ELSEVIER Regulatory Peptides 63 (1996) 171-179

REGULATORY

PEPTIDES

Immunocytochemical distribution of Locustamyoinhibiting peptide Lom-MIP) in the nervous system of Locusta migratoria.

Liliane Schoofs *, Dirk Veelaert, Jozef Vanden Broeck, Arnold De Loof Zoological Institute of the UniversiO', Naamsestraat 59, B-3000 Leuven, Belgium

Received 16 January 1996; revised 26 March 1996; accepted 26 March 1996

Abstract

Locustamyoinhibiting peptide (Lom-MIP) is one of the 4 identified myoinhibiting neuropeptides, isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion complexes of the locust, Locusta migratoria. An antiserum was raised against Lom-MIP for use in immunohistochemistry. Locustamyoinhibiting peptide-like immunoreactivity (Lom-MIP-LI) was visualized in the nervous system and peripheral organs of Locusta migratoria by means of the peroxidase-antiperoxidase method. A total of 12 specific immunoreactive neurons was found in the brain. Processes of these neurons innervate the protocerebral bridge the central body complex and distinct neuropil areas in the proto- and tritocerebrum but not in the deuterocerebrum nor in the optic lobes. The glandular cells of the corpora cardiaca, known to produce adipokinetic hormones, are contacted by Lom-MIP-LI fibers. The corpora allata were innervated by the nervus corporis allati I containing immunoreactive fibers. Lom-MIP-LI cell bodies were also found in the subesophageal ganglion, the metathoracic ganglion and the abdominal ganglia I-IV. In peripheral muscles, Lom-MIP-LI fibers innervate the heart, the oviduct, and the hindgut. In the salivary glands, Lom-MIP-LI was detected in the intracellular ductule of the parietal cells. Possible functions of Lom-MIP are discussed.

Keywords: Locustamyoinhibiting peptide. Loeusta migratoria; Corpora allata; Corpus cardiacum; Allatostatin; Juvenile hormone; Myotropin; Neuropep- tide; Salivary gland

1. Introduction

Most of the peptides identified in Locusta migratoria have been isolated on the basis of their stimulatory or inhibitory effect on insect visceral muscles (the Locusta oviduct or the Leucophaea hindgut) and were designated myotropins [1,2]. Some of the myotropins appear to be unique to insects or arthropods; others seem to be mem- bers of peptide families spanning across phyla. Members of the myotropin peptide families have been associated with a variety of physiological activities such as myotropic activities, pheromonotropic activities, diapause induction, stimulation of cuticular melanisation, diuresis (for review, see [2]) and recently allatostatic activities [3]. Some of the members appear to he important neurotransmitters present in nerve endings innervating the locust oviduct, the sali- vary glands, the male accessory glands and the heart [4], whereas others are stored in neurohaemal organs until release into the haemolymph [5].

* Corresponding author. Tel.: (32-16) 323-912; Fax: (32-16) 323-902; e-mail: liliane.schoofs @bio.kuleuven.ac.be.

One of the 21 myotropins identified in L. migratoria is locustamyoinhibiting peptide (Lom-MIP). Lom-MIP has been isolated from 9000 brain-corpora cardiaca-corpora allata-subesophageal ganglion complexes of L. migratoria. Its primary structure was identified as Ala-Trp-Gln-Asp- Leu-Asn-Ala-Gly-Trp-NH 2 [6]. Lom-MIP was found to suppress spontaneous contractions of both the hindgut and the oviduct of L. migratoria. It has the Gly-Trp-amide carboxyterminal in common with APGWamide, a penis retractor muscle inhibiting peptide identified in mollusks [7]. Until 1995, the primary structure of Lom-MIP was unique to insects. Recently, however, two and four pep- tides with high sequence similarities to Lom-MIP have been discovered in Manduca sexta [8] and in Gryllus bimaculatus [9], respectively. The recent discovery of these peptides indicates that myoinhibiting peptides or MIPs are widely distributed among insects and that yet another family of insect neuropeptides has been discovered (see Table 1).

