Comp. Biochem. Physiol.. 1975. Vol. 52A, pp. 611 to 614. Pergamon Press. Printed in Great Britain

OCCURRENCE AND POSSIBLE ADAPTIVE SIGNIFICANCE OF fl-GLUCURONIDASE

AND ARYLAMIDASE ("LEUCINE AMINOPEPTIDASE') ACTIVITY IN TWO

SPECIES OF MARINE NEMATODES

J. B. JENN1NGS* AND ALICE DEUTSCHe"

Marine Biological Laboratory, Woods Hole, MA 02543, U.S.A.

(Received 28 October 1974)

Abstract--l. In the marine nematode Monhystera denticulata fl-glucuronidase acts extra- and intra- cellularly during digestion of bacteria which constitute the bulk of the diet.

2. fl-glucuronidase could not be detected in Chromadorina germanica. This nematode is an algal feeder.

3. Production of fl-glucuronidase in M. denticulata is interpreted as a specific adaptation to a bac- terial diet.

4. Mature females of both species often show arylamidase ("leucine aminopeptidase") activity in the seminal receptacle.

5. The enzymic activity in the seminal receptacle is believed to reflect digestion and absorption of ageing sperm and is interpreted as an adaptation which increases the amount of protein available to the female for egg production.

I N T R O D U C T I O N

IT IS NOW well established that the majority of freeliv- ing marine nematodes are important primary con- sumers in the marine benthic ecosystem, feeding on diatoms, algae, yeasts and other fungi, bacteria and organic detritus (Chitwood & Timm, 1954; Perkins, 1958; Tietjen, 1967; Jennings & Colam, 1970; Tietjen & Lee, 1973). Virtually nothing is known, though, of the digestive physiology of these animals and in par- ticular no specific digestive enzymes have been identi- fied. This contrasts markedly with the situation in freshwater, terrestrial and parasitic nematodes where proteases, esterases, lipases, amylases, hyaluronidase, and a cellulase, pectinase and chitinase have been identified and, to varying extents, characterized (various studies, summarized by Lee, 1965; Fairbairn, 1969; Rogers, 1969). The specific nature of some of these hydrolases represents, without doubt, close adaptation to particular modes of life and certain types of diet and comparable adaptations could reasonably be expected among marine nematodes, especially those with specialised diets. We report here, from a comparative histoehemical study of two such species, the occurrence of two apparent adaptations of this nature, involving the enzymes fl-glucuronidase and arylamidase ("ieucine aminopeptidase").

doroidea) and Monhystera denticulata Timm (O. Monhys- teroidea). These were isolated from North Sea Harbor, Southampton, New York by Dr. J. J. Lee and cultured with their food organisms by techniques described by Lee et al. (1970) and Tietjen et al. (1970),

For histological examination specimens were fixed in Bouin's or Susa's solutions and whole mounts and sections stained by Ehrlich's haematoxylin and eosin. Spermatozoa in the seminal receptacle of the female reproductive system were demonstrated by the Feulgen method for DNA.

For histochemical studies specimens were fixed in 4% formalin in sea water (pH 6.8) for 3-5 hr at 4°C. fl-glucur- onidase was demonstrated in whole mounts using 6- bromo-2-naphthyl-fl-o-glucopyruronoside (glucuronide) as substrate and Fast Blue B salt as post coupler (Pearse, 1961). Arylamidase activity of the "leucine aminopepti- dase" type was detected by using l-leucyl /3 naphthyl- amide HCI as substrate and Garnet GBC salt as a simul- taneous coupler (Burstone & Folk, 1956), with pretreat- ment of specimens in acetone at 4°C for 15 min to remove lipids which would otherwise give false positive reactions (Gibson & Jennings, 1967).

Controls for the histochemical methods consisted of heat-inactivated specimens (held at 90°C for 5 min prior to incubation), and incubation of normal specimens in media lacking the specific substrates.

The normal food organisms of both species of nema- todes were also examined, to allow recognition of any exo- geneous enzymic reactions in the gut contents.

MATERIALS AND METHODS

The nematodes studied were the two common salt marsh species Chromadorina germanica Biitschli (Order Chroma-

* Present address: Department of Pure and Applied Zoology, University of Leeds, Leeds LS2 9JT, England.

t Present address: Department of Biology, The City College, Convent Avenue at 138th Street, New York, NY 10031, U.S.A.

RESULTS

The nematode alimentary and reproductive systems have been described and illustrated in some detail by Hyman (1951) and histological examination of Chromadorina 9ermanica and Monhystera denticulata did not reveal any features sufficiently different to merit a further description here.

