The anterior esophageal region of Schistosoma japonicum is a secretory organ
© Hong Li et al.; licensee BioMed Central. 2014
Received: 30 October 2014
Accepted: 24 November 2014
Published: 10 December 2014
The esophagus of blood-feeding schistosomes has been largely neglected although its posterior portion was designated as a gland decades ago. However, we recently showed it plays a pivotal role in blood processing. It is clearly demarcated into anterior and posterior compartments, both surrounded by a mass of cell bodies. Feeding movies revealed that erythrocytes accumulate in the anterior compartment before entering the posterior, indicating that a distinct process is executed there. We therefore investigated ultrastructural aspects and possible functions of the anterior region.
The heads of adult Schistosoma japonicum were detached and prepared for both transmission and scanning electron microscopy to define the detailed ultrastructure of the anterior esophagus. Cryosections of heads were also prepared for immunocytochemistry and confocal microscopy to define the pattern of intrinsic host antibody binding in the anterior esophageal lining.
The anterior syncytial lining of the esophagus is highly extended by long, thin corrugations of cytoplasm projecting towards the lumen. Strikingly in the male worm, the tips of the corrugations are further expanded by numerous threads of cytoplasm, producing a spaghetti-like appearance in the central lumen. Flattened, pitted cytoplasmic plates are interspersed in the tangled mass of threads. Abundant, morphologically distinct light vesicles of varied size and contents are manufactured in the cell bodies, from where they traffic through cytoplasmic connections to the corrugations and out to the tips. Clusters of vesicles accumulate in expanded tips in males, together with occasional mitochondria whilst females have more mitochondria but fewer vesicles. The membranous contents of light vesicles are secreted mainly from the tips, but also from the sides of the corrugations. They coat the surfaces and then form organised self-adherent membrane figures when shed into the lumen. Host antibody binds strongly in a characteristic pattern to the anterior esophageal lining indicating that the secretions are highly immunogenic.
We suggest that the anterior esophageal region is an independent secretory organ. The contents of light vesicles are released into the esophageal lumen via the tips of corrugation to interact with incoming blood. Our immediate task is to establish their composition and role in blood processing.
Adult schistosomes reside in the host blood stream, feeding exclusively on blood. The magnitude of this process in S. mansoni was revealed by in vivo tracer studies with Cr51-labelled erythrocytes . A female worm ingested approximately 4.4× its body volume of plasma fluid per day, the male much less (0.2×) in spite of a larger size . The blood is pumped by rapid grabbing contractions of the oral sucker, through the mouth, down a short esophagus to reach the blind ending gut caecum where terminal digestion and absorption take place. While much attention has focused on the enzymatic activities of the gut lumen and gastrodermis , the esophagus has been largely neglected. However, we recently demonstrated that it plays a pivotal role in feeding, with blood processing initiated there ,. Erythrocytes are uncoated as they pass down the esophagus  while leucocytes become tethered in the posterior region to form a plug-like mass in which they are damaged or even destroyed . Consistent with these functions, the esophagus has a complex, highly organised structure and is lined by syncytial cytoplasm, continuous with that of the tegument. The presence of a glandular structure around the posterior esophagus was established by electron microscopy more than 35 years ago -. It comprises a roughly spherical mass of cells linked to the lining syncytium by cytoplasmic connections through the muscle layer. The lining of the posterior region is expanded >25 fold in surface area by regular plate-like extensions of cytoplasm into the lumen .
