Apidae: Meliponinae) Larvae Tts Schumaker, Pt Cristofoletti, Wr Terra

Properties and compartmentalization of digestive carbohydrases and proteases in Scaptotrigona bipunctata (Apidae: Meliponinae) larvae Tts Schumaker, Pt Cristofoletti, Wr Terra

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Tts Schumaker, Pt Cristofoletti, Wr Terra. Properties and compartmentalization of digestive carbohydrases and proteases in Scaptotrigona bipunctata (Apidae: Meliponinae) larvae. Apidologie, Springer Verlag, 1993, 24 (1), pp.3-17. hal-00891051

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Properties and compartmentalization of digestive carbohydrases and proteases in Scaptotrigona bipunctata (Apidae: Meliponinae) larvae

TTS Schumaker PT Cristofoletti, WR Terra

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 20780, 01498 Sâo Paulo, Brazil

(Received 23 June 1992; accepted 29 September 1992)

Summary — Aminopeptidase (pH optimum, pHo, 7.5; enzyme relative molecular weights, Mr values: 1, 110 000; 2, 190 000; 3, 300 000), amylase (pHo 5.5, Mr values: 1, 21 000; 2, 68 000); cellobiase (pHo 5.5) and maltase (pHo 5.0, Mr values: 1, 75 000; 2, 110 000; 3, 200 000) are found in the anterior (60-80%) and posterior (20-35%) midgut contents, with minor amounts occurring in midgut cells (2-5%). Trypsin (pHo 7.0, Mr 38 000) occurs mainly in the posterior (62%) rather than in the anterior (37%) midgut contents. Maltase 1 is more active on sucrose than on maltose, the reverse being true for the other maltases. A cysteine-proteinase (pHo 5.6, Mr 79 000) was found in major amounts in the pollen grains ingested by the larvae. The results suggest that, except for a cysteine- proteinase derived from ingested pollen, all digestive enzymes originate in the midgut tissue and are most active in the luminal contents. Evidence is presented supporting the hypothesis that enzymes and nutrients diffusing through the peritrophic membrane are translocated forward by a countercurrent flux. The absence of a midgut differentiation of midgut luminal pH in S bipunctata larvae is though to be derived from putative Hymenopteran ancestors.

Scaptotrigona bipunctata / digestion / enzyme activity / Meliponinae

INTRODUCTION properties of some adult Apis mellifera digestive enzymes, such as maltase In spite of being a major insect order, the (Huber, 1975), trypsin, chymotrypsin and Hymenoptera have been the subject of 2 other endopeptidases (Giebel et al, few studies regarding digestive physiology 1971; Dahlman et al, 1978), have been (Terra, 1988, 1990). Among the Hyme- studied in some detail, whereas others noptera, Apidae is no exception. The such as lactase (Peng, 1981), have been

