Protoplasma 100, 251--265 (1979)

PROTOPLASMA 9 by Springer-Verlag 1979

The Salivary Gland Secretions of a Neotropical Bumblebee MARIA LmZA S. MELLO * a n d B. C. VIDAL UNICAMP, Campinas, Brasil Received March 12, 1979 Accepted in revised form June 18, 1979

Summary T]he larval salivary gland secretions of Bornbus atratus were studied with cytochemical and cytophysical methods. In the young feeding larvae a proteic filamentous secretion depicting striations perpendicular to the long axis of its fibrillary threads and exhibiting a wellordered macromolecular array was found. It appears not to differ from the silk secretion described for the fully grown larvae of an european Bombus species. However, in the fully grown larvae of B. atratus changes which have not yet been reported for other bees occur involving the salivary gland secretion. Two secretion types are then distinguishable. One is composed of carboxylated and sulfated acid glycosaminoglycans and glycoprotein(s) (mucous secretion), and the other has the same composition as that of the filamentous secretion of the young larvae, although differing morphologically and in terms of macromolecular al~ignment (flocculent secretion). The filamentous secretion is assumed to be involved in B. a~ratus with the spinning of the "'silken partitions" which at a relatively early stage separate the larvae reared within a common cell from one another. The mucous and flocculent secretions will participate in the cocoons which will cover the pupating larvae. The filamentous and flocculent secretions appear to contain an c~-helical fibroin, glycoprotein(s) and lipoprotein(s), but not collagen-type proteins. Keywords: Bombus atratus; Salivary gland secretion cytochemistry; Salivary gland; Silk.

1. I n t r o d u c t i o n I n insect l a r v a e silk is p r e d o m i n a n t l y a s a l i v a r y g l a n d p r o d u c t . I t is generally used to spin the cocoon which protects p u p a e during m e t a m o r p h o s i s . Special a t t e n t i o n has been focused on the silk secreted b y h y m e n o p t e r a n l a r v a e as it differs in protein composition a n d c o n f o r m a t i o n f r o m the classical lepidop t e r a n silks. While the latter exhibit pleated sheet structure for aggregates of [3- or e x t e n d e d protein chains a n d a high content of glycine residues, m o s t of the silk proteins of the h y m e n o p t e r a n g r o u p h a v e been recognized as being of a-helical f o r m (a-fibroins) a n d displaying reduced glycine and increased * Correspondence and Reprints: Department of Cell Biology, Institute of Biology, UNI(2AMP, 13100 Campinas (SP), Brazil.

0033-183X/79/0t00/0251/$

03.00

252

MARIALUIZa S. MELLOand B. C. VIDAL

content of acidic residues (RuDALL 1962, FLOWER and KENCHINOTON 1967, LucAs and RUDALL 1967, RUDALL and KENCHINGTON 1971, KENCHINGTON 1972, personal communication). On the other hand, a collagen-type protein has been specially found in silks of most members of the hymenopteran subfamily Nernatinae (RuDALL1962, LUCAS and RUDALL 1967, RUDALL and KENCHINGTON 1971). In an European bumblebee silk has been described as being organized into tactoids and fibers, both observed at the light and electron microscopic levels (FLOWERand KENCHINGTON 1967). These structures are highly oriented even in the gland's lumen, as detected with the polarizing microscope. The tactoids are produced in small numbers whereas the fibers make up the bulk of the secretion. When observed within the salivary gland lumen the fibers display a substructure in the form of rodlike bundles of fibrils (comparable to the structure of the A bands in striated muscle) which were termed fibrous bars (t~LOWERand KENCHINGTON 1967). However, observations with the polarizing microscope have indicated differences in the secretion present in the gland's lumen of larvae of a neotropical bumblebee as compared with that of the European bumblebee, also varying as a function of the larval development (M~LLO and VIDAL 1972, unpublished data). Therefore, in the present work, in an attempt to characterize the secretions of the salivary glands of a neotropical bumblebee like B o m b u s atratus, these were studied at the morphological, cytochemical, and cytophysicai levels, by means of polarization, fluorescence, and ordinary light microscopy in early and fully grown larvae.

