Major nerves in the anterior nervous system of Macracanthorhynchus hirudinaceus (Acanthocephala)

Comp. Biochem. Physiol., 1970, Vol. 37, pp. 235 to 242. Pergamon Press. Printed in Great Britain

MAJOR NERVES IN THE ANTERIOR NERVOUS SYSTEM OF M A C R A C A N T H O R H Y N C H U S H I R U D I N A C E U S (ACANTHOCEPHALA)* T. T. D U N A G A N and D. M. MILLER Department of Physiology, Southern Illinois University (Received 19 May 1970)

A b s t r a c t - - 1 . T h e anatomy of the cephalic ganglion and associated nerves in the Acanthocephalan Macracanthorhynchus hirudinaceus has been studied preliminary to use of the organ as a model system for the study of neural pathways. 2. Analysis of routine serial sections of the anterior portion of the worm revealed the existence of eighty-six cells within the cephalic ganglion itself. 3. There are six pairs of nerves with bilateral symmetry which exit from the ganglion. T h e total number of neurons contained within the six pairs of nerves is approximately seventy-two. 4. This investigation reveals that certain anatomical features are either new or different from that which has been proposed by earlier workers. Among these observations are the following: (a) T h e anterior ventral nerve does not branch but is always two separate bundles of neurons. (b) The anterior ventral nerve is not wrapped in muscle as is the lateral posterior nerve after it leaves the thick layer of the dorsal receptacle. (c) T h e paired lateral posterior nerve consists of two bundles of neurons with approximately twenty-three neurons/ bundle. (d) The lateral posterior nerve originates exclusively from the dorsal concave surface of the brain and never from the convex or ventral surface. INTRODUCTION THE NERVOUS s y s t e m o f a c a n t h o c e p h a l a differs s i g n i f i c a n t l y f r o m o t h e r h e l m i n t h s t h a t h a v e b e e n s t u d i e d . I t lacks t h e o r g a n i z a t i o n a l c o m p l e x i t y o f e i t h e r t h e c e s t o d e s or n e m a t o d e s . A s p r e s e n t l y u n d e r s t o o d , t h e a c a n t h o c e p h a l a n n e r v o u s s y s t e m consists o f an a n t e r i o r c e r e b r a l g a n g l i o n c o n t a i n i n g a s m a l l n u m b e r of cells p l u s its a s s o c i a t e d n e r v e tracts. I n t h e m a l e t h e r e is an a d d i t i o n a l p a i r of genital g a n g l i a p l u s t h e i r a s s o c i a t e d n e r v e tracts. N o s t r u c t u r e s i m i l a r to t h e c i r c u m e n t e r i c r i n g of h e l m i n t h s has b e e n f o u n d in t h e a c a n t h o c e p h a l a . T h e last d e t a i l e d s t u d i e s o f t h e m o r p h o l o g y o f t h e n e r v o u s s y s t e m of t h e a c a n t h o c e p h a l a w e r e m a d e on HamannieUa b y K i l i a n in 1932 a n d on Bolbosoma b y H a r a d a in 1931. I n b o t h cases t h e i n v e s t i g a t i o n was n o t significantly d i f f e r e n t f r o m earlier ones o n Macracanthorhynchus b y B r a n d e s (1899), a n d on Polymorphus * This work was supported by the National Institute of Health Research Grant No. RO1-NB08583 and the Graduate School Southern Illinois University through the Office of Research and Projects. 235

