PROTOPLASMA
Protoplasma 99, 147-158 (1979)
9 by Springer-Verlag 1979
A Mucous Secretion in the Malpighian Tubes of a Neotropical Bumblebee, Bombus a t r a t u s Franklin MARIA LUIZA S. MELLO ':" Department of Cell Biology, Institute of Biology, Unicamp, Campinas, Brazil Received December 15, 1978 Accepted December 18, 1978
Summary A filamentous secretion composed of carboxylated and sulfated acid glycosaminoglycans (AGAG), neutral polysaccharides, and protein(s) appears in the lumen of the Malpighian tubes of the fully grown larvae of the bumblebee, Bombus atratus Franklin. A well-ordered macromolecular array was demonstrated specially for the carboxylated AGAG components of this secretion, based on their linear dichroism and birefringence properties. It is suggested that the carboxylated AGAG macromolecules can acquire a helical conformation when present at the lumen of the organ. The mucous secretion elaborated by the Malpighian tubes of B. atratus is excreted from the larvae in the form of condensed filaments. Its function remains unclear. Globules with concentric lamination containing protein and neutral polysaccharides were seen detaching from the apical border of the epithelial cells of the Malpighian tubes of the fully grown larvae. It could not be established whether they contribute for the elaboration of the filamentous secretion. Morphologically similar globules have been assumed in some other insect groups to originate from cytolysomes. Urate crystals surrounded by a halo of calcium granules were also found in the lumen of the Malpighian tubes of B. atratus. Keywords: Bumblebee; Malpighian tubes; Mucous secretion.
1. Introduction Insect M a l p i g h i a n tubes are almost exclusively engaged in excretory mechanisms. U r i c acid, salts, a m m o n i a , pterins, riboflavin, and ions are the p r o ducts usually f o u n d in the lumen of these organs (WIcGLESWORTH 1965). F e w exceptions, however, showing the elaboration o f special secretions (glycoproteins, mucoproteins, glycosaminoglycans) b y the M a l p i g h i a n tubes h a v e also been reported. T h a t is the case o f Chrysopa flava, a neuropteran, and H y p e r a sp., a coleopteran, whose larvae m a k e small cocoons f r o m a silk * Correspondence and Reprints: Department of Cell Biology, Institute of Biology, Unicamp, 131 000, Campinas Sp., Brazil. 10" 0 0 3 3 - 1 8 3 X / 7 9 / 0 0 9 9 / 0 1 4 7 / $ 02.40
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MARIALUlZA S. MELLO
produced by transformed Malpighian tubes (LucAs and RUDALL 1967, RUDALL and KENCHINGTON 1971). The Malpighian tubes of certain Chrysomelid larvae elaborate a sticky substance which has been reported to aid progression in some species (BI~Ass 1914 apud WIGGLZSWOXT~1965) and to be used for covering egg chambers in other species (HEYMONS and LUEHMANN 1933). The Malpighian tubes of Cercopid nymphs are pointed out as producing a glycoprotein secretion, maybe related to silk (PESSON 1955, ZIEGLeR and ZIEC-LER 1958). In addition, the fully grown larvae of some halictid bees excrete faeces which become cemented by a gluey secretion ascribed to be furnished by their Malpighian tubes (HIRASHIIVIA1960). While studying with a polarizing microscope changes in the macromolecular array of the salivary gland secretions of a bumblebee (Bombus atratus) as larval development progresses, attention was drawn to a birefringent filamentous mass filling the lumen of the Malpighian tubes of this insect. This finding motivated us to undertake the present work, where morphology, composition, and cytophysical properties of products secreted by the Malpighian tubes of B. atratus are reported at different larval stages. 2. Materials and Methods Young feeding and fully grown larvae of Bombus atratus Franklin (Hymenoptera, Apoidea) [9 and 15 days-old, respectively (SAKAGAMI et al. 1967)] reared in the laboratory were used. They were fixed in 10~ formalin for 24 hours at room temperature and embedded in paraffin. Sections were cut at 7 ~tm thickness. Morphological observations were performed on sections stained with haematoxylin-eosin and also on those subjected to cytochemical tests. The observations were made with a Zeiss photomicroscope equipped for polarization microscopy and microspectrophotometry and a Zeiss microspectrofluorometer.
