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Kugler Publications, Amsterdam/Berkeley. Digestive enzyme activities and their distribution in the alimentary canal of larvae of the three extant lamprey families.
Fish Physiology and Biochemistry vol. 10 no. 1 pp 1-10 (1992) Kugler Publications, Amsterdam/Berkeley

Digestive enzyme activities and their distribution in the alimentary canal of larvae of the three extant lamprey families Max H. Cake, Ian C. Potter, Glen W. Power and Mayamin Tajbakhsh School of Biological and Environmental Sciences, Murdoch University, Murdoch, Western Australia 6150 Accepted: October 22, 1991 Keywords: larval lampreys, exocrine pancreas, lipase, amylase, chymotrypsin, trypsin, evolution

Abstract The activities of trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), lipase (EC 3.1.1.3) and amylase (EC 3.2.1.1) were measured in different regions of the alimentary tract of ammocoetes from each of the three extant lamprey families. In the southern hemisphere species Geotria australis (Geotriidae), and even more particularly Mordacia mordax (Mordaciidae), enzymatic activity was almost entirely confined to prominent diverticular extensions which arise at the oesophageal-intestinal junction. However, in the holarctic Lampetra richardsoni(Petromyzontidae), which does not possess a diverticulum, the enzymatic activity was highest in the upper anterior intestine. It is not clear whether the presence of significantly higher amylolytic and lower lipolytic activities in the diverticulum of G. australisthan in the exocrine tissue of the other two species reflects interspecific differences in the composition of their diets. The capacity of exocrine tissue extracts for chymotryptic and tryptic digestion was assayed before and after in vitro exposure to trypsin and enteropeptidase, their respective catalytic activators. Prior to exposure to these exogenous activators, both proteolytic enzymes were fully active in L. richardsoni,partially active in G. australisand totally inactive in M. mordax. Maximal chymotryptic activity was greater in M. mordax than in L. richardsoniand G. australis. In contrast, maximal tryptic activity was greater in L. richardsonithan in G. australisand was very low in M. mordax. Since trypsin is the only known activator of chymotrypsinogen, the negligible activity of trypsin in M. mordax would appear anomalous unless a trypsin inhibitor is present in the protopancreas of this species. Differences in the distribution of enzymatic activity within the alimentary tract of the three species is discussed in relation to proposed phylogenetic relationships amongst the extant lamprey families.

Introduction Lampreys, which are one of the two extant groups of agnathan (jawless) vertebrates (Hardisty 1982), are represented in the contemporary fauna by three families (Potter 1980a). All of the 36 northern hemisphere species are placed in the Petromyzontidae, whereas the four southern hemisphere species are separated into either the Geotriidae or Mor-

daciidae, each of which is represented by a single genus (Geotria and Mordacia). The life cycle of all lampreys contains a protracted larval phase, which on average lasts for three to six years, depending on the species (Potter 1980b). The larval lamprey (ammocoete) is relatively sedentary, remaining for much of the time burrowed in the soft substrata of streams and rivers. It is a microphagous feeder, extracting algae (principally

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Lampetra richardsoni

Oesophagus

Geotria australis

Right diverticulum

Bile duct Oesophagus

II]

1·1

11_1· I

II....

....

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Anterior intestine

i.

Left diverticulum

Mordacia mordax D:- 1 -

Oesophagus

Diverticulum Fig. 1. Schematic diagrams showing the morphology of the alimentary canal in ammocoetes of Lampetera richardsoni(lateral view), Geotriaaustralis(dorsal view) and Mordacia mordax (dorsal view), as modified from Potter et al. (1986). The exocrine cells are distributed in the columnar epithelium of the anterior intestine of L. richardsoni, whereas those of G. australis and M. mordax occur predominantly and exclusively in the respective diverticula of these species.

