Glycogen reserves of whole worms and their body wall, intestines, ovarian and testicular tubules of the avian intestinal nematode, Ascaridia galli, were assayed and on dry-weight basis found to be 14–20 %, except in the testicular tissue which contained only 7 % glycogen.

Segments of whole male worms or testicular tubules were found to produce more lactic acid than segments of whole female worms or ovarian tubules. The body wall and intestine of the worms had also appreciable glycolytic activity. Whereas in the segments of whole worms, male and female alike, glycolysis was more active under anaerobic conditions, no differences in glycolytic rates were seen between aerobiosis and anaerobiosis when isolated tissues were used. Exogenously added glucose did not stimulate glycolysis either aerobically or anaerobically to any extent greater than observed in the absence of glucose.

Homogenates of whole worms or their different anatomical parts were assayed for enzymic activities associated with the Embden-Meyerhof pathway of glucose assimilation. The crude extracts prior to differential centrifugation, were found to contain glycogen phosphorylase, aldokinases, pyruvate kinase, phosphatases acting on glucose 6-phosphate and hexose diphosphate and 6-phospho-gluconate dehydrogenase. The 105000 g particulate-free supernate was found to have significant activities of the following enzymes: fructokinase, phosphoglucomutase, phosphoglucoseisomerase, phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase, lactic dehydrogenase and glucose-6-phosphate dehydrogenase. This fraction was devoid of NADH or NADPH oxidase activities in the absence of added substrates.

Although there was some indication of a negative correlation between low glycogen reserves and high glycolytic activity in the testicular tubules, in general, there was no relationship between glycogen reserve and glycolytic activity on the one hand or between the rate of glycolysis and the specific activities of some of the key glycolytic enzymes in either whole worm or in tissues other than the testicular tubules.

The 105000 g supernate was fractionated with ammonium sulphate. The fraction precipitating between 25 and 80 % saturation of the salt was recovered, dialysed and chromatographed on DEAE cellulose column. By a step-wise elution schedule using increasing molarity of NaCl in tris-HCl buffer, pH 7·4, three main protein fractions were obtained representing respectively enolase, aldolase and glucose-6-phosphate dehydrogenase.

The recovery of enzyme activity after chromatography on DEAE cellulose was higher than the amount applied to the column suggesting that during the fractionation some naturally occurring inhibitors were removed. About 10- to 20-fold purification of the enzymes was achieved by anion-exchange chromatography.

Some properties of the purified enzymes were studied with respect to the affects of enzyme and substrate concentrations, temperature of preincubation and action of divalent cations, some anions, metal chelating agents and SH reagents. The Km values of enolase, aldolase and glucose-6-phosphate dehydrogenase of A. galli were 5·9 × 10−4M, 4·5 × 10−3M and 2·4 × 10−3M respectively. Glucose-6-phosphate dehydrogenase was found to be very sensitive to both heat and cold losing activity rapidly even at 43 °C or by freezing and thawing.

The SH groups of aldolase were readily blocked by pCMB and presumably by o–phenanthroline. No requirement of any divalent cations was shown by this enzyme which was, however, inhibited by borate ions.

Enolase of A. galli showed a requirement of Mg2+ for full activation. Phosphate, fluoride, EDTA, o–phenanthroline, αα-bipyridyl inhibited the enzyme. Veronal was found to inactivate the enzyme.

Glucose-6-phosphate dehydrogenase of A. galli was also found to be sensitive to SH reagents and metal chelating agents. The enzyme was activated by Co2+, Mn2+ and Mg2+.

The evidence presented indicates that segments of whole worms of A. galli or its anatomical parts are equipped with the enzymatic machinery required to mediate anaerobic breakdown of glucose and to derive energy by this mechanism.

The authors are grateful to Dr R. K. Kaushik for help in identifying the worms, to Mr P. A. George for help in the statistical analysis of the data and to Messrs A. C. Kol and S. K. Bose for skilful technical assistance.

