Ultrastructure of the protonephridial system of Polystomoides malayi Rohde and P. renschi Rohde (Monogenea: Polystomatidae)

International Journal for Parasitology, 1973, VoL 3, pp. 329-333. Pergamon Press. Printed in Great Britain

ULTRASTRUCTURE OF THE PROTONEPHRIDIAL SYSTEM OF P O L Y S T O M O I D E S MALA YI ROHDE AND P. R E N S C H I ROHDE (MONOGENEA: POLYSTOMATIDAE) KLAUS ROHDE Department of Parasitology, University of Queensland, St. Lucia, Brisbane 4067, Australia (Received 17 July 1972; amended 28 August 1972) Abstract RormF. K. 1973. Ultrastructure of the protonephridial system of Polystomoides malayi Rohde and P. renschi gohde (Monogenea: Polystomatidae). International Journal for Parasitology 3: 329-333. Polystomoides malayi and P. renschi have three types of protonephridial flames. The first type is a typical flame cell with internal and external ribs connected by a weir membrane without nephrostomes, and with internal and external leptotriches. The second type is a flame cell complex consisting of at least two flames reaching into a common cavity. The third type is a non-terminal (= lateral) flame in the protonephridial ducts, consisting of loosely arranged cilia many of which have lateral tube-like extensions and whose tips have irregularly arranged filaments gradually decreasing in number. The number of cilia in all types of flames varies. The smallest capillaries are strongly convoluted and have a smooth or slightly reticulated surface, the larger ducts have strongly reticulated walls and single cilia may be found in the cavities of the reticulum. INDEX KEY WORDS: Trematoda; Monogenea; Polystomatidae; ultrastructure; excretory system; protonephridia; flame cells; cilia. INTRODUCTION A NUMaER o f authors have examined the ultrastructure o f the protonephridial system o f parasitic platyhelminths (Kiimmel, 1958, 1960; Kruidenier, 1959; R o t h m a n , 1959, 1963; Lautenschlager & Cardell, 1961; Senft et al., 1961; Cardell, 1962; Ercoli et al., 1963; Isseroff, 1963; Pantelouris & Threadgold, 1963; Martin & Bils, 1964; Reisinger, 1964; Lumsden, 1965, 1967; Race et al., 1965; v. Bonsdorff & Telkk~, 1966; Ginetzinskaya et al., 1966; Erasmus, 1967; Gallagher & Threadgold, 1967; Morseth, 1967; Rees, 1967; Wilson, 1967; 1969; Threadgold, 1968; Howells, 1969; S a g a m o t o & Sugimura, 1969; T o n g u et al. 1970; Rohde, 1971a, b, 1972). There is, however, not a single detailed study o f the ultrastructure o f the protonephridia o f a monogenean. Only R o h d e (1971a, b) reports on a few preliminary findings in a species o f Polystomoides. In the following, the protonephridia o f two species o f Polystomoides are described. MATERIAL AND METHODS Specimens of Polystomoides malayi Rohde were collected from the urinary bladder of the Malayan tortoise Cyclemys (= Cuora) amboinensis (Daud.), specimens of t'. renschi Rohde from the oral cavity of Siebenrockiella crassicollis (Gray). Immediately after dissection they were fixed for 1½ h in 2~o OsO4 in veronal acetate (pH 7.3-7.4), and dehydrated in ethyl alcohol, isopropyl alcohol and acetone. Some specimens of 1". rnalayi were fixed in glutaraldehyde and subsequently in osmium. All micrographs are of specimens fixed in osmium only. Fixation and dehydration liquids were in an ice bath. The worms were embedded in araldite which was polymerized at an initial temperature of 50°C, gradually rising to 60°C. Sections were stained with uranyl acetate and lead citrate. Micrographs were taken with a Siemens Elmiskop 1 at 60 kV. RESULTS Electron microscopic examination revealed the presence o f three types o f flames in the protonephridia o f both species o f Polystomoides examined. The structure o f the flames, 329

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capillaries and ducts is the same in both species and no effect of the method of fixation (with or without glutaraldehyde) was found.