diatoms), micro-organisms and detritus from the water overlying its burrow (Moore and Beamish 1973; Moore and Potter 1976; Moore and Mallatt 1980). Although cells assumed to be homologous with the pancreatic exocrine (zymogen) cells of higher vertebrates have been identified in the alimentary tract of ammocoetes using both light and electron microscopy, their distribution differs markedly amongst the three lamprey families (Luppa 1964; Strahan and Maclean 1969; Hansen and Youson 1978; Epple and Brinn 1987; Hilliard and Potter 1988). In the Petromyzontidae, the cells are located within the simple columnar epithelium lining the

anterior intestine (Fig. 1). In contrast, those of the Geotriidae and Mordaciidae are largely or entirely restricted to diverticula, which extend forward from the junction of the oesophagus and the anterior intestine (Fig. 1). In the case of larval Geotria, the "exocrine pancreas" comprises a large left and smaller right diverticulum, which are essentially pronounced tubular evaginations of the anterior intestine (Hilliard and Potter 1988). The large cavities of these diverticula are thus confluent with the lumen of the intestine (Epple and Brinn 1987; Fig. 1). In ammocoetes of Mordacia, however, the corresponding exocrine tissue is confined to a single, large left diverticulum which, unlike that of

3 Geotria, is a compact and highly lobulated structure (Fig. 1). The complexity of this exocrine structure led Strahan and Maclean (1969) to describe it as a protopancreas (see also Epple and Brinn 1987). There have been few studies on the digestive enzymes of lampreys. Moreover, work on the digestive enzymes of northern hemisphere ammocoetes is confined to examining the type of proteases found in the Petromyzontidae. The predominant proteolytic enzyme in the intestine of these ammocoetes is now known to be tryptic (Barrington 1936), rather than peptic as originally thought (Alcock 1899). While the activity of the digestive enzymes of ammocoetes of southern hemisphere lampreys have been measured in Mordacia mordax and Geotriaaustralis (Strahan and Maclean 1969; Hilliard and Potter 1988), some of the results of these studies are either limited or questionable. For example, since the lipolytic and amylolytic activities were recorded in arbitrary units by Strahan and Maclean (1969), they cannot be used for making quantitative comparisons. Furthermore, Strahan and Maclean (1969) concluded that, in larval Mordaciamordax, all of the amylolytic and most of the lipolytic activity was localised in the single diverticulum, whereas proteolytic activity was confined to the intestine. The latter conclusion appears remarkable since zymogen cells are essentially absent from the intestine of this species (Strahan and Maclean 1969; Potter et al. 1986). Although Hilliard and Potter (1988) recorded high levels of amylolytic, lipolytic and tryptic activity in the diverticula of G. australis, they were unable to detect any chymotryptic activity in the same structure, a feature that may well have been due to inappropriate conditions for activating chymotrypsinogen (see Discussion). The present study was undertaken to determine whether amylolytic, lipolytic, chymotryptic and tryptic activity were present in each of the morphologically diverse sites where exocrine tissues are found in ammocoetes of the three extant lamprey families and to compare the results obtained for the different taxa. The assays were performed on material in which the intestinal lumen had been carefully flushed so that no residual secreted enzymes remained. Alternative substrates to those used previously by workers on lamprey enzymes

have been employed to assay for tryptic and chymotryptic activity. The activity of the various enzymes in the diverticula of ammocoetes of both southern hemisphere families is compared with that of different regions of the intestine to ascertain the degree to which enzymatic activity is localised in the diverticula of these two groups. The activity and site of origin of the enzymes in the three taxa are discussed in the context of proposed phylogenetic relationships amongst the living lamprey families.

Materials and methods xe-Amylase, N-benzoyl-L-tyrosine ethyl ester (BTEE), bovine enteropeptidase (EC 3.4.21.9), lipase diagnostic kits, p-tosyl-arginine methyl ester (TAME) and trypsin (porcine) were obtained from Sigma Chemical Co., St. Louis, MO, USA, and amylopectin-azure from Calbiochem Corp., La Jolla, CA, USA. Electrofishing was used to collect ammocoetes of Geotriaaustralisfrom the Donnelly River in southwestern Australia, Mordacia mordax from the Plenty River in Tasmania, and Lampetrarichardsoni from tributaries of the Willamette River in Oregon, USA. For 24 h prior to dissection, the ammocoetes were placed in a tank without substrate to ensure that most of the intestinal contents were evacuated. After animals had been anaesthetised in benzocaine and killed by decapitation, the liver and the alimentary canal (between the front of the oesophagus and the anus) were removed. The alimentary canals were then separated into diverticula (in the case of the two southern hemisphere species), and the anterior and posterior intestine (Fig. 1). The anterior intestine is the short and relatively wider region of the intestine that immediately follows the oesophagus in holarctic species and the diverticula in southern hemisphere species (Youson 1981; Hilliard and Potter 1988). Note that the whole anterior intestine of the ammocoetes of southern hemisphere lampreys was used for analyses, whereas that of the northern hemisphere species was separated on the basis of length into equal upper and lower parts, both of which are known to contain zymogen

4 Lampetrarichardsoni o Geotriaaustralis * Mordaciamordax

A

100 80 60

I

f

I

I

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I J.