Movies of S. mansoni feeding have revealed that blood ingestion is a multistep process . Erythrocytes accumulate in an anterior compartment to form a bulge before passing as a bolus to a posterior compartment. This anterior compartment, like the posterior, is surrounded by a mass of cell bodies , although it has never been designated as a gland. One reason may be that in S. mansoni the syncytial cytoplasm of the anterior lining, whilst drawn out into longitudinal folds, nevertheless contained normal tegumental inclusions, namely discoid bodies and multilaminate vesicles -; see also the SEM images in . There appear to be no equivalent studies of the S. japonicum esophagus and only two reports on the S. japonicum tegument ,. From these it is apparent that the tegument of S. japonicum differs significantly in structure from that in the more exhaustively investigated S. mansoni. Additionally, while the posterior esophagus of S. japonicum appeared similar to that in S. mansoni, it was intriguing to observe a net-like appearance of the anterior esophageal lining in confocal images. This has prompted us to undertake a complete reappraisal of the cellular organisation and potential functions of the anterior esophageal region in S. japonicum.
Animal care and all animal procedures were carried out in compliance with the Guidelines for the Care and Use of Laboratory Animals produced by the Shanghai Veterinary Research Institute. The study was approved by the Ethics Committee of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention.
Cercariae of S. japonicum were shed from naturally infected Oncomelania hupensis snails collected from the wild in Anhui Province, China. Mice were experimentally infected percutaneously with 40 or 20 cercariae using the coverslip method . Adult parasites were obtained by portal perfusion of the mice five weeks after exposure to 40 cercariae and 21 or 24 weeks after exposure to 20 cercariae, using RPMI-1640 medium buffered with 10 mM HEPES (Gibco, Life Technologies, Grand Island, NY, USA). These time points were chosen to represent newly mature adults and worms from chronically infected animals that had been subjected to prolonged exposure to immune responses. The worms were extensively washed in the same medium before tissue debris and any damaged individuals were removed under a dissecting microscope.
The structure of the anterior esophagus was examined by transmission electron microscopy (TEM). Worms were fixed overnight at 4°C in 2.5% glutaraldehyde/4% paraformaldehyde in 100 mM phosphate buffer, pH7.4. Following two washes in phosphate buffer, they were post-fixed in osmium tetroxide for 2 hours, washed in H2O and dehydrated through a water-acetone series before single embedding in Spurr’s resin. Worm heads were then cut out of the resin and mounted in a transverse or longitudinal orientation on plastic stubs. Thick sections (0.5 μm) were cut and stained with toluidine blue for examination on a light microscope to identify the location of the anterior esophagus. Thin sections (70 nm) were then cut onto grids and post-stained with saturated uranyl acetate in 50% ethanol, and Reynolds’ lead citrate solution before examination in a Tecnai 12 BioTwin microscope (FEI, Hillsboro, OR, USA). Measurements of structures in micrographs were performed using the ‘Analyzing digital images’ package (Lawrence Hall of Science, UC Berkeley, CA, USA; http://www.globalsystemsscience.org/software/download) with the intrinsic scale as calibrator. Five male and five female worms were orientated for sectioning in TS at the two sampling times. The bulk of image series were obtained from one male and one female at week 5, and again at week 21. No age-dependent structural changes were observed in these worms.
The luminal surface architecture of the esophagus was examined by scanning electron microscopy (SEM). Worms were fixed in 4% formaldehyde in PBS at room temperature. The head region was then carefully sliced transversely into thin steaks (~ 30 μm) at ×35 magnification under a Leica EZ4 stereo microscope, using vannus scissors. The steaks were washed, dehydrated through an acetone series, critical point dried, individually positioned on stubs, and sputter-coated with 7 nm gold/palladium. They were examined using a JSM 6490-LV microscope for low magnification orientation and a JSM-7600 F for high-resolution images (both Jeol, Tokyo, Japan). More than 20 head steaks were examined by SEM; the majority of images come from four specimens with good transverse orientation.