2. M a t e r i a l s and M e t h o d s Young feeding and fully grown pre-defecating larvae, about 9 and 15-days old, respectively (SAKAOAMI et al. 1967), of Bombus atratus Franklin (Hyrnenoptera, Apoidea) from an observation hive reared in the laboratory were used. They were generously supplied by Prof. R. Zuccm (Dept. of Ecology, FFCL, Ribeir~o Preto, SP). The larvae were fixed in 10% formalin for 24 hours at room temperature, rinsed in tap water for 24 hours, and embedded in paraffin wax. Sections cut at 7 btm were subjected to cytochemicai tests. Some sections were stained with haematoxylin-eosin for the observation of general morphological aspects. 2.1. Cytochemistry Proteins. A 0.1~ xylidine ponceau 3 RS (BDH, London) solution at pH 1.7 was used for the investigation of total electropositive radicals (VIDAL 1970). Controls were nitrosated for 15 hours (LILLIE 1958). Richness in lysine residues was investigated with the dansylchloride (Fluka) fluorescence method (RossELeTand Rucrt 1968). The dinitrofluorobenzene (Carlo Erba) reaction (DNFB) for e-amino acid groups according to the method of Z~t~toTVI and ENG~L (1962) was also used. Richness in hydrophobic residues was investigated on sections stained with 0.1~ solutions of 8-anilino-l-naphthalene-sulfonic acid sodium salt (ANS, Kodak-T484) in butanol or Mcllvaine buffer at pH 2.8 for 30 minutes and mounted in Eukitte (O. Kindler, Freiburg, WG) and nujol, respectively (VIDAL1978). Glycans and glycoproteins reactive to PAS. Controls underwent prior treatment with diastase and salivary amylase at 37 ~ (1 hour).

The Salivary Gland Secretions of a Neotropical Bumblebee

253

Acid glycosaminoglycans. Staining with 1if0 alcian blue (G. T. Gurr) solutions in I % acetic at pH 2.5 (LIsoN 1960) and in 0.1 iN HCI at pH 1.0 (L~v and SPICER 1964) was carried out. The reactivity to 0.025ff0 toluidine blue (Merck A.G.) solutions in 0.1 M citric acid and 0.2 M anhydrous disodium phosphate buffer at pH values of 2.5, 3.0, 3.4, 4.0, and 5.0 was also studied. Controls used: methylation for 6 hours, and saponification (20 minutes) after methylation, prior to treating the sections with the toluidine blue solutions (FlscRER and LILLIE 1954, Srlcei< and LILLIe 1959). The observations were made with a Zeiss photomicroscope equipped for phase, polarization, and fluorescence microscopies and microspectrophotornetry, and with a Zeiss microspectrofluorometer.

2.2. Polarization Microscopy and Microspectrophotometry Anisotropy was investigated in the glandular cells and in the secretory products at the lumen of the glands of stained and unstained preparations. The birefringence sign was ascertained in unstained sections using the 5%/4 S6narmont's compensator. The linear dichroism phenomenon was investigated on sections stained with the xylidine ponceau solution, by cytophotometrically determining spectral absorption curves on filaments oriented parallel (d II) and perpendicular (d.l.) to the plane of polarized light, at wavelengths from 470 to 610 nm. A Zeiss Pol-photomicroscope equipped with a O1 photometer and an EMI 6256 photomultiplier was used. Operating conditions were: Plan 40/0.65 objective, optovar 1.6, photometric diaphragm dia. = 0.16ram, field diaphragm d i a . = 0 . 3 m m , Epiplan 16/0.30 condenser, and Schott continuous monochromator filter ruler. The area of the specimen measured was therefore equal to 4.90 ~tm~.

2.3. Fluorescence Microscopy Simple visual analysis of the fluorescence of the silk gland secretion stained with dansylchloride was performed with the Zeiss photomicroscope using UV light, exciter filter I, and 410 barrier filter.