2.1. Cytochemistry Acid glycosaminoglycans. The reactivity to 0.025% toluidine blue solutions in 0.1 M citric acid and 0.2 M anhydrous disodium phosphate buffer at fixed p H (2.5 to 5.0) was studied. Methylation (6 hours) and saponification (20 minutes) after methylation were used as controls in order to identify in sections the radicais potentially responsible for the metachromatic reaction (FIscHER and LILLIE 1954, SVlCER and LILLI~ 1959). Staining with l~ alcian blue solutions at pH 1.0 (LEv and SVlCER 1964) and 2.5 (LlsoN 1960) was also employed. PAS-positive glycans and glycoproteins. Controls underwent prior treatment with diastase and salivary amylase for 1 hour at 37 ~ Proteins. The total electroposltive radicals were investigated on sections treated with a 0.1~ xylidine ponceau 3RS solution at pH 1,7 (VIDAL 1970). Controls were nitrosated for 15 hours. Calcium. A modified yon Kossa's procedure was used (BANCROFT 1967).
2.2. Polarization Microscopy and Microspectrophotometry Anisotropy was searched in stained and unstained sections. The pattern of dispersion of birefringence (toluidine blue--stained sections) and the birefringence sign were determined with k/10 Brace-K/Shler's and k/4 S6narmont's compensators.
A Mucous Secretion in the Malpighian Tubes of a Neotropical Bumblebee
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Linear dichroism was ascertained in sections treated with toluidine blue and xylidine ponceau solutions. Spectral absorption curves on the filamentous secretion oriented parallel (d I I) and perpendicular (d • to the plane of polarized light (PPL) were determined with a Zeiss Pol-photomicroscope equipped with a 01 photometer and an EMI 6256 photomultiplier. Plan 40/0.65 objective, optovar 2, photometric diaphragm dia. = 0.4 mm, field diaphragm dia. = 0.2ram, Epiplan 16/0.30 condenser, and a monochromator ruler (k from 500 to 625 nm) were used. Linear dichroism (Ad ~ d I I - - d • and dichroic ratio ( = d II/d • parameters were calculated at each wavelength. The absorption peaks and metachromatic ratios (E~, = 555 nm/EX = 625 rim; E = extinction) were also cytophotometrically calculated for toluidine blue-stained sections examined with unpolarized light. 2.3. Fluorescence Microscopy
The products present at the lumen of the Malpighian tubes were examined in unstained sections with a Zeiss microspectrophotometer equipped to perform microfluorometry with the III RS condenser. Spectral profiles of autofluorescence of some materials present at the lumen of the Malpighian tubes were determined. A monochromator ruler was placed in front of the H T V - R 446 photomultiplier. Operating conditions were: Ph 40/0.65 objective, area of the specimen measured = 12.6 ~tm~, Zeiss filter sets I (UG 1 exciter filter transmitting a k ~ 365/366 exciting light, FT 420 chromatic splitter, LP 418 barrier filter), II (B 3 exciter filter, LP 478 barrier filter, FT 460 chromatic splitter), III (BG 12 exciter filter transmitting a k = 400rim exciting light, FTS10 chromatic splitter, barrier filter 50), and IV (BG 12 exciter filter, FT 580 chromatic splitter, LP 590 barrier filter).