The thoracic skeleton and musculature of the Pupipara were studied and compared with the thorax of Musca and Glossina in order to identify the sclerites of the thorax of the Nycteribiidae. Comparative studies of the thorax and other features suggest that the Pupipara do not belong to the higher Calypterata. This comparison also supports the view which separates the Streblidae and the Nycteribiidae from the Hippoboscidae, and supplies evidence for reconsideration of the position of these two families among the Calyptera.

Gratitude is expressed to Professor O. Theodor of the Hebrew University, who supplied most of the Pupipara examined, to Professor R. Wenzel of the Field Museum of Natural History, Chicago, for species of American Streblidae and to Dr I. Paperna for the species of Glossina.

The amounts of ammonia excreted by two endoparasitic flatworms have been compared and, weight for weight, the sheep liver fluke Fasciola hepatica produces ten times as much ammonia as the rat tapeworm Hymenolepis diminuta. This is thought to reflect a difference in diet, the fluke feeding mainly on the blood and tissue proteins of its host, and the tapeworm mainly on carbohydrates from the food materials in the gut of the host. However, the addition of glucose to the saline in which the flukes are cultured produces a marked decrease in the ammonia and an increase in the lactic acid excreted, suggesting that flukes are not obligatory protein feeders but that they may utilize carbohydrate when it is available. The relatively low production of lactic acid when proteins are being metabolized suggests that the flukes are producing an alternative end-product of respiratory metabolism. It is possible that this is the neutral lipid found in the excretory system of Fasciola and the possibility of an alternative metabolic pathway for the production of such lipid is discussed. The respiratory metabolism appears to be independent of oxygen, and the possible role of oxygen in other aspects of the metabolism of the fluke is discussed.

I should like to express my sincere gratitude to Dr J. Llewellyn for his constant advice and encouragement during the course of this work and the preparation of the manuscript. I also thank Professor 0. E. Lowenstein, F.R.S., for allowing me the use of the facilities in his department during the tenure of an S.R.C. Research Studentship. Finally, my thanks are due to Mr E. Wilson, the Veterinary Officer at the Birmingham City Meat Market, for his constant co-operation in obtaining supplies of liver flukes.

The intimate cell relationships of Hymenolepis diminuta are described and occur between cells of the parenchyma, excretory system, muscle and tegument. The cell relationships are of three types: (a) a tight junction or zonula occludens; (b) a desmosome-like structure without tonofibrils or intercellular dense line; (c) a junctional complex with modified unit membranes and an intercellular region of dense granules and fibres.

This research was begun while one of us (L.T.T.) was a Visiting Lecturer in the Biology Department, Rice University, and completed in the Zoology Department, Queen's University, Belfast. We should like to thank Dr C. W. Philpott for allowing us the full use of the facilities of his electron-microscope unit and Mr W. Ferguson for photographic assistance.

(1) Light-microscopic observations were made on B. pahangi larvae at various stages of development in stained serial sections of A. aegypti thoraces, and on 8-to 9-day 2nd-stage live, unstained larvae.

(2) Six-day larvae situated in A. aegypti flight muscle, and isolated 8- to 9-day 2nd-stage larvae, were examined with the electron microscope.

(3) Results showed that host-muscle mitochondria which appeared to be morphologically intact aggregated outside the larvae at the head and tail ends and were present from 6 days onwards in the oesophagus and in the midgut. Those in the oesophagus were unchanged; some of those in the midgut were intact, others showed varying degrees of disintegration suggestive of digestion. Host-muscle mitochondria are therefore likely to be a source of nutrients for B. pahangi larvae during the last third of their intramuscular developmental phase.

Dr W. W. Macdonald, of the Liverpool School of Tropical Medicine, kindly supplied the infected mosquitoes, and offered valuable advice in the course of this work. Miss M. A. Johnson assisted with the care of the mosquitoes.

