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Bioassay and proteolytic activity of digestive enzymes from octopus saliva
Comp. Biochem. Physiol. Vol. 88B, No. 4, pp. 1117-1123, 1987
0305-0491/87 $3.00+ 0.00 © 1987 Pergamon Journals Ltd
Printed in Great Britain
BIOASSAY AND PROTEOLYTIC ACTIVITY OF DIGESTIVE ENZYMES FROM OCTOPUS SALIVA M. S. GRISLEY and P. R. BOYLE Department of Zoology, University of Aberdeen, Scotland (Received 26 March 1987)
Abstract--1. A bioassay for octopus saliva, based on detachment of crab dactylopodite flexor muscle under standard conditions, has been developed. 2. There is a direct relationship between increasing caseinolytic activity of saliva from Eledone cirrhosa and decreasing muscle detachment time. 3. Fractionation of saliva, using preparative isoelectric focusing, shows that muscle releasing activity is restricted to fractions containing proteins with high isoelectric points and maximum easeinase activity. 4. It is concluded that proteolytic enzyme(s) in octopus saliva selectively release crab muscle from attachment to the carapace. INTRODUCTION The contribution of the posterior salivary glands to the feeding mechanism of cephalopods has been the subject of research and speculation for over a century. These large paired glands in octopuses (smaller and single in squid and cuttlefish) lie within the body cavity posterior to the head, but deliver their secretion forward through a single duct which terminates within the buccal cavity in a muscular salivary papilla. The glands have several types of secretory cells (Matus, 1971), are highly innervated and have a good blood supply (Ducros, 1972a and b). Descriptions of the buccal apparatus and the relationship of the various components may be found in Halstead (1965), Young (1965), Wells (1978) and Boyle et al. (1979). Interest in the properties of the saliva produced by these glands has been prompted by particular aspects of the feeding behaviour in octopuses. Large crustacea, such as crabs, are grasped by the octopus and become paralysed within a few minutes. Thereafter, the prey, hidden by the enveloping arms and web, is disarticulated and the flesh removed leaving a clean exoskeleton which is discarded by the octopus. The carapace of crabs taken by Eledone is frequently found to be penetrated by a narrow hole (Boyle and Knobloch, 1981; Nixon and Boyle, 1982). This feature is also characteristic for molluscan prey taken by Octopus (Pilson and Taylor, 1961; Arnold and Arnold, 1969; Wodinsky, 1969; Nixon, 1979a) and the organ responsible is thought to be the tooth-tipped salivary papilla (Nixon, 1979b, 1980; Nixon and Boyle, 1982). Information on the properties of saliva, based mostly on studies of aqueous extracts of the posterior salivary glands themselves, may be categorized broadly as: (a) Venomous----especially to crustacea (Ghiretti, 1959, 1960; Dulhunty and Gage, 1971; McDonald and Cottrell, 1972; Songdahl and Shapiro, 1974; Crone et al., 1976; Cariello and Zanetti, 1977), an aspect which received additional attention after a human fatality was reported (Flecker and Cotton, 1955). (b) Pharmacological--a wide range of
chemicals is present such as acetylcholine, noradrenaline and 5-hydroxytryptamine (Erspamer and Ghiretti, 1951; Bacq et aL, 1951; Erspamer and Anastasi, 1962; Juorio and Killick, 1973), all with known actions on other tissues. Summaries of early work are given in Halstead (1965) and Russell (1965). (c) Digestive---a very early suggestion for a digestive function (Bert, 1867 in Halstead, 1965) was largely ignored although proteolytic activity was also suggested by Bacq and Ghiretti (1953). The presence of proteases and their partial characterisation using artificial substrates has been demonstrated by Morishita (1974a-c, 1978) and their involvement in external digestion suggested by Nixon (1984) who showed that surgical interference with elements of the buccal mass, including the salivary papilla, affected efficiency of cleaning of the carapace. In this paper we report on a new bioassay for octopus saliva, based on detachment of crustacean muscles from the exoskeleton. Pure saliva, collected by the envenomation technique ("milking") reported by Ballering et al. (1972) and fractions prepared by isoelectric focusing have been assayed for their ability to disrupt the musculoskeletal attachments and for proteolytic activity on a high mol. wt, non-specific chromogenic substrate. MATERIALS AND METHODS
Collection of saliva Eledone cirrhosa (Lamarck) from the coastal waters of
Aberdeen were maintained in the recycling sea-water aquarium at Aberdeen University (Boyle, 1981). Saliva was collected using the technique described by Ballering et al. (1972). The octopus is placed inside a thin plastic bag and allowed to attach itself to the inner surface. Within seconds the animal will begin biting; the beaks open and a cyclical rhythm of rasping with the radula and salivary papilla is established. This results in penetration of the bag, whereupon the papilla is fully extended and, after a few seconds delay, a fine jet of saliva is emitted (Fig. I). In addition there is often a copious production of fluid welling around the papilla which was also collected. The saliva from several animals was pooled, aliquoted and stored at -20°C. This method of envenomation or "milking" yields 25-100 #1 of saliva from each animal every one or two days.
1117
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M. S. GRISLEY and P. R. BOYLE Ampholines (4ml pH 9-11 and I ml pH 7.9) and 1 ml Triton-X to reduce precipitation. The total volume was adjusted to 100 ml and the gel was cast and set according to the evaporation data supplied with the Ultrodex powder. Following the procedure recommended by LKB the plate was subjected to a constant 30W electric current for 16-18 hr. On completion of focusing the gel was divided into 30 equal fractions using a purpose-made stainless steel grid and the pH of alternate fractions was measured using a Philips CE3 surface pH electrode. The fractions were removed and the proteins eluted from each with 3 ml 0.1 M citric acid/HCl buffer, pH 6. The eluants were aliquoted and stored at -20°C.
Fig. 1. Collection of whole from Eledone cirrhosa by envenomation--"milking". The salivary papilla projects through a hole in the polythene bag holding the animal and saliva is ejected in a fine stream.
