Age-dependent changes in structure and function of the male labial gland in Bombus terrestris

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Journal of Insect Physiology 54 (2008) 204–214

Age-dependent changes in structure and function of the male labialgland in Bombus terrestris

Jan Sobotnıka, Blanka Kalinovaa,1, Lucie Cahlıkovab, Frantisek Weydac,d,Vladimır Ptaceke, Irena Valterovaa,�

aInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 10 Prague 6, Czech RepublicbDepartment of Chemistry of Natural Compounds, Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic

cDepartment of Physiology, Institute of Entomology, Biology Centre Academy of Sciences of the Czech Republic,

Branisovska 31, 370 05 Ceske Budejovice, Czech RepublicdFaculty of Science, University of South Bohemia, 370 05 Ceske Budejovice, Czech Republic

eDepartment of Animal Physiology and Immunology, Faculty of Science, Masaryk University, Kotlarska 2, 637 11 Brno, Czech Republic

Received 11 July 2007; received in revised form 31 August 2007; accepted 4 September 2007

Dedicated to our colleague and dear fellow, the late Ondrej Blazek, for his kind humour

Abstract

The cephalic region of the labial gland in the buff-tailed bumblebee, Bombus terrestris, consists of numerous acini (formed by

associated secretory cells and a central lumen) and connecting ducts. Age-dependent changes in secretion production (both qualitative

and quantitative) are associated with changes in the amount of rough endoplasmic reticulum (RER), Golgi apparatus, and smooth

endoplasmic reticulum (SER). The main secretory organelle is RER in the youngest individuals (pharate, and less-than-a-day old males),

Golgi apparatus in 1-day-old males, and SER in males older than 2 days. Secretory cell death starts at 5 days of age, with maximal

longevity to 10 days. Pheromone production starts immediately after eclosion, with pheromone quantities increasing until day 7.

2,3-Dihydrofarnesol, the main component of the male-marking pheromone, appears in 1-day-old male glands, and reaches a maximum at

7 days of age, when its presence in the gland starts to decrease gradually. Older glands contain compounds not present in young ones.

Variation in pheromone quantity and composition are reflected sensitively in the response of the queen antennae. Though queen antennae

responded to gland extracts of all ages examined, maximum sensitivity was observed in response to extracts of glands 2–10 days old, while

extracts of older glands gradually lose their effectiveness. Both major and minor components of the labial gland secretion extract elicited

queen antennal responses suggesting that the pheromone is a multicomponent blend. Age-dependent changes in pheromone production,

accumulation and tuning of pheromone activity are all synchronized approximately with male flight from the hive.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Bumblebee; Apoptosis; Smooth endoplasmic reticulum; Male-marking pheromone; Electroantennography

1. Introduction

Premating behaviour of bumblebee males has beenstudied for a long time by different authors (Schremmer,1972; Lloyd, 1981; Bergman, 1997; Kindl et al., 1999). Thespecies can be classified into three main groups according

to this behaviour, as species exhibiting (1) patrollingbehaviour (males establish flight paths with scent markson prominent objects and fly along these routes awaitingthe appearance of females, attracted by a pheromone),(2) perching behaviour (males wait at prominent objects—perches—and dart at passing virgin queens—gynes—orother objects that resemble them), and (3) nest-waitingbehaviour (males wait for emerging gynes at a nestentrance). The patrolling behaviour is the far mostcommon type of premating behaviour between thebumblebee and cuckoo-bumblebee species. The types of

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�Corresponding author. Tel.: +420 220 183 298; fax: +420 220 183 582.

E-mail addresses: [email protected] (B. Kalinova),

[email protected] (I. Valterova).1Also to be corresponded to.


behaviour ad (1) and (2) are accompanied by scent markingeither of several spots on a flight path (patrolling type,Bergstrom et al., 1981) or of a perch (perching type,Hovorka et al., 1998).

The buff-tailed bumblebee, Bombus terrestris, is acommon European bumblebee. Like most other bumblebeespecies, males scent-mark various substrates on their flightroutes in order to attract conspecific queens for mating(Calam, 1969; Kullenberg et al., 1970; Svensson, 1980). Themarking sex pheromone is synthesized in the cephalic regionof the paired labial gland (Kullenberg et al., 1973; Agren etal., 1979) and its main components have been described(Kullenberg et al., 1970). The structure of cephalic region oflabial gland and pheromone blend composition in males ofBombus lapidarius, Bombus hypnorum, and Bombus hortor-

um with respect to the age was investigated by Agren et al.(1979). The gland consists of numerous acini and ductsconnecting particular acini. Each acinus is formed by severalsecretory cells and a large lumen, where the secretion isstored. The main secretory organelles were Golgi bodies andendoplasmic reticulum (Agren et al., 1979).

