- Email: [email protected]
A histochemical study of pollen digestion in the alimentary canal of honeybees (Apis mellifera L.)
J. Insect. Ph?‘siol. Vol. 30, No. 7. pp. 51 I-521. 1984 Printed in Great Britain. All rights reserved
Copyright f
0022.1910184$3.00+0.00 1984 Pergamon Press Ltd
A HISTOCHEMICAL STUDY OF POLLEN DIGESTION IN THE ALIMENTARY CANAL OF HONEYBEES (APIS MELLIFERA L.) L. M. Department
KLUNGNESS
of Entomology.
University
and
YING-SHIN
of California,
PENG*
Davis,
California
95616,
U.S.A.
(Rewired 2 September 1983; rerised 21 Ortober 1983) Abstract-Histochemical staining of embedded sections of the alimentary canal of honeybees indicated that carboxylated polysaccharides could be efficiently digested and absorbed. except when sequestered within unswollen and unbroken pollen grains that appeared to be impervious to enzymes of the gut. Hemicelluloses and pectic acids of the pollen wall structure underwent partial digestion, but pollen wall cellulose and sporopollenin were not digested. A separation of pollen walls from colloidal semisolids of the pollenkitt and disgorged protoplasm was apparent. The layers of slurry created by this separation remained into the rectum, and consisted primarily of saturated organic compounds which do not stain with weakly basophilic or acidophilic dyes.
Key Word Indes: Honeybee.
pollen.
alimentary
canal,
INTRODUCTION
*To whom correspondence , P 3,1,7--A
MATERIALS
AND METHODS
A total of 136 adult worker bees of the Italian race were collected for this study directly over the brood comb from a normal queen-right colony, during the early spring of 1979. The colony was fed with freezerstored corbicular pollen that had been collected the previous year in Canada. This pollen source contained a minimum of 10 species of pollen, but dandelion pollen, Taraxacum ojicinale, predominated in the sample. Since no dandelion was in bloom in the area where the colony was located, dandelion pollen was a marker for identifying recently consumed pollen. Bees used for histochemical preparation and microscopic observation were dehydrated after fixation with 2.5% gluteraldehyde in s-collidine buffer (pH 7.5). The dehydration sequence was 3&50-709/, ethanol prior to sample storage and then in 95- 1O& 1004; ethanol just prior to embedding.
____~ should
pollen digestion
pollen, consisting primarily of cellulose, and pectic acids (2) The exine is a complex structure secreted onto the outside of the exine by the tapetal cells of the anther. The sporopollenin matrix of the exine imparts rigidity and is often sculptured in complex layers and structures. The exine is often described in specific structural layers having unique nomenclature. The ectosexine, the only such layer referred to in this paper, is the outermost layer of the exine (3) The pollenkitt is a semisolid, soluble layer of lipids, proteins, and sugars, which is embedded in the ectosexine of many species of pollen. The chemistry and function of these layers of pollen is reviewed by Stanley and Linskens (1974). The purpose of this study is to apply histochemical procedures to better identify the content of the pollen during the process of digestion in the alimentary canal of the honeybee.
Since the original definitive study of honeybees to determine whether adults and larvae could utilize pollen as food (Parker, 1926), studies of the histochemistry of digestion and absorption in honeybees (Apis mellifera L.) have been scarce (Stanley and Linskens, 1974). Parker concluded that only simple proteins and sugars are available to the bee from digested pollen, but this assumption has been challenged. Snodgrass (1956) and Treherne (1967) reported that the crop had no secreting function. Barker and Lehner (1972) summarized the available literature regarding the role of the peritrophic membrane of the ventriculus in distributing digestive enzyme. Nevertheless, Joshi and Agarwal (1976) reported that 8.29, of the cholesterol consumed by honeybees was absorbed in the crop while 60% was absorbed in the ventriculus. Martinho (1975) reported that the sugar concentration varied greatly in the crops of honeybees that he sampled. He also found that digestion of pollen increased from 30 to 70”/ when pollen would swell and protrude its intine as a result of osmotic pressure. Observations of pollen viability in corbicular pollen loads have been made by Klungness, Thorp and Briggs (1984), and morphological observations of pollen in the beebread of honeybees have been recorded by Klungness and Peng (unpublished observations). We have obtained evidence indicating that pollen losses membranecontrol of osmosis during storage as beebread. But scanning electron microscopic observations of the gut of honeybees indicate that not all pollen swells and ruptures in the crop or ventriculus of Apis mellifera (unpublished observations). There are three major components of the pollen wall, namely: (1) The intine is the true cell wall of ___~
histochemistry.
be addressed. 511
512
L. M. KWNGNESS and YIN+SHIN
JB-4TM embedding reagent A (DuPont Co., Willmington, Deleware) was used to replace ethanol over 24 h of refrigeration. Specimens were then transferred to a mixture of JB-4 reagents A and B and polymerization was allowed to proceed at room temperature. The mixture was that recommended by the manufacturer as having the slowest rate of polymerization and therefore less gas is formed during the exothermal reaction. Of the 136 adult worker bees collected. 20 were randomly selected for embedding. Later 5 specimens were selected for microtomy, based on orientation of the specimen. The specimen blocks were sectioned with glass knives on a JB-4 rotary microtome (SorvallTM, DuPont Co., Newtown. Connecticut). Sections were median sagittal planes of the entire bee, and thus each section contained portions of various parts of the alimentary canal. This made comparative staining of organs possible. Ribbons of sections were created by placing a bead of contact cement (3M Co., St Paul, Minnesota) on the top and bottom of the block. These ribbons of sections were floated on water that had been placed on a microscope slide, then dried on a warming plate at approx. 40 C. which caused the sections to adhere to the glass. The following stains were used to identify the chemical components of the contents of the alimentary canal:
(I) To&dine blue 0 was used to metadistinguish chromatically carboxylated PolYsaccharides such as hemicellulose and pectic acid. from those chemicals which stain orthochromatically. or not at all in this dye (Stadelmann and Kinzel. 1972). The carboxylated polysaccharides stain red to pink. Nucleic acids, proteins and sporopollenin stain blue. Phenolics stain green. Starch, waxes, saturated cellulose and saturated lipids do not stain with this dye. A O.l”, aqueous solution of toludine blue 0 was applied to the dried sections, allowed to stain for 5 min rinsed with water, and covered with glycerine and a coverslip. This mounting method preserves metachromatic properties of the stain, but is not a permanent mount. (2) The starch iodide reaction (IKI) (Jensen, 1962) was used to locate starch and amyloids. The reagent stains starch blue to black, and amyloids yellow to red. IKI solution was made by dissolving 2 g of potassium iodide (KI) in 100 ml of water, followed by the addition of 0.2 g of iodine. The IKI solution was applied directly to the dried sections, and observations were made without rinsing the stain from the slide. Such preparations are not permanent and fade quickly. IKI stained sections were also observed with fluorescence illumination. The iodine acted as a histochemical oxidant (Lillie, 1977) to saturate resonating chemical bonds in the contents of the intestine. Resonating bonds are often the source of autofluorescence. Therefore iodine binding quenches autofluorescence where it occurs. More penetration of iodine into cellulose is known to occur when cellulose is partially hydrolyzed (Jensen, 1962). Therefore, with IKI. it was possible to
PENG
