Cyclophyllidean Cestodes Research Articles

Parthenogenesis and asexual multiplication among parasitic platyhelminths.

Among flatworms with parasitic and commensal modes of existence, parthenogenesis and asexual multiplication appear to be largely confined to the Digenea and Cestoda, the only parasitic platyhelminths that routinely utilize indirect life-cycles. Parthenogenesis is apparently restricted to a minority of adult digeneans and cestodes inhabiting their final hosts, and a survey is made of the particular modes of parthenogenesis (i.e. apomictic, automictic and generative) which are employed by such adults. Asexual (amictic) multiplication, in the form of fissioning, is demonstrated by young adults of the cyclophyllidean cestode, Mesocestoides corti, but is otherwise not exhibited by adult cestodes or digeneans, other than in the perplexing phenomenon of proglottid formation in polyzoic tapeworms. Secondary multiplication is of ubiquitous occurrence in digenean life-cycles in the form of the proliferation which takes place within sporocysts and rediae (germinal sacs) located in the first intermediate host. The controversy concerning the nature of this multiplication is reconsidered in the context of recent findings which have centred on cellular aspects. On the basis of present evidence germinal sac multiplication should be regarded as an asexual rather than a parthenogenetic process. The cestode asexual multiplication which occurs in intermediate hosts is a function of the metacestode stage of development. Metacestode proliferation is only known from about 20 species and 6 families of polyzoic cestodes with approximately half the described instances occurring in the family Taeniidae. The organization of these proliferative metacestodes, findings concerning their totipotent stem cells and the ontogeny of buds and new scolices are all reviewed. Finally, the capacity for population expansion of multiplicative larval digeneans and metacestodes are compared, while the ecological roles and the genetical consequences of both parthenogenesis and amictic multiplication in the two taxa are also examined.

On the nature of the proglottids of cestodes: a light and electron microscopic study on Taenia, Hymenolepis, and Echinococcus.

The organisation of five species of cyclophyllidean cestodes (Hymenolepis nana, H. diminuta, H. microstoma, Taenia (Hydatigera) taeniaeformis, Echinococcus multilocularis) has been studied using light and electron microscopy (SEM, TEM). It was found that there is no indication of any internal structure separating the proglottids, which are recognisable superficially. Thus in cestodes there is no metameric segmentation as in annelids. The implications of these findings on the terminology of tapeworm morphology are critically discussed.

Étude du tégument et des récepteurs sensoriels du scolex d’un plérocercoïde de Cestode Tetraphyllidea à l’aide de la microscopie électronique

The ultrastructure of bothridia and the myzorhynchus of a tetraphyllidean cestode plerocercoid is described. Two types of microtriches occur on the tegument of these organs. On the bothridium, the microtriches are lanceolated whereas on the mizorhynchus, they are filamentous. Uniciliated sensory receptors, which consist of a basal bulb contaning one or two electron-dense collars, are observed especially on the bothridia and myzorhynchus. A papilla is also described on the tegument. According to the present study, it is suggested that the rostellum of cyclophyllidean cestodes and the myzorhynchus of the tetraphyllideans are homologous. Moreover, the role in the attachment and sensory perception of the myzorhynchus is revealed.

The functional significance of scolex retraction and subsequent cyst formation in the cysticercoid larva of Hymenolepis microstoma

The cysticercoid type of larval development, involving the retraction of the scolex and the associated development of a cyst, is of widespread occurrence within cyclophyllidean cestodes, but previously its possible adaptive value has been unexplained. Scolex retraction in Hymenolepis microstoma is an active, muscular process which takes only about a minute to complete. Following its retraction the scolex develops adult structures. The cyst enclosing the scolex also differentiates further, involving the secretion of three layers of collagen fibres and the development of a myelin-like layer adjacent to the retracted scolex. The cells within the cyst, including the muscle, become condensed or are autolysed causing a decrease in thickness of the cyst and an increase in the proportion of fibrous tissue. The collagenous part of the cyst provides mechanical protection for the scolex during grinding by the teeth on entry into the mouse host. The myelin-like layer insulates the scolex from the low pH conditions of the stomach. Values of lower than pH 3·0 are lethal to newly excysted worms. The myelin-like layer loses its insulatory capacity following exposure to bile salts.

