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Changes in integument structure during the imaginalmolt of the honey bee

Moysés Elias-Neto, Michelle P.M. Soares, Márcia M.G. Bitondi

To cite this version:Moysés Elias-Neto, Michelle P.M. Soares, Márcia M.G. Bitondi. Changes in integument structureduring the imaginal molt of the honey bee. Apidologie, Springer Verlag, 2009, 40 (1), pp.29-39.�hal-00891973�

Apidologie 40 (2009) 29–39 Available online at:c© INRA/DIB-AGIB/ EDP Sciences, 2008 www.apidologie.orgDOI: 10.1051/apido:2008064

Original article

Changes in integument structure during the imaginal moltof the honey bee*

Moysés Elias-Neto, Michelle P.M. Soares, Márcia M.G. Bitondi

Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto -Universidade de São Paulo, Avenida Bandeirantes 3900, 14040-901 Ribeirão Preto, SP, Brazil

Received 3 April 2008 – Revised 2 September 2008 – Accepted 29 September 2008

Abstract – The changing pattern of developing cuticle and associated epidermis is described during theimaginal molt in the honey bee. Observations began immediately after the pupal molt, and included histo-logical analyses of the integument during apolysis and the subsequent deposition and differentiation of theadult cuticle. Apolysis coincides with a marked increase in the thickness and reorganization of the epider-mal layer, reflecting changes in cell structure. The epidermis remains thickened during the period of cuticledeposition, suggesting intense biosynthetic activity, but turns into a very thin layer during cuticle differen-tiation, clearly indicating that secretory activity for cuticle formation is terminating. The thoracic cuticledifferentiates earlier and becomes thicker than the abdominal. The observed changes in integument struc-ture provide insights that permit an improved physiological characterization for staging pupal and pharateadult development.

Apis mellifera / integument / metamorphosis / adult cuticle / exoskeleton

1. INTRODUCTION

The recent publication of the genome se-quence of the honey bee Apis mellifera L. (TheHoneybee Genome Sequencing Consortium,2006), and the finding that it has a smallernumber of genes encoding cuticular proteinsthan the Drosophila, Anopheles and Triboliumgenomes, raised questions about the evolutionof exoskeleton in social insects. The smallernumber of cuticle protein genes in the honeybee has been tentatively assigned to its socialorganization within a protective colony, a fea-ture perhaps linked to the development of aless complex cuticle. Honey bee genome dataare now motivating studies on the molecularstructure of the integument, i.e., epidermis andassociated cuticle. Three novel cuticle proteingenes were recently identified in the honey bee

Corresponding author: M. Elias-Neto,meliasneto@pg.ffclrp.usp.br* Manuscript editor: Yves Le Conte

genome (Kucharsky et al., 2007) in additionto the 28 sequences previously annotated byThe Honeybee Genome Sequencing Consor-tium (2006). We showed that expression of oneof these cuticular protein genes, AmelCPR14,was shown to be associated with metamorphicand imaginal molting events for cuticle re-newal and differentiation (Soares et al., 2007).

Studies on the expression and regulationof cuticle protein genes need to be linked tomacro and microscopic structural changes thatthe integument undergoes during molting cy-cles. Such knowledge is important in the con-text of functional genomics. However, thereare not detailed studies illustrating the trans-formations occurring in the morphology of theepidermis as the adult cuticle is synthesizedand deposited. The exception is a publicationby Thompson (1978), who used traces anddots in simple diagrams to represent the pro-gressive deposition of cuticle layers in devel-oping bees. The current study was set up tohistologically describe changes in integument

Article published by EDP Sciences

30 M. Elias-Neto et al.

during a molting cycle. We chose to focus onthe pupal-imaginal molt because it involvesthe remarkable events of cuticle pigmenta-tion and intense sclerotization needed for thedifferentiation of the adult exoskeleton. Mi-croscopical information obtained from honeybee integument sections was contrasted withmacroscopic, progressive external modifica-tions, especially pigmentation occurring in thecuticle. The derived information associates thechanging morphological pattern of the integu-ment with pupal-imaginal molt events, and isof interest for studies on molecular aspectsof exoskeleton morphogenesis, now facilitatedby the publication of the honey bee genome.In this context we also discuss a proposal tostandardize the terminology applied to the dif-ferent stages of the honey bee life cycle.