Fractionation of saliva Saliva was crudely fractionated by flat-bed preparative isoelectric focusing (PIEF) using LKB Multiphor equipment and accessories. On the basis of pilot analytical studies, pH gradients with a strong cationic bias were constructed to give optimum separation conditions. Five miUilitres of saliva was filtered (3/~m Millipore syringe filter) and added to the Ultrodex slurry together with
Crab leg bioassay The propopodite/dactylopodite segments of the walking legs of shore crabs (Carcinus maenas; mean carapace width 75 mm and wet wt 75 + 20 g), were used as the basis for the bioassay. The crabs were killed by mid-line bisection and pairs of walking legs 2-5 were removed at the basiopodite segment. The propopodite-dactylopodite joint was prepared by cutting through the extensor apodeme and the arthrodial membrane connecting the two segments. The carapace of the propopodite segment was then cut through longitudinally on both sides and the dorsal half gently removed (Fig. 2a). The dactylopodite flexor muscle, originating on the flexor apodeme and inserting on the carapace oI the ventral half of the propopodite segment, thus remained as the only connection between the two segments. As soon as the muscle preparation was completed a strip of filter paper, soaked in sea-water, was wrapped around the propopodite to act as a humidity jacket and this segment was securely clamped in a frame, leaving the dactylopodite hanging free (Fig. 2b). In the present experiments a standard load of 10 g was attached to the dactylopodite using a spring clip (total 11.6 g). A drop of glycerol was applied to the exposed muscle between the two segments to reduce desiccation. Test solutions were applied as 50 #I aliquots to the open proximal end of the propopodite segment using a Gilson micropipette. The pipette tip was used to probe gently into the dactylpodite retractor muscle to a depth of 1-2 mm to improve access of the applied sample which otherwise drained away into the humidity jacket. Time of completion of sample application was recorded and the preparation left undisturbed at room temperature (approx. 21 °C). When the dactylopodite segment detached from the propopodite the
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Octopus salivary enzymes time to dactylopodite release (DRT) was recorded to the nearest 5 sec. In preparations where the DRT was greater than 10 min, the humidity jacket was regularly remoistened with seawater. An arbitrary maximum incubation time of 60 min was allowed. This provided ample time for the more active samples to cause detachment (it approximates to the normal feeding period of Eledone). At 60 min the experiment was terminated by the addition of a further load of 50 g. When this caused dactylopodite release within 10 sec the result was recorded as "60 min + 50 g". If release did not occur within 10sec of this procedure the result was categorised as " > 6 0 min + 50 g". Whole saliva was diluted 1:1 with filtered seawater to reduce its viscosity. PIEF fractions were assayed without removing ampholytes or adjustment of concentration or pH. Control applications included filtered seawater, citric acid buffer, phosphate buffer and a solution of Ampholines in distilled water. Two groups of six legs were treated with highly purified commercial preparations of trypsin and chymotrypsin (10 mg/ml in 0.1 M phosphate buffer pH 7.0, Sigma Chemical Co.) to assess the effect of other proteolytic enzymes in the bioassay. Additional determinations of the load bearing properties of these muscle attachments were made by progressive loading of untreated preparations. Protein determination
Protein concentration was measured using Millar's (1959) modification of Lowry et al.'s technique (1951) in preference to Bradford's (1976) method which is affected by the presence of ampholytes. Bovine serum albumin (BSA) was used to construct a standard curve. Saliva and PIEF fractions required a 1 x 102-1 x 103 dilution. Assay solutions were duplicated and read at 650 nm using a Perkin-Elmer Lambda 5 dual-beam spectrophotometer.
Table 1. Total protein concentration (Lowry), proteolytic activity (Azocasein) and muscle loosening activity (daetylopodite releasing time, DRT, in the crab leg bioassay) for six different pools of Eledone saliva Bioassay Protease mean DRT trypsinequiv. Protein Saliva (N) (see) mg/ml mg/ml pool 1 235 2.40 84 (9) 350 1.88 72 (4) 2 375 1.28 38 (12) 3 410 1.16 12 (3) 4 500 1.12 16 (19) 5 670 0.88 28 (11) 6
correlation between protein concentration and D R T values or protease activity. The data for all assays on the six different saliva pools is shown graphically in Fig. 3. Assay procedures such as muscle preparation and saliva application, as well as biological factors such as crab size and stage in the moult-cycle probably account for much of the variability of individual results which increases with decreasing protease activity. Despite the variability in the data, there is a significant correlation between proteolytic activity of the pooled saliva and its muscle releasing activity ( D R T ) (Spearman's rank correlation coefficient r s = - 0 . 6 3 1 ; 58 pairs; P <0.01). Regression analysis of the 1Ogn transformed mean D R T against proteolytic activity of saliva also shows a significant correlation
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Protease determination
Of the three commercial chromogenic substrates investigated, Azocasein, Azocoll and Hide Powder Azure (Sigma Chemical Co.), Azocasein was found to be the most sensitive to salivary proteases from Eledone was subsequently used as the standard substrate. Reaction mixtures comprised 2.4mi 0.1 M phosphate buffer, pH 7.0 containing 25 mg Azocasein. At strictly timed intervals, 100gl of sample was added. After thorough mixing, reaction mixtures were incubated in a static water bath at 37°C; 10 min for whole saliva and 90 min for PIEF fractions. Hydrolysis was terminated by the addition of 2.5 ml 10% trichloroacetic acid, causing the precipitation of unhydrolysed substrate which was removed by centrifugation. The absorbance of the supernatants was read at 370 nm in the spectrophotometer against a blank in which the enzyme solution was substituted with buffer. Other controls included filtered seawater, citric acid buffer and a solution of Ampholine. All assay mixtures were duplicated. Proteolytic activity of whole saliva was standardised and expressed as trypsin-equivalents by including a series of trypsin standards (Sigma 2 x crystallised, ethanol precipitated: 0.01-1 mg/ml); measurements of PIEF fractions remain expressed as absorbance values.