Cyclophyllidean Cestodes of Australian Birds, with Three New Species

Cyclophyllidean cestodes from Australian birds are reported. Lapwingia adelaidae sp. n. from Lobibyx novaehollandae differs from L. reticulosa Singh, 1952, in having 20 rostellar hooks, each 20 u long, compared with 22 hooks, each 45 to 48 ,u long. Capiuterilepis australensis sp. n. differs from C. naja (Duj., 1845) in having rostellar hooks 40 It long, compared with 52 to 56 long, and having a very large, persistent seminal receptacle, rather than a small ephemeral one. Capiuterilepis meliphagicola sp. n. differs from C. naja in having a strobila 90 to 105 mm long, compared with 10 mm long. It differs from C. australensis in having rostellar hooks 48 to 50 long rather than 40 long, and in having a strobila twice as long. Hemiparonia bancrofti (Johnston, 1912) comb. n. is redescribed. The scolex armature of Gyrocoelia australiensis (Johnston, 1910) Johnston, 1912, is described for the first time. Also reported and briefly discussed are Anoncotaenia arhyncha Fuhrmann, 1918; A. globata (Linstow, 1879) Cohn, 1900; Pseudochoanotaenia meliphagidarum (Johnston, 1911) Matevosyan, 1953; Raillietina polychalix (Kotlan, 1921) Fuhrmann, 1924; R. cacatuina (Johnston, 1913) Fuhrmann, 1924; Infula burhini Burt, 1939; Paronia trichoglossi (Linstow, 1888) Spasskii, 1951; and Hemiparonia cacatuae (Maplestone, 1922) Baer, 1925. The following report is based on specimens collected by me in South Australia from December 1970 through March 1971. In addition, many vials of unstudied specimens were borrowed from the Helminthological Collection of The University of Adelaide. Most of the latter had been collected 30 or 40 years ago by Dr. T. H. Johnston, Mr. J. B. Cleland, or Mr. T. L. Bancroft, among others. All specimens were stained with Semichon's carmine and mounted in balsam for further study. All measurements are in microns unless otherwise

Developmental Physiology of Cestodes. IX. Cytological Characteristics of the Germinative Region of Hymenolepis diminuta

The germinative region of developing Hymenolepis diminuta is defined based on cytological characteristics and DNA synthetic activity as beginning 200 ,u posterior to the apex of the scolex, and extending posteriorly to the point of germinal primordia formation. The structure of the germinative type in this region is described. The kinetics of division in the germinative region are studied, and estimates of the duration of G1, S, and G2 of prophase are 3.0, 2.3, and 3.2 hr, respectively. The time for a complete mitotic division is estimated to be 8.5 + hr. Although proglottisation in most cyclophyllidean cestodes has been attributed to budding from the unsegmented region immediately posterior to the scolex (Wardle and McLeod, 1952; Roberts, 1961), no cytological definition or exact location has been given to the so-called germinative area. Roberts (1961) indirectly defined the germinative area of Hymenolepis diminuta as the area behind the scolex in which genital primordia are not observed. Sandeman (1959) defined the germinative area of Capsulata edenesis, a parasite of Limosa lapponica, as a posteriorly located diffuse area of the strobila from which the rest of the strobila originates. If the germinative region is indeed responsible for proglottid production, the cellular activity in this region should be significantly different from the rest of the strobila, and division indices and an index of cells involved in DNA synthesis could be used as criteria of definition. This region should also contain a cytologically identifiable type which could act as a stem cell for production of proglottids. Bell and Smyth (1958) and Wikgren (1964) have used the criterion of a mitotic index to estimate growth in trematodes and cestodes. The use of the incorporation of tritiated thymidine into DNA as an index of DNA synthesis and, therefore, as an index of replication was discussed by Clever (1967). Received for publication 30 July 1970. * This investigation was supported by Research Grant AI-06153 and Training Grant 5 TOI AI226 from the NIH, U. S. Public Health Service, Bethesda, Maryland 20014. tPresent address: Department of Biology, Notre Dame University, Notre Dame, Indiana 46556. In the present study we have investigated the cytological characteristics of the germinative region of H. diminuta and a possible germinative type within this region. The duration of the cycle of the proposed germinative has been estimated. MATERIALS AND METHODS Recovery and incubation of H. diminuta Male Holtzman rats (120 to 180 g) were inoculated with either 100 or 10 cysticercoids and worms were recovered 2, 4, and 6 to 14 days postinfection, respectively. Worms were recovered 2 and 4 days postinfection from the hosts' small intestine by scraping the intestinal mucosa with a glass microscope slide into phosphate-buffered (pH 7.4) balanced salt solution (BSS) (Roberts and Fairbairn, 1965) or into tris-maleate-buffered Ringer's solution (Read et al., 1963) with 0.1% added glucose (TMR + G; pH 7.4). The gut scrapings were examined under a dissecting microscope, and the worms were separated from the debris with a Pasteur pipette. Older worms, 6 to 14 days postinfection, were flushed from the rat small intestine with a small amount of salt solution. The worms were washed, incubated 15 min in BSS or TMR + G containing 0.4 mg streptomycin and 500 units potassium penicillin-G per ml, and then incubated in the salt solution containing an experimental substrate. All incubations were done in 25-ml Erlenmeyer flasks with a tissue-to-medium ratio of 60 mg worm fresh weight/ml incubation fluid. Incubations were at 37 C in an atmosphere of N2:CO2 (95:5). Following the incubation, the worms were washed with salt solution and fixed for cytological studies. Preparation for determination of mitotic index Worms were incubated in a concentration of colchicine for a length of time necessary to obtain the maximum number of arrested metaphase figures. Entire 2and 4-day-old worms and the ant rior 2 cm of 6to 14-day-old worms were fixed immediately in 3:1 (v:v) 70% ethanol:neutral