It is speculated that biosynthesis of marking pheromoneis hormonally controlled, as no innervation has beenreported (Agren et al., 1979). Pheromones are complexblends comprising numerous compounds (most oftenesters, alcohols, terpenes, and hydrocarbons) mixed inspecies-specific blends (Bergstrom et al., 1981; Free, 1987;reviewed by Valterova and Urbanova, 1997). Due to thedistinct species specificity of these pheromone composi-tions, they represent efficient and frequently used tool forspecies discrimination in cases where other taxonomic toolsare not sufficient (Paterson, 1985; Belles et al., 1987; Terzoet al., 2003, 2005; Rasmont et al., 2005). Additionally,individual variation in pheromone composition has beenobserved within a single species (Agren et al., 1979;Svensson and Bergstrom, 1977), which can render taxo-nomic application a difficult task. This variation can berelated to geographical origin, age, or diet of particularspecimens studied (Agren et al., 1979; Hendry, 1976;Svensson and Bergstrom, 1977).

In spite of accumulated information about pheromonecomposition in many bumblebee species (reviewed byValterova and Urbanova, 1997), it is still not clear howthe pheromone blend is formed and which compounds areessential to carry out pheromone function. Neither is itknown whether all compounds of the pheromone blend aresynthesized in the labial gland or whether some of them areformed in the labial gland from lipid precursors trans-ported from the fat body (Luxova et al., 2003). Studiesherein examined age-dependent ultrastructural and phy-siological changes associated with labial gland ontogeny inB. terrestris. Specifically: (i) gland ultrastructure andmorphology was examined using optical and transmissionelectron microscopy; (ii) pheromone blend compositionand age-dependent changes in pheromone compositionwere studied using gas chromatography and mass spectro-metry; and (iii) queen antennal response to labial gland

extracts from males of varied ages and specific componentsof labial gland extracts were measured using electroanten-nogram (EAG) and GC-EAD. None of these topics hasbeen studied in B. terrestris before.

2. Material and methods

Colony of B. terrestris (L.) was established by the knowntechnique using the two-queen cascade method (Ptacek etal., 2000) to stimulate egg laying of queens in thelaboratory. All mother queens were taken from theirnatural habitat during the searching period in order tominimize the possible negative influence of artificialconditions on their progeny. When colonies turned to themale production, the male cocoons were removed from theparental hives and left to ripen separately, supplied withquality food and the care of several workers (Ptacek, 1999).Freshly emerged males were removed and kept assortedaccording to their age. For chemical analyses, males—justemerged, 2, 4, 7, 10, 12, 17, 20, 24, and 33 days old (fivespecimens of each age)—were killed by freezing (�18 1C)and kept frozen prior to dissection (maximally 24 h). Labialglands were dissected and extracted with hexane (100 ml pergland) containing 1-bromodecane as internal standard(1.79mg/ml). Whole glands were extracted without homo-genization of the tissue. After 15min of shaking and 2 hstanding in the refrigerator (5 1C), the hexane extract wasfiltered off and stored at �18 1C prior to analyses. Eachsample was analyzed separately without further purifica-tion or concentration step.

2.1. Chemical analyses

Hexane extracts were analyzed by gas chromatograph(CE 8000, Milano, Italy) with a splitless injector (200 1C) anda mass detector (200 1C, GC–MS MD 800 Fisons, Manche-ster, UK). A DB-5MS column (30m� 0.25mm, filmthickness 0.25mm, Agilent Technologies Santa Clara, CA,USA) and helium gas (constant flow 1ml/min) were used forseparations. Initial oven temperature was 70 1C (2min delay)and was increased to 320 1C at a rate of 10 1C/min.Compound identification was based mostly on their massspectra compared to those in the National Institute ofStandards and Technology Library (NIST, USA) and on co-chromatography with synthetic or commercially availablestandards. Ethyl dodecanoate, octadecenol, and octadeca-trienol were bought from Sigma, St. Louis, MO, USA.Dihydrofarnesal, dihydrofarnesol, and geranylcitronellolwere earlier synthesized in our laboratory (Valterova et al.,1996). Tetradecanol, tetradecanal, hexadecanol, octadeca-trienal, and octadecatrienoic acid were taken from the bankof standards available in our laboratory.

