THE PROBOSCIS MECHANISM OF ACANTHOCEPHALUS RANAE

Transcription

1 J. Exp. Biol. (1966), With 1 plate and 5 text-figures Printed in Great Britain THE PROBOSCIS MECHANISM OF ACANTHOCEPHALUS RANAE BY R. A. HAMMOND Department of Zoology, University of Bristol* {Received 25 January 1966) Comparatively little is known of the activities and physiology of the Acanthocephala, despite the fact that these animals embody a number of unique structural features of great interest One particularly striking and important acanthocephalan feature is the proboscis, which can be invaginated, and its associated organs. The method of functioning of this ' proboscis apparatus' has been the subject of little direct work, although suggestions of how the system works have been made from time to time (see Hamann, 1891; Kaiser, 1893; Graybill, 1902; Rauther, 1931; Kilian, 1932; Meyer, 1933; Hyman, 1951; Van Cleave, 1952). Many of the theories put forward were based on morphological observations only. The present paper reports the results of an investigation of the activity of Acanthocephahis ranae (Schrank, 1788; Luhe, 1911), removed from the host, with particular reference to the proboscis apparatus. MATERIAL AND METHODS The acanthocephalans were obtained from common toads (Bufo bufo) with naturally acquired infections. Individual toads contained up to 65 worms, but a worm burden of 15 to 25 was more usual. Hobson (1948) reported that diluted sea water was a satisfactory medium for the in vitro maintenance of Ascaris lumbricoides. A similar medium (35 % sea water) was used for maintenance of Acanthocephalus ranae during the present work. The worms kept in it remained active and tended neither to lose nor to gain water during the period of observation (up to 8 hr.). Most observations were carried out on small male worms. These were sufficiently transparent for internal structures to be visible under the microscope. In females the mass of ovarian balls and eggs floating in the pseudocoel tended to obscure details of the internal structures. Detailed analysis of the action of the proboscis apparatus was made from photomicrographic records (35 mm. still and 16 mm. cine) of the animals' activities. A modified version of a technique originated by Pflugfelder (1949) was employed in investigating the function of the lemnisci. Pflugfelder showed that if frogs infected with A. ranae were fed on a mixture of pork fat and the stain Scharlach R, the fat globules in the lemnisci of the acanthocephalans became coloured red. Stain was not taken up by any other part of the parasite. Present address: Department of Zoology, University College, Cardiff.

2 204 R. A. HAMMOND In the present work infected toads were given two oral 2 ml. doses of 3 g./l. Scharlach R in olive oil at 24 hr. intervals. Subsequent findings agreed with those of Pflugfelder; only the fat droplets floating in the lemniscal fluid became coloured red. TW LC Text-fig. 1. Diagram of the anterior part of Acanthocephalus ranae. G, ganglion; H, hook; L, lemniscus; LC, lacunar channel; LS, ligament sac; N, neck; NR, neck retractors; P, partition; Pi?, proboscis retractor; PW, proboscis wall; R, receptacle; RE, retinaculum; RR, receptacle retractor; T, testis; TW, trunk wall. Routine histological methods were used in morphological work. Bouin, Carnoy, and 10% neutral formalin were used as fixatives. Paraffin sections were cut at thicknesses between 4/j and 10 fi. Delafield's haematoxylin and eosin, Mallory's Triple

