Metabolic changes in the medical leech Hirudo medicinalis following feeding

Camp.Biochem.Physiol.Vol. 84A, No. 1, PP. 49-55, 1986 Printed in Great Britain

0300-9629/86$3.00+ 0.00 0 1986Pergamon Press Ltd

METABOLIC CHANGES IN THE MEDICAL LEECH HIRUDO A4EDICINAUS FOLLOWING FEEDING* ERNST ZEBE, FRANZ-JOSEF ROTERS and BARBARA KAIPING Zoologisches Institut. Lehrstuhl fiir Zoophysiologie, Hindenburgplatz 55, D-4400 Miinster, FRG. Telephone: (0251) 83-3851 (Received 22 August 1985) Abstract-l. In Hirudo, feeding results in a dramatic rise of the metabolic rate as indicated by 0, consumption, excretion of NH, and elimination of ions and water, each showing a characteristic time-course. 2. Proteolytic activity is absent in the anterior alimentary system and unusually low in the intestine. In the latter it increases upon feeding and, after reaching a maximum, slowly decreases again. 3. The diverticules contain potent protease inhibitors which were not detected in the intestine. 4. Respiration, NH, excretion and the rise of proteolytic activity are inhibited by the antibiotic kanamycin.

detected the presence of aminopeptidases in the intestinal lumen and Damas (1974) reported gelatinolytic activity to be present in the salivary glands. Biising et al. (1953) found that the alimentary system of Hirudo is inhabited by one particular species of bacteria which they described as Pseudomonas hirudinis. In culture, this species was shown to inhibit the growth of other microorganisms and to initiate hemolysis and degradation of hemoglobin and of other proteins. Biising and his colleagues postulated that these bacteria alone were responsible for proteolysis in the alimentary tract of Hirudo and they reported experimental evidence for this assumption; feeding leeches blood to which they had added the antibiotic chloromycetin resulted in reduction of the elimination of water as well as in inhibition of hemolysis and breakdown of proteins. Altogether, these different results seem inconclusive and inconsistent. Therefore the processes following the ingestion of blood by Hirudo were reinvestigated. In the present paper we report on the results of experiments in which:

INTRODUCTlON leech Hirudo medicinalis is extremely resistant to starvation and, having a very low metabolic rate, can survive many months without the uptake of food. When there is an opportunity, a leech is able to ingest huge quantities of mammalian blood, increasing its body-weight 5-fold or even more. The anatomical prerequisites for taking up such large volumes are an extremely developed anterior region of the alimentary tract consisting of 10 pairs of diverticules which occupy a very large proportion of the body and, in addition, a highly extensible, but tough body-wall. Almost immediately after beginning to suck, leeches start eliminating water and ions taken up with the blood via the nephridia. This excretory activity results in highly concentrated blood stored in the diverticules. Its appearance does not change during long-term storage and microbial degradation seems to be effectively prevented. The leech utilizes the stored blood very slowly. Although in the past, leeches have been widely used in medicine, surprisingly little is known of the processes involved in preserving and storing the ingested blood and of its final digestion. Piitter (1907) studied the excretion of water, ammonia and other compounds following the uptake of blood. Diwany (1925) observed that several proteins which he injected into the crop of leeches remained there undigested. Graetz and Autrum (193 1) did not succeed in demonstrating the presence of proteolytic enzymes in the alimentary tract of Hirudo. From these results Herter (1937) concluded that protein degradation in Hirudo very likely takes place either intracellularly or by autolysis. On the other hand, Lafargue and Fayemendy (1932) claimed that leeches excreted large quantities of organic acids, especially volatile fatty acids, into the ambient water and that this was particularly evident after feeding. Jennings and Van der Lande (1967) The medical