2.4. Microspectrofluorometry Spectral emission measurements were determined on sections stained with ANS solutions using a Zeiss microspectrophotometer equipped to perform microfluorometry with the III RS condenser. A monochromator ruler was placed in front of the HTV-R 446 photomultiplier. An l-tBO-100 w stabilized mercury lamp was used as light source. Operating conditions were: Planapo 40/0.95 objective; area of the specimen measured: 78.50 ~um2; filter sets: position I ( U G 1 U V exciter filter transmitting a 5%~ 365/6nm exciting light; FT 420 chromatic splitter; LP 418 barrier filter) and position IV (BP 546/7 exciter filter transmitting a 5%= 546/7nm exciting light, or BG 12 exciter filter transmitting a k = 400 nm exciting light; FT 580 chromatic splitter; LP 590 barrier filter) (VmAL 1978).

3. R e s u l t s

The anatomical features of the silk glands of B. atratus do not remarkably differ from those reported for a bumblebee of the Northern Hemisphere (Fig. 1) (FLoxr and K~NCHINCTON1967). Aspects of the bifurcation of the glandular tubes could even be demonstrated at the histological section level (Fig. 2). However, some characteristics of the gland cells, namely the cellular protuberances and the considerable variation in cell height described for

254

MeLto et aL : The Salivary Gland Secretions of a Neotropica[ Bumbiebee

Bornbus lucorum (FLowER and K~NCmNOTON 1967) were not observed in the salivary glands of B. atratus. Furthermore, as larval development progresses, the salivary gland secretion of B. atratus exhibits changes in its com-

position, morphology, and cytophysical properties which have not yet been reported for other Apoidea species. In the young feeding larvae, the secretion present at the lumen of the salivary glands stains with xylidine ponceau (Figs. 3-6), and eosin, and is slightly PAS-positive. It is absorbant at ~. = 410-460nm when subjected to the

d

:<

F'

Fig. 1. S&ematic view of the larval salivary glands of Bombus. Only one of the two main branches of the glands is represented, d = duct; D = distal region of the glands; P = proximal zone of the gtands

DNFB reaction and fluoresces after being treated with dansylchloride solution. It is highly fluorescent when stained with ANS-butanol and ANS-McIlvaine buffer (pH 2.8) solutions. When the sections were observed with the filter set at position I, the fluorescence of the ANS-stained secretion was blue. At position IV the fluorescence was red for both dye solvent solutions. The emission profiles of the ANS-stained secretions are shown in Fig. 9. The emission values at position IV were determined relative to the emission maximum at position I, fixed as 100~ in the microspectrofluorometer. For the ANS-butanol staining condition, the emission values of the peaks observed at position IV (exciting light, )~ = 546/7 nm) were comparatively larger than those exhibited after the ANS-Mcllvaine buffer staining (Fig. 9). Tab. t summarizes results concerning the emission peaks of the ANS-stained B. atratus secretion and published data on collagen and elastin, both similarly stained (VIDA5 1978).

Fig. 2. Histological section of the salivary glands of a fully grown larva of B. atratus stained with xylidine ponceau. The arrows indicate bifurcated glandular tubes, rnt = Malpighian tube. ~, = 520 nm; • Figs. 3 and 4. Linear dichroism of the fibrillary formations of the salivary secretion of a young larva stained with xylidine ponceau. The section was cut close to the glandular duct. The plane of polarized light (PPL) is indicated (arrow). Most of the fibrillary formations in this section are shown orientated perpendicular (3) and parallel (4) to the PPL. k = 520 nm. • Fig. 5 and 6. Detail of the dichroie image of the xylidine ponceau-stained secretion of a young larva. The plane of polarized light (PPL) is indicated (arrow). Most of the filamentous formations (f) are shown orientated parallel (5) and perpendicular (6) to the PPL. The arrow under the letter f shows the preferential direction of the long axis of the filamentous secretion. The section was cut nearly longitudinal to one of the gland's tubes. k = 520nm;