3 Proboscis mechanism of Acanthocephalus ranae stain, and Weigert's haematoxylin and Ponceau S were all found to give satisfactory results. The numerous fibres of the body wall were stained very clearly by the aniline blue method of Owen (1959). Whole mounts were normally stained by the acetic acid-haematoxylin method described by Chubb (1962). 3I 005 mm. Text-fig. 2. Diagram of the trunk wall of Acanthocephalus ranae. BM, basement membrane; C, cuticle; CM, circular muscle; F, felt layer; LC, lacunar channel; LM, longitudinal muscle; N, nucleus; RF, radial fibrillar layer; S, striped layer. MORPHOLOGY The structure of the animal, particularly of the proboscis apparatus and its associated structures, was examined in detail (Text-fig. 1). Information in the literature on the structure of A. ranae is rather scattered (see Saefftigen, 1885; Hamann, 1891; Kaiser, 1893; Liihe, 1912; Rauther, 1931; Meyer, 1933; Hyman, 1951). The trunk (metasoma) has a central fluid-filled cavity in which the reproductive organs are situated. The syncytial trunk wall (Text-fig. 2) is similar to that of Polymorphus minutus (Crompton, 1963; Crompton & Lee, 1965). There is an outer cuticle beneath which lies the 'striped layer', through which run numerous pores. The 'felt layer' contains closely packed fibres running in all directions. The thick 'radial fibrillar layer', containing the nuclei, is traversed by fibres, some of which run from the basement membrane to the striped layer. The radial fibrillar layer contains a network of fluid-filled lacunar channels. These channels form a closed system within

4 206 R. A. HAMMOND the trunk wall and do not communicate with any other part of the body. Beneath the radial fibrillar layer lies the basement membrane. The body-wall is bounded internally by a circular muscle layer and a layer of longitudinal muscles. No trunk spines are present. The proboscis wall is in many respects similar to that of the trunk, although no striped layer is visible under the light microscope. The wall contains a lacunar system, but this is made up of irregular, interconnected fluid-filled spaces, and not of a definite pattern of channels. The basal part of the proboscis, which bears no hooks, is generally referred to as the 'neck'. A partition separates the trunk wall from that of the neck and proboscis. The proboscis hooks, which are arranged in a ' quincunxial' (Van Cleave, 1941) pattern, extend through the proboscis wall from the basement membrane. The cuticle of the proboscis folds in around each hook to its base, so that the blade is quite free along its entire length (Text-fig. 4). The lemnisci originate from the inner layers of the wall of the neck. They contain numerous fluid-filled spaces which communicate with those in the proboscis wall. They are completely invested by the so-called 'neck retractor muscles'. The proboscis receptacle is a double-walled muscular sac. The fibres of each muscle layer run in a clockwise spiral around the receptacle, making an angle of about 45 0 to its longitudinal axis. The proboscis and receptacle enclose a fluid-filled cavity. The proboscis retractor muscle envelops the 'cerebral ganglion', which lies near the base of the receptacle. The most conspicuous of the nerves leaving the ganglion are the large lateral nerves which penetrate the receptacle and pass, through the body cavity, to the neck retractor muscles and the body-wall. Between the receptacle and the neck retractors these nerves are enclosed in muscular sheaths the retinacula. The receptacle retractor muscle is flat and rather ribbon-like, except near the receptacle, where it closes to form a tube. The ligament sac in A. ranae, as in all palaeacanthocephalans, is single. It is attached at one end to the proboscis receptacle and at the other to the uterine bell in females, and to the genital sheath in males. The ligament sac does not persist in mature females. THE ACTIVITY OF THE PROBOSCIS APPARATUS Once the worms are detached from the intestine of their host alternate invagination and evagination of the proboscis is initiated. This activity is remarkably regular in all individuals, the proboscis becoming evaginated every sec.; activity ceases when the proboscis becomes firmly embedded in some suitable material. Once the proboscis has become attached the trunk continues to exhibit gentle bending movements. The principal phases of the cycle of proboscis activity are illustrated in Text-fig. 3 and PL 1. Contraction of the proboscis retractor, the receptacle retractor, and the longitudinal musculature of the trunk wall brings about proboscis invagination and a withdrawal of the entire proboscis apparatus within the trunk cavity (Text-fig. 3 a). An intucking of the fore-trunk also occurs during this process. The animal remains thus for sec. before a further sequence of events occurs. Contraction of the circular musculature of the trunk wall, and relaxation of the longitudinal muscles, causes the trunk to become elongated and reduced in diameter.