Adult worms recovered at 24 weeks were fixed in 4% formaldehyde in PBS for four hours and then washed several times in PBS alone. The heads were then detached just posterior to the ventral sucker, as described for SEM, transferred to optimum cutting temperature (OCT) compound and orientated horizontally with a fine needle before freezing. Seven μm cryostat sections were cut and reacted with 1:100 dilution of AF488- labelled Goat-anti mouse IgG to detect intrinsic bound host antibody or with FITC-labelled Goat-anti hamster IgG (both Invitrogen) as a control for the specificity of staining. The musculature and nuclei were visualized by staining of f-actin with a 1:100 dilution of AF555-conjugated phalloidin (Invitrogen, Molecular Probes) plus a 1:400 dilution of 4′,6-diamidino-2-phenylindole (DAPI;1 μg/ml in PBS; Sigma-Aldrich, Poole, Dorset, UK), respectively, for 30 minutes. Optical slices were obtained using a LSM-710 confocal microscope (Zeiss, Cambridge, UK). Imaging conditions were as follows: DAPI: 405 nm diode laser with 405 main beam splitter (MBS); FITC/AF488: 3 mW argon laser with 488/561/633 MBS; Phalloidin AF555: 561 nm diode laser with 488/561 MBS.
The tegument of S. japonicum has some unique structural features
The layout of the anterior esophagus reveals novel features
The anterior esophageal cell bodies manufacture a novel inclusion
Light vesicles are transported to the leading edge of the luminal corrugations
The lining of the anterior esophagus comprises a thin basal layer of cytoplasm from which numerous corrugations extend; light vesicles and mitochondria are also present. This syncytial layer is not comparable in appearance to the tegument that lines the oral sucker and covers the worm body. In places, the lining corrugations are extremely narrow (50 to 70 nm), barely wide enough to accommodate two unit membranes and a little intervening cytoplasm (Figure 4C). In wider regions (180 to 500 nm, mean 275 nm) central parallel membranes 28 nm apart, denote the presence of basal invaginations (Figure 4D), similar to those described for the plates of the posterior esophagus; these invaginations run for 40 to 60% of the distance up each corrugation from its base. In the wider segments, clusters of light vesicles are apparent and reach all the way to the leading edge, implying they must be in transit (Figure 4C). Surprisingly the light vesicles within the corrugations appear smaller (mean size 0.17 × 0.14 μm in males, 0.18 × 0.13 μm in females) than those in cell bodies and the expanded tips. Mitochondria are also evident in the cytoplasm, together with sparse discoid bodies (Figure 4D). Both debris and membranous material are present between the corrugations (Figure 4A). Plausibly this material originates from the light vesicles, some of which can be seen fusing with the bounding membranes of the corrugations in this region to release their cargo (Figure 4A and D).
Secretions are released from the expanded leading edges of the corrugations
Host antibodies bind strongly to the anterior esophagus lining
Our recent detailed study of the schistosome esophagus focused primarily on the structure and function of the posterior compartment surrounded by the esophageal gland . Based on studies with S. mansoni, the prevailing view of the anterior compartment is that its lining and associated cell bodies are simply an extension of the body surface tegument. The syncytial cytoplasm has longer folds but nevertheless contains the normal discoid bodies and multilaminate vesicles present in the body surface ,,,. The unstated assumption from these reports is that the anterior esophageal lining performs exactly the same functions as the body surface tegument, including evasion of the immune response. Our EM observations show that the anterior esophageal lining is not simply an extension of the tegument in S. japonicum. Furthermore, there are marked differences in ultrastructure of the surface tegument in the two species that have not been previously appreciated, particularly as the detailed description of the S. japonicum tegument by Sobhon & Upatham  has not attracted attention. Points of distinction in S. japonicum include: in male worms a clear division of the tegument cytoplasm into three zones; the presence of numerous ring bodies (our dark bodies); the occurrence of a system of membrane channels in the central zone. Similar but transient channels have been described in S. mansoni as the sites where multilaminate vesicles fused with the plasma membrane to release their contents that formed the membranocalyx . Our TEM images suggest that the channels likely play a similar role in S. japonicum but appear to be of a permanent nature. A more recent review  provided only low magnification images but noted the presence of “large membranous bodies of 150-200 nm diameter” in the tegument. It is reasonable to assume that these inclusions, irrespective of the terminology, represent the source of the membrane-like material that covers the tegument surface. In the anterior esophageal lining of S. japonicum the dark bodies are entirely replaced by the light vesicles whereas the discoid bodies are common to both surfaces.