2. MATERIALS AND METHODS

2.1. Honey bees

Developing and adult honey bee workers werecollected from Africanized stocks in hives main-tained at the experimental apiary of the São PauloUniversity in Ribeirão Preto, SP, Brazil. Develop-mental stages and phases were identified follow-ing criteria established by Michelette and Soares(1993). Briefly, the pupal and successive pharateadult phases were removed from sealed brood cellsand categorized in seven groups according to the ab-sence, or presence and progressive intensification ofeye color and thoracic pigmentation. Adults werecollected soon after they emerged from brood cells.Pupae, pharate adults and adults were used to in-vestigate the morphological changes in integumentshowed in Figures 1–4, and also to illustrate Fig-ure 5, which also includes 5th instar larvae and de-veloping pharate pupae. Larvae in the 5th instarwere collected from sealed brood cells at the endof the spinning phase, when their guts were com-pletely empty. Initial pharate pupae were recog-nized by the presence of exuvial fluid in the head re-gion. The next phase (intermediary pharate pupae)was identified by the presence of cephalic and tho-racic appendages, perceptible underneath the outer-most larval cuticle. The presence of a wrinkled andopaque cuticle and of a marked constriction delim-itating thorax and abdomen was used as a referenceto identify the last pharate pupal phase.

2.2. Integument preparations for lightmicroscopy

2.2.1. Histological sections

Histological sections were prepared using in-tegument dissected from the dorsal-medial regionof the thorax, and from the medial region of the3rd abdominal tergite of worker bees. At least threespecimens from each sequential phase of the pupal-imaginal development were dissected for histolog-ical sections, and the best integument preparationsare presented. After brief rinses with Ringer saline(NaCl 0.17M; KCl 0.01M; CaCl2 0.003M), fat bodyadhering to the epidermis was removed as far aspossible, and integuments were kept for 24 hoursin cold (4 ◦C) fixative (4% paraformaldehyde in0.1M phosphate buffer, pH 7.3). Next, integumentswere dehydrated in a graded ethanol series (70, 80,90 and 95% ethanol in water, v/v), and then infil-trated for 24 hours and embedded in methacrylateresin (Historesin, Leica). Sections of 5 μm werestained with methylene blue and basic fuchsin for3 minutes, followed by a rapid washing in distilledwater (Hartfelder and Steinbrück, 1997). Sectionswere mounted in Entellan (Merck) and examinedand photographed using an Axioskop II photomi-croscope (Zeiss).

2.2.2. Whole mounts

Thoracic and abdominal integuments were dis-sected from the bee dorsum, gently rinsed in Ringersaline, cleaned of excess fat body and mountedon slides using pure glycerol. These whole mountswere immediately examined and photographed. Atleast three specimens from each developmentalphase in the pupal-imaginal molting interval weredissected and the best whole mounts preparationsare shown.

3. RESULTS

3.1. Deposition and maturationof the adult cuticle in the honey bee

Sections of the thoracic and abdominal in-tegument (Figs. 1 and 2) show the gradualchanges undergone by cuticle and underlyingepidermis during a complete molting cycle.The schematic drawings shown on the left and

Honey bee integument structure during imaginal molt 31

Figure 1. Integument of the honey bee (A) before and (B) after imaginal apolysis. Thoracic (above) andabdominal (below) integument sections of a white-eyed pupa (represented at the left) and of a pink-eyedpharate adult (at the right). Schematic representations of pupa and pharate adult modified from Costa et al.(2006). cut – cuticle; ep – epidermis; n – nucleus; mf – molting fluid.

right sides of the histological sections repre-sent the developmental stages from which theintegument pieces were dissected.

