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Fluid secretion by the midgut caeca of the crab, Cancer magister
Camp.Biochem. 8
Pergamon
Phwol Vol. blA. pp. 259 to 263 Press Ltd 1980. Printedin GreatBritam
FLUID SECRETION BY THE MIDGUT CAECA OF THE CRAB, CANCER MAGISTER CHARLES W. HOLLIDAY’,
DONALD
and
L. MYKLES’,
ROBERT C. TERWILLIGER~
LAWRENCE J. DANCK&
‘The Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672. 20ak Ridge National Laboratory. P.O. Box Y, Oak Ridge, TN 37830 and ‘University of Oregon, Oregon Institute of Marine Biology, Charleston, OR 97420. U.S.A.
(Received
16 January
1980)
Abstract-l. The composition and rate of production of fluid by the anterior and posterior midgut caeca (AMC and PMC, respectively) of the crab, Cancer magister, were studied in tGL.0 and in vitro. Under both conditions these structures produced a small volume of fluid (2.2-3.4 pl/hr) which was. with the exception of sulfate ion, nearly isoionic with the serum. 2. Caecal fluid had relatively low levels of amylolytic and, less frequently, proteolytic activities. 3. The in uiuo rate of fluid production was not increased by feeding or by osmotic stress. 4. Ligation of the AMC did not impair secretion of a peritrophic membrane around the fecal strand formed after feeding. 5. It is concluded that the midgut caeca do not have a significant function in osmoregulation or formation of the peritrophic membrane and that they probably have, at best, a minor role in the digestive processes of C. magister. Further, it is suggested that the rate and direction of fluid transport by the midgut caeca may be under neurosecretory control.
MATERIALS
lNTRODUCTlON
The structure and function of the midgut caeca in decapod crustaceans have been reviewed recently by Smith (1978). Decapods possess a single posterior midgut caecum (PMC) which enters the midgut at its junction with the hindgut. In addition, brachyurans and anomurans usually possess a pair of anterior midgut caeca (AMC) which join the midgut just posterior to the pyloric stomach. The ultrastructure of the midgut caeca of the crabs, Pachygrapsus crassipes and Cancer magister, and the lobsters, Homarus americanus and H. gammarus (Mykles, 1977, 1979) resembles that of other epithelia capable of active ion transport (Berridge & Oschman, 1972). Smith (1978) has called attention to the lack of knowledge of the function of these structures. Yonge (1924) has suggested that the midgut caeca serve in nutrient uptake from the midgut. Heeg & Cannone (1966) and Dal1 (1977) have presented evidence that the midgut caeca participate in osmoregulation in the crabs, Cyclogrupsus punctatus, Plagusia chabrus and Scylla serrata. Pugh (1962, as cited by Smith, 1978) and Mykles (1979) have suggested that the midgut caeca in decapods may function in the formation of the peritrophic membrane which surrounds the fecal strand in the midgut. In the present study the role of the midgut caeca in osmoregulation and digestive processes was examined in the Dungeness crab, Cancer magister. In uioo rates of fluid secretion were measured in response to feeding or exposure to dilute seawater. In addition, ionic composition and enzymatic activity of the fluid secreted by in uiuo and in vitro preparations were determined.
AND METHODS
Large
(0.8-1.2 kg), male specimens of the crab, Cancer from commercial suppliers in Charleston, Oregon, in July and August, 1978, and were kept in running seawater (33-342,. IO-14’C) at the Oregon Institute of Marine Biology. Charleston, Oregon. The crabs were fed fish scraps at approx weekly intervals, but were starved for 48 hr prior to use in experiments. This period was sufficient to clear the gut of feces.
magister Dana, were purchased
259
In vivo studies AMC function was studied in ciao in crabs with catheters ligatured within the AMC. Crabs were secured dorsal side up in a dissecting tray and packed in ice for 20-30min. The gastric region of the carapace was dried and cleaned carefully with acetone. A I cm2 area of the carapace above and 2 cm anteriolateral to the junction of an AMC with the midgut was marked with a felt-tip pen. A thin, circular bead of hot melt glue (Thermogrip. U.S.M., Reading. PA, a thermoplastic adhesive) was put on the carapace around the marked area as a quick succession of thin spots in order not to burn the hypodermis beneath the carapace. When it had cooled, the bead of hot melt glue was secured to the carapace with r-cyanoacrylate glue (Krazy Glue. Inc., Chicago, IL). Thus secured to the carapace, the bead of hot melt glue served as a base for eventual closure of the wound with a thin layer of the same adhesive (see below). The marked area of the carapace enclosed by the bead of hot melt glue was then cut out with a high-speed hand drill. Dust from the carapace and hemolymph were washed away by a slow flow of crab Ringer solution (CR, containing, in meq/I: Na, 499, K. I I ; Ca, 25; Mg, 37; Cl. 528: SOL, 44: pH 7.8) which was controlled by a foot pedal. The wound was kept filled with CR at all times to prevent the entry of air into the heomocoele and subsequent vascular embolism. Working under a dissecting microscope, the hypodermis was reflected to expose the AMC. The AMC is
260
CHARLES W. HOLLIDAY et al.