We are particularly indebted to Mr David John for his patient work in preparing the filariae for electron-microscopic observation. Mrs J. Oliver assisted with some of the histological preparations.

We are pleased to acknowledge the financial assistance of the Royal Society towards the purchase of the ultramicrotomes used in the course of this work.

P. anoura was found in 55 % of pythons (M. spilotes variegatus) in Brisbane. Eggs contained first stage larvae after 8 days development; the first moult was completed by 10 days; the eggs were infective after 14 days. After ingestion by tadpoles, the larvae hatched from the eggs and migrated into the tissues but did not show any growth. In mice and rats the larvae migrated mainly to the liver, lungs and intestinal wall, where growth occurred to a length of 5–6 mm by 6 months after infection. The second moult was not observed, but the larvae were infective to pythons after 39–42 days development. From their structure it was assumed that after this time the larvae in mouse tissues were third-stage larvae.

Various species of Australian python, Morelia spilotes, Liasis amethistinus, L. fuscus and L. childreni, were experimentally infected with larvae of Polydelphis anoura by feeding nodules from the wall of the small intestine of experimentally infected mice and rats. The third moult occurred in the stomach within 14 days, moulting larvae at this stage measured between 3.5 and 7.0 mm long. Fourth-stage larvae occurred in all python species, the smallest being 4 mm. Growth and development of the fourth stage occurred at first in the oesophagus and stomach. Up to 2 months after infection, larvae had not grown more than 12 mm, yet they were clearly differentiated into males and females. In mouse-infected M. spilotes growth to 20 mm was reached by 154 days, but it was not until 183 days that adults were observed.

Comparison with adult specimens from P. reticulatus from Malaya confirmed the previous observation that Australian male specimens have relatively shorter spicules than male specimens from other geographical regions. Growth, development and location of larvae in mice, experimentally infected with eggs from Australian and Malayan specimens, appeared to be the same.

An account is given of the morphological characteristics of larvae of four species of the Ixodes ricinus complex, namely I. ricinus, I. persulcatus, I. pacificus and I. nipponensis. Particular reference is made to chaetotaxy and its importance in differentiating larvae of closely related species is emphasized.

We are grateful to Dr G. Owen Evans for discussion on matters on chaetotaxy.

Adult Hymenolepis diminuta were tested for their ability to transport colloids under in vitro and in vivo conditions. The tracers employed included colloidal thorium dioxide (Thorotrast), carbon (Pelikan Ink), ferritin and the 14C-labelled protein fraction from Chlorella vulgaris. Though certain of these tracers were adsorbed to the external surface of the tegument plasmalemma, no evidence for the assimilation of any of these colloids was obtained in the present study. Further, significant differences in size and structure between the granular inclusions reported earlier by Rothman (1967) as assimilated colloids and the particles present in the respective colloidal suspensions were noted. Results of this investigation are interpreted as rendering untenable previous conclusions concerning transmembranosis of colloids by tapeworms.

The fine structure and organization of the nervous system of the miracidium is described. The central ganglion consists of a mass of nerve fibres with peripherally situated cell bodies. Tracts of fibres run outward from the central mass to the muscles of the body wall and to specialized endings. The individual fibres contain several types of inclusions and synapses are common in the neuropile.

Excluding the eyespots, four kinds of specialized nerve endings are described. Two of these types terminate at the body surface and bear ‘cilia’. The other two terminate internally, one type within the body wall and the other type laterally, slightly anterior to the central ganglion. It is suggested that the function of three of the types of ending might be sensory, detecting chemical and mechanical stimuli, and the direction of gravity. The function of the fourth type of ending is problematical but might be secretory.

I am most grateful to Dr A. Robards for use of electron microscope facilities and to Mrs J. Denison for technical assistance.

