From the anther to the proctodeum: Pear (Pyrus communis) pollen digestion in Osmia cornuta larvae

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Journal of Insect Physiology 51 (2005) 749–757 www.elsevier.com/locate/jinsphys

From the anther to the proctodeum: Pear (Pyrus communis) pollen digestion in Osmia cornuta larvae Massimo Nepia, Laura Crestia, Bettina Maccagnanib,, Edith Ladurnerb, Ettore Pacinia a

b

Dipartimento di Scienze Ambientali ‘‘G. Sarfatti’’, Universita` degli Studi di Siena, Via P. A. Mattioli 4, 53100, Siena, Italy Dipartimento di Scienze e Tecnologie Agroambientali—Entomologia, Alma Mater Studiorum—Universita` di Bologna, Viale G. Fanin 42, 40127 Bologna, Italy Received 10 November 2004; received in revised form 1 March 2005; accepted 3 March 2005

Abstract Modifications of the pollen grains of Pyrus communis Linneaus that occur during the digestion by Osmia cornuta (Latreille) larvae were studied histochemically. We compared the features of the pollen grains found in the anthers, in the larval cell provisions and in the alimentary canal of the 5th instar larvae. Modifications were already evident in the provisions and consisted of protoplast protrusions through the apertures and a decrease in the number of starch-containing pollen grains. After pollen grains were ingested by the larvae, the protoplast appeared retracted from the pollen wall. Pollen digestion began in the anterior part of the midgut, where we observed: (1) disorganised intine at the apertures; (2) disappearance of DAPI staining of nuclear pollen DNA; (3) fewer pollen grains containing starch than in the anthers; (4) some empty pollen grains. Pollen grains in the proctodeum appeared extremely compressed and crushed. Some grains appeared to be unaffected by the digestive process. We hypothesise that the protrusion of the intine and of the protoplast from the apertures in bee provisions could be considered a kind of pre-treatment necessary to initiate the digestion process in the larval alimentary canal. r 2005 Elsevier Ltd. All rights reserved. Keywords: Osmia cornuta; Pyrus communis; Pollen digestion; Larval food; Protoplast protrusion; Pseudo-germination; Osmotic shock

1. Introduction Pollen is a source of free aminoacids, proteins, lipids and vitamins for a wide range of animals: marsupials, mammals, birds and arthropods, such as insects (Roulston and Cane, 2000) and mites (Fain, 1966). The pollen grain wall (sporoderm) has an outer layer (exine) composed of sporopollenin, an indigestible and chemically resistant bio-polymer (Nepi and Franchi, 2000). The mechanisms applied by pollen feeders to reach the substances contained in the pollen cytoplasm were exhaustively reviewed by Roulston and Cane (2000). One possible mechanism is the exposure of the Corresponding author. Tel.: +39 05120 96295; fax: +39 05120 96281. E-mail address: [email protected] (B. Maccagnani).

0022-1910/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2005.03.003

pollen grains to variable osmotic pressure. In the adults of the honey bee Apis mellifera Linnaeus and of the carpenter bee Xylocopa capitata Smith, the high concentration of soluble sugars in the first tract of the alimentary canal could contribute to create such a kind of osmotic gradient. In fact, in the crop of the adult carpenter bee Louw and Nicolson (1983) demonstrated the complete hydrolysis of the sucrose contained in the nectar to glucose and fructose. Roulston and Cane (2000) reported that in the very specialised proventriculus of the adult honeybee the ingested pollen, exposed to the high concentration of soluble sugars of the nectar, may experience a pre-treatment that could lead to germination or pseudo-germination. The pollen is then removed from this suspension while nectar is retained; the movements of four mobile proventricular lips allow the pollen bolus to pass into the midgut, where it experiences an abrupt osmotic shock, passing from a