While SEM has been used to characterise the architecture of the body surface in S. mansoni, and S. japonicum,,,, and the gastrodermis in S. mansoni our study is the first to use the technique for an ‘internal’ examination of S. japonicum. Strikingly, it revealed that the syncytial cytoplasm lining the anterior compartment was extended into thin corrugations up to 20 μm long in males, which terminated in threads of cytoplasm (cf. the plates of the posterior esophagus lining in S. japonicum which are up to 25 μm from base to tip). What might this remarkable cellular architecture achieve? Clearly the corrugations expand enormously the surface area of the anterior esophagus while the fringe of threads extends still further the potential interaction surface with ingested blood. Indeed it is possible, especially in the males, that the spaghetti-like threads intruding into the lumen are intended to entangle cells and retard their passage, allowing more time for interaction with esophageal secretions.
The mass of cell bodies surrounding the anterior esophagus are clearly a factory, primarily for the manufacture of light vesicles and to a lesser extent discoid bodies. If the relative proportions of the anterior and posterior cell masses are indicative of their physiological activity, then a one-third to two-thirds division of labour in the processing of ingested blood by the two compartments is likely. The heterogeneous appearance of the light vesicles themselves in the cell bodies, containing both membranous and granular material, implies a multiplicity of functions. Indeed, by analogy with the tegument it is likely they are the source of the membranous layer that coats the corrugations and is found free and aggregated in the anterior esophagus lumen. By inference from their distribution in the esophageal tissues, the light vesicles appear to traffic from their sites of manufacture at the Golgi apparatus of the cell bodies, via the cytoplasmic connections, along the lining corrugations to their tips, making these the main site for the release of vesicle contents. Given the thinness of the corrugations in many places, this traffic must occur along designated channels wide enough to accommodate the vesicles, accounting for the bunching of vesicles within the corrugations. The expanded corrugation tips of males containing clusters of vesicles are a major distinction from females with usually only a single vesicle. This could indicate that the male tips are a temporary storage site for vesicles, which accords with the recent suggestion that males feed intermittently but females continuously . Mitochondria are more prominent in the tips of females than males, suggesting a greater need for energy at this location in the former. Indeed, if the voracity of the female S. japonicum is comparable to that of S. mansoni relative to their respective males, this could amount to at least a nine-fold greater ingestion and processing of blood by the female .
We were not successful in preparing the tiny female esophagus for SEM but high-resolution images of males revealed flattened, pitted regions, < 1 μm2 in surface area, amongst the spaghetti threads. It is tempting to equate these to the expanded tips of the corrugations seen in the TEM, with the inference that the pits could represent the potential sites of vesicle fusion with the plasma membrane. However, the spacing of the pits (182 nm) and the vesicles (400 nm) do not quite match up, even allowing for some shrinkage of tissues by critical point drying for SEM .