supplied with hemolymph by a large artery which runs its length; this artery was carefully separated from the AMC for a distance of approximately 1cm by blunt dissection. A nick was made in the AMC approximately 2cm from its junction with the midgut and a small polyethylene catheter (P.E. IO) filled with CR was inserted into the lumen. The catheter was secured in place with two sutures placed around the AMC so that the arterial supply of hemolymph to the distal part of the AMC was not interrupted. The hole in the carapace was carefully closed with hot melt glue such that a dome of glue filled with CR covered the wound; the catheter extended through the dome of glue. A larger diameter tube (P.E. 50) was attached to the catheter and was in turn inserted through a hole in the screw cap of a small glass vial and glued in place in the screw cap; the cap also contained a short length of P.E. IO tubing which served as a vent. A clean, dry vial was weighed. attached to the screw cap containing the catheter and the vial was inserted in a short length of large diameter silicone rubber tubing which had been glued onto the crab’s carapace in order to hold the vial in place. The end of the catheter was approx I cm below the AMC in all preparations. This level was chosen for ease of mounting the collecting vial on the crab; both in ri~‘o and in cirro (see below) preparations could secrete fluid against a hydrostatic pressure head of approx 5 cm H,O. The crab was then secured in a shallow pan supplied with running 1001, seawater (I2 i_ 2’C) in such a manner as to allow the crab to ventilate its gills while not immersing the vial affixed to its carapace. A period of 24 hr was allowed for caecal fluid production to clear CR from the catheter. The rate of caecal fluid production was measured by manually drying the outside of the vial and reweighing it at the end of a given period of time: the volume of fluid produced was calculated assuming unit density (2 samples of caecal fluid tested were 95’: and 96”/:, water). Mortality was less than 5”,, during the 4 days following surgery and fluid production normally continued for 4-8 days after catheterization at a nearly constant rate. Samples of caecal fluid for ion and enzyme assays were taken 2448 hr after catheterization. In experiments to measure the effect of osmotic stress or feeding on the rate of fluid production. the period between 24 and 48 hr after catheterization was taken as a control rate. Osmotic stress was applied by exposing the crab to aerated 2/3 seawater (22”,,,,) in an incubation chamber at I2 k 1’C. Feeding was accomplished in the apparatus by giving the crabs l&l5 g of fish fillet. Catheterization of the PMC was not attempted: it lies beneath the highly branched superior abdominal artery and is much less accessible than the AMC. In vitro studies Fluid production by AMC and PMC was measured in with excised caeca. A 30ml sample of hemolymph was taken and allowed to clot in a glass beaker, and the crab was then killed by destruction of the thoracic ganglia. The crab was secured in a dissecting tray, the dorsal carapace was cut away with a high speed drill and the hypodermis was reflected. AMC and PMC were ligatured near their junctions with the midgut. dissected free and transferred to a small petri dish containing serum from the same crab. The petri dish was fitted with a P.E. IO catheter extending through its side 2 mm below the level of the crab serum. Midgut caeca were mounted on the catheter. ligatured in place and the petri dish was covered and placed in an incubation chamber at I2 + 1°C. The level of the catheter draining the caecum was I cm below that of the caecum. which floated in the serum. The rate of caecal fluid production was measured by noting the progress of the fluid meniscus down the catheter, which had been gravimetrically cahbrated with a known volume of distilled water. These preparations produced fluid at a slowly derirro
clining rate for 12-24 hr after catheterization. Samples for ion and enzyme assays were taken 6-12 hr after catheterization. In both in tiioo and in oitro preparations the rate of fluid production is presented as &hr/midgut caecum. Enzyme activity
Amylolytic activity of caecal fluid was measured using the starch film method of Tremblay (1962). Proteolytic activity of caecal fluid was measured by the method of Adams & Tquan (1960). using the gelatin emulsion of developed and washed (24 hr) Kodak Kodachrome 25 color transparency film. Both assays were performed by putting a small sample (S-10 ~1) of caecal fluid on the appropriate film and incubating for I hr at IS-2O’C in a watersaturated atmosphere. Lipase activity was measured using a commercially available kit (Sigma Chemical Co., St Louis, MO, Sigma Technical Bulletin No. 800).
Ion analyses Ion analysis on 5-20 ~11aliquots of caecal fluid or serum were performed as described by Hunter & Rudy (1975). Sodium, potassium and calcium were determined by flame photometry, magnesium and sulfate were measured spectrophotometrically and chloride was measured by electrometric titration with silver ion. Standard deviations for five determinations of the indicated standards were as follows: Na, 450 meq/l, k8; K 12 meqjl. iO.3; Ca. 16.0meq/l, f0.4; Mg. 30.0meq/l, kO.8: SO,, 30.0meq/l. hO.3. Because of the small amounts of caecal fluid available from any one preparation, it was not possible to perform all ion analyses on fluid from the same caecum. However, all ion levels in caecal fluid are presented with values in serum from the same preparation. The pH of caecal fluid and serum was estimated with pHydrion papers (@, Micro Essential Laboratory, Brooklyn, NY). Sodium dodecylsulfate polyacrylamide gel electrophoresis was carried out on 1.5 mm slab gels according to Studier (1973) with the discontinuous buffer system of Laemmli (I 970). Statistical
analysis
The significance of the difference between mean values was assessed using a paired t-test. Probabilities were calculated using the calculated values of t; values ~0.05 are considered significant: those GO.01 are considered highly significant.
RESULTS Secretion
of,fiuid
The rates of fluid secretion by catheterized AMC measured both in uioo with intact hemolymph circulation, and in t’itro were low and were not significantly different from one another (Table I). Thus, although the AMC are well vascularized, intact circulation is not necessary for fluid production for 612 hr PMC are larger than AMC and also showed a significantly higher rate of fluid production in vitro (Table l), The crabs used in this study weighed between 0.8 and I.2 kg; assuming a I kg crab, the total fluid output of the three midgut caeca is approx 2OOpl/day or 0.02% body weight/day. 111 vivo AMC preparations normally survived 4-8 days with a nearly constant rate of fluid production and showed no signs of swelling when dissected. The rate of in viro fluid secretion in catheterized AMC was not changed significantly by exposing the crabs to 2/3 seawater for 24 hr (Table 2). Similarly, fluid secretion by catheterized AMC remained unchanged after feeding (Table 2).
Fluid secretion Table
1. Ionic composition
by crab midgut
caeca
of caecal fluid and serum and rate of caecal fluid in catheterized PMC of C. tnagisrer
Ionic
Rate of flow (,,l/h)
Preparation
In viva
AMC
(10)
2.2to.3
(10)
(7)
2.5t0.2(7)
Serum Pl
PMC
(10)
3.4*0.3(10)
Ca
Mg
Cl
SO4
8.0*0.7(4)
29?4(5)
12.711.3(4)
503t14(4)
15*1(5)
7.5*0.3(4)
35+4(5)
15.Oi1.7( 4)
50&8(4)
44+4(5)
>0.05
70.05
>0.05
70.05
510?8(3)
11.6(2)
35+1(3)
16.6(2)
524~12(3)
17+1(3)
502+5(3)
10.2(2)
42+1(3)
19.9(2)
519t3(3)
44'1(3)
>0.05
507+3(6) 497+14(fi)
Pl
(meq/l)
496?12(5)
_
co.05
11.4cO.6(4)
31i2(4)
11.5?1.0(4)
511+14(4)
10+4(4)
9.0t0.4(4)
39?2(4)
14.4+1.0(4)
508?17(4)
42t2(4)
>0.05
Serum
Concentration
AMC and
495k14(5)
20.05
Pl
AMC
K
Na
Serum
In vitro
261
eo.05
,0.05
<0.05
.0.05
r0.05
Serum ionic levels shown for comparison were taken from the same crab or preparation. Rate of fluid production was measured 24-48 h after catheterization in in uiuo preparations and 612 hr after catheterization in in vitro preparations. Mean values + SE; numbers in parentheses indicate the number of preparations tested. ‘P = probability that mean values differ by chance alone (t-test).