In this study we describe the production of a highly specific antiserum to the carboxy-terminus of Lom-MIP, with its specificity defined by ELISA. This antiserum was

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172 L. Schoof~ et al. / Regulatoo' Peptides 63 (1996) 171-179

Table 1 Primary structures of peptides of the MIP-family isolated from Locusta migratoria (Lom-MIP), Gryllus bimaculatus (Grb-AST B 1-4) and Manduca sexta (Mas-MIP I and II)

Lom-MIP Mas-MIP I Mas-MIP II Grb-AST B 1 Grb-AST B2 Grb-AST B3 Grb-AST B4

Ala- Trp- Gln- Asp- Leu- Asn- Ala- Gly- Trp- NTI 2 Ala- Trp- Gln- Asp- Leu- Asn- Ser- Ala- Trp- NH 2

Gly- Trp- Gln- Asp- Leu- Ash- Ser - Ala- Trp- NH 2

Gly- Trp- Gln- Asp- Leu- Asn- Gly- Gly- Trp- NH 2

Gly- Trp- Arg- Asp- Leu- Asn- Gly- Gly- Trp- NH 2

Ala- Trp- Arg- Asp- Leu- Ser- Gly- Gly- Trp- NH 2

Ala- Trp- Glu- Arg- Phe- His- Gly- Ser- Trp- NH 2

used in an immunocytochemical study to localize the neuronal cell bodies that synthesize Lom-MIP in L. migra- toria. Tracing of the processes of these peptidergic neurons can give clues to the possible site of action, which can reflect a possible function of the peptide.

2. Materials and methods

2.1. Production and specificity of the antiserum

Synthetic Lom-MIP was coupled via its ",/-carboxylic acid group to bovine thyroglobulin (Sigma) using EDC (1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochlo- ride). In this way, antibodies against thyroglobulin bound Asp-Leu-Asn-Ala-Gly-Trp-NH 2 would be mainly directed against the C-terminal portion of the peptide molecule. After an incubation of 2 h, the soluble EDC urea deriva- tive, which is released as a byproduct of the conjugation reaction was separated from coupled thyroglobulin by dialysis. The bound peptide was dissolved in 1 ml of Tris-saline buffer (pH 7.4) emulsified with an equal amount of Freund's adjuvant and injected subcutaneously into New Zealand white rabbits. Three boosts were given, respectively 3, 6 and 9 weeks after initial immunization. A total of 1 mg coupled Lom-MIP was injected into each of the two rabbits for initial immunization and three boosts.

ELISA was used to test the specificity of the antiserum. Activated microtiter wells of Titertek PVC immunoassay microplates (Flow Laboratories) were coated with 100 bl of 10 p~g/ml of APGWamide [7], SchistoFLRFamide, Locustatachykinin I, Locustamyotropin II [2] and Locus- tamyoinhibin 5-13 [10] in carbonate buffer (0.05 M, pH 9.6). Subsequently, the coated wells were treated as de- scribed earlier [3].

2.2. Animals and immunohistochemical procedure

L. migratoria was raised according to Ashby [11]. Brains, corpora cardiaca-corpora allata, suboesophageal ganglia, ventral nerve cords, salivary glands, oviducts, hearts, hindguts, male accessory glands and testes of ma- ture adult locusts were fixed in situ in Bouin-Hollande's 10% sublimate. The fixed tissues were then treated as described previously [5]. Immunohistochemical staining

was accomplished by means of the PAP-method [12]. Specificity was controlled (1) by immunoabsorption of the serum (1:4000 diluted) with synthetic Lom-MIP and (2) by the processing of a series of stainings in which the various steps were omitted one by one from the regular staining sequence.

3. Results

3.1. Specificity of the Lom-MIP antiserum

An ELISA was used to test the specificity of the Lom-MIP antiserum. Fig. 1 shows that the antiserum crossreacts with Lom-MIP and to a lesser extent with

+ 0 • • ~2 0

1.00

0.80

~. 0.60

~. 0,40

m

0.20

0.00

/ T

0.00001 0.0001 0.001 0.01

Dilution (log)

Fig. 1. Enzyme-linked immunoassay. Binding of equimolar amounts of Lom-MIP (+ ) , APGWamide (O), schistoFLRFamide ( • ), Locustat- achykinin I ( . ) Locustamyotropin II ( v ) and Locustamyoinhibin s 13 (©) with Lom-MIP antiserum. B o, optical density of control (without peptide); B, optical density of sample B(,.