During pupal ecdysis, the shedding of thelarval cuticle causes the externalization of thesoft and unpigmented pupal cuticle that over-lies a thick epidermis. In this stage, the nu-clei of the epidermal cells are localized api-cally, and in the basal cytoplasm appear largeclear areas, perhaps representing stores of se-cretions to be used in the synthesis of moltingfluid and/or cuticular compounds (Fig. 1A). Afew hours later, pigments begin to appear inthe eyes of the developing pupae, changingfrom a white to a light pink coloration (com-pare schemes of developing bees at the sides ofFig. 1). This coincides with the separation ofthe pupal cuticle from the epidermis, or apol-ysis. In consequence, a space filled with molt-ing fluid is created between the detached pupalcuticle and the epidermis (Fig. 1B). Apolysisoccurs simultaneously with a reorganization inthe epidermal layer which now forms a pseu-dostratified epithelium that begins to synthe-size the adult cuticle. From this moment toadult ecdysis the developing adult is still en-closed by a pupal cuticle. Therefore, it is fre-quently referred to as a pharate adult.

The clear areas previously concentrated atthe basal cytoplasm of the epidermal cells in

newly ecdysed pupae (Fig. 1A) are now pul-verized throughout the apical cytoplasm ofepidermis in pink-eyed pharate adults, sug-gesting transport of material from the basal tothe apical cytoplasm (Figs. 2A, B arrows). Thethoracic epidermis becomes very thick in pink-eyed pharate adults. The abdominal epidermisis thinner than the thoracic and still retainsmost of the clear areas in the basal cytoplasm(Fig. 2A). Deposition of the adult cuticle is ini-tiated in pink-eyed pharate adults (Fig. 2A).In the next developmental phase, dark-pink-eyed pharate adults, a well-defined adult cu-ticle now covers the thoracic epidermis. Thedeposition of the thoracic cuticle is advancedcompared to the abdominal one (Fig. 2B). Amass of myocytes (Fig. 2B) appears below thethoracic epidermis.

The thickness of the thoracic cuticle in-creases as development goes on, although theabdominal cuticle still remains relatively thin(Fig. 2C). The pharate adult now has in-tensely pigmented brown eyes (scheme of abrown-eyed pharate adult shown at the left ofFig. 2C). Newly formed imaginal striated mus-cles are abundant below the epidermis of tho-racic sections.

In the thoracic cuticle, the onset of pig-mentation (represented by a colored mark onthe thorax of a pharate adult at the left side

32 M. Elias-Neto et al.

Figure 2. Adult cuticle deposition and maturation in developing pharate adults. Sections of thoracic andabdominal integuments are shown above and below in each figure, respectively. Beginning (A) and pro-gression (B, C) of cuticle deposition, and maturation (D–F). Pharate adults from which integuments weredissected for histological analyses are schematized at the laterals of figures. Cuticle deposition occurs inearly pharate adult phases when developmental progress is marked by a change in eye color, from pink,to dark pink, and finally to the definitive brown color (early pharate adult development represented at theleft of figures A–C). Cuticle maturation occurs during the late phases of pharate adult development, coin-ciding with cuticle pigmentation (late pharate adults with a mark on the thoracic cuticle representing theprogression of cuticle pigmentation are shown at the left of figures D–F). Arrows indicate the clear areasin the apical cytoplasm (see item 3.1). cut – cuticle; ep – epidermis; mus – muscle; fb – fat body; myo –myocytes.

