Excretory-secretory proteome of larval Schistosoma mansoni and Echinostoma caproni, two parasites of Biomphalaria glabrata

Schistosoma mansoni and Echinostoma caproni are two trematode species that use different strategies (mimicry and immunosuppression, respectively) to interfere with the snail innate immune system. Parasites excretory-secretory (ES) products have been shown to play a key role in these host-parasite immune interactions. However, they remain largely uncharacterized in larval trematodes. We developed a global proteomic approach to characterize the ES proteome of S. mansoni and E. caproni primary sporocysts. In ES products of both parasites, we found proteins involved in reactive oxygen species scavenging, glycolysis, signalling or calcium binding (superoxide dismutase Cu/Zn; glutathione S-transferase; aldo-keto-reductase; triose-phosphate isomerase; glyceraldehyde-3-phosphate dehydrogenase; aldolase, enolase, MICAL-like, calreticulin). According to their predicted functions, we propose a model in which these proteins (i) are involved in antioxidant activity, (ii) prevent hemocyte encapsulation process or (iii) favor invasion and migration of sporocysts in host tissues. These results suggest that S. mansoni and E. caproni sporocysts develope a strong immune protection during the first hours of infection giving them enough time to build up a long lasting immune evasion strategy relying on molecular mimicry or immunosuppression, respectively.     

Cellular response to Echinostoma caproni infection in Biomphalaria glabrata strains selected for susceptibility/resistance.

The Biomphalaria glabrata/Schistosoma mansoni system represented the only model available so far, for investigating snail resistance to parasites. A new host parasite model has been recently provided by selection of B. glabrata strains that are genetically resistant and susceptible to Echinostoma caproni. As a first approach in investigating resistance mechanisms in this model, we compared the hemocytic response and its effect on E. caproni development in a susceptible and a resistant strains. Histological analysis revealed that resistance does not prevent penetration or migration through the snail tissues. However, all mother sporocysts (MS) settled in the ventricle and the aorta of resistant snails were encapsulated and killed by 4 days post-exposure. MS abnormally settled in the pericardial cavity were not encapsulated and could survive for 5 days. Regardless of their level of encapsulation, all live MS observed during the first days of infection in resistant snails showed abnormal development and degeneration, raising the question of the possible role of humoral factors in the resistance to E. caproni. Finally, we observed an increased number of adherent hemocytes in the resistant as compared with the susceptible snails after parasite exposure. This different effect on circulating hemocyte subpopulations may reflect the failure of E. caproni to interfere with the hemocytic response of resistant snails.     

Comparative ultrastructure of eggs in Echinostoma paraensei, E. caproni, and E. trivolvis (Trematoda: Echinostomatidae)

The 37-collar-spined echinostomes Echinostoma paraensei, E. caproni, and E. trivolvis are digeneans that live in the intestine of small mammals and birds. Comparative studies of the eggs of these species were done using light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The egg of E. caproni was the largest of the three species studied, whereas the egg of E. trivolvis was the smallest in both length and width. The SEM study showed differences in the aboperculum region of the eggs in all three species. The TEM study showed that the eggshell of all three species consisted of three layers, but no difference in eggshell structure was observed in any species. Received: 10 September 1999 / Accepted: 9 November 1999     

High-performance thin-layer chromatographic analysis of neutral lipids and phospholipids in Biomphalaria glabrata patently infected with Echinostoma caproni

This study examined the effects of larval trematode infection on the neutral lipid and phospholipid content of Biomphalaria glabrata patently infected with the daughter rediae of Echinostoma caproni. Uninfected snails were used as matched controls. As determined by qualitative high-performance silica gel thin-layer chromatography (HPTLC), the major neutral lipids present in the whole bodies and digestive gland–gonad complexes in both snail populations were free sterols, free fatty acids, and triacylglycerols, and the major polar lipids were phosphatidylcholine and phosphatidylethanolamine. Quantitative analysis by HPTLC with visible and UV scanning reflectance densitometry showed no significant differences in the concentrations of these lipids in whole bodies of infected snails vs the controls, but the concentration of triacylglycerols in the infected digestive gland–gonad complex was significantly less than that of the uninfected. No qualitative differences in neutral lipids and phospholipids in shell or plasma samples were found between infected vs uninfected snails.     

