Schistosoma mansoni Annexin 2: Molecular characterization and immunolocalization

We here describe the cloning and characterization of the Schistosoma mansoni Annexin 2, previously identified in the tegument by proteomic studies, and as an up-regulated gene in schistosomulum stage by microarray data. In silico analysis predicts a conserved core containing four repeat domains of Annexin (ANX) and a variable N-terminal region similar to that described for mammalian isoforms. Real-time RT-PCR and Western blot analysis determined that S. mansoni Annexin 2 is significantly up-regulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages. Immunolocalization experiments and tegument membrane preparations confirmed Annexin 2 as a protein mainly localized in the tegument of schistosomula and adult worms. Furthermore, it binds to the tegument surface membranes in a calcium-dependent manner. These results suggest that S. mansoni Annexin 2 is closely associated to the tegument arrangement, being a potential target for immune intervention.     

Schistocephalus solidus: pinocytosis by the plerocercoid tegument

The possibility of pinocytosis occurring in the tegument of the plerocercoid of Schistocephalus solidus has been investigated by morphological and experimental methods. Electronmicroscopic study showed that the outer syncytial tegument contained numerous electronlucid vesicles. These vesicles had two gradients, the number of vesicles decreasing from the outer canopy region to the inner canopy and from the apical to the basal plasma membrane for any particular region of tegument. A variety of morphological modifications of the apical plasma membrane very similar to those which have been accepted as evidence of pinocytosis in other tissues were present. In vitro studies using horseradish peroxidase, ruthenium red, and lanthanum nitrate showed that all three tracers are taken up by the tegument into membrane-limited vesicles. Vesicles which contained ruthenium red occurred at the base of the tegument within a 5-min incubation period, and their contents appeared to be released into the underlying interstitial material by exocytosis.     

Lipid catabolism in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

All of the enzymes of the β-oxidation sequence have been demonstrated in the plerocercoids ofS. solidus. However, the plerocercoids could not oxidize exogenous [¹⁴C-U-]palmitate although labelled palmitate was readily taken up and encorporated into the neutral and phospholipid fractions. This suggests that despite the presence of all of the enzymes of β-oxidation, the pathway is not functional in S. solidus plerocercoids; possible roles of the β-oxidation enzymes in the metabolism of S. solidus are discussed.     

Sequential morphologic changes in adult Echinococcus granulosus during complement-mediated lysis In vitro

Herd R. P., Ko L., Weisbrode S. E. and Heath D. D. 1984. Sequential morphologic changes in adult Echinococcus granulosus during complement-mediated lysis in vitro. International Journal for Parasltology14:141–149. Sequential changes (5,10, 20, 30,40, 50 min, 1, 2, 3, 4, 5, 6, 7, 8, h) were observed by light, scanning and transmission electron microscopy after 38-day-old adult Echinococcus granulosus were exposed to 50% guinea pig serum in vitro. Early changes within 3 h included contraction of worms, fusion of microtriehes, vacuolization and vesiculization of the distal cytoplasm, followed by rupture of vesicles leading to erosion and loss of the distal cytoplasm. This was most marked in the terminal proglottid but ultimately there was complete erosion of the distal cytoplasm of all proglottids and the scolex. After 3 h there was loss of definition of organelles, apparent edema of the perinuclear cytoplasm and, in some instances, rupture of the circular muscle layer with extrusion of parenchyma. Adult tape-worms exposed to heat-inactivated complement showed none of these changes. Lysis and death of the parasite was attributed to osmotic changes subsequent to the formation of trans-membrane channels induced by complement-mediated attack of the tegument after activation of the alternate pathway by factors present in the cestode tegument.     

Structure and development of Austramphilina elongata Johnston, 1931 (Cestodaria: Amphilinidea)