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by Greeff (1864). Reviews of these publications, though very brief, have been m a d e b y H y m a n (1951), Bullock & Horridge (1965) and Nicholas (1967). I n all of these recent publications Kilian and Brandes are the most often cited authors. Nicholas (1967) indicates that Bullock & Horridge (1965) present a m u c h fuller account of the nervous system than is available in his review. Bullock and Horridge in turn present a figure originating with Brandes (1899) which also was published by H y m a n (1951) in a slightly modified form. All recent writers seem to agree that nothing of any consequence has been done since the work of Kilian (1932) and most point out that his work did not modify to any major degree the work performed earlier by Greeff (1864), Brandes (1899) and H a m a n n (1891). Although there are other publications that are concerned with other parts of the neuroanatomy of the acanthocephala, to our knowledge none have appeared since Kilian (1932) and none of these previous authors attempted to present a gross or microscopic description of the anatomy of the ganglia. A restudy of the anterior part of M. hirudinaceus was undertaken in conjunction with a study of the physiology of the anterior nervous system and its associated sensory structures. As a result of this study, we would like to make certain modifications in the accepted descriptions of the cephalic ganglion and nerve fibers originating therefrom. MATERIALS AND M E T H O D S Macracanthorhynchus hirudinaceus were obtained from the DuQuoin Packing Company and placed in Dewar Flasks along with minimal amounts of gut contents. Upon return to the laboratory, the worms were washed in several changes of 0"07 M KH2PO4 until all adhering materials were removed. Following the initial cleaning of the material, a number of different procedures were used in an attempt to obtain optimum fixation of the nervous tissue. The most satisfactory procedure consisted of the following: 1. Place the worms in 50 % glycerol at 0°C for 1 week. 2. Fix in solution "A" (0'4 ml formaldehyde, 100'0 ml water, 0"03 M bromoacetate, and 0'18 M sucrose) for 1 hr at 4°C. 3. Transfer material to solution "B" (0'4 ml formaldehyde, 100"0 ml water, 0"03 M bromoacetate, 0.18 M sucrose and 20% dimethyl sulfoxide) for 1 hr at 4°C. 4. Transfer sample into 0"3 M sucrose and 0'03 M bromoacetate at 4°C until ready to dehydrate. Routine paraffin embedding follows a brief wash of the fixed tissue with Ascaris Ringers. Sectioning at 8/, and mounting was done in the usual manner. Staining was accomplished by the standard H & E methods as well as toluidin blue, protargol-S and gold chloride. Unfortunately, with the exception of buffered toluidin blue we have never been able to obtain consistent results with any one staining procedure. RESULTS Figure 1 is the anterior region of M. hirudinaceus as originally proposed by Brandes (1899) and presented in recent reviews. T h e cephalic ganglion is represented as an eliptical mass of cells with the largest cells along the periphery about 100/, f r o m the anterior margin. It is interesting to note that H y m a n (1951) reproduces this same figure but deletes these large cells. I n fact our studies show that the largest cells are not centrally located but are found at the most posterior part of the cephalic ganglion. T w o of these cells consistently have two large welldefined nuclei in each.

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Apicalsenseorgan / l \i Anteriormedianerve l QI "iVenlralanl'eriornerve Q\ Loteralsensorybulb ~'~

.l_ateralmedial Cerebralganglion '!",e~-. \Re,inaculo ~" ~ateral poster,ornerve nerve

Fro. l . T h e a n t e r i o r region of M . hirudinaceus after B r a n d e s (1899). T h e n o m e n clature is that of Hyman (1951).

Figure 2 does not give any indication of the presence of nerves which Harada (1931) calls the anterior dorso-medialis or "Nervenfasern aus dem N. lateralis posterior". In fact, it is difficult to see why the posterior lateral nerve would penetrate the surrounding muscle layer as it moves posteriorly and then have some of its neurons re-enter the vicinity of the dorsal retractor muscle. This is especially true since peripheral to the sheath enclosing the proboscis retractor muscles are two lateral nerves which have neurons contributed by the posterior lateral nerve as well as the anterior lateral and anterior ventral nerve. In addition, Fig. 2 shows three X-sections of the proboscis retractor muscle at progressively more posterior levels. Note that in addition to the anterior medial nerve there are two groups of three neurons each which are adjacent to the anterior medial nerve as it leaves the apical sensory papillae but which progressively move more laterally (Fig. 2A, B, C) until they are completely isolated from the former nerve. Each of these groups of three neurons do not separate but enter the dorso-lateral margin of the cephalic ganglion about 60/~ from the anterior margin. It is not clear from the earlier literature if these two groups are identical with the lateral anterior nerve of Brandes' description. We believe that these two groups were overlooked in earlier descriptions and suggest they be called the anterior proboscis nerves until more is known about their function and ultimate destination. T h e anterior medial nerve branches as indicated in previous descriptions (Brandes, 1899; Kilian, 1932) and joins the cephalic ganglion along its ventral surface about 4 0 F from the anterior margin. T h u s the anterior medial nerve enters the ganglion more anteriorly than the two groups of 3 neurons each. T h e anterior ventral nerve appears as a pair of large nerve bundles at the anteriormost margin of the ganglion. Note that it can be recognized as two separate nerves