5 Proboscis mechanism of Acanthocephalus ranae 207 Antagonism between the circular and longitudinal muscles is mediated by the fluid filling the trunk cavity, which acts as a 'hydrostatic skeleton'. The trunk increases in length by up to 40 % during this activity. (c) Text-fig. 3. Operation of the proboscis, (a) Proboscis invaginated, proboscis apparatus fully withdrawn; (6) trunk maximally extended; (c) trunk extended, proboscis evaginated; (d) proboscis evaginated,, neck retractor muscles contracted. L, lemniscus; LS, ligament sac; NR, neck retractors; P, proboscis; PR, proboscis retractor; R, receptacle; RE, retinaculum; RR, receptacle retractor. Elongation of the trunk tends to stretch the receptacle retractor; normally, however, the muscle resists this tendency initially for up to 2 sec. by remaining contracted. When it relaxes the proboscis apparatus is forced forwards and the fore-trunk wall

6 208 R. A. HAMMOND unfolds (Text-fig. 3 b). The lemnisci become elongated as they are extended with the neck retractor muscles which envelop them. The phase of trunk elongation occupies 3-5 sec. As the trunk reaches its maximum length the radial fibrillar layer of the fore-trunk wall suddenly becomes very much thinner (Text-fig. 3 b). The lacunae in this region become temporarily eliminated and the fluid is forced from them to more posterior parts of the lacunar system. This phenomenon could result from the contraction of elements within the fore-trunk wall, but there is no evidence that any such contractile tissue is present. Alternatively, it may be a purely mechanical consequence of stretching of the trunk wall. If, as it appears, the fore-trunk region of the body-wall is less rigid than other regions it will be more liable to 'collapse' under mechanical stress. As the proboscis apparatus reaches the extreme anterior tip of the body the proboscis receptacle contracts. The proboscis is then forced out by hydrostatic pressure (Textfig. 3 c). The proboscis retractor, which becomes stretched, probably relaxes simultaneously with the receptacle retractor. As the proboscis wall becomes everted the hooks follow semicircular paths and finally come to lie pointing posteriorly. Evagination of the proboscis occupies 4-5 sec. The receptacle becomes reduced both in diameter and in length on contraction because of the spiral arrangement of its muscle fibres. Its reduction in volume is therefore considerable and the expulsion of the proboscis powerful. This spiral arrangement of muscle fibres has a number of other consequences. As the fibres contract the receptacle twists about its longitudinal axis, its free posterior end rotating by up to relative to the attached anterior region. In the process the receptacle retractor, the proboscis retractor, and the ligament sac become twisted. The retinacula are also affected by this rotation of the receptacle (Text-fig. 3 c). The twisting of the proboscis retractor is transmitted to a slight extent to the proboscis, the tip of which may rotate by up to 5 0 relative to its base. The animal remains in the fully extended position shown in Text-fig. 3 c for about 1 sec. The neck retractor muscles then contract and the circular muscles of the trunk wall partially relax. The proboscis remains evaginated but the proboscis apparatus is drawn inward, with part of the fore-trunk wall (Text-fig. 3 d). As this occurs fluid flows back into the lacunae of the fore-trunk wall. Van Cleave & Bullock (1950) remarked on such a withdrawal of the evaginated proboscis of Neoechtnorhynchus emydis, but considered the phenomenon abnormal. The retinacula contract simultaneously with the neck retractor muscles. The distance between the two points of attachment of each retinaculum shortens as the proboscis apparatus is drawn inwards (Text-fig. 3d). The contraction of the retinacula consequently tends to absorb much of the slack which would otherwise develop. The partial withdrawal of the proboscis apparatus (Text-fig. 3 d) also reduces the distance between the origin and insertion of the receptacle retractor. This muscle remains relaxed and shortens elastically by only 1-2 % of its extended length, and in consequence becomes slack. As it has previously been twisted by the rotation of the receptacle the muscle abruptly takes the form of a complex loop as it slackens (Textfig. 3J). The receptacle retractor develops little elastic tension when stretched. If removed from the animal it can be stretched to up to three times its contracted length and upon release shortens by only 1-2%.