2.2. Ultrastructural analyses

Pharate imagoes, 12 h, 1 day and 2-, 3-, 4-, 5-, 6-, 7-, 8-,9-, 10-, 12-, 17-, 20-, 24-, 33-day-old males with three

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individuals per age category were examined. Pharateimagoes were dissected from older cocoons with darklycoloured bumblebees inside the pupal cuticle. The heads ofbumblebees were put into a droplet of fixative (mixture of2% glutaraldehyde and 2.5% formaldehyde in 0.1Mphosphate buffer, pH 7.2) and cephalic parts of labialglands were dissected under the fixative. The glands werefixed at laboratory temperature for 1 day. Tissues werethen washed in pure 0.1M phosphate buffer and postfixedin 1.5% osmium tetroxide in the same buffer for 2 h. Afterfixation the glands were washed in bidistilled water,dehydrated in 50%, 75%, and pure ethanol, and embeddedinto Spurr resin (standard mixture). Semithin and ultrathinsections were made with a Reichert Ultracut ultramicro-tome. Semithin sections were stained with Azure II andexamined under a Carl Zeiss Amplival optical microscope(equipped with Canon EOS 300D camera). Ultrathinsections were stained with uranyl acetate and lead citrate(standard recipe) and examined under a Jeol 1011transmission electron microscope.

2.3. Electrophysiology

Using isolated queen antennae in EAG analyses, theability of labial glands extracts of males of different ages toelicit antennal response was investigated. Prior to EAGexperiments, four extracts of individual glands from malesof each age examined (new born, 2-, 4-, 7-, 10-, 12-, 17-, 20-, 24-, and 33-day-old males) were pooled to minimizeinterindividual differences. From each respective sample,0.2 gland equivalents were loaded onto a 1� 0.5 cm pieceof filter paper (F 12A, pore size 16 mm, Fisher Scientific,Waltham, MA, USA) inserted in a Pasteur pipette. Aftersolvent evaporation (15min), the loaded odour cartridgeswere sealed with paraffin and stored under �20 1C untiluse. Prior to each experiment, the odour cartridges wereallowed to equilibrate 1 h at room temperature. In EAGexperiments, stimuli were delivered by injecting an air pulse(1 s, air flow 0.8ml/min) through a respective odourcartridge into a continual air stream directed to theantenna. Each antenna was randomly subjected tostimulation by labial gland extract of each age category.Standard stimuli (1 mg of benzyl alcohol) were applied atthe beginning and the end of each experiment to determinepossible changes in antennal sensitivity during EAGrecordings. All antennal responses (maximal negativedeflection during stimulation in mV) were normalized tothese standard stimuli. EAG amplitudes are expressed in %of standard stimulus. A total of 21 antennae were used(14 queens, 21 antennae in good physiological state).

GC-EAD analyses were performed on a 5890 A Hewlett-Packard gas chromatograph equipped with a DB-5 column(30m� 0.25mm, film thickness 0.25 mm, J & W Scientific,Folsom, CA, USA). The column was split by a Graphpack3D/2 four-arm splitter. The splitter led the eluate to FIDand EAD detectors. N2 make-up gas at 20ml/min flow ratewas introduced via one arm of the splitter to compensate

the flow reduction due to splitting. Labial gland extracts(1–5 ml) were injected splitless. The GC was operated at aninitial temperature of 50 1C (2min), 30 1C/min to 270 1C(10min). The temperature of injector and FID were set to230 and 260 1C, respectively. The EAD detector consistedof queen antenna connected via two glass Ag/AgClelectrodes to universal AC/DC Probe (Syntech). EADsignal was 10� amplified. Both EAD and FID signalswere fed to a PC via the serial IDAC interface box(Syntech) and analyzed using GC-EAD software (Syntech).The antennae were exposed to compounds eluting fromGC via the Effluent Conditioner Tube (Syntech) heated to180 1C. Queens (N ¼ 4) used for GC-EAD recording werekept at low temperature (�5 1C) and high humidity untiluse. The tip of each isolated antennae was removed byexcision prior to GC-EAD analysis. Each antenna wasused only once.

3. Results

3.1. General features

The cephalic part of labial gland consists of numerousacini and connecting ducts (Fig. 1). Each acinus is formedby several secretory cells and a large lumen where thesecretion is stored. The main secretory organelles ofsecretory cells are Golgi bodies (Fig. 2e) and endoplasmicreticulum (Figs. 2a, f, and 3a). Nuclei with dispersedchromatin are large and centrally localized, up to 15 mm inthe largest dimension (Fig. 2a). Mitochondria are elon-gated (up to 2 mm), populous and evenly distributed(Figs. 2a and 3a). Microtubules form a dense mostlyapico-basally oriented network attached to the basalhemidesmosomes (not shown) that connect the secretorycells with the basement membrane (60–100 nm thick).Intercellular junctions comprise apical belt desmosomesand septate junctions; the basal parts of adjoining cells arefree (not shown). The acinar cuticular intima consists ofouter and inner epicuticle (10 and 15–30 nm thick,respectively) and two ill-defined basal layers. Basal layersare formed by diffuse chitinous fibres with differentdensities. The thickness of the cuticle increases with agefrom 200 to 600 nm in pharate males up to 1.5 mm inindividuals older than a day (Figs. 2a, e, f, 3a and b).