Parasites of the relict fauna of Ceylon. II. New species of Cyclophyllidean Cestodes from small hill-vertebrates.

Five new species of cyclophyllidean Cestodes are described from small mammals and an amphibian, collected on the Horton Plains (7,200 ft.) and in Peradeniya (1,600 ft.), in Ceylon.Four of the new species are hymenolepids, namely : Vampirolepis solisoricis from the small intestine of the Ceylon Large Shrew, Solisorex pearsoni , an endemic genus and species confined to the hills ; Vampirolepis montana and Hymenolepis sunci , from the small intestine of the Ceylon Highland Shrew, Suncus murinus montanus , an endemic subspecies of the hill country ; and Pseudhy-menolepis eisenbergi from the stomach of the same host. An unidentifiable Hymenoiepis sp. was also recovered from the small intestine of the Highland Spiny Rat, Mus (Coelomys) mayori (subgenus and species peculiar to Ceylon), from the Horton Plains.A new nematotaeniid species, Cylindrotaenia philauti , is described from the small intestine of the rhacophorid frog, Philautus variabilis , collected on the Horton Plains.

Developmental Physiology of Cestodes. VIII. Inhibition of Ribonucleic Acid Synthesis by Actinomycin-D in Developing Hymenolepis diminuta

The uptake and incorporation of actinomycin-D by Hymenolepis diminuta (Cestoda:Cyclophyllidea) was measured. Treatment of very young worms (4 days after infection of the host) with concentrations of this drug of 9.5 ILM to 15.9 /M inhibited incorporation of uridine into RNA by greater than 90%. Synthesis of DNA-dependent RNA was 100% inhibited. Inhibition was greater in the germinative and immature regions than in the mature proglottids of the strobila of 8-day-old worms. There was no effect of the drug on incorporation of thymidine into DNA. The data support the use of actinomycin-D as a tool in studying gene activity during development of H. diminuta. Actinomycin-D (AM-D) causes a block in protein synthesis by binding to the guanine bases of DNA, preventing transcription of genetic information by RNA polymerase, and, therefore, inhibiting the synthesis of DNA-dependent RNA (Carter and McCarty, 1966; Muller and Crothers, 1968). Actinomycin-D has been useful in studying control of development in various systems, e.g., developing embryos of sea urchins, amphibians, mice, and chicks (Gross and Cousineau, 1963a, b, 1964; Gross et al., 1964; Brachet et al., 1964; Brachet, 1968; Lane, 1969; Mintz, 1964; Piper and Klein, 1969). It has also been used by Bovarnick et al. (1969) to study the development of chloroplasts in Euglena and by James (1964) and Burchill (1968) to investigate regeneration in Stentor. Recently, the uptake and incorporation of this drug by sea urchin eggs and embryos and by mealy bug tissues in culture has been studied (Thaler et al., 1969; Berlowitz et al., 1969). Harris, Fodge, and Talent (pers. comm.) have used AM-D in an investigation of RNA synthesis in Hymenolepis diminuta. The strobila of H. diminuta, like most cyclophyllidean cestodes, develops by the budding of proglottids from the unsegmented region immediately posterior to the scolex (see discussion by Roberts, 1961, p. 363). Because of the central importance of this germinative region in Received for publication 2 April 1970. * This investigation was supported by Research Grant AI-06153 and Training Grant 5 TOI AI226, from the NIH, U. S. Public Health Service. t Present address: Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556. cestode development, the relation of DNAdependent RNA synthesis to protein synthesis in the region is of considerable interest The present report concerns the use of actinomycin-D as a tool in this investigation. The experiments reported here were performed on young worms recovered from the rat host at 4 days and at 8 days after infection At 4 days postinfection, H. diminuta