The presence of a central invagination within each corrugation is a feature shared with the more highly organised plates of the posterior esophagus. This arrangement is typical of transporting epithelia  and we have suggested that in the posterior esophagus, the plates might function to generate massive ion-fluxes into or out of the lumen. The same arguments can be applied to the anterior esophagus, although such a flux may not be linked directly to erythrocyte lysis since, in S. mansoni at least, that occurs in the posterior compartment . Careful measurements showing that light vesicles within the corrugations were smaller in diameter than their counterparts in the cell bodies and corrugation tips, supports the idea of an ion flux. Assuming the vesicles are acting as osmometers, then the reduction in volume suggests that in transit to the tips they pass through a region of higher osmotic potential causing them to shrink. Such a gradient could be created by a flux of ions across the corrugations, generated by transporters situated in the membranes of the basal invaginations. It must be emphasised that the light vesicles contain granular material as well as whorls of membrane indicating that the contents may serve several functions. An obvious one is release of the membranous products to coat and protect the esophageal surfaces from immune attack, dealt with below. Another may be release of the granular component comprising proteins that interact with the incoming blood cells and plasma. Expanded tips of the esophageal lining, reminiscent of the structures we describe here, have been observed in the rodent blood fluke Schistosomatium douthitti. Furthermore, the clusters of vesicles they contained were shown to be positive for acid phosphatase activity, suggesting they were lysosomes. We were unable to confirm acid phosphatase activity in the anterior esophagus in our study but this observation in a related species raises the possibility that S. japonicum is secreting lysosomal enzymes into the esophagus lumen. Certainly the morphology of the light vesicles is akin to that of primary lysosomes.
The similarity in anterior esophagus structure between S. douthitti and S. japonicum, the earliest divergent member of the genus Schistosoma, suggests that the production of light vesicles and secretion of their contents is a plesiomorphic (‘ancestral’) feature ,. It is conceivable that in more derived lineages such as S. mansoni the anterior esophagus lining has converted to essentially normal tegument but whether this is so (and how it might relate to differences in the processing of blood between S. mansoni and S. japonicum) remains to be established. However, it should be noted that S. japonicum is a zoonotic fluke that can complete its natural life cycle in seven Orders of the Mammalia  whereas S. mansoni is essentially confined to a single species of primate.
Although the dark vesicles that supply the tegument surface with its membrane-like coating are replaced by the light vesicles in the anterior esophagus, the lining still displays the multilamellar characteristics of the tegument. From this we infer that in spite of originating from a morphologically different vesicle, this secreted membranocalyx functions as an inert protective barrier in the anterior esophagus, in the same way as it does over the tegument surface. The abundance of the membranous aggregates on the surface of the lining and in the lumen may indicate a fast turnover of this secreted layer, which could reflect the effect of antibody binding and sloughing. In this respect, the strong reactivity of the anterior esophagus with host IgG is surprising. It differed from our previous observations on S. mansoni worms from mice and hamsters where there was a uniform detection of host antibody, apparently confined to the esophagus lumen . This suggests that cryosections are preferable to permeabilised whole worms for the generation of detailed high resolution images using the confocal microscope. Furthermore the body surface tegument of S. japonicum reacts only weakly for IgG implying that it is protected from antibody binding by its membranocalyx. However, if the worm has no option but to secrete proteins from specific sites on the tips of the esophageal corrugations, then such strong reactivity might be anticipated. Indeed, our high resolution images of worm head cryosections revealed that the pattern of intrinsic antibody binding on the anterior lining was punctuate, not continuously uniform. This indicates a marked heterogeneity of composition with only some points in the lining strongly recognized.
Our observations were made on worms from a murine permissive host where this bound antibody appears to cause no harm, perhaps precisely because there is rapid sloughing of targets. Nevertheless, it would be pertinent to discover the precise nature of the secretions emanating from the anterior esophageal vesicles since these are accessible to antibodies in a functional state before they can be subjected to proteolysis by the battery of hydrolases in the gut; this could make them good vaccine targets. Recently, a micro exon gene (MEG-12) has been identified as uniquely expressed in the anterior esophageal cell bodies of S.mansoni, the first such marker for these cells (Crusca et al., personal communication). Furthermore, its protein product has been implicated in the initial step of blood processing by interaction with incoming erythrocytes to destabilise the plasma membranes. Its novel site of expression in the anterior esophagus may indicate that the secretory inclusions of the lining syncytium in S. mansoni are not, after all, typical of the tegument. It would also be instructive to discover whether S. japonicum possesses a MEG-12 gene, and what other products are manufactured by the anterior esophageal cell bodies of both species.