X900

Fig. 7. Birefringence of the filamentous secretion displayed in Figs. 5 and 6. Structures resembling FLoxc~R and KENCttlNCTON'S (1967) fibrous bars are indicated (arrow). The fibrillary formations (f) have their long axis indicated (arrow under letter f). • Fig. 8. Compensation of the birefringence of Fig. 7. The fibrillary formations (f) have their long axis indicated (arrow under letter f). X 1,600

256

MARIA LUIZA S. MELLO and B. C. VIDAL

The salivary gland secretion of the young larvae does not stain with toluidine blue solutions (except at p H 5), and also appears unstained when treated with alcian blue. It is highly refractile in unstained or even stained sections studied under ordinary light microscopy, displaying a relatively homogeneous fibrillary structure (Fig. 10). Sometimes striations are seen perpendicular to the long axis of the fibrils (phase microscopy). These striations are morphologically comparable to FLowIn~ and KENCmNGTON'S (1967) fibrous bars. The secretion of the young larvae observed with the polarizing microscope exhibits anisotropic properties (Figs. 3-8, 11, and 12). A deep birefringence Table i. Emission Peaks of the Salivary Gland Secretion of Young Larvae of B. atratus Stained With ANS Solutions. W a v e l e n g t h s of the excitation light beam: 365/6 n m (A), 400 n m (B), 546/7 n m (C). E = emission values Dye solvents

Butanol

Mcllvaine buffer at p H 2.8

Materials

Emission peaks (~ in nm) Filter set I Filter set IV

Fluorescence ratio (X 100)

(A)

(B)

(C)

Bombus salivary

470

605-610

gland secretion collagen bundles o elastin fibers ~

61.9 + 1.4 [E;t = 470(A)/EX ~ 700(12 18.0 _+ 0.6 [E;k = 470(A)/EX = 610((

470 470

---

700 610 1 ---

Bombus salivary

480

610

gland secretion collagen bundles ~ elastin fibers "~

15.3 -t- 0.8 [ E x : 4 7 0 ( A ) / E x ~ 700((2 1.9 __. 0.4 [E x = 470 ( A ) / E x = 610(C

520 460-470

700 610 * ---

Secondary peak.

---

-~ VmAL 1978.

is seen even in unstained sections. These properties support the hypothesis of a fibrillary structure making up the salivary secretion at the relatively early stage of the larval development. The striations are also birefringent (Figs. 7, 8, and 12). They are distinguishable upon the fibrillary formations themselves mainly when their bireffingence is compensated (Figs. 7 and 8). The fibers exhibit a positive birefringence sign with respect to their long axis. The striations show a positive birefringence with respect to their right angle position (i.e., across the bars), however, this birefringence is compensated with an angle of 30 ~ larger than that of the fibrillary formations (S6narmont's compensator). The fluorescence displayed by the secretion stained with dansylchloride and ANS solutions was also birefringent. In sections stained with xylidine ponceau a linear dichroism phenomenon was visually and cytophotometrically detected on the secretion (Figs. 3-6 and 14). Its sign is positive (A d = d , - - d. ; d , ~ da. ) with respect to the long axis of the fibrillary formations of the secretion analyzed near the glandular duct (Figs. 3 and 4); the A d maximum is placed at ~ = 520 nm and A d secon-

The Salivary Gland Secretions of a Neotropical Bumblebee

257

d a r y peaks are f o u n d at k = 490 and 550 n m (Fig. 14). H i g h dichroic ratio values ( d n / d • 1.70) are attained at k = 520, 530, a n d 5 7 0 - 5 9 0 nm. Dichroism was also observed at the level of the striations (Figs. 5 a n d 6). Tactoids were n o t observed in the s a l i v a r y gland secretion o f B. a t r a t u s . E