localize in the alimentary canal the areas where hydrolysis of the pollen wall occurred. (3) A solution of 0. I “; hrotnophennl blue in ethanol was used to stain protein and other acidophilic components of the intestinal contents. This dye stains the amino groups of protein (Davenport. 1960). although it is not highly specific to protein. Its colour is a function of pH of the staining environment and may vary from red to blue. This reagent was applied to dry sections and allowed to stain for 15 min before rinsing with water. Glycerine was used as the mounting medium for microscopic observation. Bromophenol blue has fluorescent properties, which can be used to better determine the amount of stain bound in the specimen. It is a metachromatic dye which changes from yellow to red, as the concentration of the bound dye increases. (4) The basic dye, phrtwsq~iunin was reported to be a strongly acidophilic dye (Moore, 1933). We used this dye to stain acid contents of the intestine because they had little or no chemical reactivity with the weakly acidophilic dyes. The reagent was mixed in a contrast stain contammg 1.o”,, of phenosafranin and 1.0’; of bromophenol blue in 30”” ethanol and 68”,, glycerine. This counterstaining reagent was applied to dry sections, which were then placed on a warming plate for several hours to achieve staining of the undigested components of the intestinal contents. All photomicrographs. includng fluorescence, were taken soon after slide preparation, in order to obtain uniform results. For normal tungsten light photomicrography, EktachromeTM 50 (Eastman Kodak, Rochester. New York) was used. Ektachrome200 was used for fluorescence photomicrography. Fluorescence light microscopy was achieved with a Zeiss Standard microscope equipped with a 200 W mercury vapour lamp for transmitted illumination. Filter combinations varied with the stain: (1) for autofluorescence of the pollen walls, both with and without IKI stain, an UGI excitation filter, a BG38 red-absorption filter and the 490-nanometer barrier filter were used (2) for hromophenol blue fluorescence. a FITC dichroic excitation filter, the heat reflection filter and the 530-nanometer barrier filter were used. Where comparative views of different zones of the alimentary canal were photographed, considerable care was taken to ensure equal exposure conditions. All such sets of photomicrographs had been made on the same slides, thus assuring that the same staining procedure and dye concentration were compared.
RESULTS
Starches and umyloids Potassium iodide when used to stain pollen amyloplasts, indicated that starch was quickly digested in the ventriculus if it was either released from the pollen grains, or the amylases that are secreted into the intestine, were able to penetrate the pollen membrane. Starch that was retained within unruptured impervious pollen grains, was not digested by the time the pollen reaches the rectum. This was clearly demonstrated in Figs 14.
Histochemical
study
Nucleic acids, proteins and lipoprotein membranes Bromophenol blue, which complexes with amino groups, stained proteins of pollen protoplasm and/or nuclear components brown to red in normal light and orange to red with fluorescence. These acidophilic components were observed throughout the alimentary canal of the bee (Figs 9-12). In Fig. 10, large pollen grains were seen that had retained starch amyloplasts. This was confirmed by IKI reagent. which stained the starch blue (Fig. 2) and by toluidine blue 0. The latter stain showed that only the amyloplast membrane stained pink and blue, whereas the starch granule remained unstained by this dye (Fig. 16). With bromophenol blue viewed in fluorescent light, the amyloplast membrane was red but no stain was taken into the starch granule (Fig. 10). Therefore, these three stains indicated that starch and membrane components of some pollen species were not visibly affected by the digestive enzymes of the intestine. It was observed that acidophilic compounds were present in the intestinal slurry around the pollen, but that these were not the predominant component of the slurry. With bromophenol blue staining of acidophilic compounds (Figs 12 and 20) and with toluidine blue 0 staining of basophilic compounds (Fig. 18) it was apparent protoplasmic and nuclear components of some pollen remained undigested in the rectum. although most pollen contents were removed. Carboxylated polysaccharides of pollenkitt Rapid digestion of carboxylated polysaccharides was seen in longitudinal sections of the ventriculus, where the pink-stained polysaccharides diminish rapidly toward the posterior (Fig. 15). The enlarged image of the anterior portion of the section (Fig. 16) showed the carboxylated polysaccharides components of the intine and exine and peritrophic membranes, as well as the presence of extra-cellular polysaccharide in the lumen. Figure 17 showed that much of the extracellular polysaccharide was removed from the lumen shortly after entering the ventriculus. Whereas within the anterior rectum (Fig. 18), little to no carboxylated polysaccharide remained, except that associated with the pollen intine (hemicellulose and possibly residual pectic acids). The pollenkitt of T. ofiicinale pollen had been observed to be mostly separated from the exine in the crop (Fig. 13). Some traces of pink-stained polysaccharides of the pollenkitt were present in the crop, but the copious quantity of pollenkitt that is normally embedded in the ectosexine of T. oficinale had obviously been displaced. Some regions of the ventriculus were observed to contain very few pollen grains. In these areas it was sometimes observed that the plane of section through the diameter of the ventriculus contained a slurry containing little or no pollen. A fraction of that slurry (Fig. 14) appears to be layered with other acidophilic or neutral compounds which do not bind toluidine blue 0. Counterstaining the slurry with bromophenol blue, confirmed the presence of acidophilic compounds in the slurry. These bases were probably of protoplasmic origin. The apparently empty space between the pollen in
of pollen
digestion
513
the rectum (Figs 18-20) was a slurry which contained the unstained and undigested components of pollenkitt and disgorged pollen cytoplasm. The slurry had no affinity for the weakly basic dye toluidine blue 0. but reacted very slowly in a warm environment with the strongly basic dye, phenosafranin. Only a portion of the slurry stained with the acid dye, bromophenol blue (Fig. 20). Certain areas of the lining of the posterior rectum stained pink and dark blue (Fig. 19). High magnification observations indicated that these were associated with bacteria. Pollen wall components The autofluorescence of the pollen wall. before and after IKI staining, gave evidence of a change in the chemical nature of the pollen wall as it passed through the honeybee’s digestive tract. Before IKI stain was applied to the sections, autofluorescence of the pollen wall appeared to be similar throughout the crop and the entire intestine. After iodine was added as an oxidant, the autofluorescence of the pollen wall decreased from the anterior to posterior region of the gut (Figs 5-8). This chemical change in the pollen wall is also suggested by the bromophenol blue staining method, when appropriate fluorescence filters were used (Figs 9-12). There was an increased absorption of this fluorescent dye (red) by the pollen wall, as it progressed through the gut. Bromophenol blue, used as a fluorescent dye, confirms an alteration in pollen wall chemistry as it passes through the intestine. In the crop, the pollen wall is impervious to the stain. Therefore, the yellowgreen autofluorescence of the wall is seen in the crop with this stain and appropriate filter combination (Fig. 9). In the anterior ventriculus some of the dye is bound to the pollen wall. giving it an orange fluorescence (Fig. 10). As more bromophenol blue binds to the pollen wall in the posterior ventriculus (Fig. 11) and the rectum (Fig. 12) the fluorescence of the dye takes on more red tone. It cannot be determined by these histochemical tests whether the dye is actually binding to the cell wall components themselves or whether other components of the intestinal slurry are more able to penetrate the wall as digestion proceeds, in which case they would bind the dye. In either case a change in the cell wall structure is indicated. Staining with toluidine blue 0 indicated that partial digestion of pollen wall components did occur. In particular, the carboxylated polysaccharides of the intine were partially digested by the gut (Figs 16, 18 and 19). These polysaccharides are primarily pectic acids and hemicellulose. Figure 16 shows a magnified image of the pollen in the ventriculus. Although red stained sugars and pollenkitt components are seen outside of the pollen, the red layer, inside and next to the blue stained exine wall, is the intine. This consists primarily of cellulose, hemicellulose and pectic acids (Stanley and Linksens 1974). Figures 18 and 19 show pollen in the anterior and posterior rectum respectively, after staining with toluidine blue 0. It is apparent that, though some carboxylated polysaccharides remain in the intine, much of the red staining is gone, indicating partial digestion of the intine components.