consists of scolex, germinative area, and a few immature proglottids; organogeny (but not segmentation) has just begun, and the worms are about 5 mm long (Roberts, 1961). By comparison, H. diminuta at 8 days postinfection has 600 to 700 immature and 100 to 300 mature proglottids; gametogenesis is actively proceeding in the terminal proglottids, but embryogenesis has yet to start (Roberts, 1961). MATERIALS AND METHODS Recovery and incubation of H. diminuta Male Holtzman rats (120 to 180 g) were inoculated with either 500 cysticercoids or 10 cysticercoids per rat for recovery and incubation of the worms at 4 days and 8 days postinfection, respectively. The worms were removed from the rat small intestines into Krebs-Ringer's salt solution (phosphate buffer, pH 7.4), then washed and sterilized according to the method described for in vitro cultivation (Roberts and Mong, 1969). The basal incubation medium to which experimental substrates were added was a Hanks' solution with added glucose (as used by Roberts and Mong, 1969, for the cultivation fluid overlay). The worms were incubated for 2, 4, or 8 hr in 25-ml Erlenmeyer flasks at 37 C, under a gas phase of nitrogen/carbon dioxide (95/5), and a shaking rate of 160 excursions/min. One milliliter of medium was provided per 60 mg worm fresh weight. After incubation, the worms were washed 8 times in clean Hanks' solution and either fixed for autoradio-

Electron Microscopy of Postembryonic Stages of the Tapeworm, Hymenolepis citelli

The morphology of precysticercoids of the tapeworm, Hymenolepis citelli, was studied at the ultrastructural level and has provided baseline data which may be applicable to other species of the family Hymenolepididae. Differences in cellular morphology between the developmental stages and those of the hatched oncosphere are described. Histogenesis of some major tissue types is discussed. Microvilli, structurally unlike those of the adult, are found on the periphery of the organism. The host response to the postembryonic stages is indicated by beetle hemocyte attachment to the cestode microvilli. Degeneration of these hemocytes occurs with time and may be related to contact with the microvilli. The structural characteristics of cestode postembryonic stages have received little current attention, particularly with more advanced technology. Studies by Voge (1956, 1960a, 1961) have helped to elucidate the development of the postembryonic stages of Hymenolepis diminuta and H. citelli, and have provided data on which the present study is based. Due to the limitations of the light microscope and the cellular density of these small organisms, a considerable amount of structural detail remains unresolved. Braten (1968a, b) has described the fine structure of the larval stages of the pseudophyllid, Diphyllobothrium latum. Race et al. (1965) studied developing stages of the taenioid cestode, Multiceps serialis, and Morseth (1967) investigated the morphology of the hydatid cyst and protoscoleces of Echinococcus granulosus. However, little has been done to describe the cytomorphogenesis of tissues which lead to the formation of adult structures of a cyclophyllidean cestode. The adult stages of the hymenolepids have been used extensively for biochemical and ultrastructural studies but the general morphology of developmental stages has not been investigated at the utrastructural level. This study describes the cell types present in 3and 5-day developmental stages of Hymenolepis citelli with the hope of establishing a pattern of morphogenesis applicable to other cestodes of the group. Incorporated are also observations of the cellular interaction between Received for publication 12 March 1970. * This investigation was supported by the NIH Fellowship 7 F02 AI40-01A1 from NIAID. the host, Tribolium confusum, and the para-

