New Aspects of Acanthocephalan Lacunar System as Revealed in Anatomical Modeling by Corrosion Cast Method

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1 Proc. Helminthol. Soc. Wash. 52(2), 1985, pp New Aspects of Acanthocephalan Lacunar System as Revealed in Anatomical Modeling by Corrosion Cast Method DONALD M. MILLER AND TOMMY T. DUNAGAN Department of Physiology and Pharmacology, School of Medicine, Southern Illinois University, Carbondale, Illinois ABSTRACT: In order to study the acanthocephalan lacunar system we previously constructed a series of twodimensional representations utilizing india ink injection, glycerol clearing, and transmission photography. Threedimensional models were then produced by stereological extrapolation of the photographic images. We now describe a technique that produces a permanent three-dimensional model. The lacunar system is injected with partially polymerized colored butyrate monomer which is allowed to fully polymerize in situ. The tissues are then disintegrated with potassium hydroxide leaving a pliable replica of the original lacunar system that is suitable for photography. New aspects of the acanthocephalan lacunar system, which have not previously been reported, have been revealed by these corrosion cast models. We conclude from this study that the lacunar system is a very effective fluid transport system and possibly serves as a hydrostatic skeleton. Early attempts (Miller and Dunagan, 1977) to examine the electrophysiology of acanthocephalan muscle cells revealed their unique tubular nature as part of the lacunar system. Previously (Miller and Dunagan, 1976, 1978), models were made of the lacunar system utilizing india ink injection, glycerol clearing and transmission photography. Interpretation of these produced three-dimensional representations of the lacunar canalicular system in Macracanthorhynchus hirudinaceus and Oligacanthorhynchus tortuosa. Preparations upon which these models were based were not permanent and left much for interpretation. Nevertheless, they did indicate that the lacunar system of the Acanthocephala was a vastly more complex system than had previously been described. We now report the outlining of the lacunar system by means of corrosion modeling (Batson, 1935). This method has allowed construction of permanent models of the system, which may be colored (Narat et al., 1936) to add clarity to interpretation. The flow of injected material also indicates patterns of circulation around the system, and has disclosed outlines of additional features not previously described in the lacunar system. Materials and Methods Male and female Acanthocephala, Macracanthorhynchus hirudinaceus were obtained from Swift Fresh Meats Company, East St. Louis, Illinois, and transported to the laboratory in Dewar flasks containing a small amount of gut contents. Afterwards, the worms were transferred into a physiological saline solution (Denbo and Miller, 1975). The injectate was a partially polymerized monomer, Batson's # 17 kit obtained from Polysciences Inc., to which coloring dyes (10 mg/ ml), promoter (2 drops/200 ml), and catalyst (1 ml/ 200 ml) were added. The injection was performed with disposable 5-ml syringes and #26 hypodermic needles. After injection, manual pressure was applied at the injection site to offset contractile activity of the worm and to keep the plastic inside until polymerization occurred. Once the plastic had polymerized, parasite tissues were macerated at 50 C using technical potassium hydroxide (340 g to 1,000 ml of water). Tissues were removed from the solution every 5 min and rinsed in water. Dissolution of tegument occurred in the first 15 or 20 min leaving the muscle tissue intact. Fibrous connective tissues were the last to be macerated and required hr of processing. Photography was ac complished using a Leitz Orthomat photomicroscope. Results OVERALL SYSTEM: Monomer injected into any channel eventually flowed into all connecting channels and canals (Figs. 1, 2). However, contractions of the worm tended to squeeze the monomer back out through the injection hole. Therefore, it was necessary to hold pressure on the area of injection until the plastic polymerized. In addition, it was noted that when plastic monomer was injected into the medial longitudinal channel, flow in the posterior direction was much easier than in the anterior direction. Attempts to drain the worm of lacunar fluid one day prior to injection did not produce satisfactory results. Apparently, the collapsed channels tended to fuse as a result of the drainage. This was also the case if we removed most of the fluid osmotically by putting worms in a very concentrated brine solution prior to injection. However, a secondary opening of the lacunar system that allowed fluid to drain and reduce back pressure, did expedite the injection process.