7 Proboscis mechanism of Acanthocephalus ranae 209 The neck retractor muscles compress the lemnisci as they contract. The lemnisci shorten with these muscles and become more globular in shape. The more globular the lemnisci become the more effectively are they squeezed. Fluid is forced from the spaces within them to the lacunar spaces of the proboscis wall. The flow of the lemniscal fluid can be traced conveniently by intra-vitam staining with Scharlach R of the fat droplets which float in it. It was found that fluid from the Text-fig. 4. Longitudinal section of the proboscis wall of Acanthocephalus ranae. (a) Before and (6) after the lacunar spaces are distended by the influx of fluid from the lemnisci. BM, Basement membrane; C, cuticle; FS, fluid-filled space; H, hook; M, muscular layer. lemnisci flows only to the wall of the praesoma. No fluid flows from the lemnisci until the neck retractors contract, by which time the proboscis is always fully evaginated. There is no evidence that any contractile tissue is present within the lemnisci which might aid in the expulsion of fluid. There appears to be no valve mechanism controlling the flow of fluid between the lemnisci and the praesoma wall. As fluid flows into it from the lemnisci, the proboscis wall becomes distended (Text-fig. 4). The cuticle and outer subcuticular layers are forced outwards, so that the cuticle becomes firmly pressed against the blades of the hooks. The hooks themselves do not move outwards, as they arise from the basement membrane. The worms remain with the neck retractors contracted and the proboscis evaginated

8 2IO R. A. HAMMOND for up to 70 % of the time occupied by each cycle of activity. During this time local contractions of its longitudinal and circular muscles cause the trunk to bend gently from side to side. These movements cause local redistribution of fluid within the lacunar channels. At the onset of the next stage of activity the proboscis, while remaining evaginated, often becomes completely ensheathed by the fore-trunk. This is due to complete relaxation of the muscles of the trunk wall, which allows the neck retractor muscles to shorten fully. The proboscis retractor and receptacle retractor muscles then contract simultaneously, and the receptacle relaxes. The proboscis becomes rapidly drawn into 0-50 k.. H c*"'p E Time (gee.) S Text-fig. 5. Contraction and relaxation of the receptacle retractor muscle. Analysis from cine film exposed at 64 ft./sec. A-B, Muscle remains at its resting length, but slack. B-C, Muscle contracting, slack being absorbed. C-D, Arrest of shortening as the muscle becomes taut. D E, Completion of contraction. The muscle now exerts a pull on the receptacle. E F, Muscle stretched as a result of trunk elongation due to contraction of the circular musculature. F-G, The receptacle retractor remains contracted and resists the elongation of the trunk. G-H, Relaxation of the receptacle retractor. the receptacle, which expands as this occurs, and the entire proboscis apparatus is pulled within the trunk cavity (Text-fig. 3 a). The quincunxial arrangement of the proboscis hooks allows them to become closely packed as the proboscis becomes invaginated. As the receptacle relaxes and expands to accommodate the proboscis, its free end rotates in the opposite direction from that in which it rotates on contraction. The proboscis retractor and receptacle retractor muscles, and the ligament sac, untwist. As the proboscis becomes invaginated fluid is forced back from its wall to the lemnisci. Relaxation of the neck retractors and retinacula is simultaneous, or nearly so, with the contraction of the proboscis and receptacle retractors. The longitudinal muscles of the trunk wall contract and the animal shortens. In male worms the ligament sac shortens, apparently elastically, as the proboscis