L. Schools et al . / Regulato~" Peptides 63 (1996) 171-179 173

APGWamide, but not with the other peptides of the locust tested. For the primary antiserum, a final dilution of 1:4000 appeared to be appropriate for an optimal im- munoreaction on tissue sections. No staining was observed when anti-Lom-MIP serum was replaced either by serum previously inactivated with Lom-MIP or with control serum. Likewise no staining occurred when the secondary antibody, PAP-antibody or DAB, respectively, were omit- ted from the immunohistochemical procedure.

3.2. Immunohistochemical detection of Lom-MIP-like im- munoreactive material in the central nervous system

The data presented below are derived from paraffin sections. In whole-mounts the resolution was not suffi- cient, due to the limited number of small immunolabeled neurons. In whole mounts these small cell bodies were hard to distinguish. The number of Lom-MIP-like im- munoreactive (Lom-MIP-LI) neurons in the locust brain is rather small. The distribution of Lom-MIP-LI cell bodies is

summarized in Fig. 2. Immunoreactive cell bodies were found in the brain (protocerebrum, tritocerebrum), in the subesophageal ganglion, the metathoracic ganglion and the abdominal ganglia. In the anterior protocerebrum, 6 Lom- MIP-LI neurons were detected in the pars intercerebralis (Fig. 3a). More posteriorly, one neuron was detected in each hemisphere of the lateral protocerebrum (Fig. 3b). These neurons are located near the ocellar nerves and most likely innervate the glandular cells of the corpora cardiaca.

As seen in Fig. 2, there is a widespread distribution of Lom-MIP-LI fibers in the locust midbrain, including the proto- and tritocerebrum. The deuterocerebrum was devoid of immunolabeled fibers. Extensive immunolabeling of arborizing Lom-MIP-LI fibers can be seen in the central body complex, including the protocerebral bridge (Fig. 3c). In the central complex (Fig. 4a,b), Lom-MIP-LI fibers are seen in the upper and lower (also referred to as the fanshaped body and its ellipsoid region, respectively) divi- sions of the central body, in the protocerebral bridge, in the median and lateral accessory lobes and the noduli. At

MEal Ac NV1

NV3 NV4 NV5

Ab3MN

aMN

B E

" i~coe Ablll SOG

N H O - ~ e e ~ AC

• " ~" StNV

Abl l

AbIV

A b V

Fig. 2. Distribution of Lom-MIP immunoreactive cell bodies and nerve fibers in the brain (A,B), subesophageal ganglion (C) and ventral nerve cord (D,E,F) of Locusta migratoria, seen in frontal view. Anterior portion of the brain is shown in (A) and the posterior part is displayed in (B). Abbreviations: Ab l -V, abdominal ganglia I-V; AC, anterior connectives; AL, antennal lobe; bL, beta lobe; aMN, anterior median nerve; CA, mushroom body calyx; CB, central body; COC, circumesophageal connectives; LAL, lateral accessory lobe; Ld, lower division of central body; MAL, median accessory lobe; META, metathoracic ganglion; MN, median nerve; NHO, neurohaemal organ; NV 1-5, nerves 1-5; PC, posterior connectives; PDC, posterior deuterocerebrum; SOG, subesophageal ganglion; StNV, sternal nerve; Tc, tritocerebrum; TgNV, tergal nerve; Ud, upper division of central body.

174 L. Schoofs et al. / Regulatory Peptides 63 (1996) 171-179

least some of these immunoreactive fibers are derived from scattered small cell bodies (6) dorsal to the protocere- bral bridge (Fig. 3c). The Lom-MIP-LI fibers form inter- connections between the central complex neuropil and surrounding non-glomerular neuropils, i.e., the dorsal pro- tocerebral neuropil, adjacent to the pars intercerebralis (Fig. 3d). A dense distribution of Lom-MIP immunoreac- tive fibers was also seen in the non-glomerular neuropils surrounding the mushroom bodies. In this area, numerous dense fibers and varicosities are revealed; they have a beaded appearance and show a strongly stained branching pattern. No immunoreaction could be observed either in the c~- and [3-lobes, the peduncle, the lateral calyces or in the globuli cells of the mushroom bodies.

