ORIGINAL PAPER

The antennal sensilla of Melipona quadrifasciata (Hymenoptera:Apidae: Meliponini): a study of different sexes and castes

Samira Veiga Ravaiano & Ríudo de Paiva Ferreira &

Lucio Antonio de Oliveira Campos &

Gustavo Ferreira Martins

Received: 18 March 2014 /Revised: 30 April 2014 /Accepted: 5 May 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The sensilla of insects are integumental units thatplay a role as sensory structures and are crucial for the per-ception of stimuli and for communication. In this study, wecompared the antennal sensilla of females (workers andqueens), males (haploid (n) and diploid (2n)), and queen-likemales (QLMs, resulting from 2n males after juvenile hormone(JH) treatment) in the stingless bee Melipona quadrifasciata.Images of the dorsal antenna surfaces were acquired using ascanning electron microscope. As reported for other hyme-nopterans, this species exhibits a heterogeneous sensillar dis-tribution along the antennae. Thirteen different types of sen-silla were found in the antennae of M. quadrifasciata:trichodea (subtypes I to VI), chaetica (subtypes I and II),placodea, basiconica, ampullacea, coeloconica, andcoelocapitula. Sensilla trichodea I were the most abundant,followed by sensilla placodea, which might function in olfac-tory perception. Sensilla basiconica, sensilla chaetica I, sen-silla coeloconica, and sensilla ampullacea were found exclu-sively in females. In terms of the composition and size of thesensilla, the antennae of QLMsmost closely resemble those ofthe 2n male, although QLMs exhibit a queen phenotype. Thisstudy represents the first comparative analysis of the antennalsensilla of M. quadrifasciata. The differences found in the

type and amount of sensilla between the castes and sexes arediscussed based on the presumed sensillary functions.

Keywords Antenna .Melipona quadrifasciata . Sensilla .

Stingless bee . Scanning electronmicroscopy

Introduction

The antennae of insects contain several sensory units knownas sensilla (Saïd et al. 2003; Yan et al. 2010), and the type,abundance, and distribution of which vary among species(Faucheux et al. 2006). The distribution and type of sensillamight also vary according to insect behavior, sex, and geo-graphical distribution (Chen et al. 2003). Sensilla can bedistinguished based on their morphology and function. Mor-phologically, sensilla are categorized as trichodea, basiconica,placodea, campaniformia, coeloconica, chaetica, orampullacea. Functionally, sensilla are categorized asmechano-, chemo-, thermo-, or hygro-receptors and areCO2-sensitive (reviewed in Chapman et al. 2012; Gillott2005; Nakanishi et al. 2009).

In eusocial insects, antennal sensilla are crucial for mating,foraging, recognition, and communication between colonymembers (Renthal et al. 2003; Lambin et al. 2005). Furthermore,in eusocial insects, the type and number of sensilla vary accord-ing to sex and caste. For instance, male Apis mellifera exhibitmore sensilla placodea than workers. These sensilla are believedto helpmales to encounter females during nuptial flights becauseincreased numbers of sensilla placodea enhance the olfactoryacuity of the males (Stort and Moraes-Alves 1999).

Stingless bees belong to the tribe Meliponini (Hymenop-tera, Apidae) and are among the most common pollinators intropical and subtropical regions, pollinating both wild andcultivated plants (Heard 1999; Antonini et al. 2006). Despitetheir ecological and economic importance, certain species of

Communicated by: Sven Thatje

Electronic supplementary material The online version of this article(doi:10.1007/s00114-014-1184-0) contains supplementary material,which is available to authorized users.

S. V. Ravaiano :G. F. MartinsDepartamento de Entomologia, Universidade Federal de Viçosa(UFV), 36570-900 Viçosa, Minas Gerais, Brazil

R. d. P. Ferreira : L. A. d. O. Campos :G. F. Martins (*)Departamento de Biologia Geral, Universidade Federal de Viçosa(UFV), 36570-900 Viçosa, Minas Gerais, Brazile-mail: gmartins@ufv.br

NaturwissenschaftenDOI 10.1007/s00114-014-1184-0

stingless bees in Brazil (including the genus Melipona) arethreatened by the fragmentation and loss of their ecologicalniches and by the intensive use of pesticides (Freitas et al.2009).