Fluid composition With the exception of sulfate ion, the caecal fluid from both in uiuo and in vitro preparations had an ionic composition nearly identical to that of serum (Table 1). Magnesium and potassium were significantly lower and higher, respectively, than serum in some in uitro preparations, but sulfate ion levels were much less than serum levels to a highly significant degree in all preparations. It seems likely that the low concentration of sulfate is due to the large size of the ion and its consequently slow rate of diffusion from hemolymph into the caecal fluid. The pH of the fluid produced by in uiuo and in vitro preparations was z 7-8 ; that of the serum was 18. Fluid from in uiuo and in vitro AMC and PMC preparations was examined for enzymatic activity (Table 3). Amylolytic activity was the most consistently present activity in in uiuo preparations (8 of 9 tested), proteolytic activity was present in approxi-
Table
2. Effect of osmotic fluid production
mately half (4 of 9 tested) of the preparations and no lipolytic activity was detected in the four preparations tested. Fluid from a few in vitro preparations was also tested for amylolytic and proteolytic activity and the results were roughly comparable (Table 3). In preliminary experiments both the amylase and protease in the caecal fluid were found to be heatlabile; boiling the fluid for 1 min eliminated enzymatic activity. Further, protein was found in caecal fluid and preliminary molecular weight determinations using SDS gel electrophoresis indicate. that a major protein of MW -‘41,000 is present in the fluid. However, the relative activities of these enzymes (as estimated by the time required at 20-C to digest a starch or gelatin film) were much less than those of the stomach fluid in four of five preparations tested. Secretion of peritrophic membrane
(VI/h)
Osmotic
stress
Feeding
(6)
(5)
significantly
stress and feeding on the rate of in riro caecal by cannulated AMC in C. magister
Control rate of fluid production Treatment
If the AMC participate
Rate for 24 h after treatment (ul/h)
Pl
2.2 t 0.5
2.4 f 0.4
>0.05
2.3 t 0.3
2.2
>0.05
* 0.3
AMGC were cannulated, the crabs were allowed to recover for 24 hr and a 24 hr control rate of fluid production was established. Crabs were then immersed in 2/3 seawater or fed and the rate of fluid production was measured for an additional 24 hr. Mean values + SE; numbers in parentheses indicate the number of preparations. ‘P = probability that mean values differ by chance alone (t-test).
in the forma-
262
CHARLES
W. HOLLIDAYet al.
Table 3. Enzymatic activities of caecal fluid
Amylolytic Preparation
+
In viva AMC --
a
In vitro AMC -__ PMC
2 2
activity
Proteolytic
from C.
activity
tnagister Lipolytic
+
4
5
1
0
4
0
1
2
1
Signs indicate the presence (+) or absence (-) number of preparations tested. tion of the peritrophic membrane, then ligation of these structures should impair or eliminate its formation. Both AMC of four starved crabs were ligated near their junctions with the midgut and the crabs were fed 10-15 g of fish fillet. Four starved crabs were sham-operated and similarly fed. An apparently normal peritrophic membrane was present around the feces voided 18-24 hr after feeding in both groups of crabs, indicating that the AMC do not participate significantly in the formation of the peritrophic membrane. DISXSSION Feeding and digestion Pugh (1962, as cited by Smith, 1978) and Mykles (1979) suggest that midgut caeca may participate in secretion of the peritrophic membrane. However, ligation of Cancer magister AMC had no effect on formation of the peritrophic membrane. It could be argued that the intact PMC in experimental crabs produced the membrane, but this seems unlikely, as a peritrophic membrane is present around the fecal strand throughout the entire length of the midgut in intact crabs. Mykles (1979) has described membrane-bound, pleomorphic vesicles in the apical cytoplasm of epithelial cells of C. magister, Homarus americatms and H. gammarus midgut and midgut caeca, and has suggested that these vesicles contain material from which the peritrophic membrane is made. The results of the present investigation are consistent with this interpretation. Ligation of AMC would not prevent the fornation of peritrophic membrane because the lining of the midgut proper would continue to synthesize and secrete peritrophic membrane materials. The midgut caeca of C. magister produce enzymes that may act in the posterior part of the midgut (Table 3). It is unlikely that the enzymatic activity is derived from midgut fluid forced into the AMC before catheterization because fluid produced by itt ciuo preparations continued to show enzymatic activity for several days after catheterization. Digestive processes in decapod crustaceans are thought to take place in the stomach and highly-branched midgut gland (hepatopancreas), anterior to the junction of the midgut caeca with the midgut (van Weel, 1974). Thus, the digestive role of the small volume of relatively lowactivity fluid produced by the midgut caeca is questionable. However. it is possible that enzymes are secreted by the midgut caeca in an inactive form and subsequently activated in the midgut. If so, it is dificult to understand why the fecal strand is exposed to
activity
+
of activity:
0
numbers
4
indicate the
enzymes after it has been consolidated and invested with a peritrophic membrane: the membrane would probably deter mixing of the enzymes with their presumed substrates in the fecal strand. Yonge (1924) has suggested that the midgut caeca serve in nutrient uptake from the midgut. Dal1 (1967) and Smith (1978) found no food material in midgut caeca of several crustacean species, indicating the fluid flows out of the caeca into the midgut and not in the opposite direction. Smith (1978) also reported that carbon particles ingested with food never appear in the midgut caeca of three species of crabs. The results of the present study and the findings of Dal1 (1967) and Smith (1978) are not consistent with Yonge’s (1924) hypothesis. However. there are instances where fecal material is found within midgut caeca. Mykles (unpublished observations) and Bayer et al. (1979) have frequently found fecal material in the PMC of the lobster, H. americamts. Osmoregulatiotl andfluid