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RESULTS Six different pools o f Eledone saliva (50% in filtered seawater) were tested for muscle loosening activity in the crab leg bioassay. Table 1 shows that when the dactylopodite detachment times ( D R T ) for the saliva pools are ranked, values for their proteolytic activity on the Azocasein substrate fall into rank order such that increasing D R T ' s correlate with decreasing protease activity. There is no apparent
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Fig. 3. Results for six different pools of Eledone saliva tested for muscle releasing activity using the crab leg bioassay. Time to dactylopodite release (DRT in sec-ordinate) is plotted against protease activity (nag trypsin equivalents/ml--abscissa) measured using Azocasein. ( 0 , individual assays; &, mean DRT for each saliva pool)
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Fig. 4(a). Protein concentrations (Lowry--mg/ml, • . . . . . • ) and proteolytic activity (Azocasein-absorbance at 370 rim, • A) for 30 fractions of Eledone saliva separated by preparative is•electric focusing (PIEF) using ampholytes in the pH range 7-11 ( O - - - O ) . Fig. 4(b). Muscle releasing activity of the saliva fractions in 3(a), expressed as dactylopodite releasing time (DRT in rain) determined by the crab leg bioassay. The number of occasions on which the terminal additional load of 50 g applied at 60 min caused release is shown in row A (60 + 50 g). The number of occasions on which this additional 50 g failed to cause dactylopodite release is shown in row B ( > 6 0 + 50g). (R ~ = 84.8; P < 0.01) and for the equation: In y = a + b x yields the values: x In y b a
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proteolytic activity (trypsin equiv./ml) log transformed mean D R T --0.000570 (slope) 6.83 (y intercept)
The protein concentration and protease activity of all 30 fractions from a single preparative is•electric focusing (PIEF) fractionation of saliva, are shown in
Fig. 4a; the plotted values are the means of duplicate assays and the individual values varied by less than 1 mg/ml and 0.05 absorbance units respectively. Although only a preparative step in protein purification, this technique reveals at least two distinct areas of proteolytic activity (fractions 8-10 and 20-27) and two minor ones (fractions 15 and 17). The data for muscle loosening activity of the same 30 fractions falls into three categories (see methodology); preparations in which the dactylopodite was released under standard load within 60 rain are individually plotted as actual times in Fig. 4b; the
Octopus salivary enzymes
1121
The most recent and comprehensive account of the numbers of preparations which released at 60 min with an additional 50 g load (60 min + 50 g) are listed physico-chemical characteristics of salivary enzymes in row A of Fig. 4b; the numbers of those which failed from an octopod is to be found in Morishita's review to release at all after this treatment ( > 60 rain + 50 g) (1978) of the properties of enzyme extracts from the are listed in row B of Fig. 4b. The figure contains data posterior salivary glands (PSG's) and "digestive collated from experiments performed on five different juice" of O. vulgaris. While Morishita gives values for days and the dashed line delimits the graphical mol. wt, pI, pH optima and artificial substrate values. All crab legs treated with the range of control specificities for proteases, no functional inferences solutions described in the methodology fell into the other than those of conventional digestion are made. > 6 0 m i n + 50g category and showed no sign of It is interesting that of the eight (possibly nine) caseinolytic enzymes he identifies in PSG extracts, muscle loosening. The major peak of proteolytic activity (fractions only three can also be traced in the gut. While factors 20-27 in Fig. 4a), with a pI of approx. 9, corresponds such as enzyme lability and dilution may account for exactly with distribution of muscle loosening activity this, it may also show the inherent difficulties in between PIEF fractions (Fig. 4b). In addition, the interpreting enzymic activity of tissue extracts; in this reduction in proteolytic activity of fractions on either instance, not all of these proteases may be present in side of fractions 23 and 24 is associated with an normal saliva. Out results suggest that, assuming no increase in DRT, suggesting that the bioassay result great interspecies differences, the nature of the enzymic activity and the numbers of different proteases is directly related to protease activity. From the 60 rain + 50 g data in Fig. 4b (row A) in freely expressed Eledone saliva fall within the range it is inferred that fractions 15 and 17 also possess of those recorded from salivary gland extracts and muscle loosening properties, as indicated by the "digestive juices" by Morishita for Octopus vulgaris. Certain similarities with Morishita's Octopus prooccasional detachment achieved with additional load, although they were apparently too weak to cause teases emerge from the present results. All the prodactylopodite detachment within 60 min at standard teases described by Morishita (1978) are caseinolytic, load. In contrast, the proteolytic activity detected in and while this may not be true for those in Eledone fractions 8-10 caused no muscle loosening on any of saliva, the enzymes apparently responsible for muscle the 14 occasions on which they were tested, despite loosening, as well as at least one other protease, having Azocaseinase activity comparable to other exhibited strong activity on a casein-based substrate. fractions with the capacity to cause muscle release The highest concentration of protease(s) (or perhaps those with the greatest casein specificity) occurred in (e.g. fractions 20 and 28). Control experiments in which untreated dac- PIEF fractions at around pH 9; this high isoelectric tylopodite flexor preparations were loaded in- point is also consistent with pI data for Octopus crementally showed that at least 600 g was required proteases. Preliminary experiments in which highly before the muscle was torn away. The standard 10 g purified digestive enzymes from standard laboratory and terminal 50 g load regimes used in these experi- sources (10 mg/ml soutions in phosphate buffer) were ments therefore represent less than 10% of the nor- applied to the crab leg bioassay showed that trypsin mal load-bearing properties of the average crab leg and chymotrypsin were also capable of causing some muscle loosening. This suggests that the salivary preparation. Of the legs treated with commercial enzyme prepa- enzymes of Eledone are of this general type and rations all but 2 fell into the 60min + 50g DRT consequently similar in this respect to the majority of category. Two legs treated with a 10 mg/ml solution proteases from the PSG of Octopus (Morishita, of chymotrypsin had DRT's of 30 min 40 sec and 1978). We detected four peaks of caseinolytic activity 36 min 15 sec respectively. following PIEF fractionation of saliva, of which the major peak and possibly two minor ones, possessed muscle loosening properties. In its preparative form DISCUSSION this separation technique is not very sensitive and it In this study we have demonstrated directly the is likely that the activity encompassed by fractions muscle loosening properties of Eledone saliva and 20-28 comprises more than one protease. If activity shown that proteolytic enzymes are the components is expressed relative to total protein concentration of most likely to be responsible for this effect. These individual fractions, these fractions appear to comfindings have special significance since no study of prise two peaks. Casein may not be a suitable subnaturally produced saliva has been made previously strate for detecting all the proteases present in this and it has only been possible to infer the role of complex mixture of proteins, but it is interesting that proteases in external digestion from the hydrolytic the majority of the proteolytic activity we identified nature of gland extracts (Morishita, 1974a-c, 1978) is associated with muscle-loosening in vitro, consisand the secretions of isolated glands (Bacq and tent with the effect we have observed when saliva of Ghiretti, 1953; Ghiretti, 1960); Ballering et aL, (1972) this species is injected into live crabs. The question arises as to whether this activity is deduced the proteolytic nature of the saliva milked from Octopus apollyon but made no detailed studies. specific to musculo-skeletal attachments. Nixon In addition, Our bioassay provides a realistic tool for (1984) points out that lumps of muscle tissue are still the in vitro study of the function of these enzymes, recogniseable in the crop several hours after ingestion both isolated and as whole saliva, without having to of a meal and we can confirm this observation which resort to surgical interference with the feeding appa- implies an enzyme specificity for detachment of musculo-sketetal attachments. Our detection of proratus of octopus (Nixon, 1984).