611

612 J.B. JENNINGS AND ALICE DEUTSCH

.

7~

Fig. 1. Monhystera denticulata. Whole mount showing fl-glucuronidase activity (black) in gland cells (gc) at the junction of the oesophagus and intestine, and in the cells of the intestinal wall. The oeso- phagus, and algae and bacteria in the background, show no reaction. Post-coupling method as modified

by Pearse (1961). x 120.

fl-o lucuronidase This enzyme occurs in the intestine of adult speci-

mens of both sexes ofM. denticulata (Fig. 1). It could not be demonstrated in the larval stages of this spe- cies nor in any stage of the life cycle of C. oermanica.

In adult M. denticulata strong positive reactions were given by a group of gland cells which form a cone-like structure at the extreme anterior end of the intestine at its junction with the oesophagus. The merocrine glandular nature of these ceils was con- firmed from histological examination and from this, and their position, it is deduced that secretions from these cells are concerned with extracellular digestion in the lumen of the intestine. The gland cells show a positive enzymic reaction at all times, indicating a continual synthesis and discharge of enzyme. This is in accord with the nutritional habits of the nema- tode which feeds almost entirely and continually on bacteria, continuous low level secretion of digestive enzymes being a characteristic feature of continuous microphagous feeders (Jennings, 1972).

The wall of the intestine posterior to the gland cells consists of a single layer of cuboidal to columnar cells which have brown pigment granules scattered throughout their cytoplasm. This showed consistently a positive reaction for fl-glucuronidase but it was not possible to distinguish any reaction in the pigment granules, due to their intrinsic colouration. It is not known, therefore, whether these represent sites of in- tracellular digestion or simply accumulations of the end products of such a process. The intestinal cells showed no evidence of holocrine or merocrine score-

tory activity and it is concluded that the enzymic acti- vity demonstrated in them indicates intracellular digestion of material absorbed from the gut lumen. The bacteria upon which M. denticulata feeds lack any demonstrable fl-glucuronidase activity, smear preparations giving negative results when tested histo- chemically, so that all the enzymic activity present in the nematode's intestine must be endogenous.

No reaction was found in any part of the rectum.

Ar ylamidase

Strong arylamidase ("leucine aminopeptidase") acti- vity was found in the walls and contents of the seminal receptacles of most of those adult female specimens of C. ~ermanica and M. denticulata whose uteri contained maturing fertilised eggs (Figs. 2 and 3). No reactions were found in any other parts of the body of females of either species, nor were any observed in the juvenile females or in males of any age.

The arylamidase reaction was clearly originating in the walls of the seminal receptacle and spreading in- wards into the sperm contained within its lumen. Over-incubation (beyond the 1-2 hr normally employed) produced some artefact staining of areas around the seminal receptacle but this was not speci- fic and was obviously due to diffusion of the reaction product.

The seminal receptacle of the nematode reproduc- tive system is difficult to locate when normal some- what unspecific histological methods are used but ap- plication of the Feulgen technique, specific for DNA,

C . ~ - ; / _ ~ - . ' ~ ~ ' ~ , : ~ , , . ~ , ~ " , ~v - ~ .', . . , '~ ' - ' : . , '~ , . ,

][ Fig. 2. Chromadorina germanica. Whole mount showing arylamidase ("leucine aminopeptidase") activity in the seminal receptacle (sr) of the female reproductive system. Burstone & Folk (1956) method.

x 200.

Hydrolases in two marine nematodes 613

" - : ~ - ~ ~ . ~ i . ' ':i::~ ~ ............ S i" . : - ~ : ~

• m , 4 : ~ ' :

Fig. 3. Monhystera denticulata. As Fig. 2. x 200..

readily revealed its lOcation in both species and con- firmed that it was the site at which the arylamidase reaction was occurring.

DISCUSSION

The differential occurrence of the enzymes fl-glu- curonidase and arylamidase ("leucine aminopepti- dase") in the two species of marine nematodes examined can be interpreted as probable adaptations to the diets and modes of life of these two animals.