Although the structures of the antennae of several speciesof Meliponini have been previously studied (Moraes andCruz-Landim 1972; Stort and Barelli 1981; Johnson and

Howard 1987; Stort 2002; Nascimento et al. 2013), differ-ences in their morphology and distribution according to sexand caste remain poorly understood. In this study, the antennalsensilla of individual Melipona quadrifasciata of differentsexes and castes, including 2n (diploid) males and queen-like males (QLMs, derived from 2n males after juvenilehormone (JH) treatment), are described and compared bothqualitatively and quantitatively.

Materials and methods

Biological samples

Colonies of M. quadrifasciata were collected in the city ofViçosa (MG, Brazil) and were maintained at the ExperimentalApiary at the Federal University of Viçosa for use in theexperiments. Brood chambers containing larvae were re-moved from the hives, transferred to the laboratory, and main-tained in an incubator (28 °C; 95±65 % relative humidity)until the adults emerged. Antennae were collected fromworkers, n (haploid) and 2n (diploid) males, and queens at 1to 5 days after emergence.

For the production of 2n males, newly emerged n malesand queens (brothers and sisters) were separated until sexualmaturation, after which they were housed together andallowed to mate inside Petri dishes. After copulation, thequeens were marked with automotive paint and transferredto Petri dishes containing honey syrup and fermented pollen,together with newly emerged workers. After 5 to 10 days, thequeen and the workers were transferred into a wooden box(16×16×13 cm) containing wax, food, and workers from a

Fig. 1 SEM images showing general views of the dorsal surface ofM. quadrifasciata antennae with scape (S), pedicel (P), and flagellum(F). a Aworker (W) and a queen (Q) antenna with 10 flagellomeres. b Aqueen-like male (QLM) antenna with 11 flagellomeres

Fig. 2 SEM images showing apanoramic view of the dorsalsurface of the M. quadrifasciataantenna. a A portion of a queenantenna showing the scape,pedicel, and flagellomere 1 (F1).b Flagellomere 8 (F8) andflagellomere 9 (F9) of a workerbee

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colony of M. quadrifasciata. These boxes were maintainedwithin the incubator for 1 month, during which time they werecleaned; the bees were provided with food and newly emergedworkers every 5 days (Ferreira 2011).

When the queens became physogastric, they were trans-ferred to field colonies (one per colony) that were previouslyorphaned (i.e., without a queen). Brood chambers containinglarvae were taken from these colonies for karyotype analysisto confirm the production of 2n males.

Fifth-instar larvae received a topical application of 2 μl ofJH III (Sigma-Aldrich) (1 μg/μL acetone) using a 1-μlmicrocapillary tube (Drummond). After the application ofJH, the larvae were transferred to artificial cells made ofhoneybee wax that was placed into the wells of polyethylenemicroplates (flat-bottom, 24-well plates). Each larval cell wasmaintained within a microplate well, and the artificial combs

were cleaned daily to prevent the accumulation of feces(Ferreira 2011).

Individuals were maintained in the incubator until theirdevelopment was complete. The QLMs were identified bythe presence of male genitalia and bodily features that arecharacteristic of queen bees (i.e., small body size, no yellowstripes on the terga, and a small head relative to the length ofthe abdomen).

Analyses of the antenna

The right antennae were removed from five specimens of eachclass of individuals (i.e., queens, workers, n and 2nmales, andQLMs) of M. quadrifasciata for scanning electron microsco-py (SEM). The samples were fixed in 2.5 % glutaraldehyde incacodylate buffer (0.1 M, pH 7.2, and 3 % sucrose).

Fig. 3 Sensilla of theM. quadrifasciata antenna. aSensilla trichodea I (ST I),sensillum trichodeum (S II),sensillum basiconicum (SB), andsensillum placodeum (SP) of aqueen bee. b Sensilla trichodea III(ST III) and sensillum trichodeumIVof a 2n male bee withlongitudinal grooves arranged in ahelical fashion (arrowhead). cThe tip of flagellomere 10 of aqueen bee depicting sensillatrichodea V (ST V) and VI (STVI), SP, sensillum ampullaceum(SA), SB, and sensillacoelocapitulum (SCp)

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The samples were washed in phosphate-buffered saline(PBS, 0.1 M), dehydrated in an ascending series of acetonesolutions (50 to 100 %), dried in a critical point dryer,mounted on aluminum stubs, and metallized using a sputter-coating device. The samples were visualized using a LEOVP1430 SEM at Núcleo de Microscopia e Microanálise/UFV.