1122
M. S. GRISLEYand P. R. BOYLE
teolytic activity in components of saliva with apparently no muscle loosening capacity is consistent with the idea that only some of the enzymes have this function. With the type of data obtained from the bioassay in its present form the minimum DRT values for a saliva sample are of particular interest, since they represent the full potential activity of the enzyme under the prevailing experimental conditions. Longer dactylopodite release time probably result from the influence of uncontrolled physical and biological variables; with refinement of the assay it should be possible to reduce the variability of these data and we expect that this will result in the mean values of multiple assays approaching minimum values (Figs 3 and 4b), rather than a generalized contraction of the range of values. Unlike the Azocasein assay, which yields easily reproducible results, the variability of the bioassay data precludes the derivation of an absolute value for muscle loosening activity for a given concentration of enzyme. A realistic use of these kinds of data is in the form of a dose-response curve, where the time taken for 50% of a standard number of preparations to show muscle detachment is taken as the measure of activity for a sample of saliva, expressed as DRTs0. Although Azocasein is susceptible to hydrolysis by these enzymes, the bioassay described is the only method available for testing for the presence, specificity and characteristics of muscle-loosening activity in Eledone saliva and it should allow a functional comparison of salivary proteases from other cephalopods. Koueta and Boucaud-Camou (1986) have identified only a single protease in extracts of the posterior salivary gland of Sepia oflicinalis L. although there appears to be extensive esterase activity in the same material. We are confident of the presence of several muscle loosening and proteolytic enzymes in Eledone saliva and Morishita (1978) describes as many as eight in PSG extracts of Octopus. The comparative lack of proteases in Sepia (Koueta and Boucaud-Camou, 1986) may be associated with the marked differences in feeding behaviour between decapods and octopods (Boucher-Rodoni et al., 1986). The bioassay should permit us to identify components of saliva with respect to their in vivo function and with minor modifications could be adapted to examine the effects of normal capture and handling by octopods on crustacean prey. This simple yet effective assay may allow an insight to the mechanisms of cephalopod feeding behaviour that other methods have not provided. SUMMARY
A bioassay for the muscle-releasing properties of octopus saliva is described, based upon the detachment of the dactylopodite flexor muscle from its origin on the carapace of the propopodite joint of the walking leg of the shore crab Carcinus maenas. Under standard conditions of leg preparation and applied load, the time for muscle detachment is recorded as a measure of enzyme activity. For whole saliva from the octopus Eledone cirrhosa (obtained by envenomation, "milking"), increasing proteolytic activ-
ity (measured on a chromogenic casein substrate) correlated with decreasing dactylopodite releasing times (DRT). Assay of Eledone saliva fractions obtained by preparative isoelectric focusing showed that muscle detachment activity was restricted to those fractions containing most of the caseinolytic activity. It is concluded that the ability of octopus saliva to selectively release crab muscle from the carapace, prior to ingestion, is principlaly due to protease(s) in the saliva. Acknowledgements--We are grateful to the NERC for financial support, to Mrs Gillian Robertson for assistance with laboratory techniques and to Professor G. Gooday for reading the manuscript. REFERENCES
Arnold J. M. and Arnold K. O. (1969) Some aspects of hole-boring predation by Octopus vulgaris. Am. Zool. 9, 991-996. Bacq Z. M., Fischer P. and Ghiretti F. (1951) Action de la 5-hydroxytryptamine chez les cephalopodes. Archs lnt. Physiol. 59, 165-171. Bacq Z. M. and Ghiretti F. (1953) Physiologic des glandes salivaires post6rieures des Cephalopodes Octopodes isol~s et perfus~es/n vitro. Pubbl. Staz. Zool. Napoli 24(3), 267-277. Ballering R. B., Jalving M. A., Ven Tresca D. A., Hallacher L. E., Tomlinson J. T. and Wobber D. R. (1972) Octopus envenomation through a plastic bag via a salivary proboscis. Toxicon 10, 245-248. Boucher-Rodoni R., Boucaud-Camou E. and Mangold K. (1986) Feeding and Digestion. In Cephalopod Life Cycles, (Edited by Boyle P. R.), Vol. 2, pp. 85-108. Academic Press, London. Boyle P. R. (1981) Methods for the aquarium maintenance of the common octopus of British waters, Eledone cirrhosa. Lab. Anita. 15, 327-331. Boyle P. R. and Knobloch D. (1981) Hole boring of crustacean prey by the octopus Eledone cirrhosa (Mollusca, Cephalopoda). J. Zool. Lond. 193, 1-10. Boyle P. R., Mangold K. and Froesch D. (1979) The mandibular movements of Octopus vulgaris. J. Zool. Lond. 188, 53-67. Bradford M. IVl.(1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye binding. Analyt. Biochem. 72, 248-254. Cariello L. and Zanetti L. (1977) ~- and /~-cephalotoxin: two paralysing proteins from posterior salivary glands of Octopus vulgaris. Comp. Biochem. Physiol. 57C, 169-173. Crone H. D., Leake B., Jarvis M. W. and Freeman S. E. (1976) On the nature of "Maculotoxin", a toxin from the blue-ringed octopus (Hapalochlaena maculosa). Toxicon 14, 423-426. Ducros C. (1972a) Etude ultrastructurale de l'innervation des glandes salivaires post6rieures chez Octopus vulgaris--I. Les troncs nerveux du canal salivaire. Z. Zellforsch. Mikrosk. Anat. 132, 35-49. Ducros C. (1972b) Etude ultrastructurale de l'innervation des glandes salivaires post6rieures chez Octopus vulgaris--II. Innervation de la musculature du canal des glandes. Z. Zellforsch. Mikrosk. Anat. 132, 51-65. Dulhunty A. and Gage P. W. (1971) Selective effects of an octopus toxin on action potentials J. Physiol. 218, 433-445. Erspamer V. and Anastasi A. (1962) Structure and pharmacological actions of eledoisin, the active endecapeptide of the posterior salivary glands of Eledone. Experientia 18, 58. Erspamer V. and Ghiretti F. (1951) The action of enteramine on the heart of molluscs. J. Physiol. 115, 470-481.