The schistosome esophagus is not simply a muscular tube that conveys blood from the oral cavity to the gut for digestion. In a previous study we showed that it is was divided into anterior and posterior compartments, with the ball of cell bodies that comprise the gland around the latter portion secreting proteins into the lumen to interact with incoming blood. We now show that, in S. japonicum at least, the smaller ball of cells around the anterior compartment also serves as a gland. The secretory vesicles manufactured in this region have distinct morphology from those of the posterior, suggesting a different composition. The cytoplasmic lining of the anterior compartment has a spaghetti-like appearance that may allow it to entangle incoming cells to increase their interaction time with the secretions. We suggest that the two masses of cell bodies should be designated the anterior and posterior esophageal glands in recognition of this distinction in morphology. We also show that the anterior compartment is the site for intense binding of host antibody, indicating that these secretions are highly immunogenic. Work is in progress to define the proteins manufactured in the anterior gland in order to understand better the role of the esophagus in blood feeding.
LXH was funded by the National Natural Science Foundation of China, grant No. 31100637. CJP was funded by National Natural Science Foundation of China grant No. 81371841 and National S&T Major Program grant No. 2012ZX10004-201. We thank Mr. Yuxin Xu of the National Institute of Parasitic Diseases, for technical help with the infection and recovery of parasites.
- Lawrence JD: The ingestion of red blood cells by Schistosoma mansoni. J Parasitol. 1973, 59: 60-63. 10.2307/3278572.View ArticlePubMedGoogle Scholar
- Skelly PJ, Da’dara AA, Li XH, Castro-Borges W, Wilson RA: Schistosome Feeding and Regurgitation. PLoS Pathog. 2014, 10 (8): e1004246-10.1371/journal.ppat.1004246.PubMed CentralView ArticlePubMedGoogle Scholar
- Kasny M, Mikes L, Hampl V, Dvorak J, Caffrey CR, Dalton JP, Horak P: Peptidases of trematodes. Adv Parasitol. 2009, 69: 205-297. 10.1016/S0065-308X(09)69004-7.View ArticlePubMedGoogle Scholar
- Tort J, Brindley PJ, Knox D, Wolfe KH, Dalton JP: Proteinases and associated genes of parasitic helminths. Adv Parasitol. 1999, 43: 161-266. 10.1016/S0065-308X(08)60243-2.View ArticlePubMedGoogle Scholar
- Hall SL, Braschi S, Truscott M, Mathieson W, Cesari IM, Wilson RA: Insights into blood feeding by schistosomes from a proteomic analysis of worm vomitus. Mol Biochem Parasitol. 2011, 179 (1): 18-29. 10.1016/j.molbiopara.2011.05.002.View ArticlePubMedGoogle Scholar
- Li XH, de Castro-Borges W, Parker-Manuel S, Vance GM, Demarco R, Neves LX, Evans GJ, Wilson RA: The schistosome oesophageal gland: initiator of blood processing. PLoS Neglect Trop Dis. 2013, 7 (7): e2337-10.1371/journal.pntd.0002337.View ArticleGoogle Scholar
- Morris GP, Threadgold LT: Ultrastructure of the tegument of adult Schistosoma mansoni. J Parasitol. 1968, 54 (1): 15-27. 10.2307/3276867.View ArticlePubMedGoogle Scholar
- Spence IM, Silk MH:Ultrastructural studies of the blood fluke-- Schistosoma mansoni . IV. The digestive system. S Afr J Med Sci. 1970, 35 (3): 93-112.PubMedGoogle Scholar
- Dike SC: Ultrastructure of the esophageal region in Schistosoma mansoni. Am J Trop Med Hyg. 1971, 20 (4): 552-568.PubMedGoogle Scholar
