International Journal for Parasitology.

1975. Vol. 5. pp. 293-300. Pergamon Press. Printed in Great Britain.

COMPARISON OF SURFACE TOPOGRAPHY OF THREE SPECIES OF ~I~~Y~~~~~~~~ZU~ (CESTODA, PSEUDOPHYLLIDEA) BY SCANNING ELECTRON MICROSCOPY University of Oslo, Zoological Museum, Sarsgt. 1, Oslo 5, Norway (Received 16 Augusst 1974;

revised

23 Ocrober 1974)

Abstract-ANnEasEN K. 1975. Comparison of surface topography of three species of Diphyllobothrium (Cestoda, Pseudophyllidea) by scanning electron microscopy. International Journal for Parasitology 5: 293-300. Plerocercoids of different sizes as well as adult worms of D. dendriticum, D. latum and D. ditremum were studied using scanning electron microscopy (S.E.M.). In the plerocercoids there were found distinct differences in appearance and length of microtriches between these three species, while the microtriches of adult worms were more similar. A regional difference in microtrix appearance was found in the larvae of D. ditremum and D. dendriticum. This was not apparent with S.E.M. in adult worms. The length of ‘body’ microtriches in D. dendriticum varied with the length of the larvae. The topo~pby of the genital atrium of mature and gravid proglottids in adult worms of these three species is also described. KEY WORDS: Diphyllobothrium dendriticum; Diphyllobothrium latum: Diphyllobothrium scanning electron microscopy; plerocercoid; adult worm; proglottid; microtriches; genital atrium; ‘nipples’ or papillae. INDEX

ditremum;

INTRODUCTION

STUDIESbased on the relatively low resolution and magnification of light microscopy, showed microvilli or microtriches on the tegumental surface in some species of tapeworms while others were believed to be lacking these. The absence or length of microtriches were, with the light microscope, used as taxonomic characters for some D@&y@obotkrillm species (Bylund, 1973 ; Halvorsen, 1970; KuhJow, 1953; Wikgren, 1964). Transmission electron microscope studies have, however, revealed that all cestode species hitherto examined possess microtriches in the larval as well as in the adult stage and that they are probably of universal occurrence (B&ten, 1968a; Jha & Smyth, 1969, 1971; Lumsden, 1966; Morseth, 1966; Yamane, 1968). Studies on some species of pseudophy~lid~ tapeworms indicate, however, that there are differences in shape and density of microtriches between larvae and adult worms (Braten, 1968a; Grammeltvedt, 1973 ; Yamane, 1968) and between different species (Charles & Orr, 1968). Lately scanning electron microscope studies of cestodes have appeared and shown that this is a suitable method for obtaining information regarding the complex thr~-dimensional relationship of surface structures. Berger & Mettrick (1971) described the size, shape and number of microtriches in different parts of adult worms of three ~y~eno~ep~s species, while

Ubelaker, Allison & Specian (1973) gave a description of the surface topography of ~~~~nole~~~ diminuta. The present paper gives the results of a comparative S.E.M. study of plerocercoids and adult worms of R. dendritium (Nitzsch, 1824), D. latum (L. 1758) and D. ditremum (Creplin, 1825). MATERIALS AND METHODS D. dendriticum plerocercoids were obtained from trout (Saimo trutfa), D. d~tremum plerocercoids from char (Salveiinus alp&us and D. Iatum larvae were found in perch (Percajkkxtilis) and pike (Esox lucius). The plerocercoids

were killed in form01 saline (4 per cent form01 in 1 per cent saline) and fixed in the same medium for 24 h. They were subsequently transferred to 70 per cent alcohol. The treatment for S.E.M. was as follows: the larvae were deyhdrated in an ascending series of alcohol and ether and air dried, positioned on metal specimen stubs, rotary-coted with carbon gold palladium and examined with a Stereoscan, MKIIA (Cambridge Instrument Co.). The length of larvae given was calculated from fixed individuals. Adult worms of all three species were obtained from experimentally infected golden hamsters (Mesocrisetus aura&s). The hamsters were each fed, via a stomach tube under light ether anaesthesia, with 5-10 plerocercoids of either of the three Diphy~lobothrium species. The hamsters were killed 4-5 days after the first eggs were found in faecal samples, cut open, the small intestine was removed and flushed with 1 per cent saline (room temperature), After a few minutes in several changes of saline, the worms were killed and fixed in the

293

294.