(1) The metabolic rate and the rate of NH, excretion were monitored during several weeks after feeding; (2) Changes in the contents of anterior part of the alimentary system and the excretion of electrolytes were investigated; (3) The activity of proteolytic enzymes in the anterior alimentary system and in the intestinum were assayed after various periods following the ingestion of blood or serum. We also attempted to assess the significance of microorganisms by studying the effect of the antibiotic kanamycin on these processes. MATERIALS AND METHODS Medical leeches were purchased from a commercial supplier. They were kept in chlorine-free tap water at room temperature (1525°C). The water was changed at least once

*Dedicated to Professor B. Rensch on the occasion of his 85th birthday. C.B.P. WA-Ll

49

50

ERNST ZEBE et

a week. Feeding was carried out by letting the leeches attach themselves to an animal hide which was used (inside out) to close the opening of a plastic bottle fixed upside down and, through perforations, filled with lukewarm blood or serum. The leeches (0.8-1.5 g in weight), starved for at least 4 months, would start sucking eagerly and ingested &8 ml of blood or serum until, after 20 or 30mir1, they detached themselves. The concentration of kanamycin (monosulfate, Sigma) was 1 mg/ml serum. This did not influence the volume of serum ingested. Injection of saline into the anterior alimentary system was executed by means of a syringe with a finely drawn tip of PVC tubing replacing the injection needle which was gently forced into the mouth and pharynx. For incubation, 6-8 individuals were transferred to bottles containing 200 ml of distilled water to which 1 ml/l artificial sea water had been added. Usually the water was changed daily. Aliquots were stored in a freezer until they were used for analyses. In some experiments the measurements were carried out instantaneously. No difference was found between the data obtained from fresh or stored samples. NH, and urea were estimated according to Gutmann and Bergmeyer (1974). Oxygen consumption was monitored in an open system in which the water leaving a respiratory chamber passed an oxygen electrode (microprocessor oxygen indicator model 2609, Orbisphere Laboratories, outfitted with electrode No. 2118). Velocity of perfusion was 600ml/hr and the temperature was 18°C. The conductivity of the incubation water was measured daily using a voltmeter WLW type LF 91 with a KLEl/T monitor attached. Subsequently the water was replaced. The contents of the anterior alimentary system were collected by making the leeches vomit. This is easily evoked by putting a few drops of saturated NaCl solution on their rear-end, which causes strong contractions of the body-wall, and by gently pressing the body. The blood extruded from the mouth was collected, frozen and stored. Samples of this blood were analyzed for iron, sodium, potassium, calcium and magnesium by means of an atomic absorption spectrometer (Unicam SP 1900). For estimating proteolytic and inhibitory activity in the contents of diverticules and intestine the leeches were sacrificed, opened dorsally by a longitudinal cut and pinned down on a dissecting platform. After removing the bodyfluid by blotting, the intestine was dissected out and transferred to a preweighted vessel containing a known volume of 50 mmole/l TRIS/HCl, pH 7.6. After weighing the intestine was cut into several pieces and the contents were removed by thoroughly mixing and washing. The mixture was centrifuged, the sediment discarded and the supematant used in the assays. After dissection of the intestine the posterior diverticules were opened by a short cut and the contents removed by means of a pipette. Except for using 103 mmole/l NaCl the extraction of the diverticules was as described for the intestine. The extracts were stored at - 70°C without loss of activity. For the tests the extracts of the intestine were used, either undiluted or diluted up to 20-fold, whereas the extracts of the diverticules were diluted lo- to 200-fold. The assays of proteolytic activity with synthetic substrates were performed photometrically at 25°C: benzoyl-isoleucylglutamyl-glycyl-arginine-nitroanilide (BIGGANA), according to Geiger and Fritz (1984); succinyl-alanyl-alanyl-prolylphenylalanine-nitroanilide (SAPPNA), according to Geiger (1984) and leucin-nitroanilide (LeuNA) according to Appel (1974). The conditions of the azocasein test were essentially as described by Langner (1982). Bovin hemoglobin was labelled with [‘%]cyanate (potassium salt) as follows. 50 milligrams Hb were dissolved in 475 ~1 Hi0 and filled up to I.0 ml by adding TRIS/HCl, 0.5 mole/l, pH 8.5. The solution was dialyzed against 0.25 mole/l TRIS/HCI, pH 8.5 using an ultra-thimble (UH

al.