t

1,00

/-,20

440

/

/~60

480

500

520

540

S6O

580

600

8;~0

54.0

660

680

700

nm

Fig. 9. Emission profiles of the salivary gland secretions of young feeding larvae of B. atratus stained with ANS--butanol ( ~ ) and ANS--Mcllvaine buffer (pH 2.8) (. . . . and . . . . . . ) solutions. 9 = filter set I (k of the excitation light beam = 365/6 nm); O = filter set IV (k of the excitation light beam = 400 nm); 5( = filter set IV (k of the excitation light beam = 546/7 nm). The emission values at the various wavelengths were evaluated relative to the maximal emission value obtained with filter set I and calibrated as 100%. In the case of the curve ... the sensitivity of the microspectrofluorimeter amplifier was enlarged with the aim of determining the correct positions of the emission peaks (filter set IV). Each point in the curves is the arithmetic mean of 5 measurements I n the fully g r o w n predefecating larvae the salivary gland secretion presenting cytochemical properties a n d intense birefringence like those depicted b y y o u n g larvae is m o s t l y confined to the center o f the glandular lumen. It generally appears s u r r o u n d e d b y another t y p e of secretion (Figs. 13, 15, a n d 20). This one is finely filamentous and displays a v e r y faint birefringence (Figs. 13, 16,

258

MAI~IALUIZA S, MELLO and B. C. VIDAL

Fig. 10, Approximate longitudinal section of part of a glandular tube of the salivary glands of a young feeding larva stained with haematoxylin-eosin. The filamentous secretion (s) reacts with eosin. 2~ = 550 nm; X570 Fig. 11. Polarization view of Fig. 10. The secretion (s) is deeply birefringent. X 700 Fig. 12. Birefringence view of the salivary secretion in a cross section of the glands of a young larva stained with xylidine ponceau. Arrows indicate striations resembling "fibrous bars". X510 Fig. 13. Polarization view of a salivary gland section from a fully grown larva stained with haematoxylin-eosin. Birefringence is intense in the flocculent secretion (f) and very light in the mucous filaments (rn). Birefringent granulous masses appear to be eliminated from the gland cells and to cross the mucous layer (arrows). X 260

The Salivary Gland Secretions of a Neotropical Bumblebee

259

and 18) which is positive with respect to the long axis of the fibrils. Sometimes at certain points it appears disrupted and resembles bubble-like formations (Figs. 15 and 17). It stains with haematoxylin (Fig. 21) and xylidine ponceau, being in this case devoid of linear dichroism properties. This secretion displays pale fluorescence when treated with the dansylchloride and ANS solutions, and stains very slightly with DNFB. On the other hand, it deeply responds to PAS reaction (after diastase or amylase digestion) (Figs. 15

I"O001E .750 .500

j~176

\

x

\\

.250

z,60

480

500

520

540

560

580

600

nm

Fig. 14. Spectral absorption curves of the salivary gland secretion of young B. a t r a t u s larvae stained with the xylidine ponceau solution at pl-t 1.7. The fibrillary formations were orientated parallel (d II) and perpendicular ( d j . ) to the plane of polarized light. Linear dichroism = A d = d l l - - d J." Each point in the curves is the arithmetic mean of 5 measureme:nts

and 17) and alcian blue staining methods, and stains metachromatically with toluidine blue solutions above a pH of 3.0 (Fig. 20). The basophilia is abolished by methylation and partly recovered after saponification followed by toluidine blue staining at pH ~ 4.0. The mentioned cytochemical data highlight the mucous composition of this secretion. Dichroism could not be evaluated in toluidine blue stained sections due to difficulties concerning the orientation of the entangled filaments of the secretion and their relatively pale staining intensity. In the sections stained with alcian blue solutions the thin

Fig. 15. Salivary glands of a fully grown predefecating larva subjected to PAS reaction. The mucous secretion (m) is strongly stained whereas the flocculent mass (f) appears very slightly stained, k = 540 nm; • 190 Fig. 16. Polarization view of Fig. 15. Birefringence is predominant at the flocculent secretion (f) but also occurs, although with lesser intensity, in the mucous secretion fin) and gland cells (arrows). • 190 Fig. 17. Detail from Fig. 15. f = Flocculent secretion; m = mucous secretion, k -- 535 rim; • Fig. 18. Section of a fully grown larva stained with the alcian blue solution at p H 2.5 and observed with crossed polarizer and analyzer. Birefringence is observed in the flocculent (f) and mucous (m) secretions and glandular cells (arrows). X 256