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hypertonic environment into a hypotonic one. This sudden osmotic shock may cause the pollen grains to burst, or their pores to open with subsequent release of the protoplasm; this was primarily described as a prerequisite for pollen digestion in adult honey bees by Kroon et al. (1974). Peng et al. (1985, 1986) restricted the efficiency of such a mechanism to thin-walled pollen grains, since they found slow pollen degradation along the alimentary canal of species with thick pollen wall, like alfalfa (Medicago sativa Linnaeus) and dandelion (Taraxacum officinale Weber) pollen. They assumed that the swelling of the pores due to the difference in the osmotic pressure during the pollen transit from the crop to the midgut facilitates the enzymatic weakening of the intine. They also hypothesised that this process is of crucial importance for allowing the extrusion of the cytoplasm. Additionally, Human and Nicolson (2003) suggested that mechanisms other than osmotic shock are involved in pollen digestion in the adult honeybee. Starch is a common carbohydrate reserve in pollen grains and its presence and physico-chemical properties vary according to plant species (Franchi et al., 1996). Ripe pollen during presentation and dispersal may regulate water loss or intake by de-polymerising starch to simple sugars or polymerising simple sugars to starch, respectively (thus increasing or decreasing the osmotic pressure in the cytoplasm) according to the environmental conditions that it experienced (Pacini, 2000). Starch is hardly digested by adult honey bees (Klungness and Peng, 1984) and larvae (Bertholf, 1927). In contrast, larvae of solitary bees that are oligolectic on plants having starchy pollen can remove starch from the grains very efficiently (Simpson and Neff, 1983). Among insects, solitary bee larvae are direct pollen consumers. They do not possess a crop where nectar and pollen can be retained, like in the adult honeybee, but the consumed pollen is part of a provision containing variable amounts of nectar (Roubik, 1989; Dobson and Peng, 1997; Ladurner et al., 1999; Roulston and Cane, 2000). Osmia cornuta (Hymenoptera Megachilidae) is a candidate commercially managed pollinator, especially on early flowering and protected crops (Krunic et al., 1995; Pinzauti et al., 1997; Ladurner et al., 2002; Maccagnani et al., 2003a, b; 2005, Vicens and Bosch, 2000; Bosch and Kemp, 2002). Females of this cavitynesting species produce around 30 larval cells, each containing a pollen-nectar based provision on which a single egg is laid (Bosch, 1994; Maccagnani et al., 2003b). After a few days the first instar larva hatches, but it remains inside the egg chorion; within 12 h, while completing the hatching from the egg chorion, it moults. The small apodal 2nd instar larva starts feeding on the maternal provision, and continues until the end of the 5th larval stage (Torchio, 1989). Previous studies on pollen digestion by Osmia bees concerned comparisons between pollen in the provisions

and in the faeces (Sua´rez-Cervera et al., 1994; Nepi et al., 1997) without considering the intermediate steps of pollen digestion in the alimentary canal. In this study we compare the histochemical features of pollen grains in the anthers, in O. cornuta pollen provisions, and in the alimentary canal (midgut and proctodeum) of O. cornuta larvae, in order to gain a more complete insight of pollen grain modifications occurring during the digestive process.

2. Materials and methods Eggs with their maternal pollen provisions, produced by O. cornuta females actively foraging in a pear orchard (Pyrus communis Linneaus, variety ‘‘Abate Fete´l’’), were individually transferred from their original nests (pieces of reeds Arundo donax Linneaus) into gelatine capsules (type ‘‘000’’). They were housed in an incubator at temperature 20 1C, R.H. 70%, and L:D ¼ 0:24. When the larvae had reached the 5th instar, they were transferred in aceto-alcoholic formaldehyde. The larval digestive tracts were removed, fixed (5% glutharaldehyde in phosphate buffer at pH 6.9), dehydrated in an ethanol series and embedded in Technovit 7100 (Heraeus Kulzer GmbH). Serial sections (3–5 mm) of the anterior, median and posterior parts of the midgut, and of the proctodeum were obtained with a LKB 8800 III ultramicrotome. Sections were stained with the following histochemical reagents (Nepi and Franchi, 2000): (1) PAS (Periodic Acid—Schiff reaction) for total insoluble polysaccharides; (2) Auramine O for sporopollenin (exine); (3) Calcofluor White for cellulose (intine); (4) Coomassie Blue for total proteins; (5) DAPI for DNA. The same fixation and staining procedure was used for samples of pollen from anthers and from O. cornuta provisions. 2.1. Cytological pollen features and pollen counts Preliminary observations on untreated pollen from anthers and from O. cornuta provisions were performed by adding a drop of immersion oil and observing under phase contrast. These observations allowed us to discriminate four morphological pollen types according to the cytological features of the pollen grains: unmodified, with protoplast protrusion from the apertures, with protoplast retraction from the pollen wall, and empty pollen grains. The stained semi-thin sections from anterior, median, posterior midgut, and proctodeum (five replicates per intestine tract randomly selected from three different