The life cycle and structure of the larva of Austramphilina elongata using light-microscopy, scanning and transmission electron microscopy are described. Eggs are round and non-operculate. Larvae hatch in freshwater and penetrate through the cuticle of juvenile crayfish, Cherax destructor, and of freshwater shrimps, Paratya australiensis and Atya (= Atyoida) sp., shedding their ciliated epidermis. In the last two hosts, development to the infective stage does not occur. In crayfish, larvae grow and reach the infective stage. Turtles, Chelodina longicollis, become infected by eating infected crayfish. Larvae penetrate through the oesophageal wall of the turtle and migrate toward the coelom, where maturation occurs. The free-swimming larva has a syncytial epidermis which covers most of the body except for the posterior region bearing the hooks. It is loosely attached to a thin underlying tegument, which is connected to ‘insunk’ nucleated cell bodies. It forms a thick surface layer in the posterior region. There are three flame cells on each side of the body and two postero-lateral excretory pores. There are no lateral flames. The weir apparatus of the flame cell has the structure typical of parasitic platyhelminths. The smaller capillaries have a smooth surface, that of the terminal ducts is covered by numerous microvilli. Three types of penetration glands open anteriorly. There are five pairs of hooks; one median ‘normal’, two submedian halberd-shaped, and two lateral serrate. Hook are not lost, they are arranged around the gonopore of the adult. Frontal glands opening into the proboscis were found in the anterior part of the body in all stages examined. Infective stages in crayfish have developing reproductive organs and ducts. The tegument of the adult has many microvilli.     

Expression of gonadotropin subunits in roach (Rutilus rutilus, Cyprinidae) infected with plerocercoids of the tapeworm Ligula intestinalis (Cestoda)

Plerocercoids of the tapeworm Ligula intestinalis (Cestoda: Bothriocephalidea) have been reported to inhibit gametogenesis of their intermediate fish hosts. However, mechanistic studies are rare and the proximate cues leading to impaired reproduction still remain unknown. In the present study we investigated the effects of infection by L. intestinalis on reproductive parameters of roach (Rutilus rutilus, Cyprinidae), a common fish host of this parasite. Field studies on roach demonstrated that in both genders infection prevented gonad development. As revealed by quantitative PCR, infection was accompanied by essentially lower pituitary expression of follicle-stimulating hormone β-subunit (FSHβ) and luteinizing hormone β-subunit (LHβ) mRNA compared with uninfected roach, providing clear evidence for gonadotropin-insufficiency as the cause of arrested gametogenesis. Under controlled laboratory conditions infected roach showed lower mRNA levels of FSHβ but not of LHβ, despite histology revealing similar gonad stages as in uninfected conspecifics. These findings indicate the involvement of FSH rather than LH in mediating effects of infection early during gonad development in roach. Moreover, the impact of L. intestinalis on reproductive parameters of roach appeared to be independent of the parasite burden. Together, these data provide valuable information on the role of FSH and LH as mediators of parasite-induced sterilization in a vertebrate and implicate the selective inhibition of host reproduction by L. intestinalis as a natural source of endocrine disruption in fish.     

The in vitro culture and tegumental dynamics of the plerocercoid of Ligula intestinalis (Cestoda: Pseudophyllidea)

Hoole D. and Arme C. 1985. The in vitro culture and tegumental dynamics of the plerocercoid of Ligula intestinalis (Cestoda: Pseudophyllidea). International Journal for Parasitology14: 609–615. An in vitro system for maintaining Ligula has been developed and used in an autoradiographic study on the tegumental dynamics. Parasite tissue was cultured in teleost Ringer, Eagle's medium, M199 medium and modified Leibovitz' medium. Small membrane-bound bodies within the microthrix border increased in number in all media, and abnormal mitochondria were found in the distal tegument in teleost Ringer, M199 and Eagle's. Autophagy occurred after 24 h culture in teleost Ringer. Ultrastructural and physiological (¹⁴C-D-glucose uptake) evidence suggests that parasite tissue maintained in modified Leibovitz' medium remains viable for at least 24 h. The turnover rate of the surface components of L. intestinalis in this medium is approx. 12–24 h.     

Exposed proteins of the Schistosoma japonicum tegument

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.     

Fasciola gigantica: Anthelmintic effect of the aqueous extract of Artocarpus lakoocha