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same way as previously described for the larvae. Before dehydration each worm was sectioned into the following regions: the scolex and neck, immature proglottids, mature proglottids, gravid proglottids, and the posterior end or ‘tail’ of the strobila. Except for the scoIeces, which were dehydrated and mounted intact, small pieces (2-3 proglattids) were cut out of each of the regions mentioned above. Twelve plerocercoids of different sizes and 10 adult worms of each species were examined, In addition one adult D. de~dr~~~~~rn from common gull (Lams canus) and one adult D. dendriticum from the arctic fox (Alopex Zagopus)were also examined. The measurements of microtriches given in the present paper are approximations as the microtriches do not tie flat and their base probably is not exposed. To illustrate the discrepancy between S.E.M. observations and Transmission E.M. observations two T.E.M. pictures from an adult D. dendrif~cumare inciuded in the present paper. OBSERVATIONS Pierocercoids D. dendriticum. A gradual increase in microthrix length was found from the smallest (2.5 mm) to the largest (40 mm) plerocercoids investigated. Small larvae possessed slender microtriches (Fig. 1) which observed with S.E.M. appeared to be 3-4 pm long while the ‘body’ of the biggest plerocercoids studied was covered with slender microtriches which appeared to be 7-10 Hrn long (Fig. 2). The scolex region, however, possessed shorter (2-3 pm) and not so slender microtriches (Fig. 3). D. ditremum. The ‘body’ of D. ditremum plerocercoids of all sizes investigated (2-10 mm) possessed very long (IS-17 pm) and slender microtriches (Fig. 6). In small larvae (about 2 mm) both the ‘body’ and the scolex were covered with such microtriches (Fig. 4), while in larger larvae (Fig. 5) the microtriches of the scolex appeared shorter and more densely arranged (Fig. 7). D. latum. Plerocercoids of D. latum possessed short (2-3 pm long) microtriches (Fig. 8) although with a slight increase in length from small (3 mm) to larger (IO mm) larvae. No clear regional difference could be observed with S.E.M. between ‘body’ and scolex microtriches. Adult worms

D. dendriticum, D. ditremum and D. latum. Initially, D. dendriticum was investigated, The tegument of the scolex was covered with microtriches which very much resemble those on the scolex of large larvae (Fig. 3) although being more densely arranged. The inner surface of each bothrium also possessed m~crotriches (Fig. 10). These microtriches were somewhat longer and had a more slender appearance than those seen elsewhere on the scolex. The neck region, immature, and mature proglottids were covered with conical and posteriorly directed microtriches (Fig. 9). On gravid segments,

I.J.P. VOL.5.1975

however, there was an increasing degree of disorder

in the arrangment of microtriches, while the surface of the posterior-most part in the majority of worms examined was in a stage of dissolvment with no microtriches being apparent (Fig. 11). On account of the individual variance, no difference in shape and length of microtriches could be found between worms developed in hamster and those from common gull and arctic fox. Little difference in shape and length of microtriches and their arrangement along the strobilar length existed between adult worms of D. dendriticum D. ditremum and D. latum. Tke external morphologv ofgenitatalia

In mature and gravid segments the genital atrium was situated on an elevation densely covered with ‘nipples’ or papillae (Figs. 12 & 13). This elevation had an eliptical shape in D. dendriticum and a circular shape in D. ditremum while in D. /alum it was either round or elliptic depending on the length of segments in the investigated worm [worms belonging to type A or B (Andersen, 1973)). In many segments an extended cirrus was seen (Fig. 12) and its surface was densely covered with microtriches (Fig. 14). A ‘cloud’ of spermatozoa was sometimes observed around the extruded cirrus. The spermatozoa were of primitive type with a bent head and a split tail which was longitudinally striped. DISCUSSION Several descriptions of the complex ultrastructure of cestode tegument are available (e.g. B&en, 1968a, b; Grammeltvedt, 1973; Jha & Smyth, 1971; Morseth, 1966; Yamane, 1968). The microtriches are each divided into two clearly distinguishable regions; a shaft and a base (Jha & Smyth, 1969). In S.E.M. investigations probably only the shaft is visible. To illustrate this, Figs. 15 & 16 are included in the present paper. These are Transmission E.M. sections from a mature segment and the scolex outside of an adult D. dendriticum. There appears to be a distinct difference in length of the microtrix base between these two regions while the shafts seem to be more similar. Observed with S.E.M. the microtriches of these two regions appeared similar. S.E.M. observations, therefore, only reveal differences existing in the electron dense spine or shaft of the microtrix. On the other hand, such differences (differences in length and shape of microtriches shafts) should be easier recognized with S.E.M. as no sectioning angle has to be considered. From transmission electron microscope studies on pseudophyllid~n tapeworms many different shapes of microtriches are described (e.g. Charles & Orr, 1968; Grammeltvedt, 1973; Yamane, 1968). The present S.E.M. study shows that in D. dendriticum and D. ditremum there is a change in the shape and length of microtriches during

Surface topography of Diphyllobothrium

I.I.P. VOL.5. 1975

FIG.