Honey bee integument structure during imaginal molt 33

Figure 3. Pattern of cuticle pigmentation during pharate adult development. Pharate adults from whichthe integuments were dissected for whole mount preparation are schematized at the left of whole mounts.Thoracic and abdominal integuments are shown above and below in each figure, respectively. Cuticle ofpharate adults in initial development (A–C) is devoid of pigments. Pigment formation initiates earlier inthorax than in abdomen (D–F) at the second half of the pharate adult development. Branched setae protrudefrom setal sockets in the thoracic and abdominal cuticles.

of Fig. 2D) apparently marks the transitionfrom cuticle deposition to its differentiation.As thoracic cuticle pigmentation intensifies(as represented in the schemes at the leftside of Figs. 2E, F), the epidermis becomesvery thin, mainly in the thorax, where it canhardly be visualized. Epidermis is also con-verted into a thin layer in the abdominal region(Figs. 2E, F).

Thoracic muscles that initially were onlyloosely associated with the integument(Fig. 2D), now become tightly connected(Figs. 2E, F). At the end of the pharate adultdevelopment (Figs. 2E, F), the thoracic cuticleis characterized by two differentially stained

layers (pink and blue) suggesting distinctchemical compositions. Both layers are partof the procuticle, which is the major cuticlecomponent formed mainly by interlacedproteins and chitin.

Figure 3 shows the onset and progress ofcuticle pigmentation in whole mount prepa-rations. Between the pink-eyed (Figs. 3A, B)and the early brown-eyed pharate adult phases(Fig. 3C), pigments could not yet be discernedmacroscopically in the thoracic or abdominalcuticle, but they became detectable in the tho-racic cuticle of the subsequent pharate adultphase (Fig. 3D), with a rapid increase in theamount of pigmented areas as development

34 M. Elias-Neto et al.

Figure 4. Pattern of cuticle pigmentation and sclerotization in adults. Whole mount preparations (at the left)and histological sections (at the right) of thoracic (above) and abdominal (below) integuments of an adultworker bee (shown in the center of the figure). cut – cuticle; ep – epidermis; mus – muscle.

advances (Figs. 3E, F). Pigmentation initiateslater in abdominal cuticle, only when the tho-rax is already darkened (compare Figs. 3D, E).

Cuticle tanning also continues in the honeybee after adult ecdysis. Whole mount prepara-tions of adult cuticle (Fig. 4, left side) clearlyshow that it is much darker in comparison tothe cuticle of later pharate adults (Fig. 3F).In histological sections of the adult cuticle wecould observe the presence of a very dark layer(Fig. 4, right side), not seen in the cuticle oflater pharate adults (Fig. 2F).

4. DISCUSSION

4.1. Changes in honey bee integumentduring a molting cycle

The temporal sequence of integument sec-tions revealed considerable changes in epider-mis and cuticle structure during the imagi-nal molt. Pupal epidermis contains large clearareas in the basal cytoplasm, but follow-ing pupal/adult apolysis, clear inclusions alsoappeared pulverized throughout the apical cy-toplasm. Studies carried out in the epidermisof a lepidopteran, Calpodes ethlius, revealedapical and basal peptide trafficking routes for

secretion into the cuticle and hemolymph, re-spectively. However, it appears that basally lo-calized peptides may be firstly secreted api-cally, and then endocytosed into vesicles to betransported via a basal route to the hemolymph(Locke, 2003). Our preparations do not makepossible to identify the nature and destinationof the material forming the clear areas in theepidermal cell cytoplasm. Considering that anew cuticle is being deposited following apol-ysis, it is possible that such material is beingtransported from the basal to the apical cyto-plasm to be used in cuticle formation. Alter-natively, as this material is abundantly presentin epidermal cells before and during formationof the ecdysial space, it is possible that it isdischarged to fill this space as molting fluid.At a molting stage that precedes the detach-ment of epidermis in the crab Carcinus mae-nas, similar clear areas appear at the apicesof the epidermal cells. They were identifiedas membrane-bounded large vacuoles openedat the cell surface, and their profiles stronglysuggested that they contribute to molting fluidformation (Compère et al., 1998).