0

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ACTIVATION TIME (min) Fig. 2. The time course for the activation of trypsinogen in the intestinal diverticula of ammocoetes of Geotriaaustralis and Mordacia mordax and in the upper anterior intestine of larval Lampetria richardsoni following activation with exogenous enteropeptidase. Data in this and Figs. 3-6 are based on three or four batches of approximately 20 animals.

cells (Hansen and Youson 1978). The liver was included in these studies to act as a control. Residual food material and bile were flushed from the gut using either cold 1.0 mM HCI (for proteolytic activity) or 0.9% NaCl (for lipolytic and amylolytic activities). Each of the different components of the alimentary canal and the whole liver of twenty animals of each species were pooled. Three or four batches of the tissues were individually homogenised in either 1.0 mM HCI or 0.9% NaCl using a Hula teflon-glass hand homogeniser. The preparations were then centrifuged at 210 kPa (165,000 x g max.) in a Beckman Airfuge, to provide the cytosolic fraction.

Assay for digestive enzyme activities The chymotrypsinogen of lamprey tissues was activated by exposing aliquots of cytosol (250 /il) to trypsin (10 Atg.ml- 1) in Tris-HCI buffer (30 mM, pH 7.8) containing CaCI 2 (37.5 mM) and then incubating at 20 0C for up to 120 min. Cytosol ex-

tracts were also exposed to the trypsinogen activator enteropeptidase (0.25 mg.ml-1) in Tris-HCI buffer (17 mM, pH 8.1) containing CaC1 2 (4.3 mM) and incubated at 20 0C for up to 45 min. Throughout the course of these incubations, aliquots were removed and assayed for chymotryptic and tryptic activity (Hummel 1959). The activities are expressed as moless substrate hydrolyzed per min per g wet weight of tissue. Amylase was assayed by measuring the solubilization of amylopectin-azure, using a modification of the method of Hall et al. (1970). The activity was then standardized against an a-amylase standard curve and expressed as ztmoles of maltose liberated from starch per min per g wet weight of tissue. Lipase was assayed titrimetrically using a Sigma diagnostic kit based on the method of Tietz and Fiereck (1966). The activity is expressed in Sigma-Tietz units per g wet weight of tissue. N.B. 280 International units corresponds to 1.0 Sigma-Tietz unit. Two way Analysis of Variance was used to test whether, in the case of each digestive enzyme, the mean activity differed amongst the main exocrine

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A Lampeta richardsoni o Geotda austalis

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Fig. 3. The time course for the activation of chymotrypsinogen in the intestinal diverticula of ammocoetes of Geotria australisand Mordacia mordax and in the upper anterior intestine of larval Lampetra richardsoni following activation with exogenous trypsin.

tissues of L. richardsoni, G. australisand M. mordax. Where significant differences occurred, Tukey's test was employed to ascertain which means were significant at p < 0.05. All significant differences are recorded in the Results.

Results Activation of protease zymogens Trypsinogen in the cytosol of the upper anterior intestine of L. richardsoniwas maximally activated prior to exposure to exogenous enteropeptidase (Fig. 2). However, tryptic activity in the diverticula of G. australiswas only 35% active and required a 30 min exposure to exogenous enteropeptidase to achieve full activity (Fig. 2). The amount of tryptic activity was very low in the diverticulum of M. mordax, even after exposure to exogenous enteropeptidase for 45 min (Fig. 2). Chymotrypsinogen in the cytosol of the upper anterior intestine of L. richardsoniwas maximally activated prior to exposure to exogenous trypsin (Fig. 3). The chymotrypsinogen in the diverticula of G. australiswas 73o% activated and required only 15