295

FIG. 1. D. dendriticum plerocercoid (2.5 mm) ‘body’ microtriches. FIG. 2. D. dendriticum plerocercoid (30 mm) ‘body’ microtriches. 3. D. dendriticumplerocercoid (30 mm) microtriches from the scolex region.

develop pment fr om plerocercoid to adult worms. This clIal lge is especially pronounced between the ‘body’ of the PIerocercoid and the strobila of adult worms (1‘igs. 2 & 9). The length of ‘body’ micro-

triches in D. de’ndriticttm larvae t:ended 3 ilncrc:ase from about 3 to 10 pm with the b ngth aI pr oba rbly the age of the plerocercoids (Figs .2& Wli lile the ‘body’ of all D. ditremum larvae illdepen :nt of: size

Fto. 4. B. ~j~~e~~~~pterocercclid (23 mm) scolex. Fio. 5. L).dj~~~~~~pterocercoid (4 mm). The circles indicate the regions from which Figs. 6 & 7 were obtained. Fro. 6, D. d~~r~~u~ pler~rcoid (4 mm} ‘bvdy* microtriches. Fio. 7. D. ~jr~e~~~ ~ieroce~co~d(4 mm) micrvtri~es from the scolex region, posse:ssed long (15-I 7 pm) and slender microtriches (Fig. 6). The microthriches of the strobila of adult worn IS of these two species were clearly different from those seen on the plerocercoids in being only

about 2 pm long and more densely arranged (‘Figs, 2, 6 & 9). The microtriches of the scolex re:gion, however, did not change so significantly from plerocercoids to adults as the largest larvae here

I.I.P. VOL.5. 1975

Surface topography of Diphyllobothrium

FIG. 8. D. latum plerocercoid (8 mm) microtriches from the scolex region. Fro. 9. D. dendriticum adult worm, microtriches of immature proglottid. FIO. PO. D. dendriticum adult worm, microtrich~s seen on the inner surface of a bothrium. FIG. Il. D. de~rjt~cum adult worm, surface topography of the posterior most part of the strobila, pOSSei ssed shorter and more adult like microtric~es (Figs. 3 8%7). All L). ~u~~~ p~erocercoids investigated possessed

short (about 2 pm long) microtriches, they bt :ing also more uniform all over the plerocercoid (F ‘ig. Q No clear difference in microthrix length be :tween

KARIN ANDERSEN

UP. VOL. 5. 1975

FIG. 12. D. hum adult worm, genital atrium from gravid proglottid. FIG. 13. D. dendriticum adult worm, ‘nipples’ or papillae from the genital atrium. FIG. 14. D. ditremum adult worm, cirrus. adult worm, Transmission E.M. of seolex tegument (scolex outside). M.b. = Microtrix base, MS. = Microtrix shaft. FIG. 16. D. dendritic~m adult worm, Transmission E.M. of strobila tegument (mature segment). M.b. = Micro&ix base, M.S. = Microtrix shaft. FIG. 15. D. dend&icum

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Surface topography of Diphyllobothrium