The sensilla from the dorsal surface of the antennal flagel-lum were classified according to previously described mor-phological criteria (Marques-Silva et al. 2006; Nakanishi et al.2009; Frasnelli et al. 2010) and separately counted withinfixed antennal areas (230×230 pixels) using the AdobePhotoshop CS program. Mean values of the total number ofsensilla for each antenna/each class of individual were con-sidered for statistical analysis (except for the following sen-silla: coeloconicum, ampullaceum, and coelocapitulum,which were found in very small numbers). Images of antennaewere acquired with fixed magnification (×240) and resolution(72 pixels/inch) (see example in Supplementary Fig.).

We measured the length of the sensilla using the Image-ProPlus program (Media Cybernetics). Mean values of sensillalength (μm) for each antenna/each class of individuals wereconsidered for statistical analysis (except for the followingsensilla: coeloconicum, ampullaceum, and coelocapitulum,for the same reason cited above). These values were comparedusing an ANOVA, a Tukey’s test, or (if there were only two

variables) a Student’s t test. The results were consideredstatistically significant if p<0.05.

The Morisita similarity index was used to analyze similari-ties between the antennae of individuals based on the type andnumber of sensilla (Morisita 1959; Krebs and Kenney 2002).

Results and discussion

The antennae of M. quadrifasciata are geniculate and com-posed of a scape, a pedicel, and a flagellum. The flagellum isdivided into 10 and 11 flagellomeres in females and males,respectively (Figs. 1 and 2). Not surprisingly, this overallorganization is similar to that of the antennae of several othereusocial bees (Esslen and Kaissling 1976; Nascimento et al.2013) and ants (Marques-Silva et al. 2006; Nakanishi et al.2009; Mysore et al. 2010; Euzébio et al. 2013).

The number and type of antennal sensilla vary amongeusocial Hymenoptera. For example, in A. mellifera(Frasnelli et al. 2010) and Melipona scutellaris (Nascimentoet al. 2013) there are six and seven types of sensilla, respec-tively. In the ants Solenopsis invicta (Renthal et al. 2003) andCamponotus japonicus (Nakanishi et al. 2009) there are fiveand eight types of sensilla, respectively. Additionally, the

Fig. 4 Sensilla of theM. quadrifasciata antenna. aSensillum placodeum of a 2nmale. b Sensillum basiconicum ofa worker with tiny pores(arrowheads). c Sensillumchaeticum I of a worker (inset, anarrowhead indicates the pore-likestructure at the sensillum tip of aqueen). d Sensillumcoeloconicum (SCo) and sensillaampullacea (SA) proximal to eachother in the flagellomere 10 of aqueen. f Sensillumcoelocapitulum in the tip offlagellomere 10 of a queen

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following 13 types of sensilla are found in the antennae ofM. quadrifasciata: trichodea (ST, subtypes I to VI) (Figs. 3a–c), placodea (SP) (Figs. 3c and 4a), basiconica (SB)(Figs. 3a, c and 4b), chaetica (SCa, subtypes I and II)(Fig. 4c), coeloconica (SCo), ampullacea (SA) (Fig. 4d), andcoelocapitular (SCp) (Fig. 3c and 4e).

Similar to A. mellifera (Stort and Barelli 1981; Gupta1992), the ST are polymorphic and are the most abundanttype of sensillum found in M. quadrifasciata. According totheir morphology, the ST ofM. quadrifasciata can be separat-ed into subtypes I to VI and are inserted into small circulardepressions within the antennal cuticles (Fig. 3a, c). Interest-ingly, in bees, there are more subtypes of ST than in ants. Forexample, inM. quadrifasciata and bees of the tribe Emphorini(Galvani et al. 2012) six and four subtypes of ST are found,respectively, whereas only two subtypes are found inC. japonicus and S. invicta (Nakanishi et al. 2009; Renthalet al. 2003).