min exposure to exogenous trypsin to achieve full activity. In contrast, the chymotrypsinogen in the diverticulum of M. mordax was totally inactive and required 120 min exposure to trypsin for full activation (Fig. 3). Trypsin and chymotrypsin activities Mean tryptic activity in the diverticula of G. australis was 38.7 units.g- (Fig. 4), exceeding by over six times that recorded in the anterior intestine of this species. Mean tryptic activity in the diverticulum of M. mordax was only 3.5 units.g- l (Fig. 4). There was negligible activity in the anterior intestine of this species. In contrast, activity in the upper anterior intestine of L. richardsoni was 90.2 units.g- , a value that far exceeded the 3.2 units.g- recorded in the lower end of the anterior intestine (Fig. 4). Negligible tryptic activity was recorded in the posterior intestine and liver of G. australis, M. mordax and L. richardsoni. Tryptic activity was significantly greater in the upper anterior intestine of L. richardsonithan in the diverticula of G. australis, which in turn was significantly greater than in the diverticulum of M. mordax.

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Geotria australis

Mordacia mordax

Lampetra richardsoni

ulum of M. mordax was 60.8 units.g- 1. Essentially no chymotryptic activity was found in the anterior intestine of this species (Fig. 4). Mean chymotryptic activity recorded in the upper anterior intestine of L. richardsoniwas 40.8 units.g- 1, approximately fifteen times the activity in the lower anterior intestine. Chymotryptic activity was negligible in the posterior intestine and liver of all three species. Mean chymotryptic activity was significantly greater in the diverticulum of M. mordax than in the upper anterior intestine of L. richardsoni, which in turn was significantly greater than in the diverticula of G. australis.

100 80

Lipase and amylase

60 40 20 0

L___

.__i_ D A P L

D A P L

Al A2

P

L

Fig. 4. Mean maximum activity of trypsin and chymotrypsin + I SEM in different regions of the alimentary tract of ammocoetes of Geotriaaustralis, Mordaciamordax and Lampetra richardsoni.In this and Figs. 5 and 6, D = diverticulum, A = anterior intestine, A = upper anterior intestine, A2 = lower anterior intestine, P = posterior intestine, L = liver.

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Geotria austrlis

Mordacia mordax

Lampetra richardsoni

D A P L

D A P L

AlA

120 100

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40 20 0 2

PL

Fig. 5. Mean maximum activity of lipase + 1 SEM in different regions of the alimentary tract of ammocoetes of Geotria australis, Mordacia mordax and Lampetra richardsoni.

Mean chymotryptic activity in the diverticula of G. australiswas 23.2 units.g - 1, which is approximately six times the activity in its anterior intestine (Fig. 4). Mean chymotryptic activity in the divertic-

Mean lipolytic activity in the diverticula of G. australis was 65.2 units.g-l, which is approximately

fourteen times that of the anterior intestine (Fig. 5). The mean activity of lipase in the diverticulum of M. mordax was 92.5 units.g-1. Negligible lipase activity was recorded in the anterior intestine of this species (Fig. 5). The mean activity of lipase in the upper anterior intestine of L. richardsoniwas 115.7 units.g-l, which is approximately thirteen times

greater than in the lower anterior intestine. Lipolytic activity was negligible in the posterior intestine and liver of all three species. Mean lipolytic activity in the upper anterior intestine of L. richardsoni was significantly higher than in the diverticula of G. australisbut not in that of M. mordax. Mean lipolytic activity did not differ significantly between the diverticula of the two southern hemisphere species. The mean amylolytic activity in the diverticula of G. australis was 604.1 units.g-1, which was 25 times greater than in the anterior intestine (Fig. 6). The mean amylolytic activity in the diverticulum of M. mordax was 256.0 units.g-l, whereas the activity was negligible in the anterior intestine of this species. Mean amylolytic activity in the upper anterior intestine of L. richardsoni was 274.4 units.g- 1, nine times greater than in its lower anterior intestine. Amylolytic activity in the posterior intestine and liver of all three species was negligible. Mean amylolytic activity was significantly higher

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3 ;E3

Geotia austrafis

Mordacia mordax

D A P L

D A P L

Lanpetra richardsoni

iis 200 E E

0 Al A2

P

L

Fig. 6. Mean maximum activity of amylase + 1 SEM in different regions of the alimentary tract of ammocoetes of Geotriaaustralis, Mordacia mordax and Lampetra richardsoni.

in the diverticula of G. australisthan in either the diverticulum of M. mordax or the upper anterior intestine of L. richardsoni.