3. R e s u l t s Deeply birefringent spherulites with a radial striation are evident at the lumen of the Malpighian tubes in young and fully grown larvae of B. atratus, as detected with the polarizing microscope (Figs. 1, 3, and 4). The birefringence of the spherulites is positive parallel to their radius. The spherulites did not stain with anyone of the dyes used in this work. A dark halo appears surrounding the spherulites when the sections are subjected to yon Kossa's method for calcium. In the fully grown larvae highly ordered filaments make up the bulk of the secretion filling the lumen of the Malpighian tube along most of its length (Figs. 2, 3, and 6-9). The filaments traverse the lumen of the Malpighian tube, and are attached to the cellular upper border (Figs. 2 and 3). They stain strongly with haematoxylin (Fig. 3). The spherulites are generally seen surrounded at least in part by the filamentous network in the fully grown larvae (Figs. 2 and 3). Only the spherulites display autofluorescence. Their spectral emission profiles resemble those exhibited by uric acid crystals (Fig. 5). In unstained sections, the filamentous secretion shows positive birefringence, i.e., maximum refractive index (y) parallel to long axis of the strands. The filamentous secretion stains deeply with toluidine blue solutions even at low pH (2.5) (Fig. 6), displaying a metachromatic basophilia. The basophilia is completely abolished by methylation and partly recovered after saponification. The absorption peaks for the toluidine blue-stained secretion were 10"*
150
MARIA LUIZA S. MELLO
always found at the ~. = 540-550 nm region ('I metachromasy). The metachromatic ratios were especially high ( ~ 2.3) after staining at pH 4.0. Slightly smaller ratios were exhibited by sections methylated and saponified
Fig. 1. Strongly birefringent spherulites (s) present in the lumen of the Matpighian tubes in a young larva of B. atratus. Crossed polarizer-analyzer. ;'<615 Fig. 2. Intense birefringence in the fibrillary strands which traverse the lumen of the Malpighian tubes (MT) of a fully grown larva stained with a toluidine blue solution at pH 4. Orientation of the largest refractive index (~) (in this case, ne) of the secretion is indicated. Anisotropy is also observed in the peritrophic membrane (pm) aposed to the midgut cells (rag). s = spherulite. Crossed polarizer-analyzer. X 190 Fig. 3. The filamentous secretion spans the lumen of the Malpighian tube in a fully grown larva and appears attached to the apical border of the ceils (black arrows). One spherulite (white arrow) is also observed. Haematoxylin-eosin staining. ~. = 540 nm. X 300 Fig. 4. Detail of a spherulite in a fully grown larva. Crossed polarizer-analyzer. X 680
prior to staining ( ~ 1.90). In the sections stained with toluidine blue at pH 4.0 a strong birefringence was observed (Fig. 2). The birefringent material is rather granular than filamentous (Fig. 2). Birefringence values varying as
A Mucous Secretion in the Malpighian Tubes of a Neotropical Bumblebee
151
a function of the wavelength of the incident light (dispersion of birefringence) were detected on the unstained filamentous secretion. In the case of the filaments stained with toluidine blue solution, the dispersion of birefringence was found to be anomalous (-- ADB), the birefringence sign changing as a function of the wavelength. In the present case it was negative for light of wavelengths over 520 nm and positive for light of wavelengths under this Emission (%1 100.
III
80.
50
/.0:
20.