absorption at tnolr
C. magister is a moderately good osmoregulator in media as dilute as 30-50x seawater (Hunter & Rudy, 1975) and the urinary rate in 7.5% seawater is -774 body weight/day (Holliday, 1977). indicating that the crab is experiencing a large osmotic influx of water in dilute media. The results of the present study indicate that the midgut caeca of C. magister produce a fluid which, with the exception of sulfate ion, is essentially isoionic with the serum. Even if the midgut caeca produced an extremely dilute fluid, the rate of fluid production is much too low (2.0.02?< body weight/day) to make a significant contribution to osmoregulation. The rate of fluid production did not increase with osmotic stress, a finding that is not consistent with the hypothesis that the caeca function in osmoregulation (Heeg & Cannone, 1966; Dali, 1967). Mykles (1977) reported that there were no significant changes in the width of lateral intercellular spaces in PMC from Pachygrapsus crassipes adapted to low salinity. This provides indirect evidence that the rate of fluid secretion did not increase and is consistent with our observations of C. magisrer. It should be noted that the midgut caeca of the crabs, P. crassiprs and C. magisrer and the lobsters, H. americanus and H. gammarus. are innervated by putative neurosecretory axons (Mykles. 1977, 1979). The gross anatomy of the nervous supply to the AMC is not known and thus it is possible that the catheterization procedure used in the present study resulted in the transection of the AMC nerves. We suggest that the rate (and direction. see below) of fluid production
Fluid secretion
by crab midgut
may be controlled by release of neurosecretory material from these axons. Thus, it is possible that only basal rates of fluid secretion were measured in the present study. Mykles (1979) has shown that the midgut caeca of several decapods, including C. rnagistvr are ultrastructurally similar to the midgut proper. which is known to absorb fluid at an increased rate at ecdysis (Mykles & Ahearn, 1978; Mykles, 1980). It is possible that the midgut caeca amplify the absorptive surface area of the midgut, and thus permit the animal to absorb greater quantities of fluid at ecdysis. Although the results of the present study are not consistent with the hypotheses that the midgut caeca have a significant function in osmoregulation (Heeg & Cannone, 1966; Dali, 1967) or in digestive processes (Yonge, 1924; Pugh. 1962; Mykles, 1979), the function of these structures is still largely unknown. We suggest that the midgut caeca may have two functions: (1) fluid production during most of the moult cycle (present study) and (2) fluid absorption from the midgut at ecdysis (Mykles & Ahearn. 1978; Mykles, 1980). Thus, the decapod midgut caecum may prove to be an interesting tissue in which fluid transport may proceed in either direction. The presence of putative neurosecretory axons in the midgut and midgut caeca of several decapods (Mykles, 1977, 1979; Smith, 1978) suggests that the rate and, perhaps, direction of fluid transport may be under neurosecretory control. Acknow[edyenlenrs-This study was supported in part by a grant from The Lerner Fund for Marine Research of the American Museum of Natural History to CWH, NSF grant PCM 76-20948 to RCT. NSF grant PCM 75-16345 to Howard A. Bern. Department of Zoology, University of California Berkeley and a Sea Grant traineeship to DLM through the Aqualculture Project. Bodega Marine Laboratory, Bodega, CA. We thank Drs Stephen Vigna and Joan D. Ferraris for their comments on the original manuscript. REFERENCES ADAMSC. W. M. & TLQAN N. A. (1960) The histochemical demonstration of proteases by a gelatine-silver film substrate. J. Histochem. C~rochem. 9. 469472.
caeca
263
BAYER R. C.. GALLAGHER M. L., LEAVITT D. F. & RITrENBURG J. H. (1979) A radiographic study of the lobster (Homarus americanus) alimentary canal. Proc. /f&k Ann. Meer. World Maricult. Sot. 10, 19-21. BERRIDGE M. J. and OSCHMAN J. L. (1972) Trurlrporrirxg Epithelia. Academic Press, New York. DALL W. (1967) Hypo-osmotic regulation in Crustacea. Camp. Biochem. Physiol. 21, 653-678. HEEG J. & CANNONE A. J. (1966) Osmoregulation by means of a hitherto unsuspected organ in two grapsid crabs. Zool. Africuna 2, 127-I 29. HOLLIDAY C. W. (1977) A new method for measuring the
urinary rate of a brachyuran crab. Camp.
Biochem.
P/I!,-
siof. 58A, 119-120.
HUNTER K. C. and RUIIY P. P. (1975) Osmotic regulation in the Dungeness crab, Catlcer mugister (Dana). Camp. hiochem. Physiol. 51A. 439447. LAEMMLI U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Nut~oc 227, 68s-685. MYKLES D. L. (1977) The ultrastructure of the posterior midgut caecum of Pachygrapsus crnssipes (decapoda. Brachvura) adapted to low salinity. Tiss. Cell 9. 681 69 I MYKLES-D. i. (1939) Ultrastructure-of alimentary epithelia of lobsters, Homarus umericunus and H. gummaru.s. and crab, Cancer magisrer. Zoomorphologie. 92, 20 1~2 15. MYKLES D. L. (1980) The mechanism of fluid absorption at ecdysis in the American lobster. Homurus umrrictmx~. J. exp. Biol. 84. 89-101. MYKLES D. L. & AHEARN G. A. (1978) Changes in fluid transport across the perfused midgut of the freshwater prawn, Macrohrachium rosenhergii, during the molting cycle. Camp. Biochem. Physiol. 61A. 643-645. PUGH J. E. (1962) A contribution toward a knowledge of the hind-gut of fiddler crabs (Decapoda, Grapsidae). Trans. Am. microsc. Sot. 81. 309-320. SMITH R. 1. (1978) The midgut caeca and the limits of the hind-gut of brachyura: a clarification. Cru,\raceu~~cr 35. 196-205. STUDIER F. W. (1973) Analysis of bacteriophage T, earl!