Comparative ultrastructure study of lamellar gastrodermal projections in Echinostoma paraensei, E. caproni, and E. trivolvis (Trematoda: Echinostomatidae)

A comparative ultrastructure study using transmission electron microscopy (TEM) of ultrathin sections, freeze-fracture replication, and scanning electron microscopy (SEM) was conducted on the gastrodermal lamellar microvilli in adults of Echinostoma paraensei, E. caproni, and E. trivolvis. Lamellar projections observed by SEM were mainly planiform and rhomboidal or paddle-shaped, and they were commonly seen in the three species. The distal margins of these lamellae were mainly smooth in E. paraensei, whereas filiform or digitiform extensions in the margins occurred occasionally in E. caproni and frequently in E. trivolvis. Freeze-fracture TEM elucidated significant interspecific differences in the numbers of intramembranous particles (IMP) per square micrometer; the numbers of IMP were significantly lower in E. trivolvis than in either E. caproni or E. paraensei. Thus, ultrastructural differences in the gastrodermal lamellae of adult echinostomes will be a taxonomically useful criterion for distinguishing species of Echinostoma. Received: 8 March 1999 / Accepted: 25 March 1999     

Effects of various larval digeneans on the calcium carbonate content of the shells of Helisoma trivolvis, Biomphalaria glabrata, and Physa sp.

The calcium carbonate concentrations in the shells of Helisoma trivolvis and Physa sp. naturally infected with larval trematodes and Biomphalaria glabrata experimentally infected with larval trematodes were analyzed quantitatively. The larval trematode-snail relationships studied were H. trivolvis infected with larval Echinostoma trivolvis and Physa sp. infected with various larval digeneans, and B. glabrata infected with Echinostoma caproni or Schistosoma mansoni. The calcium carbonate concentrations of the shells of infected snails and uninfected cohorts and of the water in which the snails were maintained were determined by ion exchange chromatography. No significant differences in the calcium carbonate concentrations of shells of infected versus uninfected snails were found. The shells of B. glabrata infected with E. caproni contained significantly less calcium carbonate than the shells of uninfected B. glabrata. The hypercalcification hypothesis, i.e., larval trematodes induce an increase in the calcium concentrations in the shells of their snail hosts, was not upheld in any of the snail-larval digenean systems studied herein.     

Effects of Echinostoma caproni miracidia dose on the neutral and polar lipids of Biomphalaria glabrata as determined by high-performance thin-layer chromatography

The effects of a 5 versus 25 miracidia exposure of Echinostoma caproni on the lipid composition of Biomphalaria glabrata was studied using high performance thin layer chromatography (HPTLC)-densitometry. A 50 miracidia dose was not used because such a high level of exposure caused severe snail mortality by 3 weeks post-exposure (PE). Lipids were determined in the digestive-gland gonad complex (DGG) of the exposed snails and in the uninfected matched controls at 2 and 4 weeks PE. Extraction of lipids from DGGs was carried out by the Folch method with chloroform-methanol (2:1), and extracts were analyzed on Analtech HPTLC-HLF pre-adsorbent silica gel plates with measurement of separated bands using a CAMAG Scanner 3. For neutral lipids the mobile phase was petroleum ether-diethyl ether-glacial acetic acid (80:20:1) and the detection reagent was 5% ethanolic phosphoric acid, and for polar lipids chloroform-methanol-deionized water (65:25:4) mobile phase and 10% cupric sulfate in 8% phosphoric acid detection reagent were used. No significant differences in the concentrations of free sterols, free fatty acids, triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine were seen at 2 weeks PE in any of the groups. At 4 weeks PE, the free fatty acid concentration increased significantly in the snails exposed to 25 miracidia compared to that of the 5 miracidia/snail group or the controls. Elevation of the free fatty acid fraction in the high dose snail group suggested that some changes occurred in the lipid metabolism of the snails in that group as a function of miracidia dose.     