L. M. KLUNGNESS and YING-SHIN PENG
514 DISCUSSION
The presence of a slurry containing no pollen raises questions about the separation of materials in the digestive process. The origin of the components cannot be resolved histochemically. The acidophilic components might have been the enzyme protein secreted from the ventricular epithelium. but was more probably the protoplasmic protein from the ruptured pollen. The presence of the acidophilic compounds in the rectum suggests the occurrence of incomplete digestion and/or absorption of those compounds, having basic reactive groups to bind bromophenol blue (Figs 12 and 20). In addition to the suggestion that the protoplasmic content of ruptured pollen might be physically separated from the pollen, there was also indication that the pollenkitt components might have been physically separated from the pollen by the action of the proventriculus. This separation process caused the formation of layers of slurry in the gut. These layers contained no pollen walls within them (Fig. 14). These layers also persisted into the rectum. though histochemical indications of changes in the chemistry of the slurry appeared at different positions in the intestine. This observation confirms scanning electron micrographic studies of Klungness and Peng (unpublished observations), which suggested that the proventriculus separates food components. This process may cause layering in the food bolus, rather that a homogeneous food mixture. This suggests that the proventriculus can writhe with considerable force when the ventriculus is nearly empty. Under these conditions. the fluid dynamics would probably be such that only large and firm particulates like pollen could be impeded by the comb of the proventriculus. This would cause pollen to collect in the ventriculus. but pollenkitt and protoplasm of ruptured pollen grains would flow through the comb of the proventriculus, into the crop. As the ventriculus becomes engorged with the food bolus. the intestinal muscles cannot generate such a high flow rate through the proventriculus. The fluid dynamics of this slower flow might allow the comb of the proventriculus to impede
Plate 1. (Fig. 1). A median sagittal section of the abdomen, exposing the crop of a honeybee was stained with IKI reagent. Blue-stained starch granules (s) and yellow stained amyloids (ubiquitous) were found inside and outside of the pollen. The pollen shown are of several unidentified species not including T. c@cinale. x 175. (Fig. 2) An enlarged view of Fig. 1 illustrated that the starch granules outside of the pollen were released into the slurry because some of the pollen grains had ruptured. Broken pollen walls (r) are not found in all grains of the larger pollen species. x 280. (Fig. 3) A median sagittal section of the abdomen, exposing the posterior midgut of the honeybee, illustrated that extracellular starch granules had been thoroughly digested. The starch granules retained within the unruptured pollen were not digested, as indicated by the IKI blue staining (s). x 175. (Fig. 4) A median sagittal section of the abdomen, exposing the rectum, illustrated that starch (s) remained in some pollen grains that did not rupture. x 175. (Fig. 5) A median
the passage of viscous as well as particulate food in the slurry. The result of such changes in flow dynamics would be the formation of alternate layers of pollen and slurry in the food bolus. The histochemistry of the slurry also supported this hypothesis. since the slurry did contain the carboxylated polysaccharides (stained pink in Fig. 14) and the lipids (unstained) that have been reported to occur in pollenkitt of Compositae (George 1982). This suggested that the slurry, by the time it reached the rectum, consisted primarily of complex saturated macromolecules with few reactive sights to bind the dyes, Because the chemistry of the pollenkitt is known (Stanley and Linskens. 1974). it is reasonable to conclude that the soluble and digestible portions of the pollenkitt are stained at their reactive binding sites. These are also the more likely compounds to be reduced to small molecules that would be absorbed by the gut wall. There are pollenkitt components that would not have many exposed reactive sites and would not be readily broken down by the digestive enzymes. Therefore, we suggest that it is probable that the undigested slurry came primarily from the pollenkitt components which had not been digested by the time they reached the rectum. Although it was previously reported that the bees could not digest the components of the pollen walls (Stanley and Linskens. 1974) the combined histochemical evidence suggests that the chemistry of the wall changes as it proceeds through the digestive tract. Apparently the cellulose structure is altered in such a way that it can incorporate more iodine. The iodine oxidized unsaturated bonds thus diminishing the autofluorescence (a phenomenon which occurs primarily in resonating unsaturated organic chemicals). Bromophenol blue was also bound to pollen walls more readily as pollen proceeded through the midgut of the honeybee. Whether the alteration is the result of an actual digestion and extraction of cell wall components cannot be determined histochemically. but there is sufficient histochemical evidence that the alteration occurred (Figs 5-8). In conclusion the present study indicated that pollen which ruptures in the gut is more likely to be
sagittal section of the abdomen, exposing the crop stained with IKI was photographed with fluorescence micrography. The cellulose and sporopollenin of the exine (e) and intine (i) of several species of pollen fluoresced brightly. T. o@icinale (D) showed less fluorescence of the ectosexine (ec) than of the intine. x 280. (Fig. 6) A median sagittal section of the abdomen, exposing the anterior ventriculus was stained and observed under identical conditions as in Fig. 5. A decline in pollen wall fluorescence, due to the incorporation of iodine into the chemical structure of the pollen wall. is indicated. x 280. (Fig. 7) A median sagittal section of the abdomen, exposing the anterior rectum, indicates that most of the autofluorescence of the pollen wall was quenched by iodine binding, as described in Figs 5 and 6. x 280. (Fig. 8) A median sagittal section of the abdomen. exposing the posterior rectum, indicated complete loss of fluorescence of the pollen wall due to iodine binding. x 280.