RNAs and proteins on slab gels.
J.
rnolec.
Biol.
79.
237-248.
TREMBLAYG. (1963) The localisation of amylase activit) in tissue sections by a starch-film method. J. Hi~rochem. Cyrochem. 11. 202-206. WEEL P. B. VAN (1974) Hepatopancreas? Contp. Biochem. Physiol. 47, 1-9. YONGF C. M. (1924) The mechanism of feeding and digchtion in Nrpltrops rlorwgicus. J. KYp. Biol. 1. 343 -3X9.
Pergamon
Phwol Vol. blA. pp. 259 to 263 Press Ltd 1980. Printedin GreatBritam
FLUID SECRETION BY THE MIDGUT CAECA OF THE CRAB, CANCER MAGISTER CHARLES W. HOLLIDAY’,
DONALD
and
L. MYKLES’,
ROBERT C. TERWILLIGER~
LAWRENCE J. DANCK&
‘The Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672. 20ak Ridge National Laboratory. P.O. Box Y, Oak Ridge, TN 37830 and ‘University of Oregon, Oregon Institute of Marine Biology, Charleston, OR 97420. U.S.A.
(Received
16 January
1980)
Abstract-l. The composition and rate of production of fluid by the anterior and posterior midgut caeca (AMC and PMC, respectively) of the crab, Cancer magister, were studied in tGL.0 and in vitro. Under both conditions these structures produced a small volume of fluid (2.2-3.4 pl/hr) which was. with the exception of sulfate ion, nearly isoionic with the serum. 2. Caecal fluid had relatively low levels of amylolytic and, less frequently, proteolytic activities. 3. The in uiuo rate of fluid production was not increased by feeding or by osmotic stress. 4. Ligation of the AMC did not impair secretion of a peritrophic membrane around the fecal strand formed after feeding. 5. It is concluded that the midgut caeca do not have a significant function in osmoregulation or formation of the peritrophic membrane and that they probably have, at best, a minor role in the digestive processes of C. magister. Further, it is suggested that the rate and direction of fluid transport by the midgut caeca may be under neurosecretory control.
MATERIALS
lNTRODUCTlON
The structure and function of the midgut caeca in decapod crustaceans have been reviewed recently by Smith (1978). Decapods possess a single posterior midgut caecum (PMC) which enters the midgut at its junction with the hindgut. In addition, brachyurans and anomurans usually possess a pair of anterior midgut caeca (AMC) which join the midgut just posterior to the pyloric stomach. The ultrastructure of the midgut caeca of the crabs, Pachygrapsus crassipes and Cancer magister, and the lobsters, Homarus americanus and H. gammarus (Mykles, 1977, 1979) resembles that of other epithelia capable of active ion transport (Berridge & Oschman, 1972). Smith (1978) has called attention to the lack of knowledge of the function of these structures. Yonge (1924) has suggested that the midgut caeca serve in nutrient uptake from the midgut. Heeg & Cannone (1966) and Dal1 (1977) have presented evidence that the midgut caeca participate in osmoregulation in the crabs, Cyclogrupsus punctatus, Plagusia chabrus and Scylla serrata. Pugh (1962, as cited by Smith, 1978) and Mykles (1979) have suggested that the midgut caeca in decapods may function in the formation of the peritrophic membrane which surrounds the fecal strand in the midgut. In the present study the role of the midgut caeca in osmoregulation and digestive processes was examined in the Dungeness crab, Cancer magister. In uioo rates of fluid secretion were measured in response to feeding or exposure to dilute seawater. In addition, ionic composition and enzymatic activity of the fluid secreted by in uiuo and in vitro preparations were determined.
AND METHODS
Large
(0.8-1.2 kg), male specimens of the crab, Cancer from commercial suppliers in Charleston, Oregon, in July and August, 1978, and were kept in running seawater (33-342,. IO-14’C) at the Oregon Institute of Marine Biology. Charleston, Oregon. The crabs were fed fish scraps at approx weekly intervals, but were starved for 48 hr prior to use in experiments. This period was sufficient to clear the gut of feces.
magister Dana, were purchased
259
In vivo studies AMC function was studied in ciao in crabs with catheters ligatured within the AMC. Crabs were secured dorsal side up in a dissecting tray and packed in ice for 20-30min. The gastric region of the carapace was dried and cleaned carefully with acetone. A I cm2 area of the carapace above and 2 cm anteriolateral to the junction of an AMC with the midgut was marked with a felt-tip pen. A thin, circular bead of hot melt glue (Thermogrip. U.S.M., Reading. PA, a thermoplastic adhesive) was put on the carapace around the marked area as a quick succession of thin spots in order not to burn the hypodermis beneath the carapace. When it had cooled, the bead of hot melt glue was secured to the carapace with r-cyanoacrylate glue (Krazy Glue. Inc., Chicago, IL). Thus secured to the carapace, the bead of hot melt glue served as a base for eventual closure of the wound with a thin layer of the same adhesive (see below). The marked area of the carapace enclosed by the bead of hot melt glue was then cut out with a high-speed hand drill. Dust from the carapace and hemolymph were washed away by a slow flow of crab Ringer solution (CR, containing, in meq/I: Na, 499, K. I I ; Ca, 25; Mg, 37; Cl. 528: SOL, 44: pH 7.8) which was controlled by a foot pedal. The wound was kept filled with CR at all times to prevent the entry of air into the heomocoele and subsequent vascular embolism. Working under a dissecting microscope, the hypodermis was reflected to expose the AMC. The AMC is
260
CHARLES W. HOLLIDAY et al.