Effects of Echinostoma trivolvis (trematoda) infection on metallic ions in the host snail Helisoma trivolvis (gastropoda)

Flame and graphite-furnace atomic absorption spectrometry and inductively coupled plasma atomic emission spectrometry were used to study the metallic ions in the digestive gland-gonad complex (DGG) of Helisoma trivolvis snails infected with the daughter rediae of Echinostoma trivolvis and in uninfected DGG. Seven metals were found to be present in infected and uninfected DGG at concentrations above the detection limits of the analytical methods. Of these, sodium was present in significantly higher amounts (Student’s t-test, confidence level of 95%) in the infected versus uninfected DGG; magnesium and manganese occurred in significantly lower amounts in the infected DGG. Our results were compared with those from a Bulgarian study in which neutron activation analysis was used to determine elements in Lymnaea stagnalis snails infected with the intramolluscan stages of the 37-collar-spined echinostome E. revolutum. The Bulgarian study also reported a significant elevation of sodium but reduction of zinc in the hepatopancreas (i.e., digestive gland) of infected snails. Other differences between the two studies are discussed.     

Maintenance of the life cycle of Echinostoma trivolvis (Trematoda) in dexamethasone-treated ICR mice and laboratory-raised Helisoma trivolvis (Gastropoda)

 Echinostoma trivolvis is a ubiquitous 37-collar-spined echinostome found in aquatic birds and mammals and in the planorbid snail Helisoma trivolvis. This echinostome has not been cycled continously in the laboratory. The present report provides details on the continuous life cycle of E. trivolvis in dexamethasone-treated ICR mice and laboratory-raised H. trivolvis snails. Previous attempts to obtain patent adult of E. trivolvis in mice hosts failed because of worm rejection within 2 weeks of infection. ICR mice infected with encysted metacercariae and injected with 2 mg/kg dexamethasone daily for 28 days yielded gravid worms that produced 250–500 eggs/worm at 21 and 28 days postinfection (p.i.). Miracidia derived from these eggs or eggs containing fully developed miracidia were capable of infecting 3- to 5-mm shell-diameter, laboratory-reared H. trivolvis snails. These snails released cercariae by 35 days p.i. Cercariae encysted in the kidney-pericardium of the snails. Encysted metacercariae could be excysted in vitro in an alkaline trypsin-bile salts medium or in vivo when fed to domestic chicks. Received: 13 May 1996 / Accepted: 25 June 1996     

Rapid expulsion of the intestinal trematodes Echinostoma trivolvis and E. caproni from C3H/HeN mice after infection with Nippostrongylus brasiliensis

 The number of goblet cells in the small intestines of C3H/HeN mice increased rapidly following their infection with about 500 third-stage larvae (L₃) of the intestinal nematode Nippostrongylus brasiliensis. The number of goblet cells reached its peak on day 9 postinfection (p.i.). Worm burdens in the hosts’ small intestines were determined following a challenge infection with encysted metacercariae of the intestinal trematodes Echinostoma trivolvis or E. caproni on days 8 and 16 after primary infections with N. brasiliensis. All metacercariae of E. trivolvis or E. caproni used to challenge the hosts on day 8 p.i. were expelled. Considerable numbers of E. trivolvis (48.6%) and E. caproni (67.1%) remained in the intestines of hosts challenged with these echinostomes on day 16 p.i. All the controls used for E. trivolvis and E. caproni infections without primary infections with N. brasiliensis showed recovery rates greater than 70%. An enzyme-linked immunosorbent assay (ELISA) showed that the IgM titer rose remarkably and plateaued on day 11 p.i. No marked rise in the IgG or IgA titer occurred during the experiment. These results indicate that mucins increased by hyperplastic goblet cells associated with primary infections with N. brasiliensis are responsible for a rapid expulsion of the worms of the challenge infection with E. trivolvis or E. caproni from the mouse host. Received: 10 January 1996 / Accepted: 10 April 1996     