Plate 1.
515
Plate 2. (Fig. 9) A median sagittal section of the abdomen, exposing the crop stained with bromophenol blue was photographed with fluorescence micrography. The cellulose and sporopollenin of pollen wall autofluoresced yellowgreen. The acidophilic components, stained with bromophenol blue, fluoresced orange to red depending on the quantity of dye bound. Cytoplasm (c) and nuclei (n) were seen in unruptured pollen, and red debris around the pollen were acidophilic compounds either of pollenkitt or protoplasmic origin. In T. ofjicinale pollen (D) there was a difference in fluorescence between the intine (i) and the exine with its elaborate ectosexine structure (ec). x 200. (Fig. 10) A median sagittal section of the abdomen, exposing the anterior ventriculus, stained and photographed as in Fig. 9, illustrated that a change in the chemistry of the pollen wall had occurred, causing the walls of fluoresce orange. At this point in the intestine, there was little difference in the amount of cytoplasm (c), and nuclei (n) retained in unruptured pollen grains. x 200. (Fig. 11) A median sagittal section of the abdomen, exposing the posterior ventriculus, indicated that more bromophenol blue was concentrated in
516
the wall. Cytoplasm (c) was still seen in unruptured pollen grains, and acidophilic particulates (a) were observed in layers through the slurry devoid of pollen walls. x 200. (Fig. 12) A median sagittal section of the abdomen, exposing the rectum, illustrated that acidophilic compounds (a) remaining in the slurry and the pollen wall, are bound to the pollen wall, or were undissolved in water or ethanol during specimen preparation. x 200. (Fig. 13) A median sagittal section of the abdomen, exposing the crop, was stained with toluidine blue 0. Carboxylated polysaccharides, including pollenkitt components on the outside of the pollen, and hemicehulose and pectic acids of the intine on the inside of pollen, all stained pink to red. Sporopollenin of the exine, as well as proteins and nucleic acids stained blue. x 125. (Fig. 14) A median sagittal section of the abdomen, exposing a cross-sectional view of the anterior ventriculus, illustrated that the bolus sometimes consisted of layers of slurry. The slurry lacked pollen and contained layers of carboxylated polysaccharide (cp) and unstained semisolids, probably of pollenkitt origin. Nectar sugars dissolved away over the long period of storage in ethanol. x 54
Plate 2.
517
Plate 3. (Fig. 15) A median sagittal section of the abdomen, exposing a longitudinal section of a turn in the anterior ventriculus, illustrated the progressive absorption of carboxylated polysaccharide (pink to red) by the midgut. The anterior portion (to right of image) contains more polysaccharides (red) than- the postehor portion (to left of imane). x 125. (Fig. 16) Enlaraed view of the anterior portTon of the vent&ulus in Fig- 15 showed the exine (e), intine (i), peritrophic membranes (pm) and the presence of carboxylated polysaccharides (cp) in the slurry around the pollen. The red stained slurry could be pollenkitt or cytoplasm from ruptured pollen (r), through unruptured pollen did contain little evidence of carboxylated polysaccharides in their retained cytoplasm (c). x 312. (Fig. 17) An enlarged image of the posterior portion of the ventricular section, shown in Fig. 15, illustrated that much of the carboxylated polysaccharide (cp) in the slurry around the pollen had been absorbed by the intestine. Traces of carboxylated poly-
518
saccharides remained on the ectosexine of T. oficinale pollen (D). x 312. (Fig. 18) A median sagittal section of the abdomen, exposing the anterior rectum, illustrated the absence of the carboxylated polysaccharides (red), except in association with the intine components (undigested hemicellulose and pectic acid). The unstained slurry (us) between the pollen did not stain with toluidine blue 0. x 125. (Fig. 19) A sagittal median section of the abdomen, exposing the folded lining of the posterior rectum, contained areas stained dark blue with toluidine blue 0. This staining was associated with bacteria. x 125 (Fig. 20) A median s&ittal section of the abdomen, exposing the rectum stained with bromophenol blue, confirmed that the slurry (us) (unstained with toluidine blue) contained acidophilic components (a). Some pollens also still remain small amounts of acidophilic components which are probably nuclei, or protoplasm. x 125.
Plate 3.
519
Histochemical
study
digested than pollen which does not. Digestion of pollen by honeybees is not complete, but starches, sugars. pectic acids, proteins and nucleic acids are efficiently digested and absorbed, if they are released from the pollen. Sporopollenin, cellulose, waxes and perhaps some complex proteins can not be digested by honeybees.
REFERENCES
Barker R. .I. and Lehner Y. (1972) A look at the honey bee gut functions. Am. Bee J. 112(9) 336338. Davenport H. A. (1960) Histological and Histochemical Techniques. 401 pp. W. B. Saunders C., Philidelphia. Jensen W. A. (1962) Botanical Histochemistry. 409 pp. W. H. Freeman and Co., San Francisco. Joshi M. and Agarwal H. C. (1976) Site of cholesterol absorption in some insects. J. Insect Phvsiol. 23,403404. George D. L. (1982) Self-compatabihty and Autogamy of Hybrids and their Parents in Sunflower (Helianthus annuus L.) Thesis, University of California, Davis. Klungness L. M.. Thorp R. W. and Briggs D. (1984) Field testing the germinability of almond (Prunus dulcis) pollen. J. Hori. Sri. S(2) 229-235.
of pollen digestion
521
Klungness L. M., and Peng Y. S. (1982) A scanning electron microscopic study of the homogeneity and condition of pollen collected by honeybees (Apis melltjera L.). J. Apic. Res. 22(4). Lillie R. D. (1979) Biological Stains (Ed. by Conn H. J.). 692 pp; 9th edn. The William and Wilkins. Baltimore. Martinho M. R. (1975) Contribuicao Estudo da Digestao do Grao de Polen em Melipona quadrifasciata anthidioides Lepelertier (Hymenoptera, Apidae, Meliponinae). Tese de Mestrado, Ribeirao Preto, Sao Paulo. 67 pp. Moore E. J. (1933) The use of phenosafranin for staining fungi on culture media or in a host tissue. Science 77, 23-24. Parker R. L. (1926) The collection and utilization of pollen by the honeybee. Cornell Unio., Agric. Exp. Sm.. Mem. 98, l-55. Snodgrass R. E. (1956) Anatomy and Phvsiolog,v qf the Honeybee. pp. 168-200 McGraw Hill, New York. Stadelmann E. J. and Kinzel H. (1972) Vital staining of plant cells. Methods in Cell Phwiologv (Ed. by Prescott D. M.) ch. 10. Vol. 5. Stanley R. G. and Linskens H. F. (1974) Pollen Biolog_v Biochemistry and Munagement. 307 pp. Springer, New York. Treherne J. E. (1967). Gut absorption. A. Rer. Ent. 12, 43-58.
Copyright f
0022.1910184$3.00+0.00 1984 Pergamon Press Ltd
A HISTOCHEMICAL STUDY OF POLLEN DIGESTION IN THE ALIMENTARY CANAL OF HONEYBEES (APIS MELLIFERA L.) L. M. Department
KLUNGNESS
of Entomology.
University
and
YING-SHIN
of California,
PENG*
Davis,
California
95616,
U.S.A.
(Rewired 2 September 1983; rerised 21 Ortober 1983) Abstract-Histochemical staining of embedded sections of the alimentary canal of honeybees indicated that carboxylated polysaccharides could be efficiently digested and absorbed. except when sequestered within unswollen and unbroken pollen grains that appeared to be impervious to enzymes of the gut. Hemicelluloses and pectic acids of the pollen wall structure underwent partial digestion, but pollen wall cellulose and sporopollenin were not digested. A separation of pollen walls from colloidal semisolids of the pollenkitt and disgorged protoplasm was apparent. The layers of slurry created by this separation remained into the rectum, and consisted primarily of saturated organic compounds which do not stain with weakly basophilic or acidophilic dyes.
Key Word Indes: Honeybee.
pollen.
alimentary
canal,
INTRODUCTION
*To whom correspondence , P 3,1,7--A
MATERIALS
AND METHODS
A total of 136 adult worker bees of the Italian race were collected for this study directly over the brood comb from a normal queen-right colony, during the early spring of 1979. The colony was fed with freezerstored corbicular pollen that had been collected the previous year in Canada. This pollen source contained a minimum of 10 species of pollen, but dandelion pollen, Taraxacum ojicinale, predominated in the sample. Since no dandelion was in bloom in the area where the colony was located, dandelion pollen was a marker for identifying recently consumed pollen. Bees used for histochemical preparation and microscopic observation were dehydrated after fixation with 2.5% gluteraldehyde in s-collidine buffer (pH 7.5). The dehydration sequence was 3&50-709/, ethanol prior to sample storage and then in 95- 1O& 1004; ethanol just prior to embedding.
____~ should
pollen digestion
pollen, consisting primarily of cellulose, and pectic acids (2) The exine is a complex structure secreted onto the outside of the exine by the tapetal cells of the anther. The sporopollenin matrix of the exine imparts rigidity and is often sculptured in complex layers and structures. The exine is often described in specific structural layers having unique nomenclature. The ectosexine, the only such layer referred to in this paper, is the outermost layer of the exine (3) The pollenkitt is a semisolid, soluble layer of lipids, proteins, and sugars, which is embedded in the ectosexine of many species of pollen. The chemistry and function of these layers of pollen is reviewed by Stanley and Linskens (1974). The purpose of this study is to apply histochemical procedures to better identify the content of the pollen during the process of digestion in the alimentary canal of the honeybee.
Since the original definitive study of honeybees to determine whether adults and larvae could utilize pollen as food (Parker, 1926), studies of the histochemistry of digestion and absorption in honeybees (Apis mellifera L.) have been scarce (Stanley and Linskens, 1974). Parker concluded that only simple proteins and sugars are available to the bee from digested pollen, but this assumption has been challenged. Snodgrass (1956) and Treherne (1967) reported that the crop had no secreting function. Barker and Lehner (1972) summarized the available literature regarding the role of the peritrophic membrane of the ventriculus in distributing digestive enzyme. Nevertheless, Joshi and Agarwal (1976) reported that 8.29, of the cholesterol consumed by honeybees was absorbed in the crop while 60% was absorbed in the ventriculus. Martinho (1975) reported that the sugar concentration varied greatly in the crops of honeybees that he sampled. He also found that digestion of pollen increased from 30 to 70”/ when pollen would swell and protrude its intine as a result of osmotic pressure. Observations of pollen viability in corbicular pollen loads have been made by Klungness, Thorp and Briggs (1984), and morphological observations of pollen in the beebread of honeybees have been recorded by Klungness and Peng (unpublished observations). We have obtained evidence indicating that pollen losses membranecontrol of osmosis during storage as beebread. But scanning electron microscopic observations of the gut of honeybees indicate that not all pollen swells and ruptures in the crop or ventriculus of Apis mellifera (unpublished observations). There are three major components of the pollen wall, namely: (1) The intine is the true cell wall of ___~
histochemistry.
be addressed. 511
512
L. M. KWNGNESS and YIN+SHIN
JB-4TM embedding reagent A (DuPont Co., Willmington, Deleware) was used to replace ethanol over 24 h of refrigeration. Specimens were then transferred to a mixture of JB-4 reagents A and B and polymerization was allowed to proceed at room temperature. The mixture was that recommended by the manufacturer as having the slowest rate of polymerization and therefore less gas is formed during the exothermal reaction. Of the 136 adult worker bees collected. 20 were randomly selected for embedding. Later 5 specimens were selected for microtomy, based on orientation of the specimen. The specimen blocks were sectioned with glass knives on a JB-4 rotary microtome (SorvallTM, DuPont Co., Newtown. Connecticut). Sections were median sagittal planes of the entire bee, and thus each section contained portions of various parts of the alimentary canal. This made comparative staining of organs possible. Ribbons of sections were created by placing a bead of contact cement (3M Co., St Paul, Minnesota) on the top and bottom of the block. These ribbons of sections were floated on water that had been placed on a microscope slide, then dried on a warming plate at approx. 40 C. which caused the sections to adhere to the glass. The following stains were used to identify the chemical components of the contents of the alimentary canal:
(I) To&dine blue 0 was used to metadistinguish chromatically carboxylated PolYsaccharides such as hemicellulose and pectic acid. from those chemicals which stain orthochromatically. or not at all in this dye (Stadelmann and Kinzel. 1972). The carboxylated polysaccharides stain red to pink. Nucleic acids, proteins and sporopollenin stain blue. Phenolics stain green. Starch, waxes, saturated cellulose and saturated lipids do not stain with this dye. A O.l”, aqueous solution of toludine blue 0 was applied to the dried sections, allowed to stain for 5 min rinsed with water, and covered with glycerine and a coverslip. This mounting method preserves metachromatic properties of the stain, but is not a permanent mount. (2) The starch iodide reaction (IKI) (Jensen, 1962) was used to locate starch and amyloids. The reagent stains starch blue to black, and amyloids yellow to red. IKI solution was made by dissolving 2 g of potassium iodide (KI) in 100 ml of water, followed by the addition of 0.2 g of iodine. The IKI solution was applied directly to the dried sections, and observations were made without rinsing the stain from the slide. Such preparations are not permanent and fade quickly. IKI stained sections were also observed with fluorescence illumination. The iodine acted as a histochemical oxidant (Lillie, 1977) to saturate resonating chemical bonds in the contents of the intestine. Resonating bonds are often the source of autofluorescence. Therefore iodine binding quenches autofluorescence where it occurs. More penetration of iodine into cellulose is known to occur when cellulose is partially hydrolyzed (Jensen, 1962). Therefore, with IKI. it was possible to
PENG