Octopus salivary enzymes Reeker H. and Cotton B. C. (1955) Fatal bite from octopus. Med. J. Aust. H 42(9), 329-331. Ghiretti F. (1959) Cephalotoxin: the erab-paralysing agent of the posterior salivary glands of cephalopods. Nature, London 183, 1192-1193. Ghiretti F. (1960) Toxicity of octopus saliva against crustaeea. A. N.Y. Acad. Sci. 90, 726-741. Halstead B. W. (1965) Poisonous and Venomous Marine Animals of the World, Vol. I, pp. 663-770. Juorio A. V. and Killick S. W. (1973) The distribution of monoamines and some of their acid metabolites in the posterior salivary glands and viscera of some cephalopods. Comp. Biochem. Physiol. 44A, 1059-1067. Koueta N. and Boucaud-Camou E. (1986) Comparative study of the secretion of the posterior salivary glands of deeapod cephalopods--I. Sepia officinalis L. Cell. Molec. Biol. 32, 333-341. Lowry O. H., Rosebrough N. J., Farr A. L. and Randell R. J. (1951) Protein measurement with the Folin Phenol reagent. J. biol. Chem. 193, 265-275. McDonald N. M. and Cottrell G. A. (1972) Purification and mode of action of toxin from Eledone cirrosa. Comp. Gen. Pharmac. 3, 243-248. Matus A. I. (1971) Fine structure of the posterior salivary gland of Eledone cirrosa and Octopus vulgaris. Z. Zellforsch. Mikrosk. Anat. 122, 111-121. Millar G. L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 31, 964. Morishita T. (1974a) Participation in digestion by the proteolytic enzymes of the posterior salivary glands in octopus---II. Isolation and purification of the proteolytic enzymes from the posterior salivary glands. Bull. Jap. Soc. Fish. 40(6), 601-607 (in Japanese). Morishita T. (1974b) Participation in digestion by the proteolytic enzymes of the posterior salivary glands in octopus---Ill. Some properties of purified enzymes from
C.B.P. 88/4B.--G
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the posterior salivary glands. Bull. Jap. Soc. Fish. 40(9), 917-925 (in Japanese). Morishita T. (1974c) Participation in digestion by the prot¢olytic enzymes of the posterior salivary gland in octopus--IV. Purification and some properties of proteolytic enzymes from digestive juice. Bull. Jap. Soc. Fish. 40(9), 927-936 (in Japanese). Morishita T. (1978) Studies on the protein digestive enzyme of octopus, O. vulgaris (Cuvier). Bull. Fac. Fish. Univ. 5, 197-282 (in Japanese). Nixon M. (1979a) Hole-boring in shells by Octopus vulgaris Cuvier in the mediterranean. Malacologia 18, 431-443. Nixon M. (1979b) Has Octopus vulgaris a second radula? J. Zool., Lond. 187, 291-296. Nixon M. (1980) The salivary papilla of Octopus as an accessory radula for drilling shells. J. Zool. Lond. 190, 53-57. Nixon M. (1984) Is there external digestion by Octopus? J. Zool. Lond. 202, 441--447. Nixon M. and Boyle P. R. (1982) Hole-drilling in crustaceans by Eledone cirrhosa (Mollusca: Cephalopoda). J. Zool., Lond. 196, 439 444. Prison M. E. Q. and Taylor P. B. (1961) Hole-drilling by Octopus. Science N. Y. 134, 1366-1368. Russell F. E. (1965) Marine toxins and venomous and poisonous marine animals. Adv. mar. Biol. 3, 255-384. Songdahl J. H. and Shapiro B. I. (1974) Purification and composition of a toxin from the posterior salivary gland of Octopus dofleini. Toxicon 12, 109-115. Wells M. J. (1978) Octopus Physiology and Behaviour of an Advanced Invertebrate, p. 417 Chapman and Hall, London. Wodinsky J. (1969) Penetration of the shell and feeding on gastropods by Octopus. Am. Zool. 9, 997-1010. Young J. Z. (1965) The buceal nervous system of Octopus. Phil. Trans. R. Soc. B, 249, 27-43.
0305-0491/87 $3.00+ 0.00 © 1987 Pergamon Journals Ltd
Printed in Great Britain
BIOASSAY AND PROTEOLYTIC ACTIVITY OF DIGESTIVE ENZYMES FROM OCTOPUS SALIVA M. S. GRISLEY and P. R. BOYLE Department of Zoology, University of Aberdeen, Scotland (Received 26 March 1987)
Abstract--1. A bioassay for octopus saliva, based on detachment of crab dactylopodite flexor muscle under standard conditions, has been developed. 2. There is a direct relationship between increasing caseinolytic activity of saliva from Eledone cirrhosa and decreasing muscle detachment time. 3. Fractionation of saliva, using preparative isoelectric focusing, shows that muscle releasing activity is restricted to fractions containing proteins with high isoelectric points and maximum easeinase activity. 4. It is concluded that proteolytic enzyme(s) in octopus saliva selectively release crab muscle from attachment to the carapace. INTRODUCTION The contribution of the posterior salivary glands to the feeding mechanism of cephalopods has been the subject of research and speculation for over a century. These large paired glands in octopuses (smaller and single in squid and cuttlefish) lie within the body cavity posterior to the head, but deliver their secretion forward through a single duct which terminates within the buccal cavity in a muscular salivary papilla. The glands have several types of secretory cells (Matus, 1971), are highly innervated and have a good blood supply (Ducros, 1972a and b). Descriptions of the buccal apparatus and the relationship of the various components may be found in Halstead (1965), Young (1965), Wells (1978) and Boyle et al. (1979). Interest in the properties of the saliva produced by these glands has been prompted by particular aspects of the feeding behaviour in octopuses. Large crustacea, such as crabs, are grasped by the octopus and become paralysed within a few minutes. Thereafter, the prey, hidden by the enveloping arms and web, is disarticulated and the flesh removed leaving a clean exoskeleton which is discarded by the octopus. The carapace of crabs taken by Eledone is frequently found to be penetrated by a narrow hole (Boyle and Knobloch, 1981; Nixon and Boyle, 1982). This feature is also characteristic for molluscan prey taken by Octopus (Pilson and Taylor, 1961; Arnold and Arnold, 1969; Wodinsky, 1969; Nixon, 1979a) and the organ responsible is thought to be the tooth-tipped salivary papilla (Nixon, 1979b, 1980; Nixon and Boyle, 1982). Information on the properties of saliva, based mostly on studies of aqueous extracts of the posterior salivary glands themselves, may be categorized broadly as: (a) Venomous----especially to crustacea (Ghiretti, 1959, 1960; Dulhunty and Gage, 1971; McDonald and Cottrell, 1972; Songdahl and Shapiro, 1974; Crone et al., 1976; Cariello and Zanetti, 1977), an aspect which received additional attention after a human fatality was reported (Flecker and Cotton, 1955). (b) Pharmacological--a wide range of