1. Single flame cells This is the most common type of flame which corresponds closely to that demonstrated in other parasitic platyhelminths. The nucleus is usually lateral, beside the cilia, but may also be located below the basal bodies of the cilia. The cell body has many long and irregularly branched processes, and microtubuli and vacuoles are prominent (Figs. 1 & 2). Ciliary rootlets, basal bodies and cilia have the typical structure (Figs. 1-3). The cilia are tightly packed and the lateral ones may be incomplete, sometimes containing only a few filaments. The part of the flame cell above the basal bodies forms a widened cavity enclosed in a typical weir apparatus. The weir apparatus consists of internal and external ribs which are connected by a complete weir membrane. Examination of numerous oblique and transverse sections at this level did not show any pores (nephrostomes) in the membrane. There is a multiple membrane between some of the ribs. Also present are outgrowths of the internal ribs, the internal leptotriches, and outgrowths of the external ribs, the external leptotriches (Figs. 1 & 3). The wall of the flame cell above the level of the basal bodies has a zonula adhaerens along its whole length.

2. Flame cell complexes Beside single flame cells, flame cell complexes were found which consist of two flames reaching into a common cavity (Fig. 5). It cannot be excluded that further flames join the complex at different levels, though in sections through several flame cell complexes more than two flames were never seen. One of the flames has a typical weir apparatus with internal and external ribs and leptotriches, the other is probably a nonterminal flame in a capillary. But the possibility cannot be ruled out that the latter is a second typical flame cell resembling a lateral non-terminal flame because it is sectioned near its tip. Sections, some of them serial, through several flame cell complexes in Polystomoides malayi and P. renschi were examined, and in some two nuclei belonging to the two flames were found and one of the flames reached into the middle of the cavity of the other (compare Fig. 12 in Rohde, 1971 a, which is a diagram of one of several serial sections through a flame cell complex). Thus, it can be excluded that the structures seen are sections through a convoluted flame at different levels.

3. Non-terminal (lateral)flames This type of flame is found in all but the smallest capillaries, and in the protonephridial ducts. The cilia composing the flames are very loosely arranged (Figs. 6 & 7), and some show deviations from the typical ciliary structure with the 9 q- 2 pattern. There may be additional filaments (Fig. 7, in four of the cilia), cytoplasmic tube-like formations may run along parts of the cilia (Figs. 6-8), and the pattern of filaments becomes irregular with increasing reduction in their number towards the tips of the cilia (Fig. 8). The number of cilia varies strongly in all three types of flames. In single flame cells the following numbers were counted: 27, 43, 48, 49, 49, 51, 53, 55.

4. Capillaries and ducts The smallest capillaries are strongly convoluted (Figs. 1 & 4) and their wall is usually smooth with some vacuoles (Figs, 1, 4 & 5). The larger capillaries and ducts (and some small capillaries) have strongly reticulated walls (Figs. 6-8) and single cilia may be scattered in the cavities of the retieulum (Fig. 6). The non-terminal flames are anchored in the walls by

FIG 1 Polystomotdes renscht 1 s through single flame cell FIG 2 Polystomotdes renscht 1 s through basal part o f cdla o f single flame cell FIG 3 Polystomotdes malayt Obhque section through basal part o f single flame cell [IJP f p 330]

FIc, 4 Polygtomotdes Jenscht Secnon through convoluted capillary near flame t.ell FI(, 5 Poll stomotde~ tens¢ht Secnon through flame cell complex

FIG 6 Polystomotdes renschl t s through large protonephndlal duct with non-terminal flame FIG 7 Polystomotdes renschl t s through medium-sized protonephndlal duct with non-terminal flame Some basal bodies and rootlets of clha can be seen FIG 8 Polystomoldes renscht t s through cd~a of non-terminal flame Most o h a secttoned near their tips