100/25, Schleicher and Schiill) and the volume reduced to 200 ~1 in a vacuum. “C-cyanate (0.25 mCi) was dissolved in 30 ~1 of this solution and continuously shaken for 24 hr at room temuerature. Thereafter 50 vl TRISIHCI. 25 mmolell. pH 8.5, ias added and the mixiure was’ kept’ for anothk; 24 hr at 4°C. It was transferred to a Sephadex G-25 column, 0.5 x 2Ocm, equilibrated with TRIS/HCl lOmmole/l, pH 7.4 and eluted in 250 ~1 fractions with the above TRIS/HCl. The fractions containing [“C]Hb were collected and stored at -20°C. The 14C-Hb test of proteolytic activity was carried out as follows. The solution (100 ~1,2% in TRIS/HCl, 50 mmole/l, pH 7.6) was adjusted with 14C-Hb to 200,000 cpm/ml, and 50 ~1 of the above TRIS/HCl and 10-50 ~1 extract were added. The solution was then incubated for 60min with continuous shaking at 25°C. The proteins were precipitated by adding 300 ~1 TCA, 0.3 mole/l. After 10 min standing the mixture was centrifuged for 10min at 16,000g. A 200~1 aliquot of the supematant was used for measuring the radioactivity. Inhibitory activity was investigated using standard solutions of bovine trypsin and chymotrypsin according to Geiger and Fritz (1984) and Geiger (1984). RESULTS Respiratory rate

After feeding, the metabolism of Hirudo is dominated by the processes which eliminate the inorganic ions and water ingested to restore the osmotic balance, as is also evident from the reduction of weight. This is reflected by the respiratory rate which rose dramatically immediately after feeding to reach a peak within 24 hr (serum), or after 4-5 days (blood). Thereafter it declined until, after several days, it became virtually constant at a level 4- to S-fold above that of unfed controls (Figs 1 and 2). The increase of the respiratory rate was extreme in leeches fed serum, but the level established after the initial peak was similar in the groups fed serum and in those fed blood. In a parallel experiment the antibiotic kanamycin was added to the serum, prior to feeding the leeches, to inhibit the growth of bacteria in the alimentary tract. Figure 2 shows how the oxygen consumption was affected in this group. Although the respiratory rate was maximal immediately after feeding, it

Fig. 1. Weight (+), daily 0, consumption (0) and NH, excretion (a----0) in leeches at various intervals after the ingestion of blood. (Mean values of five groups each with six individuals. 0, consumption was monitored continuously using six individuals kept together in a respiration vessel.) Temp. 19°C.

Metabolic changes in Hirudo following feeding

20% 180

160 xY1.40 I

6o unfed

20 -+ 8

4

12

+---+

16 20

2AYs20

Fig. 2. Daily 0, consumption by leeches at various intervals after the ingestion of serum (0) or serum and kanamycin (0) and by unfed controls (+). (Mean values of six animals kept together in a respiration vessel.) Temp. 19°C. dropped rapidly thereafter and became steady at a level much lower than in the other groups; however, it was still about twice that of the unfed controls. In an attempt to estimate the energy cost of osmotic and

ion regulation an additional group of leeches was injected with 0.15 M NaCl solution into the crop because the animals would not ingest it voluntarily. The injection evoked an immediate rise of the oxygen consumption to a peak value about eight times that prior to the injection. But thereafter the respiratory rate decreased rapidly and after approximately 24 hr it became similar to the rate prior to the injection. Excretion of NH,

Another

indicator

of the metabolic

activity

in

Hirudo is the rate of NH, excretion. NH3 appearing

in the water in which leeches were incubated may originate from both NH3 and urea ingested with blood or serum and/or from the degradation of amino acids arising from the digestion of proteins. NH, excretion after feeding was monitored continuously for several weeks. Its course was found to be similar to that of oxygen uptake (Fig. 3). It rose 7- to 8-fold above the value of unfed controls within 24 hr, was at a maximum after 4 or 8 days, and then decreased again, at first rapidly, but later on, very slowly. The changes in the rate of NH, excretion were particularly evident in animals fed serum as compared to those fed blood.