ST I are lance-shaped and bend toward the tip of theantenna (Fig. 3a). These sensilla are the most numerous(sub)type of sensillum in M. quadrifasciata (Table 1) andare uniformly distributed along the flagellomeres (Fig. 2b),except in flagellomere 1 (F1), where they are not observed.The number of ST I is decreased in queens, whereas thenumber of ST I does not differ significantly (p<0.05) amongthe other types of individuals (Table 1). ST I are larger inworkers and smaller in n and 2n males (p<0.05) (Table 2). STI are similar to “trichoid sensilla-2” of the parasitoidMetaphycus parasaissetiae (Hymenoptera: Encyrtidae)(Zhou et al. 2013), which is also known as a chemoreceptor.These putative olfactory sensilla are believed to help individ-uals to find food sources and to recognize colony members(Zacharuk 1985; Shanbhag et al. 1999). Males of the genusMelipona also forage for short periods immediately afterleaving the colony, which might explain the increased numberof ST I in their antenna (Velthuis et al. 2005).

Similar to ST I, the basal portions of ST II bend toward thetip of the antenna, and ST II are uniformly distributed alongthe flagellomeres, except in F1; however, ST II are thinnerthan ST I (Fig. 3a). The number of ST II is greater inM. quadrifasciata worker bees than in queen and male bees(i.e., n, 2n, and QLM) (Table 1), and ST II are larger in femalesthan in males (p<0.05) (Table 2). The sickle-shaped (flattenedlaterally) ST III are exclusively found in the latter half of F1(Fig. 3b). ST IV exhibit longitudinal grooves in a helicalarrangement (Fig. 3b), are exclusive to F1, and are concen-trated near the pedicel (Fig. 2a). The number of ST IV doesnot differ among individuals (p>0.05) (Table 1). Moreover,ST IVare longest in n males and queens, followed by workers,QLM, and 2n males (Table 2). Both ST II and III are found inthe tribe Emphorini (Galvani et al. 2012); ST IVare found inparasitoid wasps belonging to the Braconidae and Eupelmidae(Ochieng et al. 2000; Das et al. 2011; Meng et al. 2012), in T

able1

Num

bersof

sensillain

theantennaof

M.quadrifa

sciata

Classof

individual

Num

bersof

sensilla

STI

STII

STIII

STIV

STV

STVI

SCaI

SCaII

SPSB

Worker

960.00

(±222.56)b

216.00

(±30.14)

a48.60(±12.75)

a42.80(±9.03)a

110.00

(±15.13)

a26.66(±2.88)a

11.20(±1.48)a

–500.40

(±123.26)a

41.40(±4.33)a

Queen

666.60

(±123.78)a

152.80

(±16.57)

b52.80(±11.84)

a36.20(±6.97)a

81.33(±14.57)

b27.66(±8.08)a

21.60(±7.56)b

–508.00

(±154.16)a

58.00(±1.78)b

nmale

1,184.60

(±163.32)b

94.20(±30.95)

c77.60(±22.28)

a42.80(±20.81)

a48.66(±3.21)c

18.00(±3.60)b

–23.60(±4.56)a

1,099.00

(±131.29)b

–

2nmale

1,213.00

(±48.76)

b98.20(±14.82)

c116.80

(±12.71)

b56.00(±24.08)

a30.50(±2.12)c

13.00(±1.41)c

–29.00(±2.54)a

1,186.60

(±46.75)

b–

QLM

975.80

(±58.37)

b81.60(±23.75)

c94.20(±27.23)

b31.40(±15.5)a

46.50(±4.65)c

17.50(±5.19)b

–24.40(±6.87)a

1,087.20

(±61.19)

b–

Different

lettersin

thesamecolumnindicatesignificantd

ifferences

(ANOVA,p

<0.05)

QLM

queen-lik

emale,ST

sensillum

trichodeum

,SCasensillum

chaeticum

,SPsensillum

placodeum,SBsensillum

basiconicum

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bumblebees (Shang et al. 2010), in female wasps Ooencyrtusphongi (Encyrtidae) (Xi 2011), and inMacrocentrus cingulum

(Braconidae) (Ahmed et al. 2013), and are considered mech-anoreceptors (van Baaren et al. 1999).

