HAL Id: hal-00891471https://hal.archives-ouvertes.fr/hal-00891471

Submitted on 1 Jan 1997

HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.

Occurrence of a prothoracicotropic hormone-like peptidein the developing nervous system of the honey bee (Apis

mellifera L)Zl Paulino Simões, Ic Boleli, K Hartfelder

To cite this version:Zl Paulino Simões, Ic Boleli, K Hartfelder. Occurrence of a prothoracicotropic hormone-like peptidein the developing nervous system of the honey bee (Apis mellifera L). Apidologie, Springer Verlag,1997, 28 (6), pp.399-409. <hal-00891471>

Original article

Occurrence of a prothoracicotropic hormone-likepeptide in the developing nervous system

of the honey bee (Apis mellifera L)

ZL Paulino Simões IC Boleli K Hartfelder

1 Faculdade de Filosofia Ciências e Letras de Ribeirâo Preto, Universidade de Sâo Paulo,Av Bandeirantes 3900, 14040-901, Ribeirão Preto, SP, Brazil;

2 Zoologisches Institut, Universität Tübingen, Auf der Morgenstelle 28,D-72076 Tübingen, Germany

(Received 21 July 1997; accepted 27 September 1997)

Summary &mdash; An antibody generated against prothoracicotropic hormone (PTTH) of the silkwormBombyx mori reacted with distinct sets of cells in the central nervous system and also in the retrocerebralcomplex of Apis mellifera larvae and pupae. Neurons expressing a Bombyx-PTTH-like peptide werefirst detected in the spinning stage of the last larval instar. Five distinct clusters of brain neuronscomprising a total of 20-24 neurons were stained in this larval stage, in addition to a pair of somatalocated in the labial neuromere and in the first thoracic ganglion. With progressing metamorphosisthis pattern changed. In white-eyed pupal brains only two neurons in the dorsal protocerebrum con-tinued to express a PTTH-like peptide, whereas in the suboesophageal ganglion each neuromerenow contained a pair of immunoreactive neurons. No axonal PTTH immunoreactivity was detected.

PTTH / insect neuropeptide / corpora allata / insect metamorphosis / Apis mellifera

INTRODUCTION

Insect postembryonic development is coor-dinated by precise regulation of the releaseof neurosecretory brain peptides, in partic-ular prothoracicotropic hormone (PTTH).After release into the haemolymph, PTTH

activates the prothoracic glands to synthesizeecdysteroids that in turn induce and syn-chronize molting and metamorphosis in theepidermis and other target tissues (Bollen-bacher and Granger, 1985; Bollenbacher etal, 1993). Structural and functional analy-sis of PTTH has most successfully been per-

* Correspondence and reprintsTel: (55) 16 6023805; fax: (55) 16 6336482; e-mail: zlpsimoe@usp.br

formed in lepidopteran insects, especiallythe silkworm, Bombyx mori, where severalprothoracicotropic peptides have beencloned and sequenced [for a review seeIshizaki and Suzuki (1992)], and expressionpatterns of the PTTH gene have been inves-tigated (Adachi et al, 1994). Bombyx PTTHis now considered to be a new member ofthe vertebrate growth factor superfamily(Noguti et al, 1995). The physiological roleof PTTH in insect development has mainlybeen investigated using the tobacco horn-worm, Manduca sexta, as a model (Gilbert,1989). In this moth, PTTH has been physi-cally characterized (Muehleisen et al, 1994),and a partial amino acid sequence has beenobtained, which showed sequence homol-ogy with cellular retinoid binding proteins,but surprisingly not with Bombyx PTTH(Muehleisen et al, 1993). While differing attheir active sites, Bombyx and ManducaPTTH nevertheless appear to be immuno-

logically related at their N-terminus

(Rybczynski et al, 1996). Prothoracicotropicpeptides have also been partially purifiednot only in other lepidopterans, such as theEuropean cornborer, Ostrinia nubilalis (Gel-man et al, 1992), and the gypsy moth,Lymantria dispar (Kelly et al, 1995), butalso in the cockroach, Periplaneta ameri-cana (Richter, 1992), and in Drosophilamelanogaster (Pak et al, 1992).