* Correspondence and reprints poorly characterized. Only the midgut MATERIALS AND METHODS compartmentalization of trypsin has been studied. This enzyme occurs in midgut cells and in luminal spaces inside (endo- Animals peritrophic) and outside (ectoperitrophic) the membrane and peritrophic (Moritz Scaptotrigona bipunctata (Lepeletier, 1836) (Hy- Crailsheim, 1987; Jimenez and Gilliam, menoptera: Apidae: Meliponinae) combs with 1989). 5th instar larvae were collected from free-flying The absorption sites for leucine and colonies. The 5th instar is the last larval instar (Cruz-Landim and Mello, lar- glucose have been found in the anterior stage 1981). Only vae with midgut containing ample food and two-thirds of the ventriculus honeybee showing no connection between the midgut and (Crailsheim, 1988a, b). These data, to- the hindgut (hindgut lacking food) were used in gether with a detailed ultrastructural and the determinations. cytochemical study of adult Apis mellifera midgut, led Jimenez and Gilliam (1990) to propose that in these bees enzymes pH of gut contents and nutrients diffusing through the peri- membrane are translocated for- trophic S bipunctata larvae were immobilized by placing ward by a counter-current flux (endo- them on crushed ice and were then dissected in ectoperitrophic circulation), as previously cold 231 mM NaCl. The rinsed midguts were described for other insects (see review in transferred to a glass slide and sectioned in 3 of same Terra, 1990). In spite of these reports, a parts approximately the length. To the detailed model for the digestion by adult contents of each section was added 20 μl of a 10-fold dilution of a universal pH indicator (pH bees of the material extruded from or 4-10) or 20 μl of 0.04% methyl red. The result- on is not present the walls of pollen grains ing colored solutions were compared with suita- available. Moreover, digestive physiology ble standards. in larval bees is still being investigated, and only the properties of trypsin and chymotrypsin have been determined to some Preparation of samples of pollen extent in larval bees (Dahlman et al, and of gut sections 1978). In this paper we describe the distribu- Samples (67 mg) of pollen collected by S bi- tion and properties of several hydrolases punctata were suspended in 1 ml of 5 mM cit- occurring in different midgut regions of rate-sodium phosphate buffer pH 5.6 containing Scaptotrigona bipunctata (Apidae, Meli- 3 mM EDTA (for abbreviations, see table I) and 1.5 mM DTT. The were poninae) larvae. The results suggest that suspensions ruptured with a sonicator semimicroprobe (Branson 250), for a derived except cysteine-proteinase with output set at 3 using 3 pulses of 30-s each from ingested pollen, all digestive en- at 10-s intervals. The sonicates were then ho- zymes originate in the midgut tissue and mogenized in a Potter-Elvehjem homogenizer, are most active in the luminal contents. In passed through a 100-μm pore size nylon mesh addition, evidence is presented suggest- and centrifuged at 100 000 g for 60 min at 4 °C. The resulting supernatants were used as pollen ing the existence of an endo- enzyme sources. ectoperitrophic circulation of digestive en- Larvae were dissected as described above. zymes and nutrients, and that the larval After the removal of the midgut, the tissue and bees have lost a differentiation of midgut the peritrophic membrane with contents were midgut luminal pH hypothetically present pulled apart and sectioned in an anterior and a in Hymenopteran ancestors. posterior region. After being thoroughly rinsed with 231 mM NaCl, midgut tissue was homoge- (Mr 64 500) and bovine liver catalase (Mr nized in double-distilled water using a Potter- 232 000) as reference standards. Recoveries Elvehjem homogenizer, and then passed (%) of the activities applied to the gradients through a 100-μm pore size nylon mesh. Peri- were: aminopeptidase, 30-35; amylase, 30-50; trophic membranes and contents were homoge- maltase, 8-15; cysteine-proteinase, 50-60; to- nized in the same manner as midgut prepara- tal proteinase, 40-60; trypsin, 40-60. tions without previously rinsing in saline solution. Larval bodies from which the guts had been removed were rinsed, homogenized and Hydrolase assays passed through a nylon mesh as described for and determination midgut tissue. The resulting filtrates from larval protein body homogenates were than centrifuged at 10 000 for 10 min at 4 °C and the g superna- Protein was determined according to Bradford tants used as an source. Membrane- enzyme (1976) using ovalbumin as a standard. Enzymat- bound and soluble were determined in enzymes ic assays were carried out as described in midgut tissue by homogenizing in water with the table I. aid of a Potter-Elvehjem homogenizer and, after centrifugation of the homogenates at 100 000 g for 60 min at 4 °C, the resulting supernatants (soluble proteins) and pellets (mem- RESULTS brane-bound proteins) were assayed for several enzymes. All enzymes assayed could be stored for at least 1 month at -20 °C without a noticea- Luminal pH and distribution ble change in their activities. of hydrolases in midgut

