D. J. Ameel

THE EARLY DEVELOPMENT OF THE DAUGHTER SPOROCYSTS OF THE STRIGEOIDEA (TREMATODA)

appear to be differences between species. However, there are rarely fewer than 12 or more than 30 in each sporocyst. The largest embryos of the germinal masses are at the ends and the production of free embryos in both mother and daughter sporocysts appears to be accomplished only by their breaking off. They are replaced by the growth of other embryos which in their turn separate from the masses; new embryos appear to be constantly formed from the germinal cells which persist in the germinal masses and multiply throughout the reproductive life of the germinal sacs. This mechanism for the multiplication of germinal cells in the strigeoid group produces in the mother sporocyst a sufficient number of daughters to fill completely the digestive gland of the snail host, and in the daughters the enormous numbers of cercariae which escape during the life of an infection. Up to the pres

Studies on a microsporidian hyperparasite of strigeoid trematodes. II. Experimental transmission.

In the first paper of this series (Cort, Hussey and Ameel, 1960) we reported on investigations made during the summers of 1956, 1957, and 1958 on the prevalence and effect on the parasitized larval trematodes of a microsporidian hyperparasite of the genus Nosema, which is found in a large proportion of the strigeoid infections in the snails of the region around the University of Michigan Biological Station near the northern tip of the southern peninsula of Michigan. We also carried out series of experiments designed to solve some of the problems involved in the transmission of this species of Nosema. As in the first paper of this series, we will refer to this microsporidian hyperparasite as Nosema sp. and will not try to determine its exact specific relationships until more details of its life cycle have been worked out. In a preliminary experiment done during the summer of 1956 we tried to find out what happened to the spores of Nosema that were ingested by snails that harbored no trematode infections. Several small laboratory raised juveniles of Physa parkeri were placed in small dishes with enormous numbers of the spores of Nosema sp. They were seen to ingest the spores during the period they were in the dishes with them, so that very large numbers must have entered their digestive tracts. Examinations were made of 2 of these snails, one after 36 hours and the other after 72 hours. Although their tissues were carefully teased apart and examined under the high power of the microscope, no trace of the spores or of anything recognizable as developmental stages of the microsporidian was found. Two more of the snails were killed and fixed for sectioning, one at 36 and the other at 72 hours after exposure to the spores. In examination under high power of the microscope of sections of the whole bodies of these snails no trace of the spores or of anything recognizable as a stage of Nosema sp. was found. Rather late in the summer of 1956 an experiment was performed in which snails harboring larval strigeoids were exposed to very large numbers of the spores of Nosema sp. During the summer of 1957 four more experiments of this same type were carried out, in which it was determined how long it took experimental infections to produce mature spores. In one of these experiments it was demonstrated that the metacercariae (tetracotyles) of Cotylurus flabelliformis could be infected with Nosema sp. Two further experiments in the summer of 1958 showed that the spores could survive at least a year and that several different species of larval strigeoids could be hyperinfected experimentally with this microsporidian.

Further studies on the development of the sporocyst stages of plagiorchiid trematodes.

As a part of a program of investigations on the embryology of the digenetic trematodes, a study was made, during the summer of 1941 (Cort and Olivier, 1943b), at the University of Michigan Biological Station of the development of the sporocyst stages of a common plagiorchiid, Plagiorchis muris Tanabe, 1922. The material was obtained from immature natural infections in juveniles and adults of Stagnicola emarginata angulata (Sowerby) from a beach where a very high incidence of P. muris was known to occur. Only late stages of the mother sporocyst of P. muris were found. They are entirely different from any mother sporocysts previously described for this group. They are oval, irregularly disc-shaped masses, 0.5 to 1.7 mm in largest diameter, which are made up of large numbers (about 300 to 500) of closely packed daughter sporocyst embryos (I.c., Figs. 1 and 2). These masses are discrete structures firmly attached to the outside of the snails intestine, and may occur anywhere along its whole length. Each daughter sporocyst in the mass is surrounded by an outer coat of irregular cells. In fact, the mother sporocyst, at this stage, has no special outer wall, but is composed entirely of the daughters, which appear to be held together by the cells of their outer coats which form the matrix of the mass. This outer coat of the daughter sporocysts persists throughout their whole life. It has been described for mature daughter sporocysts of a number of species of plagiorchiids, and has been called the paletot. In the largest mother sporocysts the daughters are elongate and mobile when freed. Each contains cercarial embryos of different stages of development, and a single large discrete germ-mass (l.c., Fig. 13). It was found that when the mother sporocysts are located on those portions of the snails intestine in front of the digestive gland, the daughters break away from the mother and migrate to the digestive gland and other organs of the snail, leaving behind no trace of the mother. When the daughter sporocysts cease migrating they become firmly attached to the snails tissue, grow thicker and appear crowded with cercarial embryos. Soon, thickened areas of the true sporocyst wall appear at their ends, which frequently