Immunocytochemical identification ofPTTH-immunoreactive cells and their axonshas considerably improved our knowledgeon the structural organization of these neu-rons in the neuroendocrine axis of insects

(O’Brien et al, 1988; Mizoguchi et al, 1990;Westbrook et al, 1991; Goltzené et al, 1992;Zitnan et al, 1993; Dai et al, 1994), andadvanced our understanding of the devel-opment of the insect central nervous sys-tem (Westbrook and Bollenbacher, 1990).Furthermore, they provided information onthe programming of diapause, an importantaspect of an insect’s life cycle. In Manducasexta, diapause is induced by a reprogram-ing of a photoperiodic clock (Bowen et al,

1984). This eventually postpones, appar-ently by glia-neuron signaling (Hartfelderet al, 1994), the release of PTTH from theprothoracicotropic neurons.

Embedding postembryonic developmentinto an insect’s life history, thus appears tobe an important aspect of PTTH function.Not surprisingly, therefore, peptides con-trolling activity patterns in the endocrinesystem are also hypothesized to play a rolein another evolutionary highly successfullife history strategy, namely reproductivedivision of labor in social insects resultingfrom the differentiation of morphologicallydistinct castes during metamorphosis. Ecdys-teroid titers have been shown to be caste-

specifically modulated in the honey bee(Rachinsky et al, 1990), with differences inecdysteroid titer reflecting a caste-specificprogram of prothoracic gland activity (Hart-felder, 1993). Since topical applications ofsynthetic juvenile hormone to early fifth-instar worker larvae induced a shift in the

developmental profile of the ecdysteroidtiter from a worker- to a queen-like pattern(Rachinsky and Engels, 1995), and sinceecdysteroids have been shown to control theexpression of specific proteins in the devel-oping honey bee ovary (Hartfelder et al,1995), understanding the regulation of pro-thoracic gland activity becomes an importantissue in caste development. In this study,we report the immunocytochemical identi-fication of neurons expressing a PTTH-likepeptide in the central nervous system andthe retrocerebral complex of the honey beeduring larval and early pupal development.

MATERIALS AND METHODS

Animals and tissue preparation

Last instar larvae and white-eyed pupae wereobtained from colonies of Apis mellifera kept inour experimental apiaries at Tübingen andRibeirão Preto. Entire ventral nerve cords, or

brains with the retrocerebral complex and sub-oesophageal ganglion remaining attached weredissected in sterile saline solution, and immedi-ately fixed in Bouin’s fixative for 2 h at roomtemperature. After fixation, the tissues wererinsed overnight in 0.05 M PBS (phosphate-buffer 0.05 M, pH 7.4, containing 0.15 M NaCland 0.1% sodium azide), dehydrated, and embed-ded in paraplast. Serial 10-&mu;m sections weremounted on gelatin-coated slide, deparaffinizedin xylene, and rehydrated in a graded alcoholseries for immunocytochemical localization ofPTTH.

Immunocytochemistry

Neurons expressing a PTTH-like peptide wereidentified using an antiserum generated in rabbitagainst the N-terminal amino acid sequence ofBombyx mori PTTH (Mizoguchi et al, 1990).Immunocytochemical staining was performedby a peroxidase-antiperoxidase reaction.

Sections were first immersed in 3% H2O2 in100% methanol for 5 min to remove endogenousperoxidase activity. After this treatment, sectionswere washed in PBS-B-T (PBS containing 0.1%BSA and 0.2% Triton X-100), Bombyx mori-PTTH antiserum (diluted 1:200) PBS-B-T) wasadded to the sections for 16 h at 4 °C. Followingthree short PBS-B-T rinses, sections were firstincubated for 60 min at room temperature with a

pig-anti-rabbit IgG antiserum (DAKO A/S,Glostrup, Denmark), and after being washed inPBS-B-T for 10 min they were incubated withperoxidase-antiperoxidase (PAP) complex(DAKO) for 60 min. Following two short rinsesin PBS-B-T and in Tris-HCl (0.05M, pH 7.6),sections were reacted with 0.05% 3.3’-diaminobenzidine tetrahydrochoride (SIGMA)diluted in Tris-HCl containing 0.01% imidazole,0.4% NiCl2 and 0.015% H2O2. The reaction wasterminated after 2-6 min by rinsing the slides inPBS. Alcohol-dehydrated sections were mountedin Entellan (SERVA). Specificity of theimmunoreaction was checked in control experi-ments in which the first antibody specific forPTTH was omitted. Specimens were viewed andphotographed on a Zeiss photomicroscopeequipped with differential-interference-contrastoptics. Drawings were made with a camera lucidaattachment.