The pH of S bipunctata larval midgut con- Polyacrylamide gel electrophoresis tents was found to decrease slightly along the midgut as follows (mean ± SEM; N = 10): anterior midgut, 6.0 ± 0.1; middle mid- Electrophoresis was carried out in gels of differ- 5.7 ± 0.2; 5.6 ± 0.1. ent concentrations as described by Hedrick and gut, posterior midgut, Smith (1968), using the system of Davis (1964), However, the differences found were in glass tubes of 5-mm id and 100 mm length. small, and may not be significant. Other details have been described elsewhere Pollen is known to contain digestive en- and Ferreira, of the (Terra 1983). Recovery and aminopeptidase activities applied to the gels zymes (Grogan Hunt, 1979). Enzymat- ic were therefore on was in the range of 30-40%. assays performed pollen masses (taken from S bipunctata pollen pots) equivalent to the S bipunctata Density-gradient ultracentrifugation midgut fresh-weight (6.7 ± 0.2 mg/animal, mean ± SEM; N = 20). The activities found were < 5% of the activities displayed in of 1.5 Samples (0.2 ml) preparations containing table II. The role of pollen hydrolases in lar- mg bovine hemoglobin and 50 μg bovine liver val was thus catalase were layered on top of 10-ml linear digestion discounted, except glycerol gradients (10-30%, W/v) made up in for that a cysteine-proteinase (see below). 50 mM citrate-sodium phosphate pH 5.6, un- Larval S bipunctata bodies from which the less otherwise specified. Centrifugation and col- guts had been removed were homoge- lection of fractions were performed as de- nized instead of their salivary glands alone scribed and previously (Terra Ferreira, 1983). because of the difficulty in dissecting these Molecular relative weight values of en- (Mr) The amount of each in zymes assayed in the fractions were calculated glands. hydrolase by the method of Martin and Ames (1961), us- these homogenates was always < 5% of ing sedimentation rates of bovine hemoglobin the activity found in the midgut. Digestive enzymes are found in high S bipunctata digestive enzymes are re- amounts in S bipunctata midgut contents, markably stable in the presence of their whereas only minor amounts are recov- own proteases. ered from midgut cells (table II). Except for The existence of soluble and mem- trypsin, which predominates in posterior brane-bound activities for each cellular en- midgut contents, all the other enzymes as- zyme was investigated. Rinsed midgut tis- sayed occur mostly in the anterior midgut sues were homogenized in water and, contents. In cells, amylase and maltase after centrifuging the homogenates at are mainly found in the anterior region; the 100 000 g for 60 min at 4 °C, the resulting other enzymes are uniformly distributed supernatant and pellet was assayed for throughout the tissue (table II). Enzyme as- several enzymes. The following activities says and protein determinations carried (% total activity) were found in the super- out in the different sections amounted to natant (the remaining activities were recov- 75-85% of similar determinations carried ered in the pellets) (mean ± SEM; N = 4): out in whole midguts (data not shown). aminopeptidase, 38 ± 4; amylase, 25.8 ± This suggests that small amounts of mate- 0.5; cellobiase, 86 ± 8; maltase, 28 ± 5. rial were lost during dissections and that Trypsin was not investigated due to its there seem to be no activators or inhibitors very low activity in tissue. The results sug- in the midgut cells or contents which affect gest that, except for cellobiase, tissue en- S bipunctata digestive enzymes. This also zymes are mostly membrane-bound, this suggests that there are no enzymes that being perhaps associated with the microvil- are restricted to the ectoperitrophic space. lar membranes. Attempts to confirm this by The activity of all digestive enzymes stud- using known insect microvillar enzyme ied in S bipunctata midgut homogenates markers such as γ-glutamyl transferase was proportional to a time period of at (Espinoza-Fuentes and Terra, 1987) and least 12 h. This supports the assertion that alkaline phosphatase (Ferreira and Terra, 1980; Santos and Terra, 1984; Ferreira et Properties of midgut proteases al, 1988) were unsuccessful. About 45% of the glutamyl transferase and 95% of al- S bipunctata proteolytic activity with azoca- kaline phosphatase activities were found sein as substrate displays a peak ≈ pH 7.0, in the luminal contents of S bipunc- midgut which is decreased in the presence of SBTI, tata (data not shown). Furthermore, most and a shoulder ≈ pH 6, which is increased of these activities are in the soluble frac- in the presence of EDTA plus DTT (fig 2). tion. Thus, these enzymes cannot serve as midgut microvillar markers in the larval bee. S bipunctata membrane-bound enzymes were not investigated further due to their low activity.