Two large cell bodies (Fig. 5a) were found on the lateral internal margin of each tritocerebral lobe , which may be part of the 'ventral giants cluster' [13]. Their processes intermingle in the tritocerebral neuropil. Im- munopositive axons connecting the tritocerebrum to the subesoephageal ganglion, can be seen in the circume- sophageal connectives (Fig. 5b).

In the optic lobes no Lom-MIP-LI neurons nor fibers could be observed.

Lom-MIP-LI fibers were observed in the nervi corporis cardiaci (NCC II) nerves to the corpora cardiaca (Fig. 6a,b). The median tract (NCCI) was left unstained (Fig. 3d). Within the corpora cardiaca, an extensive distribution of varicose immunoreactive fibers was detected within the glandular part (CCg). However, in some corpora cardiaca investigated, immunoreactive fibers were restricted to the peripheral zone of the CCg (Fig. 6b), while in other CCg the immunoreactive fibers enter the CCg surrounding the adipokinetic cells (Fig. 6a). This indicates that the labeling is dependent on the physiological conditions of the animal.

Immunopositive fibers were also found in the nervi corporis allati I (NCA I) connecting the CC tot the CA and arborizing there (Fig. 6a,c,d). No immunoreactivity was detected in the NCA II connecting the CA to the suboe- sophageal ganglion. The intrinsic cells of the CA were unstained.

In the subesophageal ganglion, two dorsal midline cell bodies give rise to immunoreactive axons in the median nerve, leading to a neurohaemal organ (Fig. 7a). In the three thoracic and abdominal ganglia, scarce, however, intensively labeled fibers were detected in the neuropils (Fig. 7b-e). The dorsal intermediate tract which runs

~ ! ~ i ! : ~ i ¸̧ :i ii ̧ iii~ ̧ ~ < !ii kj~q~ii! ~ !!!~ii:!i~

Fig. 3. Frontal sections of central part of Locusta brain. (a) Group of immunoreactive cell bodies in pars intercerebralis (PI); Ca, calyx of mushroom body (X 500). (b) Densely stained single immunoreactive cell in pars lateralis (PL), (X 500). (c) Only weakly stained immunoreactive fibers were found in the protocerebral bridge (PB) ( x 600). Note immunoreactive cell body (arrow) innervating the PB fibers. (d) Immunoreactive processes in the protocerebral

neuropil (P). The median tract (MT) was unstained (X 500).

L. Schoofs et al./Regulatory Peptides 63 (1996) 171-179 175

racic ganglion. In the free abdominal ganglia, a cluster of _+ 6 dorsal midline cell bodies send axons to the anterior median neurohaemal organs. Two immunoreactive poste- rior-lateral cells have axons in the sternal nerve (Fig. 7d). The immunoreactive endings (see next section) in the lateral heart nerve might be derived from these neurons. The terminal abdominal ganglion does not contain im- munoreactive cell bodies.

3.3. Immunohistochemical detection of Lom-MIP-like im- munoreactit,e material in peripheral organs

Fig. 4. Lom-MIP-LI fibers in central portion of protocerebrum (× 600). (a) Densely packed immunoreactive fibers in the upper (ud) and lower division (ld) of the central body (CB) and in the median accessory lobe (MAL). The fiber connection between the central body and the left lateral accessory lobe (LAL) can be seen at arrow. (b) More posterior section of central body. Fiber connection with right lateral accessory lobe at arrow.

longitudinally through all three ganglia contains im- munoreactive fibers. Dorsal median cell bodies (3) were only found in the abdominal neuromeres of the metatho- racic ganglion (Fig. 7b), but not in the pro- and mesotho-

In the peripheral muscles investigated (Fig. 8), Lom- MIP-LI fibers were detected in nerves innervating the oviduct, the heart and the hindgut. In the parietal cells of the salivary glands, Lom-MIP-LI material was found in the bifurcating ductule, of which both arms surround the nu- cleus. The testis and male accessory glands were devoid of Lom-MIP-LI material.