RESULTS

The presence of a potentially prothoraci-cotropic peptide in the brain-retrocerebralcomplex and the ventral ganglia of honeybee (Apis mellifera) larvae and pupae cor-responded to the reactivity to an antibodyraised against the 30-kDa PTTH of the silk-worm, Bombyx mori (Mizoguchi et al,1990). It was only at the end of the spin-ning phase in the fifth larval instar that neu-rons started to exhibit PTTH immunoreac-

tivity for the first time (fig 1). In this stage,a PTTH-like peptide was expressed in 20-24somata in the brain. Based on analyses ofserially sectioned brains of worker larvae,these neurons could be grouped into fivedistinct sets in each brain hemisphere(fig 2A). Cluster 1 consists of a singlePTTH-immunoreactive neuron located inthe dorsal protocerebrum dorsally to theupper anterior fold of the lamina gan-glionaris. A group of three neurons repre-senting cluster 2 is in a more medial positionin the protocerebrum, between the laminaand medulla of the expanding optic lobesand adjacent to the developing mushroombody calces. Cluster 3 is less well definedand consists of 2-4 neurons distributed alongthe lateral margin of the optic lobe. Twoneurons situated in the ventromedial proto-cerebrum between the oesophageal foramenand the protocerebral bridge were denomi-nated as cluster 4. Finally, cluster 5 consistsof two PTTH-immunoreactive somata thatare located in the deutocerebrum close tothe developing antennal structures.

In addition to the somata in the larval

brain, PTTH-immunoreactive cells werealso detected in the suboesophageal gan-glion (fig 1D). Serial sectioning revealedthat there are two bilaterally symmetric cells expressing a PTTH-like peptide in the labialneuromere (fig 2B). In the segmental gangliaof the ventral nerve cord, it was only in theprothoracic ganglion that we could detectPTTH-immunoreactive cells (figs 1E and2C). These were found located in a similar

position as the somata in the preceding labialneuromere.

With the first metamorphic molt this pic-ture changed quite dramatically. First, thetotal number of PTTH-immunoreactive cellsin the brain was reduced from 24 neuronsin the brain of spinning-phase larvae to onlytwo neurons in the brain of white-eyedpupae (fig 3). Whether these neurons cor-respond to the cluster 1 somata of the lar-val brain awaits further experimental anal-ysis, because with the extension of the opticlobes and growth of the mushroom bodies,the pupal PTTH-immunoreactive neurons

could have acquired a more medial positionin the dorsal protocerebrum, laterally in thepars intercerebralis. The number of PTTH-immunoreactive neurons in the suboe-

sophageal ganglion also changed after thepupal molt. In addition to a pair of somata inthe labial neuromere, neurons in corre-

sponding postions in the maxillary andmandibular neuromeres also acquired thisphenotype.

Since we could not detect PTTH-immunoreactive material in axons descend-

ing from the immunostained brain somatawe were unable to map the architecture of

these neurons. Hence, information regardingrelease sites of a PTTH-like peptide into thehemolymph from a retrocerebral neurohemalorgan could not be obtained in this study.In serial sections of the corpora cardiacaand corpora allata, we were, however, ableto detect a small number of PTTH-immunoreactive cells located in a bridgestructure that connects the paired corporaallata ventrally of the oesophagus (fig I F).These cells were found directly adjacent tothe corpora allata, close to the anterior endof the aorta.

DISCUSSION

Neurons expressing a PTTH-like peptidewere identified in the brain, the suboe-sophageal neuromeres, and the prothoracicganglion of last instar larval and pupal honeybees. Due to the peptide’s well-documentedcentral function in controlling molting cyclesand metamorphosis in the Lepidoptera, com-parative analyses on occurrence and distri-bution of PTTH-like peptides in the centralnervous system of different orders of insectscould shed light on possible functions of

neurons expressing related peptides in spe-cific insects.