Properties of midgut carbohydrases

The pH optima of S bipunctata larval midgut carbohydrases are: amylase, 5.0; cellobiase, 5.5 and maltase, 5.0 (data not shown). Amylase activities sediment as proteins of Mr 21 000 ± 800 (minor peak) and 68 000 ± 600 (major peak) (fig 1 A). Amylase assays with and without chloride addition were carried out on midgut homogenates before and after ultracentrifugation in glycerol gradients, with identical results (data not shown). Thus S bipunctata larval amylase is not activated by chloride. The sedimentation profile of maltase activity displays peaks corresponding to the following Mr values: 1) 75 000 ± 7 000; 2) 110 000 ± 20 000; 3) 200 000 ± 30 000 (fig 1 B). Sucrase displays a major peak, which sediments as peak 1 of maltase, and one shoulder which sediments with peak 2 of maltase (compare fig 1 B,C). The results suggest that maltase 1 is more active on sucrose than on maltose, the reverse being true for maltases 2 and 3. It is possible that maltase 3 is a dimer of maltase 2. Recovery of amylase and maltase activities after electrophoretic separation in polyacrylamide gels was too low to permit further study with these techniques. Cellobiase was not studied by electrophoresis and ultracentrifugation due to its low activity. molecule with Mr 79 000 ± 5 000; it does not appear if the gradient and assay media does not contain EDTA and DTT (data not shown); peak 2 is able to hydrolyze BANA (compare figs 3A, C), and is inhibited by pHMB (fig 3C). These results, together with previous data, strongly suggest that peak 1 is a trypsin-like enzyme, whereas peak 2 is a cysteine-proteinase. Trypsin is absent (data not shown), whereas cysteine-proteinase is present in the pollen ingested by the larvae (fig 3C). Thus, only trypsin should be synthesized by S bipunctata larvae. Further study of S bipunctata proteinases using electrophoretic techniques was prevented due to their poor recoveries after the runs. S bipunctata midgut aminopeptidase activity has a pH optimum of 7.5 (data not shown), sediments as a major enzyme (aminopeptidase 2) with Mr 190 000 ± 20 000 and a minor enzyme with Mr 300 000 ± 9 000 (aminopeptidase 3) (fig 4B). After electrophoresis, only one molecular form of aminopeptidase is detected (fig 4A) in each of several polyacrylamide gel concentrations (fig 4C). Aminopepti- dase Mr value calculated from electropho- This suggests the existence of a major trypsin-like proteinase and a minor cysteine-proteinase. The occurrence of a major trypsin-like enzyme was confirmed with the use of the trypsin substrate BAPA. As shown in table III, SBTI abolishes BAPA hydrolysis and the presence of EDTA plus DTT do not affect this hydrolysis. The existence of a cysteine proteinase was supported by the finding that azocasein hydrolysis at pH 5.6 is not abolished by SBTI and that even in the presence of this com- pound EDTA plus DTT are able to in- crease azocasein hydrolysis (table III). Figure 3A and B shows that the major azocasein hydrolase (peak 1) is active upon BAPA, is inhibited by SBTI and sediments as molecule with an Mr of 38 000 ± 2 000. Peak 2 in figure 3A sediments as a retic data is 110 000 (aminopeptidase 1) (fig 4D). This value is ≈ 50% the Mr value of aminopeptidase 2, resolved by ultracentrifugation, if one takes into account the SEM found in these determinations. Ami- nopeptidase 2 has ≈ two-thirds the Mr value of aminopeptidase 3. These results suggest that there is only one molecular species of aminopeptidase in S bipunctata midgut with Mr 110 000, and that it is able to form dimers and trimers.