2 $20

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serum + km.

_o_o-o-----o

-.-----n unfed

*-__+-+-+~+-*

I., L

8

12

16

20

21

28

32

,

36

Fig. 3. Daily excretion of NH, by leeches at various intervals after the ingestion of serum (0) or serum and kanamycin (0) and by unfed controls (+). (Mean values of six groups, each with six individuals.) Temp. 19°C.

51

Ingestion of serum plus kanamycin resulted in initial rates of NH3 excretion similar to that observed in the absence of the antibiotic (Fig. 3). However, after 3 or 4 days, a rapid and steep decrease occurred. Finally, the excretion rate became constant at a very low level, approximately twice that of unfed leeches. In leeches injected with saline, NH, excretion rose only slightly. In another experiment it was checked whether NH3 excretion was due to oxidative processes, i.e. how it is affected by changing from aerobic to anaerobic metabolism. Groups of leeches were incubated alternately under normoxic and under anoxic conditions. The result was striking: under anoxia the rate of excretion dropped to one-third or one-quarter of that value measured on the preceding day. It was restored to the previous level when oxygen was readmitted. The excretion of urea was also measured in addition to NH,. It was found to be limited to a very short period immediately after feeding. A peak was reached on the second day, but on the third day it was almost zero. Thereafter urea excretion was insignificant or lacking. In view of the report by Lafargue and Fayemendy (1932) it seemed important to analyze the incubation water for metabolites excreted by the leeches. However, only traces of amino acids and acetate were detected. Excretion of electrolytes In order to investigate how the metabolic rate and the rate of NH, excretion are correlated with the processes of ionic and osmotic regulation, the appearance of electrolytes in the ambient water was followed by monitoring its conductivity. In all cases conduc-

tivity was found to be at a maximum on the day following feeding or injecting saline. Although the absolute values differed due to the different volumes of blood, serum or saline ingested or injected, the time-course was always similar (Fig. 4). The quantity of electrolytes excreted became constant after 2-4 days depending on the volume of fluid taken up by the leech, indicating that a new ionic and osmotic balance was established. This was corroborated by analyses of the contents of the anterior alimentary system carried out at various intervals after the ingestion of blood. As shown in Fig. 5, the dry matter of the ingested blood rose 3-fold within 2 days, whereas the concentration of sodium dropped correspondingly. After this initial phase both parameters were found to change only slightly. In contrast, the concentration of potassium decreased slowly and steadily for several weeks. Calcium (not shown) was found to change in a manner similar to that of sodium, while magnesium (not shown) declined slowly, like potassium. All results reported thus far indicate that the ingestion of large volumes of hyperosmotic fluid induces a phase of maximal metabolic activity to establish a new osmotic balance. This is attained within one or more days, depending on the quantity of fluid taken up. After this phase of “concentrating” the ingested fluid the metabolic rate decreased somewhat, but it was maintained at a high, and approximately constant, level for periods of many weeks.

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6

7

8

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Fig. 4. Conductivity of the water in which leeches were kept for 24 hr at various intervals after the ingestion of blood (+ ) or serum (0) or after the injection of 0.154mole/l NaCl (0). The dimension of the ordinate (NaCl equ./g fresh wt x 24 hr) is the sum of all electrolytes excreted resulting in the conductivity equivalent to NaCl solutions of respective concentrations. (Mean values of six groups, each with six individuals.) Temp. 19°C. Proteolytic activity

We tried to investigate this directly by assaying the activity of proteolytic enzymes in the anterior and posterior parts of the alimentary system. All attempts to demonstrate the presence of proteolytic activity in the contents of the diverticules had negative results. Instead, the presence of potent inhibitors was detected, which completely inhibited the activities of standard solutions of trypsin or chymotrypsin.