larvae and adult worms of D. la&m could be revealed with S&M. The microtriches of adult worms appeared, however, to be more slender and somewhat more densely arranged (Figs. 8 & 9). Taking account of the individual variation and compared with the plerocercoids, there was little difference in shape and length of microtriches or their arrangement along the strobilar length among adult worms of D. dendriticum, D. latum and D. ditremum. Grammeltvedt (1973) who studied plerocercoids and ad& worms of D. de~r~ticum by Transmission EM. found that the shape of microtriches varied within each plerocercoid. Three types of microtriches are reported from a single section; the procercoid type (thorn-like shape, base about O-3 pm and tip about I.4 pm); the plerocercoid type (base 0.7-0.8 l.trn and tip at least 7 pm); the intermediate type (some with short bases and long tips, others with longer bases and shorter tips). These three types are not d~stin~ishable with S.E.M. (Figs. 1 & 2). Grammeltvedt (1973), however, reports no difference in microtriches appearance between larvae of different sizes and states that no regional difference exists neither in the plerocercoids nor in adult worms (adult worms had microtriches with a base of about 1 pm length and the shaft was up to 2 pm long). This corresponds only to a certain extent with the present S.E.M. observations. In the larvae, especially the largest, there was a distinct regional difference in shape and length of microtriches between the ‘body’ and the scolex (Figs. 2 & 3). The length of body microtriches varied with plero~r~id size (Figs. 1 & 2). Except for the microtriches seen on the inner bothrium surface (Fig. lo), no regional difference in microtriches length or appearance could be observed in adult worms using S.E.M. Figures 15 & 16, however, indicate that a difference in the length of microtrix base exists between the scolex and the strobila. This is also reported by Yamane (1968) for Diplj$lobothrium erinacei. Figure 16 in the present paper is found to be in accordance with the findings of Grammeltvedt (1973, Fig. 13). B&ten (19686) investigated D. latum and found a change in shape and density of microtriches from the plerocercoid to the adult worm; the proximal part of the microtrix had increased considerabIy in length and the distal part became more slender. This corresponds with the present observations as the proximal part of the microtrix is not seen with S.E.M. The increase in microtriches density from plerocercoids to adults (Figs. 8 & 9) was in the present study, however, not so outstanding as reported by B&ten (19686) (from 4-5 per pm% in the ‘larvae to 20-30 per pme in adults). From ~yrne~ole~~s spp. Berger & Mettrick (1971) described polymorphism of microtriches along the strobilar length. This was not observed in the present study of Di~hy~~obot~rium. Neither were the scoleces equipped with special branched microtriches as

299

reported for Hyrne~o~ep~sdim~nuta by Jha & Smyth (1971). The function of the microtriches is often discussed and the general opinion seems to be that these st~ctures increase the surface area for adsorption and secretion and aid the worm in maintaining its position in the host’s gut (Lumsden, 1966; Lee, 1972; Morseth, 1966; Smyth, 1969, 1972). However, S.E.M. investigations contribute little to solve these problems. B&en (1968a) indiated that as the microtriches form an almost continuous covering of the worm, the distal part of the microtriches may be involved in protecting the worm against host reaction. In gravid segments of the present investigated worms there was an increasing tendency towards a higher degree of disorder in the arrangement of microtriches, while the posteriormost part of the strobila, in the majority of worms examined, was in a stage of dissolvment and no microtriches were visible any longer (Fig. 11). In the posterior part of adult Dj~hyllobothrium pseudoapolysis takes place, the uterus has then shed the fertilized eggs and protection against host reaction no longer seems so necessary. No difference in length or apqearance of microtriches could be seen with S.E.M. between adult worms of D. dentriticum, D. latum and D. ditremum. D. den~it~cum may, however, be separated from D. d~tremum by the shape of the elevation and arrangement of ‘nipples’ or papillae around the genital atrium, while D. latum exhibited greater variations depending on the length of segments in the investigated worm [worms of type A or B (Andersen, 1973)]. Around the extruded cirri and in the vaginal pores spermatozoa were sometimes observed (Fig. 12). The insemination process in cestodes is unknown. Both self impregnation of adjacent proglottids of the same strobila or impregnation of different strobilae, are believed to occur @myth, 1969). This insemination is probably connected with a copulation process resulting in an internal deposition of spermatozoa. The ‘nipples’ or papillae around the genital atrium (Fig. 12) may have a function in this process. Transmission E.M. studies on these structures are in progress. According to earlier ~nvesti~tions on Dip~~ylfobothrium populations (Andersen, 1972; Hafvorsen & Andersen, 1974) adult worms when found in the same host tend to be attached near to each other and to have their strobilae lying side by side or even coiled round each other. This may assist in enhancing cross fertilization between different

the chance

of

worms of the

same species. ~ckno~~edge~nts-I

am grateful to Professor Odd Halvorsen for assistance with preparation of the manuscript, to Dr. John Brittain for his help with the English and to Anne SchjGnhaug and Eva Jenssen for technical assistance. This work was mainly carried out at the