Our results show that apolysis is marked byan increase in the thickness of the epidermallayer, which also change cell shape. In fact, ininsects in general, apolysis has been associated

Honey bee integument structure during imaginal molt 35

Figure 5. Metamorphic and imaginal molts in the honey bee. (A) Life stages (larval, pupal and imagi-nal) are delimited by ecdyses (arrowheads) and defined by the outermost cuticle; (B) Ontogenetic instars(larva, pupa and adult) is delimited by apolyses (arrows) and defined by the innermost cuticle (adhered tothe epidermis). During the pharate period, which is extended from apolysis to ecdysis, the new cuticle issynthesized and differentiates underneath the cuticle of the preceding instar; (C) Photographs of the de-velopmental series: larva, pharate pupae, pupa, pharate adults and adult; (D) Schematic representation ofpharate adults showing the morphological attributes (eye color and thoracic pigmentation) used to recog-nize successive developmental phases. To facilitate comparisons, pharate adult representations are the sameshown in the sides of Figures 1, 2 and 3.

with mitosis and expansion of the volume ofepidermal cells (Hepburn, 1985). Followingthe period of cuticle deposition during the firsthalf of pharate adult development in workerbees, the epidermis becomes very thin indicat-ing that its biosynthetic activity for cuticle for-mation is subsiding. This change in morpho-logical pattern reflects the transition from theperiod of biosynthesis of the adult cuticle to itsdifferentiation, which is visually marked by aprogressive pigmentation.

As cuticle is deposited in the dark-pink-eyed pharate adults, a mass of muscle cellprecursors (myocytes) appears in the thoracicsections. In brown-eyed pharate adults, mus-cle formation is in progress and fibers de-rived from myocytes come in contact withthe epidermis for attachment on the cuticle.The thoracic integument sections of develop-ing pharate adults (see Fig. 2) show the re-building of the thoracic musculature in sub-stitution of the larval, which is completelyhistolyzed during honey bee metamorphosis(Oertel, 1930). Muscle fibers are not seen inabdominal (see Fig. 2) sections because histo-

logical preparations did not include the inter-segmental grooves where the abdominal longi-tudinal muscle fibers are attached (Snodgrass,1956), but only the medial region of the seg-ment (3rd tergite), deprived of attached mus-cles.

Cuticle is a multilayered structure formedby an outermost envelope (which serves toprotect the epidermis from molting fluid en-zymes during cuticle renewal), an interme-diary chitin-free epicuticle and the most in-ternal procuticle, the thickest layer, made upmainly of proteins and chitin (Locke, 2001;Willis et al., 2005). This layer is in part se-creted during the pharate stage, and in part af-ter ecdysis. Pre-ecdysially secreted procuticleis termed exocuticle, and the post-ecdysial se-cretion forms the endocuticle (Andersen et al.,1995). Our integument preparations do notpermit us to distinguish the thinner envelopeand the epicuticle, but the pink and blue lay-ers seen in histological sections (Figs. 1, 2) areboth components of the procuticle. The greatincrease in procuticle thickness during honeybee pharate adult development indicates that a

36 M. Elias-Neto et al.

very major part is deposited before adult ecd-ysis, mainly during the first half of pharate de-velopment.

We observed that the process of procuticlemelanization, which accompanies the harden-ing (sclerotization) of the newly forming ex-oskeleton, continues and intensifies even afterthe eclosion of the adult. This is in accordancewith studies on chemical and mechanical prop-erties of the honey bee cuticle (Thompson andHepburn, 1978; Andersen et al., 1981), whichdemonstrated that both deposition of materi-als, including melanin, and reactions for cu-ticle stabilization continue well into the adultstage. However, when the degree of sclerotiza-tion was estimated by measuring the amountsof ketocatechols released from abdominal cu-ticle by acid hydrolysis, the highest amountwas found in the late pharate adults, and theselevels did not increase after eclosion (An-dersen et al., 1981). Although ketocatecholquantification is a useful method to measuresclerotization, it only quantifies ketocatecholsderived from the sclerotizing precursors N-acetyldopamine and N-β-alanyldopamine. It islikely that other molecules have a role as cuti-cle sclerotization precursors (Andersen, 2005).