MELLO et al.: The Salivary Gland Secretions of a Neotropicai Bumblebee

261

Fig. 19. Polarization light view of a salivary gland section from a fully grown larva stained with xylidine ponceau, f = flocculent secretion; m = mucous secretion. • 350 Fig. 20. Section of a fully grown larva where the mucous salivary gland secretion (rn) appears stained after treatment with the toluidine blue solution at p H 4. f flocculent secretion. ~ = 560 nm; • Fig. 21. Very posterior zone of the salivary glands of a fully grown larva stained with haematoxylin-eosin. One of the sections of the glandular tubes (arrow) shows only mucous secretion at its lumen, f = flocculent secretion; m = mucous secretion, k = 540 nm; 5<200 Fig. 22. Birefringent eosin-stained granules (arrows) are being extruded across the cellular border and the mucous secretion layer (m) in the anterior zone of the salivary glands of a fully grown larva. They will make up part of the flocculent secretion (f). The section is nearly longitudinal to part of a glandular tube. • 770

262

MARIALUIZA S. MELLO and B. C. VIDAL

filaments which make up the mucous secretion, showed a red interference color. The deeply birefringent secretion which is still present in the salivary glands of the fully grown larvae looses its filamentous appearance, looking like a flocculent mass (Figs. 13, 16, 18, and 19). It is also devoid of dichroism phenomena after the xylidine ponceau staining. Some granulous aggregates with the same cytochemical and cytophysical properties of this flocculent secretion are eventually observed where the mucous mass generally aposes to the glandular cell border (Figs. 13, 18, 19, and 22). On the other hand, close to the gland opening, the mucous secretion can be observed either inside or concentrically surrounding the flocculent secretion, when cross sections of the glands are studied. The mucous secretion in this stage is apparently eliminated from the glands in an intimate association with the flocculent secretion. The mucous secretion is extruded from the cells at the posterior region of the salivary glands (Fig. 21). Cross sections were seen where this secretion almost completely fills in the glandular lumen (Fig. 21). The flocculent secretion is also present at the posterior region of the glands (Fig. 21). However, extrusion of flocculent eosin-positive birefringent masses were also observed in the cell border of the anterior zone of the glands, close to the glandular duct (Fig. 22). These particles seem to be part of a new wave of flocculent secretion penetrating the mucous layer which in addition surrounds the inner flocculent mass. 4. D i s c u s s i o n

The filamentous silk secretion of the young larvae of B. atratus does not appear to differ morphologically from that described for the fully grown larvae of B. lucorum (FLowER and KENCmNGTON 1967). In addition, their birefringence patterns are alike. However, subsequent changes in the salivary gland secretion of the fully grown larvae of B. atratus have not yet been reported for other Apoidea. These changes are concerned with structure and array of the protein secretion at the gland's lumen, and release of an oriented mucous product from the glandular cells. The mucous secretion was cytochemically demonstrated to be composed of carboxylated and sulfated acid glycosaminoglycans, and glycoprotein(s). Acid glycosaminoglycans and PASpositive glycans have also been reported in the secretion of the larval salivary glands of the stingless bee, Melipona quadrifasciata (M~LLO and VIDAL 1971). In this case, they precede (early 2nd instar) or appear simultaneous (late 3rd instar) to the onset of the structural protein secretion which is stored in the gland's lumen and is extruded from the glands at the end of the larval phase (MELLO and VIDAL 1971). However, the mucous secretion is not morphologically distinguishable from the silk protein filaments in M. quadrifasciata, differing from what occurs in B. atratus.