3.2. Ontogeny of the cephalic part of the labial gland

The acini of pharate imagoes are flat with minute lumina(opposing cuticles are close together, see Figs. 1a and 2a).The secretory cells are voluminous (about 20 mm from thebase to the apex and up to 30 mm long). Apical plasmamembrane forms short and spaced microvilli (Fig. 2a)equipped with apical plaques attached to the cuticle. Thebasal invaginations may reach up to 4 mm into the cell. Atthe cell bases, the formation of pinocytotic vesicles can beseen frequently (Fig. 2c). The dominant organelle ofsecretory cells of this stage is rough endoplasmic reticulum

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(RER) which is mostly tubular but around the cell nucleusthe RER forms flat cisternae (Fig. 2a). Golgi bodies arepresent sporadically. Quite characteristic are lipid dropletsfreely distributed in cytoplasm predominantly at the cellbases (Figs. 2a and b). The droplets are usually about 1 mmin diameter (rarely up to 4 mm). Adipocytes are oftenintermingled with acini having tight contact with particularsecretory cells in pharate imagoes (Figs. 1a and 2b).

In 12 h and 1-day-old males, the acini are still flat but thefirst secretion appears in the lumen (Fig. 1b). The apicalplasma membrane is plain and touching the cuticularintima (Fig. 2e). The pinocytosis is less abundant incomparison to pharate imagoes. RER starts to disappearand Golgi bodies become numerous (Fig. 2e). Theyproduce small lucent vesicles of heterogeneous content.After exocytose, their content accumulates between the cell

apex and the cuticle (Fig. 2f). Smooth endoplasmicreticulum (SER) starts to be formed in males of this age(Fig. 2e). Occurrence of adipocytes among acini wasobserved rarely in 12 h and 1-day-old males, and neverobserved in older males.The secretion volume in the lumen rapidly increases and

the size of secretory cells decreases starting at age day 2(Fig. 1c). The cells elongate (up to 40 mm) at the expense oftheir width (usually about 10 mm at the 4th day of age).Development of different acini desynchronizes but the cellswithin one acinus usually exhibit similar development.Plasma membrane invaginates apically, and these folds canreach 1–2 mm (Fig. 2f). Moderately dense basal layer (up to30 nm) occurs to strengthen the apical plasma membrane(Figs. 2d and f). The pinocytosis rate slows down and stopscompletely in males older than 3 days. RER becomes rare

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Fig. 1. Development of acini in the cephalic region of the labial gland in pharate, 1-, 8-, 17-, and 33-day-old male Bombus terrestris: (1a) development of

acini in the pharate imago. Note the adipocytes located among the acini; (1b) development of acini in a 1-day-old male; (1c) development of acini in an 8-

day-old male. Note the difference between living (right side of the panel) and dead (left side of the panel) secretory cells; (1d) development of acini in a 17-

day-old male. All secretory cells are dead, but the amount of secretion is relatively large; (1e) development of acini in a 33-day-old male. Note the minute

volumes of secretion inside the acini. Bar represents 200mm in all panels. (Abbreviations: A, adipocyte; al, acinar lumen; gd, labial gland duct; hc,

haemocoel; tr, trachea.)

J. Sobotnık et al. / Journal of Insect Physiology 54 (2008) 204–214 207


and cisternal RER disappears completely. Groups ofribosomes remain scattered among SER tubules (Fig. 2f).The number of Golgi bodies decreases, however somelucent vesicles produced by Golgi apparatus are stillpresent in the cytoplasm. The dominant organelle ofsecretory cells is SER formed by apico-basally orientedtubules. The tubules reach the apical membrane andapparently penetrate through strengthened basal layer(Fig. 2d). SER produces moderately electron-dense (lipid-like) secretion that first accumulates between the cell apexand the cuticle and later also in the cytoplasm. Some

mitochondria are apparently associated with secretiondroplets (inset in Fig. 3a). The volume of acinar lumenreaches maximum approximately in the 5th day of adultlife and then slowly decreases. At the age of 5 days, the firstapoptic cells (see Fig. 1c) were observed. The process of celldeath starts in filling SER with secretion, disintegration ofSER (Fig. 3a) and accumulation of secretion in the form ofdroplets located freely in the cytoplasm (Fig. 3b). Theseevents are followed by swelling of the mitochondria andchromatin compaction (content of nucleus changes intoheavy electron-dense condensations and the volume of