KARENANDERSEN

300 Electron Microscopical Unit for Biological University of Oslo, Blindern, Oslo 3, Norway.

Sciences,

REFERENCES ANDERSENK. 1972. Studies of the helminth fauna of Norway XXI: The influence of population size (intensity of infection) on morphological characters in DiohvJlobothrium dendriticum Nitzsch in the aolden hamster (Mesocrisetus aurafus Waterhouse).- Norwegian Journal of Zoology 20: l-7. ANDER~ENK. 1973. Studies of the helminth fauna of Norway XXXII: The primary strobila in Diphytlobothrjum Cobbold. Studies on the Development of primary strobilae in D. dendriticum (Nitzsch), D. Zatum (L.) and D. ditremum (Creplin). Norwegian Journal of Zoology 21: 341-350. BERGER J. & METTRICKD. F. 1971. Microtrical polymorphism among hymenolepid tapeworms as seen by scanning electron microscopy. Transactions of the American Microscopicrrl Society 90: 393-403. BRATEN T. 1968a. An electron microscope study of the te~ent and associated structures of the procercoid of Di~hy~~obothr~uml&urn (L.). Zeitschr~ft fur Parasitenkunde 30: 95-103. BRATENT. 196Sb. The fine structure of the tegument of Diphyllobothrium iatum (L.). A comparison of the plerocercoid and adult stages. Zeitschrift fur Parasitenkunde30: 104-112. BYLUNDG. 1973. Observations on the taxonomic status and the biology of Diphyllobothrium ditremum (Creplin, 1825) = D. osmeri (von Linstow, 1878). Acta Academiae Aboensis, Ser. B. 33: l-18. CHARLESG. H. & ORR T. S. C. 1968. Comparative fine structure of outer tegument of Ligtda ~ntestina~~sand Schistocephalus solidus. Experimental Parasitology 22: 137-149. GRAMMELTVEDTA.1973. Differentiation of the tegumcnt and associated structures in Diphyllobothrium dendriticum Nitzsch (1824) (Cestoda: Pseudophyllidea). An electron microscopical study. hrternational Journal for Parasitology 3: 321-327. HALVORSEN0. 1970. Studies of the helminth fauna of Norway XV: On the taxonomy and biology of plerocercoids of Diphy~iobothr~um Cobbold, 1858 (Cestoda, Pseudophyllidea) from North-western Europe. Nytt Magasin for Zoologi 18: 113-174.

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HALVORSEN0. & ANDERSON K. 1974. Some effects of population density in infections of D~phyl~obothr~um dendriticum (Nitzsch) in golden hamster (Mesocriset~ aurattis) and common gull (Larus canus). Parasitology 69: 1499160. JHA R. K. & SMYTHJ. D. 1969. Echinococcus granulosus: Ultrastructure of microtriches. Experimental Parasitology 25: 232-244. JHA R. K. & SMYTHJ. D. 1971. Ultrastructure of the rostallar tegument of Echinococcus granulosus with special reference to biogenesis of mitochondria. ~nternatjonal Journal for Parasitology 1: 169-177. KUHLOW F. 1953. Bau und D~fferentjal diagnose heimischer ~~~~yZ~obothrium-Pler~rcoide. Zeitschrift fur Tropenmedizin und Parasitotogie 4: 186-202. LEE D. L. 1972. The structure of the helminth cuticle. Advances in Parasitology 10: 347-379. LUMSDENR. D. 1966. Cytological studies on the absorptive surface of cestodes I. The fine structure of the strobilar integument. Zeitschrift fir Parasitenkunde 27: 355-382. MORSETHD. J. 1966. The fine structure of the teg~ent of adult Ech~n#coccus granulosus, Tuenia hydatigena and Taeniapisiformis. The Journal of Parasitofogy 52: 1074-1085. SMYTH J. D. 1969. The Physiology of Cestodes, 279 pp. Oliver & Boyd. Edinburgh. SMYTHJ. D. 1972. Changes in the digestive absorptive surface of cestodes during larval/adult differentiation. In Functional Aspects of Parasite Surfaces, Symposia for the British Society for Parasitology, Vol. 10 (Edited by TAYLORA, E. & MULLERR.), pp. 41-70. UBELAKERJ. E., ALLISON,U. F. & SPECIANR. D. 1973. Surface topography of Hymenolepis diminuta by scanning electron microscopy. The Journal of Parasitology 59: 667-671. WIKGREN B.-J. 1964. Notes on the taxonomy and occurrence of plerocercoids of Diphyllobothrium dendriticum Nitzsch, 1824, and D. osmeri (v. Linstow, 1878). Societas Scienf~arum Fennica, Comment~iones B~o~ogicffeXX VII, 6: I-26. YAMANEY. 1968. On the fine structure of ~~hyflobothrium erinacei with special reference to the tegument. Yonago Acta Medica 12: 169-181.