The effect of the crude extract of Artocarpus lakoocha (70% composition is 2,4,3′,5′- tetrahydroxystilbene -THS) on adult Fasciola gigantica was evaluated after incubating the parasites in M-199 medium containing 250, 500, 750 and 1000 μg/ml of the crude extract, or triclabendazole (TCZ) at the concentrations of 80 and 175 μg/ml as the positive control, for 3, 6, 12 and 24 h, using relative motility (RM) assay and observation by scanning electron microscope (SEM). Decreased contraction and motility were first observed after 3 h incubation with TCZ at the concentration 80 and 175 μg/ml. TCZ markedly reduced the parasite’s motility at the concentration of 175 μg/ml at 6 h, and killed the worms after 12 h exposure. The crude extract of A. lakoocha at all concentrations reduced the parasite’s motility similar to TCZ at 3 h incubation. In 250 and 500 μg/ml of the crude extract, the values were decreased from 3 to 12 h, then they were stable between 12 and 24 h and reduced to the level approximately 30-40% of the control. At 750 and 1000 μg/ml concentrations the crude extract rapidly reduced the RM values from the start to 12 h and killed the parasites between 12 and 24 h incubation. The crude extract also inhibited the larval migration by 75% and 100% at the concentrations of 250-500 and 750-1000 μg/ml, respectively. TCZ and the crude extract caused sequentially changes in the tegument including swelling, followed by blebbings that later ruptured, leading to the erosion and desquamation of the tegument syncytium. As the result, lesion was formed which exposed the basal lamina. The damage appeared more severe on the dorsal than the ventral surface, and earlier on the anterior part and lateral margins when compared to the posterior part. The severity and rapidity of the damages were enhanced with increasing concentration of the crude extract. Hence, the crude extract of A. lakoocha, may exert its fasciolicidal effect against adult F. gigantica by initially causing the tegumental damage.     

The non-native solitary ascidian Ciona intestinalis (L.) depresses species richness

Non-native ascidians are a dominant feature of many sessile marine communities throughout the world and may have negative effects on species diversity. We tested effects of the non-native Ciona intestinalis on the sessile invertebrate community in San Francisco Bay, where it occurs in dense aggregations. In particular, we compared species richness between PVC panels from which C. intestinalis were experimentally removed to panels with naturally dense C. intestinalis growth, using fouling panels of four sizes (between 49 cm² and 1177 cm²) to measure the effect of C. intestinalis recruitment on species-area relationships. We initially deployed 120 fouling panels (30 of each size) at a site known to have dense populations of C. intestinalis, assigning these to three different treatments: (1) Experimental removal, whereby new recruits of C. intestinalis were removed on a weekly basis, pulling panels out of the water for a short time period to do so; (2) Manipulated control, whereby panels were removed from the water each week (as in the experimental removal) but without C. intestinalis removal; and (3) Unmanipulated control, which remained in the water throughout the experiment. After 4 months, all of the panels were collected and analyzed to estimate species richness and relative abundance (percent cover) of sessile invertebrates and of C. intestinalis. Across all panels, species richness was negatively correlated with C. intestinalis abundance. The removal of C. intestinalis produced communities with significantly higher species richness than the controls. The overall species composition of treated and control panels was also distinctly different, with many species occurring more often in the absence of C. intestinalis, while others occurred more often on C. intestinalis-dominated panels. These data suggest that C. intestinalis both depress local species diversity and alter community assembly processes to fundamentally change sessile community composition.     

The genetic structure of the A mating-type locus of Lentinula edodes

The Shiitake mushroom, Lentinula edodes (Berk.) Pegler is a tetrapolar basidiomycete with two unlinked mating-type loci, commonly called the A and B loci. Identifying the mating-types in shiitake is important for enhancing the breeding and cultivation of this economically-important edible mushroom. Here, we identified the A mating-type locus from the first draft genome sequence of L. edodes and characterized multiple alleles from different monokaryotic strains. Two intron-length polymorphism markers were developed to facilitate rapid molecular determination of A mating-type. L. edodes sequences were compared with those of known tetrapolar and bipolar basidiomycete species. The A mating-type genes are conserved at the homeodomain region across the order Agaricales. However, we observed unique genomic organization of the locus in L. edodes which exhibits atypical gene order and multiple repetitive elements around its A locus. To our knowledge, this is the first known exception among Homobasidiomycetes, in which the mitochondrial intermediate peptidase (mip) gene is not closely linked to A locus.     

The ultrastructure of the tegument and the digestive caeca of in vitro cultured metacercariae of Fasciola hepatica

Davies C. 1978. The ultrastructure of the tegument and digestive caeca of in vitro cultured metacercariae of Fasciola hepatica. International Journal for Parasitology8: 197–206. The ultrastructure of the tegument and digestive caeca of metacercariae of Fasciola hepatica grown in vitro in two different media is described and compared with the development of these two systems during maturation in vivo. Although the tegument of metacercariae grown in Medium RC showed no development, that of flukes cultured in Medium CS began to produce T-1 and T-2 granules typical of the liver phase of development in vivo. The gastrodermal cells showed some degree of conversion to an adult-like morphology in vitro with the production of typical secretory granules, a limited amount of orientation of the GER and the development of junctional complexes with adjacent parenchyma cells—this was particularly evident in flukes from Medium CS. The growth achieved in each of the culture media is correlated to the degree of development of the tegument and the digestive caeca.     