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Frequently a major problem arises when the processes concerned with the digestion of food components are studied namely, the lack of a suitable reference. This lack is because the contents of the alimentary system are altered in qualitative as well as in quantitative terms as the degradation of the macromolecules proceeds. In the leech a constant ratio was found to exist between the weight of the intestine and that of the remaining body from which the contents of the alimentary system and the body fluids had been removed. The mean weight of the intestine (including its contents) was 1.5 + 0.5% of the restbody (N = 86). The volume of the intestine contents seemed to be rather invariable and independent of the quantity and quality of the ingested food. Therefore, the weight of the intestine was used as a reference to which data obtained were related. Proteolytic activity of the intestine was assayed using several substrates: r4C-hemoglobin and azocasein for estimating general proteolysis, and the synthetic compounds BIGGANA, SAPPNA and LeuNA, which are specific for tryptic, chymotryptic and amino peptidase activity, respectively. Degradation of r4C-hemoglobin and SAPPNA was always distinct, i.e. even in leeches starving for several months, whereas hydrolysis of the other substrates occurred at very low rates in these cases, sometimes close to the limit of detection. It was important to dissect the intestine particularly carefully to avoid its contamination by the contents of the diverticules, since even traces of these were sufficient to completely inhibit proteolysis in the extracts of the intestine. On the other hand, standard solutions of trypsin and chymotrypsin were always fully active in the presence of preparations of the intestine. This demonstrates that, contrary to the anterior alimentary tract, they did not contain inhibitors. In several experiments the activity of proteolytic enzymes was assayed at different intervals after feeding the leeches blood, serum or serum plus kanamycin. As Fig. 6 demonstrates, the rate of hydrolysis tested rose strikingly after ingestion. Peak activity was observed after 2-4 weeks. Thereafter proteolytic activity decreased slowly. The extent of these changes, as well as the time-course, were somewhat different and clearly dependent of the substrate used. This is evident when Figs 6a to e are compared. Generally a wide individual variation was found. Therefore, differences between the leeches fed blood and those fed serum are not considered significant. In contrast, proteolytic activity in leeches fed serum plus kanamycin remained at the low level characteristic of starving animals. Instead of an immediate rise after feeding, a slight increase was observed to occur after several weeks. DISCUSSION

Fig. 5. Dry matter (+) and concentration of sodium (O), potassium (0) and iron (V) in the contents of the crop (diverticules) at various intervals after the ingestion of blood. (Mean values of 3-5 animals. Iron data are multiplied b; 10 to adjust them to the dimensions of the ordinate.)

When a leech ingests 5-8 times its own weight of mammalian blood which is by 100 mosmoles/l hyperosmotic to its own body-fluid, an enormous osmotic stress must arise. To establish a new osmotic balance and to reduce its volume an individual of 1 g weight would have to eliminate 0.3-0.4 mmole of NaCl and, in addition, 2-3 ml of water. The processes involved in osmotic and volume regulation seem to be initiated

Metabolic changes in Hirudo following feeding while the leech is still sucking. This is indicated by a clear and colorless fluid frequently appearing on the sides of its body which, very likely, results from an enormously increased activity of its nephridia. As Zerbst-BorolIlca (1973) and Wenning et al. (1980) have demonstrated, the osmolarity of the body fluid of a leech rises substantially immediately after injecting hyperosmotic saline into its crop, and the rate of urine production measured by catheterization of nephridia in situ then increases I-fold within 1 hr. The urine of Hirudo was always found to be hypoosmotic to its body fluid. According to the results described above osmotic and volume regulation are accomplished in less than 24 hr after taking up a large meal hacmoglobin

64 azocasein

@I SAPPNA

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9

III

012

I

.4

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weeks

BIGGANA “I

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4 II\

,I 30 ?ii

Jfy , I-4-.lca.