Viewed from this perspective, certainPTTH-immunoreactive cells in the larvaland pupal honey bee brain could be consid-ered as homologs to the functionally anddevelopmentally better described PTTHneurons in moths. Homolgy of the two largePTTH-immunoreactive neurons (cluster 1)in the larval and pupal honey bee proto-cerebrum with the lateral neurosecretorygroup III (L-NSC III) neurons of Manducasexta (Westbrook et al, 1991, 1993), andwith the corresponding neurons in Bombyxmori (Mizoguchi et al, 1990) is suggestedby their localization within the brain, as wellas by the fact that they are the only neuronsin the dorsal protocerebrum that continu-ally produce a PTTH-like peptide. Timing ofappearence of this peptide in the dorsolateralneurons, coincides with increases in the

hemolymph ecdysteroid titer (Rachinsky etal, 1990), as well as in prothoracic glandactivity in honey bee larvae (Hartfelder,1993). Since, however, we could not detectaxonal PTTH-immunoreactive material inthe honey bee brain, functional equivalence

with the lepidopteran prothoracictropic neu-rons is not established, and caution is war-ranted when proposing a role of these neu-rons in the control of ecdysteroid synthesisin the honey bee prothoracic gland. Con-clusions from interspecific comparisons onPTTH function are further complicated bythe apparent lack of conservation of the pep-tide’s structure; Bombyx PTTH, for example,does not stimulate ecdysteroid secretion inManduca sexta prothoracic gland prepara-tions (Muehleisen et al, 1993), even thoughBombycidae and Sphingidae are fairlyclosely related lepidopteran families. Recentelegant experiments by Rybczynski et al(1996) using immuno in vitro assays, how-ever, showed that the antibody against Bom-byx PTTH recognizes Manduca PTTH, andblocks its effects on prothoracic glands.Rybczynski et al (1996) suggest that Bombyxand Manduca PTTH share sequence homol-

ogy at the terminus, but that the putativereceptor binding sites may have diverged,so that the two peptides do not exhibit func-tional interspecific activity. This class ofpeptide may thus be more conserved thanexpected, a fact which is also supported bythe detection of PTTH-immunoreactive neu-rons with the Bombyx PTTH antibody ininsects other than lepidopterans (Zitnan et al,1993), and by the recent three-dimensionalmodels of Bombyx PTTH (Noguti et al,1995).

Functional interpretations from neu-roanatomical evidence have to take intoaccount the possibilty that a number of neu-ropeptides can be coexpressed in function-ally identified neurons. In insects, primeexamples for peptide coexpression are the L-NSC III of Manduca sexta, where a 28-kDa

peptide and Bombyx PTTH are expressedin addition to authentic Manduca PTTH

(Gray et al, 1993, 1994). Similar observa-tions have been made for the ventromedial

neurosecretory cells (VM-NSC). The VM-NSC of Manduca sexta are known to controleclosion activity (Copenhaver and Truman,1986). They express, however, not only

eclosion hormone but also show immunore-

activity against Manduca sexta PTTH(Westbrook et al, 1993). In the honey beelarval brain, two bilaterally symmetric pairsof PTTH-immunoreactive neurons weredetected in the ventromedial protocerebrum(cluster 4 neurons). By position and tempo-ral caracteristics of PTTH-like peptideexpression, these neurons could be

homologs of the Manduca VM-NSC.

The number of PTTH-immunoreactiveneurons in the honey bee brain was observedto change during development. With theprogression of metamorphosis, only oneneuron per brain hemisphere continued toexpress the PTTH phenotype indicating ces-sation of the peptide’s expression in manypupal neurons. Cessation of the synthesisof a particular neuropeptide has been demon-strated for Manduca neurons coexpressingbursicon and a cardioacceleratory peptide(Tublitz and Sylvester, 1990). Similarly,Westbrook et al (1993) report a number ofneurons other than the well-described L-NSC III and the VM-NSC that temporarilyexpress a PTTH-immunoreactive peptide inthe Manduca sexta central nervous system.Interestingly, somata that by position andtheir transitory pattern of PTTH expressioncorrespond to the Manduca neurons couldalso be detected in the honey bee brain.These are the far lateral protocerebral neu-rons (cluster 3), as well as the PTTH-immunoreactive somata in the suboe-