Light and scanning electron microscopic observations of the eggs,daughter rediae,cercariae, and encysted metacercariae ofEchinostoma trivolvis andE. caproni

Light microscopy (LM) and scanning electron microscopy (SEM) were used to determine interspecific differences between various larval stages of 2 allopatric species of 37-collar-spined echinostomes,Echinostoma caproni andE. trivolvis. The abopercular and embryo diameters of the eggs ofE. trivolvis were significantly different from those ofE. caproni. The abopercular knobs ofE. caproni andE. trivolvis have shallow and deep infoldings in the eggshells, respectively. Interspecific differences were noted in the tegument, tegumentary papillae, and spherical bodies around the mouth and in the papilliform process of the two rediae. The cercaria ofE. caproni has one ventral tail fin-fold, whereas that ofE. trivolvis has two; both species have two dorsal tail fin-folds. The outer metacercarial cyst diameter ofE. caproni was significantly greater than that ofE. trivolvis. E. caproni has a smoother outer cyst wall than doesE. trivolvis.     

Direct and indirect antagonism between Paryphostomum segregatum and Echinostoma paraensei in the snail Biomphalaria glabrata

Summary Antagonism between the Brazilian parasites Paryphostomum segregatum Dietz and Echinostoma paraensei Lie and Basch was studied in the snail Biomphalaria glabrata (Say). Snails harbouring E. paraensei were easily super-infected with P. segregatum. Developing P. segregatum rediae killed and ate E. paraensei larvae, eventually clearing the snails of the second parasite except for metacercariae formed in the viscera. The indirect antagonism exerted by E. paraensei upon the invading P. segregatum larvae delayed their development. Double infections were readily obtained by simultaneous exposure of the snails to both parasites, but E. paraensei larvae developed in the ventricle for a limited period only. When E. paraensei were superimposed upon P. segregatum, double infections developed only in snails whose second exposure was within 8 days and E. paraensei larvae then developed in the heart for a limited period only.

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Zusammenfassung Der Antagonismus zwischen der brasilianischen Parasitenart Paryphostomum segregatum Dietz und Echinostoma paraensei Lie und Basch wurde in der Schnecke Biomphalaria glabrata (Say) studiert. Schnecken, die E. paraensei beherbergen, lassen sich leicht mit P. segregatum superinfizieren. In der Entwicklung begriffene Redien von P. segregatum töten und fressen E. paraensei-Larven, wobei die Schnecke unter Umständen von dem zweiten Parasiten völlig befreit wurde, es seien denn Metacercarien vorhanden, die sich in den Eingeweiden ausgebildet haben. Der indirekte Antagonismus, durch E. paraensei verursacht, wirkt sich auf die P. segregatum-Larven aus und stört ihre Entwicklung. Doppelinfektionen werden erzielt bei gleichzeitiger Exposition der Schnecken mit beiden Parasitenarten, aber E. paraensei-Larven entwickeln sich im Ventrikel nur eine begrenzte Zeitspanne. Wenn E. paraensei superinfiziert wird von P. segregatum, dann entwickelt sich eine Doppelinfektion nur in Schnecken, bei denen die zweite Exposition innerhalb von 8 Tagen erfolgte und E. paraensei-Larven sich dann nur im Herzen für eine begrenzte Zeitspanne entwickeln.