Of 414 Bermuda fishes, 148 belonging to 13 species harboured Digenea and 11 belonging to 6 species Aspidogastrea, Acanthocephala or nematodes. In a previous paper 69 of these fishes belonging to 20 species were found to contain cestodes. The number of infected species was greatest in the case of cestodes and the number of infected individuals in the case of Digenea. The latter is probably due to the closer proximity, for reasonably long periods, of the various hosts in the life-cycles. Of the 17 species of Digenea 3 are new host records, 3 new locality records and 4 new host and new locality records. New records were found, also, in the other groups of helminths. The digenean fauna of Bermuda fishes most closely resembles that of Dry Tortugas, Florida. Digenea and cestodes were not usually found in the same families of fishes. Cestode larvae were abundant in the deep-water Serranidae and Lutianidae which had no Digenea, the latter being common in Clupeidae, Hae-mulidae and Belonidae, which do not harbour cestodes and which occur, mostly, in the inner reefs and shallows.

The functional morphology of the apical disk, tentacles and rhynchus of Alcicornis carangis is described. The tentacles are invaginable and concerned with attachment.

I am most grateful to Dr W. H. Sutcliffe Jun., Director of the Bermuda Biological Station, for the excellent facilities provided and to The Shell Grants Committee and The National Science Foundation for grants which made the work possible. I would also like to express my gratitude to all those who kindly helped in the capture of fishes and to Mrs M. Fisher for technical assistance.

The ultrastructure of the body wall of the third-stage larva of Haemonckus placei was studied. The cuticle was found to consist of eight layers: a thin outer layer, a membrane-bounded layer, an electron-dense layer, a thin irregular layer, an inner cortical layer, a matrix layer, a striated layer and a fibril layer. Interposed between the inner cortex and matrix were two transverse fibres.

The region between the fibril layers and the contractile part of the muscle cells was occupied by the hypodermis, which enlarged to form the dorsal, ventral and lateral cords. Within the cords lay hypodermal cells, nerves, crystalline inclusions and an excretory canal.

The sarcoplasmic part of the muscle cells was rich in glycogen and contained numerous mitochondria. Myofibrils of two types were present in the contractile part of the cell.

I am grateful to Dr D. W. Brocklesby for his help and advice and to Mr E. Harness for the production and supply of third-stage larvae. I would also like to thank Dr D. L. Lee and Mr W. G. MacMillan for helpful discussions.

Electron-microscope investigations on the outer layers of parasitic platyhel-minths have so far neglected the monogeneans and have been confined to the endoparasitic digeneans and cestodes (Braten, 1968; Burton, 1966; Charles & Orr, 1968; Erasmus, 1967; Lumsden, 1966; Morris & Threadgold, 1967; Race, Larsh, Esch & Martin, 1965; Rothman, 1968; Threadgold, 1965). For this reason the covering layer of monogeneans is still referred to as a ‘cuticle’ even though it is unlikely to be so since the surface of the endoparasitic platyhelminths has been shown to be a living cytoplasmic ‘epidermis’ (see Lee, 1966) which, as pointed out by Erasmus (1967) and Lumsden (1966), constitutes a metabolically active inter-face with host tissues and fluids. Two main considerations prompted the present investigations of the covering layer of monogeneans. First, it was hoped that a comparison of the covering layer of these ectoparasites with that of the digeneans and cestodes might help to define particular specializations of this layer associated specifically with the ectoparasitic or endoparasitic habit; secondly, because ectoparasitism can, in an evolutionary sense, be considered as a transitional state between a free-living and an endoparasitic existence (see Llewellyn, 1965), it was felt that this work on monogeneans could assist an understanding of the evolution of the platyhelminth outer layer.

1. The weight of blood ingested by all stages of H. anatolicum anatolicum has been determined and from the results a direct relationship appears to exist between the weights of replete adults and their blood content. In engorged nymphs the relationship was subject to some variations, while in fully fed larvae no such correlation was apparent, indicating the ingestion of a large proportion of extravascular fluids.