Table 2 Sizes of sensilla in the antenna ofM. quadrifasciata

Class ofindividual

Sizes of sensilla (μm)

ST I ST II ST III ST IV ST V ST VI SCa I SCa II SP SB SC SA SCp

Worker 15.14(±1.38)a 13.75(±1.13)a 26.75(±2.20)a 17.40(±2.21)a 19.63(±1.25)b 20.81(±2.18)b 17.10(±3.09)a – 15.29(±1.42)a 8.93(±1.14)a 1.72(±0.40) 0.52(±0.15) 4.88(±0.12)

Queen 13.78(±1.39)b 15.04(±2.48)a 26.67(±3.79)a 18.04(±2.10)b 30.08(±4.73)a 32.42(±4.17)a 18.69(±2.10)a – 13.64(±1.01)b 9.23(±2.02)a 2.51(±0.08) 0.73(±0.17) 4.59(±0.29)

n male 6.80(±0.69)c 7.82(±0.70)b 13.77(±0.80)b 18.51(±2.46)b 19.26(±2.22)b 21.62(±2.17)b – 8.43(±1.35)a 8.09(±0.64)c – – – –

2n male 6.10(±0.93)c 7.35(±1.01)b 13.94(±0.85)b 12.74(±2.13)c 19.07(±2.61)b 20.09(±1.57)b – 8.55(±1.20)a 8.63(±1.16)c – – – 3.26(±0.09)

QLM 8.26(±0.51)d 8.81(±0.68)b 16.54(±1.80)b 15.50(±2.75)a 19.09(±1.87)b 27. 00(±8.46)b – 9.06(±1.00)a 9.31(±0.95)c – – – 4.06(±1.23)

Different letters in the same column indicate significant differences (ANOVA, p < 0.05)

QLM queen-like male, ST sensillum trichodeum, SCa sensillum chaeticum, SP sensillum placodeum, SB sensillum basiconicum, SCo sensillumcoeloconicum, SA sensillum ampullaceum, SCp sensillum campaniformium

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ST V are found only in the distal quarter of the lastflagellomere, and their tips tilt toward the tip of the antenna(Fig. 3a). These sensilla are more abundant in workers,followed by queens, n males, QLM, and 2n males (Table 1).Similar to ST V, ST VI are observed at the tip of the lastflagellomere, although they are straight and perpendicular tothe antennal surface (Fig. 3c). ST VI are more abundant infemales, followed by n males, QLM, and 2n males (Table 1)and are larger in queens than in males and workers (Table 2).ST V have been observed in female A. mellifera (Al Ghamdi2006; Zakaria and Allam 2007; Frasnelli et al. 2010),M. quadrifasciata and Scaptotrigona postica (Cruz-Landim2009). ST VI have also been observed in M. scutellaris bees(Nascimento et al. 2013); as in the case of ST V, no functionhas yet been identified for these sensilla.

SP are oval plates that are bound by a line-shapedgroove and have a slight depression in their center(Figs. 3a and 4a). SP are the second most abundant typeof sensilla in M. quadrifasciata, and there are almosttwice as many SP in males than in females (Table 1).Additionally, the largest SP are found in workers, and thesmallest are found in n males (p<0.05) (Table 2). SP areamong the most abundant types of sensilla present in bees(McIndoo 1914 cited in Gramacho et al. 2003; Ochienget al. 2000; Cruz-Landim 2009). Increased amounts of SPare associated with enhanced olfactory acuity in Bombusterrestris (Apidae) bees (Spaethe et al. 2007). In addition,these sensilla are believed to facilitate the detection offemale pheromones due to the increased olfactory acuityconferred by their increased numbers in males (Stort andMoraes-Alves 1999; Ochieng et al. 2000).

SB are also olfactory sensilla (Mustaparta 1973; vanBaaren et al. 1999; Ozaki et al. 2005); in M. quadrifasciata,SB appear as short and thick hairs with rounded tips and verytiny pores (Figs. 3a, c and 4b), occur in the centralflagellomeres of females, and are bigger and more numerousin queen bees (p<0.05) (Tables 1 and 2). SB have beendescribed as chemoreceptors for cuticular hydrocarbons,allowing the recognition of nestmates in ants (Ozaki et al.2005; Nakanishi et al. 2009). Although this hypothesis re-mains to be confirmed inMeliponini, these sensilla might alsohave a similar function in M. quadrifasciata.

SCa most often occur proximal to the sides of the antennaeand exhibit a slight upward bend at the tip. Two subtypes ofSCa exist: SCa I, with an apical pore-like cavity, and SCa II,without this structure. SCa I are found in females (Fig. 4c),whereas SCa II are found in males (not shown). SCa I aremore numerous in queens than in workers (p<0.05) but do notdiffer in size (p>0.05) (Table 1). The slight bending mightfacilitate the contact of these contact chemoreceptors withobjects and surfaces (Nakanishi et al. 2009) and, consequent-ly, might aid the recognition of nestmates by workers (Inoueet al. 1999; Stort 2002).