chemicals is present such as acetylcholine, noradrenaline and 5-hydroxytryptamine (Erspamer and Ghiretti, 1951; Bacq et aL, 1951; Erspamer and Anastasi, 1962; Juorio and Killick, 1973), all with known actions on other tissues. Summaries of early work are given in Halstead (1965) and Russell (1965). (c) Digestive---a very early suggestion for a digestive function (Bert, 1867 in Halstead, 1965) was largely ignored although proteolytic activity was also suggested by Bacq and Ghiretti (1953). The presence of proteases and their partial characterisation using artificial substrates has been demonstrated by Morishita (1974a-c, 1978) and their involvement in external digestion suggested by Nixon (1984) who showed that surgical interference with elements of the buccal mass, including the salivary papilla, affected efficiency of cleaning of the carapace. In this paper we report on a new bioassay for octopus saliva, based on detachment of crustacean muscles from the exoskeleton. Pure saliva, collected by the envenomation technique ("milking") reported by Ballering et al. (1972) and fractions prepared by isoelectric focusing have been assayed for their ability to disrupt the musculoskeletal attachments and for proteolytic activity on a high mol. wt, non-specific chromogenic substrate. MATERIALS AND METHODS
Collection of saliva Eledone cirrhosa (Lamarck) from the coastal waters of
Aberdeen were maintained in the recycling sea-water aquarium at Aberdeen University (Boyle, 1981). Saliva was collected using the technique described by Ballering et al. (1972). The octopus is placed inside a thin plastic bag and allowed to attach itself to the inner surface. Within seconds the animal will begin biting; the beaks open and a cyclical rhythm of rasping with the radula and salivary papilla is established. This results in penetration of the bag, whereupon the papilla is fully extended and, after a few seconds delay, a fine jet of saliva is emitted (Fig. I). In addition there is often a copious production of fluid welling around the papilla which was also collected. The saliva from several animals was pooled, aliquoted and stored at -20°C. This method of envenomation or "milking" yields 25-100 #1 of saliva from each animal every one or two days.
1117
1118
M. S. GRISLEY and P. R. BOYLE Ampholines (4ml pH 9-11 and I ml pH 7.9) and 1 ml Triton-X to reduce precipitation. The total volume was adjusted to 100 ml and the gel was cast and set according to the evaporation data supplied with the Ultrodex powder. Following the procedure recommended by LKB the plate was subjected to a constant 30W electric current for 16-18 hr. On completion of focusing the gel was divided into 30 equal fractions using a purpose-made stainless steel grid and the pH of alternate fractions was measured using a Philips CE3 surface pH electrode. The fractions were removed and the proteins eluted from each with 3 ml 0.1 M citric acid/HCl buffer, pH 6. The eluants were aliquoted and stored at -20°C.
Fig. 1. Collection of whole from Eledone cirrhosa by envenomation--"milking". The salivary papilla projects through a hole in the polythene bag holding the animal and saliva is ejected in a fine stream.
Fractionation of saliva Saliva was crudely fractionated by flat-bed preparative isoelectric focusing (PIEF) using LKB Multiphor equipment and accessories. On the basis of pilot analytical studies, pH gradients with a strong cationic bias were constructed to give optimum separation conditions. Five miUilitres of saliva was filtered (3/~m Millipore syringe filter) and added to the Ultrodex slurry together with
Crab leg bioassay The propopodite/dactylopodite segments of the walking legs of shore crabs (Carcinus maenas; mean carapace width 75 mm and wet wt 75 + 20 g), were used as the basis for the bioassay. The crabs were killed by mid-line bisection and pairs of walking legs 2-5 were removed at the basiopodite segment. The propopodite-dactylopodite joint was prepared by cutting through the extensor apodeme and the arthrodial membrane connecting the two segments. The carapace of the propopodite segment was then cut through longitudinally on both sides and the dorsal half gently removed (Fig. 2a). The dactylopodite flexor muscle, originating on the flexor apodeme and inserting on the carapace oI the ventral half of the propopodite segment, thus remained as the only connection between the two segments. As soon as the muscle preparation was completed a strip of filter paper, soaked in sea-water, was wrapped around the propopodite to act as a humidity jacket and this segment was securely clamped in a frame, leaving the dactylopodite hanging free (Fig. 2b). In the present experiments a standard load of 10 g was attached to the dactylopodite using a spring clip (total 11.6 g). A drop of glycerol was applied to the exposed muscle between the two segments to reduce desiccation. Test solutions were applied as 50 #I aliquots to the open proximal end of the propopodite segment using a Gilson micropipette. The pipette tip was used to probe gently into the dactylpodite retractor muscle to a depth of 1-2 mm to improve access of the applied sample which otherwise drained away into the humidity jacket. Time of completion of sample application was recorded and the preparation left undisturbed at room temperature (approx. 21 °C). When the dactylopodite segment detached from the propopodite the
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1119
Octopus salivary enzymes time to dactylopodite release (DRT) was recorded to the nearest 5 sec. In preparations where the DRT was greater than 10 min, the humidity jacket was regularly remoistened with seawater. An arbitrary maximum incubation time of 60 min was allowed. This provided ample time for the more active samples to cause detachment (it approximates to the normal feeding period of Eledone). At 60 min the experiment was terminated by the addition of a further load of 50 g. When this caused dactylopodite release within 10 sec the result was recorded as "60 min + 50 g". If release did not occur within 10sec of this procedure the result was categorised as " > 6 0 min + 50 g". Whole saliva was diluted 1:1 with filtered seawater to reduce its viscosity. PIEF fractions were assayed without removing ampholytes or adjustment of concentration or pH. Control applications included filtered seawater, citric acid buffer, phosphate buffer and a solution of Ampholines in distilled water. Two groups of six legs were treated with highly purified commercial preparations of trypsin and chymotrypsin (10 mg/ml in 0.1 M phosphate buffer pH 7.0, Sigma Chemical Co.) to assess the effect of other proteolytic enzymes in the bioassay. Additional determinations of the load bearing properties of these muscle attachments were made by progressive loading of untreated preparations. Protein determination
Protein concentration was measured using Millar's (1959) modification of Lowry et al.'s technique (1951) in preference to Bradford's (1976) method which is affected by the presence of ampholytes. Bovine serum albumin (BSA) was used to construct a standard curve. Saliva and PIEF fractions required a 1 x 102-1 x 103 dilution. Assay solutions were duplicated and read at 650 nm using a Perkin-Elmer Lambda 5 dual-beam spectrophotometer.
