Heterochrony of cuticular differentiation in eusocialcorbiculate bees
Moyss ELIAS-NETO1, Ana L. O. NASCIMENTO1, Ana M. BONETTI2,Fabio S. NASCIMENTO1, Sidnei MATEUS1, Carlos A. GARFALO1,
Mrcia M. G. BITONDI1
1Departamento de Biologia; Faculdade de Filosofia, Cincias e Letras de Ribeiro Preto, Universidade de SoPaulo, Av. Bandeirantes 3900, 14040-901 Ribeirao Preto, Sao Paulo, Brazil
2Instituto de Gentica e Bioqumica, Universidade Federal de Uberlndia, Rua Acre, bloco 2E, 38400-902Uberlandia, Minas Gerais, Brazil
Received 28 June 2013 Revised 15 October 2013 Accepted 30 October 2013
Abstract The exoskeleton (cuticle) of insects varies widely in shape, biomechanical properties, andfunctions, which are inherent to the biological species and developmental stage, besides showing a wealth ofarchitectural specializations and nuances in the different body regions. The morphological pattern ofexoskeleton maturation was studied in eusocial and solitary bees, including species of all three eusocial tribesof corbiculate bees as follows: Apini, Meliponini, and Bombini. The results showed striking differences in therate of cuticle maturation between the solitary bees that leave the nest soon after the adult ecdysis, and theeusocial bees that take longer to leave the colony for foraging activities. There was a clear delay in the post-ecdysial cuticle tanning (melanization and sclerotization) in the eusocial bee species in comparison to thesolitary species, suggesting adaptation to sociality and to the protective environment of the colony. Suchheterochrony of cuticle maturation seems a conserved ontogenetic trait related to the way of life in corbiculatebees. The data generated a basic framework of exoskeleton maturation in corbiculate bees, under ontogeneticand evolutionary approaches, and give experimental support for further research on adaptation to the colonyenvironment.
exoskeleton / tanning / eusociality / Apinae / solitary bees
The cuticular exoskeleton is a multifunction-al structure because in addition to the support itprovides the insects, it allows locomotion andmovements, protects against predators, andprevents desiccation, besides functioning in themechanical digestion carried out by mouthparts,
and in the sensorial perception via antennae andsensorial setae (Hepburn 1985).
Exoskeleton differentiation is a remarkableevent of insect ontogenesis and occurs througha process known as tanning, meaning theprogressive darkening (melanization) and hard-ening (sclerotization) of the newly-secretedcuticle (Andersen 2005). Ontogenetically, twobasic types of exoskeleton can be identified inthe honey bees, the flexible and colorless(unpigmented) larval and pupal exoskeletons,and the rigid (highly sclerotized) and pigmentedadult exoskeleton. Melanin pigments are depos-ited into the adult cuticle, which becomesincreasingly sclerotized, as it differentiates
Corresponding author: M. Elias-Neto,email@example.com;M. Bitondi, firstname.lastname@example.orgManuscript editor: Monique Gauthier
Apidologie Original article* INRA, DIB and Springer-Verlag France, 2013DOI: 10.1007/s13592-013-0254-1
underneath the pupal cuticle. In the honey bee,melanization starts in the developing adultcuticle 80 h after pupal cuticle apolysis, ap-proximately (Michelette and Soares 1993).Apolysis progressively occurs along the anteri-orposterior body axis, and adult cuticle depo-sition and tanning follow this pattern. Thus,cuticle tanning is first seen in the head andthorax. The abdomen is weakly pigmented andsclerotized until close to the adult ecdysis.
Cuticular melanization and sclerotizationoccur simultaneously through the same biosyn-thetic pathway starting with hydroxylation oftyrosine to dopa, followed by a step requiringthe catalytic activity of dopa-decarboxylase fordopamine production. Dopamine is a precursorin the synthesis of melanin, catalyzed byphenoloxidases, and in the synthesis ofsclerotizing agents. Sclerotization starts whenN-acetyldopamine and N--alanyldopamine areoxidized to quinones by the action ofphenoloxidases and laccases. Quinones thenreact with cuticular proteins, thus stabilizingcuticle structure. These reactions can occurbefore the ecdysis (pre-ecdysial sclerotization)or after ecdysis (post-ecdysial sclerotization)(review in Andersen 2010). In the honeybee,the cuticle deposited before the adult ecdysis isless sclerotized than the post-ecdysial cuticle(Andersen et al. 1981) thus indicating thatsclerotization proceeds throughout part of theadult stage. A detailed description of themorphogenesis of the adult integument (epider-mis and its associated cuticle) in the honeybee,using conventional histology and light micros-copy, evidenced that exoskeleton maturationcontinues and is intensified after ecdysis to theadult stage (Elias-Neto et al. 2009).