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Fig. 2. Secretory cell morphology in the cephalic region of the labial gland in pharate, 1, and 3-day-old male Bombus terrestris: (2a) secretory cell in the

acinus of the pharate imago. Lipid droplets in the cytoplasm are marked with asterisks. Bar represents 5mm; (2b) basal part of a secretory cell in the

pharate imago showing the tight contact with adipocyte. Note the extensive system of basal invaginations and lipid droplet (asterisk) in the cytoplasm of

secretory cell. Bar represents 2mm; (2c) detailed view of the base of a secretory cell in pharate imago showing formation of pinocytotic vesicles. Bar

represents 200 nm; (2d) detailed view on the apex of secretory cell in a 3-day-old male. Note the apico-basally oriented tubules of SER, of which two

(arrowhead) penetrate through strengthened basal layer of plasma membrane (arrow). Bar represents 200 nm; (2e) apical part of a secretory cell in a 1-day-

old male. Note that the apical plasma membrane (marked with arrowhead) is flat, RER and Golgi apparatus are still present, and SER occurs only locally.

Bar represents 2 mm; (2f) apical part of a secretory cell in a 3-day-old male. Note that ribosomes (marked with arrowheads) are detached from ER and

clustered among SER tubules. The secretion produced by Golgi apparatus is located between the secretory cell and the cuticle. Bar represents 2mm.

(Abbreviations: A, adipocyte; al, acinar lumen; c, cuticle; ga, Golgi apparatus; hc, haemocoel; ld, lipid droplet; m, mitochondria; n, nucleus; rer, rough

endoplasmic reticulum; ser, smooth endoplasmic reticulum.)

J. Sobotnık et al. / Journal of Insect Physiology 54 (2008) 204–214208


nucleus decreases; Fig. 3c). The last living cells can be seenin glands of 10-day-old males. The processes of secretorycell remnant disintegration continue (Figs. 1c–e and 3d).Relatively small amount of remnants are observed inglands older then 33 days, with some droplets of secretionstill present (Fig. 1e).

3.3. Composition of the labial gland secretion and changes

with age

The composition of 4-day-old male labial gland secretionin B. terrestris is listed in Table 1. Four-day-old male labialgland extract elicited maximal EAG queen antennal responseand thus was selected for labial gland secretion compositionGC–MS analyses. Only compounds present in amounts

above 0.1% are listed in Table 1. The pheromone blend isdominated by the isoprenoid compounds 2,3-dihydrofarne-sol and geranylcitronellol. Remaining compounds are fattyacid esters and alcohols. Table 1 gives mean values andstandard deviations from four males’ glands.The compositions of the labial gland secretions differ

with age. Fig. 4 shows GC–MS analyses (reconstructedTIC chromatogram) of male labial gland extracts (newlyborn, 4, 12, 17, 20, and 33 days old). Both quantitativeand qualitative changes in the composition of malelabial secretion were observed. The most remarkableage-dependent change in quantity was observed with2,3-dihydrofarnesol (Fig. 4, peak 3). The amount of2,3-dihydrofarnesol was undetectable in young glands(newly born males), rapidly increased up to 4–7 days of

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Fig. 3. Apoptosis in secretory cells in the cephalic region of the labial gland in older male Bombus terrestris: (3a) secretory cell of acinus in a 5-day-old

male. Note the disintegration of SER. Inset shows mitochondria placed in the droplet of secretion. Bar represents 5mm (500nm in inset); (3b) secretory cell

of acinus in a 5-day-old male. Note the plentiful release of secretion from living cell. Bar represents 5mm; (3c) dead secretory cell of an acinus in a 5-day-

old male. Note the increased decomposition of dispersed chromatin and numerous remnants of mitochondria (arrowheads). Bar represents 5 mm; (3d) dead

secretory cell of acinus in a 17-day-old male. Note that the amount of cell remnants has decreased but some secretion is still present in the remaining cells.

Arrowhead marks remnant of mitochondrion. Bar represents 5mm. (Abbreviations: al, acinar lumen; c, cuticle; dm, decomposing mitochondria; dn,

decomposing nucleus; hc, haemocoel; m, mitochondria; s, droplets of secretion; ser, smooth endoplasmic reticulum.)



the age, and then gradually decreased close to zero in olderglands. In older glands, high quantities of dihydrofarnesyldodecanoate, hexadecyl dodecanoate, and geranylcitronel-lyl dodecanoate were detected (peaks 10–12). Thesecompounds were absent in glands of very young males.