2. It was found that all instars concentrate the imbibed blood by the elimination of water. In larvae the concentration is by a factor of at least 5·25, in nymphs 3·4, in females 2·1 and in males 1·4.

3. A correlation was found between the external colour of fully engorged larvae and the quantity of blood imbibed.

I wish to thank Dr R. P. Chaudhuri for providing the original specimens of H. anatolicum anatolicum from which the culture was started, and Professor Don. R. Arthur for reading the manuscript and for his advice and assistance.

The results of ultrastructural studies and histochemical tests on the capsule of Trichinella spiralis are given. The capsule wall is shown to consist of two layers, an outer region of tightly compacted fibrils and an inner region of fibrils dispersed through matrix cytoplasm. Histochemical tests show the capsule wall to consist mainly of collagen and cystine. The appearance of the matrix, which is packed with mitochondria, rough endoplasmic reticulum, smooth reticulum and Golgi complexes, together with the histochemical reaction to the five enzymes tested, strongly suggests that the matrix is a region of high metabolic activity. Although there are several very large nuclei in the matrix there is no evidence of cell walls; consequently the matrix is a syncytium.

I wish to thank the A.R.C. for financial support while this work was carried out. I should also like to thank Dr D. L. Lee for his advice on many occasions and the late Professor Boyd of the Anatomy Department, University of Cambridge, for the use of the electron microscope in that Department.

Leucocytozoon sakharoffi of English rooks (Corvus f. frugilegus) underwent sporogony in female Simulium (Eusimulium) angustitarse, the cycle taking 3–4 days. Sporozoites from the salivary glands of S. angustitarse, both naturally and experimentally infected, gave rise to L. sakharoffi infections when injected to aparasitaemic rooks, with a minimum pre-patent period of 8 days.

I am very grateful to the following persons, for invaluable help in many ways: Mr C. Swan and Mr A. Ponchaud (Ministry of Agriculture, Fisheries and Food); Dr J. Ash, Mr N. A. Wood and Mr M. Holland (Game Research Association); Mr N. A. Hancock (Smith, Kline and French); Mr N. Biddlecombe (Breamore Estate); Dr L. Davies (Durham University); Professor D. S. Bertram, Mr E. J. Blackie, Miss L. Selden, Miss J. Freeman and Mr P. Mellor (London School of Hygiene and Tropical Medicine).

This work was generously supported by a grant from the Natural Environment Research Council.

Cortisone acetate, given to mice on two days during a primary infection with the nematode Trichuris muris, suppressed the development of acquired immunity to the parasite. The proportion of mice in which suppression occurred was greatest when the drug was given (at 2.5 mg per mouse) on days 14 and 15 of the infection. Suppression of the response prevented the elimination of larvae, which otherwise occurs during the third week, and allowed the parasite population to mature and survive for at least a further 3 weeks. Similar treatment given during a challenge infection in resistant mice prevented the elimination of the challenge larvae for 2 or 3 days only. Mice in which cortisone treatment had prevented the elimination of a primary infection were not only found to be fully susceptible to subsequent challenge, but showed no development of immunity to the challenge infection itself. This long-lasting suppression of the immune response to T. muris is discussed and the possibility of the induction of a specific immunological unresponsiveness to the parasite is considered.

Studies have been made of the growth and excystment of a trematode, Acanthoparyphium spinulosum, in chicks fed on various denned diets.

Day-old chicks were fed metacercariae and maintained on deficient as well as complete rations for periods up to 20 days. The excystment rate was determined by the number of worms that were recovered from the metacercariae. The largest number of worms were recovered from chicks that received a normal diet with starch as the sole carbohydrate source. The smallest percentage of parasites was found in birds that had received a normal diet with sucrose serving as the carbohydrate source. About 25 % were recovered from chicks fed several other diets.