Life History of the North American Lung Fluke of Mammals

On July 29, 1931, a collection of snails of the species Pomatiopsis lapidaria taken near Ann Arbor, Michigan, yielded several individuals shedding microcercous cercariae closely resembling the cercaria of the human lung fluke, Paragonimus. Numerous cercariae were placed in a dish with parasite-free crayfishes. They soon disappeared from sight and an examination of the ventral side of a crayfish revealed numerous cercariae penetrating through the thin chitin of the tail at the union of the segments. About twenty hours after exposure, eleven cercariae were found in the heart muscles of one of these crayfishes. Later examination of other crayfishes of the same lot revealed similar infections. The cercariae did not encyst immediately, but individuals were found one to two days after infection completely enclosed in a very thin membrane. After a four weeks sojourn in the crayfishes, encysted metacercariae were recovered that agreed in every respect with proven Paragonimus metacercariae found in naturally infected crayfishes of the same region. Although this cercaria differs in certain details from Kobayashis figures and descriptions of the Asiatic lung fluke, there are many points of similarity. A comparison of the work of Kobayashi (1921) and of Faust (1929) on the same species shows several discrepancies. My material agrees much more closely with Kobayashis figures than with Fausts. Measurements in microns were made of fifteen specimens killed in 5% formalin and slightly flattened under a number one cover slip. The averages are as follows: total length, 178; width, 93; tail, 15 by 14 (often perfectly spherical); oral sucker, 48 (usually slightly elongated anteriorly); stylet, 39 by 7; acetabulum, 47 by 34. The cercaria is completely covered with spines of about equal length which are most conspicuous on the posterior portions of the body and tail. The group of large spines located on the tip of the tail is particularly conspicuous, for the remainder of the tail is sparsely covered with minute spines. Penetration glands surround the lateral and anterior edges of the acetabulum but usually do not extend beyond its posterior border. These can be differentiated into two types by their affinities for neutral red: a lateral group of four large cells on each side of the body which have a great affinity for this stain, and a median mass of six smaller cells which stain poorly. Four large ducts leave each group of

Variations in infections of Diplostomum flexicaudum (Cort and Brooks, 1928) in snail intermediate hosts of different sizes.

Diplostomum flexicaudun (Cort and Brooks, 1928) is by far the commonest trematode species in the snails of the Douglas Lake region of northern Michigan. Its cercaria was described by Cort and Brooks (1928) and its life cycle was worked out experimentally by van Haitsma (1931). The metacercaria was described by Hughes and Berkhaut (1929) as Diplostomulum gigas from the lens of the eyes of the common sucker, Catostomus commersonnii. The adult is a parasite in the small intestine of the herring gull, Larus argentatus. The cercaria of this species is very common in the beach snails, Stagnicola emarginata angulata (Sowerby) and S. e. canadensis (Sowerby). It has also been found in the Douglas Lake region in Lymnaea stagnalis appressa Say, L. s. perampla Walker, Stagnicola exilis (Lea), Stagnicola palustris elodes (Say) and Fossaria abrussa (Say). Since its snail hosts vary so greatly in size, D. flexicaudum is ideal for the study of the adaptation of a trematode species to intermediate hosts of different sizes. The cercariae of this species are very characteristic in structure and behavior, and there is not the slightest evidence that we have been dealing in this study with more than one species or with varieties of a species. Measurements of cercariae did not show size differences related to the size of the intermediate hosts. The slight variations that we found in the different lots, which were measured, appeared to be due to differences in the killing procedures, since some lots from a large host were slightly smaller than others from much smaller ones.

The Distribution of Paragonimus.