SCo and SA are found exclusively in females. These sen-silla are small, appearing as pores within the cuticle; the poresare larger in SCo (1.72 μm in diameter in workers and2.51 μm in diameter in queens) than in SA (0.52 μm indiameter in workers and 0.73 μm in diameter in queens)(Figs. 3c and 4d). These two sensilla appear adjacent to eachother on the right side of the end of F10. SCo are also foundproximal to the side of the antenna but can be observed fromF2 to F10, although they are more abundant near the tip of theantenna . The loca t ion of SCo is conse rved inM. quadrifasciata and other Apidae (Moraes and Cruz-Landim 1972; Stort 2002) as well as in the ant Atta sexdens(Kleineidam et al. 2000).

SCo are hygro- and thermoreceptors (Rebora et al. 2007).In M. quadrifasciata, SCo are most likely important inworkers and queens for the perception of humidity, allowingthe insects to detect the most appropriate weather conditionsfor foraging and mating (Kleineidam and Tautz 1996;Weidenmuller et al. 2002). SA are considered sensitive toCO2, humidity, and/or temperature, which are important fac-tors for nest maintenance in eusocial insects (Kuwabara andTakeda 1956; Kleineidam et al. 2000).

The pin-like SCp are located in the center of rounded andshallow depressions, and they are found exclusively near theright side of the antennal tip of all classes of individuals(Figs. 3c and 4e). SCp are larger in females (Table 2), andthere are 8–10 SCp/individual. These sensilla are hygro- andthermoreceptors (Steinbrecht 1989) and have been reported inthe ant C. japonicus (Nakanishi et al. 2009) and in honeybees(Yokohari 1983).

JH regulates the differentiation of castes inMeliponini bees(Kerr et al. 1975; Wyatt 1992; reviewed by Klowden 2007).Accordingly, individuals with high concentrations of JH ma-ture into queens during postembryonic development (Asencotand Lensky 1988; Hartfelder et al. 2006). It has been shownthat the topical application of JH to the larvae of 2n males ofM. quadrifasciata can produce individuals with a queen

Fig. 5 A dendrogram based on values for the Morisita index (Morisita1959) showing the similarity of the antennae of different classes ofM. quadrifasciata. Method of cluster analysis: UPGMA (Krebs andKenney 2002). QLM queen-like male

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phenotype (Camargo 1977). In this study, we showed that,despite the acquisition of a queen phenotype after JH treat-ment, QLMs have male antennae, which are thin and contain11 flagellomeres (Fig. 1b). In addition, the dorsal topographyof the antennae in QLMs resembles that of males. Further-more, QLMs do not have SCa I, SB, SCo, or SA, and thenumber of SP in QLMs is similar to that in males (Table 1).Figure 5 presents a dendrogram, based on the Morisita simi-larity index, showing that females are grouped separately frommales.

Work e r and ma l e b ee s , i n c l ud i ng t ho s e o fM. quadrifasciata, are known to exhibit similarities to eachother while differing from queen bees in terms of cuticularhydrocarbon composition (Abdalla et al. 2003; Nunes et al.2009; Borges et al. 2012). The most probable explanation ofthis difference is that JH increases the expression of femini-zation genes in queens, promoting their differentiation fromthe workers. Thus, workers are phenotypically more similar tomales (Kerr et al. 2004). In contrast, we have shown thatM. quadrifasciataworkers are more similar to queens in termsof the numbers and types of antennal sensilla. Thus, thetreatment of fifth-instar larvae with JH does not change thenumber of flagellomeres or the composition of sensilla inQLMs.

In this study, the antennal sensilla of M. quadrifasciatahave been characterized in detail. We investigated variationsin sensillar distribution, size, and number along the antenna,enabling an improved understanding of sexual dimorphismand the division of labor in bees. Thus, the study of themorphology, number, and size of these structures is importantfor the differentiation of sexes and castes in this eusocial insectand provides an important step toward the further study of themechanisms of communication in social insects.

Acknowledgments We acknowledge the Núcleo de Microscopia eMicroanálises, Universidade Federal de Viçosa, Minas Gerais, for tech-nical assistance. Financial support was provided by Coordenação deAperfeiçoamento de Pessoal de Nível Superior (CAPES).

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