sophageal neuromeres and in the prothoracicganglion. Expression of the PTTH peptide inthese neurons, which do not show any appar-ent relationship to the neuroendocrine axiscontrolling prothoracic gland activity in themoth, has raised the possibility that PTTHmay, in addition to its known steroidogenicfunction, play additional roles, includingneurotransmitter and neuromodulatory func-tions, at different stages in the insect’s lifecycle (Bollenbacher et al, 1993). The timepattern of PTTH expression observed inhoney bee development by neurons situatedin the brain, suboesophageal ganglion and

prothoracic ganglion provides additionalsupport to this hypothesis.

A strict temporal regulation in the expres-sion pattern of a Bombyx-PTTH-like pep-tide has also been observed in Drosophilamelanogaster (Zitnan et al, 1993). PTTH-immunoreactive neurons were not onlydetected in the dorsolateral protocerebrum ofDrosophila but also in the suboesophageal,thoracic, and even in the abdominal neu-romeres. The striking parallels in neu-roanatomical organization and develop-mental programing of PTTH expression inDrosophila with the lepidopteran specieshave now been further corroborated by ourfindings in a third group of holometabolousinsects, a hymenopteran species. The generalpicture emerging for the organization ofpeptidergic neurons expressing a BombyxPTTH-like peptide in these holometabolansclearly differs from the situation describedfor the hemimetabolous orthopteran, Locustamigratoria, where a large number of smallpeptidergic neurons in the pars intercere-bralis showed PTTH-immunoreactivity(Goltzené et al, 1992).

The lack of PTTH-immunoreactive mate-rial in axons of the honey bee brain makes itdifficult to assess which of the neurons

expressing a Bombyx PTTH-like peptideproject into the corpora cardiaca or corporaallata. Absence of axonal immunostaining,however, fits into an emerging picture ofpostembryonic differentiation of the honeybee brain, and corresponds to findingsrecently obtained for a biogenic amine(Boleli et al, 1995) which is capable of stim-ulating juvenile hormone release in vitrofrom corpora allata of Apis mellifera larvae(Rachinsky, 1994). Interestingly, the PTTH-immunoreactive cells in the ventral bridgeconnecting the corpora allata were found ina position quite similar to serotonin-immunoreactive cells (Boleli et al, 1995).Their products could, thus, be directlyreleased into the hemolymph to activate pro-thoracic gland projections that terminate in

the vicinity of the corpora allata (Boleli,unpublished). The question, thus, becomeswhether a fully functional neuroendocrineaxis is actually present in honey bee larvaeand pupae, or whether neurosecretory cellsin the vicinity of the corpora allata functionas regulators of gland activity.

ACKNOWLEDGMENTS

We thank A Mizoguchi (Nagoya, Japan) forkindly providing the antiserum against Bombyxmori PTTH. This research was financially sup-ported by the Deutsche Forschungsgemeinschaft(grant Ha 1625/1-4), and by a CAPES/DAAD-scholarship to ICB.

Résumé &mdash; Présence d’un peptide ana-logue de l’hormone prothoracotrope dansle système nerveux en cours de dévelop-pement de l’abeille (Apis mellifera L).L’expression et la sécrétion de l’hormoneprothoracotrope (PTTH) par les cellules neu-rosécrétrices de la pars intercerebralisjouent un rôle clé dans la mue et la méta-morphose des insectes. Chez les insecteshautement sociaux, la différenciation descastes fait partie intégrante de la métamor-phose et est régulée par une modulation spé-cifique à la caste des teneurs en hormonejuvénile et en ecdystéroïde. Pour com-prendre la structure de la voie neuroendo-crine chez l’insecte social, nous avonsrecherché la présence d’un peptide poten-tiellement prothoracotrope dans le cerveau,le complexe rétrocérébral et les ganglionsventraux des larves et des nymphesd’abeille, à l’aide d’un anticorps dirigécontre la PTTH de 30 kDa du ver à soie,Bombyx mori (Mizoguchi et al, 1990).