3.4. EAG and GC-EAD

Gland extracts from males of all ages examined elicitedEAG responses in queen antennae (Fig. 5). The EAGactivities increased in response to extracts from newborn to4-day-old males and then decreased gradually whenstimulated by gland extracts dissected from males olderthen 7 days. EAG responses to extracts of 33-day-oldglands elicited only 30% of maximal EAG reaction toextract of 4-day-old glands. The bars represent mean andstandard error from 21 successful antennal recordings.GC-EAD responses to labial gland extract of 7-day-old

male B. terrestris were recorded from isolated queenantennae. Several compounds elicited antennal responses(Fig. 6). The GC-EAD active compounds were identified as

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Table 1

Composition of labial gland extract from 4-day-old male Bombus terrestris

Compound Relative proportions (%; N ¼ 4)

Mean S.D.

Ethyl dodecanoate 14.4 1.2

2,3-Dihydrofarnesal 0.6 0.4

2,3-Dihydrofarnesol 57.9 4.6

Hexadecanol 1.6 0.3

Octadecatrienol 1.8 0.5

Octadecenol 0.1 0.1

Henicosane 0.2 0.1

Geranylcitronellol 13.3 4.5

Tricosene 0.1 0.1

Icosenol 6.1 3.4

Tricosane 0.9 0.9

Docosenol 0.4 0.1

Pentacosane 0.1 0.1

Heptacosene 0.3 0.2

Geranylcitronellyl butyrate 0.5 0.6

Nonacosene 1.1 1.9

2,3-Dihydrofarnesyl tetradecenoate 0.2 0.2

Hexadecyl dodecanoate 0.5 0.8

252015 10

0 days

4 days

12 days

17 days

20 days

33 days

IS

1

2

3

45

687

9

1011

12

time (min)

Fig. 4. Chromatograms of labial gland extracts from 0-, 4-, 12-, 17-, 20-, and 33-day-old male Bombus terrestris: IS, 1-bromodecane; 1, ethyl dodecanoate;

2, 2,3-dihydrofarnesal; 3, 2,3-dihydrofarnesol; 4, hexadecanol; 5, octadecatrienol; 6, geranylcitronellol; 7, icosenol; 8, tricosane; 9, docosenol; 10,

dihydrofarnesyl dodecanoate; 11, hexadecyl dodecanoate; 12, geranylcitronellyl dodecanoate.

J. Sobotnık et al. / Journal of Insect Physiology 54 (2008) 204–214210


ethyl dodecanoate, dihydrofarnesal, dihydrofarnesol, hexa-decanol, octadecatrienol, and geranylcitronellol. Amongminor components, tetradecanal, tetradecanol, octadecatrie-nal, and octadecenoic acid elicited minor EAD responses.

4. Discussion

4.1. Labial gland structure

The basic arrangement and ultrastructural organizationof the cephalic region of the labial gland in male B.

terrestris in general corresponds with those alreadydescribed for other bumble bee species (Agren, 1975;Agren et al., 1979). The thoracic part of labial gland in B.

terrestris is not associated with pheromone production.The secretion produced here is proteinaceous and it isreleased in saliva (Sobotnık et al., unpublished).

The cephalic part of labial gland represents the largest andthe most important exocrine organ in B. terrestris males. Itconsists of hundreds of acini with secretory cells producing ahigh amount of secretion (up to milligrams per single male).Particular cells of a single acinus yield to the similar timing ofchanges, but the development of different acini starts to bedifferent each other from the 2nd day of male life. We expectthat this desynchronization is due to exhaustion ofpheromone precursors and/or energy resources. This ob-servation differs from that made by Agren et al. inB. lapidarius, B. hypnorum, and B. hortorum, in whichfunctional differentiation of individual cells within a singleacinus was observed. The biological significance of suchasynchronous development is not known; but the possibilitythat particular acini might be involved in production ofdifferent pheromone components cannot be excluded. Labialgland secretion begins immediately after eclosion. The

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0

100

200

300

400

500

600

0 2 4 7 10 12 17 20 24 33 air std

EA

G r

esp

on

se

(%

std

)

male age (days)

air std

Fig. 5. EAG responses of Bombus terrestris queen antennae to labial gland extracts from 0-, 2-, 4-, 7-, 10-, 12-, 17-, 20-, 24-, and 33-day-old male Bombus

terrestris. Data represent mean7S.E.; N ¼ 21.