0124

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53

1

4

15

/

weeks

a

30 “w

(4 Fig. 6. Proteolytic activity in the intestine of leeches at various intervals after the ingestion of blood (a), serum (0) or serum plus kanamycin (A) as assayed with [W]hemoglobin (a) azocasein, (b) SAPPNA, (c) BIGGANA, (d) and LeuNA (e). (Mean values of 46 extracts.)

of blood and within 48 hr after ingesting serum, respectively. This phase of eliminating salt and water and concentrating the contents of the diverticules coincides with an enormous increase of oxygen consumption reflecting a corresponding rise in the rate of energy utilization. We have attempted to calculate the cost of energy paid for osmotic and ion regulation by comparing the respiratory rate of unfed leeches with that of the same animals after injecting them with a certain volume of 0.15 M NaCl solution under the presumption that only the processes of excreting salt and water are responsible for the additional oxygen uptake. In one specific experiment eight leeches (fresh weight 8.62 g) together consumed 9.61 ml of oxygen during 30 hr. After injecting a total of 8.84 ml of a 0.154 M solution of NaCl this group took up 21.40 ml of O2 during the 30 hr following the injection, or 11.8 ml of O2 in excess of the basal respiration. Thus the ratio of NaCl excreted to additional O2 consumed is 2.61. If 1 mole of Or is equivalent to 6 moles of ATP produced, this would mean utilization of 2.3 moles of ATP per mole of NaCl. Essentially the same values were obtained when the experiment was repeated with additional groups of leeches. Osmotic and volume regulation is accompanied by the excretion of waste products ingested with the blood. This is especially evident when the appearance of urea in the ambient water is followed. Maximum quantities were measured on the second day after taking up serum. Urea excretion dropped to a very low value on the third day and remained insignificant thereafter. In contrast, excretion of NHr, which also starts rising immediately after a meal, continues to do so until it reaches a peak after several days. The quantity of NH, excreted in this period is much larger than that presumably ingested with blood or serum. Even if all urea present had been degraded this would not be enough to account for the total NH, excreted. It is concluded, therefore, that a large proportion of the NH, appearing in the ambient water must be derived from the breakdown of components of the ingested blood or serum. This is corroborated by the observation that the rate of NH, excretion remained at a high level for several weeks. Its time course resembles that of the respiratory rate. Therefore, very likely,

54

ERNSTZEBE

oxidative processes are involved in the production of NH3 which, in addition, was shown to drop substantially in the absence of oxygen. At present it is difficult to tell from which substrate(s) NH3 excreted soon after the “concentrating phase” might be derived. Does it result from the initiation of proteolysis in the intestine or from deamination of amino acids or other small molecules absorbed by the epithelia lining the anterior part of the alimentary system? Employing several standard methods we were unable to demonstrate the presence of proteases in the diverticules. Although it cannot be ruled out that certain specific enzymes, such as dipeptidases, might have remained undetected because of the limited number of substrates used, a large-scale degradation of proteins in the anterior alimentary system seems very unlikely. Highly effective inhibitors were shown to be present there. The occurrence of several different protease inhibitors in extracts of whole leeches, in addition to the well known thrombin-specific hirudine, was discovered by Fritz et al. (1969). Subsequently named bdellins and eglins, respectively, these inhibitors have since been studied thoroughly by Fritz and his colleagues mainly with respect to their biochemical and medical aspects (Fritz et al., 1971; Seemiiller et al., 1980, 1981). Their biological role, however, still remains obscure at present. Possibly, they prevent the degradation of blood proteins by bacteria and, therefore, are responsible for the conservation of the blood during several months of storage. In contrast, we succeeded in definitely demonstrating that the intestine contains proteolytic activity. This requires, however, an especially cautious dissection, since the posterior pair of diverticules is situated very close to the intestine and, therefore, can be damaged easily, which would result in contamination and, consequently, inhibition of the latter. This may explain why previous investigators failed to detect proteolytic activity in the alimentary system of Hirudo. In addition, sensitive assaying methods have to be employed because in starving leeches, the activity is very low. Ingestion of blood or serum resulted in a marked increase of protease activity and maximum values were found between 2 and 3 weeks afterwards. This rather slow rise indicates that it was probably due to de nova synthesis of enzymes which, in addition, was different in extent when measured with tryptic, chymotryptic and aminopeptidase substrates. Apparently, the respective enzymes were secreted in different proportions. Altogether the activities found in the intestine seem high enough to account for the slow degradation of the blood stored in the diverticules. The maximum level of proteolytic activity in the intestine, however, was not maintained. Instead a slow decrease was observed until, after several weeks, the values measured were similar to those characteristic of starving leeches, although the diverticules still contained stored blood and, in addition, the respiratory rate and the rate of NH, excretion remained several times greater than the basal (starvation) level. It is important to note that no inhibitors were found in the intestine as opposed to the anterior alimentary

