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International audience; Polystomes (Monogenea: Polystomatidae) of freshwater turtles are currently represented by five genera, namely Neopolystoma, Polystomoides, Polystomoidella, Uropolystomoides and Uteropolystomoides. These parasites can infect the urinary, oral and/or the conjunctival sac systems of their hosts, showing strict site specificity. A recent phylogenetic study showed that the two most diverse genera within chelonian polystomes, i.e. Neopolystoma and Polystomoides, are not monophyletic. Furthermore, polystomes infecting the conjunctival sacs of their host, except for one species, formed a robust lineage. A fusiform egg shape has been reported for conjunctival sac polystomes and it was assumed that this characteristic could be a good character for the systematics of polystomes. Our objective in the present work was, therefore, to study more in depth the morphology of polystomes collected from the conjunctival sacs of chelonians to find characters defining a putative new genus. To achieve this objective, more specimens were collected in 2018 and 2019 from turtles sampled in North Carolina and Florida (USA) to extend taxon sampling for the phylogenetic analysis. Morphological characters of relevant polystome specimens were re-examined from several collections from Asia, Australia, Europe, South Africa, South America and North America. Based on a Bayesian tree inferred from the analysis of four concatenated genes, namely 12S, 18S, 28S and COI, polystomes found in the conjunctival sacs were grouped in three distinct lineages, the first one including a single species infecting an Australian pleurodire turtle; the second one including eleven species infecting cryptodire turtles of South America, North America and Asia; and the last one including a single species infecting a softshell cryptodire turtle of North America. Based on observations of live specimens by Dr. Sylvie Pichelin and our morphological analysis, the conjunctival sac polystomes from Australian turtles are small, cannot extend their body significantly, have a spherical ovary and egg, have a large genital bulb and possess latero-ventral vaginae at the level of the testis. Based on observations of live specimens and morphological analysis of whole mounted specimens, polystomes of the second lineage share the following morphological characteristics: the ability to stretch out and double their length, a long oval ovary, a separate egg-cell-maturation-chamber, fusiform to diamond-shaped eggs with acute tips, small genital bulb and vaginae peripheral on the side of the body at the level of the testis. The polystome species of the third lineage occupies a basal position, has the ability to stretch out and possess an elongated ovary, a large fusiform egg with rounded tips, a small genital bulb and small latero-ventral vaginae at the level of the ovary. These three distinct conjunctival sac polystome lineages are herein described as separate new genera, Aussietrema, Fornixtrema and Apaloneotrema, respectively.

An in vitro study has been carried out to monitor changes to the female reproductive system in adult triclabendazole (TCBZ)-resistant Fasciola hepatica following treatment with a commercial preparation of myrrh ("Mirazid"). Flukes were immersed for 6 h and 24 h in myrrh extract at a concentration of 200 µg/ml, then processed for histological and transmission electron microscope (TEM) examination of the uterus, Mehlis' gland, ovary and vitellaria. Egg production had become abnormal at 6 h post-treatment (pt), with the uterine lumen being filled with free vitelline cells and masses of shell protein material; few eggs were present. At 24 h pt, no eggs were present. Distinct changes to the ovary and Mehlis' gland were only observed after 24 h incubation in Mirazid. The ovary contained numbers of apoptotic oogonia and oocytes. In the Mehlis' gland, the S1 cells were disorganised and the processes from them were vacuolated, although the disruption was not significant. More severe changes were observed in the vitelline cells and follicles. After 6 h incubation in Mirazid, although the gross organisation of the vitelline follicles appeared to be normal, nuclear changes indicative of the early stages of apoptosis were observed in the stem cells and shell protein production by the mature cells had decreased. At 24 h pt, a distinct shift in cell population was evident, with the follicles containing mainly mature cells and spaces were present between the cells. The shell globule clusters in the mature cells were disorganised. In more severely-affected follicles, cells were seen to be breaking down, with karyolytic nuclei and disintegrating cytoplasm. Overall, the results have shown that exposure to Mirazid treatment had a severe impact on egg production by TCBZ-resistant flukes, an effect that was mediated by disruption of the vitelline cells and of the mechanism co-ordinating egg formation in the ootype.

The objectif in the present study is description of a new species of Sphincteristomum Oshmarin, Mamaev & Parukhin 1961, named Sphincteristomum mediterraneae, which is described in Oran bay, Algeria. It parasitizes the intestine of naturally infected Pagellus erythrinus, Teleostei, Sparidae. The Pagellus erythrinus is in important food fish in the Mediterranean. This genus belongs to the family Apocreadiidae, sub family Schistorchiinae, characterized by an obvious oral muscular sphincter, into a large oral sucker, a disc-shaped acetabulum under the intestinal bifurcation, an ocular pigment scattered in the forebody, a sequence ovary and two testes in tandem and a I-shaped excretory bladder. In the new species, the digestive apparatus displays a hexagonal peculiar aperture, followed by a longitudinal slit, a waved pharynx, two long and broad caeca, with a thick internal wall of endothelial cells. Each caecum has a sub terminal anus. The female genital apparatus displays a transversally elongated ovary, under the acetabulum. On the side of the ovary, there is a small Mehlis gland. Under the ovary, there are two transverse converging vitelloducts. Lateral and extracaecal vitelline follicles, are spread near the caeca. They converge in the hindbody. The relatively short uterus is preovarian, mainly. It contains few large yellow eggs. Its distal part is creased transversally, just before ending in genital atrium. The male genital apparatus shows two lobed heart-shaped testes, post ovarian, in tandem. From the anterior edge of each testis, two spermiducts climb up and converge into a naked, curved or sinuous seminal vesicle, followed by a short prostate surrounded by few prostatic cells, and a narrow ejaculatory duct, opening in genital atrium. The genital pore is close to the acetabulum and the intestinal bifurcation. No cirrus pouch. The excretory system shows a relatively short tubular I-shaped bladder, reaching the inferior level of the second testis. Two thin excretory arms are seen until the forebody of the worms. The terminal excretory pore is closed by a small sphincter. In the same P. eryrthrinus, immature worms devoid of genitalia, young worms with genitalia devoid of eggs and ovigerous worms, live together with senile specimens. These last ones show still, oral sucker, diffuse vitelline follicles and very small and scattered ocular pigment.

Lambs infected with the Cullompton triclabendazole (TCBZ)-susceptible isolate of Fasciola hepatica were treated with TCBZ at a dosage of 10 mg/kg at 16 weeks post-infection. Adult flukes were recovered from the liver at 3 h, 24 h, 48 h and 60 h post-treatment (pt). They were processed for histological analysis of the uterus, Mehlis’ gland, vitellaria, ovary and testis. At 3 h pt, the flukes were essentially similar to the controls and were producing normal eggs. Egg production had ceased by 24 h pt. At this time period, the cells of the Mehlis’ gland showed some evidence of vacuolation, but otherwise were relatively normal. A shift in the population of vitelline cells towards mature cells was observed at 24 h pt, and this trend continued at later time-periods. It was accompanied by a breakdown of the cells and the presence of apoptotic bodies. Marked changes to the ovary were first noted at 48 h pt, as evidenced by vacuolation and the presence of apoptotic bodies. Some disruption to the testis was seen at 24 h pt, with a reduction in the population of spermatogenic cells, the appearance of apoptotic bodies and some peripheral vacuolation of the tubules. These abnormalities increased in severity with longer time periods pt. The results bring forward the time-line of cessation of egg production by 24 h, demonstrating that this process is affected very rapidly pt.

Eight indoor-reared cross-bred sheep with no prior exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Twelve weeks after infection, sheep were treated with 10 mg/kg triclabendazole. Two sheep were euthanised per time period; at 48 h, 72 h and 96 h post-treatment (pt). Two untreated control sheep were euthanised at 96 h pt. Flukes were recovered from the liver and, if present, from the gall bladder of the sheep. They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system; specifically, the uterus, vitelline follicles, Mehlis’ gland and ovary. Over the 4-day post-treatment period, there was a progressive reduction in the number of oogonia and oocytes in the ovary and evidence of apoptosis. Vacuolation and a decrease in the number of Mehlis’ gland cells were observed from 48 h pt onwards and disruption of the normal role of the gland in egg formation was evident. The vitelline follicles showed a gradual decrease in size and became vacuolated; the population structure in each follicle changed to be one consisting mainly of mature cells and the production of shell protein material declined. The follicle became disorganised as the cells broke down and released their contents into the lumen of the follicle. While the uterus appeared to contain eggs at 48 h pt in whole-mount specimens, no properly-formed eggs were observed in histological sections. By 96 h pt, the uterus was completely devoid of eggs. Overall, egg production was seen to be severely affected by TCBZ treatment and flukes were incapable of producing normal eggs within 2 days of treatment. The implications of this in terms of the epidemiology of the disease are discussed.

The ‘secretory pathway’ of the Mehlis' gland secretion as well as the maturation of the secretory granules are described. The secretions produced by the Mehlis' gland and the distal ootype in Schistosoma mansoni were identified as neutral glycoproteins. They were both periodate reactive, stained with phosphotungstic acid at low pH, and sensitive to the proteolytic action of papain. The secretions did not strain with both the low-iron diamine and the high-iron diamine methods for the presence of acidic glycoconjugates, and they did not contain a detectable level of sulphydryl groups. The possible role of the secretion from the Mehlis' gland in egg-shell formation is discussed.

The ancyrocephaline monogenean gill parasite Cichlidogyrus halli typicus has a short oviduct/ovo-vitelline duct separated from the germarium and from the ootype by sphincter muscles. The chamber so-formed contains spermatozoa, the axonemal ends of which appear to be attached in an intimate way between lamellated extensions of the chamber lining. This arrangement may facilitate fertilization by retaining an oocyte temporarily in close contact with the free nucleated ends of the spermatozoa, i.e. the short oviduct/ovo-vitelline duct may serve as a fertilization chamber. There are two distinct types of Mehlis' gland opening at the base of the ootype and the latter has a cellular, non-secretory lining which is not penetrated by gland ducts.

Cardicola ambrosioi n. sp. (Digenea: Sanguinicolidae) is found in the blood vessels of liver and gills of the Brazilian flathead, Percophis brasiliensis Quoy and Gaimard, 1824 (Pisces: Percophidae), from Mar del Plata, Argentina. Among the 13 known species within Cardicola Short, 1953, the new species closely resembles Cardicola coridodacis Manter, 1954, from which it is distinguished by having a relatively shorter oesophagus, the vitellaria extending anteriorly to the nerve commissure, rather than to the end of anterior caeca, the female pore located closer to male pore, the latter situated medially instead of laterally and by possessing a larger Mehlis gland and a smaller seminal vesicle.

Light microscopy of the amphistomes Orthocoelium scoliocoelium clearly reveals that there are clear differences in the Mehlis’ gland of immature and mature worms. The Mehlis’ glands were neither fully formed nor functional in immature worm. Contrary to this, mature amphistomes had prominent structure, fully formed and functional Mehlis’ gland. Therefore, the present study is great significance from medico-veterinary standpoint Comparative Histology of the Mehlis’ Gland of Immature and Mature Orthocoelium Scoliocoelium (Trematoda: Digenea) INTRODUCTI ON Comparison of the histology and histochemistry of diverse tissues of the amphistomes receive little attention (Sharma et al., 1981; Sharma and Hora, 1983; Gupta et al., 1987 Swarnakar and Sharma 1992 and Swarnakar & Soni 2004). The comparison of the light microscope’ histology of Mehlis’ glands of immature and mature flukes as well as tissues of different adult flukes are hardly available. Even the ultrastructure of reproductive ducts and accessory glands of a few trematodes have been studied (Hanumantha-Rao, 1959; Burton, 1967; Threadgold and Irwin, 1970; Spence and Silk, 1971; Orido, 1991; Swarnakar and Sharma, 1997 and Swarnakar, 2010). In view of the lacuna indicated above in the study of immature and mature amphistomes, it was decided to undertake comparative light microscope histology of Mehlis glands of Orthocoelium scoliocoelium infecting the rumen of water buffaloes. MATERIALS AND METHODS The amphistomes Orthocoelium scoliocoelium were collected from rumen of the freshly slaughtered buffalo (Bubalus bubalis) at local zoo abattoir in Udaipur. Mature worms were retrieved between July to September and immature worms from the remaining months of the year. The amphistomes were washed several times in normal saline water, fixed for 24 hrs. in alcoholic Bouin’s fixative, washed in running tap water for at least 24 hrs, dehydrated in ascending series of alcohols, cleared in xylene, in filtered with and embedded in wax, sectioned at 6 μm and stained in Harris haematoxylin and eosin for general histology. RESULTS Mehlis gland of O. scoliocoelium is madeup of two types of secretory unicellular gland cells. They have been named as S1 and S2 cells. These cells are radially arranged around the ootype. The S1 type gland cells are comparatively larger in size and number and located away from the ootype forming the periphery of a Mehlis’ gland. Whereas the S2 type cells are few in number, oval in shape, smaller in size and located near the ootype. Both the cell types are supported by parenchymatous cells. S1 and S2 type of cells discharge their secretion into the lumen of ootype through their ducts. The duct is funnel shaped. The gland opens into funnel like part of the duct which then narrows, becomes tubular, penetrate the musculature of ootype and open into its lumen. The wall of the ootype is composed of syncytial tegument which bears a striated luminal border towards the lumen. The syncytial lining of ootype is supported externally by musculature. It consists of outer circular and inner longitudinal muscle layers. In case of immature worm musculature is highly developed as a result of which the lumen of ootype becomes considerably narrower (Fig. 1). The structure of Mehlis’ gland of immature and mature worm exhibits differences in them. S1 and S2 gland cells are observed equal in size and numbers in Mehlis’ gland of immature worm; the parenchymalous cells between them appear quite prominent and also larger in number. In mature worms, on the contrary the S2 gland cells and parenchymal cells are few in number and smaller in size. While S1 gland cells are larger in size (Pear ‘shaped) and abounded in number (Fig. 2). The Mehlis gland of mature worm is thus larger in size and secretory in nature, whereas in immature stages, it is smaller in size and nonfunctional. DISCUSSION The Mehlis’ gland of O. scoliocoelium is composed of two cell types S1 and S2 in a mature worm. The S1 cells are quite large and pear shaped and they also differ in staining properties with S2 cell type. The S2 type cells are few in number and located near the wall of ootype. In this respect the Mehlis’ gland of Orthocoelium scoliocoelium differ from Ceylonocotyl scoliocoelium in which the Mehlis’ gland has been reported to be composed of single types of cells (Sharma et al., 1981 and Swarnakar and Soni 2004). The present observation however, is in full agreement in thsi regard with the Mehlis’ gland of Fasciola hepatica (Threadgold and Irwin, 1970), Haematolocchus medioplexus (Burton, 1967) and Mehlis’ gland in the lung fluke, Paragonimus ohirai (Orido, 1991). The function of Mehlis’ gland in (i) the formation of membranes to enclose the oocyte and vitelline cells (Hanumantha-Rao,1959); (ii) lubrication of the capsule filled uterus or stimulation of spermatozoa (Burton,1967); (iii) finally, one or other of the secretions may play a part in the actual tanning process, which results in a highly resistant egg shell, either by triggering off the enzymatic process that results in tanning or adding some essential cofactors for the process (Threadgold and Irwin, 1970). Information is limited to a few trematodes such as fasciola (Threadgold and Irwin, 1970; Haewatoloechus (Burton, 1967) and Schistosoma (Spence and Silk, 1971). In immature worm the Mehlis’ gland is not well developed as is evident from its histology. The S1 and S2 type of cells are almost equal in number and they have not reached the functional stage to elaborate the secretory material. REFERENCE Burton, P.R. (1967). Fine structure of the reproductive system of a frog lung fuke. I Mehlis’ gland and associated ducts. J. Parasitol. 53, 540-555. Gupta, B. C., Parshad, VR. and Guraya, SS. 1987. Morphological and histochemical observations on the vitelline cells of developing and adult Paramphistomum cervi (Trematoda: Digenea). ICMR Advanced Centre in Reproduction, Department of Zoology, Punjab Agricultural University, Ludhiana, India. J Helminthol. Dec; 61(4): 297-305. Hanumanth-Rao, K 1959. Histochemistry of Mehlis' gland and egg shell formation in the liver fluke Fasciola hepatica. L. Experimentia 15: 464 – 465. Orido, Y. 1991. Ultrastructure of Mehlis' gland in the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae). J. Morphol. Jan. 207(1): 9 – 16. Swarnakar, G. and Sharma, P.N. 1992. Comparison of the histochemical distribution of non-enzymatic substances in the reproductive tissues of Cotylophoron cotylophorum. J. of Parasitol. and Appli. An. Biol. 1, 143-149. Sharma, P. N. Mandawat, S. and Sharma, A. N. (1981). Cytochemistry of Mehlis’ gland in ceylonocotyle scoliocoelium. J. of Helminthology. 55, 141148. Sharma, P. N. and Hora (1983). Role of oesophageal gland in the digestive physiology of two rumen amphistomes Orthocoelium scoliocoelium and Paramphistomum cervi, Journal of Helminthology. 57, 11-20. Swarnakar, G. 2010. Ultrastructural study on spermatogenesis in rumen amphistome Orthocoelium scoliocoelium (Amphistome: Digenea), International J. of The Bioscan. 5(3), 547 – 553.

Mehlis' gland of a digenetic trematode, Paragonimus ohirai, is composed of two types of secretory cells, DB and CB. The less abundant type (DB) produces dense bodies, with the cytoplasm characterized by greatly distended cisternae of rough endoplasmic reticulum. The other type (CB) synthesizes clear, vesicular bodies. Its cytoplasm contains numerous mitochondria, rough endoplasmic reticulum with narrow cisternae, and abundant Golgi complexes. Processes of the two cell types converge on the ootype-proximal uterine wall, pass through the epithelium, and finally open into the lumen. These proximal processes contain longitudinally arranged microtubules whose luminal ends are anchored to the epithelium by ring-form septate desmosomes. According to the distribution of the two types of processes, three different zones (DB, mixed, and CB) can be recognized within the epithelia. As the CB processes enter the lumen predominantly beyond the uterine valve region, this cell may produce secretions required for egg shell maturation or hardening. The role of DB cells (which enter the lumen more commonly in the ootype near the oviduct) remains unknown.

Histochemical techniques were used to study the chemical composition of Mehlis’ gland secretion in Ceylonocotyle scoliocoelium. The results suggest the secretion to be lipoproteinous in nature, containing several hydrolytic and oxidative enzymes. In tests for nine hydrolytic enzymes only seven were detected in relation to gland cells, ihe two enzymes not showing any reactivity being glucose-6-phosphatase and 5-Nucleotidase. Alkaline phosphatase and ATPase have been associated with the permeability and transport of precursors of synthetic material from the surrounding parenchyma and with eliminating the waste products from the gland: ACPase in (i) generating a favourable milieu in the gland for the synthesis of secretory material and (ii) in regulating secretion and its over production; TPPase in elaborating the secretion, esterases in the metabolism of the gland. Intense SDH and MAO activities were related to high synthetic activity of the gland cells. Phenol oxidase—a key enzyme involved in tanning was absent.

Austramphilina elongata Johnston, 1931 were collected from the freshwater turtle Chelondina longicollis taken in New South Wales, Australia. The morphology and histochemistry of the oogenotop is described with emphasis on egg shell formation. The vitelline gland granules compose most of the early egg shell; the granules stain positively with PAS, bromophenol blue and Heidenhains iron haematoxylin. There is a weak reaction for phenolase. The Mehlis gland secretory granules are PAS-positive. The first formed egg shells stain positively with PAS, bromophenol blue and Heidenhains iron haematoxylin; there is a weak phenolase reaction. The significance of these reactions is discussed.

Mehlis' gland of both Diplozoon paradoxum and Calicotyle kroyeri is composed of two cell types that taper to form ducts opening into the lumen of the ootype. The cells are invested with fibrous interstitial material and form a close structural relationship with surrounding parenchyma. The most prevalent cell type, the S 1 cell, is characterized by an extensive GER with narrow cisternae containing a finely granular material, and numerous Golgi stacks involved in the formation of multi-vesicular secretory bodies. In the S 2 cells the GER cisternae are greatly distended with a finely filamentous material and the Golgi give rise to dense secretory bodies with a packed fibrous appearance. There are species differences in the fine structure of the secretory bodies and these may reflect differences in the chemical composition of the glands. The ducts of the glands are lined with microtubules and are anchored to the ootype epithelium by septate desmosomes. They convey the secretory products to the ootype where they are released, apparently by exocytosis involving membrane fusion, into the lumen. The ootype is lined by a highly folded cellular epithelium which in D. paradoxum is ciliated. The cells contain profiles of GER and Golgi complexes and produce a third type of secretion which is also discharged into the ootype lumen.

The female reproductive organs (ovary, vitellaria, and Mehlis' gland) of the digenetic trematode Halipegus eccentricus were studied by transmission electron microscopy. Oocytes entered diplotene while in the ovary and produced cortical granules and lipid bodies. Vitelline cells produced large amounts of eggshell protein but no yolk bodies. Two types of Mehlis' gland secretory cells were present, distinguishable by the morphology of their rough endoplasmic reticulum, Golgi bodies, and secretory bodies, and by the persistence of recognizable secretory material within the ootype lumen after exocytosis. In an attempt to standardize the nomenclature regarding the cell types of the Mehlis' gland, a classification that takes into account these four criteria is proposed. Two basic types of Golgi body organization were noted for the cells of the female reproductive system: a stack of flattened cisternae (Mehlis' gland alpha cells) and spherical Golgi bodies with vesicular cisternae (oocytes, vitelline cells, and Mehlis' gland beta cells).

The metacercarial stage of Gymnophalloides tokiensis has a mid-ventral pit which was mistaken in the original description for a wide genital pore. The genital pore is small, on the anterior edge of the ventral sucker, and not surrounded by prominent muscle fibers. Other corrections of the original description include paired, compact vitellaria (referred to as Mehlis gland), a bipartite (not globular) seminal vesicle, and an ovary smaller than the testes; no eye spots or a seminal receptacle are present.Yamaguti's (1971) synonymy of Lacunovermis Ching, 1965 with Gymnophalloides is rejected because of the differences in the location and type of genital pore. In contrast to G. tokiensis, both L. conspicuus and L. macomae (Lebour, 1908) Loos-Frank, 1970 have a wide genital pore that opens some distance from the ventral sucker and both ventral pit and genital pore are surrounded by strong muscle fibers.Two of Yamaguti's (1971) footnotes about gymnophallid trematodes are corrected. In my 1965 paper, I did not synonymize Gymnophalloides with Meiogymnophallus but replaced Gymnophalloides as emended by James (1964) with the name Meiogymnophallus. I did not synonymize Gymnophallus oidemiae, now M. minutus (Cobbold, 1859) Bowers and James, 1967, with G. somateriae but regard them as distinct species.

Evidence obtained would suggest that the processes involved in egg formation in Fasciola hepatica occur in the following sequence. The egg constituents, namely an ovum and some vitelline cells, pass through the proximal ootype and as they do so they are smeared by the secretions of Mehlis' gland which have accumulated there. A temporary interface is set up between the Mehlis' gland secretion and the fluid which surrounds the egg constituents. Shell globules are released by the vitelline cells and coalesce on the inner aspect of the interface. At the same time some of the Mehlis' secretion diffuses across the interface, thereby bringing about the dissociation of the interface. It is suggested that Mehlis' gland secretion brings about the fusion of the shell layer. During the initial stages of the process the developing egg is supported by the cells of the ootype epithelium. Later the egg passes into a wider, more distal part of the ootype where the process of shell deposition is continued. When complete, or almost complete, the egg passes into the uterus where changes indiciating the initial process of tanning are seen to take place. A thin, very uniform layer of Mehlis' gland secretion can still be identified on the surface of mature eggs.

The oogenotop was studied using certain histochemical tests and the contributions of the various parts to the egg were noted. The Mehlis gland and the uterine epithelium make large contributions to the egg. Both the inner and outer envelopes are of reduced importance. A photographic record of oogenesis is presented showing it follows previous descriptions for the Cestoda. The first cleavage is unequal. Shell granules from the oocyte are carried to the macromeres where they are used in the formation of the outer capsule. Hatching is standard for eggs with a membraneous inner capsule. Most infections are characterized by one female and one male worm. Microsporidians reduce the number of sperm produced and probably limit the production of oncospheres.

Amphilina bipunctata Riser, 1948 were collected from the white sturgeon Acipenser transmontanus from the Columbia River, USA. The morphology and histochemistry of the oogenotop of Amphilina as they pertain to egg formation are described. The vitelline gland granules secrete the main part of the egg shell. These shell granules are positive for basic proteins, SS groups, and there is a weak reaction for phenolase. The Mehlis gland granules are positive for PAS. The first-formed egg shell is positive for basic proteins and it is weakly positive for phenolase. These egg shells are negative for SS groups and PAS. Conjectures concerning the significance of these data are made.

1. 1. When exposed in vitro to C 14 proline male schistosoma become much more heavily than do females. 2. 2. The areas of concentration in the male are the integument and sub-integument, the paraoesophageal region, the gut and the testes. 3. 3. In the female local accumulation in the posterior ovary and, to a lesser extent, in the mehlis gland was noted. 4. 4. The vitelline gland and duct fail to tag heavily on exposure; the female gut also shows minimal proline uptake. 5. 5. Proline may play a special metabolic role in either the paraoesophageal musculature or in the nearby neural ganglionic ring. 6. 6. Speculations concerning functional implications of these findings are given.

Mehlis' gland of F. hepatica is composed of two secretory cell types supported by parenchymal cells. The products of the secretory cells are carried by means of cytoplasmic extensions into the ootype. The most prevalent type of secretory cell is termed S 1 type. It possesses many narrow granular endoplasmic cisternae and numerous Golgi complexes. The secretory bodies of this cell resemble slightly curved sausages and have a filamentous content radiating from a central core. The other secretory cell type is termed S2 type. It is characterized by distended granular endoplasmic cisternae and numerous Golgi complexes which give rise to round dense secretory bodies with a crystalline or packed fibrous appearance. Both types of secretory cells are penetrated deeply by invaginations of interstitial material. The cytoplasmic extensions from the cells are lined with microtubules and anchored to the epithelial cells lining the ootype by septate desmosomes. The secretory bodies of both cell types are released into the lumen of the ootype, where they disintegrate.

Histochemische Untersuchungen der Mehlischen Druse vonFasciola hepatica erweisen deren Phospholipidabsonderung. Es wird auf die Moglichkeit einer engen Bezichung des Phospholipids zu Lezithin und seine enzymatische Aktivierung zur Herauslosung von Eischalenprokursoren hingewiesen.

The cells of Mehlis's gland in the liver fluke (Fasciola hepatica L.) have been studied with light and electron microscopy. The gland contained large cells in the periphery and small cells in the centre. The large cells presented morphological features characteristic of secretory cells, viz. large nucleoli, cytoplasmic basophilia, a well developed endoplasmic reticulum, abundant ribosomes, and secretory granules in different stages of development. The mitochondria were moderately frequent. Several small groups of vesicles and membraneous lamellae reminiscent of Golgi complexes occurred randomly in the cytoplasm. These structures were associated with mitochondria and secretory granules. Evidence of alterations and conversion into a cell debris was observed, and a process of holocrine secretion was suggested.

reproductive system of the trematodes and cestodes, and consists of a series of glandular structures surrounding the ootype, the tube connecting the oviduct to the uterus. These glands vary in number and arrangement in the different groups and species. This gland is particularly well developed in the liver fluke, F. hepatica, where its huge size has made it difficult to appreciate its relationship with the complicated system of ducts embedded in it. In consequence there has been a conflict in interpretation (Stephenson, 1947, Yosufzai, 1953) and the purpose of this paper has been to attempt some clarification.

Egg shell formation for Parastrigea mexicanus is described. Eleven histochemical tests were used to elucidate the process and to characterize the oogenotop. The egg is formed in the ootype (a region of high secretory activity); an oocyte and 12-17 vitelline cells are enclosed by a coalescence of vitelline shell globules. The vitelline cells also contribute copious amounts of glycogen and RNA. There was no direct histochemical evidence for Mehlis or uterine gland function; however, secretory functions were inferred from morphology and histochemical reactions. The basic mechanism of egg shell formation in P. mexicanus closely follows that of Fasciola, but it differs in some regards from the process described for the latter trematode. Helminthologists have studied egg shell formation in trematodes for 82 years now and their efforts have concentrated on widely separated taxa. In spite of this long period of time and the many investigators involved, only a few of the more than 100 families of digenetic trematodes have been studied in this regard. Our approach has been a comparative one and to date, we have completed studies on representatives from the following families: Fellodistomidae, Hasstilesiidae, Hemiuridae, Microphallidae, Notocotylidae, Pronocephalidae, and Syncoelidae. Fasciola hepatica has received the greatest attention; Haematoloechus has been studied in some detail also. Egg shell formation in the digenetic trematodes appears to follow a generalized mechanism which has been elucidated by a number of authors and reviewed by Smyth & Clegg (1959). Since that date, a number of papers have described variations on the central theme. Recently, Ebrahimzadeh (1966) studied 27 species (from ten families) on a comparative basis and he thus established a standard of excellence for students of egg shell formation to follow. During the course of other studies on parasites of the black-necked stilt, the author collected a large number of Parastrigea mexicanus Coil, 1955 (Strigeidae). Although the strigeoids have received much attention concerning their life cycles, it has been only recently that details of their morphology have been studied; many of these have emphasized the holdfast organ (Erasmus, 1968; and others). There are certain striking features of these worms which warrant further studythe large amount of tissue devoted to vitelline production, the extensive Mehlis gland tissue, the extremely large egg, and the nature of the uterus. P. mexicanus provides excellent material for study. The worm is short, compact, and easily handled; sectioned material can be prepared without difficulty. Microdissections are difficult, however, due to the muscular body wall and the heavy cuticle. This is the fifth report in our series of comparative studies on egg shell formation in digenetic trematodes. 1This investigation was supported by Public Health Service Grants AI 05145 and GM 12587. 2The author is indebted to Dr. Clarence Cottam and Mr. Caleb Glazener, both of the Welder Wildlife Foundation, for their help in providing facilities, equipment, and supplies for the early parts of this study. TRANS. AMER. MICROSC. Soc. 88(1): 127-135. 1969. This content downloaded from 207.46.13.21 on Tue, 27 Sep 2016 05:13:10 UTC All use subject to http://about.jstor.org/terms 128 TRANS. AMER. MICROSC. SOC., VOL. 88, NO. 1, JANUARY 1969

Observations on the functional anatomy of the ducts associated with Mehlis' gland were made in five species of echinostomes: Echinostoma malayanum, E. lindoense, E. audyi, Hypoderaeum dingeri, and a 43-spined, undescribed Echinoparyphium species. In all five species the structure of the Mehlis' gland complex is similar. There is an ovicapt in the form of a chamber which regulates the passage of the ova from the ovary into the oviduct. Ciliated epithelium lines the ovicapt, the oviduct distal to the ovicapt, and the proximal part of Laurer's canal. The movement of the cilia is directed toward Laurer's canal from which excess sperm and vitelline granules are discharged at the dorsal surface. Spermatozoa are stored in the uterine seminal receptacle in the absence of a seminal receptacle. The Mehlis' gland complex of echinostomes is best studied in living worms because the ducts and organs associated with it are not clearly visible in mounted specimens. Moreover, the gland region is often filled with eggs, making a detailed study extremely difficult or impossible. Descriptions are often incomplete and sometimes inaccurate. This paper reports observations on the Mehlis' gland complex in living specimens of five species (three genera) of echinostomes. MATERIALS AND METHODS The following species of echinostomes were studied: Echinostoma nmalayanum Leiper, 1911; E. lindoense Sandground and Bonne, 1940; E. audyi Lie and Umathevy, 1965; Hypoderaeum dingeri Lie, 1964; and an undescribed 43-spined Echinoparyphium species which will be named later when its complete life cycle is described. An unlimited supply of living worms was maintained in the laboratory for life-cycle studies. E. malayanum was obtained from rats, the other four species from pigeons and ducklings. Young adult worms with few eggs in the uterus were preferred. The Echinoparyphium species is the easiest to study since the worm is small and seldom contains many eggs; H. dingeri is the most difficult to examine due to thickness of the worm and density of Mehlis' gland. The living worm is examined dorsal side up in a drop of physiological saline under a cover slip. Received for publication 12 August 1964. * This work was supported by the University of California International Center for Medical Research and Training (The Hooper Foundation, San Francisco School of Medicine) with Research Grant GM-11329 from the Office of International Research, NIH, U. S. Public Health Service. t Present address: Hooper Foundation, University of California Medical Center, San Francisco. When movement of the worm is restricted and the position is suitable, ducts and organs in the Mehlis' gland region can be seen. An oil-immersion lens is required for detailed observation of certain parts.