In the honey bee, the rates of depositionand maturation of the thoracic and abdomi-nal cuticles are very distinct. Thoracic cuticleis deposited earlier than the abdominal one,and rapidly forms a thick layer whereas theabdominal cuticle is deposited more slowly,and is thin. Thoracic and abdominal cuti-cles have different origins and this may ex-plain their distinct rate of development andmaturation. In Drosophila melanogaster, itis well established that imaginal discs origi-nate the thoracic cuticle and histoblast nestsin abdomen give rise to the abdominal cuti-cle (Fristrom and Fristrom, 1993). The factthat the honey bee abdominal cuticle is thin-ner than the thoracic one was previouslyobserved by Thompson (1978) who illus-trated cuticle layers deposition using simpleschematic diagrams. The sequential histolog-ical sections shown here detail the transforma-tions occurring in epidermis as the adult cuti-cle is deposited and differentiates, thus addingnew information on the changing pattern of

epidermis and cuticle during pupal and pharateadult development.

4.2. Considerations on the terminologyused to describe honey bee life cyclestages

The life cycle of the honey bee has beendescribed under different perspectives, allhaving in common suggestions or propos-als of criteria for the recognition of sequen-tial phases of postembryonic development(Bertholf, 1925; Oertel, 1930; Myser, 1954;Snodgrass, 1956; Jay, 1963; Thompson, 1978;Rembold et al., 1980; Michelette and Soares,1993; Nunes-Silva et al., 2006). Notwith-standing, there is still some confusion andinconsistency regarding the terminology usedto describe honey bee stages. Based on thesequential integument sections and wholemounts shown in the current work, Figure 5includes a proposal to make such descriptionsmore uniform or at least clearer. This figureassociates the sequential changes in externalmorphology with the events (apolysis and ecd-ysis) of pupal and imaginal molts and ex-plains the terminology used here to discrim-inate the developmental phases of the honeybee worker. It is clear in this figure that theterm stage is related to the time interval be-tween two subsequent ecdyses (indicated byarrowheads in Fig. 5), i.e., the shedding of thatportion of the outermost cuticle which cannotbe broken down by chitinases and other en-zymes from the molting fluid. The stage (lar-val, pupal or adult) is defined by the type of theoutermost cuticle. Consequently this definitionof stage does not take into consideration thatmajor components of the cuticle of the subse-quent stage are being synthesized beneath andunder the protective cover of the old cuticle.

Apolysis signalizes the beginning of thesynthesis of a new cuticle and, for many insectphysiologists it is a more appropriate parame-ter to delimitate developmental phases. This isclearly expressed by Jones (1978), for whomsubsequent apolyses (indicated by arrows inFig. 5) delimitate an instar, which is definedby the innermost (new) cuticle adhering to theepidermis, even while the instar is still hidden

Honey bee integument structure during imaginal molt 37

(pharate) beneath the old cuticle that soon willbe shed. Although apolysis is a key event inarthropod molting cycle, its utilization as amarker of instar transition has been largely ne-glected in the honey bee literature, especiallywhen referring to the pupal-adult transition.Thompson (1978) pioneered the use of apol-ysis as a marker to separate pupal from adultdevelopment in honey bees. As show in Fig-ure 5, we also consider the importance of apol-ysis and favor the use of the term “pharate” be-cause it best describes the postapolytic stageswhen a new cuticle is being synthesized be-neath the old one.