et al.

system. Evidently those present in the stored blood must have become ineffective before or while passing into the intestine. In an attempt to inhibit the growth of microorganisms in the alimentary system the antibiotic kanamycin was added to the serum prior to the ingestion on some experiments. This proved to be very effective as no bacteria could be detected in those leeches whereas normally their presence is easily observed. In leeches fed serum plus kanamycin the respiratory rate and the rate of NH3 excretion during the Iirst 2 or 3 days rose, essentially similar to the animals fed plain serum. Obviously, the processes involved in restoring the osmotic balance and in volume regulation, which are restricted to this phase, were not affected by the antibiotic. Only afterwards an effect of kanamycin became evident, as oxygen consumption and NH, excretion dropped to a level distinctly lower than that in leeches fed serum (but still twice that of starved animals). In addition, the rise of proteolytic activity evoked by ingestion failed to appear in leeches that had obtained kanamycin. These effects may be caused partially, directly or indirectly, by the elimination of the bacteria which probably share, more or less, in the consumption of oxygen and the production of NH, measured in untreated leeches. However, it seems difficult to understand how the failure in the rise of proteolytic activity in the intestinum could be brought about by the inhibition of bacterial growth in the anterior alimentary system. An unspecified effect of kanamycin cannot be ruled out. In bacteria the antibiotic is known to inhibit protein synthesis, but perhaps it also affects the cells of Hirudo. Therefore, it seems rash to conclude that the effects of kanamycin are solely due to the elimination of bacteria. Further investigations will be necessary to elucidate the precise relations between the leech and the bacteria inhabiting its anterior alimentary system. Acknowledgements-The investigation was supported by a grant from the Deutsche Forschungsgemeinschaft (Ze 40/15-l and 2). The authors are indebted to Dr F. J. Austenfeld for carrying out the analyses with the atomic absorption spectrometer.

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Seemiiller U., Eulitz M., Fritz H. and Strobl A. (1980) Structure of the elastasecathepsin H inhibitor of the leech Hirudo medicinalis. Hoppe-Seyler’s Z. physiol. Chem. 361, 1841-1846. Seemiiller U., Fritz H. and Eulitz M. (1981) Eglin: elastasecathepsin G inhibitor from leeches. In Method in Enzymology, Vol. 80, pp. 804-816. Academic Press, New York. Stark G. R. (1965) Reactions of cyanate with functional groups of proteins. III. Reactions with amino and carboxylgroups. Biochemisfry 4, 103&1036. Wenning A., Zerbst-Borollka I. and Bazin B. (1980) Water and salt excretion in the leech (Hirudo medicinalis L.). J. camp. Physiol.

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