localize in the alimentary canal the areas where hydrolysis of the pollen wall occurred. (3) A solution of 0. I “; hrotnophennl blue in ethanol was used to stain protein and other acidophilic components of the intestinal contents. This dye stains the amino groups of protein (Davenport. 1960). although it is not highly specific to protein. Its colour is a function of pH of the staining environment and may vary from red to blue. This reagent was applied to dry sections and allowed to stain for 15 min before rinsing with water. Glycerine was used as the mounting medium for microscopic observation. Bromophenol blue has fluorescent properties, which can be used to better determine the amount of stain bound in the specimen. It is a metachromatic dye which changes from yellow to red, as the concentration of the bound dye increases. (4) The basic dye, phrtwsq~iunin was reported to be a strongly acidophilic dye (Moore, 1933). We used this dye to stain acid contents of the intestine because they had little or no chemical reactivity with the weakly acidophilic dyes. The reagent was mixed in a contrast stain contammg 1.o”,, of phenosafranin and 1.0’; of bromophenol blue in 30”” ethanol and 68”,, glycerine. This counterstaining reagent was applied to dry sections, which were then placed on a warming plate for several hours to achieve staining of the undigested components of the intestinal contents. All photomicrographs. includng fluorescence, were taken soon after slide preparation, in order to obtain uniform results. For normal tungsten light photomicrography, EktachromeTM 50 (Eastman Kodak, Rochester. New York) was used. Ektachrome200 was used for fluorescence photomicrography. Fluorescence light microscopy was achieved with a Zeiss Standard microscope equipped with a 200 W mercury vapour lamp for transmitted illumination. Filter combinations varied with the stain: (1) for autofluorescence of the pollen walls, both with and without IKI stain, an UGI excitation filter, a BG38 red-absorption filter and the 490-nanometer barrier filter were used (2) for hromophenol blue fluorescence. a FITC dichroic excitation filter, the heat reflection filter and the 530-nanometer barrier filter were used. Where comparative views of different zones of the alimentary canal were photographed, considerable care was taken to ensure equal exposure conditions. All such sets of photomicrographs had been made on the same slides, thus assuring that the same staining procedure and dye concentration were compared.
RESULTS
Starches and umyloids Potassium iodide when used to stain pollen amyloplasts, indicated that starch was quickly digested in the ventriculus if it was either released from the pollen grains, or the amylases that are secreted into the intestine, were able to penetrate the pollen membrane. Starch that was retained within unruptured impervious pollen grains, was not digested by the time the pollen reaches the rectum. This was clearly demonstrated in Figs 14.
Histochemical
study
Nucleic acids, proteins and lipoprotein membranes Bromophenol blue, which complexes with amino groups, stained proteins of pollen protoplasm and/or nuclear components brown to red in normal light and orange to red with fluorescence. These acidophilic components were observed throughout the alimentary canal of the bee (Figs 9-12). In Fig. 10, large pollen grains were seen that had retained starch amyloplasts. This was confirmed by IKI reagent. which stained the starch blue (Fig. 2) and by toluidine blue 0. The latter stain showed that only the amyloplast membrane stained pink and blue, whereas the starch granule remained unstained by this dye (Fig. 16). With bromophenol blue viewed in fluorescent light, the amyloplast membrane was red but no stain was taken into the starch granule (Fig. 10). Therefore, these three stains indicated that starch and membrane components of some pollen species were not visibly affected by the digestive enzymes of the intestine. It was observed that acidophilic compounds were present in the intestinal slurry around the pollen, but that these were not the predominant component of the slurry. With bromophenol blue staining of acidophilic compounds (Figs 12 and 20) and with toluidine blue 0 staining of basophilic compounds (Fig. 18) it was apparent protoplasmic and nuclear components of some pollen remained undigested in the rectum. although most pollen contents were removed. Carboxylated polysaccharides of pollenkitt Rapid digestion of carboxylated polysaccharides was seen in longitudinal sections of the ventriculus, where the pink-stained polysaccharides diminish rapidly toward the posterior (Fig. 15). The enlarged image of the anterior portion of the section (Fig. 16) showed the carboxylated polysaccharides components of the intine and exine and peritrophic membranes, as well as the presence of extra-cellular polysaccharide in the lumen. Figure 17 showed that much of the extracellular polysaccharide was removed from the lumen shortly after entering the ventriculus. Whereas within the anterior rectum (Fig. 18), little to no carboxylated polysaccharide remained, except that associated with the pollen intine (hemicellulose and possibly residual pectic acids). The pollenkitt of T. ofiicinale pollen had been observed to be mostly separated from the exine in the crop (Fig. 13). Some traces of pink-stained polysaccharides of the pollenkitt were present in the crop, but the copious quantity of pollenkitt that is normally embedded in the ectosexine of T. oficinale had obviously been displaced. Some regions of the ventriculus were observed to contain very few pollen grains. In these areas it was sometimes observed that the plane of section through the diameter of the ventriculus contained a slurry containing little or no pollen. A fraction of that slurry (Fig. 14) appears to be layered with other acidophilic or neutral compounds which do not bind toluidine blue 0. Counterstaining the slurry with bromophenol blue, confirmed the presence of acidophilic compounds in the slurry. These bases were probably of protoplasmic origin. The apparently empty space between the pollen in
of pollen
digestion
513
the rectum (Figs 18-20) was a slurry which contained the unstained and undigested components of pollenkitt and disgorged pollen cytoplasm. The slurry had no affinity for the weakly basic dye toluidine blue 0. but reacted very slowly in a warm environment with the strongly basic dye, phenosafranin. Only a portion of the slurry stained with the acid dye, bromophenol blue (Fig. 20). Certain areas of the lining of the posterior rectum stained pink and dark blue (Fig. 19). High magnification observations indicated that these were associated with bacteria. Pollen wall components The autofluorescence of the pollen wall. before and after IKI staining, gave evidence of a change in the chemical nature of the pollen wall as it passed through the honeybee’s digestive tract. Before IKI stain was applied to the sections, autofluorescence of the pollen wall appeared to be similar throughout the crop and the entire intestine. After iodine was added as an oxidant, the autofluorescence of the pollen wall decreased from the anterior to posterior region of the gut (Figs 5-8). This chemical change in the pollen wall is also suggested by the bromophenol blue staining method, when appropriate fluorescence filters were used (Figs 9-12). There was an increased absorption of this fluorescent dye (red) by the pollen wall, as it progressed through the gut. Bromophenol blue, used as a fluorescent dye, confirms an alteration in pollen wall chemistry as it passes through the intestine. In the crop, the pollen wall is impervious to the stain. Therefore, the yellowgreen autofluorescence of the wall is seen in the crop with this stain and appropriate filter combination (Fig. 9). In the anterior ventriculus some of the dye is bound to the pollen wall. giving it an orange fluorescence (Fig. 10). As more bromophenol blue binds to the pollen wall in the posterior ventriculus (Fig. 11) and the rectum (Fig. 12) the fluorescence of the dye takes on more red tone. It cannot be determined by these histochemical tests whether the dye is actually binding to the cell wall components themselves or whether other components of the intestinal slurry are more able to penetrate the wall as digestion proceeds, in which case they would bind the dye. In either case a change in the cell wall structure is indicated. Staining with toluidine blue 0 indicated that partial digestion of pollen wall components did occur. In particular, the carboxylated polysaccharides of the intine were partially digested by the gut (Figs 16, 18 and 19). These polysaccharides are primarily pectic acids and hemicellulose. Figure 16 shows a magnified image of the pollen in the ventriculus. Although red stained sugars and pollenkitt components are seen outside of the pollen, the red layer, inside and next to the blue stained exine wall, is the intine. This consists primarily of cellulose, hemicellulose and pectic acids (Stanley and Linksens 1974). Figures 18 and 19 show pollen in the anterior and posterior rectum respectively, after staining with toluidine blue 0. It is apparent that, though some carboxylated polysaccharides remain in the intine, much of the red staining is gone, indicating partial digestion of the intine components.
