. R. and tKASI K A. (lY62) Neuroxcrctlon ni the emhryo and later stages of the cockroach F’~~t?y/trnc,rcr ~r,,~<,r~ic~rr,itr (L. I. hlr>,n. Sac,. Ei&x,r. 12, 349 369. L~'~s(‘HI:K M. and Exit.l MAXN F. (1960) Histologischc und experimentelle Ilntersuchungen iiber die Aus~i~ung detMetamorphose bei L~,~rc~op/~[~~r~ rn~/t/(,rl~cs.J. Irw~t P/z 1 .sb,l. 5, 240 25x. MASOY C. A. (1973) New fcaturcs of the hrain rctrocercbra1 neurocndocrinc complex of the locust .Sc/~isfoc,c,~r/ rc~(,tr (&udder). %. Z~,//firr\cl~. ,%fihr~~\l~.+lmrt.141. 19 32. Mosc-oN1-Br-Ks.4RoINI P. (19641 Morpholoplc de la glande ventrale et dc la glande prothoraciquc che7 ley hlattes et les termites. .AIIII. E~lt/o~~~.25, (Suppl.). 71 7X. NIJHWT H. F. (1975)Anonal pathways in the hram retrocerebral ncuroendocrine complex of ,\!(rr~tfuc,tr wxtu (L.1 (Lcpidoptcra: Sphingtdac). /III. J. Irlrcc I .2/cwph. Ildy~~~d ENGELMAXN

4, 579 53%.

nervous

PWA

system.

I. The

peripheral

distribution

t hol-aclc nc~-VCYof the adult cockroach, Pzriphneru ,<‘t,,lt,. Illll. 1’111.‘5,I(‘. 4tt1. 52, 695.-710.

of the trmrr-

R. M.. T\\~EDLE C. D.. and COHEN M. J. (1971) Branching of central neurons: Intracellular cobalt injection for light and electron microscopy. Sci~rlct~. W&I. 176. 412 414. SCtiARRkR B. (1952) Neurosecretion. XI. The eflects of nerve section on the intercerebralis+zardiacumallatum system of the insect L,c1rcopl7cwu mdrrcw. Biol. Bull.. W’m/.\ Hole 102, 261 272. SC IIARKI.K B. 11964) Histophyslological studies on the corpus allatum of Lcxopktrutr mcrdotrc. IV. Ultrastructure during normal activity cycle. Z. Zc~llfi~rsclr. Lfihsk. .4rur1. 62, I2 1%. T>K~K N. M. and BELL E. M. (1974) The intensification of cobalt-tilled neurone proliles using a modification of Timm’c sultidr silver method. Brc/irl Res. 73, 151~ 155. WFnTt-tn,\~) A. T. (1971) The innervation of the salivary gland in the American cockroach: light and electron microscopic observations. J. Morph. 135. 483- 506. WiLtrv R. B. (1961) The morphology of the stomodeal nervous sytem in Pwipltrwttr mwricuntr (L.) and other Blattarla. J. ,\lorp,h. 108, 719 162. \‘\\I,~~,\LI T. and NAKkItAStiIT. 11959) The cficcts of potas\~um and xxhum tons on the resttng and action potent~ala of the cockroach giant axon. J. Inset PI~~xiol. 3. 1% 15x. PIIMAU