DISCUSSION

Origin and properties of S bipunctata larval digestive enzymes

Based on their morphological and histochemical data, Cruz-Landim and Mello (1981) argued that Scaptotrigona postica salivary glands were involved in lubricating the food to be ingested and, at the end of the larval stage, in secreting a silken co- coon. Our results lend support to their pro- posal, since digestive enzymes in salivary glands accounted for < 5% of the activity found in midguts. Thus it is possible that the activity found is a result of contamina- tion during dissection. The only significant digestive enzyme found in pollen is a cysteine-proteinase (fig 3C), which accounts for ≈ 25% of the total proteolytic activity found in S bipunctata midgut, as determined at the midgut luminal pH (fig 3A). Our data thus showed that digestion is ac- complished in S bipunctata midguts under the action of midgut enzymes, with the help of a pollen cysteine-proteinase. The properties of amylase, trypsin and aminopeptidase from S bipunctata are similar to those from other insects (see references in Vonk and Western, 1984; Apple- baum, 1985; Ferreira and Terra, 1986; Baker, 1989; Lemos and Terra, 1992). Nevertheless, S bipunctata amylase is not activated by chloride, like some other in- fructosidase (EC 3.2.1.26) seems to occur sect amylases (Baker, 1989; and referenc- in S bipunctata. It should be noted that sues therein), and in contrast to most animal crose is a major constituent of nectar (Per- amylases (Vonk and Western, 1984). cival, 1961), used by the worker bee to There are in S bipunctata at least 2 mal- prepare honey (which still contains su- tases (α-glucosidase, EC 3.2.1.20) with pH crose), and pollen (Todd and Bretherick, optima and Mr values similar to those 1942). The absence of β-fructosidase con- found in other insects (see references in trasts with enzymological data obtained for Baker, 1991; Jordão and Terra, 1991). other insects, such as the Lepidoptera, One of the S bipunctata maltases hydrolyz- which, like bees, are major consumers of es sucrose better than maltose. No β- nectar at the adult stage. Lepidoptera hydrolyze sucrose from leaves (larvae) or ingested food backward, thereby distend- from nectar (adults) with the aid of a β- ing the posterior midgut. The possibility fructosidase (Santos and Terra, 1986; Ter- that digestive enzymes are inactivated at ra et al, 1987). This finding lends further the posterior midgut has no experimental support to the assertion (Terra, 1988, support. There appear to be no activators 1990) that studies regarding digestive or inhibitors in midgut tissue or contents morphology, physiology and enzymology which affect S bipunctata digestive en- should include phylogenetic in addition to zymes, and these enzymes are stable in dietary considerations. the presence of S bipunctata midgut proteases. It is possible, then, that S bipunctata shows an endo-ectoperitrophic circula- Spatial organization of digestion tion of digestive enzymes similar to that in S larvae bipunctata described for other insects (for reviews see Terra, 1988; 1990). This circulation results Meliponinae worker bees provision the from a countercurrent flux which depends brood cells with a mixture of pollen, honey on the secretion of fluid at posterior midgut and glandular secretion. After the queen and its absorption back at the anterior mid- has laid an egg inside the cell, the workers gut. The countercurrent flux displaces to close it (Sakagami et al, 1965). The pollen the anterior midgut the enzymes, and prod- and nectar used by S bipunctata workers ucts of digestion, which are able to diffuse come mostly from Eucalyptus flowers (Ra- from the endoperitrophic space to the ecto- malho, 1990). The larvae feed on the cell peritrophic