La présence d’un peptide analogue, laPTTH, a été détectée dans le cerveau, lesneuromères sub&oelig;sophagiens et le ganglionprothoracique chez les larves de dernierstade et les nymphes d’abeilles. À la fin dela phase de filage du cocon, 20 à 24 neu-rones exprimaient dans le cerveau le pep-

tide analogue à la PTTH. Le nombre de neu-rones immunoréactifs à la PTTH dans lecerveau varie au cours du développement.Après la nymphose, un seul neurone parhémisphère continue à exprimer le phéno-type PTTH, ce qui indique la fin de l’expres-sion du peptide dans de nombreux neuronesnymphaux. Dans le ganglion sub&oelig;sopha-gien aussi, le nombre de neurones immu-noréactifs à la PTTH change après la nym-phose. Le pattern de leurs projections n’apu être tracé car nous n’avons pas pu détec-ter de matériel immunoréactif à la PTTHdans les axones qui partent des corps cellu-laires du cerveau immunomarqué. La ques-tion reste ouverte de savoir si les neuronesexercent une fonction régulatrice dans lamétamorphose de l’abeille et le détermi-nisme des castes ou s’ils acquièrent plustard des fonctions encore inconnues. Ladétection des cellules immunoréactives à laPTTH dans le voisinage des corpora allatasuggère cependant la possibilité que le pep-tide soit mis directement en circulation dans

l’hémolymphe et que la synthèse de l’ecdys-téroïde soit stimulée dans les glandes pro-thoraciques proches.

Apis mellifera / neuropeptide / métamor-phose / hormone prothoracotrope

Zusammenfassung &mdash; Vorkommen einesPTTH-ähnlichen Peptids im sich ent-wickelnden Nervensystem der Honigbiene(Apis mellifera L). Expression und Aus-schüttung des prothorakotropen Hormons(PTTH) durch neurosekretorische Zellen inder Pars intercrebrales hat eine Schlüssel-funktion bei der Häutung und der Meta-morphose von Insekten. Bei den hoch-euso-zialen Insekten ist die Ausbildung derunterschiedlichen Kasten-Phänotypen in dieMetamorphose integriert und wird durchkastenspezifische Modulation der Juvenil-hormon- und Ecdysteroid-Titer reguliert.Um die Struktur der neuroendokrinen Achsebei sozialen Insekten zu verstehen, unter-

suchten wir das Vorkommen eines Peptidsmit möglicherweise prothorakotroper Funk-tion im Gehirn, dem retrocerebralen Kom-plex und den Ventralganglien von Larvenund Puppen der Honigbiene (Apis melliferaL). Die immuncytochemischen Analysenwurden mit einem spezifischen Antikörpergegen das 30 kDa PTTH des Seidenspin-ners Bombyx mori durchgeführt (Mizogu-chi et al, 1990).

Ein PTTH-ähnliches Peptid lie&szlig; sich im

Gehirn, in den Neuromeren des Unter-schlundganglions und im Prothorakalgang-lion nachweisen. Am Ende der larvalen

Spinnphase exprimierten im Bienengehirn20-24 Neurone ein PTTH ähnliches Peptid.Die Zahl der Neurone mit positiver PTTHImmunreaktion änderte sich im weiterenVerlauf der Entwicklung. Nach der Pup-penhäutung zeigte nur noch ein Paar Neu-rone pro Hirnhälfte eine PTTH Immunreak-tion. Nach der Puppenhäutung scheintdemzufolge in den meisten ehemals PTTH-positiven Neuronen die Peptidexpressionvon PTTH eingestellt zu werden. Die Zahlder PTTH-immunreaktiven Neurone im

Unterschlundganglion änderte sich ebenfallsnach der Puppenhäutung. Das Projektions-muster der PTTH-exprimierenden Neuronekonnte jedoch nicht aufkärt werden, da wir inden Axonen dieser Neurone im Gehirn und

Unterschlundganglion kein PTTH-immun-reaktives Material nachweisen konnten. Obdiese Neurone eine regulatorische Funktionbei der Metamorphose und Kastenentwick-lung der Honigbienen haben, oder ob siespäter eventuell andere Funktionen über-nehmen, ist nicht geklärt. Der Nachweis vonPTTH-immunreaktiven Zellen in der Näheder Corpora allata weist jedoch auf die Mög-lichkeit einer Freisetzung solcher Peptide indie Hämolymphe hin, die dann die Ecdy-steroid -Synthese in den benachbarten Pro-thoraxdrüsen stimulieren könnten.