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from the time it leaves the ganglion. Each bundle can be seen to consist of approximately seven neurons (Fig. 3). These nerve bundles separate about 35/~ anterior to their origin into two branches which gradually move ventrally into the wall of the receptacle. Once the bundle of nerves has entered the wall, they move toward the dorsal surface and then laterally in opposite directions. As they move apart, each bundle of neurons gradually crosses the receptacle wall until they reach the large lateral tracts innervating the proboscis musculature. These lateral tracts are composed in part of neurons from the anterior lateral nerve which goes more ventrally before it too is directed laterally. Cells in the anterior part of the ganglion provide most of these neurons but it also appears that ganglion cells remote from the exit of the nerve may contribute neurons. On the other hand, some of the ganglion cells appear to be multiaxonal in structure and contribute to different nerves. An example of this is indicated in Fig. 4 which shows what appears to be a multiaxonal cell containing two large nuclei. A survey of other phyla indicated that binucleate ganglion cells are unique to the Acanthocephala. Notice that one branch of the cell extends along the ventral surface for some distance until it can no longer be recognized. The second branch goes anterior-mesad becoming indistinct shortly after leaving the cell body. The anterior lateral nerve is seen to originate about 35/x posterior (as paired nerves) to the anterior-most margin of the ganglion. It is important to note, however, that they have been depicted as single nerves by previous authors, whereas Fig. 3 shows them to contain at least two neurons in each nerve. Kilian (1932) in his discussion adds important information and partially clarifies the description of Brandes (1899) in this regard. However, he considered the anterior median nerve which innervates the apical sensory papillae as being the only nerve which is "wrapped in the proboscis retractor muscle". The shape of the cephalic ganglion resembles a cupped hand (Fig. 5) whose concave surface (according to Hyman, 1951) is the dorsal part of the animal. Hyman (1951) states that the position of this ganglion is one of the best ways to determine dorsoventrality. According to her, the central surface would presumably always be the convex surface of the ganglion which is adjacent to the wall of the proboscis receptacle. We have followed her views as to which surface is dorsal. The shape of the ganglion varies somewhat depending on the degree of retraction of the proboscis but not to the degree expected. The main changes occurring during contraction are seen in sagital sections. Contrast the model in Fig. 5 with the section seen in Fig. 4. These changes do not affect cell position which stays the same in relation to the surrounding cells. Since some cells have more than one nucleus, the number of cells is not necessarily the same as the number of nuclei. Kochanowski (1924) pointed out that as early as 1892, Borgstrom described binucleate cells in acanthocephalan ganglia. Obviously, a nuclear count would only indicate the maximum possible cell number. In this instance we have never counted more than ninety nuclei. If there are only two binucleate cells in the cephalic ganglion, then it would be relatively simple to arrive at the number of cells. Kilian (1932), in a review of the earlier work of Kaiser (1893)

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and Brandes (1899), supports Kaiser's view that this ganglion contains 86 cells. (We are not prepared at this time to modify that number until we can relate most cell connections.) The size of the cephalic ganglion represented by the model in Fig. 5 is 270 F long x 250/x wide x 50/x thick. We have observed very little difference in the size and shape of the ganglion regardless of the size of the worm. However, we have not examined serial sections of worms smaller than three inches in length.