In the larval-pupal molt, the period betweenthe separation (apolysis) of the old (larval)cuticle from the epidermis until the sheddingof this cuticle is frequently called prepupal.One can also propose the utilization of theterm “pharate pupa”, instead of the inappro-priate “pre-pupa”. According to Snodgrass(1956): “The so-called propupa... is merely thepupa in its early developmental stages withinthe moulted skin of the larva” (“moulted”is the term that the author used to informthat apolysis has already occurred). Later, thiswas repeated “When the last larval cuticle ismoulted, the insect is no longer a larva, thoughbefore ecdysis it is still within the larval cuti-cle” (Snodgrass, 1960). Except for Thompson(1978), who used “pharate pupa” to describethis phase, all other authors listed above (inthe first paragraph of this section) have pre-ferred to use the terms prepupa or pro-pupa.Jay (1963) actually used both terms, prepupaand pharate pupa synonymously.

The pupal-adult transition is more compli-cated in terms of cuticle events, since apolysisof the pupal cuticle from the underlying epi-dermis already occurs when eye coloration isgradually changing from white. From a cuti-cle point of view, the pink-eyed pupa wouldthus mark the end of the pupal instar, being fol-lowed by the pharate adult instar during whichthe new imaginal cuticle is laid down and grad-ually gains in pigmentation. However, this de-veloping adult still covered by the pupal cuti-cle is frequently designated as a pupa.

Following this line of argument, the termpharate adult should be preferred over the term

pupa to designate developing adult bees stillinvolved by the pupal cuticle.

As mentioned above, apolysis and the on-set of the synthesis of the adult cuticle wereverified in pink-eyed pharate adults. Pre-cisely at this developmental phase (then calledpink-eyed pupae) a peak of ecdysteroids wasdemonstrated in hemolymph using radioim-munoassay (Pinto et al., 2002). Therefore,a morphological character, pink eyes, wasdefinitively associated with the beginning ofthe production of a new cuticle, an event trig-gered by elevated ecdysteroid levels.

The data presented here and the discussionof terminology applied to the stages of thehoney bee life cycle should be useful to futurestudies on physiology, biochemistry, genetics,and functional genomics related to moltingevents and adult exoskeleton formation.

ACKNOWLEDGEMENTS

Financial support was provided by a grant fromFAPESP (05/03926-5) and by a FAPESP fellowshipto M. Elias-Neto (05/03301-5). We thank K. Hart-felder for critically reading a previous version of themanuscript. We also thank Z.L.P. Simões for inter-esting suggestions regarding histology techniques,and L.R. Aguiar for technical assistance in the api-ary.

Modifications dans la structure du tégument aucours de la mue imaginale de l’Abeille domes-tique.

Apis mellifera / métamorphose / exosquelette /cuticule / tégument / histologie

Zusammenfassung – Änderungen in der Inte-gumentstruktur im Verlauf der Imaginalhäu-tung der Honigbiene. Zur Unterstützung von Un-tersuchungen zur differentiellen Expression vonCuticula-Proteine kodierenden Genen beschreibenwir hier die Dynamik der Bildung der Adultcuti-cula. Wir konzentrieren uns hier auf die Häutungvon der Puppe zur Imago, da diese bemerkenswer-te Prozesse der Cuticulapigmentierung und Skle-rotisierung beinhaltet, die für die Differenzierungdes adulten Exoskeletts wichtig sind. Integument-stücke wurden aus dem Thorax und dem Abdo-men entnommen, in Ringerlösung gewaschen undin Paraformaldehyd fixiert. Nach der Entwässerung