- Ernst SC: Biochemical and cytochemical studies of digestive-absorptive functions of esophagus, cecum, and tegument in Schistosoma mansioni: acid phosphatase and tracer studies. J Parasitol. 1975, 61 (4): 633-647. 10.2307/3279456.View ArticlePubMedGoogle Scholar
- Sobhon P, Upatham ES: Snail hosts, Life cycle, and tegumental structure of oriental schistosomes. 1990, World Health Organisation, GenevaGoogle Scholar
- Gobert GN, Stenzel DJ, McManus DP, Jones MK: The ultrastructural architecture of the adult Schistosoma japonicum tegument. Int J Parasitol. 2003, 33 (14): 1561-1575. 10.1016/S0020-7519(03)00255-8.View ArticlePubMedGoogle Scholar
- Yason CV, Novilla MN: Clinical and pathological features of experimental Schistosoma japonicum infection in pigs. Vet Parasitol. 1984, 17: 47-64. 10.1016/0304-4017(84)90064-5.View ArticlePubMedGoogle Scholar
- Skelly PJ, Wilson RA: Making sense of the schistosome surface. Adv Parasitol. 2006, 63: 185-284. 10.1016/S0065-308X(06)63003-0.View ArticlePubMedGoogle Scholar
- Silk MH, Spence IM, Buchi B: Observations of Schistosoma mansoni blood flukes in the scanning electron microscope. S Afr J Med Sci. 1970, 35 (1): 23-29.PubMedGoogle Scholar
- Wilson RA, Barnes PE: The formation and turnover of the membranocalyx on the tegument of Schistosoma mansoni. Parasitology. 1977, 74 (1): 61-71. 10.1017/S0031182000047533.View ArticlePubMedGoogle Scholar
- Wilson RA, Barnes PE: An in vitro investigation of dynamic processes occurring in the schistosome tegument, using compounds known to disrupt secretory processes. Parasitology. 1974, 68 (2): 259-270.PubMedGoogle Scholar
- Voge M, Price Z, Bruckner DA: Changes in tegumental surface during development of Schistosoma mansoni. J Parasitol. 1978, 64 (4): 585-592. 10.2307/3279937.View ArticlePubMedGoogle Scholar
- Sakamoto K, Ishii Y: Scanning electron microscope observations on adult Schistosoma japonicum. J Parasitol. 1977, 63 (3): 407-412. 10.2307/3279988.View ArticlePubMedGoogle Scholar
- Xu LH, Zhou SJ, Lian WN, Mao MZ, Yu YF: Electron microscopic observations of tegumental damage in adult Schistosoma japonicum after in vivo treatment with levo-praziquantel. Chinese Med J. 1994, 107 (10): 771-774.Google Scholar
- Nordestgaard BG, Rostgaard J: Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes. J Microsc. 1985, 137 (2): 189-207. 10.1111/j.1365-2818.1985.tb02577.x.View ArticlePubMedGoogle Scholar
- Bogitsh BJ, Shannon WA: Cytochemical and biochemical observations on the digestive tracts of digenetic trematodes. 8. Acid phosphatase activity in Schistosoma mansoni and Schistosomatium douthitti. Exp Parasitol. 1971, 29: 337-347. 10.1016/0014-4894(71)90041-5.View ArticleGoogle Scholar
- Snyder SD: Phylogeny and paraphyly among tetrapod blood flukes (Digenea: Schistosomatidae and Spirorchiidae). Int J Parasitol. 2004, 34 (12): 1385-1392. 10.1016/j.ijpara.2004.08.006.View ArticlePubMedGoogle Scholar
- Webster BL, Littlewood DT: Mitochondrial gene order change in Schistosoma (Platyhelminthes: Digenea: Schistosomatidae). Int J Parasitol. 2012, 42 (3): 313-321. 10.1016/j.ijpara.2012.02.001.View ArticlePubMedGoogle Scholar
- He YX, Salafsky B, Ramaswamy K: Host–parasite relationships of Schistosoma japonicum in mammalian hosts. Trends Parasitol. 2001, 17 (7): 320-324. 10.1016/S1471-4922(01)01904-3.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.