space. Although the composi- food and at the end of the 5th larval instar, tion of pollen varies widely, major compo- their midguts become enormously distend- nents are protein, sugars (sucrose and ed (mainly at their distal ends) with undi- monosaccharides), carbohydrate associat- gested food residues, which are not evac- ed with the pollen wall and, among the mi- uated until just before pupation (Cruz- nor components, there is starch (Todd and Landim and Mello, 1981). This is charac- Bretherick, 1942). This it is probable that teristic of these bees, as well as of all the as the pollen food passes along the mid- other larvae of Hymenoptera Apocrita gut, sucrose, starch and the resulting oligo- (Richards and Davies, 1977), wherein the saccharides are hydrolyzed, thus permit- midgut is closed at its posterior end, and ting the corresponding carbohydrases to remains unconnected to the hindgut until diffuse through the peritrophic membrane. pupation. Once ingested by the larvae, the Protein, nevertheless, probably remains in pollen grains probably burst and open at substantial amounts associated with the the pores, owing to the presumed low os- pollen in the posterior midgut. The fact that motic pressure of the midgut, thereby re- the protein present in the posterior luminal sulting in the extrusion of protoplasm as contents is much higher than ,in the anteri- described in Apis mellifera adults (Kroon or agrees with this view (table II). As a con- et al, 1974; Klungness and Peng, 1984). sequence, it is reasonable to suppose that Digestive enzymes, except for trypsin, trypsin remains bound in substantial occur mainly in S bipunctata anterior mid- amounts to pollen protein in the posterior gut contents. This is at first sight an unex- midgut and, as such is prevented from dif- pected finding. Even if digestive enzymes fusing through the peritrophic membrane. are secreted in the anterior midgut, they Polypeptides, resulting from the action of are expected to be translocated to the trypsin on protein, are probably able to dif- posterior midgut, as peristalsis moves the fuse through the peritrophic membrane together with aminopeptidase. Diffusion ly in posterior midgut, the same is not true through the peritrophic membrane de- for aminopeptidase. Hence, the observed pends on the size of the translocating enzyme distribution is derived from the hy- molecules and the diameter of peritrophic pothetical ancestral condition. Some Hy- membrane pores. The larger S bipunctata menoptera acquire digestive enzymes, digestive enzymes (maltase 2 and mono- with vital roles by ingesting fungi (Martin, meric aminopeptidase) have diameters of 1987). Although S bipunctata larvae ac- 8 nm, as interpolated in a plot of log (Mr) quired a cysteine-proteinase by ingesting against Stoke’s radius for 11 proteins (de- pollen grains, the role of this enzyme tails in Terra and Ferreira, 1983). These seems to be secondary in relation to the proteins penetrate the S bipunctata endo- bee’s proteinase. Thus, the acquisition of peritrophic space, as judged by the finding this proteinase does not appear to repre- (results not shown) that ultracentrifugation sent a significant phylogenetic character. profiles of midgut contents are similar to those obtained from midgut homogenates (figs 1, 4A). Thus, S bipunctata larval peri- ACKNOWLEDGMENTS trophic membrane has pores of at least 8 nm diameter. Pore sizes of the peritrophic We are much indebted to VL Imperatriz- membranes from well-studied insects vary Fonseca and A Kleinert-Giovannini from the Bio- from 7.0-8.0 nm (Terra, 1990). sciences Institute, University of São Paulo, for introducing us to Scaptotrigona bipunctata, to C Ferreira for helpful discussion and to LY Nak- for technical assistance. This work by