The life cycle of Proteocephalus fluviatilis Bangham (Cestoda) from smallmouth bass, Micropterus dolomieui Lacépède

In all, 1176 smallmouth bass (Micropterus dolomieui), ranging from 1.0 to 46.7 cm in fork length, were examined for Proteocephalus fluviatilis Bangham. Of bass more than 7.6 cm in length, 67% were infected whereas 16% of bass fry less than 7.6 cm in length contained these cestodes. Plerocercoids, segmented, and early gravid P. fluviatilis occurred throughout the sampling period from May into October, but egg shedding worms were not found until June when the surface temperature of the lake approached 15 °C. Gravid P. fluviatilis were up to three times larger in Ontario than in Ohio. The egg was described, and structures similar to "penetration glands" or "epidermal glands" reported from cyclophyllidean cestodes were found in the oncosphere. Plerocercoids of P. fluviatilis developed in mature female Cyclops bicuspidatus, C. vernalis, C. scutifer, and Tropocyclops prasinus; development from egg to plerocercoid I required approximately 18 to 21 days at 18 °C. Plerocercoid I infected smallmouth bass fry directly from the copepod, increased in size to plerocercoid II, then segmented and matured without leaving the gut. Plerocercoids, segmented but not gravid worms, and scoleces from gravid worms were transferred from one bass to another and continued growth and differentiation in the second host. Plerocercoids survived as long as 6 days in fish other than bass, but segmented worms did not survive longer than 24 hours.

Histochemical Studies of Esterases in the Cuticle and Nerve Cords of Four Cyclophyllidean Cestodes

The histochemical localization of nonspecific (simple) esterase and cholinesterase and the effects of esterase inhibitors on these enzymes have been studied in Hymenolepis diminuta, H. nana, Dipylidium caninum, and Hydatigera taeniaeformis. The enzymes are primarily localized in the cuticle and nervous system, but with quantitative and qualitative differences from species to species. Inhibition studies suggest that the enzyme hydrolyzing both acetate and thiocholine substrates is an eserine-, fluoride-, and organic phosphate-sensitive cholinesterase. Partial inhibition by a strong ammonium base and hydrolysis of two different choline esters indicate that both acetylcholinesterase and pseudocholinesterase are involved. During a recent histochemical survey of certain tapeworms (Waitz and Schardein, 1964), reactions to nonspecific esterase and cholinesterase histochemical techniques varied in intensity and localization in the several species. This paper deals with an analysis of these esterases in several cestode species through the use of various known esterase inhibitors. Esterases, particularly of the cholinesterase type, have been demonstrated in homogenates of several cestode species (Read and Simmons, 1963). The localization of nonspecific esterase and cholinesterase in cestodes has been demonstrated histochemically in Hymenolepis diminuta, H. citelli, H. microstoma, and Hydatigera taeniaeformis by Lee et al. (1963), and in Anoplocephala perfoliata by Lee and Tatchell (1964). By use of eserine and E-600 as inhibitors, these workers demonstrated esterases in the cuticle, parenchyma, excretory system, and in portions of the reproductive system, as well as cholinesterases in the cuticle, parenchyma, nervous system, and reproductive system. Species' differences in the distribution of these

Comparative Histochemical Studies on Cestodes I. The Distribution of Glycogen in Hymenolepis diminuta and Raillietina cesticillus

Since numerous physiological studies with endoparasitic organisms have shown considerable differences, often between taxonomically closely related forms, a comparative histochemical study was conducted on 2 cyclophyllidean cestodes to determine what differences, if any, exist in the distribution of certain substances in their bodies and, if possible, to determine the significance of these substances in the metabolism of the tapewormns. Hymenolepis dininuta (Rudolphi, 1819) and Raillietina cesticillus (Molin, 1858) were particularly well-suited for this purpose since they were readily available and since concurrent studies on their metabolism were being made in the same laboratory.