400
Z.20
z.40
450
Z.80
500
520
51.0
560
580
600
620
" 5/.0
560
680
700
nrn
Fig. 5. Emission profiles of the filamentous secretion of the Malpighian tubes of fully grown larvae of B. a t r a t u s (O) and of crystals of a uric acid standard (O). The emission values at the various wavelengths were evaluated relative to the maximal emission value obtained with the Zeiss filter set III and calibrated as 100~ Each point in the curves is the arithmetic mean of 5 measurements
value (Fig. 10). This situation persisted in preparations methylated and saponified prior to staining (Fig. 10). Negative linear dichroism (d• ~ d IJ) even visually detected was found in the filaments stained with the toluidine blue solution at pH 4.0 (Figs. 7 and 8). The dichroic ratios which most depart from value 1. were found at k -- 540 nm (Tab. 1). No dichroism was found in the secretion stained with toluidine blue solutions at pH lower than 4.0. These results remained practically unchanged when the sections were methylated
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MARIALUtZAS. MELLO
and saponified prior to staining (Tab. 1). The curves plotted with linear dichroism (A d) values varying as a function of the wavelength values, depicted one peak at k = 550 nm, that is, close to the wavelength of the ADB inflection point (~ = 520 nm) (Fig. 10). This has been interpreted as a Cotton effect-like phenomenon (VmAL 1972). The filamentous secretion also stains strongly with the alcian blue solutions (greenish-blue colour) (Figs. 9 and 15) and responds positively to PAS reaction
Fig. 6. The filamentous secretion (s) stains metachromatically with the toluidine blue solution at pt-t 2.5. )~ = 550 nm. )<370 Figs. 7 and 8. Visual observation of linear dichroism in the secretion filaments stained with the toluidine blue solution at pH 4. The filaments (arrows) are oriented perpendicular (Fig. 7) and parallel (Fig. 8) to the plane of polarized light (PPL).)~ = 560 nm.• 250 Fig. 9. Filamentous secretion (s) stained with alcian blue at pH 1.0. )~ -- 590 nm.• 245 after enzyme treatments. The filaments stained with the alcian blue solution at p H 2.5 acquire an intense birefringence with a red interference colour. The cytoplasm of the Malpighian tube cells also appears birefringent after the alcian blue staining at p H 2.5. The filaments stain very faintly with the xylidine ponceau solution. No dichroism was observed in this case, although birefringence was enhanced. Nitrosation prevented staining with the xylidine ponceau solution. The filamentous secretion elaborated in the Malpighian tubes of B. atratus retains its well-ordered array while going through the hindgut and finally outside of the larvae, becoming increasingly condensed (Fig. 16).
A Mucous Secretion in the Maipighian Tubes of a Neotropicai Bumblebee
153
In the lumen of the Malpighian tubes of the fully grown larvae, concomitant to or independent of the appearance of the filamentous secretion (Figs. 11-15) globules were seen which stain with xylidine ponceau and respond positively to PAS. They apparently detach from the apical zones of the epithelial cells of the organ (Figs. 12 and 13). These globules are not birefringent and do not (nm)
20.
10~
II II II iI
i,i I .
.
/.80
.
.
IIII 520
500
.
.
.
.
540
.
.
560
.
.
.
580
), into) 600
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-10,-
\
,
........... j->-'-,.p;.,_Z. -.100
-20.-
-.200 ~ e i ; i
/
" ction) Ad (extln
Fig. 10. Linear dichroism spectral curves (--) and anomalous dispersion of birefringence ( - - - ) of the filamentous secretion of the Malpighian tubes in fully grown larvae of B. a t r a t u s stained with a toluidine blue solution at pH 4.0 preceded by methylation plus saponification procedures ( 0 ) or not (O). Each point in the curves is the arithmetic mean of 5 measurements
positively respond to yon Kossa's method for calcium. Some of the globules show their core less reactive to the dye molecules than their "cortical" layer (Figs. 12 and 13), whereas others depict a homogeneously positive staining (Fig. 11). They do not stain with toluidine blue or aMan blue solutions (Figs. 14 and 15). However, when they appear immersed in the filamentous secretion, they are encircled by a relatively strong toluidine blue or alcian blue-stained material which is probably part of the mentioned secretion (Figs. 14 and 15).