2 222 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY 1 Figures 1-4. Photomicrographs of injected preparations. 1. Photomicrograph near the anterior end of the worm. Note branching of medial longitudinal channel into two limbs (>< 40). 2. Photomicrograph of a corrosion model in which the medial longitudinal channel has been injected and the partially polymerized monomer has

3 OF WASHINGTON, VOLUME 52, NUMBER 2, JULY Observations of contractile activity of fresh worms indicated that there was alternate constriction of longitudinal and circular muscle sets interrupted by relaxation phases. This action tended to move lacunar fluid alternatively into the circular and then longitudinal channels of the lacunar system. Indeed, stretching the worm after injection of colored monomer tended to move material preferentially into tegumental canals. The channels identified by this technique corresponded for the most part but not entirely to previous findings (Miller and Dunagan, 1976, 1977, 1978). MUSCULAR CHANNELS: Models produced when monomer was injected into the medial longitudinal channel agreed with previous scanning electron microscope studies (Miller and Dunagan, 1978): channels in the longitudinal, circular, and rete muscles were patent and irregularly interconnected (Figs. 1, 2). Radial connections to longitudinal muscles alternate sides (Figs. 3, 4). MEDIAL LONGITUDINAL CHANNELS: Corrosion modeling has demonstrated that the medial longitudinal channel alternates its connections with the inner muscular layer of the body wall. Within these connection areas, radial canals arise leading to both muscle and more externally to primary ring canals. At each end of the worm the medial longitudinal channels branch and invade the body wall (Fig. 1). After they pass through the body wall they form smaller longitudinal channels that double back in the tegument and are termed secondary longitudinal channels. These channels interconnect with other channels in the tegument by way of the primary ring canals. SECONDARY RING CANALS: In agreement with a previous study of injected dyes (Miller and Dunagan, 1976) secondary ring canals were discerned, but with this technique these canals were discovered to be entirely within the tegument. They, like the primary ring canals, also connect with the medial longitudinal channel by means of short radial canals (Figs. 3-5), however radial canals to secondary ring canals do not alternate and they outnumber the primary ring canal connections an average of four to one. HYPODERMAL CANALS: Photomicrographs of MEDIAL LONGITUDINAL CHANNEL Figure 5. Schematic diagram outlining relationship of medial longitudinal channel to primary and secondary ring canals. Note that radial canals to primary ring canals alternate whereas those to secondary ring canals do not, and the ratio of secondary to primary ring canals is approximately 4:1. lacunar models demonstrated an extensive invasion of outer tegument layers by the lacunar tubular system that reached to within microns of the surface (Figs. 6-10). Interestingly enough, the three-dimensional structure of these connections, which extend from one secondary ring canal to another, is not that of a tube but rather that of a tortuous irregular sinus (Figs. 9, 11) whose morphology varies with the stretching of the body wall. We previously termed these connections hypodermal canals (Miller and Dunagan, 1976). When the longitudinal muscles are contracted, these hypodermal canals are very tortuous whereas when the same muscles are relaxed they appear straightened. HYPODERMAL DUCTS: This name was applied to the small outward extensions observed in previous work (Miller and Dunagan, 1976). Our results provide evidence that hypodermal ducts are small projections on the hypodermal canals' periphery which reach through the radial layer of the tegument (Figs. 6-10). In high magnification flowed into other tubules of the lacunar system. MLC = Medial longitudinal channel, PRC = primary ring canal, SRC = secondary ring canal, and LMC = longitudinal muscle channel (x 100). 3. Enlargement of medial longitudinal channel area from Figure 2 detailing the numerous radial canals that conduct fluid to primary and secondary ring canals (x 400). 4. Further enlargement of medial longitudinal channel and radial canals (x 1,000).