9 Proboscis mechanism of Acanthocephalus ranae 211 apparatus is withdrawn. It does not appear to contain contractile tissue. The ligament sac keeps the reproductive organs, in particular the testes, in a central position within the body cavity. The contraction of the receptacle retractor muscle is very striking as the loop into which it is formed while relaxed is suddenly pulled straight. Analysis of cin6 film has shown that contraction of this muscle is most rapid while the loop is being straightened; as the muscle becomes taut and begins to exert a pull on the proboscis apparatus the rate of contraction slows momentarily (Fig. 5). Once the proboscis has been invaginated and the proboscis apparatus as a whole withdrawn, a further cycle of trunk extension and proboscis evagination is initiated. DISCUSSION Two hydraulic systems are involved in the movements of Acanthocephalus ranae: the central cavity of the trunk and the cavity enclosed by the proboscis and receptacle. These two systems are independent in that changes of hydrostatic pressure in one do not directly affect the working of the other. As Clark (1964) has pointed out the Acanthocephala differ from the majority of the pseudocoelomates in possessing 'multiple, independent hydrostatic systems'. The presence of a lacunar system in the walls of the trunk and praesoma is probably associated with the passage of materials through the surface of the worm, and with their distribution within the body. General movements of the trunk cause the lacunar fluid to flow to some extent, but the periodic elimination of fluid from the wall of the fore-trunk is far more effective in this respect. The activity of the proboscis apparatus, and consequently of the fore-trunk 'pump', ceases when the proboscis becomes attached. It would be of interest, therefore, to know how often the worms become detached while within the host, and for what periods they remain so. A number of theories concerning the function of the lemnisci have been advanced (see Meyer, 1933), but these have been based on scanty observations. The chief theories of lemniscal function are as follows: (a) The lemnisci may aid in the evagination of the proboscis and serve as reservoirs for the fluid of its lacunar system (Hamann, 1891). The latter function is stated by Hyman (1951) to be the 'accepted explanation of the function of the lemnisci'. (b) Substances of nutritional importance may enter the main part of the body cavity by diffusing across the surface of the lemnisci (see Kaiser, 1893; Baer, 1961). (c) The lemnisci may be excretory organs (Greef, 1864; Bullock, 1949). (d) The lemnisci may be concerned in the uptake and subsequent metabolic fate of materials absorbed from the intestine of the host (Meyer, 1933; Pflugfelder, 1949; Bullock, 1949; Crompton & Lee, 1965). In the present work it has been shown that fluid flows from the lemnisci to the proboscis wall only after the proboscis has become evaginated. The lemnisci cannot, therefore, aid in the evagination of the proboscis. Graybill (1902) reached a similar conclusion. The passage of Scharlach R from the intestine of the host to the lemnisci suggests that these organs are more than mere hydraulic reservoirs. The value to the animal of the receptacle retractor muscle is not immediately obvious. It has been found, however, that without the restraint normally imposed by