qedial n e r v e

d e f t e r nerve

:)oscis n e r v e l a t e r a l nerve

medial

nerve

sterior

nerve

lglion

FIG. 5. A model of the cephalic ganglion of M. hirudinaceus showing the entry of the major nerves. This is a dorsal lateral view showing the concave surface. DISCUSSION It is apparent from the studies presented in this report that investigators have not attempted to verify the results of earlier studies. Each has simply referred to the previous author and assumed that the limited results were due to a poorly defined system in the parasite. It is true, of course, that the pig acanthocephalan is difficult to properly fix and section because of the nature of the body wall and this has contributed to the confusion. However, the small number of cells in the cephalic ganglion offers investigators a unique opportunity to correlate function with behavior. When compared with other organisms, even among the invertebrates, the cephalic ganglion has very few cells. The circumoral ring in Ascaris sp. alone contains 162 nerve cells (Goldschmidt, 1908-9). Even the rotifer possesses 183 ganglionic cells in the brain (Martini, 1912) and cestodes are known to have very elaborate nervous systems (Bullock & Horridge, 1965). This simplified system with its limited number of known sense receptors (located in an area suitable for dissection) is a distinct advantage in the examination of the function of the nervous system.

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Most of the previous investigators have relied heavily on the work of Hamann (1891), Greeff (1864) and Brandes (1899). More recent authors such as Kilian (1932) and Harada (1931) mention these, but it is unclear Low Kilian (1932) failed to make note of some of the nerves that leave the cephalic ganglion since many of the diagrams of sections he presented are very clear. He mentions only five peripherial nerves originating from the cephalic ganglion and of this group only the two big bundles of fibers (lateral posterior nerves) are directed away from the ganglion in a posterior direction. Kilian indicates (p. 316) that they originate from the lateral and the dorsal concave surface of the ganglion whereas we have only observed them originating from the dorsal surface. Kilian also states that the anterior median nerve is wrapped in the proboscis retractor muscle. We believe that it is not wrapped in this muscle but is a "free" nerve that connects the apical sensory organ to the cephalic ganglion and passes alongside the proboscis retractor muscles or in the company of them. It is certainly not wrapped in the same way that the bundles of the lateral posterior nerves are enclosed after they leave the thick wall of the dorsal receptacle. Dr. R. J. Gee (personal communication) has information which caused him to question the existence of this specific nerve particularly as illustrated in existing drawings. However, we believe this tissue to be a nerve and not something else. It is always present and can easily be seen in cross section of the neck region of the worm. Furthermore, it takes up the toluidin blue stain in the same manner as the other neural tissues. Brandes (1899) indicated that this nerve divides prior to reaching the cephalic ganglion and also illustrated the anterior ventral nerve dividing some distance anterior to the ganglion with a branch going to each side of the neck region. Unfortunately Kilian does not discuss the division of either of these nerves. He was certainly aware of this literature since he referred to it on other occassions. One can only conclude that he must have agreed with the earlier descriptions. In all fairness to Kilian the reader is reminded that he worked on a different species from that reported here. However, a free translation of his comments on p. 320 ( " I n concluding this short description of the nervous system of Hamanniella microcephala, it should be noted that M. hirudinaceus is the best suited specimen to employ in studying the nervous system (especially the fine structure) since its nerve system is the most extensive and agrees in many respects to that of H. microcephala") and again on p. 324 ("Also the structure of the nervous system of G. echinodiscus does not deviate from the characteristic features of H. microcephala and M. hirudinaceus") would indicate that he considered the nervous system to differ only in smaller details and not in the number or arrangement of the neurons leaving the cephalic ganglion. Our observations indicate that the anterior ventral nerves are not a branch of a single nerve but are two separate nerves which leave the ganglion very close together. They later diverge and are easily observed as separate bundles of neurons. Likewise, the anterior medial nerve originates as paired neurons which appear to unite some distance anterior to the ganglion. These paired neurons leave the ganglion along the ventro-lateral surface about 35/z from the anteriormost margin.