Discussion Distribution of enzyme activities The activities of the digestive enzymes determined in the present study of larval lampreys were largely confined to those regions of the alimentary canal in which the presumed zymogen cells are most abundant (Strahan and Maclean 1969; Hansen and Youson 1978; Youson 1981; Potter et al. 1986; Hilliard and Potter 1988). These regions are the anterior intestine in L. richardsoniand the intestinal diverticula in G. australisand M. mordax. Although all four enzymes exhibited some limited activity in the upper anterior intestine in G. australis, their activity in this region of the alimentary tract of M. mordax was negligible. This is consistent with the observation that, in contrast to the situation in M. mordax, zymogen cells are present in the extreme upper region of the anterior intestine of G. australis(Hilliard and Potter 1988). Chymotrypsin and trypsin This study has provided the first record of chymotryptic activity in the digestive system of am-

mocoetes and shows that it occurs in representatives of all three lamprey families (Fig. 4). The inability of Hilliard and Potter (1988) to detect this enzyme in their study of the diverticula of G. australis can be attributed to the fact that they used a nonspecific substrate (Ravin et al. 1954) and included an ovomucoid trypsin inhibitor which would have prevented chymotrypsinogen activation. The pattern of activation of chymotrypsinogen differed markedly amongst the exocrine tissues of the three species (Figs 2, 3). Thus, at the extremes, the chymotrypsin of L. richardsoniwas fully active before exposure to exogenous trypsin, whereas that of M. mordax was totally inactive initially and required exposure to trypsin for 120 minutes for full activation. The above difference probably reflects differences in the location of the exocrine cells. Thus, in L. richardsoni, the exocrine cells occur in the lining of the intestine where, as in gnathostomatous fish (Kapoor et al. 1975) and other vertebrates (Vonk and Western 1984), enteropeptidasesecreting cells would also be expected to be found. Since enteropeptidase, which activates trypsin in the alimentary canal, is therefore presumably present in the upper anterior intestine of L. richardsoni, a combined presence of trypsinogen and its catalytic activator enteropeptidase would account for the full activation we have recorded for trypsinogen in this region of the alimentary tract of L. richardsoni. The resultant presence of trypsin would in turn account for the full activation of chymotrypsinogen in this tissue. However, since the zymogen cells of M. mordax are located in a discrete structure, which is not a site for digestion, they occupy a region where, as in the pancreas of higher vertebrates (Vonk and Western 1984), enteropeptidase would not be expected to be found. An absence of enteropeptidase in the diverticulum of M. mordax would account for the complete lack of chymotryptic activity in the cytosol of this structure prior to the addition of exogenous trypsin. Although the activity of chymotrypsin is relatively high in the protopancreas of M. mordax, the activity of trypsin in this organ is negligible, even after exposure to enteropeptidase. This lack of tryptic activity, which contrasts with the situation in the anterior intestine of L. richardsoniand the diver-

8 ticulum of G. australis, is surprising in view of the fact that the chymotrypsinogen of this species is activated by exogenous trypsin. Since trypsin is the only known activator of chymotrypsinogen, the negligible tryptic activity in M. mordax would thus appear anomalous, unless it is due to the presence of a trypsin inhibitor. If such an inhibitor is present, it would provide a parallel with the pancreas of higher vertebrates (Rinderknecht 1986), and would thus likewise protect this discrete lobulated structure from the effects of its own proteolytic secretions. Alternatively, the substrate specificity of the trypsin of M. mordax may differ from that of both L. richardsoniand G. australis. If such a difference does exist, it could have precluded the utilization of the arginine ester, TAME, employed in this investigation. Our inability to detect tryptic activity in the diverticulum of M. mordax parallels the results of Strahan and Maclean (1969). However, these latter workers did record some tryptic activity in the anterior and posterior intestine of larval M. mordax. Since zymogen cells are essentially restricted to the protopancreas in larval M. mordax, and we found virtually no tryptic activity in the intestine after careful flushing, we suggest that the activity Strahan and Maclean recorded in the intestine was the result of the retention of a small amount of tryptic enzyme through an incomplete flushing of the intestine. Such a conclusion would be in agreement with the view proposed earlier, namely that trypsinogen secreted by the protopancreas in M. mordax is only activated after entering the intestine and becoming exposed to enteropeptidase. The situation regarding chymotrypsin in G. australis is intermediate between that of L. richardsoni and M. mordax, in that the chymotryptic activity exhibited prior to the addition of exogenous trypsin was 27% below the maximum it subsequently reached after full activation. As the lining of the diverticula of G. australisis essentially a continuation of the same epithelium as the intestine (Fig. 1), it is thus likely that, in contrast to the situation with the more complex diverticulum of M. mordax, some of its cells may also secrete enteropeptidase. It also seems reasonable to conclude that in G. australis some enteropeptidase would be likely to pass