Symbols of electron mlcrographs BG--basal granule, C--capdlary, EL---external leptotnches, F--flame, IL--mternal leptotrlches,

M--membrane of we~r apparatus, M U--muscle, N--nucleus, NF--non-termmal flame, R--Rootlets of o h a , ZA--znnula adhaeren~

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means of rootlets which form a dense mass (Fig. 7). The wall of the smaller capillaries is formed by a single cell, that of larger ducts by several cells, as indicated by the presence of a single (Fig. 7, left) or several zonulae adhaerentes. DISCUSSION The flame cells of all parasitic platyhelminths examined have a similar structure with some minor differences only. Apical processes of the ciliary membranes which anchor the tips of the cilia in the cytoplasm of the flame cell, have been demonstrated only in the aspidogastrid Multicotyle purvisi Dawes (Rohde, 1971a, 1972). They were not found in Polystomoides, though a number of sections through the tips of flame cells were carefully examined for these structures. Whether they are characteristic of all aspidogastrids, has to be shown by further studies; Digenea should be re-examined as they are closely related to the Aspidogastrea and the possibility exists that apical processes of the ciliary membranes have been overlooked. In none of the electron micrographs of flame cells of several genera of cestodes can external leptotriches be recognized (Lumsden, 1965, 1967; Race et al., 1965; v. Bonsdorff & Telkk~i, 1966; Morseth, 1967; Howells, 1969; Sakamoto & Sugimura, 1969.)On the other hand, all larval and adult Digenea and the aspidogastdd Multicotyle purvisi have not only internal, but also external leptotriches (Kiimmel, 1958, 1960; Senft et al., 1961; Cardell, 1962; Pantelouris & Threadgold, 1963; Reisinger, 1964; Rees, 1967; Wilson, 1969; Rohde, 1971a, 1972). External leptotriches were also demonstrated in Polystomoides malayi and P. renschi. The answer to the question whether this finding is of any phylogenetic significance, must be postponed until more species have been examined. A very remarkable and rather consistent difference between the major groups of parasitic platyhelminths appears to be the structure of the walls of their protonephridial ducts. In all groups, the surface of the ducts is enlarged, but this is achieved in different ways. In all the cestode genera examined, enlargement is brought about by microvilli (Rothman, 1963; Morseth, 1967, Howell, 1969). In cercarlae and adults of Digenea surface enlargement is due to numerous lamellae (Kruidenier, 1959; Lautenschlager & Cardell, 1961; Senti et al., 1961 ; Gallagher & Threadgold, 1967; Threadgold, 1968), which are apparently absent only in the strigeoid Cyathocotyle bushiensis Khan (Erasmus, 1967). Exactly the same kind of lamellae is present in the larval and adult aspidogastrid Multicotyle purvisi (see Rohde, 1971 a, b, 1972), which is closely related to the Digenea. Digenea and Multicotyle correspond also in the presence of a connective tissue sheath around the capillaries and ducts, which has not been demonstrated in any of the other parasitic platyhelminths (compare discussion by Rohde, 1971b. In Polystomoides spp. a third type of surface enlargement by means of an extensive reticulum was shown. If studies of other species of Monogenea should yield the same result, it would lend strong support to the view that Monogenea are phylogenetically completely different from the Cestoda and the Digenea + Aspidogastrea. The presence of flame cell complexes in Polystomoides is probably without phylogenetic significance as they can be derived from flame cells which are located close to ciliated capillaries. Different habitats (Polystomoides malayi in the urinary bladder, P. renschi in the pharyngeal cavity of tortoises) do not result in a different structure of the protonephridia.

Acknowledgements--Thisstudy was made during tenure of a Universityof Queensland Research Fellowship. I wtsh to thank Prof. J. F. A. Sprent for working facilities at the Department of Parasitology, and Mr. J. V. Hardy for the pernussmnto work at the Electron MicroscopeUmt, Universityof Queensland, Brmbane.

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Fasciolahepatica.