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larvae) and the stained pollen samples from anthers and provisions (five replicates each) were observed under a light microscope. The frequencies of the above-mentioned morphological pollen types and of the starch containing pollen grains were calculated. Observations concerning proctodeum sections have only a descriptive value, because the extreme deformation of the pollen grains in this digestive tract did not allow detailed counting and observation. The differences in the frequencies of pollen grains in each morphological class across five environments (anther, larval provision, anterior midgut, median midgut, posterior midgut) were statistically analysed by means of one-way ANOVAs. The Bonferroni multiple comparison procedure (Po0:05) was used to discriminate among the means (Statisticas 6.0; Stasoft, 1993). The same statistical approach was applied to compare the frequencies of starch containing pollen grains in the five environments. Means are reported with their standard error, unless indicated otherwise.

3. Results 3.1. Structural and cytochemical pollen grain modifications Pear pollen grains collected from the anthers presented the typical features of the species, being tricolpate, prolate and medium-small in size (40  60 mm). Due to dehydration naturally occurring in anthers, the pollen wall was folded into the apertures (Fig. 1A). The intine had a linear, compact profile all around its surface (Fig. 1B and C). Pollen from larval provisions had a more distended wall and most of the grains showed protoplast protrusion from the apertures (Figs. 1D and E) suggesting a higher water content, if compared to the pollen collected from anthers. The intine presented a more irregular profile, especially near the apertures (Fig. 1F). Pollen ingested by O. cornuta larvae underwent profound structural and cytochemical modifications. The most conspicuous modification, already evident in the anterior midgut, was the retraction of the protoplast from the pollen wall (Fig. 2A, see also Fig. 2E). Some pollen grains were void of protoplasm (Fig. 2A). Also modifications of the intine started already in the anterior midgut: its compact fibrils became disorganised and loose near the apertures (Fig. 2B) where the intine appeared undulated. Nuclear DNA could no longer be observed by DAPI staining (Fig. 2C). Cytological modifications of the pollen grains continued to appear as they proceeded through the midgut (Figs. 2D–H). In the posterior midgut, the protoplast of some pollen grains appeared to extrude through the apertures (Figs. 2D and E). Remnants of protoplasm

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were present in the lumen (Fig. 2D). Pollen grains containing starch were the least modified and were intensely coloured by PAS, while those with retracted protoplast and protruding intine were stained by Comassie Blue but not by PAS (Fig. 2E). Intine modifications extended to the inter-apertural regions, and some grains were void of intine in the posterior midgut (Fig. 2F). The pollen grains were progressively emptied while advancing through the digestive tract, and most of them were almost completely deprived of protoplasm as they reached the proctodeum (Figs. 2G and H). Here, the pollen grains appeared compressed and deformed so that it was not possible to classify them according to the morphological classes defined by the preliminary observations. Anyway, it was possible to recognise a few unmodified grains (Fig. 2G). Some pollen grains lacked intine, which was very loose and undulated in all the other ones (Fig. 2H). The exine was the only component of the pollen grain that did not undergo any evident change under light microscope and after staining with Auramine O. It showed no breaks except the natural apertures. The grains containing starch were the least modified and did not appear compressed (Fig. 2G). Proteins were gradually broken down while the pollen grains proceeded through the digestive tract. A small proportion of grains still stained weakly for proteins in the proctodeum. 3.2. Frequency of the morphological pollen types and of the starch containing pollen grains Unmodified pollen grains constituted the large majority of the grains in the anthers. No significant differences in the percentages of unmodified pollen grains among anterior, median, posterior midgut emerged (F ¼ 669:7; Po0:001) (Fig. 3A). The percentage of pollen grains with protoplast protrusion was only 12.873.7% in the anthers, but it increased to 81.575.1% in O. cornuta provisions, being then very low in the larval alimentary canal, without any significant difference among the different parts of the midgut (F ¼ 630:8; Po0:001) (Fig. 3B). Pollen grains with retracted protoplast were observed only in the midgut: the percentage was highest in the anterior midgut (59.971.3%), decreased in the median midgut (45.574.1%) and increased again in the posterior midgut (51.971.6%) (F ¼ 993:8; Po0:001) (Fig. 3C). Empty pollen grains were present in all the environments. Percent empty pollen grains was lowest in anthers and bee provisions, increased in the anterior and median midgut (30.171.1% and 42.774.83%, respectively), slightly decreased in the posterior midgut (36.970.6%) (F ¼ 297:0; Po0:001) (Fig. 3D). Significant differences were found in the mean frequencies of starch containing grains during the pollen