38 M. Elias-Neto et al.

wurden sie in Methacrylharz eingebettet. Histologi-sche Schnitte wurden mittels Methylenblau und ba-sischem Fuchsin gefärbt. Wir fertigten auch Ganz-präparate an, die in Glyzerin montiert wurden.Epidermiszellen von frischgehäuteten weissäugigenPuppen zeigen einen apikal gelagerten Kern undein basalseitiges Cytoplasma mit klaren Bereichen(Abb. 1A). Das eigentliche Puppenstadium ist sehrkurz, da die Apolyse bereits einsetzt, sobald dieAugen der Puppen eine leicht rosa Färbung zeigen(Abb. 1B) Mit der Apolyse geht eine Umorganisa-tion der Epidermis in ein pseudostratifiziertes Epi-thel und dem Beginn der Synthese der Adultcuticulaeinher. Während die thorakale Epidermis von rosa-äugigen Pharatadulten bereits vergleichsweise dickist, ist die abdominal Epidermis noch dünn und we-nig differenziert (Abb. 2A).Die klaren Bereiche, die in frischgehäuteten Pup-pen zunächst auf die Basalseite der Epidermiszel-len beschränkt waren sind jetzt auch im apikalenBereich klar zu sehen, was auf Materialtransport-vorgänge von der basalen zur apikalen Epithelseitehindeutet (Pfeile in Abb. 2A und B). In Puppen mitdunkelrosa gefärbten Augen ist die Ablagerung derthorakalen im Vergleich zur abdominalen Cuticulabereits weit fortgeschritten (Abb. 2B). Bei braunäu-gigen Pharatadulten ist der Thorax von einer dickenCuticula bedeckt (Abb. 2C), während auch hier dieabdominal Cuticula noch dünn ist. (Abb. 2C). Unterder thorakalen Cuticula ist jetzt bereits die gestreif-te Imaginalmuskulatur zu sehen, und der Beginn derCuticulapigmentierung markiert den Übergang vonder Phase der Cuticulaablagerung zur Differenzie-rung. Einhergehend mit der Cuticulapigmentierung(Abb. 2E und F) wird die Epidermis dünner, nichtnur im Thorax sondern auch im Abdomen, was aufdas Ende der Biosynthesephase der Cuticulabildunghinweist (Abb. 2E und F). Die Thoraxmuskulatur,die zunächst nur lose mit dem Integument assoziiertwar (Abb. 2D), gewinnt jetzt einen festen Anschluss(Abb. 2E und F).Der Beginn und Verlauf der Cuticulapigmentie-rung konnte in den Ganzpräparaten verfolgt werden(Abb. 3). In der Cuticula von rosa-äugigen oder frü-hen braunäugigen Pharatadulten ist in der Cuticulanoch keine Pigmentierung zu sehen (Abb. 3 A-C).Die Pigmentierung beginnt in der Cuticula in derdarauffolgenden pharatadulten Phase (Abb. 3D) undzeigt eine rasche Ausweitung der pigmentierten Be-reiche (Abb. 3E und F). In der abdominalen Cuti-cula beginnt die Pigmentierung später. Sowohl dieGanzpräparate als auch die histologischen Schnitte(Abb. 4, linke und rechte Seite) zeigen klar, dass dieCuticula der Imagines stärker pigmentiert ist als dieder späten Pharatadultphase (Abb. 2F und 3F).In dieser Zeitverlaufsserie zeigen die histologischenSchnitte klar die weitreichenden Veränderungen inder Integumentstruktur im Häutungszyklus auf undliefern damit neue Informationen über die Dynamikder Veränderungen der Epidermisstruktur und zumCuticulaaufbau vor und während der Apolyse und

in den darauffolgenden Phasen der pharatadultenEntwicklung, die mit der adulten Ecdysis endet. DieVeränderung in der Integumentstruktur geht ein-her mit der Entwicklung der äusseren Morphologieund den Häutungsereignissen. Diese Information istvon Interesse für Untersuchungen zu genetischen,biochemischen und physiologischen Aspekten desAufbaus des adulten Exoskeletts. Die gegenwärtigeArbeit enthält ausserdem einen Vorschlag zur Ver-einheitlichung der Terminologie, die den Verlaufdes Häutungszyklus bei Bienen beschreibt.

Apis mellifera / Integument / Metamorphose /Adultcuticula / Exoskelett

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