10 212 R. A. HAMMOND the receptacle retractor when contracted, the free end of the receptacle no longer acts as a rigid point of attachment for the proboscis retractor. Thus the tip of the receptacle buckles inwards when the proboscis retractor contracts, and the proboscis cannot be effectively withdrawn. Although the mechanism of proboscis operation seems surprisingly complex, it appears to be highly efficient. Evagination of the proboscis, during which this organ normally penetrates the intestinal wall of the host, is rapid and forceful. SUMMARY 1. A brief description of the proboscis apparatus and body-wall of Acanthocephalus ranae (Schrank, 1788; Liihe, 1911) has been given. 2. Two hydrostatic systems are involved in the movements of the animal. That of the trunk controls the position of the proboscis apparatus as a whole. The proboscis is operated by the separate hydraulic system of the proboscis apparatus. 3. Contraction of the neck retractor muscles forces fluid from the lemnisci to the proboscis wall, which becomes distended in consequence. 4. It has been shown that the lemnisci do not aid in the evagination of the proboscis. Their function may not be solely to act as hydraulic reservoirs. I wish to thank Prof. J. E. Harris for the provision of laboratory facilities and Dr H. D. Crofton for his advice and criticism during the course of this work. I am grateful to Prof. James Brough and Dr D. A. Erasmus for reading and criticizing the manuscript. REFERENCES BAER, J. C. (1961). Embranchement des Acanthocephales. In Grasse, TrcdU de Zoologie, Tome IV, BULLOCK, W. L. (1949). Hiatochemical studies on the acanthocephala: II. The distribution of glycogen and fatty subrtancei. J. Morph. 84, CHUBB, J. C. (1962). Acetic acid as a diluent and dehydrant in the preparation of whole, stained helminths. Stain Tech. 37, CLARK, R. B. (1964). Dynamics in Metazoan Evolution, pp. x Oxford. CROMPTON, D. W. T. (1963). Morphological and histochemical observations on Polymorphus minutut (Goeze, 1782), with special reference to the body wall. Paraiitology, 53, CROMPTON, D. W. T. & LEE, D. L. (1965). The fine structure of the body wall of Polymorphus nrinutus (Goeze, 1782) (Acanthocephala). Parasitology, 55, GRAYBILL, H. W. (1902). Some points in the structure of the Acanthocephala. Trans. Amer. Micr. Soc. 33, GREEF, R. (1864). Untersuchungen Uber den Bau und die Naturgeschichte von Echinorhynchus miliarius, Zenker (E. polymorphus). Arch. Naturgesch. 30, Bd. 1, HAMANN, O. (1891). Monographic der Acanthocephalen (Echinorhynchen). Jena Z. Naturw. 35, HOBSON, A. D. (1948). The physiology and cultivation in artificial media of nematodes parasitic in the alimentary tract of animals. Parasitology, 38, HYMAN, L. H. (1951). The Invertebrates. III. Acanthocephala, Aschelminthes and Entoprocts, pp. vii New York. KAISER, J. E. (1893). Die Acanthocephalen und ihre Entwickelung. Bibl. zool. 11, Heft 7. KILJAN, R. (1932). Zur Morphologie und Systematik der Gigantorhynchidae. Z. toiss. Zool. 141, LOHE, M. (191a). Zur Kenntnis der Acanthocephalen. Zool. Jb. (Suppl.), 15, MEYER, A. (1933). Acanthocephala. Bronris Klassen, 4, Abt. 2, Buch 2, Lief 1 and 2. OWEN, G. (1959). A new method for staining connective tissue fibres, with a note on Liang's method for nerve fibres. Quart. J. Micr. Set. 100,

11 Journal of Experimental Biology, Vol. 45, No. 2 Plate 1 tw tw. 0 5 mm. R. A. HAMMOND (Faring p. 213)

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13 Proboscis mechanism of Acanthocephlus ranae 213 PFLUGFELDER, O. (1949). Histophysiologische Untersuchungen fiber die Fettresorption darmloser Parasiten: Die Funktion der Lemnisken der Acanthocephalan. Z. Paraiitenk. 14, RAUTHER, M. (1931). Sechste Klasse des Cladus Nemathelminthes: Acanthocephala = Kratzwurmer. In Kukenthal, W., Handbuch der Zoologie, pp , Berlin and Leipzig. SAEFFTIGEN, A. (1885). Zur Organisation der Echinorhynchen. Morph. Jb. io, VAN CLEAVE, H. J. (1941). Hook patterns on the acanthocephalan proboscis. Quart. Rev. Biol. 16, VAN CLEAVE, H. J. (1952). Some host-parasite relationships of the Acanthocephala, with special reference to the organs of attachment. Expl Parasit. 1, VAN CLEAVE, H. J. & BULLOCK, W. L. (1950). Morphology of Neoechinorhynchui emydis, a typical representative of the Eoacanthocephala. I. The praesoma. Trans. Amer. Micr. Soc. 69, EXPLANATION OF PLATE Proboscis evagination in Acanthocephalus ranae. (1) Proboscis invaginated, trunk maximally short and thick. (2) Proboscis invaginated, trunk maximally long and thin. (3) Proboscis evaginated, trunk maximally long and thin. (4) Proboscis evaginated, neck retractor muscles contracted. /, Lemniscus; p, proboscis; r, receptacle; rr, receptacle retractor; t, testis; to, trunk wall. 14 Exp. Biol. 4J, 3

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