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Supported by the University of California International Center for Medical Research and Training (UC ICMRT, Hooper Foundation, San Francisco School of Medicine) with Research Grants TW 00144 from the Office of International Research and AI 07054 from the Institute of Allergy and Infectious Diseases, NIH, U.S. Public Health Service; and by Rockefeller Foundation grant GA-MNS-6654.     

Antagonism between two species of echinostomes (Paryphostomum segregatum and Echinostoma lindoense) in the snail Biomphalaria glabrata

Summary The snail Biomphalaria glabrata (Say) was infected with two species of echinostomes of Brazilian origin: Paryphostomum segregatum Dietz and Echinostoma lindoense Sandground and Bonne. Snails harboring E. lindoense were easily superinfected with P. segregatum. Developing P. segregatum rediae consumed E. lindoense larvae and eventually cleared the snails of this species except for metacercarial cysts. E. lindoense rediae were unable to consume P. segregatum larvae, but they exerted indirect antagonism resulting in delayed development of P. segregatum. Snails simultaneously exposed to the two species of miracidia developed double infections, but E. lindoense larvae were ultimately destroyed in the snails. Snails infected with P. segregatum were difficult to superinfect with E. lindoense. Double infections developed only when the exposure interval was 10 days or less. P. segregatum rediae consumed E. lindoense larvae in these double infections.

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Zusammenfassung Die Schneckenart Biomphalaria glabrata (Say) wurde mit zwei Echinostomatiden-Arten aus Brasilien: Paryphostomum segregatum Dietz und Echinostoma lindoense Sandground und Bonne infiziert. Schnecken, die schon E. lindoense beherbergten, konnten leicht mit P. segregatum superinfiziert werden. Die heranwachsende Redie von P. segregatum verzehrt die Larven von E. lindoense und befreit die Schnecke von dieser Trematoden-Art, mit Ausnahme der Metacercarien-Cysten. Die Redien von E. lindoense hingegen sind nicht in der Lage, die Entwicklungsstadien von P. segregatum zu verzehren, aber sie üben einen indirekten Antagonismus aus, der sich in einer verzögerten Entwicklung von P. segregatum zeigt. Werden Schnecken gleichzeitig Mirazidien beider Trematoden-Arten ausgesetzt, dann gehen beide Infektionen an; aber die Larven von E. lindoense werden letztlich in den Schnecken vernichtet. Schnecken, die schon mit P. segregatum infiziert sind, lassen sich nur schwer mit E. lindoense superinfizieren. Beide Infektionen entwickeln sich nur, wenn der Zeitraum zwischen den beiden Infektionen 10 Tage oder weniger beträgt. Auch bei diesen Doppelinfektionen verzehren die Redien von P. segregatum die Larvenstadien von E. lindoense.

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Supported by the University of California International Center for Medical Research and Training (UC ICMRT, Hooper Foundation, San Francisco School of Medicine) with Research Grants TW 00144 from the Office of International Research, and AI 07054 from the Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service.

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Medical student, University of California School of Medicine, San Francisco.     

Glutamate as a putative neurotransmitter in the buccal central pattern generator of Helisoma trivolvis.

1. The effects of L-glutamate superfusion over identified neurons within the buccal ganglia of Helisoma trivolvis were examined. Glutamate mirrored the effect of activity of subunit 2 (S2) of the tripartite feeding central pattern generator (CPG) on S2 postsynaptic neurons. Neurons that are excited by S2 are depolarized by glutamate, whereas neurons that are inhibited by S2 are hyperpolarized by glutamate. Glutamate also stimulated rhythmic S2 activity. 2. Different glutamate agonists could mimic specific components of the effects of glutamate on buccal neurons. Kainate produced depolarizations in neurons that receive S2 excitatory postsynaptic potentials (EPSPs) and activated rhythmic S2 activity. Quisqualate produced hyperpolarizations in neurons that receive S2 inhibitory postsynaptic potentials (IPSPs). 3. The non-N-methyl-D-aspartate glutamate receptor antagonist cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked the effects of S2 EPSPs and depolarizations produced by application of glutamate and kainate, but was ineffective in blocking S2 IPSPs or hyperpolarizations produced by application of glutamate and quisqualate. 4. These data support the hypothesis that glutamate is the transmitter of S2 of the feeding CPG in Helisoma, acting at CNQX-sensitive kainate-like receptors at excitatory synapses and CNQX-insensitive quisqualate-like receptors at inhibitory synapses.     