The Salivary Gland Secretionsof a NeotropicalBumblebee 263 Although the early filamentous and the flocculent secretions of B. atratus are predominantly composed of protein(s), a small amount of PAS-positive glycans (part of a glycoprotein?) is also present. The filamentous secretion is spun relatively early in the larval development and will probably make up the silken partitions which at the 13 day age separate the larvae reared within a common cell from one another (SAKAGAMI et al. 1967). The protective cocoons which will cover the pupating larvae start to be observed only when the larvae are 14-15 days old (SAKAGAMIet aI. 1967). Therefore, it is expected that the composition of the silken partitions differs from that of the cocoons, considering the nature of the secretions for the early and fully grown larvae. In fact, they differ in texture. The silken partitions are thin and semi-transparent, whereas cocoons are thick, tough, and opaque (SAKAGAMI1978, personal communication). The protein(s) present in the filamentous secretion of the young larvae of B. atratus, although exhibiting positive birefringence, positive dichroism after the xylidine ponceau staining, and a substantial amount of lysine residues available to DNFB and dansylchloride stainings is not a collagen-type. The shape of the linear dichroism (A d) curve of Bombus secretion differs from that obtained for collagen, both stained with xylidine ponceau solutions (VIDAL 1970, 1979). The fluorescence displayed by the secretion stained with the ANS solutions exhibits emission characteristics which differ significantly from those of collagen (VIDAL 1978). They are close to patterns displayed by elastin [blue fluorescence for both butanol and Mcllvaine buffer dye solvents (VIDAL 1978)], probably because the silk secretion of B. atratus contains a substantial amount of hydrophobic residues available for ANS binding. In fact, the percentage of hydrophobic amino acid residues in the a-fibroin of four species of Vespidae and Apldae analyzed biochemically is not small as compared with that of the silkworm fibroin (LucAs and RUDALL 1967) and of the ligamentum nuchae elastin (WHITEe t aI. 1964). In addition, work in progress reveals that phospholipid radicals are also present in this secretion, probably as part of a lipoprotein, and certainly contributing to the emission pattern obtained after the ANS staining. The characteristics of the red fluorescence exhibited by the ANS-stained filamentous secretion (filter set IV) may be related to a change in conformation of the stained substrate and in the rigidity of the ANS molecules as a function of the fluorescence probe solvent types (PENZ~R 1972). The fact that the fluorescence with ANS (and dansylchloride) is birefringent indicates the well-ordered disposition of the dye-binding sites. Also the protein(s) present in the flocculent secretion is not a collagen-type as it has the same fluorescence pattern and other cytochemical characteristics of the proteins making up the filamentous secretion of the young larvae. The finding of protein(s) other than collagen-types in the salivary gland secretions of B. atratus is in agreement with the observation that the silks of four [18

Protoplasma100/3--4

264

MARIALUIZA S, MELLO and B. C. VIDAL

species of the Aculeate hymenopteran group display c,-helical fibroin-type proteins (LucAs and RUDALL 1967). The reactivity of the filamentous and flocculent secretions to eosin is certainly due to their large content of acidic residues, usually present in the a-helical silks (RuDALL and K~NCmNOTON 1971). The dichroism detected in the filamentous secretion of B. atratus after the xylidine ponceau staining indicates an ordered alignment of the stained protein threads (VI3AL 1970). Dichroism and birefringence data give support to the idea that the protein fibrous molecules lie side-by-side in a specially ordered fashion, giving rise to the regular "banding" observed in the secretion at the gland's lumen, and resembling the observations reported for B. lucorum (FLowER and KENCHINGTON 1967). Although the flocculent secretion does not differ in composition from the filamentous secretion, observations with the polarizing microscope indicate that proteins in the former undergo some change in their macromolecular array as compared with that of the latter. This would be assumed to facilitate some kind of interaction of the flocculent secretion with the oriented filaments of the mucous secretion. In fact, close to the glandular ducts, the flocculent and mucous secretions form concentric layers closely aposed to each other. Work in progress, however, reveals that they make part of separate layers in the cocoons. It is interesting that the salivary gland mucous secretion appears simultaneously with the onset of a mucous secretion in the Malpighian tubes of same B. atratus larvae (MELLO 1979). The mucous secretion produced in the Malpighian tubes is extruded with a highly oriented array from the larvae apparently at the same time as do salivary gland flocculent and mucous secretions, but its function remains to be solved (MEtLO 1979). Although tactoids have not been observed in the secretion of B. atratus, their occurrence is not to be neglected as even in B. lucorum they appear in small numbers and vary as a function of the gland region and larval age (FLOWER and K~NCHINGTON 1967).