4 224 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY HD

5 OF WASHINGTON, VOLUME 52, NUMBER 2, JULY micrographs (Fig. 10) they appear to be fine, sometimes branched projections (or tufts) from the hypodermal canals which extend to just under the tegument surface. Discussion A FLUID TRANSPORT SYSTEM: We consider the lacunar system of the body wall of the acanthocephalan which we have modeled and described herein to be a well-defined "fluid transport system." A fluid transport system (LaBarbera and Vogel, 1982) has been defined as "any system in which internal bulk convection of a fluid functions to reduce diffusion distances between points within an organism or between a point within the organism and the external environment." The definition of a "fluid transport system" has been accepted rather than the use of any variant of "circulatory system" because the latter may imply the presence of a mesodermal lining. In previous studies on the tegument utilizing transmission electron microscopy, lacunar canals were found to be surrounded by membranes in Polymorphus minutus (Crompton and Lee, 1965), but were not in Moniliformis dubius (Nicholas and Mercer, 1965). This important point needs clarification. If all the canalicular elements of the lacunar system are membrane limited, then perhaps there is no difference between a canal and a channel. But if certain parts in the tegument are not membrane limited, the terminology should be changed accordingly. Energy for movement of fluids within the acanthocephalan body results from contraction of muscle sets. Acanthocephalans thus have a distributed macropump system which is closed. As such, it follows the usual rules for such systems as previously defined by LaBarbera and Vogel (1982): "(1) While diffusion is always used for short distance transport, it is augmented by bulk flow of fluid for any long distance transport. (2) Transport systems use both large and small pipes small pipes at exchange sites and large HYPODERMAL^ L/i V*- \H fj EXTERNAL SURFACE OF TEGUMENT Figure 12. Schematic diagram of lacunar system within the tegument. Hypodermal canals appear straighter when the worm is extended. Hypodermal canals are much more numerous than shown in diagram. pipes for moving fluid from one exchange site to another. (3) Total cross sectional area of smaller pipes greatly exceeds that of larger pipes, so that velocity in smaller pipes is less than that in larger ones." A HYDROSTATIC SKELETON: Acanthocephalans drained of their lacunar fluid are very flaccid and lose their general contractile activity. Thus the lacunar system clearly functions as a hydrostatic skeleton for the worm. Also, we conclude that lacunar channels tend to fuse (i.e., become harder to inject) whenever lacunar fluid is removed or the worm is dehydrated. This and the fact that fixation of the worm usually results in drastic muscle contraction would account for difficulties in studying this system of canals with normal histological techniques. CIRCULATION DYNAMICS: As diagrammed in Figure 13 the structure of the system allows fluid communication between the outermost tegmental tufts and the innermost rete system and medial longitudinal channel. In summary, this study has clarified the form and location of some of the previously named canals of the lacunar system, pointed out the Figures Photomicrographs of a corrosion model of the tegumental system. 6. Low magnification photomicrograph of the model contains representations of primary ring canals (PRC), hypodermal canals (HC), and barely visible are hypodermal ducts (HD) (x 100). 7. Enlargement of tegumental model taken from a side angle at the edge. At this magnification hypodermal ducts are more apparent on top of the hypodermal canals (x 250). 8. Further enlargement of tegumental model taken from a side angle at the edge. At this magnification hypodermal ducts are evident on top of the hypodermal canals (xl60). 9. Close-up photomicrograph of a single hypodermal canal and the hypodermal duct extensions from it (x 400). 10. Side view of hypodermal canal and ducts (x!60). 11. High power magnification of hypodermal duct extensions (x 1,000).

6 226 MEDIAL LONGITUDINAL CHANNEL RETE SYSTEM r / SECONDARY RING CANAL Q n EXTERNAL SURFACE OF TEGUMENT Figure 13. Diagrammatic stereogram of body wall lacunar system showing interconnection of most of the canals and channels that allow for circulation of lacunar fluids throughout body wall. This stereogram is a composite of Figures 5 and 12. Rete system and most longitudinal muscles have been eliminated from right side of the medial longitudinal channel to allow showing the presence of hypodermal canals and ducts underneath in the tegument. Dark lines and spaces indicate interconnections within lacunar system. extensive interconnection between all of the canalicular elements, identified the possible circular nature of flow within the system, clarified the functional nature of the lacunar system within the acanthocephalan, and demonstrated the use of corrosion modeling in an endoparasitic worm. We speculate that the injection technique described would work for the study of transport systems that must exist within other large, relatively fast-growing endoparasites (e.g., tapeworms), which by virtue of their size would be diffusion limited in their growth. Acknowledgments This study was supported by the School of Medicine, Southern Illinois University at Carbondale. Literature Cited Batson, O. V A new material for corrosion preparations. Science 81:519. Crompton, D. W. T., and D. S. Lee The fine structure of the body wall of Po/ymorp/jws minutus (Goeze 1782) (Acanthocephala). Parasitology 53: 357_364. Denbo, J. R., and D. M. Miller Ionic and osmotic regulation in an acanthocephalan. Trans Acad. Sci. 68: LaBarbera, M., and S. Vogel The design of fluid transport systems in organisms. Am. Scientist 70: Miller, D. M., and T. T. Dunagan Body wall organization of the acanthocephalan, Macracanthorhynchus hirudinaceus: a reexamination of the lacunar system. Proc. Helminthol. Soc. Wash. 43: , and The lacunar system and tubular muscles in Acanthocephala. Proc. Helminthol. Soc. Wash. 44: , and Organization of the lacunar system in the acanthocephajan, Oligacanthorhynchus tortuosa. J. Parasitol. 64: Narat, J. K., J. A. Loef, and M. Narat On the preparation of multi-colored corrosion specimens. Anat. Rec. 64:155. Nicholas, W. L., and E. H. Mercer The ultrastructure of the tegument of Moniliformis dubius (Acanthocephala). Quart. J. Microsc. Sci. 106: