Histochemical and ultrastructural observations on digestion in Tetrameres fissispina Diesing, 1861 (Nematoda : Spiruridea) with special reference to intracellular digestion

ZnternationafJournuifor Parasitology, 1973, Vol. 3, pp. 157-164. Pergamon Press. Printed in Great Brituin

HISTOCHEMICAL AND ULTRASTRUCTURAL OBSERVATIONS ON DIGESTION IN ~~~~~~~~ ~~~~2s~~~~ DIESING, 1861 (NEMATODA : SPIRURIDEA) WITH SPECIAL REFERENCE TO INTRACELLULAR DIGESTION J. RILEY* Dep~ment

of Zoology, The University, Leeds LS2 9JT

(Received 27 Marc& 1972; in amendedform 2 June 1972)

RIL.EY,J. 1973. Histochemical and ultrastructural observations on digestion in Tetrameres fissispinu (Diesing, 1861) (Nematoda : Spiruridea) with special reference to intracellular digestion. Z~ter~t~ona~Jownaf for Parasitology, 3: 157-164. A large proportion of the blood ingested by Tetr~re~ &&@ta is digested ext~~llul~ly to haematin. The probable site of extracellular haemoglobin degradation is the glycocalyx of the microvilli which may carry adsorbed enzymes functional in contact digestion. A smaller proportion of the haemoglobin released from haemolysed erythrocytes is eodocytosed in an unchanged state by isolated groups of absorptive cells. In the latter, haemoglobin-containing phagosomes apparently fuse with primary lysosomes ultimately to produce large, heterogeneous, multiple phagolysosomes (digestive complexes). Lipid droplets produced during digestion are extruded from these at intervals. Haemosiderin is the end-product of ~~acellul~ haemoglobin breakdown-the differences in residues of the extracellular and in~cellul~ processes ~ematin and haemosiderin) reflecting differences in the two enzyme systems employed. Haemosiderin is accumulated as sphaerocrystals in dilated cisternae of the ER. It is suggested that the purpose of intracellular digestion is to provide a source of ferric ions (in the form of haemosiderin)for the

biosynthesisof endogenoushaemoglobinwhich the extracellulardegradationof haemoglobin cannot supply. INDEX KEY WORDS: nematode; Tetrameres ~~sis~ina; digestion; intracellular digestion; contact digestion; haemoglobin; endogenous haemo~obin; haemoside~n; sphaerocrystals; lysosomes; phagosomes.

INTRODUCTION MOST evidence concerning digestion in nematodes points to an extracellular process followed by absorption of simple food materials from the intestine (Lee, 1965). More recent ultrastructural studies on the intestinal epithelium of nematodes have largely supported this. Pinocytosis (which is indicative of intracellular digestion) has been observed only rarely: Sheffield (1964) demonstrated it in Ascaris suum, as did Andreasson (1968) in Ancylostoma caninum and Colam (1971a, b) in Rhabdias bufonisand Cyathostoma lark Although in all these cases this process concerned only a very small proportion of the ingested food its occurrence in certain nematodes may be highly sig~~cant. In this respect three facts emerged from Colam’s work on R. bufonis which are of particular relevance to the present paper: (a) R. bufonis possesses endogenous haemoglobin, (b) haemosiderin results from intracellular digestion, in contrast to the haematin produced extracellularly, and (c) endogenous haemoglobin is synthesized by gastrodermal cells. Apparently then, in this species intracellular digestion is associated in some way with haemoglobin biosynthesis. The present investigation was undertaken to establish the digestive pattern of another species of haematophagous nematode possessing a haemoglobin respiratory pigment. Tetrumeres jisssispina Diesing 1861, a common parasite of the proventriculus of a wide * Present address: Department

of Biological Sciences, The University, Dundee DDl 4HN, Scotland. 157

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variety of bird hosts, was seIected after preliminary studies had established the probability of intracellular digestion in this species. It was hoped that the information obtained from this study would shed more light on the relationship between intracellular digestion of haemoglobin and the possession of a haemoglobin respiratory pigment.

MATERIALS

AND

METHODS

Adult female ~etrameres~ss~~pi~a Diesing 1861 were obtained from the proventricular glands of Herring gulls (Larus apeentatus) shot in the East and West Ridings of Yorkshire. A total of forty parasites were used in this study. For histological examination living worms were dissected out of the stomach wall and fixed immediately in 10% formol-saline for 24 h. The gut was then removed and fixed for a further 1 h before dehydration in graded alcohols, clearing in xylol and embedding in para& wax. Sections were cut at 8 pm, and stained in Mallory’s trichrome, by Perl’s and Turnbull’s techniques for the demonstration of ferric and ferrous iron, and by the Haematoxylin-Lake method for haemosiderin. Acid phosphatase was visualized using the azo dye method of Burstone (1958) after gut material had been fixed in 10 per cent buffered formalin at 4”C, dehydrated in graded acetones at 4”C, cleared at room temperature, and embedded in 45°C melting point parafhn wax. For electron microscopy whole active specimens were fixed at 3 % glut~aldehyde for 4-S h, after which the gut was dissected out and fixed for a further 1 h. Gut material was then washed twice in cacodylate buffer postfixed for 1 h in 1% osmium tetroxide, washed in Verona1 buffer and dehydrated in graded ethanols. Material was embedded in Shell Epikote Resin (epon 812). Acid-phosphatase was visualized ultrastructurally using the lead nitrate method of Gomori (1952). After glutaraldehyde fixation tissue was incubated for O-5 h in a medium containing IO-’ M sodium-~-gly~rophosphate, 4 x 10m3 M lead nitrate and 75 % sucrose, buffered to DH 5-2 with 5 x 10e2 M acetate buffer. The incubation was carried out at 40°C and substrate was omitted from the controls. Material was then rinsed in cacodylate buffer at 4°C and postflxed etc. as above. Sections were cut on a Cambridge (Huxley) microtome and mounted on formvar-coated grids. Staining was in uranyl acetate followed by lead citrate. Sections incubated for acid phosphatase activity were examined unstained. All were viewed in an AEI EM6B electron microscope. Some sections, cut at 0.3 pm, were stained in Azure II and examined by means of light microscopy.

The structureof thegut

RESULTS

The part muscular, part glandular oesophagus extends well into the swollen mid-body region and is followed by the sac-like intestine which measures up to 4 mm in length and 25 mm wide. The intestine communicates posteriorly with a short rectum. The walls of the intestine are markedly infolded and convoluted and consist of a single layer of cells, bounded by a prominent basement lamella, 0.64 pm thick. The gut of all the worms examined was always grossly distended and full of granular, brown-black crystals of haematin. Very occasionally haemolysing erythrocytes were also present and in one specimen a faint reaction for haemoglobin could be detected in the gut contents.

61 dc g ger gv h hb I

Key to Lettering Figs. l-12 basement lamella 1.v lysosome (primary) digestive complex m mitochondria Golgi complex I?ZV microviili granular endoplasmic reticulum phagosome P Golgi vesicle ply phagolysosome haematin rb residual body haemoglobin SP sphaerocrystal lipid droplet SV secretory vesicle & gut lumen

FIG. 1. A light micrograph of a section through a fold of gastrodermal cells showing black, granular crystals of haematin in the gut lumen forming a dense cap to each cell. Clusters of iron-positive spheres occur in the basal regions of these cells (arrows). Perl’s, Neutral red. Xl000

FIG. 2. An epon section of the gastrodermis showing endogenous haemoglobin in the haemocoel. Note the presence of dense, irregular digestive complexes in the basal halfs of most cells and granular haematin deposits between the microvilli. Azure II. x 900

[T.J.P.JCP. 1581

Fro. 3, An electron micrograph showing detail of three gastrodermal cells resting on a uniform basement lamella. Erythrocyte envelopes are visible in the gut lumen along with scattered crystals of haematin. The cell cytoplasm carries numerous dense mitochondria, scattered lipid droplets and broken, scattered profiles of GER. Note the large heterogeneous digestive complexes dominating the basal cytoplasm of the central cell. Four sphaerocrystal-containing cisternal profiles are arrowed. x 2500

FIG. 4. Detail of the apex of an absorbing ceil showing haemoglobin

adhering to the microvilh. The glycocalyx is readily visible around isolated microvilli. x 15000 Fro. 5. Detail of an absorbing cell showing haemoglobin-containing phagosomes streaming into the distal cytoplasm. A dense terminal bar delimits the apical junction between two cells. Note the dense homogeneous precipitates of haemoglobin investing the microvilli. x 33,000 FIG. 6. A later stage absorbing cell showing accummulated phagosomes of endocytosed haemoglobin under the brush border. Note the scattered mitochondria and the whorls of granular endoplasmic reticulum. The central mass of irregular granules is composed of phagolysosomes some already showing lipid material in the matrix. Lysosomes (arrows) are indicated. Centres of intense Golgi activity surround the phagolysosome field. x 8000

‘IG. 7. Detail of a single digestive complex showing lipid droplets, miscellaneous vesic:les, membrane remnants and lysosomal residues. A lipid droplet, probably extruded from the complex is present in the cytoplasm. x 38,000 ‘IG. 8. A micrograph showing the early stages of sphaerocrystal formation. The smerller Jhaerocrystal (arrowed) can be seen to be ‘condensing’ out of the dense subunits contai ned Gth the dilated cisternae. Note that each is contained within a single dilated profile deri ved from the endoplasmic reticulum. x 34,000

FIG. 9. Detail of the apical cytoplasm of a gastrodermal cell showing the breakdown of normal profiles of endoplasmic reticulum into numerous rough-surfaced vesicles. A particularly clear example of this is arrowed. Note the moderately dense content of some of the free vesicles. x 18,000 FIG. 10. An electron micrograph showing acid phosphatase activity in the glycocalyx of the microvilli and the lateral cell membranes. x 5000 FIG. 11. Detail of three digestive complexes incubated for acid phosphatase activity. Note the distribution of reaction production throughout the matrix of these inclusions. A single, free lysosome, loaded with reaction product is arrowed. x 15,000

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Gastrodermal cells varied between 10-20 pm in height and 20-28 pm wide and bore distally a prominent brush border. The distal surface of these cells is convex and the basement lamella forms inpushings between cells, giving the gastrodermis, as seen in section, a beaded appearance (Fig. 2). The centrally located nucleus, 4-6 pm dia., contains a prominent nucleolus. Under the electron microscope the internal organization of gastrodermal cells is exceedingly complex (Fig. 3). Prominent terminal bars indicate the apical junctions of adjacent cells and consists of a uniform intercellular space containing a dense substance confined by double membranes. Lateral cell membranes show little or no interdigitation; complex folding is limited to the basement membrane, the folds of which extend a short distance into the cell. The microvilli projecting into the gut lumen are 100-130 nm dia. and each contains a central core of fibrils which barely penetrate the plasma cap. There is no terminal web. Throughout the cytoplasm are masses of dense, multicristate mitochondria, spherical or ovoid in profile but some more markedly elongate. The endoplasmic reticulum of these cells commonly exists as a few isolated profiles, studded with ribosomes, dispersed in the cytoplasm. Occasionally the ER is organized into concentric whorls or stacks (Fig. 6) consisting of narrow, regular cisternal profiles. These ‘ergastoplasmic nebenkerns’ probably represent sites of synthesizing activity within the cells. This degree of organization is uncommon and in the majority of cells narrow cisternae have broken down to form vast numbers of rough-surfaced vesicles 0.1-0.4 ,um dia. (Fig. 9). This process can be so complete that no ‘normal’ profiles of ER remain. Clear or moderately dense vesicles, derived from the breakdown of the ER but devoid of ribosomes, are observed in the cytoplasm where they are often closely associated with the apical plasmalemma (Fig. 9). Clusters of dense aglycogen particles are evident after lead staining and lipid droplets O-5-2.5 pm dia. are also a conspicuous feature of these cells. Most gastrodermal cells contain large, very irregular, heterogeneous inclusions which are easily distinguished by light microscopy where they appear as light-brown pigmented bodies usually located in the basal half of the cell (Fig. 3). These inclusions are known to be sites of intracellular digestion and throughout the following account are termed multiple phagolysosomes or digestive complexes. They range from 2 to 20 pm dia., show a very irregular profile and are bounded by single or multiple membranes (Fig. 7). Droplets of lipid material are present in the granular matrix of these complexes together with numerous miscellaneous residues, some probably lysosomal in origin, and myelin figures. In addition to the above organelles a number of small, electron-opaque bodies occur which contain varying amounts of reaction product in cells incubated for acid phosphatase activity. They are most frequently located around the digestive complexes but can occur in clusters throughout the cell. These inclusions, measuring O-2-0.5 pm dia., are primary lysosomes which are morphologically identical to the virgin lysosomes described by Moe et al. (1965) from epithelial cells of the rat intestine. The lysosomes are round, ovoid or disc shaped and occasionally are curved to form cup-shaped bodies. There was some evidence to suggest the larger, more irregular primary lysosomes can arise by the fusion of smaller ones. All are surrounded by conspicuous single or multiple membrane systems and contain a dense, homogeneous or finely granular ground substance. Also present in the immediate vicinity of the digestive complexes are spherical dense bodies, which stain positively for inorganic iron using Perl’s, and Turnbull’s techniques on sections prepared for the light microscope (Fig. 1). Several forms are recognizable. Some spheres are very intensely staining and measure up to 1 pm dia., though most lie in the

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range 0.3-0.5 pm. Others are larger, up to 2.2 pm dia., less intensely staining and contain an iron-positive core closely applied to one side of the enclosing vesicle. All these inclusions stain positively when treated with the Haematoxylin-Lake method for haemosiderin, thus at least a proportion of their iron content is present in this simple organic form. The ultrastructural appearance of these spherical inclusions corresponds precisely to the above. The dense cores of these bodies are located within dilated sacs of GER (Fig. 8). A proportion of these dense bodies are typically sphaerocrystalline and appear as concentric rings of granular material, whereas others are denser, and in some the concentric lamellations are completely obscured. Yet another stage is represented by a clear vesicle, devoid of ribosomes, containing a dense core of material (Fig. 3). From light microscope evidence it is reasonable to propose that the electron opacity of these sphaerocrystals is due, at least in part, to the presence of haemosiderin. The organization of the ER breaks down at the onset of sphaerocrystal formation so that each is contained within a single dilated profile. Small Golgi complexes, consisting of three to five flattened or vacuolated sacs occur throughout the cells associated with short lengths of GER. When active these complexes produce large numbers of dense vesicles some of which have been observed in close proximity to lysosomes. There was some evidence to suggest that Golgi vesicles may fuse with these lysosomes. The microvilli of the lumenal surface of all the gastrodermal cells of T..fissispina showed a very strong reaction for acid phosphatase which was limited to the finely granular outer covering of each microvillus-the glycocalyx (Fig. 10). The lateral cell membranes and the infoldings of the basal plasmalemma contained further deposits. A more diffuse reaction product was present in the distal cytoplasm of many cells. Reaction product was also visualized in the matrix of the primary lysosomes and in the digestive complexes, probably as a result of the fusion of the latter bodies with lysosomes.

Undoubtedly the vast majority of ingested haemoglobin is digested extracellularly to haematin. Elongate, cigar-shaped crystals of haematin were observed very infrequently in some gastrodermal cells and it seems too remote a possibility that this could contribute significantly to the lumenal haematin. Haemolysis of ingested erythrocytes is evidently very rapid as only rarely were intact erythrocytes observed. Every gas~ode~al cell carried a heavy black cap of haematin which suggests that a proportion of the haematin may actually be formed between the microvilli (Fig. 1). The problem of the entry of haemoglobin into gastrodermal cells proved unusually difficult to resolve, if for no other reason than it was only seen in two of the forty nematode intestines sectioned during this study. This process was only visualized ultrastructurally: no intracellul~ reaction for haemoglobin or DNA was demo~trated at the light microscope level. Pinocytosis was always limited to a small, isolated, group of cells which was apparently primed in some way to attract haemoglobin preferentially which adhered to their microvilli (Figs. 4-6). Precisely how this haemoglobin was attracted to these cells out of the vastness of the haemotin-occluded gut is obscure. It is perhaps worth re-emphasizing that contiguous cells showed no such activity and often contained digestive complexes known to be at a relatively late stage of the in~ace~ul~ digestive cycle. The dense, homogeneous mass of haemoglobin adhering to, and often completely surrounded by, the microvilli of absorbing cells (Fig. 4) enters the cells by pinocytosis (Fig. 5) as evidenced by the large numbers of dense, membrane bound vesicles under the apical plasmalemma and in the distal cytoplasm.

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The stages in digestion from a vesicle of endocytosed haemoglobin to a digestive complex was relatively easy to follow as the various stages were rapid, and the entire sequence could be visualized within a single cell (Fig. 6). Haemoglobin-containing phagosomes apparently fuse with each other and with primary lysosomes to form incipient digestive complexes (phagolysosomes) (Fig. 6). Small Golgi vesicles often invest phagosomes and may fuse with them. Aggregations of phagolysosomes, some showing lipid droplets in their matrix, liberated by presumed intracellular digestion, undergo a series of multiple fusions which finally result in the formation of the large digestive complexes (multiple phagolysosomes) up to 20 pm dia. During this process many more lysosomes fuse with, and discharge their contents into, the complexes until ultimately the complexes are effectively residual bodies. As lipid droplets form as a by-product of haemoglobin degradation they are extruded at intervals (Fig. 7). Most of the cytoplasmic lipid droplets apparently form in this manner so that when intracellular digestion is completed all the remains in the residual body are myelin figures, miscellaneous vesicles, lysosome remnants and residual lipid material. Residual bodies, much reduced in size, have been observed close to the apical plasmalemma. From certain micrographs it was apparent that they are eliminated from gastrodermal cells into the gut lumen. Haemosiderin-containing sphaerocrystals only arise after the formation of digestive complexes. Presumably haemosiderin, a relatively simple degradation product of haemoglobin is released from the digestive complexes into the cytoplasm, from which it is removed and concentrated by the ER. The time of appearance, and the position of the sphaerocrystals supports this hypothesis, and as far as can be seen the only source of intracellular haemosiderin is the digestive complexes. No iron was detectable in the latter under the light microscope which suggests that haemosiderin is lost from the complexes immediately after its formation. The foregoing events are summarized diagrammatically in Fig. 12. DISCUSSION

It has been shown that the bulk of the ingested haemoglobin is degraded extracellularly to haematin. Colam (1971a, b 8~ c), from studies on the digestive physiology of three nematode species, suggested that enzymes adsorbed on to the glycocalyx of the microvilli function in contact digestion, a mechanism first proposed by Ugolev (1965). Colam was able to demonstrate several proteolytic enzymes in the glycocalyx, and this evidence, together with the appearance of pigment residues between the microvilli, strongly suggested that contact digestion accounted for at least the terminal stages of extracellular digestion. The glycocalyx coat of the microvilli of T. Jissispina shows very strong acid phosphatase activity and it is likely that other enzymes are present in this region. This hypothesis is supported by the occurrence of dense precipitates of haematin between the microvilli which suggests that it is actually formed at this site. The marked affinity of the microvillar layer for haemoglobin (Fig. 5) is perhaps an indication that all haemoglobin, once it is released from haemolysed erythrocytes, is adsorbed on to the brush border. The ensuing pattern of digestion would then depend upon the physiological state of the cell. Should the microvilli be devoid of adsorbed enzymes, the haemoglobin would be endocytosed unchanged. However where a full complement of proteolytic enzymes is present the microvilli would be able to function by contact digestion thereby producing inter-microvillar haematin. There are two possible sources of enzymes functional in extracellular digestion, the oesophageal secretions and the vesicles derived from the breakdown of highly organized stacks or whorls of GER (Fig. 12). These vesicles, often containing a moderately dense,

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FIG. 12. A-H. FIG. 12. A diagrammatic representation of intracellular sequence of digestion in a single gastrodermal cell of Tetrameres fissispina (not to scale). Pathways known with certainty are represented by unbroken arrows; predicted pathways are represented by broken arrows. A. The breakdown of erg~topl~mic nebenkerns into rout-surface vesicles. Certain of the latter lose their ribosomes and may be secreted through the apical plasmalemma into the gut lumen. B. Intense Golgi activity producing numerous vesicles. Some of these vesicles fuse with, and may even form, lysosomes. Others fuse with phagosomes of endocytosed haemoglobin. C. Haemolysed haemoglobin in the microvilli entering the cell by endocytosis. The resulting phagosomes fuse with each other, with Golgi vesicles, and with lysosomes to form incipient phagolysosomes. D. A collection of phagolysosom~, some already showing lipid droplets in their matrix liberated during the intracellular digestion. E. Sphaerocrystal formation inside sacs of dilated endopIasmic reticulum. The inorganic iron content of the haemosiderin of these sphaerocrystals is thought to be derived from phagolysosomal digestion. F. A multiple phagolysosome or digestive complex showing the lipid, membrane, and lysosome components of its matrix. Lysosomes continue to fuse with complexes at this stage. G. Lipid droplets being extruded from a residual body which is a spent digestive complex at the terminal stage of digestion. H. A residual body being eliminated through the apical plasmalemma.

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flocculent content, were observed in close proximity to the apical plasmale~a though their discharge was never observed. Staubli et al. (1966) described very similar events to account for the extracellular digestion of haemoglobin in the mosquito Aedes uegyptilikewise these authors failed to detect vesicle discharge but they noted the disappearance of many of the vesicles after a blood meal and also that the erythrocytes closest to the gut cells haemolysed first. Thus extracellular digestion in T. fissispina produces haematin, a very stable but metabolically useless end-product, whereas intracellular digestion results in the formation of haemosiderin. The latter is thought to be ferric oxide or hydroxide associated with a protein carrier. Extracellular haemoglobin degradation to haematin merely requires the enzymatic splitting of the haeme and globin fractions, the haeme group then spontaneously oxidizing the haematin. In order to obtain haemosiderin however, the porphyrin ring of the haeme moiety must be ruptured before the separation into haeme and globin molecules (Lemberg & Legge, 1949). Obviously the enzyme systems employed in the two processes must be different. This hypothesis was first proposed by Colam (1971a) from studies on the digestive physiology of Rhabdias bufonis, a nematode showing an almost identical overall pattern of digestion to T.Jissispina. It is known from investigations on Ascaris lumbricoides that dietary haemoglobin is broken down and resynthesized by the worm before entering the pseudocoelomic fluid (Smith & Lee, 1963). Both R. bufonis and T. jissispina possess endogeneous haemoglobin. In the former this is found in the reproductive system, hypodermis and body wall muscle and in T. ~ss~spina haemoglobin forms a major component of the pseudocoelomic fluid. Obviously iron is an essential prerequisite for haemoglobin synthesis and, as the only source of iron is haemosiderin, it is very probable that the elaborate and involved process of intracellular digestion in these two species has been evolved primarily to provide a source of ferric ions for the biosynthesis of endogenous haemoglobin. Colam (1971a) actually observed the passage of haemoglobin-containing Golgi vesicles through the basal lamella of gastrodermal cells of R. bufonis into the pseudocoel. This haemoglobin could only have been synthesized in the gastrodermis. There was no evidence as to the fate of the haemosiderin sphaerocrystals in T. Jissispina, but from light microscope observations it appears that the granules are not accumulated and stored in the gastrodermis as in many other nematode species, but rather are utilized relatively soon after their appearance, Although these two species have probably evolved intracellular digestion for the same reason the mechanisms involved differ. In R. bufo~is haemosiderin was retained within the body of the phagolysosome whereas in T. ~ssispina ferric ions in the form of haemosiderin were released from multiple phagolysosomes and concentrated within the GER as sphaerocrystals. Why this difference should exist is unclear. Yet another variation on this basic theme was reported by Riley (1972) who studied digestion in a haematophagous pentastomid Reighardia sternae. In this case limited intracellular digestion was accompanied by a concentration of haemosiderin in heterolysosomes. These heterolysosomes were not directly concerned in intracellular digestion but apparently served only to concentrate toxic haemosiderin released into the cytoplasm from phagolysosomes. Apparently in this particular pentastomid a source of iron was not the reason for intracellular digestion, and consequently it was tentatively suggested that some other specific metabolite was required by the pentastomid which the extracellular degradation of haemoglobin to haematin could not supply. Almost all the ul~as~uc~al studies on the gastrodermis of nematodes have revealed pigment granules [summarized by Lee (1969)], although in nearly all these cases the nature PARA. 3/2--o

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and function of the granules is unknown. Granules are variously referred to as lamellar bodies or lysosomes although in only two instances has the lysosomal nature of pigment granules been definitely established (Colam, 197la, b)_ Clearly the whole question of intracellular digestion and pigment granules in nematodes wouId repay future investigation. REFERENCES ANDREA~~~N J. 1968. Fine structure of the intestine of the nematodes, Ancylostoma caninum and Phocanema deciuiens. Zeitschifr fiir ParasitenKunde. 30: 318-336. BURST~NEM. S. 19~8.-Hist~hemical demons~at~on of acid phosphatase with naphthol AS-phosphates. Journal of the National Cancer Institute 21: 523-539. COLAMJ. B. 1971(a). Studies on gut ultrastructure and digestive physiology in Rhabdias bufonis and R sphaerocephala (Nematoda: Rhabditida). ParasitoIogy 62: 247-258. COLAMJ. B. (1971(b). Studies on gut ultrastructure and digestive physiology in Cyathostoma lari (Nematoda: Strongylida). Parasitology 62: 273-283. COLAMJ. B. (1971(c). Studies on gut ultrastructure and digestive physiology in Cosmocerca ornata (Nematoda: Ascaridida). Parasitology 62: 259-272. GOMORIG. 1952. Micruscooic Histochemistr~. Uni~e~ity of Chicago Press. Chicano. LEE C. C. 1969. Ancylostotk canirmm: fine sbucture of intestinal ep%helium: Expe&ental Parasitology 24: 336-347. LEE D. L. 1965. Thephysiology of nematodes. Oliver & Boyd, Edinburgh. LEMBERG R. & LEGGEJ. W. 1949. Hematin Compounds and Bile Pigments. Their Constitution, Metabolism and Function. Interscience, New York. MOE H., ROSTGAARDJ. & BEHNKE0. 1965. On the morphology and origin of virgin lysosomes in the intestinal epithelium of the rat. Journ& of U~tr~tract~~ Research 12: 396-403. RILEY J. 1972. His&chemical and ultr~tructu~l observations on feeding and digestion in Reighardia sternae (Pentastomida: Cephalobaenida). Journal of Zoology 167: 307-318. SHEFFIELDH. G. 1964. Electron microscope studies on the intestinal epithelium of Ascariz sum. Journal of Parasitology 50: 365-379. SMITHM. H. & LEE D. L. 1963. Metabolism of haemoglobin and haematin compounds in Ascaris lumbricaides. Proceedings of the Royal Society, Series B 151: 234-257. STAUBLIW., Fk~woon~ T. A. & SUIXR J. 1966. Structural modification of the endoplasmic reticulum of midgut epithelial cells of mosquitoes in relation to blood intake. Journal a%Microscopic 5: 189-204. UCOLEVA. M. 1965. Membrane (contact) digestion. PhysioIogical Review 45: 555-595.