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Fig. 1. Pollen of Pyrus communis cv. ‘‘Abate Fete`l’’ collected from anther (A–C) and bee provision (D–F). (A) Untreated fresh pollen collected from anthers is tricolpate, prolate (40  60 mm). Pollen wall is folded into the aperture (arrow). (B) Cross section of pollen grains from anthers stained with PAS+Coomassie Blue. Amiloplasts (dark dots) are present in 78% of the pollen grains. (C) Cross section of a pollen grain from anthers stained with Calcofluor White. The brightly fluorescent intine appears compact and with a linear profile. (D) Untreated fresh pollen collected from bee provision has a more distended wall compared to pollen collected from anther (see Fig. 1A). Some grains show the protoplast protruding from the apertures (arrow). (E) Cross section of pollen grains from bee provision stained with PAS+Coomassie Blue. Amyloplasts (dark dots) are present in 42% of the pollen grains. Some grains show the protoplast protruding from the apertures (arrows). (F) Cross section of pollen grains from bee provision stained with Calcofluor White. Intine has an irregular profile especially in the apertural regions (arrows). Scale bars A–F ¼ 15 mm.

digestion (F ¼ 50:7; Po0:001) (Fig. 4). In fact, starch was present in 77.772.97% of the pollen grains sampled from anthers, but its presence was already significantly reduced in the provision (42.074.2%). The percentage of starch-containing grains did not significantly decrease upon entering the larval anterior midgut (37.673.5%), but, along with its passage through the alimentary canal, it was reduced to lower values in the median midgut (21.971.6%) posterior midgut (Fig. 4). In the proctodeum, only about 4% of the grains contained starch, and most of the compressed and

crushed pollen (73.070.6%).

grains

appeared

to

be

emptied

4. Discussion 4.1. Structural and cytochemical pollen grain modifications in the provision Intine and protoplast protrusion from the apertures in bee provisions, protoplast’s retraction in the midgut and

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Fig. 2. Sections of the midgut (A–E) and proctodeum (F–H) of Osmia cornuta larvae. (A) Pollen grains in the anterior part of the midgut stained with PAS+Coomassie Blue. The pollen protoplast is retracted from the pollen wall. Intine is still protruding from the apertures (arrows). Some pollen grains are void of protoplasm (asterisks). (B) Pollen grains in the anterior part of the midgut stained with Calcofluor White. Intine is loose and undulated in the apertural regions (arrow). (C) Pollen grains in the anterior part of the midgut stained with DAPI. Fluorescent nuclei are evident in the epithelium and muscular fibrils of the larva but not in the pollen grains. (D) Pollen grains in the posterior part of the midgut stained with PAS+Coomassie Blue. The pollen protoplast is retracted from the pollen wall and in some grains it extrudes through the apertures (arrow). Remnants of pollen protoplasm are present in the lumen (asterisk). (E) Pollen grains in the posterior part of the midgut stained with PAS+Coomassie Blue. Starch-containing pollen grains (asterisk) are intensely PAS positive and appear unmodified. In most of the pollen grains the intine protrudes through the apertures (arrows) and a condensed cytoplasm only stained by Commassie Blue. (F) Pollen grains in the posterior part of the midgut stained with Calcofluor White. The intine modifications extend to the inter-apertural regions and some grains are void of intine (asterisk). (G) Pollen grains in the proctodeum stained with PAS+Coomassie Blue. Pollen grains are compressed and deformed. Very few are apparently unaffected by the digestive process (asterisk). The starch-containing grains are the least modified and their protoplast do not protrude through the apertures. Remnants of protoplasm are still present inside pollen grains. (H) Pollen grains in the proctodeum stained Calcofluor White. Intine of pollen is very undulated and loose in all its parts. Some grains are void of intine (asterisk). Scale bars A–H ¼ 15 mm. E ¼ epithelium of the midgut; MF ¼ muscular fibril; P ¼ pollen grain; p ¼ pollen protoplast.