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I t is interesting to note that the m o s t anterior muscles o f these helminths frequently have outer sheaths that stain in a m a n n e r similar to that of the nerves. I t m a y very well be that there is no clear distinction between the muscles and nerves. T h e muscles m a y possess properties similar to those of their nerves. T h e lateral posterior nerve is a large bundle of neurons arising from the concave surface of the ganglion. I t consists of approximately 23 nerve tracts per bundle. However, it is clear that the n u m b e r of tracts varies depending on the level of sectioning. T h i s nerve is enclosed by three muscle fibers (Retinacula) which are frequently organized in such a fashion as to leave one side of the bundle covered only b y the muscle sheath. T h e anterior lateralis and the anterior proboscis nerve contain more than one neuron. T h e anterior lateralis appears to have between 6 and 9 tracts whereas the anterior proboscis always contains 3 neurons. T h e anterior lateral nerve of Bullock & Horridge (1965) and the lateral anterior nerve of H y m a n (1951) m a y be the same nerve which we are calling the anterior proboscis nerve. W e are not using their nomenclaure in this instance because they refer to a single neuron and it is clear f r o m Fig. 3 that there are three neurons. T h e i r original illustrations are not sufficiently detailed to enable a comparison. Both Bullock & Horridge, and H y m a n take their information f r o m Brandes (1899) but their illustrations and nomenclature differ f r o m one another significantly. Kilian (1932) states "the lateral anterior nerves originate from the lateral edges of the ganglion. T h e y innervate the protrusores laterales. T h e lateral medial nerves which innervate the muscles of the receptaculum also originate close to the lateral anterior nerves." Since Kilian does not give an illustration of this, it is not possible to determine where he places the origin of these nerves. Indeed, the existing terminology and illustrations are very confusing. W e have tried to use the terms used by Brandes (1899) rather than those by Kilian (1932) or H y m a n (1951). BIBLIOGRAPHY BRANDES G. (1899) Das Nervensystem der als Nemathelminthen zusammengefassten Wurmtypen. Abhandl. Naturf. Gesellsch. Halle, 21, 271-299. BULLOCK T. H. & HORRIDGEG. A. (1965) Structure and Function in the Nervous System of Invertebrates, Vol. I. Freeman, San Francisco. GOLDSCHMIOT R. (1908) Das Nervensystem yon Ascaris lumbricoides und megalocephala. Ein Versuch in den Aufbau eines einfachen Nervensystems einzudringen. 1. Tell. Z. wiss. Zool. 90, 73-136. GOLDSCHMIDT R. (1909) Das Nervensystem von Ascaris lumbricoides und megalocephala. Ein Versuch in den Aufbau eines einfachen Nervensystems einzudringen. 1. Teil Z. wiss. Zool. 90, 306-357. GREEFF R. (1864) Untersuchgen fiber den Bau und die Naturgeschichte yon Echinorhynchus miliarius Zenker (E. polymorphus). Arch. Naturg., Berlin, 30, Bd. J. 1, 98-140. HAMANN O. (1891) Monographie der Acanthocephalan (Echinorhychen). Ihre Entwickelung, Histogenie, Anatomie, nebst Beitr~gen zur Systematik und Biologie. Jenaische Ztschr. Naturwiss. 25, 113-231. HARADA I. (1931) Das Nervensystem yon Bolbosoma turbinella (Dies.). Japan J. Zool. 3, 161-199.

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HYMAN L. H. (1951) The Invertebrates. Vol. I I I : Acanthocephala, Aschelminthes, and Entoprocta. McGraw-Hill, New York. KAISER J. E. (1893a) Die Acanthocephalen und ihre Entwickelung. 1. Theil, 3. Cassel. (LEUCKARTK. G. F. R. & CHUN C., Biblioth. Zool. Heft 7.) KAISER J. E. (1893b) Die Acanthocephalen u n d ihre Entwickelung. 2. Theil. Cassell. (LEUCKARTK. G. F. R. & CHUN C. BibIioth. Zool. Heft 7.) KILIAN R. (1932) Zur Morphologie und Systematik der Giganthorhynchidae (Acanthocephala). Z. z~'iss.Zool. 141,246-345. KOCHANOWSKIJ. (1924) Anatomie microscopique du syst6me nerveux du Giganthorhynchus hirudinaceus Pall. C. r. Soc. Biol., Paris 91,711-713. MARTINI E. (1912) Studien fiber die Konstanz histologischer E l e m e n t e - - I I I . Hydatina senta. Z. zz,iss. Zool., 102, 425-645. NICHOLAS W. L. (1967) The Biology of Acanthocephala. Advances in Parasitology, p. 209. Academic Press, New York. Key Word Index--Acanthocephalan C N S ; Macracanthorhynchus hirudinaceus ; neurones; nerves.