6 1 2 3 4 5

a

b

c,d

Fig. 6. GC-EAD responses by queen Bombus terrestris antennae (N ¼ 2) to labial gland extracts from 7-day-old male Bombus terrestris. The upper

chromatogram represents FID response, while two lower traces represent queen antennal responses from two independent GC-EAD recordings. The

active components are as follows: 1, ethyl dodecanoate; 2, 2,3-dihydrofarnesal; 3, 2,3-dihydrofarnesol; 4, hexadecanol; 5, octadecatrienol; 6,

geranylcitronellol (major chromatographic peaks); a, tetradecanal; b, tetradecanol; c, octadecatrienal; d, octadecatrienoic acid (minor chromatographic

peaks).



production reaches its maximum at about the 6th posteclo-sion day and stops about the 10th day of the adult life. Thenthe secretion volume in labial gland gradually decreases dueto male-marking behaviour and reaches zero in older males.Similarly to Agren et al. (1979), we have not observedinnervation of labial gland; therefore we speculate thatsecretion release is due to increased pressure caused either bychewing muscles or hemolymph pressure.

The quantitative changes in volume of secretion and ofsecretory cells correspond well with profound changes ofsecretory cells ultrastructure. The most important changewas observed in endoplasmic reticulum. In pharate males,only RER is present in the secretory cells. Since RER isregarded as the site of proteins biosynthesis (Becker et al.,2006), proteins necessary for pheromone biosynthesis arelikely formed at this particular period. The majority ofRER breaks down by the 1st posteclosion day and RER isthen replaced with SER. Soon after, the first quantum ofpheromone is synthesized. Similar transformations ofendoplasmatic retiulum were observed in young labialglands of bumble bee species B. lapidarius, B. hypnorum,and B. hortorum (Agren et al., 1979). Our data clearly showthat SER is responsible for production of lipid-likesecretion. SER is for a long time known to produce fattyand oily products (Fawcett, 1966), and based on all dataavailable we assume that in male B. terrestris labial gland,SER is responsible for male-marking pheromone biosynth-esis similarly to other exocrine glands in many insectspecies (for reviews see Percy-Cunningham and MacDo-nald, 1987; Tillman et al., 1999). On the other hand, therole of Golgi apparatus in pheromone production is notclear. Our data indicate that maximum secretion of Golgiapparatus occurs during the 1st post eclosion day, i.e., afew days earlier then that SER activity peak. The materialproduced by Golgi apparatus is enclosed in membrane-bound heterogeneous vesicles that were observed to bepresent in secretory cells long after Golgi apparatusdisappearance (rarely up to the 5th day of life). Primaryfunction of the Golgi complex is to process (e.g., to modify,sort, pack and distribute) protein and lipid macromolecules(Moreau et al., 1990; Becker et al., 2006). Agren et al.(1979) suppose that the ‘‘prosecretion’’ produced by SERbecomes condensed or otherwise manipulated by Golgivesicles and then it is transported to the apical infoldings ofsecretory cells where it passes the apical borders into thelumen of the acinus. Our observation is not compatiblewith Agren’s general suggestions since (i) we observed thatSER produces, releases, and later accumulates the secretionthat is quite different from the secretion produced by Golgiapparatus (e.g., droplets located freely in the cytoplasm vs.heterogeneous secretion enclosed in membrane); (ii) thepeak of Golgi apparatus activity precedes the peak of SERactivity (Golgi apparatus was not observed in glands olderthan 3 days). Some insight into the question of whether andhow the Golgi complex is involved in pheromonebiosynthesis might provide detailed evaluation of changesof individual pheromone components during ontogenesis.

The presence of dense basal layer strengthening theapical plasma membrane and the occurrence of apoptosisat the end of secretion production are two particularlyintriguing findings herein. The ultrastructural evidence ofapoptosis in labial glands of B. terrestris (namelydisintegration of SER and nucleus, and swelling ofmitochondria) is both similar to, e.g., Bowen et al. (1996)or Silva-Zacarin et al. (in press) and different from, e.g.,Sreng (1998) or Hacker (2000) previous observations. Thefirst apoptic cells appear in male labial gland at 5 days. Thelast living secretory cells persist in the gland up to the ageof 10 days and then the pheromone production ceases.Occurrence of apoptosis in the inter-moult period is aunique feature, because it is usually connected withmetamorphosic activities (e.g., elimination of larval organs,differentiation of adult organs, rebuilding of tissues) (for areview see Hacker, 2000). Contrary to B. terrestris,