Table 1. Total protein concentration (Lowry), proteolytic activity (Azocasein) and muscle loosening activity (daetylopodite releasing time, DRT, in the crab leg bioassay) for six different pools of Eledone saliva Bioassay Protease mean DRT trypsinequiv. Protein Saliva (N) (see) mg/ml mg/ml pool 1 235 2.40 84 (9) 350 1.88 72 (4) 2 375 1.28 38 (12) 3 410 1.16 12 (3) 4 500 1.12 16 (19) 5 670 0.88 28 (11) 6
correlation between protein concentration and D R T values or protease activity. The data for all assays on the six different saliva pools is shown graphically in Fig. 3. Assay procedures such as muscle preparation and saliva application, as well as biological factors such as crab size and stage in the moult-cycle probably account for much of the variability of individual results which increases with decreasing protease activity. Despite the variability in the data, there is a significant correlation between proteolytic activity of the pooled saliva and its muscle releasing activity ( D R T ) (Spearman's rank correlation coefficient r s = - 0 . 6 3 1 ; 58 pairs; P <0.01). Regression analysis of the 1Ogn transformed mean D R T against proteolytic activity of saliva also shows a significant correlation
1200.
Protease determination
Of the three commercial chromogenic substrates investigated, Azocasein, Azocoll and Hide Powder Azure (Sigma Chemical Co.), Azocasein was found to be the most sensitive to salivary proteases from Eledone was subsequently used as the standard substrate. Reaction mixtures comprised 2.4mi 0.1 M phosphate buffer, pH 7.0 containing 25 mg Azocasein. At strictly timed intervals, 100gl of sample was added. After thorough mixing, reaction mixtures were incubated in a static water bath at 37°C; 10 min for whole saliva and 90 min for PIEF fractions. Hydrolysis was terminated by the addition of 2.5 ml 10% trichloroacetic acid, causing the precipitation of unhydrolysed substrate which was removed by centrifugation. The absorbance of the supernatants was read at 370 nm in the spectrophotometer against a blank in which the enzyme solution was substituted with buffer. Other controls included filtered seawater, citric acid buffer and a solution of Ampholine. All assay mixtures were duplicated. Proteolytic activity of whole saliva was standardised and expressed as trypsin-equivalents by including a series of trypsin standards (Sigma 2 x crystallised, ethanol precipitated: 0.01-1 mg/ml); measurements of PIEF fractions remain expressed as absorbance values.
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RESULTS Six different pools o f Eledone saliva (50% in filtered seawater) were tested for muscle loosening activity in the crab leg bioassay. Table 1 shows that when the dactylopodite detachment times ( D R T ) for the saliva pools are ranked, values for their proteolytic activity on the Azocasein substrate fall into rank order such that increasing D R T ' s correlate with decreasing protease activity. There is no apparent
;
i
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Fig. 3. Results for six different pools of Eledone saliva tested for muscle releasing activity using the crab leg bioassay. Time to dactylopodite release (DRT in sec-ordinate) is plotted against protease activity (nag trypsin equivalents/ml--abscissa) measured using Azocasein. ( 0 , individual assays; &, mean DRT for each saliva pool)
1120
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Fig. 4(a). Protein concentrations (Lowry--mg/ml, • . . . . . • ) and proteolytic activity (Azocasein-absorbance at 370 rim, • A) for 30 fractions of Eledone saliva separated by preparative is•electric focusing (PIEF) using ampholytes in the pH range 7-11 ( O - - - O ) . Fig. 4(b). Muscle releasing activity of the saliva fractions in 3(a), expressed as dactylopodite releasing time (DRT in rain) determined by the crab leg bioassay. The number of occasions on which the terminal additional load of 50 g applied at 60 min caused release is shown in row A (60 + 50 g). The number of occasions on which this additional 50 g failed to cause dactylopodite release is shown in row B ( > 6 0 + 50g). (R ~ = 84.8; P < 0.01) and for the equation: In y = a + b x yields the values: x In y b a
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proteolytic activity (trypsin equiv./ml) log transformed mean D R T --0.000570 (slope) 6.83 (y intercept)
The protein concentration and protease activity of all 30 fractions from a single preparative is•electric focusing (PIEF) fractionation of saliva, are shown in
Fig. 4a; the plotted values are the means of duplicate assays and the individual values varied by less than 1 mg/ml and 0.05 absorbance units respectively. Although only a preparative step in protein purification, this technique reveals at least two distinct areas of proteolytic activity (fractions 8-10 and 20-27) and two minor ones (fractions 15 and 17). The data for muscle loosening activity of the same 30 fractions falls into three categories (see methodology); preparations in which the dactylopodite was released under standard load within 60 rain are individually plotted as actual times in Fig. 4b; the
Octopus salivary enzymes
1121
The most recent and comprehensive account of the numbers of preparations which released at 60 min with an additional 50 g load (60 min + 50 g) are listed physico-chemical characteristics of salivary enzymes in row A of Fig. 4b; the numbers of those which failed from an octopod is to be found in Morishita's review to release at all after this treatment ( > 60 rain + 50 g) (1978) of the properties of enzyme extracts from the are listed in row B of Fig. 4b. The figure contains data posterior salivary glands (PSG's) and "digestive collated from experiments performed on five different juice" of O. vulgaris. While Morishita gives values for days and the dashed line delimits the graphical mol. wt, pI, pH optima and artificial substrate values. All crab legs treated with the range of control specificities for proteases, no functional inferences solutions described in the methodology fell into the other than those of conventional digestion are made. > 6 0 m i n + 50g category and showed no sign of It is interesting that of the eight (possibly nine) caseinolytic enzymes he identifies in PSG extracts, muscle loosening. The major peak of proteolytic activity (fractions only three can also be traced in the gut. While factors 20-27 in Fig. 4a), with a pI of approx. 9, corresponds such as enzyme lability and dilution may account for exactly with distribution of muscle loosening activity this, it may also show the inherent difficulties in between PIEF fractions (Fig. 4b). In addition, the interpreting enzymic activity of tissue extracts; in this reduction in proteolytic activity of fractions on either instance, not all of these proteases may be present in side of fractions 23 and 24 is associated with an normal saliva. Out results suggest that, assuming no increase in DRT, suggesting that the bioassay result great interspecies differences, the nature of the enzymic activity and the numbers of different proteases is directly related to protease activity. From the 60 rain + 50 g data in Fig. 4b (row A) in freely expressed Eledone saliva fall within the range it is inferred that fractions 15 and 17 also possess of those recorded from salivary gland extracts and muscle loosening properties, as indicated by the "digestive juices" by Morishita for Octopus vulgaris. Certain similarities with Morishita's Octopus prooccasional detachment achieved with additional load, although they were apparently too weak to cause teases emerge from the present results. All the prodactylopodite detachment within 60 min at standard teases described by Morishita (1978) are