Acknowledgements The authors are indebted to Prof. SHOICHI SAKAGAMI and Dr. C. GAROFALO for valuable information on biology of Bombus atratm and to Prof. RoY BI~UNS for helping with the English. The support of F.A.P.E.S.P. (grants nr. 70/371 and 72/313) is gratefully acknowledged.

References FISCHER, E. I~., LILLIE, R. D., 1954: The effect of methylation on basophilia. J. Histochem. Cytochem. 2, 81--87. FLOWER, N. E., KENCHINGTON, W., 1967: Studies on insect fibrous proteins: the larval silk of Apis, Bombus and Vespa. J. roy. micr. Soc. 86, 297--310. LEv, R., SPICER, S. S., 1964: Specific staining of sulphate groups with alcian blue at low pH. J. Histochem. Cytochem. 12, 309. LILLI~, R. D., 1958: Acetylation and nitrosation of tissue amines in histochemistry. J. Histochem. Cytochem. 6, 352--362.

The Salivary Gland Secretions of a Neotropical Bumblebee

265

LisoN, L., 1960: Histochimie et Cytochimie Animales. Paris: Gauthier-Villars. LUCAS, F., RUDALL,K. M., 1967: Extracellular fibrous proteins: the silks. In: Comprehensive Biochemistry 26, 475--558 (M. FLOt~KINand E. H. STOTZ, ed.). Amsterdam: Elsevier Publ. MrLLO, M. L. S., 1979: A mucous secretion in the Malpighian tubes of a neotropical bumblebee, Bornbus atratus Franklin. Protoplasma 99, 147--158. - - VmAL, B. C., 1971: Histochemical and histophysical aspects of silk secretion in Melipona quadrifasciata (Hym., Apoidea). Z. Zellforsch. 118, 555--569. P~NZER, G. R., 1972: I-anilinonaphthalene-8-sulphonate. The dependence of emission spectra on molecular conformation studied by fluorescence and proton-magnetic resonance. Eur. J. Biochem. 25, 218--228. ROSSELET, A., R~.TCrt,F., 1968: Cytofluorometric determination of lysine with dansyldaloride. J. Histochem. Cytochem. 16, 459--466. RUDALL, K. M., 1962: Silk and other cocoon proteins. In: Comparative Biochemistry 4, 397--433. New York-London: Academic Press. --K:E'SC~ING'roN, W., 1971: Arthropod silks: the problem of fibrous proteins in animal tissues. Ann. Rev. Entom. 16, 73--96. SAKAGAMgS. F., AKAHU~A,Y., Zucc~tg R., 1967: Nest architecture and brood development in a ueotropical bumble-bee, Bombus atratus. Insectes sociaux 14, 389--414. SPICER, S. S., LILLIE, R. D., 1959: Saponification as a means of selectively reversing the methylation blockage of tissue basophilia. J. Histochem. Cytochem. 7, 123--125. VmAL, B. C., 1970: Dichroism on collagen bundles stained with xylidine ponceau 2 R. Ann. Histochim. 15, 289--296. 1978: The use of the fluorescent probe 8-anilinonaphthalene sulfate (ANS) for collagen and elastin histochemistry. J. Histochem. Cytochem. 26, 196--201. 1979: Feixes de coldgeno: detec~o e quantifica~.o de ordem macromolecular. I. Dicrofsmo corn corantes azoicos sulfatados (Orange G, Xylidine Ponceau e Sirius Red). Citric. Cult. (in press). WHITr, A., HANDLER, P., SMITH, E. L., 1964: Principles of Biochemistry. New York: McGraw-Hill, Inc. Zrl~LOTTg E., ENCEL, M. B., 1962: The reactivity of proteins of some connective tissues and epithelial structures with 2,4-dinirrofluorobenzene. J. Histochem. Cytochem. 10, 537--546. -

-

-

-

18"