crushing of most pollen grains in the proctodeum were the main modifications occurring during the digestive process. Morphological, physiological and biochemical modifications were already evident in O. cornuta provisions. Since the percentages of unmodified and protruded pollen grains in the different parts of the midgut were comparable, protoplast protrusion must have occurred only in the provision. Protrusion of pollen protoplast through the apertures has been observed in provisions of other Osmia species (Sua´rez-

Cervera et al., 1994), but not in honey bee provisions (Klungness and Peng, 1983; Sala-Llinares et al., 1992). Wall distension and protrusion of the protoplast from the apertures may be related to an increase in water content with respect to the pollen obtained from anthers, probably because of the presence of nectar, which is added by Osmia spp. females to the provisions in appreciable quantity (Torchio, 1985; Torchio et al., 1987; Bosch, 1994; Seidelmann, 1995; Ladurner et al., 1999). The protrusion of the protoplast through the

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100

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Starch-containing pollen grains 100

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With protruding protoplast 100

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Post.midgut Med.midgut

Environments

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0 With retracted protoplast 100 80

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Fig. 4. Percentage of pear (Pyrus communis cv. ‘Abate Fetel’) starchcontaining pollen grains in the anthers, bee provisions and in the different parts of the midgut. Different letters indicate statistically significant differences (Po0:05) according to ANOVA followed by Bonferroni’s test. Data are presented as mean7standard error.

%

Cytomorphological features of the pollen grains

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Post.midgut

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Fig. 3. (A–D) Percentages of pear (Pyrus communis cv. ‘Abate Fetel’) pollen grains of the four cyto-morphological classes in anthers, bee provisions and in the different parts of the midgut. In each series different letters indicate statistically significant differences (Po0:05) according to ANOVA followed by Bonferroni’s test. Data are presented as mean7standard error.

apertures resembled that of germinating pollen although there is not the formation of a real pollen tube. Pollen of many plant species germinates in media containing 10% sucrose and micronutrients. Furthermore, if pollen is soaked in 10% sucrose solution and then placed in acid, it may form instant pollen tubes, and the so-called pseudo-germination process occurs (Linskens and Mulleneers, 1967). Certain animals extract the pollen contents using this process (Roulston and Cane, 2000). Adding nectar to the pollen, to prepare the provision the larva will feed on, may initiate germination or pseudogermination. The morphological and biochemical changes occurring in O. cornuta pollen provisions, might be necessary for ‘‘activating’’ the pollen grains for the digestive process. Pollen in the anther is normally very dehydrated, its water content being generally below