The trematodes developed and grew well in vitamin A deficient chicks; there was a large number of intra-uterine ova observed in worms from these hosts. Acanthoparyphium spinulosum from chicks fed a ‘no vitamin D’ diet grew at a normal rate until the 6th day; after this they grew more slowly than 6-day-old worms in chicks fed the ‘no vitamin A’ diet.

The lengths of worms from birds deprived of all vitamins differed little from those of the parasites recovered from the chicks deficient in vitamin D. The largest worms were obtained from chicks fed a complete diet with starch as the sole carbohydrate source.

Parasites from chicks fed a diet 50 % deficient in carbohydrates (with sucrose as the carbohydrate) developed normally.

Worms also developed in chicks fed a ‘no protein’ diet with starch and a ‘no protein’ diet with sucrose. A deleterious effect on ova production was observed in worms from both these groups.

Metacercariae developed in chicks fed a ‘no carbohydrate’ diet, but no mature worms were recovered due to the reduced life of the host. Starvation of the hosts produced small worms containing few intra-uterine ova.

The growth curve of A.spinulosum was plotted and compared with Echino-paryphium flexum.

This investigation was supported in part by U.S. Public Health Service Grant UI-00202, and by a grant-in-aid of research by the Society of the Sigma Xi, New Haven, Connecticut.

The first marsupial demodicid, Demodex antechini, is described with notes on its life cycle, sexual dimorphism and pathology. This species is remarkably similar except for the marked sexual dimorphism, to demodicids of eutherian mammals. This suggests an early origin and a long evolutionary history of parallelism within the genus.

Our thanks to Patricia Woolley, Waite Agricultural Institute, Glen Osmond, Australia, for the specimens used in preparing this paper. We would also like to thank Clifford Desch for the photography and the drawings. The work reported here was supported in part by NSF Grant (GB-3516).

The structure and function of the vacuoles in the eggs of Schistosoma mansoni is unknown. In this study the eggs have been immersed in serum albumin and examined by phase-contrast optics. Under these conditions the vacuoles exhibit a definite internal structure, suggesting that they may be or may have been cellular. Histochemical staining techniques to detect the nature of the vacuolar contents revealed that the vacuoles stained heavily with iodine and gave a positive PAS reaction. This suggested the presence of carbohydrate. Stains for nucleic acids, protein and lipid were not taken up by the vacuoles. The egg shell stained with o–toluidine blue and with basic fuchsin uniformly, except for a thin irregular band which might indicate a possible line of weakness. The vacuolar membranes seem to be similar to the vitelline membrane in permeability to hydroxyl ions and it is possible that these membranes have a common origin. Plasmolysis studies with salts, urea, glycerol and sucrose showed that the vacuolar membranes have about the same permeability properties to these compounds as the other egg membranes. Glycerol treatment of the eggs until equilibrium is attained, followed by transfer of the eggs to water or saline results in a hatching of the eggs. In this, a dead mira-cidium is extruded from the shell. Similar treatment of eggs equilibrated with sucrose does not result in this hatching process. It is postulated that glycerol solubilizes a factor which aids the hatching process.

I should like to thank the following: Dr S. A. Ibrahim, in whose department this work was carried out; Dr S. Dawood of the Stack Laboratory, Khartoum, who kindly made his microscope available for use; Mr J. R. Lauder for valuable discussions and suggestions; and many young patients in Khartoum Civil Hospital for stool samples and smiles.

Fertilization phenomena has been observed in the malaria parasite of the Himalayan Flying Squirrel with blood showing heavy infection. The blood was obtained on the slide, put in the moist chamber, dried and stained. Two types of microgametes could be distinguished. One of these two types were found in various stages of the process of fertilization. More than one microgamete were often found to take part in fertilizing a single macrogamete. This is the first recorded case of polyspermy in malaria parasites.

Thanks are due to Mr Nandalal Pau for his help in preparing the Plate incorporated in this paper.

This investigation received financial support from the World Health Organization.