There is a clear cut difference in the diets and it seems highly significant that fl-glucuronidase can only be demonstrated in the gut of Monhystera denticulata, which is predominantly a bacterial feeder. While there can be no doubt that other enzymes are also con- cerned in digestion, fl-glucuronidase appears to play a major part in both extracellular and intracellular digestive processes. This enzyme hydrolyses com- ponents of mucopolysaccharides and may well be im- portant in digesting the polysaccharide coat of the nematode's food organisms.

fl-glucuronidase is produced in the gut of the aber- rant polychaetes H istriobdella homari and Ctenodrilus serratus (Jennings, unpublished work), both of which include a large proportion of bacteria in their diet and it may well be that this enzyme is a characteristic component of the digestive secretions of molt bac- terial feeders.

The intracellular phase of digestion in M. denticu- lata must follow absorption of soluble partially digested materials from the gut lumen, and not pha- gocytosis of discrete particles, as food vacuoles were never seen in the intestinal cells. This procedure does occur elsewhere in the lower invertebrates (Jennings, 1968a) and, indeed, has been reported in other nema- todes (Colam, 1971).

The absence of fl-glucuronidase from the larval stages of M. denticulata may indicate that these are not feeding but are dependent on reserves derived from the egg. In many nematodes the larvae do not feed during moulting or when preparing to moult (Rogers & Sommerville, 1969) and Tietjen & Lee (1972) have shown that larval M. denticulata moult, in fact, four times within 4-6 days of hatching.

The histochemical reaction for arylamidase, using l-leucyl fl naphthylamide HCI as substrate, was ori- ginally regarded as a specific test for leucine amino- peptidase (Burstone & Folk, 1956) but it has been used subsequently as a convenient and valid test for general exopeptidase activity (Jennings, 1962; Gibson & Jennings, 1967; Jennings, 1968b). The occurrence in the seminal receptacles o f C. germanica and M. denticulata of an enzyme, or enzymes, which give a positive reaction to the Burstone & Folk technique

is interpreted, therefore, as an indication that some measure of proteolysis is occurring at these sites. The natural substrate for such proteolysis could, presum- ably, only be the sperm which are stored there and this deduction is supported by suggestions from Chit- wood & Chitwood (1950) and Hyman (1951) that ageing sperm are phagocytosed from the seminal receptacle of the nematode Ascaris and subsequently digested. This phenomenon certainly occurs in the temnocephalid flatworms where excess sperm donated by a sexual partner and stored in the vesicula resor- biens, a chamber closely resembling the nematode seminal receptacle in origin, position and function, are digested and absorbed (Jennings, 1968b). Further, in the temnocephalids the vesicula resorbiens and its contents show precisely the same histochemical reac- tions for arylamidase as are given by the seminal receptacles of C. germanica and M. denticulata. Resorption of sperm in nematodes is not unknown and occurs, for example, in male Nippostrongylus bra- siliensis affected by the immune response of the host (Lee, 1969).

The use of arylamidase or "leucine aminopepti- dase" for digestion of non-dietary protein has been

'reported elsewhere in the nematodes. Davey & Sau (1967) and Roggen et al. (1967) describe its involve- ment in moulting in Phocanema and Xiphinema and Rogers (1965) found it in the ex-sheathing fluids of larval Haemonchus and Trichostroru3ylus. Its more or- thodox use in digestion of dietary protein has also been reported, in Ascaris (Lee, 1962) and Cyathostoma (Colam, 1971). Lee also reports its occurrence in the excretory canals and hypodermis of Ascaris.

Digestion and absorption of sperm in C. germanica and M. denticulata may be linked with the low pro- tein diet of these two nematodes. Adult females will be actively synthesing protein for egg production and it seems likely that ageing sperm present in the seminal receptacle from an earlier copulation are being used as a supplementary supply of protein. In this context it is of considerable interest that Tietjen & Lee (1972) report that M. denticulata copulates two or three times and produces about 60 eggs during a life span of some 34 days. Thus digested aged sperm may well be replaced by fresh ones received in later copulations and these will be available to fertilise further batches of eggs.

Acknowledaements--We thank Professor D. L. Lee for constructive criticism of the manuscript, Dr. J. J. Lee for providing the nematodes and Dr. J. H. Tietjen for identify- ing C. germanica. The work was supported in part by SRC grant B/RG 2016/4 to J. B. Jennings and NSF Grant GA 33388 to Dr. J. J. Lee. It was carried out in association with the 1974 Experimental Invertebrate Zoology Course

614 J.B. JENNINGS AND ALICE DEUTSCH

at the Marine Biological Laboratory, Woods Hole, Massa- chusetts.

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Key Word Index--Adaptation to diet; arylamidase; fl- glucuronidase; digestion; "leucine aminopeptidase"; nema- tode; reproductive system; seminal receptacle; sperm.