L. M. KLUNGNESS and YING-SHIN PENG
514 DISCUSSION
The presence of a slurry containing no pollen raises questions about the separation of materials in the digestive process. The origin of the components cannot be resolved histochemically. The acidophilic components might have been the enzyme protein secreted from the ventricular epithelium. but was more probably the protoplasmic protein from the ruptured pollen. The presence of the acidophilic compounds in the rectum suggests the occurrence of incomplete digestion and/or absorption of those compounds, having basic reactive groups to bind bromophenol blue (Figs 12 and 20). In addition to the suggestion that the protoplasmic content of ruptured pollen might be physically separated from the pollen, there was also indication that the pollenkitt components might have been physically separated from the pollen by the action of the proventriculus. This separation process caused the formation of layers of slurry in the gut. These layers contained no pollen walls within them (Fig. 14). These layers also persisted into the rectum. though histochemical indications of changes in the chemistry of the slurry appeared at different positions in the intestine. This observation confirms scanning electron micrographic studies of Klungness and Peng (unpublished observations), which suggested that the proventriculus separates food components. This process may cause layering in the food bolus, rather that a homogeneous food mixture. This suggests that the proventriculus can writhe with considerable force when the ventriculus is nearly empty. Under these conditions. the fluid dynamics would probably be such that only large and firm particulates like pollen could be impeded by the comb of the proventriculus. This would cause pollen to collect in the ventriculus. but pollenkitt and protoplasm of ruptured pollen grains would flow through the comb of the proventriculus, into the crop. As the ventriculus becomes engorged with the food bolus. the intestinal muscles cannot generate such a high flow rate through the proventriculus. The fluid dynamics of this slower flow might allow the comb of the proventriculus to impede
Plate 1. (Fig. 1). A median sagittal section of the abdomen, exposing the crop of a honeybee was stained with IKI reagent. Blue-stained starch granules (s) and yellow stained amyloids (ubiquitous) were found inside and outside of the pollen. The pollen shown are of several unidentified species not including T. c@cinale. x 175. (Fig. 2) An enlarged view of Fig. 1 illustrated that the starch granules outside of the pollen were released into the slurry because some of the pollen grains had ruptured. Broken pollen walls (r) are not found in all grains of the larger pollen species. x 280. (Fig. 3) A median sagittal section of the abdomen, exposing the posterior midgut of the honeybee, illustrated that extracellular starch granules had been thoroughly digested. The starch granules retained within the unruptured pollen were not digested, as indicated by the IKI blue staining (s). x 175. (Fig. 4) A median sagittal section of the abdomen, exposing the rectum, illustrated that starch (s) remained in some pollen grains that did not rupture. x 175. (Fig. 5) A median
the passage of viscous as well as particulate food in the slurry. The result of such changes in flow dynamics would be the formation of alternate layers of pollen and slurry in the food bolus. The histochemistry of the slurry also supported this hypothesis. since the slurry did contain the carboxylated polysaccharides (stained pink in Fig. 14) and the lipids (unstained) that have been reported to occur in pollenkitt of Compositae (George 1982). This suggested that the slurry, by the time it reached the rectum, consisted primarily of complex saturated macromolecules with few reactive sights to bind the dyes, Because the chemistry of the pollenkitt is known (Stanley and Linskens. 1974). it is reasonable to conclude that the soluble and digestible portions of the pollenkitt are stained at their reactive binding sites. These are also the more likely compounds to be reduced to small molecules that would be absorbed by the gut wall. There are pollenkitt components that would not have many exposed reactive sites and would not be readily broken down by the digestive enzymes. Therefore, we suggest that it is probable that the undigested slurry came primarily from the pollenkitt components which had not been digested by the time they reached the rectum. Although it was previously reported that the bees could not digest the components of the pollen walls (Stanley and Linskens. 1974) the combined histochemical evidence suggests that the chemistry of the wall changes as it proceeds through the digestive tract. Apparently the cellulose structure is altered in such a way that it can incorporate more iodine. The iodine oxidized unsaturated bonds thus diminishing the autofluorescence (a phenomenon which occurs primarily in resonating unsaturated organic chemicals). Bromophenol blue was also bound to pollen walls more readily as pollen proceeded through the midgut of the honeybee. Whether the alteration is the result of an actual digestion and extraction of cell wall components cannot be determined histochemically. but there is sufficient histochemical evidence that the alteration occurred (Figs 5-8). In conclusion the present study indicated that pollen which ruptures in the gut is more likely to be
sagittal section of the abdomen, exposing the crop stained with IKI was photographed with fluorescence micrography. The cellulose and sporopollenin of the exine (e) and intine (i) of several species of pollen fluoresced brightly. T. o@icinale (D) showed less fluorescence of the ectosexine (ec) than of the intine. x 280. (Fig. 6) A median sagittal section of the abdomen, exposing the anterior ventriculus was stained and observed under identical conditions as in Fig. 5. A decline in pollen wall fluorescence, due to the incorporation of iodine into the chemical structure of the pollen wall. is indicated. x 280. (Fig. 7) A median sagittal section of the abdomen, exposing the anterior rectum, indicates that most of the autofluorescence of the pollen wall was quenched by iodine binding, as described in Figs 5 and 6. x 280. (Fig. 8) A median sagittal section of the abdomen. exposing the posterior rectum, indicated complete loss of fluorescence of the pollen wall due to iodine binding. x 280.