larval bees: abayashi Digestion was supported by the Brazilian Research Agen- considerations evolutionary cies FAPESP, CNPq and FINEP. TTSS is a post-doctoral trainee on leave from the Biomedi- cal Sciences Institute, University of São Paulo; Based on the literature and clad- following PTC is an undergraduate fellow of the CNPq, istic-like Terra techniques (Cracraft, 1974), and WRT is a staff member of the Biochemistry (1990) proposed that in relation to their di- Department and research fellow of the CNPq. gestive physiology, Hymenopteran ancestors displayed the following characteristics: 1) polymer, oligomer and dimer hydrolases Résumé — Propriétés et compartimen- that were free and small (< 8 nm in diame- tation de l’activité des carbohydrases et ter) and thus able to pass through the peri- des protéases dans l’intestin moyen trophic membrane; 2) endo-ectoperitrophic des larves de Scaptotrigona bipunctata circulation of digestive enzymes caused by (Apidae, Meliponinae). Le pH du contenu the secretion of fluid in the posterior mid- de l’intestin moyen des larves de S bipunc- gut and its absorption in the anterior mid- tata a été déterminé à l’aide d’indicateurs gut; 3) differentiation of an acidic anterior de pH. Les résultats obtenus sont les sui- midgut (with high carbohydrase activity) vants : partie antérieure de l’intestin and an alkaline posterior midgut (with high moyen : 6,0; partie médiane : 5,7; partie protease activity). Derived characters postérieure : 5,6. Des dosages enzymati- found among larval Apocrita include a mid- ques ont été faits sur des homogénats de gut closed at its rear end. We may specu- tissus et de contenus de l’intestin moyen, late that bees (Apidae) display as a de- sur des homogénats de corps de larves rived character the loss of a clear midgut dont on avait extrait le tube digestif et sur differentiation of luminal. pH and enzyme des sonicats de grains de pollen récoltés distribution. Although trypsin is found main- par S bipunctata. Les enzymes testés et les substrats utilisés sont les suivants : protéinase du pollen (pHO 5,6; Mr : aminopeptidase sur L-leucine-p- 79 000). L’amylase n’est pas activée par le nitroanilide; amylase sur amidon; cellobia- chlorure et la maltase 1 est plus active sur se sur cellobiose; maltase sur maltose; le saccharose que sur le maltose, l’inverse protéinase totale sur azocaséine; cystéi- étant valable pour les autres maltoses. ne-protéinase sur N-benzoyl-L-arginine-β- À la lumière des travaux faits sur les au- naphtylamide (testée également en mesu- tres insectes, les résultats soutiennent rant l’augmentation de la protéinase totale l’hypothèse suivante : la majeure partie de addition d’acide après éthylènediamineté- la digestion a lieu à l’intérieur de la mem- et de traacétique dithiothreitol); trypsine brane péritrophique, les enzymes et les ali- sur N-benzoyl-DL-arginine-p-nitroanilide. ments diffusent à travers elle et sont trans- Les activités enzymatiques trouvées portés vers l’avant par un flux à contre- dans les corps dépourvus de tube digestif courant. Si l’on retrouve la trypsine en plus (y compris les glandes salivaires) et dans grandes quantités dans le contenu de la les grains de pollen (sauf pour une cystéi- partie postérieure de l’intestin moyen, c’est ne-protéinase) sont inférieures à 5% des probablement parce qu’elle reste liée à la mêmes activités mesurées dans les intes- protéine du pollen en excès. On estime tins moyens. Le rôle des hydrolases des que l’absence d’une différentiation du pH glandes salivaires et du pollen dans la di- du lumen dans l’intestin moyen chez les gestion larvaire (sauf pour la cystéine- larves de S bipunctata serait dérivée des protéinase du pollen) n’a donc pas été pris ancêtres supposés des hyménoptères. en