PTTH, Insekten-Neuropeptide / Corporaallata / Insektenmetamorphose / Apis mel-lifera

REFERENCES

Adachi T, Yamada T, Iwami M, Kataoka H, SuzukiA, Ishizaki H (1994) Structure and expression ofthe gene for the prothoracicotropic hormone of thesilkmoth Bombyx mori. Eur J Biochem 220, 633-643

Boleli IC, Hartfelder K, Simoes Z.L.P (1995) Sero-tonin-like immunoreactivity in the central nervoussystem of honey bee (Apis mellifera) larvae andpupae. Zoology 99, 58-67

Bollenbacher WE, Granger NA (1985) Endocrinologyof the prothoracicotropic hormone. In: ComparativeInsect Physiology, Biochemistry, and Pharmacol-ogy (GA Kerkut, LI Gilbert, eds), vol 7, PergamonPress, New York, 109-151

Bollenbacher WE, Gray RS, Muehleisen DP, ReganSA, Westbrook AL (1993) The biology of the pro-thoracicotropic hormone peptidergic neurons in aninsect. Am Zool 33, 316-323

Bowen MF, Saunders DS, Bollenbacher WE, Gilbert LI(1984) In vitro reprogramming of the photoperi-odic clock in an insect brain-retrocerebral com-

plex. Proc Natl Acad Sci, USA, 81, 5881-5884

Copenhaver PF, Truman JW (1986) Identification ofcerebral neurosecretory cells that contain eclosionhormone in the moth Manduca sexta. J Neurosci 6,1738-1747

Dai J-D, Mizoguchi, A Gilbert LI (1994) Immunore-activity of neurosecretory granules in the brain-retrocerebral complex of Manduca sexta toheterologous antibodies against Bombyx protho-racicotropic hormone and bombyxin. InvertebrReprod Dev 26, 187-196

Gelman DB, Thyagaraja BS, Kelly TJ, Masler EP, BellRA, Borkovec AB (1992) Prothoracicotropic hor-mone levels in brains of the European corn borer,Ostrinia nubilalis: diapause vs the non-diapausestate. J Insect Physiol 38, 383-395

Gilbert LI (1989) The endocrine control of molting:the tobacco hornworm, Manduca sexta, as a modelsystem. In: Ecdyson (J Koolman, ed), Thieme,Stuttgart, 448-471

Goltzené F, Holder F, Charlet M, Meister M, Oka T(1992) Immunocytochemical localization of Bom-byx-PTTH-like molecules in neurosecretory cells ofthe brain of the migratory locust, Locusta migra-toria. Cell Tissue Res 269, 133-140

Gray RS, Muehleisen DP, Katahira EJ, BollenbacherWE (1993). A 28-kDa neuropeptide from Mand-uca sexta: relationship to insect prothoracicotropichormone. Cell Mol Neurobiol 13, 39-57

Gray RS, Muehleisen DP, Katahira EJ, BollenbacherWE (1994) The prothoracicotropic hormone(PTTH) of the commercial silkmoth, Bombyx mori,in the CNS of the tobacco hornworm, Manducasexta. Peptides 15, 777-822

Hartfelder K (1993) Structure and function of the pro-thoracic gland in honey bee (Apis mellifera L)development. Invertebr Reprod Dev 23, 59-74

Hartfelder K, Hanton WK, Bollenbacher WE (1994)Diapause-dependent changes in prothoracicotropichormone-producing neurons of the tobacco horn-worm Manduca sexta. Cell Tissue Res 277, 68-78

Hartfelder K, Köstlin K, Hepperle C (1995) Ecdys-teroid-dependent protein synthesis in caste-specificdevelopement of the larval honey bee ovary. Roux’sArch Dev Biol 205, 73-80