Plate 1.
515
Plate 2. (Fig. 9) A median sagittal section of the abdomen, exposing the crop stained with bromophenol blue was photographed with fluorescence micrography. The cellulose and sporopollenin of pollen wall autofluoresced yellowgreen. The acidophilic components, stained with bromophenol blue, fluoresced orange to red depending on the quantity of dye bound. Cytoplasm (c) and nuclei (n) were seen in unruptured pollen, and red debris around the pollen were acidophilic compounds either of pollenkitt or protoplasmic origin. In T. ofjicinale pollen (D) there was a difference in fluorescence between the intine (i) and the exine with its elaborate ectosexine structure (ec). x 200. (Fig. 10) A median sagittal section of the abdomen, exposing the anterior ventriculus, stained and photographed as in Fig. 9, illustrated that a change in the chemistry of the pollen wall had occurred, causing the walls of fluoresce orange. At this point in the intestine, there was little difference in the amount of cytoplasm (c), and nuclei (n) retained in unruptured pollen grains. x 200. (Fig. 11) A median sagittal section of the abdomen, exposing the posterior ventriculus, indicated that more bromophenol blue was concentrated in
516
the wall. Cytoplasm (c) was still seen in unruptured pollen grains, and acidophilic particulates (a) were observed in layers through the slurry devoid of pollen walls. x 200. (Fig. 12) A median sagittal section of the abdomen, exposing the rectum, illustrated that acidophilic compounds (a) remaining in the slurry and the pollen wall, are bound to the pollen wall, or were undissolved in water or ethanol during specimen preparation. x 200. (Fig. 13) A median sagittal section of the abdomen, exposing the crop, was stained with toluidine blue 0. Carboxylated polysaccharides, including pollenkitt components on the outside of the pollen, and hemicehulose and pectic acids of the intine on the inside of pollen, all stained pink to red. Sporopollenin of the exine, as well as proteins and nucleic acids stained blue. x 125. (Fig. 14) A median sagittal section of the abdomen, exposing a cross-sectional view of the anterior ventriculus, illustrated that the bolus sometimes consisted of layers of slurry. The slurry lacked pollen and contained layers of carboxylated polysaccharide (cp) and unstained semisolids, probably of pollenkitt origin. Nectar sugars dissolved away over the long period of storage in ethanol. x 54
Plate 2.
517
Plate 3. (Fig. 15) A median sagittal section of the abdomen, exposing a longitudinal section of a turn in the anterior ventriculus, illustrated the progressive absorption of carboxylated polysaccharide (pink to red) by the midgut. The anterior portion (to right of image) contains more polysaccharides (red) than- the postehor portion (to left of imane). x 125. (Fig. 16) Enlaraed view of the anterior portTon of the vent&ulus in Fig- 15 showed the exine (e), intine (i), peritrophic membranes (pm) and the presence of carboxylated polysaccharides (cp) in the slurry around the pollen. The red stained slurry could be pollenkitt or cytoplasm from ruptured pollen (r), through unruptured pollen did contain little evidence of carboxylated polysaccharides in their retained cytoplasm (c). x 312. (Fig. 17) An enlarged image of the posterior portion of the ventricular section, shown in Fig. 15, illustrated that much of the carboxylated polysaccharide (cp) in the slurry around the pollen had been absorbed by the intestine. Traces of carboxylated poly-
518
saccharides remained on the ectosexine of T. oficinale pollen (D). x 312. (Fig. 18) A median sagittal section of the abdomen, exposing the anterior rectum, illustrated the absence of the carboxylated polysaccharides (red), except in association with the intine components (undigested hemicellulose and pectic acid). The unstained slurry (us) between the pollen did not stain with toluidine blue 0. x 125. (Fig. 19) A sagittal median section of the abdomen, exposing the folded lining of the posterior rectum, contained areas stained dark blue with toluidine blue 0. This staining was associated with bacteria. x 125 (Fig. 20) A median s&ittal section of the abdomen, exposing the rectum stained with bromophenol blue, confirmed that the slurry (us) (unstained with toluidine blue) contained acidophilic components (a). Some pollens also still remain small amounts of acidophilic components which are probably nuclei, or protoplasm. x 125.
Plate 3.
519
Histochemical
study
digested than pollen which does not. Digestion of pollen by honeybees is not complete, but starches, sugars. pectic acids, proteins and nucleic acids are efficiently digested and absorbed, if they are released from the pollen. Sporopollenin, cellulose, waxes and perhaps some complex proteins can not be digested by honeybees.
REFERENCES
Barker R. .I. and Lehner Y. (1972) A look at the honey bee gut functions. Am. Bee J. 112(9) 336338. Davenport H. A. (1960) Histological and Histochemical Techniques. 401 pp. W. B. Saunders C., Philidelphia. Jensen W. A. (1962) Botanical Histochemistry. 409 pp. W. H. Freeman and Co., San Francisco. Joshi M. and Agarwal H. C. (1976) Site of cholesterol absorption in some insects. J. Insect Phvsiol. 23,403404. George D. L. (1982) Self-compatabihty and Autogamy of Hybrids and their Parents in Sunflower (Helianthus annuus L.) Thesis, University of California, Davis. Klungness L. M.. Thorp R. W. and Briggs D. (1984) Field testing the germinability of almond (Prunus dulcis) pollen. J. Hori. Sri. S(2) 229-235.
of pollen digestion
521
Klungness L. M., and Peng Y. S. (1982) A scanning electron microscopic study of the homogeneity and condition of pollen collected by honeybees (Apis melltjera L.). J. Apic. Res. 22(4). Lillie R. D. (1979) Biological Stains (Ed. by Conn H. J.). 692 pp; 9th edn. The William and Wilkins. Baltimore. Martinho M. R. (1975) Contribuicao Estudo da Digestao do Grao de Polen em Melipona quadrifasciata anthidioides Lepelertier (Hymenoptera, Apidae, Meliponinae). Tese de Mestrado, Ribeirao Preto, Sao Paulo. 67 pp. Moore E. J. (1933) The use of phenosafranin for staining fungi on culture media or in a host tissue. Science 77, 23-24. Parker R. L. (1926) The collection and utilization of pollen by the honeybee. Cornell Unio., Agric. Exp. Sm.. Mem. 98, l-55. Snodgrass R. E. (1956) Anatomy and Phvsiolog,v qf the Honeybee. pp. 168-200 McGraw Hill, New York. Stadelmann E. J. and Kinzel H. (1972) Vital staining of plant cells. Methods in Cell Phwiologv (Ed. by Prescott D. M.) ch. 10. Vol. 5. Stanley R. G. and Linskens H. F. (1974) Pollen Biolog_v Biochemistry and Munagement. 307 pp. Springer, New York. Treherne J. E. (1967). Gut absorption. A. Rer. Ent. 12, 43-58.