We have observed that the rate of post-ecdysial cuticle tanning differs among beespecies. This variation in the temporal patternof exoskeleton tanning, or heterochrony, mayrepresent an adaptive mechanism related to thebee lifestyle. Heterochrony is defined as anyevolutionary change in the temporal pattern ofexpression of a certain characteristic (West-Eberhard 2003), meaning the shift (acceleration
or delay) in the relative timing of developmentalevents. Heterochrony is a phenomenon of greatimportance in the evolution, both in the frequencyof occurrence and the origin of significant evolu-tionary changes (Gould 1977). New approachesand tools for the study of heterochrony at cellularand molecular levels have provided opportunitiesto a deeper understanding on the basis of the shiftsin the timing of ontogenetic events (Smith 2003).
In this context, the evolution of insect socialityrepresents remarkable scenery for testing hypothe-ses related to adaptive mechanisms of exoskeletonmaturation. The protective environment of thecolony possibly has led to a loosening of theselective pressures over the cuticular differentia-tion, which enables, for instance, the emergence ofadults with a relatively immature exoskeleton.Complete maturation is only attained several dayslater, when the eusocial bees become foragers. Incontrast, the solitary bees leave the nest soon afterthe emergence from the brood cells, and thisevidently demands a fully mature exoskeleton.Therefore, morphological markers of exoskeletontanning, such as the dynamics of melanization andsclerotization, are useful to investigate whether thecomplexity of the evolution of eusociality includedthe slowdown in the rate of exoskeletonmaturation.
Co r b i c u l a t e b e e s (Ho l ome t a bo l a ,Hymenoptera, Apidae) are a monophyleticgroup (Michener 2007), supported by a seriesof synapomorphies, including the presence of acorbicula (basket-shaped structure in the fe-male tibia which is used for pollen transport).Such bee group forms a distinct clade withinApinae (Apidae). It includes four tribes asfollows: Euglossini (orchid bees), Bombini(bumblebees), Apini (honey bees), andMeliponini (stingless bees). Euglossini spe-cies are solitary, communal or primitivelysocial; Bombini species are primitively euso-cial (except for the Psithyrus, which comprisessocial parasites) (Silveira et al. 2002);Meliponini and Apini show true sociality(eusociality) (Michener 2007).
Except for a few studies in Apis mellifera(Thompson 1978; Elias-Neto et al. 2009), othercorbiculate bee species have not been studied asregards to developmental morphology of the
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exoskeleton, despite a series of reports (com-piled in Cruz-Landim and Abdalla 2002) oncharacterization of exocrine glands associatedwith the epidermis and cuticle.
Species of the bee tribes above mentioned wereused to investigate whether the temporal shift incuticle tanning reflects the evolutionary transition toeusociality. In a comparative approach, the purposeof the present workwas to investigate the hypothesisthat the heterochrony of cuticular maturation is aconserved ontogenetic phenomenon related to thesocial environment in corbiculate bees.
2. MATERIALS AND METHODS
This study included species of all three eusocialtribes of corbiculate bees as follows: Apini (A.mellifera), Meliponini (Frieseomelitta varia,Tetragonisca angustula, Scaptotrigona aff. depilis,Melipona scutellaris), and Bombini (Bombus morio).The outgroup consisted of the solitary bees Centrisanalis (Centridini) and Tetrapedia diversipes(Tetrapediini). Females (workers) of these specieswere collected soon after the adult ecdysis (newly-emerged bees) and when they were foraging.
The bees were collected at the Campus of the SoPaulo University in Ribeiro Preto. The species A.mellifera, F. varia, T. angustula, S. aff. depilis, and M.scutellaris were obtained in the experimental apiary/meliponary of the Department of Genetics. Specimens ofB. morio were collected from a colony at the Laboratoryof Ecology, Department of Biology, where trap-nests ofC. analis and T. diversipes are also maintained.
The criteria for the selection of the species werebased on the representativeness of different evolu-tionary lineages. Previous studies on the biology ofMeliponini, Bombini, and solitary species, whichenabled their maintenance and collection (Camillo etal. 1995; Nogueira-Neto 1997; Jesus and Garfalo2000; Goulson 2006), also influenced our choice.
2.2. Integument sections
Histological sections were prepared using theintegument dissected from the dorsal region of thethorax of newly-emerged and forager bees. The
thoracic sections were made in a longitudinal plane,cutting the dorsal portion of the thorax (includingpronotum, mesonotum, and metanotum) at the mid-line. The cuticle pieces were fixed for 24 h at 4 C in4 % paraformaldehyde in phosphate buffer (0.1 M,pH 7.3). This was followed by dehydration insuccessively higher concentrations (70, 80, 90, and95 %) of ethanol in water (v/v) for 30 min in eachsolution. The cuticles were then embedded inmethacrylate resin. Sections of 5 m were obtainedwith the Microm HM200 Ergostar Microtome, andwere stained with methylene blue and basic fuchsinfor 3 min, followed by a rapid washing in distilledwater. Sections were mounted in Entellan (Merck)and examined and photographed using an opticalmicroscope (Axioskop II photomicroscope, Zeiss). Atleast three samples of each developmental stage wereused in the histological preparations. The best-qualityphotographs (mesonotum) were presented.