4. Discussion

The antiserum raised to Lom-MIP has been shown to react with Lom-MIP and to a lesser extent with APG- Wamide [7], a neuropeptide isolated from the snail Lym- nea stagnalis, which has a Ala-X-Gly-Trp-amide car- boxyterminal in common with Lom-MIP. The antiserum does not crossreact with peptides unrelated to Lom-MIP, such as Lom-TK I, Lom-MT I, locustakinin [2] and Lom- MIH 5-13 [10]. Although there is a small sequence similar- ity in the carboxyterminus with AKH, which ends in Gly-Trp-amide, the antiserum does not crossreact with AKH, since the glandular cells of the corpus cardiacum were immunonegative. Consequently, the antiserum em- ployed here is capable of labeling Lom-MIP in tissue

Fig. 5. Cross-sections (× 600) of tritocerebrum (a) and tritocerebral connectives (b) of Locusta brain. (a) The two large immunoreactive cell bodies (arrow) may be part of the cluster of 'ventral giants (VB1-3) described by Williams (1975). (b) Intensely labeled immunoreactive fiber in tritocerebral connective (arrow).

176 L. Schoofs et al. / Regulatory Peptides 63 (1996) 171-179

sections, but not any of the known unrelated peptides of the locust.

The number of Lom-MIP-LI neurons in the locust brain is quite small. The distribution pattern of Lom-MIP-LI neurons differs from that of other peptide containing neu- rons mapped in the locust brain with antisera to locustamy- otropins [5], locustakinin and locustatachykinins [14], al- latostatins [ 15], locustamyoinhibin 5-13 [ 10] and crustacean cardioactive peptide [16]. It can, however, not be excluded that Lom-MIP-LI material is localized in very small sub- sets of the above peptide containing neurons.

A unique Lom-MIP-LI immunolabeling is demonstrated in the pars lateralis. It reveals a new category of lateral neurosecretory cells, distinct from the allatostatin-LI im- munoreactive cells [15]. The Lom-MIP-LI cells in the pars lateralis present collaterals in the protocerebral neuropiles and, in addition, processes which project within the NCC II and end in the glandular part of the CC. Thus these Lom-MIP-LI endings are localized in close vicinity with the cells which synthesize the adipokinetic hormones [17]. This interesting anatomical Lom-MIP-LI immunolabeling raised the question of the AKH release from the glandular cells. Octopamine and cAMP have been shown to induce release of AKH from isolated corpora cardiaca of Locusta migratoria [18]. Since no specific octopamine immuno- reactivity could be detected in the pars lateralis, nor in the corpus cardiacum [19] and since data from a study by

Passier et al. [20] did not support the stimulating action of octopamine on adipokinetic hormone secretion from the corpus cardiacum, the latter authors concluded that oc- topamine is not the neuroactive compound that initiates the release of AKH. More recently, Locustatachykinin-I, has been shown to induce the release of AKH-I from isolated corpora cardiaca in a dose-dependent manner [21]. Five natural analogs of locustatachykinins were originally iso- lated as myotropic peptides [22,23]. They stimulate con- traction of the Leucophaea hindgut and of the Locusta foregut and oviduct in vitro. Immunocytochemical map- ping of the Lom-TKs in the locust also indicated that these peptides may function as AKH releasing factors. Lom-TK immunoreactive fibers contact the glandular cells of the corpus cardiacum of Locusta migratoria. The immunore- active pattern of Lom-TK-LI immunoreactive fibers in the glandular part is different from the presently detected pattern of axons immunoreactive to the Lom-MIP anti- serum, which seems to be more extensive. This may indicate that Lom-MIP might be a candidate inhibitor in the control of the release of AKH. Future studies will investigate a possible role of Lom-MIP in the regulation of AKH-release.

We also demonstrated the presence of axons in the corpora allata. This might indicate that Lom-MIP is in- volved in the regulation of JH-biosynthesis, of which the nervous control has been demonstrated recently [24]. Lom-

Fig. 6. Corpora cardiaca (CC)-corpora allata (CA). (a) Immunoreactive processes in nervus corporis cardiaci II (NCC II) and numerous immunoreactive endings (arrows) in glandular part (CCg). Immunoreactive fibers in nervus corporis allati I (NCA I) and endings in corpus allatum (CA) (X 500). (b) Glandular part (CCg) of CC of another locust. Immunoreactive fibers only at periphery (× 500). (c,d) Corpora allata with NCAI and immunoreactive endings (arrows) ( x 800).