secretory cells of labial glands in B. lapidarius,B. hypnorum, and B. hortorum remain alive for the entiremale bumblebee lifespan; though a decrease in cell volumeis observed in older individuals.Findings herein indicate a link between the primary

metabolism and pheromone biosynthesis in the 1st days ofmale bumblebee adulthood: (i) close association betweensecretory cells and adipocytes occurring to the 1st day ofmale life; (ii) presence of lipid droplets in the secretory cellsof pharate imagoes; and (iii) active transport of materialfrom hemolymph to the secretory cells via pinocytosis atthe cell bases in males younger than 3 days. Recently, arelationship between the composition of the pheromoneand fatty acids in triacylglycerols of the fat body has beenreported (Cvacka et al., 2006; Luxova et al., 2003). Thesepreliminary results suggest that pheromone production isclosely associated with the primary metabolism of thebumblebee. Our findings have a practical implication forbiosynthetic studies, especially for those comparing de novo

synthesis to that occurring from lipid precursors trans-ported from the fat body (Luxova et al., 2003).Labial glands in male B. terrestris are active and produce

pheromone in young individuals with production dimin-ishing with age. Thus, the ability of older males to marktheir territories depends fully on pheromone producedearlier and stored in the gland. Pheromone productionstarts after male emergence from the cocoons. Males donot leave the nest immediately after eclosion. Rather, theymay stay in the nest up to 7 days (Hoffer, 1882; Free andButler, 1959). During this time, pheromone production andaccumulation reaches its maximum. The energy forpheromone synthesis is fully provided by the colony, sincemales in the nest are fed by other members of the colony.

4.2. Chemical analysis of pheromone blend and biological

activity of individual components

B. terrestris male-marking pheromone is a complexmixture composed of two different chemical groups,isoprenoids and fatty acid derivatives (Table 1). These



different groups are produced by different biosyntheticpathways (Morgan, 2004). The major compounds elicitedEAD responses in queen antennae. However, several minorcomponents were antennally active, suggesting that thespecificity of male-marking pheromone is maintained by amulticomponent mixture rather then by one or two majorcomponents. This conclusion is contradictory to the workof Bergman (1997), who considered major components ofthe secretion to convey species-specific sex pheromonefunction (e.g., dihydrofarnesol in B. terrestris). The majorcompound, 2,3-dihydrofarnesol, however, exhibited thelargest age-dependent quantitative changes relative to allcompounds found in labial gland secretion. This impliesthat 2,3-dihydrofarnesol is the most important (though notsufficient) for the pheromone function. Larger moleculessuch as terpenic esters of fatty acids, present only in glandsof very old males (33 days), did not elicit EAG responses.These compounds may be formed in the gland by enzymesfrom terpenic alcohols and fatty acids present in glands ofyounger males. Their biological significance remainsunknown.

EAG experiments on queen antennae revealed thatvolume equivalents of extracts of labial gland of differentages elicited different biological activity. EAG activitycopied the qualitative and quantitative changes in the labialgland with highest activity peak for glands of 4–7 old malesand with minimum EAG responses for gland extracts ofvery old males (33 days). EAG activity was significant incomparison with solvent even in glands immediately afteremergence. The EAG results tightly correspond withultrastructural changes and chemical analysis, namely thetime course of excretion accumulation and depletion. Thecontribution of qualitative and/or quantitative changes topheromone blend activity cannot be determined based onour experiments. EAG represents a sum of receptorpotentials of all olfactory receptor cells activated by astimulus. The EAG amplitude reflects both quantity andquality. In spite of these limitations, our results as a wholeclearly show that males between 4 and 7 days of age are thefittest with respect to pre-mating activity.

Acknowledgements

We would like to thank the staff of the Laboratory ofElectron Microscopy (Faculty of Sciences, Charles Uni-versity, Prague) for their kind help. Our thanks also belongto Mrs. Jitka Pflegerova (Institute of Entomology,Academy of Sciences of the Czech Republic, CeskeBudejovice) for preparation of samples for TEM, OldrichHovorka (IOCB, Prague) for dissecting labial glands forchemical analyses and EAG, Prof. Johan Billen (KatholicUniversity, Leuven, Belgium) for consultation over severalobservations, and Prof. Jan Zd’arek (IOCB, Prague) forcritical reading of this manuscript. Financial supports fromthe Ministry of Education of the Czech Republic (Projectno. 2B06007) and the Grant Agency of the Academy of

Sciences of the Czech Republic (Grant no. IAA4055403)are gratefully acknowledged.

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Age-dependent changes in structure and function of the male labial gland in Bombus terrestris

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