2.3. Whole mounts
Whole mounts were also prepared with the dorsalportion of the thorax (pronotum, mesonotum, andmetanotum). The thoracic integument samples weregently rinsed in Ringer saline, cleaned of excess fatbody and muscles, and mounted on slides using pureglycerol. The whole mounts were immediatelyexamined in the Axioskop II photomicroscope andphotographed. At least three samples were obtainedfor each stage of development. The best-qualityphotographs (mesonotum) were shown.
Forager bees from the eusocial species A.mellifera, F. varia, T. angustula, S. aff. depilis,M. scutellaris, and B. morio showed in a lesseror greater degree, depending on the species, adarker and stiffer cuticular exoskeleton thannewly-emerged bees. Such difference was notobserved in the solitary bee species (C. analisand T. diversipes) (Figure 1). These intra- andinterspecific comparisons highlighted that incontrast to the solitary bees, the exoskeleton ofeusocial bees takes longer to differentiate as acompletely mature structure.
Cuticle differentiation in eusocial bees
To a deeper approach on this issue, the thoraciccuticle was then dissected from newly emerged
and forager bees of the eusocial and solitaryspecies, and prepared for microscopy analysis
Figure 1. Evolutionary relationships of eusocial corbiculate bees (continuous line) and solitary bees (dottedline) according to strongly corroborated phylogenetic hypotheses (Michener 2007). Side-views of newly-emerged (left) and forager (right) females. Newly-emerged and forager bees in each eusocial species showcontrasting exoskeleton differentiation, a phenomenon which was not observed in solitary bees.
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(Figures 2 and 3). Both preparationssagittalsections (Figure 2) and whole mounts(Figure 3)emphasized the morphological dif-ferences between the cuticle of the newly-emerged and forager bees in the eusocial species,
both in the intensity of pigmentation and in theprocess of sclerotization. The cuticle proved to bealways much more melanized and rigid in theforagers (Figure 2af). It is lacking in the newly-emerged F. varia, T. angustula, and S. aff. depilis,
Figure 2. Sagittal sections of the dorsal thoracic exoskeleton of newly-emerged (left) and forager (right) bees ofeusocial and solitary species. The distinct morphological patterns of the newly-emerged and forager eusocialbees (af) were not observed in the solitary bees (g, h). Fuchsine and methylene blue highlighted the degrees ofcuticle differentiation, the reddish pink color (arrowheads) evidencing the non-differentiated cuticle. The black(melanized) layers (arrows) indicate the intense sclerotization of the mature cuticle.
Cuticle differentiation in eusocial bees
the melanized superficial layer, which is onlyevident in the foragers. In spite of the presence ofsuch a layer in the cuticle of newly-emerged beesof the other eusocial species, M. scutellaris, A.mellifera, and B. morio (Figure 2df, arrows), it iscomparatively thicker in the foragers of thesespecies. Themelanized surface of the cuticle is notalways well-delimited in the stained cross sec-tions, thus making the comparison less obvious insome species, likeB. morio (Figure 2f). Even so, itis clear that a completely differentiated (mature)cuticle occurs in the eusocial bees only severaldays after the emergence. In contrast, the solitaryspecies, C. analis and T. diversipes, collected atthe emergence or when they were foraging,showed very similar cuticle patterns, which arecharacterized by a thick melanized layer(Figure 2g, h).
In addition to the differences in melanization,cuticle sections of the newly-emerged eusocialbees showed a pink-reddish layer that varies inthickness among the different species (Figure 2af,arrowheads). This layer undergoes intense modi-fications in its chemical properties during cuticularmaturation as deduced by the change in its color inforagers of the eusocial species. The pink-reddishcolor thus marks the yet undifferentiated layer ofthe cuticle.
In the whole mount preparations (Figure 3),there was also a clear distinction between thecuticular patterns of the newly-emerged andforager bees of the eusocial species (Figure 3af),which was not observed in the solitary species(Figure 3g, h). F. varia, T. angustula, and S. aff.depilis showed the highest degree of cuticularmaturation heterochrony (Figure 3ac). Foragersof these species have a brownish cuticle with ahomogeneous distribution of pigments, whereas inthe newly-emerged bees, pigmentation was con-centrated in the setae and setal sockets.
Compared to F. varia, T. a...