154
MARIA LUIZA S. MELLO
Figs. 11-13. Globules of several sizes are present at the lumen of the Malpighian tubes of t:ulIy grown larvae after staining with xytidine ponceau at p H 1.7. The globules shown in Figs. 12 and 13 exhibit unstained cores differing from those seen in Fig. 11. Early stages of globule extrusion are indicated (arrows) in Fig. 12. k = 54Ohm. Fig. 11. X560; Fig. 12. • Fig. 13. • Fig. 14. Globules apparently extruded through the cell border in the Malpighian tube of a fully grown larva do not stain with the toluidine blue solution at pH 4. On the other hand, staining is observed in the filamentous secretion (s). k = 560 n m . • 530 Fig. 15. An alcian blue staining is shown in the filamentous secretion (s), specially close to the epithelial ceil border (b) and encircling globules (arrows) at the organ lumen. (pH 2,5; k = 580 nm). X580 Fig. 16. The well-ordered filamentous secretion elaborated in the Malpighian tubes migrates to the hindgut (hg) and is finally excreted (arrow). Haematoxylin-eosin staining. ~. = 560 nm. • 42
A Mucous Secretion in the M a l p i g h i a n Tubes of a N e o t r o p i c a l Bumblebee
155
An attempt to localize whether a specific site of secretion exists for the different lumen contents (filaments, uric acid, and globules) along the tubule was unsuccessful. Table 1. Dichroic Ratios for the Filamentous Secretion of the Malpighian Tubes of B. atratus
Stained With the Toluidine Blue Solution at pH 4.0 and Preceded by Methylation Plus Saponification (II) or Not (I). N u m b e r of m e a s u r e m e n t s = five I
II
)~
I
II
(nm)
(X 4- S)
(X q- S)
(nm)
(X 4- S)
( X 4- S)
510 520 530 540 550 560
0,91 0.80 0.76 0.71 0.78 0.82
0.83 0.76 0.72 0.68 0.71 0.76
570 580 590 600 610
0.80 0.82 0.78 0.75 0.78
0.80 0.78 0.80 0.82 0.76
+ + + + + +
0.05 0.05 0.04 0.02 0.03 0.04
+ + + + + +
0.05 0.06 0.03 0.03 0.04 0.05
+ + + + +
0.05 0.06 0.06 0.04 0.04
+ + + + +
0.06 0.05 0.06 0,06 0.04
4. D i s c u s s i o n
The cytochemical data indicate that the secretion present as filaments in the lumen of the Malpighian tubes of the fully grown larvae of Bombus atratus contains sulfated and carboxylated glycosaminoglycans, neutral polysaccharides, and protein(s) (LlsoN 1960, VIDAL 1970). It can be considered a mucous secretion. Its PAS-positive neutral polysaccharides may well be associated with the protein(s) (glycoprotein). The deep metachromatic staining of the secretion with the toluidine blue solution at p H 2.5 is probably due to sialyl residues (LisoN 1960). The anisotropic properties of the filamentous secretion indicates high molecular orientation of some of its components. The carboxylated AGAG macromolecules are much more well-ordered in the filaments than are the sulfated ones. This is deduced from the dichroism properties displayed by the filamentous secretion stained with toluidine blue only at p H 4.0 and which remain practically unchanged when the sections are subjected to methylation plus saponification prior to staining (VIDAL 1963, M•LLO and VIDAL 1973). In addition, a red birefringence colour was found in the filamentous secretion stained with the alcian blue solution at p H 2.5 and not in that stained at p H 1.0. The latter staining condition reveals occurrence exclusively of sulfated AGAG (LEv and SI'ICER 1964) whereas the former potentially stains sulfated and carboxylated AGAG (LlsoN 1960). Considering that no birefringence was observed in the secretion stained at p H 1.0, macromolecularly oriented carboxylated AGAG are responsible for the cytophysical phenomenon involved (VIDAL1977--personal communication). The finding of a negative linear dichroism after the toluidine blue staining indicates that the substrate which binds the dye molecules is mostly parallel to the long axis of the filaments ( V I D A L 1963, VIDAL and MELLO 1970). The detection of a Cotton effect-like phenomenon (VIDAL 1972) in
156
MARIALulzA S. MELLO