Differential expression of FREP genes in two strains of Biomphalaria glabrata following exposure to the digenetic trematodes Schistosoma mansoni and Echinostoma paraensei

Fibrinogen-related proteins (FREPs) are hypothesized to function in non-self-recognition in the snail Biomphalaria glabrata. To investigate this assumption, the expression of four members of the FREP gene family was studied using quantitative PCR at 0.5-16 days following exposure of M line and BS-90 strain B. glabrata to Echinostoma paraensei and Schistosoma mansoni. Both strains react to, but fail to eliminate E. paraensei. Only the BS-90 strain is immunologically resistant to S. mansoni. Both snail strains responded to E. paraensei with significantly elevated expression of FREP 2 and 4. Following exposure to S. mansoni, resistant BS-90 snails showed an increase in expression of FREP 2 and 4 (57-fold and 4.5-fold increase, respectively), susceptible M line snails did not display a FREP response. Expression of FREP 3 and 7 was not significantly elevated in any snail/trematode combination. These expression profiles support the hypothesis that some FREPs play a role in the anti-trematode responses in B. glabrata.     

Chemicotaxonomic study of the genus Echinostoma: comparison of a strain isolated in the Cameroon (E. sp.) and 2 African species (E. caproni and E. togoensis)

A comparative enzymatic study is carried out on adults of three species of the genus Echinostoma (E. caproni, E. togoensis, E. sp.). Amongst 23 enzymes tested by starch gel electrophoresis, Pgm and Pgi are characterised by different zymogrammes in each species. Agarose and polyacrylamide isoelectrophoresis focusing define isoenzyme genetic structures: Pgm is coded by three loci, Pgi is coded by one locus. Pgm polymorphism allows us to extend this work to other experimental or natural stocks.     

Effects of a diet deficient in vitamins A, D, and E on infectivity, growth, and development of Echinostoma trivolvis in domestic chicks

The effects of a diet deficient in fat-soluble vitamins on infectivity, growth, and development of Echinostoma trivolvis in domestic chicks were studied. The vitamin-deficient diet (experimental) contained 21% protein, but lacked vitamins A, D, and E. The control diet was isocaloric, contained more than 18% protein, and a normal vitamin complement. There were no significant differences in chick infectivity or worm recovery at 7, 10, or 14 days postinfection (p.i.) from hosts on either diet. Likewise, there were no significant differences in chick weights at 7, 10, or 14 days p.i. in hosts maintained on either diet. However, worm weights and body and organ dimensions were significantly greater at 10 and 14 days p.i. in worms from control versus experimental hosts; worms from experimental hosts never became ovigerous. Our results support Beaver's speculation that E. trivolvis develop poorly in avian hosts lacking fat-soluble vitamins. Received: 12 March 1999 / Accepted: 4 May 1999     

The effect of dexamethasone on the course ofEchinostoma caproni andE. trivolvis infections in the golden hamster (Mesocricetus auratus)

Golden hamsters (Mesocricetus auratus) were given intramuscular injections of dexamethasone and infected withEchinostoma caproni orE. trivolvis. All animals were necropsied on day 14 postinfection. Dexamethasone treatment at high doses resulted in increased parasite recovery. Decreased total white blood cell counts and decreased relative splenic weights were observed in corticosteroid-treated hamsters. Dexamethasone-treated animals also demonstrated significantly lower mean parasite dry weights forE. caproni. Specific serum IgG against the parasites was not detected in corticosteroid-treated hamsters on day 14 postinfection.