A brief resume of the results of the study on the distribution of Paragonimus was published in the American Journal of Hygiene by one of us (Ameel, I934). However, it has been deemed advisable to place the complete data at the disposal of those who may be interested in furthering the knowledge of the distribution of this parasite in North America. Though material was secured from many parts of the United States and from several regions in Canada by means of expeditions and purchase, the actual area covered is relatively small. The genus Paragonimus has almost world-wide distribution and for the sake of completeness, the published records are briefly summarized here. Authentic reports for Asia are as follows: Formosa (Manson, I88I; Kubo, 1912), Japan (Baelz, I880), Korea (Kobayashi, I918), New Guinea (Cilento and Backhouse, 1927), Indo-China (Salomon and Neveu, 19I7), Philippine Islands (Musgrave, I907), India (Cobbold, 1859; Vevers, I923), Malay States (Vevers, I923), Sumatra (Weber, 1891), Java (Noto-Soediro, 1930), China (Maxwell, 1931; Wu, 1935 and I936). Faust (1929) also lists Siam. Onorato (1920) published what appears to be an authentic record of a human case of paragonimiasis from Africa. The patient, a resident of Tripoli, had never been further from home than Tunisia. The symptoms described were similar to those of paragonimiasis and the measurements of eggs recovered in the sputum were within the range of Paragonimus. After the onset of the disease, the patient suffered from epileptiform attacks which Onorato believed might be due to the presence of worms in the brain. About 7 years after he first began to suffer from the disease, the patient died, but, unfortunately, no autopsy was performed. Onorato and also Salomon and Neveu (1917) casually mentioned a case of paragonimiasis in an Arab reported by R. Wurtz but no reference was given. A copy of the report which was included in an unpublished thesis by M. Crespin presented in I908 and deposited in the University of Paris was secured through the kindness of Prof. E. Brumpt. The patient, a native of Cayor, had spent io years in Senegal and Dahomey in the military service. In addition, he had resided a year on the ivory coast and 5 years in Madagascar. Attacks of hemoptysis began 4 months

The life history of Nudacotyle novicia Barker, 1916 (Trematoda : Notocotylidae).

The greater part of the work on the life history of Nudacotyle novicia was undertaken at the University of Michigan Biological Station during the summers of 1939 and 1940. Several phases of the study lack completeness, but since further work has not been possible the data at hand are being made available. Infection experiments involving the feeding of metacercariae of Cercaria mnarilli Ameel, 1939, to various birds and mammals yielded adult specimens of N. novicia from the bile duct of the meadow mouse, Microtus pennsylvanicus pennsylvanicus. Formerly this species had been reported only from the small intestine of the muskrat, Ondatra zibethica zibethica. Subsequently infection experiments with the snail host provided an insight into the early development of this trematode in the intermediate host. Daughter redia and cercaria.-The daughter redia and cercaria, C. marilli, from the naturally infected snail host, Pomatiopsis lapidaria, have been describedpreviously (Ameel, 1939). The simple sac-like daughter rediae (Fig. 7) occur in the digestive gland of the snail. The body is pigmented and the mouth is terminal, opening immediately into a muscular pharynx. The gut is moderately voluminous and long, extending to the posterior third of the body. It is easily identified under low magnification because of its contents of reddish brown food particles. There seemed to be a relatively small number of daughter rediae per snail. Of a collection of ten infected snails, the number of rediae recovered ranged from 8.to 39 with an average of 24. C. marilli (Fig. 8) is a trioculate monostome cercaria which occurred in 1.2 per cent of the snails collected at Ann Arbor, Michigan, and in 55 per cent of the snails collected near Marion, Iowa. Both body and tail are capable of considerable extension and contraction. Living cercariae under a supported cover glass measured as follows: body extended, 0.42 mm; contracted, 0.21 mm; tail extended, 0.56 mm; contracted, 0.28 mm. Naturally emerged cercariae have three eyespots but most of the immature cercariae lack the median eyespot. The body is opaque and is filled with brown pigment granules and cystogenous cells packed with rod-like cystogenous material. Spineless posterolateral locomotor organs are present. C. marilli resembles the cercaria of Quinqueserialis quinqueserialis (Barker and Laughlin) described by Herber (1942). However, the excretory concretions in the excretory bladder of this species are small but numerous, but those of C. marilli are large and relatively few. The rmetacercaria.-When collections of P. lapidaria are placed in small bottles or stender dishes containing water, the cercariae emerge in numbers during the evening, night, and early morning, and encyst after a brief period of activity. Some

THE GERMINAL DEVELOPMENT IN THE DAUGHTER REDIAE OF AN OPHTHALMO-XIPHIDIOCERCARIA FROM POMATIOPSIS LAPIDARIA*

ALLOCREADIIDAE. In fact, recent work indicates that this family should include only species with ophthalmo-xiphidiocercariae (Hopkins, 1934; Cable and Hunninen, 1942). The structure of cercariae of this type, especially the presence of a stylet and stylet glands, suggests that this family is related to the PLAGIORCHIOIDEA. We might go even further and suggest the hypothesis that this group on account of the presence of eye-spots in the cercariae and their development in rediae is more primitive than the PLAGIORCHIOIDEA, and may be close to the ancestral type from which the true plagiorchiids developed. This possibility made it of special interest to determine whether the germinal development in the daughter rediae of Cercaria pomatiopsidis showed any resemblance to that in the daughter sporocysts of the plagiorchiids. During the summer of 1948 we had the opportunity of examining 1282 specimens of Pomatiopsis lapidaria collected near Ann Arbor, Michigan, of which 37 or about 3.0 per cent were infected with C. pomatiopsidis. Most of the infections were mature or old but a few immature daughter rediae were available for study.