Involvement of lectin pathway activation in the complement killing of Giardia intestinalis

Giardia intestinalis (syn. G. lamblia, G. duodenalis) is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. In humans, the clinical effects of Giardia infection range from the asymptomatic carrier state to a severe malabsorption syndrome possibly due to different virulence of the Giardia strain, the number of cysts ingested, the age of the host, and the state of the host immune system at the time of infection.The question about how G. intestinalis is controlled by the organism remains unanswered. Here, we investigated the role of the complement system and in particular, the lectin pathway during Giardia infections. We present the first evidence that G. intestinalis activate the complement lectin pathway and in doing so participate in eradication of the parasite. We detected rapid binding of mannan-binding lectin, H-ficolin and L-ficolin to the surface of G. intestinalis trophozoites and normal human serum depleted of these molecules failed to kill the parasites. Our finding provides insight into the role of lectin pathway in the control of G. intestinalis and about the nature of surface components of parasite.     

Cotugnia digonopora: Transport of leucine

The uptake of leucine through the tegument of Cotugnia digonopora, a cestode found in the fowl intestine, occurs by a process of active transport. The K_t of transport is 0.87 mM and the V_max is 0.223 μmol/min/g. Uptake of the amino acid is competitively inhibited by valine (K_t = 1.30 mM). Potassium cyanide and 2,4-dinitrophenol do not completely block the entry of leucine into the parasite.     

Effects of infection of EGFP-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for humoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage 1 h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians.     

Hymenolepis microstoma: Ultrastructural localization of adenyl cyclase in the tegument

Peroxidase and phosphatase activities have been reported to be localized in the tegument of adult hymenolepidid tapeworms. In order to localize adenyl cyclase (EC 4.6.1.1) activity in the tegument of mature and gravid sections of Hymenolepis microstoma, 5-adenyl-imidodiphosphate was used as a substrate, and lead was used as a capturing agent. Results indicate that adenyl cyclase activity is present in the crypts between the microtriches of the mature sections and that activity is absent from the gravid sections.     

Fasciola gigantica: Histology of the digestive tract and the expression of cathepsin L

The digestive tract of Fasciola gigantica is composed of the oral sucker, buccal tube, pharynx, esophagus, and caecum. The tegumental-type epithelium lines the first four parts of the digestive tract while the caecal-type epithelium lines the remaining parts from the caecal bifurcation. The caecal-epithelial cells are classified into 3 types according to their staining properties and ultrastructural characteristics, as related to the amount of food contents in the caecal lumen. All caecal-type epithelial cells synthesize and secrete cathepsin L, a major group of enzymes in the digestive tract, as detected by in situ hybridization and immunolocalization. Moreover, the secreted cathepsin L is also adsorbed on the outer surface of the tegument and the glycocalyx coating of the surface of the tegument, whereas the tegumental cells and tegumental syncytium covering the parasite’s body and lining the proximal part of the digestive tract exhibit no in situ hybridization signal and immunostaining for cathepsin L.     

Ultrastructural aspects of early immune damage to Taenia crassiceps metacestodes

Siebert, A. E. Jr., Good A. H. and Simmons J. E. 1978. Ultrastructural aspects of early immune damage to Taenia crassiceps metacestodes. International Journal for Parasitology8: 45–53. Changes in the ultrastructure of the tegument and subtegumental cells of intraperitoneally implanted Taenia crassiceps metacestodes were studied by transmission electron microscopy over a 4-week period. Death of metacestodes without involvement of host inflammatory cells is indicated initially by vacuolization of the larval tegument followed by loss of the tegument and subsequent death of the larvae. Changes in the tegument involve loss of the glycocalyx, reduction in the numbers of mitochondria and microtriches present, and loss of secretory capacity. Subtegumental cells show an accumulation of secretory vacuoles and marked disruption of nuclear morphology. Tegument damage is attributed to a complement-mediated lysis of the outer tegument membrane and death of the larvae probably results from loss of tegument function.