supplied with hemolymph by a large artery which runs its length; this artery was carefully separated from the AMC for a distance of approximately 1cm by blunt dissection. A nick was made in the AMC approximately 2cm from its junction with the midgut and a small polyethylene catheter (P.E. IO) filled with CR was inserted into the lumen. The catheter was secured in place with two sutures placed around the AMC so that the arterial supply of hemolymph to the distal part of the AMC was not interrupted. The hole in the carapace was carefully closed with hot melt glue such that a dome of glue filled with CR covered the wound; the catheter extended through the dome of glue. A larger diameter tube (P.E. 50) was attached to the catheter and was in turn inserted through a hole in the screw cap of a small glass vial and glued in place in the screw cap; the cap also contained a short length of P.E. IO tubing which served as a vent. A clean, dry vial was weighed. attached to the screw cap containing the catheter and the vial was inserted in a short length of large diameter silicone rubber tubing which had been glued onto the crab’s carapace in order to hold the vial in place. The end of the catheter was approx I cm below the AMC in all preparations. This level was chosen for ease of mounting the collecting vial on the crab; both in ri~‘o and in cirro (see below) preparations could secrete fluid against a hydrostatic pressure head of approx 5 cm H,O. The crab was then secured in a shallow pan supplied with running 1001, seawater (I2 i_ 2’C) in such a manner as to allow the crab to ventilate its gills while not immersing the vial affixed to its carapace. A period of 24 hr was allowed for caecal fluid production to clear CR from the catheter. The rate of caecal fluid production was measured by manually drying the outside of the vial and reweighing it at the end of a given period of time: the volume of fluid produced was calculated assuming unit density (2 samples of caecal fluid tested were 95’: and 96”/:, water). Mortality was less than 5”,, during the 4 days following surgery and fluid production normally continued for 4-8 days after catheterization at a nearly constant rate. Samples of caecal fluid for ion and enzyme assays were taken 2448 hr after catheterization. In experiments to measure the effect of osmotic stress or feeding on the rate of fluid production. the period between 24 and 48 hr after catheterization was taken as a control rate. Osmotic stress was applied by exposing the crab to aerated 2/3 seawater (22”,,,,) in an incubation chamber at I2 k 1’C. Feeding was accomplished in the apparatus by giving the crabs l&l5 g of fish fillet. Catheterization of the PMC was not attempted: it lies beneath the highly branched superior abdominal artery and is much less accessible than the AMC. In vitro studies Fluid production by AMC and PMC was measured in with excised caeca. A 30ml sample of hemolymph was taken and allowed to clot in a glass beaker, and the crab was then killed by destruction of the thoracic ganglia. The crab was secured in a dissecting tray, the dorsal carapace was cut away with a high speed drill and the hypodermis was reflected. AMC and PMC were ligatured near their junctions with the midgut. dissected free and transferred to a small petri dish containing serum from the same crab. The petri dish was fitted with a P.E. IO catheter extending through its side 2 mm below the level of the crab serum. Midgut caeca were mounted on the catheter. ligatured in place and the petri dish was covered and placed in an incubation chamber at I2 + 1°C. The level of the catheter draining the caecum was I cm below that of the caecum. which floated in the serum. The rate of caecal fluid production was measured by noting the progress of the fluid meniscus down the catheter, which had been gravimetrically cahbrated with a known volume of distilled water. These preparations produced fluid at a slowly derirro
clining rate for 12-24 hr after catheterization. Samples for ion and enzyme assays were taken 6-12 hr after catheterization. In both in tiioo and in oitro preparations the rate of fluid production is presented as &hr/midgut caecum. Enzyme activity
Amylolytic activity of caecal fluid was measured using the starch film method of Tremblay (1962). Proteolytic activity of caecal fluid was measured by the method of Adams & Tquan (1960). using the gelatin emulsion of developed and washed (24 hr) Kodak Kodachrome 25 color transparency film. Both assays were performed by putting a small sample (S-10 ~1) of caecal fluid on the appropriate film and incubating for I hr at IS-2O’C in a watersaturated atmosphere. Lipase activity was measured using a commercially available kit (Sigma Chemical Co., St Louis, MO, Sigma Technical Bulletin No. 800).
Ion analyses Ion analysis on 5-20 ~11aliquots of caecal fluid or serum were performed as described by Hunter & Rudy (1975). Sodium, potassium and calcium were determined by flame photometry, magnesium and sulfate were measured spectrophotometrically and chloride was measured by electrometric titration with silver ion. Standard deviations for five determinations of the indicated standards were as follows: Na, 450 meq/l, k8; K 12 meqjl. iO.3; Ca. 16.0meq/l, f0.4; Mg. 30.0meq/l, kO.8: SO,, 30.0meq/l. hO.3. Because of the small amounts of caecal fluid available from any one preparation, it was not possible to perform all ion analyses on fluid from the same caecum. However, all ion levels in caecal fluid are presented with values in serum from the same preparation. The pH of caecal fluid and serum was estimated with pHydrion papers (@, Micro Essential Laboratory, Brooklyn, NY). Sodium dodecylsulfate polyacrylamide gel electrophoresis was carried out on 1.5 mm slab gels according to Studier (1973) with the discontinuous buffer system of Laemmli (I 970). Statistical
analysis
The significance of the difference between mean values was assessed using a paired t-test. Probabilities were calculated using the calculated values of t; values ~0.05 are considered significant: those GO.01 are considered highly significant.
RESULTS Secretion
of,fiuid
The rates of fluid secretion by catheterized AMC measured both in uioo with intact hemolymph circulation, and in t’itro were low and were not significantly different from one another (Table I). Thus, although the AMC are well vascularized, intact circulation is not necessary for fluid production for 612 hr PMC are larger than AMC and also showed a significantly higher rate of fluid production in vitro (Table l), The crabs used in this study weighed between 0.8 and I.2 kg; assuming a I kg crab, the total fluid output of the three midgut caeca is approx 2OOpl/day or 0.02% body weight/day. 111 vivo AMC preparations normally survived 4-8 days with a nearly constant rate of fluid production and showed no signs of swelling when dissected. The rate of in viro fluid secretion in catheterized AMC was not changed significantly by exposing the crabs to 2/3 seawater for 24 hr (Table 2). Similarly, fluid secretion by catheterized AMC remained unchanged after feeding (Table 2).