Ishizaki H, Suzuki A (1992) Brain secretory peptidesof the silkworm, Bombyx mori: Prothoracicotropichormone and bombyxin. In: Progress in BrainResearch (J Joose, RM Buijs, FJH Tilders, eds),vol 92, Elsevier, Amsterdam, 1-14

Kelly TJ, Thyagaraja BS, Bell RA, Masler EP (1995)A novel low molecular weight ecdysiotropin inpost-diapause, pre-hatch eggs of the gypsy moth,Lymantria dispar L. (Lepidoptera: Lymantriidae).Regulat Peptides 57, 253-261

Mizoguchi A, Oka T, Kataoka H, Nagasawa H, SuzukiA, Ishizaki H (1990) Immunohistochemical local-ization of prothoracicotrpic hormone-producingneurosecretory cells in the brain of Bombyx mori.Dev Growth Diff 32, 591-598

Muehleisen DP, Gray RS, Katahira EJ, Thomas MK,Bollenbacher WE (1993) Immunoaffinity purifi-cation of the neuropeptide prothoracicotropic hor-mone from Manduca sexta. Peptides 14, 531-541

Muehleisen DP, Katahira EJ, Bollenbacher WE (1994)Physical characteristics of the cerebral big protho-racicotropic hormone from Manduca sexta. Expe-rientia 50, 159-163

Noguti T, Adachi-Yamada T, Katagiri T, Kawakami A,Iwami M, Ishibashi J, Kataoka H, Suzuki A, GoM, Ishizaki H (1995) Insect prothoracicotropic hor-mone: a new member of the vertebrate growth fac-tor superfamily. FEBS Lett 376, 251-256

O’Brien MA, Katahira EJ, Flanagan TR, Arnold LW,Haughton G, Bollenbacher WE (1988) A mono-clonal antibody to the insect prothoracicotropichormone. J Neurosci 8, 3247-3257

Pak JW, Chung KW, Lee CC, Namkoong KY, Kool-man J (1992) Evidence for multiple forms of theprothoracicotropic hormone in Drosophilamelanogaster and indication of a new function. JInsect Physiol 38, 167-176

Rachinsky A (1994) Octopamine and serotonin influ-ence on corpora allata activity in honey bee (Apismellifera) larvae. J Insect Physiol 40, 549-554

Rachinsky A, Engels W (1995) Caste development inhoneybees (Apis mellifera): juvenile hormone turnson ecdysteroids. Naturwissenschaften 82, 378-379

Rachinsky A, Strambi C, Strambi A, Hartfelder K( 1990) Caste and metamorphosis: hemolymph titersof juvenile hormone and ecdysteroids in last instarhoney bee. Gen Comp Endocrinol 79, 31-38

Rembold H (1987) Caste-specific modulation ofjuve-nile hormone titers in Apis mellifera. Insect Biochem17, 1003-1006

Richter K (1992) Prothoracicotropic activity in thebrain of the cockroach Periplaneta americana. JInsect Physiol 38, 349-355

Rybcynski R, Mizoguchi A, Gilbert LI (1996) Bom-byx and Manduca prothoracicotropic hormones: animmunological test for relatedness. Gen CompEndocrinol 102, 247-254

Tublitz NJ, Sylvester AW (1990) Postembryonic alter-ation of transmitter phenotype in individually iden-tified peptidergic neurons. J Neurosci 10, 161-168

Westbrook AL, Bollenbacher WE (1990) The devel-opment of identified neursecretory cells in the

tobacco hornworm, Manduca sexta. Dev Biol 140,291-299

Westbrook AL, Haire ME, Kier WM, BollenbacherWE ( 1991 ) Three-dimensional achiteture of iden-tified cerebral neurosecretory cells in an insect. J

Morphol 208, 161-174

Westbrook AL, Regan SA, Bollenbacher WE (1993)Developmental expression of the prothoracicotropichormone in the CNS of the tobacco hornwormManduca sexta. J Comp Neurol 327, 1-16

Zitnan D, Sehnal F, Brian PJ (1993) Neurons produc-ing specific neuropeptides in the central nervoussystem of normal and pupariation-delayedDrosophila. Dev Biol 156, 117-135