compte. Les enzymes digestives sont présents en grandes quantités dans le Scaptotrigona bipunctata / digestion / contenu de l’intestin moyen de S bipuncta- activité enzymatique / Meliponinae ta, alors qu’on ne les trouve qu’en quanti- tés mineures dans les cellules de l’intestin moyen (tableau II). Mise à part la trypsine, Zusammenfassung — Eigenschaften prédominante dans le contenu de la partie und Aktivitätsverteilung von Carbohy- postérieure de l’intestin moyen, toutes les drasen und Proteasen im Mitteldarm der autres enzymes testées sont principale- Larven von Scaptotrigona bipunctata ment présentes dans le contenu de la par- (Apidae, Meliponinae). Der pH Wert des tie antérieure. L’amylase et la maltase se larvalen Mitteldarms von S bipunctata trouvent principalement dans les cellules wurde an verschiedenen Stellen mit fol- de la région antérieure, les autres enzy- gendem Ergebnis bestimmt: Im vorderen mes étant distribuées uniformément le Drittel wurde ein pH von 6,0 gemessen, im long du tissu (tableau II). mittleren Teil 5,7 und im hinteren 5,6. Les valeurs optimales de pH pour Enzymtests wurden mit Homogenaten l’activité enzymatique (pHO) et les poids aus Mitteldarmgewebe, Mitteldarminhalt moléculaires relatifs des enzymes (Mr), und von Larven ohne Darm durchgeführt. déterminés par ultracentrifugation avec Tests wurden auch an mit Ultraschall be- gradient de densité, sont les suivants : handelten Pollenkörnern vorgenommen, aminopeptidase (pHO : 7,5; Mr : 1, die von diesen Bienen gesammelt worden 110 000; 2, 190 000; 3, 300 000), amylase waren. Folgende Enzyme wurden mit fol- (pHO 5,5; Mr : 1, 21 000; 2, 68 000), cello- genden Substraten getestet: Aminopepti- biase (pHO 5,5), maltase (pHO 5,0; Mr : 1, dase mit L-Leucin-p-Nitroanilid; Amylase 75 000; 2, 110 000; 3, 200 000), trypsine mit Stärke; Zellobiase mit Zellobiose; Mal- (pHO 7,5; Mr : 38 000), et cystéine- tase mit Maltose (Malzzucker); Gesamt- Proteinasen mit Azocasein; Cystein- se die Hypothese, daß die Verdauung Proteinase mit N-Benzoyl-L-Arginin-β- hauptsächlich innerhalb der peritrophen Naphthylamide (außerdem wurde die Zu- Membran stattfindet, daß Enzyme und nahme an Gesamt-Proteinasen nach Nährstoffe durch die peritrophe Membran Zugabe von Ethylen-Diamin-tetra- diffundieren und daß die Enzyme in einem Essigsäure und Dithiothreitol getestet); Kreislauf vom hinteren Teil des Mittel- Trypsin mit N-Benzoyl-DL-Arginin-p- darms wieder nach vorne transportiert Nitroanilid. werden (endo-ectoperitrophische Zirkula- Die Enzymaktivität im Larvenhomoge- tion). nat ohne Darm (aber mit Speicheldrüse) Trypsin wurde in größeren Mengen im und in Pollenkörnern (mit Ausnahme von hinteren Teil des Mitteldarminhalts nachge- Cystein-Proteinase) betrug weniger als 5% wiesen. Wahrscheinlich bleibt es an über- im Vergleich zur Aktivität des Mitteldarms. schüssigem Polleneiweiß gebunden. Das Demnach spielen die Hydrolasen der Spei- Fehlen einer Differenzierung des pH cheldrüse und des Pollen (mit Ausnahme Wertes innerhalb des Darmlumens bei der der Cystein-Proteinase der Pollen) keine Larve von S bipunctata wird als Abstam- Rolle für die larvale Verdauung. mung von angenommenen ursprünglichen Größere Mengen von Verdauungsenzy- Hymenopteren gedeutet. men wurden im Mitteldarminhalt nachgewiesen, während nur kleinere Mengen im Scaptotrigona bipunctata / Verdauung / Mitteldarmgewebe gefunden wurden (Ta- Enzymaktivität / Meliponinae belle II). Außer Trypsin, das in erhöhten Mengen im hinteren Drittel des Mitteldarms vorkommt, verteilen sich die anderen gete- REFERENCES steten Enzyme über den gesamten Mittel- darminhalt. Im Mitteldarmgewebe fanden Applebaum SW (1985) Biochemistry of diges- sich Amylase und Maltase vor allem in der tion. 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