from the intestine into the lumen of the diverticulum. Both views would be consistent with the fact that trypsinogen and chymyotrypsinogen are both partially activated in G. australis. The above conclusions are consistent with the observation that in selachian fishes, which possess a discrete pancreas, the proteolytic enzymes require activation (Prahl and Neurath 1966). For example, their chymotrypsinogen was inactive until exposed to exogenous trypsin, and it subsequently did not reach maximal activity until 240 min after such exposure. It is also relevant that in the case of the lungfish, Protopterusaethiopicus, which also has a discrete pancreas, the proteolytic enzymes in pancreatic extracts are in the inactive form (Reeck et al. 1970) and therefore are presumably only activated after they have reached the intestine and become exposed to the enteropeptidase-dependent activation cascade. Furthermore, this species possesses a pancreatic trypsin inhibitor, which presumably protects this organ from autolysis.

Lipolytic and amylolytic activities The present study has demonstrated for the first time that lipolytic and amylolytic activities are present in the main exocrine region of the larvae of northern hemisphere lampreys. The detection of lipase and amylase in the diverticula of M. mordax and G. australis parallels the respective results of Strahan and Maclean (1969) and Hilliard and Potter (1988). Our study has shown that, in comparison with the activities in the main exocrine tissues of both L. richardsoniand M. mordax, those of lipase and amylase in the diverticula of G. australisare significantly lower and higher, respectively. There are insufficient comparative data on the microphagous diet of the ammocoetes of these species to elucidate whether these reflect variations in the composition of their food.

Proposed phylogenetic trends The distribution of zymogen cells in the epithelial lining of the anterior intestine, as found in the am-

9 mocoetes of the Petromyzontidae, has long been considered to represent the most primitive vertebrate arrangement of exocrine tissue (Brachet 1897; Barrington 1972). In contrast, the localisation of zymogen cells in the compact and lobulated diverticulum of M. mordax can be regarded as an extreme specialisation towards a discrete exocrine pancreas. It is thus relevant that the southern hemisphere lamprey M. mordax is considered to have evolved from a stock similar to that of the Ichthyomyzon species of the northern hemisphere (Hubbs 1952; Potter and Hilliard 1987), a group which do not possess a protopancreas (Hilliard and Potter 1988). Thus, the protopancreas of Mordacia is considered to have evolved independently of the compact exocrine component of the higher vertebrate pancreas. It is also possible to envisage that, during the evolution of a protopancreas, the exocrine cells of ammocoetes first became concentrated in a diverticulum similar to that of the large left diverticulum of G. australisand that this structure subsequently became transformed into a compact exocrine protopancreas such as is now found in larval M. mordax.

Acknowledgements Our gratitude is expressed to Mr. M. Feldwick and Mr. L. Pen for technical assistance, and to Mr. A. Fink and Mr. J. Koehn for help with collecting animals. We are also indebted to the staff of the Inland Fisheries Commission (Tasmania) and Professor C. Bond of Oregon State University (USA). Thanks are expressed to Professor J.H. Youson for criticisms of the text. Financial support was provided by the Australian Research Council.

References cited Alcock, R. 1899. On proteid digestion in Ammocoetes. J. Anat. 33: 612-637. Barrington, E.J.W. 1936. Proteolytic digestion and the problem of the pancreas in the ammocoete larvae of Lampetraplaneri. Proc. Roy. Soc., Ser. B 121: 221-232. Barrington, E.J.W. 1972. The pancreas and intestine. In The Biology of Lampreys. Vol. 2, pp.135-169. Edited by M.W. Hardisty and .C. Potter. Academic Press, London.

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