10% (Franchi et al., 2002). In this dehydrated state, the pollen wall is folded in the aperture regions and the exine, an indigestible layer, is the only pollen wall component exposed to the external environment. When nectar is added to the pollen stored in the provision, the pollen grain rehydrates, and the intine becomes exposed through the apertures. We hypothesise that without this crucial modification, occurring only during the storage of the pollen in the provision, the intine could not protrude and the exine would prevent any subsequent enzymatic action from taking place in the larval alimentary canal. Other active substances could play a role in the pollen digestion process in the solitary bee larvae, such as mother-derived secretions produced by the female and added to the provision (Ladurner et al., 1999), (HeroinDelauney, 1966). Even though nothing is known about their composition, glandular secretions might be similar to those of honey bees, which produce saccharase, among the others (White, 1963; Maurizio, 1968; Nepi et al., 1997), and could be involved in the pre-treatment of the pollen (Ladurner et al., 1999). The possible role of commensalistic microbial fauna of the larval cell in inducing biochemical changes in pollen-nectar provision remains to be investigated. 4.2. Structural and cytochemical pollen grain modifications in the midgut Pollen grains in the anterior midgut of O. cornuta larvae showed the protoplast retraction from the pollen wall (Fig. 2A), and no appreciable increase in the percentage of pollen grains showing this modification throughout the midgut was observed. This observation could be interpreted as a consequence of an enzymatic action on the protruded protoplast, which could cause it

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to collapse. Nevertheless, in this case, the release of the cytoplasm should have produced an irregular shape, with the cytoplasm flowing through the apertures. On the contrary, in our observations, the protoplast presented a round contour and a central position with respect to the pollen wall which is hardly attributable to an enzymatic digestion. Moreover, the space between the protoplast and the pollen wall did not appear to be coloured either with PAS (polysaccharides) or with Comassie Blue (proteins), thus appearing empty (see Fig. 2E). These findings seem to suggest that the pollen grains were exposed to a sudden change in osmotic pressure as soon as they entered the alimentary canal, resembling the passage from a hypotonic environment (provision) to a hypertonic environment (midgut). Perhaps, it could be hypothesised that a rapid water absorption immediately after the introduction of the bolus in the larval alimentary canal determines the concentration of the soluble sugars, thus creating a hypertonic environment with respect to the provision, to which also the hydrolysis of the starch and other sugars could contribute. Such a kind of osmotic gradient would be opposite to the one reported by Kroon et al. (1974) in the adult honeybee, where, during the transit from the honey sac to the midgut the pollen grain passes from a hyper- to a hypotonic environment. These authors, then, envisaged an increase in the volume, leading to pressure which caused the pollen grains to burst, or the cytoplasm to stream out through the apertures. In a study on the digestion of dandelion pollen in the adult honeybee, Peng et al. (1985) attributed to the change in the osmotic pressure a role in weakening the intine that was subsequently digested by the bee’s enzymes. In fact, these authors did not observe the formation of extensive germination tubes, though they described pollen grains to occur with slightly misshapen intine and also the gradual decrease in the protoplasmic volume, which they explained to be the result of the protoplasm’s exudation from the pores. Dobson and Peng (1997) reported a gradual release of the protoplasm by extrusion through the apertural regions of the pollen grain in Chelostoma florisomne Linnaeus (Hymenoptera Megachilidae) larvae. Evidence for a contribution of proteolytic enzymes located in the microstructure of the pollen wall to the total midgut protease activity in pollen-eating adult honeybees was provided by Grogan and Hunt (1979), while Crailsheim (1990) reported that only little proteolytic activity was found in the midgut tissue, and most in the endoperitrophic space where pollen boluses are transported. Under this hypothesis, it is possible that any kind of osmotic gradient could promote pollen digestion. A general action of weakening of the pollen wall could also explain the deformation of the pollen grains in the proctodeum, where pollen grains appeared strongly