A morphological and histochemical study of egg formation in a cyclophyllidean cestode.

1. The morphology and histochemistry of the genitalia concerned in the formation of the egg in Multiceps smyihi n.sp. are described.2. The vitelline cells are devoid of the phenolic protein globules, which occur in the vitelline cells of the Pseudophyllidea; this result is correlated with the absence of a true external egg-shell.3. The hardened embryophore (often mistakenly termed the egg-shell) gives none of the reactions of sclerotin (tanned protein) or chitin, but gives a strong P.F.A.S. reaction for disulphide bonds, suggesting that it is a ‘keratin’ type of protein.4. The cells of Mehlis' gland secrete a material which is intensely P.A.S.-positive and is not digested by saliva, α-amylase or hyaluronidase. This suggests that it may be secreting a mucopolysaccharide or muco- or glycoprotein.

THE PHYSIOLOGY OF TAPEWORMS

Summary1. Cestodes have a high carbohydrate content in the form of glycogen which is stored mainly in the parenchyma. The proportion of ether‐extractable material is also high and consists mainly of phospholipids. The protein content–probably mainly scleroproteins–is unusually low, being less than the sum of the carbohydrate and fat contents.2. The following physico‐chemical properties of cestodes are known: (a) the osmotic pressure of the parasites is in general lower than that of the surrounding host tissue or fluid; (b) the cuticle is freely permeable to water; (c) the internal fluid of cysticercoids is merely a transudate of the host serum; (d) cestodes have a pH tolerance range of approximately 4–11.3. Owing to the failure to establish aseptic conditions attempts to cultivate cestodes outside the host body have until lately been unsuccessful. Recent work has shown that once asepsis is obtained it is possible to cultivate plerocercoid larvae in vitro for periods up to 10 months at room temperatures. By raising the temperature of cultivation to 400 C, larvae can be induced to become sexually mature strobilae in vitro. This technique should allow of considerable advances in our knowledge of cestode physiology.4. The evidence suggests that immunity to digestion of cyclophyllidean cestodes from the host digestive juices is due to the protective action of the cuticle, and that anti‐enzymes are not produced. Adult worms are quite resistant to alkaline pancreatic juice in which they are normally bathed. Digestion of the strobila can, however, be obtained by subjecting the cuticle to both acid and alkaline baths previous to enzymatic action; this effect may be due to the interaction of these substances at the cuticle surface, opening up the surface and allowing the enzymes to act. When the eggs are eaten by the intermediate host, the embryophore surrounding the onchosphere will only dissolve after immersion in dilute acid followed by alkali; consequently, the onchosphere can only be freed in the intestine and not in the stomach. When u cysticercus is swallowed by the final host similar factors operate. The scolex of the cysticercus is invaginated into a ‘sleeve’ which protects it completely from the action of the acid gastric juice. On reaching the duodenum, the presence of bile stimulates the scolex to evaginate, and the bladder (which receives both acid gastric and alkaline pancreatic juice) is digested, whereas the scolex which has received the latter only, remains undigested and elongates into a typical strobila.5. Knowledge of metabolism is scanty. It is known that tapeworms can utilize glucose in quantity; and that the excess glucose is stored in the parenchyma in the form of glycogen. Plerocercoid larvae of pseudophyllidean cestodes can, however, undergo complete maturation in vitro in glucose‐free media and it is supposed that cestodes may be capable of utilizing complex carbohydrate‐protein compounds. The metabolic rate in warm‐blooded hosts is high and the glycogen reserve falls rapidly to about one‐eleventh of its initial level after 20 hr. of host starvation. The glycogen is probably decomposed anaerobically with fatty acids as by‐products; cestodes can, however, utilize oxygen if it is offered to them.6. There is as yet no evidence to indicate that cestodes have any fat requirements.7. Little is known regarding protein metabolism; absence of protein in the host diet has no apparent effect on the metabolism of the worm.8. The nutritional requirements may not be as simple as is generally believed, and there is some evidence that vitamins–notably vitamin G–may be essential for normal cestode development.