L. Schools et al. / Regulator), Peptides 63 (1996) 171-179 177

MIP inhibits in vitro juvenile hormone biosynthesis by corpora allata from 3-day-virgin female locusts with a threshold concentration of 10 -6 M (unpublished results). When the CA were still attached to the brain-CC-complex, JH biosynthesis is low and no effect on JH biosynthesis could be observed. The four nonapeptides recently identi- fied in Gryllus bimaculatus, (and designated as Grb-Ast B1-4) are potent inhibitors of JH III biosynthesis by corpora allata from 3-day-old virgin females of G. bimacu- latus using an in vitro bioassay [9]. These four peptides exhibit high sequence homologies to Lom-MIP (Table 1). Synthetic Lom-MIP showed a half-maximal inhibitory ac- tivity on JH-III biosynthesis of crickets at a threshold concentration of 10 -8 M [9]. At this concentration the natural Gryllus peptides display maximal inhibition of JH III biosynthesis, indicating that Lom-MIP and the cricket

Grb-AST-B 1-4 bind with equal efficiency to the allatal receptor but that Lom-MIP causes a weaker response [9].

Lom-MIP was found to suppress the contractions of the locust oviduct in vitro [6]. This finding together with the present immunohistochemical observation of Lom-MIP-LI immunoreactive nerves, innervating the oviduct, suggests that Lom-MIP has probably a reproductive function. Lom- MIP may also be involved in the efferent regulation of muscle activity of the hindgut, hereby increasing the transit time of the gut contents. Indeed, the presence of im- munoreactive fibers in the musculature of the locust hindgut accords with the inhibitory effect of Lom-MIP on the hindgut muscle contraction [6] and with the inhibition of ileal peristalsis of the Mas-MIPs in Manduca sexta [8].

The acini of the salivary glands of the locust consist of two cell types, the central cells that produce secretory

Fig, 7. Lom-MIP-LI in subesophageal ganglion and ventral nerve cord (X 500). (a) Cross-section through subesophageal ganglion with Lom-MIP immunoreactive fibers in median nerve (MN) and neurohaemal organ (NHO). (b,c) Metathoracic ganglion; (b) two immunoreactive dorsal midline cells in abdominal neuromere; (c) scarce immunoreactive fibers in neuropil; (d,e) abdominal ganglion; (d) immunoreactive lateral posterior cell body (arrows). Note immunoreactive fiber in posterior (sternal) nerve (SN). (e) lmmunoreactive arborisations in neuropil.

178 L. Schoofs et al. / Regulatory Peptides 63 (1996) 171-179

Fig. 8. (a) Lom-MIP-LI processes in musculature of Locusta oviduct (arrow) ( X 500). (b,c) ( X 500) Immunoreactive fibers (arrow) innervating the heart (b) and hindgut. (d) Lom-MIP-LI material in bifurcating ductules of parietal cells in acinus (AC) of salivary gland, sd, salivary duct ( X 800).

granules and the parietal cells that do not produce any

secretion [25]. The parietal cells contain an intracellular ductule of which the narrowest ramifications connect with

a vacuolar system belonging to the reticulum. It is in these

ductules that Lom-MIP-L1 material was detected. This type of cells allows processes of active transport as is the case

for the cells of the spermathecae that also contain a ductule. Whether this immunoreactive material is authentic

Lom-MIP remains to be investigated. Nevertheless, it is the first demonstration of peptide-like material in the

ductules of these cells. The presence of similar MIPs in both orthopteran and

lepidopteran species suggests that this peptide family may

be widely distributed among the lnsecta. The peptide appears to have been well conserved during evolution and

possesses a number of functions.

Acknowledgements

We are grateful to Mr Roger Jonckers for insect rearing, to Mrs Julie Puttemans and Mrs Heidi Vanderstukken for photography and Mr. Luc Vanden Bosch and Mr. Louis Freson for technical assistance. This research project was sponsored by the E.C. project N ° TS3* CT 930208. D. Veelaert thanks the Belgian 'Vlaams Instituut voor de

Bevordering van het Wetenschappelijk Technologisch On-

derzoek in de Industrie' (I.W.T.) for providing a Ph.D. scholarship.

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