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misshapen and compressed. In fact, only those grains still containing protoplasm maintained their typical shape. 4.3. Pollen digestion efficiency The pollen grain modifications occurring in the provision, which cause the protoplast to be exposed at the apertures and the weakening of the pollen wall in the anterior part of the midgut, could allow the larval enzymes to start the digestive process. The extreme deformation of the pollen grains in the proctodeum did not allow a quantitative evaluation of the pollen digestion efficiency according to Human and Nicolson (2003). Nevertheless, O. cornuta larvae efficiently digested all the components of pear pollen except the exine’s sporopollenin. Pollen digestion efficiency varies with the pollen source and the pollen consumer. In O. tricornis, O. cornuta, O. latreillei and O. rufa larvae, the degree to which pollen is digested depends on intine thickness (Sua´rez-Cervera et al., 1994). The intine consists mainly of polysaccharides, cellulose and pectic substances being the main components (Nepi and Franchi, 2000). Adult worker honeybees and larvae of C. florisomne are not able to digest cellulose (Peng et al., 1986; Dobson and Peng, 1997). Our results and the observations reported by Sua´rez-Cervera et al. (1994) show that O. cornuta larvae are able to alter the cellulosic fibrillar component of the intine, and we observed that cellulose was completely removed from some grains in the proctodeum (Fig. 2G). The decrease in the PAS staining indicated a degradation of other polysaccharides present in the cytoplasm. According to Simpson and Neff (1983), larvae of solitary bees which are oligolectic on plants having starchy pollen can remove the starch from the grains very efficiently. This is supported by our observations in O. cornuta larvae where the decrease in starch content already started in the provision and continued along the alimentary tract. Pollen starch content’s decrease is also reported for C. florisomne larvae fed on Ranunculus sp. pollen (Dobson and Peng, 1997). Our observations suggest that the main decrease in pollen starch content takes place in the provision and it is likely due to a physiological activation of starch hydrolysis by the pollen itself rather than to an enzymatic degradation by enzymes coming from outside sources. When pollen is mixed with nectar in the provision (Ladurner et al., 1999) the increased sugar content of this environment activates starch de-polymerisation to simple sugars in order to prevent water loss. On the contrary, starch polymerisation was demonstrated to occur in Lilium longiflorum Thunb. pollen after a few minutes in a germination medium (Rosen, 1971). The decrease in the number of starch-containing pollen grains in the proctodeum was probably not due to starch hydrolysis

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but to the protoplasm’s extrusion occurring when the pollen grains collapsed. Some starch-containing pollen grains were unmodified throughout the alimentary canal and their number remained constant in the midgut, suggesting that they were not affected by the digestive process, as was also found in guts of adult honey bees (Klungness and Peng, 1984). Unmodified starch-containing pollen grains were also found in the provision, where they did not show protoplast protrusion through the apertures. There could be two hypotheses to explain the behaviour of these pollen grains: (1) they degenerated in a late stage of pollen development and, being dead, they were not able to respond to the stimuli they experienced in the provision and in the midgut of O. cornuta larvae; (2) they were in equilibrium with the environment they encountered in the provision so that there was no protrusion of the protoplast through the apertures, which we hypothesise to be essential for pollen digestion in the larval alimentary canal. Differences in the physiological state of ripe pollen grains are likely to exist because of a certain grade of asynchrony of their development after meiosis (Heslop-Harrison 1968; Franchi and Pacini, 1980). The absence of DNA fluorescence after staining with DAPI in pollen grains in the anterior midgut may suggest a modification of the tertiary and quaternary structure of the macromolecule rather than its complete degradation. If a role has to be found for commensalistic microorganisms in the pre-treatment of the provision, more research is necessary on the activity of endosimbionts in enhancing digestion efficiency in the alimentary canal. To this point, several researchers enlightened the important role played by a rich microflora in the mid and hind gut of A. mellifera overwintering workers (Tysset et al., 1969) whose enzymatic characterisation is not substantially different from that of the microflora found in man, in other Vertebrates and Invertebrates. Cruz Landim (1996) found an extensive bacterial flora (all bacilliform) in the ileum of Melipona quadrifasciata anthidioides (Hymenoptera Apidae). The bacterial flora was arranged perpendicularly close to the ileal wall and around food material, suggesting a direct contribution to the degradation of pollen grains.

Acknowledgements We are grateful to Prof. H.H.W. Velthuis for his careful reading of the manuscript, to Dr M. Pinzauti and Dr A. Felicioli for their suggestions and support on O. cornuta rearing, to Donato Tesoriero and Giovanni Di Benedetto for their precious help. The research was conducted within the A.M.A. National Research Project (B.H.E. Bees, Honeybees, Environment) and financially

supported by the Italian Ministry for Agricultural Policy. Contribution no. 241.

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