Fluid secretion Table
1. Ionic composition
by crab midgut
caeca
of caecal fluid and serum and rate of caecal fluid in catheterized PMC of C. tnagisrer
Ionic
Rate of flow (,,l/h)
Preparation
In viva
AMC
(10)
2.2to.3
(10)
(7)
2.5t0.2(7)
Serum Pl
PMC
(10)
3.4*0.3(10)
Ca
Mg
Cl
SO4
8.0*0.7(4)
29?4(5)
12.711.3(4)
503t14(4)
15*1(5)
7.5*0.3(4)
35+4(5)
15.Oi1.7( 4)
50&8(4)
44+4(5)
>0.05
70.05
>0.05
70.05
510?8(3)
11.6(2)
35+1(3)
16.6(2)
524~12(3)
17+1(3)
502+5(3)
10.2(2)
42+1(3)
19.9(2)
519t3(3)
44'1(3)
>0.05
507+3(6) 497+14(fi)
Pl
(meq/l)
496?12(5)
_
co.05
11.4cO.6(4)
31i2(4)
11.5?1.0(4)
511+14(4)
10+4(4)
9.0t0.4(4)
39?2(4)
14.4+1.0(4)
508?17(4)
42t2(4)
>0.05
Serum
Concentration
AMC and
495k14(5)
20.05
Pl
AMC
K
Na
Serum
In vitro
261
eo.05
,0.05
<0.05
.0.05
r0.05
Serum ionic levels shown for comparison were taken from the same crab or preparation. Rate of fluid production was measured 24-48 h after catheterization in in uiuo preparations and 612 hr after catheterization in in vitro preparations. Mean values + SE; numbers in parentheses indicate the number of preparations tested. ‘P = probability that mean values differ by chance alone (t-test).
Fluid composition With the exception of sulfate ion, the caecal fluid from both in uiuo and in vitro preparations had an ionic composition nearly identical to that of serum (Table 1). Magnesium and potassium were significantly lower and higher, respectively, than serum in some in uitro preparations, but sulfate ion levels were much less than serum levels to a highly significant degree in all preparations. It seems likely that the low concentration of sulfate is due to the large size of the ion and its consequently slow rate of diffusion from hemolymph into the caecal fluid. The pH of the fluid produced by in uiuo and in vitro preparations was z 7-8 ; that of the serum was 18. Fluid from in uiuo and in vitro AMC and PMC preparations was examined for enzymatic activity (Table 3). Amylolytic activity was the most consistently present activity in in uiuo preparations (8 of 9 tested), proteolytic activity was present in approxi-
Table
2. Effect of osmotic fluid production
mately half (4 of 9 tested) of the preparations and no lipolytic activity was detected in the four preparations tested. Fluid from a few in vitro preparations was also tested for amylolytic and proteolytic activity and the results were roughly comparable (Table 3). In preliminary experiments both the amylase and protease in the caecal fluid were found to be heatlabile; boiling the fluid for 1 min eliminated enzymatic activity. Further, protein was found in caecal fluid and preliminary molecular weight determinations using SDS gel electrophoresis indicate. that a major protein of MW -‘41,000 is present in the fluid. However, the relative activities of these enzymes (as estimated by the time required at 20-C to digest a starch or gelatin film) were much less than those of the stomach fluid in four of five preparations tested. Secretion of peritrophic membrane
(VI/h)
Osmotic
stress
Feeding
(6)
(5)
significantly
stress and feeding on the rate of in riro caecal by cannulated AMC in C. magister
Control rate of fluid production Treatment
If the AMC participate
Rate for 24 h after treatment (ul/h)
Pl
2.2 t 0.5
2.4 f 0.4
>0.05
2.3 t 0.3
2.2
>0.05
* 0.3
AMGC were cannulated, the crabs were allowed to recover for 24 hr and a 24 hr control rate of fluid production was established. Crabs were then immersed in 2/3 seawater or fed and the rate of fluid production was measured for an additional 24 hr. Mean values + SE; numbers in parentheses indicate the number of preparations. ‘P = probability that mean values differ by chance alone (t-test).
in the forma-
262
CHARLES
W. HOLLIDAYet al.
Table 3. Enzymatic activities of caecal fluid
Amylolytic Preparation
+
In viva AMC --
a
In vitro AMC -__ PMC
2 2
activity
Proteolytic
from C.
activity
tnagister Lipolytic
+
4
5
1
0
4
0
1
2
1
Signs indicate the presence (+) or absence (-) number of preparations tested. tion of the peritrophic membrane, then ligation of these structures should impair or eliminate its formation. Both AMC of four starved crabs were ligated near their junctions with the midgut and the crabs were fed 10-15 g of fish fillet. Four starved crabs were sham-operated and similarly fed. An apparently normal peritrophic membrane was present around the feces voided 18-24 hr after feeding in both groups of crabs, indicating that the AMC do not participate significantly in the formation of the peritrophic membrane. DISXSSION Feeding and digestion Pugh (1962, as cited by Smith, 1978) and Mykles (1979) suggest that midgut caeca may participate in secretion of the peritrophic membrane. However, ligation of Cancer magister AMC had no effect on formation of the peritrophic membrane. It could be argued that the intact PMC in experimental crabs produced the membrane, but this seems unlikely, as a peritrophic membrane is present around the fecal strand throughout the entire length of the midgut in intact crabs. Mykles (1979) has described membrane-bound, pleomorphic vesicles in the apical cytoplasm of epithelial cells of C. magister, Homarus americatms and H. gammarus midgut and midgut caeca, and has suggested that these vesicles contain material from which the peritrophic membrane is made. The results of the present investigation are consistent with this interpretation. Ligation of AMC would not prevent the fornation of peritrophic membrane because the lining of the midgut proper would continue to synthesize and secrete peritrophic membrane materials. The midgut caeca of C. magister produce enzymes that may act in the posterior part of the midgut (Table 3). It is unlikely that the enzymatic activity is derived from midgut fluid forced into the AMC before catheterization because fluid produced by itt ciuo preparations continued to show enzymatic activity for several days after catheterization. Digestive processes in decapod crustaceans are thought to take place in the stomach and highly-branched midgut gland (hepatopancreas), anterior to the junction of the midgut caeca with the midgut (van Weel, 1974). Thus, the digestive role of the small volume of relatively lowactivity fluid produced by the midgut caeca is questionable. However. it is possible that enzymes are secreted by the midgut caeca in an inactive form and subsequently activated in the midgut. If so, it is dificult to understand why the fecal strand is exposed to
activity
+
of activity:
0
numbers
4
indicate the
enzymes after it has been consolidated and invested with a peritrophic membrane: the membrane would probably deter mixing of the enzymes with their presumed substrates in the fecal strand. Yonge (1924) has suggested that the midgut caeca serve in nutrient uptake from the midgut. Dal1 (1967) and Smith (1978) found no food material in midgut caeca of several crustacean species, indicating the fluid flows out of the caeca into the midgut and not in the opposite direction. Smith (1978) also reported that carbon particles ingested with food never appear in the midgut caeca of three species of crabs. The results of the present study and the findings of Dal1 (1967) and Smith (1978) are not consistent with Yonge’s (1924) hypothesis. However. there are instances where fecal material is found within midgut caeca. Mykles (unpublished observations) and Bayer et al. (1979) have frequently found fecal material in the PMC of the lobster, H. americamts. Osmoregulatiotl andfluid
absorption at tnolr
C. magister is a moderately good osmoregulator in media as dilute as 30-50x seawater (Hunter & Rudy, 1975) and the urinary rate in 7.5% seawater is -774 body weight/day (Holliday, 1977). indicating that the crab is experiencing a large osmotic influx of water in dilute media. The results of the present study indicate that the midgut caeca of C. magister produce a fluid which, with the exception of sulfate ion, is essentially isoionic with the serum. Even if the midgut caeca produced an extremely dilute fluid, the rate of fluid production is much too low (2.0.02?< body weight/day) to make a significant contribution to osmoregulation. The rate of fluid production did not increase with osmotic stress, a finding that is not consistent with the hypothesis that the caeca function in osmoregulation (Heeg & Cannone, 1966; Dali, 1967). Mykles (1977) reported that there were no significant changes in the width of lateral intercellular spaces in PMC from Pachygrapsus crassipes adapted to low salinity. This provides indirect evidence that the rate of fluid secretion did not increase and is consistent with our observations of C. magisrer. It should be noted that the midgut caeca of the crabs, P. crassiprs and C. magisrer and the lobsters, H. americanus and H. gammarus. are innervated by putative neurosecretory axons (Mykles. 1977, 1979). The gross anatomy of the nervous supply to the AMC is not known and thus it is possible that the catheterization procedure used in the present study resulted in the transection of the AMC nerves. We suggest that the rate (and direction. see below) of fluid production
Fluid secretion
by crab midgut
may be controlled by release of neurosecretory material from these axons. Thus, it is possible that only basal rates of fluid secretion were measured in the present study. Mykles (1979) has shown that the midgut caeca of several decapods, including C. rnagistvr are ultrastructurally similar to the midgut proper. which is known to absorb fluid at an increased rate at ecdysis (Mykles & Ahearn, 1978; Mykles, 1980). It is possible that the midgut caeca amplify the absorptive surface area of the midgut, and thus permit the animal to absorb greater quantities of fluid at ecdysis. Although the results of the present study are not consistent with the hypotheses that the midgut caeca have a significant function in osmoregulation (Heeg & Cannone, 1966; Dali, 1967) or in digestive processes (Yonge, 1924; Pugh. 1962; Mykles, 1979), the function of these structures is still largely unknown. We suggest that the midgut caeca may have two functions: (1) fluid production during most of the moult cycle (present study) and (2) fluid absorption from the midgut at ecdysis (Mykles & Ahearn. 1978; Mykles, 1980). Thus, the decapod midgut caecum may prove to be an interesting tissue in which fluid transport may proceed in either direction. The presence of putative neurosecretory axons in the midgut and midgut caeca of several decapods (Mykles, 1977, 1979; Smith, 1978) suggests that the rate and, perhaps, direction of fluid transport may be under neurosecretory control. Acknow[edyenlenrs-This study was supported in part by a grant from The Lerner Fund for Marine Research of the American Museum of Natural History to CWH, NSF grant PCM 76-20948 to RCT. NSF grant PCM 75-16345 to Howard A. Bern. Department of Zoology, University of California Berkeley and a Sea Grant traineeship to DLM through the Aqualculture Project. Bodega Marine Laboratory, Bodega, CA. We thank Drs Stephen Vigna and Joan D. Ferraris for their comments on the original manuscript. REFERENCES ADAMSC. W. M. & TLQAN N. A. (1960) The histochemical demonstration of proteases by a gelatine-silver film substrate. J. Histochem. C~rochem. 9. 469472.
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263
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HUNTER K. C. and RUIIY P. P. (1975) Osmotic regulation in the Dungeness crab, Catlcer mugister (Dana). Camp. hiochem. Physiol. 51A. 439447. LAEMMLI U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Nut~oc 227, 68s-685. MYKLES D. L. (1977) The ultrastructure of the posterior midgut caecum of Pachygrapsus crnssipes (decapoda. Brachvura) adapted to low salinity. Tiss. Cell 9. 681 69 I MYKLES-D. i. (1939) Ultrastructure-of alimentary epithelia of lobsters, Homarus umericunus and H. gummaru.s. and crab, Cancer magisrer. Zoomorphologie. 92, 20 1~2 15. MYKLES D. L. (1980) The mechanism of fluid absorption at ecdysis in the American lobster. Homurus umrrictmx~. J. exp. Biol. 84. 89-101. MYKLES D. L. & AHEARN G. A. (1978) Changes in fluid transport across the perfused midgut of the freshwater prawn, Macrohrachium rosenhergii, during the molting cycle. Camp. Biochem. Physiol. 61A. 643-645. PUGH J. E. (1962) A contribution toward a knowledge of the hind-gut of fiddler crabs (Decapoda, Grapsidae). Trans. Am. microsc. Sot. 81. 309-320. SMITH R. 1. (1978) The midgut caeca and the limits of the hind-gut of brachyura: a clarification. Cru,\raceu~~cr 35. 196-205. STUDIER F. W. (1973) Analysis of bacteriophage T, earl!
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TREMBLAYG. (1963) The localisation of amylase activit) in tissue sections by a starch-film method. J. Hi~rochem. Cyrochem. 11. 202-206. WEEL P. B. VAN (1974) Hepatopancreas? Contp. Biochem. Physiol. 47, 1-9. YONGF C. M. (1924) The mechanism of feeding and digchtion in Nrpltrops rlorwgicus. J. KYp. Biol. 1. 343 -3X9.