caseinolytic, load. In contrast, the proteolytic activity detected in and while this may not be true for those in Eledone fractions 8-10 caused no muscle loosening on any of saliva, the enzymes apparently responsible for muscle the 14 occasions on which they were tested, despite loosening, as well as at least one other protease, having Azocaseinase activity comparable to other exhibited strong activity on a casein-based substrate. fractions with the capacity to cause muscle release The highest concentration of protease(s) (or perhaps those with the greatest casein specificity) occurred in (e.g. fractions 20 and 28). Control experiments in which untreated dac- PIEF fractions at around pH 9; this high isoelectric tylopodite flexor preparations were loaded in- point is also consistent with pI data for Octopus crementally showed that at least 600 g was required proteases. Preliminary experiments in which highly before the muscle was torn away. The standard 10 g purified digestive enzymes from standard laboratory and terminal 50 g load regimes used in these experi- sources (10 mg/ml soutions in phosphate buffer) were ments therefore represent less than 10% of the nor- applied to the crab leg bioassay showed that trypsin mal load-bearing properties of the average crab leg and chymotrypsin were also capable of causing some muscle loosening. This suggests that the salivary preparation. Of the legs treated with commercial enzyme prepa- enzymes of Eledone are of this general type and rations all but 2 fell into the 60min + 50g DRT consequently similar in this respect to the majority of category. Two legs treated with a 10 mg/ml solution proteases from the PSG of Octopus (Morishita, of chymotrypsin had DRT's of 30 min 40 sec and 1978). We detected four peaks of caseinolytic activity 36 min 15 sec respectively. following PIEF fractionation of saliva, of which the major peak and possibly two minor ones, possessed muscle loosening properties. In its preparative form DISCUSSION this separation technique is not very sensitive and it In this study we have demonstrated directly the is likely that the activity encompassed by fractions muscle loosening properties of Eledone saliva and 20-28 comprises more than one protease. If activity shown that proteolytic enzymes are the components is expressed relative to total protein concentration of most likely to be responsible for this effect. These individual fractions, these fractions appear to comfindings have special significance since no study of prise two peaks. Casein may not be a suitable subnaturally produced saliva has been made previously strate for detecting all the proteases present in this and it has only been possible to infer the role of complex mixture of proteins, but it is interesting that proteases in external digestion from the hydrolytic the majority of the proteolytic activity we identified nature of gland extracts (Morishita, 1974a-c, 1978) is associated with muscle-loosening in vitro, consisand the secretions of isolated glands (Bacq and tent with the effect we have observed when saliva of Ghiretti, 1953; Ghiretti, 1960); Ballering et aL, (1972) this species is injected into live crabs. The question arises as to whether this activity is deduced the proteolytic nature of the saliva milked from Octopus apollyon but made no detailed studies. specific to musculo-skeletal attachments. Nixon In addition, Our bioassay provides a realistic tool for (1984) points out that lumps of muscle tissue are still the in vitro study of the function of these enzymes, recogniseable in the crop several hours after ingestion both isolated and as whole saliva, without having to of a meal and we can confirm this observation which resort to surgical interference with the feeding appa- implies an enzyme specificity for detachment of musculo-sketetal attachments. Our detection of proratus of octopus (Nixon, 1984).
1122
M. S. GRISLEYand P. R. BOYLE
teolytic activity in components of saliva with apparently no muscle loosening capacity is consistent with the idea that only some of the enzymes have this function. With the type of data obtained from the bioassay in its present form the minimum DRT values for a saliva sample are of particular interest, since they represent the full potential activity of the enzyme under the prevailing experimental conditions. Longer dactylopodite release time probably result from the influence of uncontrolled physical and biological variables; with refinement of the assay it should be possible to reduce the variability of these data and we expect that this will result in the mean values of multiple assays approaching minimum values (Figs 3 and 4b), rather than a generalized contraction of the range of values. Unlike the Azocasein assay, which yields easily reproducible results, the variability of the bioassay data precludes the derivation of an absolute value for muscle loosening activity for a given concentration of enzyme. A realistic use of these kinds of data is in the form of a dose-response curve, where the time taken for 50% of a standard number of preparations to show muscle detachment is taken as the measure of activity for a sample of saliva, expressed as DRTs0. Although Azocasein is susceptible to hydrolysis by these enzymes, the bioassay described is the only method available for testing for the presence, specificity and characteristics of muscle-loosening activity in Eledone saliva and it should allow a functional comparison of salivary proteases from other cephalopods. Koueta and Boucaud-Camou (1986) have identified only a single protease in extracts of the posterior salivary gland of Sepia oflicinalis L. although there appears to be extensive esterase activity in the same material. We are confident of the presence of several muscle loosening and proteolytic enzymes in Eledone saliva and Morishita (1978) describes as many as eight in PSG extracts of Octopus. The comparative lack of proteases in Sepia (Koueta and Boucaud-Camou, 1986) may be associated with the marked differences in feeding behaviour between decapods and octopods (Boucher-Rodoni et al., 1986). The bioassay should permit us to identify components of saliva with respect to their in vivo function and with minor modifications could be adapted to examine the effects of normal capture and handling by octopods on crustacean prey. This simple yet effective assay may allow an insight to the mechanisms of cephalopod feeding behaviour that other methods have not provided. SUMMARY
A bioassay for the muscle-releasing properties of octopus saliva is described, based upon the detachment of the dactylopodite flexor muscle from its origin on the carapace of the propopodite joint of the walking leg of the shore crab Carcinus maenas. Under standard conditions of leg preparation and applied load, the time for muscle detachment is recorded as a measure of enzyme activity. For whole saliva from the octopus Eledone cirrhosa (obtained by envenomation, "milking"), increasing proteolytic activ-
ity (measured on a chromogenic casein substrate) correlated with decreasing dactylopodite releasing times (DRT). Assay of Eledone saliva fractions obtained by preparative isoelectric focusing showed that muscle detachment activity was restricted to those fractions containing most of the caseinolytic activity. It is concluded that the ability of octopus saliva to selectively release crab muscle from the carapace, prior to ingestion, is principlaly due to protease(s) in the saliva. Acknowledgements--We are grateful to the NERC for financial support, to Mrs Gillian Robertson for assistance with laboratory techniques and to Professor G. Gooday for reading the manuscript. REFERENCES
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