the secretion stained with toluidine blue solutions at special p H conditions is suggestive that the stained substrate (carboxylated AGAG) can acquire a helical conformation. This resembles findings on the mucus produced by vertebrate goblet cells, although the anisotropy abtained after toluidine blue staining in the latter is certainly due to sulfated AGAG (MusY et al. 1972, VIDAL and MELLO 1974). The protein molecules of the mucous secretion of the Malpighian tubes of B. atratus are not remarkedly oriented with respect to one of the axes of the filaments as dichroism was not seen after staining with xylidine ponceau (VIDAL 1970). The high level of the macromolecular array depicted by some of the components of the fiiamentous secretion at the lumen of the Matpighian tubes and out of the fully grown larvae of B. atratus is most likely concerned with the function performed by this secretion. There is a single report on bees (Halictus ajfinis) showing that the Malpighian tubes can also function as a secretory organ, producing a substance which will cement the faeces excreted by the insect (HIRASHIMA 1960). In other insect groups, data have been published that show unusual participation of a Malpighian tube secretion in the elaboration of cocoons (LucAs and RUDALL 1967, RUDALL and KENCHINGTON 1971). In the present work, however, the function of the well-ordered mucous secretion of the Malpighian tubes of B. atratus remains unclear. This secretion is present only in the fully grown larvae, coinciding in time with the elaboration of a mucous secretion by the silk glands of same insects (MI~LLO and VIDAL 1978) and the very start of the spinning of the cocoons which will cover the pupating larvae (NAKAGAMIe t al. 1967). Whether the Malpighian tube secretion plays a part in the elaboration of these cocoons, it could not be established. While the spherulites present in the Malpighian tubes during the larval phase represent urate crystals deduced mainly from their fluorescence emission profiles, the globules with concentric lamination present in the fully grown larvae look like the "concretions" described in an european Bombus species at the EM level (MEYER 1957, BERKALOFF 1958). The globules present in Bombus atratus, however, contrary to Berkaloff's hypothesis, are devoid of both uric acid and calcium. They contain protein and neutral polysaccharides, components which have also been found in "concretions" of the Malpighian tube and midgut cells of Musca domestica (SoHAL et aI. 1976, 1977). Morphologically similar bodies apparently originate from cytolysomes in the Malpighian tubes of Bombus and other insect species (M~YER 1957, WIGOLESWORTH and SALPETER 1962, MELLO and IDOLDER 1977). The various sized globules which do not depict the classical concentric lamination may be another type of material under extrusion or simply an artifact (WIGGLESWORTH1965). None of the globules was proven to be directly engaged in the production of the filamentous secretion or contain birefringent material.
A Mucous Secretion in the Malpighian Tubes of a Neotropical Bumblebee
157
Acknowledgements The author is indebted to Prof. R. ZUCCHI for generously providing her with the bumblebees. She is also grateful to Prof. H. FoRe, sT for a generous gift of uric acid crystals used as fluorescence standards. This work was supported by F.A.P.E.S.P. (Brazil, grants n. 70/371 and 72/313).
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VIDAL, B. C., 1970: Dichroism in collagen bundles stained with xylidine ponceau 2R. Ann. Histochim. 15, 289--296. - - 1972: Anormal dispersion of birefringence, linear dichroism, and relationships with ORD (Extrinsic Cotton Effect). Histochemie 30, 102--107. - - M r r r o , M. L. S., 1970: Absorption spectral curves of dichroism on collagen bundles. Histochemie 23, 176--179. - - - - 1974: Macromolecular conformation of the colon mucus as revealed by detection of anisotropic phenomena. Ann. Histochim. 19, 151--156. WIG6LESXCORTH, V. B., 1965: The principles of insect physiology. London: Methuen and Co. Ltd. -SALPETER, M. M., 1962: Histology of the Malpighian tubules in Rhodnius protixus Stal (Hemiptera). J. Insect. Physiol. 8, 299--307. ZiEorrl~, H., ZIrGLrr~, I., 1958: Uber die Zusammensetzung des Zikadenschaumes. Z. vergl. Physiol. 40, 549--555.
Herausgeber, Eigentiimer und Verleger: Springer-Verlag, M~51kerbastei 5, A-1011 Wien. -- Ffir den Textteil verantwortlich: Dr. Wilhelm Schwabl, M61kerbastei 5, A-1011 Wien. -- F[ir den Anzeigenteil verantwortlich: Mag. Bruno Schweder, MiSlkerbastei 5, A-1011 Wien Druek: Adolf Holzhausens Nfg., Kandlgasse 19-21, A-1070 Wien Printed in Austria