Is species identification of Echinostoma revolutum using mitochondrial DNA barcoding feasible with high-resolution melting analysis?

The taxonomic evaluation of Echinostoma species is controversial. Echinostoma species are recognized as complex, leading to problems associated with accurate identification of these species. The aim of this study was to test the feasibility of using DNA barcoding of cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) conjugated with high-resolution melting (HRM) analysis to identify Echinostoma revolutum. HRM using COI and ND1 was unable to differentiate between species in the "revolutum complex" but did distinguish between two isolates of 37-collar-spined echinostome species, including E. revolutum (Asian lineage) and Echinostoma sp. A from different genera, e.g., Hypoderaeum conoideum, Haplorchoides mehrai, Fasciola gigantica, and Thapariella anastomusa, based on the Tm values derived from HRM analysis. Through phylogenetic analysis, a new clade of the cryptic species known as Echinostoma sp. A was identified. In addition, we found that the E. revolutum clade of ND1 phylogeny obtained from the Thailand strain was from a different lineage than the Eurasian lineage. These findings reveal the complexity of the clade, which is composed of 37-collar-spined echinostome species found in Southeast Asia. Taken together, the systematic aspects of the complex revolutum group are in need of extensive investigation by integrating morphological, biological, and molecular features in order to clarify them, particularly in Southeast Asia.

Definitive host influences the proteomic profile of excretory/secretory products of the trematode Echinostoma caproni.

BACKGROUND: Echinostoma caproni is an intestinal trematode extensively used as experimental model for the study of factors that determine the course of intestinal helminth infections, since this markedly depends on the host species. Although the host-dependent mechanisms for either chronic establishment or early parasite rejection have been broadly studied, little is known regarding the parasite response against different host environments. METHODS: To identify host-dependent differentially expressed proteins, a comparative proteomic analysis of the excretory/secretory products released from E. caproni adults, isolated from hosts displaying different compatibility with this trematode, was performed. RESULTS: A total of 19 differential protein spots were identified (14 overexpressed in mice and 5 overexpressed in rats). The establishment of chronic infections in mice is mainly associated with the overexpression by adult worms of antioxidant and detoxifying enzymes (e.g. glutathione S-transferase, hydroxyacylglutathione hydrolase, thiopurine S-transferase, etc.) and metabolic enzymes like enolase, leucine aminopeptidase or malate dehydrogenase. However, the overexpression of cathepsin L and the structural protein actin observed in worms isolated from rats seems not to be effective for the colonization of the intestinal mucosa of this host. CONCLUSIONS: The observed differences suggest that protein expression and/or release is modulated by the local environment generated inside the host and provide useful insights in regards to the resistance mechanisms developed by parasites to ensure their long-term survival.

Identification of proteins in excretory/secretory extracts of Echinostoma friedi (Trematoda) from chronic and acute infections.

In the present study, we describe the investigation of Echinostoma friedi excretory/secretory products using a proteomic approach combined with the use of heterologous antibodies. We have identified 18 protein spots corresponding to ten proteins, including cytoskeletal proteins like actin, tropomyosin, and paramyosin; glycolytic enzymes like enolase, glyceraldehyde 3P dehydrogenase, and aldolase; detoxifying enzymes like GSTs; and stress proteins like heat shock protein (Hsp) 70. Among these proteins, both actin and, to a lesser extent, Hsp70, exhibited differential expression patterns between chronic and acute infections in the Echinostoma-rodent model, suggesting that these proteins may play a role in the survival within the host.

Effects of hypotonicity on amino acid release in adult Echinostoma caproni as determined by thin layer chromatography.

Thin layer chromatography (TLC) was used to analyze the amino acids in worm incubates isotonic and hypotonic to the intestinal habitat of adult Echinostoma caproni and to analyze the free pool amino acids of these trematodes after incubation. Qualitative analysis revealed the presence of histidine, lysine, alanine, and proline in all samples of incubate and worm tissue. Quantification of histidine and lysine by TLC with densitometry gave mean concentrations of 24.1 micro g histidine/g worm per ml incubate in Locke's solution and 195.0 micro g lysine/g worm per ml incubate in deioinized (DI) water. Quantification of histidine and lysine in the worm tissue gave mean weight percents of 0.0587 and 0.0263, respectively, in worms incubated in Locke's solution and 0.0229 and 0.0163, respectively, for worms incubated in DI water. Our findings suggest that E. caproni adults may leak amino acids for osmoregulation in hypotonic environments.

Neutral lipids in cercariae, encysted metacercariae, and rediae of Echinostoma caproni.

High performance thin-layer chromatography (HPTLC) was used to analyse the neutral lipids in the rediae, cercariae, and encysted metacercariae of Echinostoma caproni from Biomphalaria glabrata snails. Visual observations of the chromatograms showed that the most abundant lipid fraction in all stages was free sterol. Quantification of the free sterol revealed mean weights of 2.7 +/- 0.64 ng per redia, 0.53 +/- 0.023 ng per cercaria, and 0.081 +/- 0.0098 ng per encysted metacercaria. Oil Red O staining of the larval stages confirmed the presence of lipids within the rediae and cercariae but did not show lipids in the encysted metacercariae. The dimunition in neutral lipids from the cercarial to the encysted metacercarial stage does not support a previous observation that fat increases in successive phases of the digenean life cycle.

Effects of diet on the lipid composition of Echinostoma caproni (Trematoda) in ICR mice.

High-performance thin-layer chromatography (HPTLC) was used to determine neutral lipids and phospholipids in the intestinal trematode Echinostoma caproni from experimentally infected ICR mice fed a high-fat diet (hen's egg yolk) as compared with worms from mice fed a standard laboratory diet. Worms were removed from the hosts at 2, 3, and 4 weeks postinfection (p.i.). Analysis by TLC-densitometry showed significantly greater amounts of triacylglycerols and free sterols at 2, 3, and 4 weeks p.i. in worms from mice on the high-fat diet as compared with worms from mice on the standard laboratory diet. Significantly greater amounts of phosphatidylcholine and phosphatidylethanolamine were found in worms from mice on the high-fat diet as compared with worms from those on the standard diet at 2 weeks p.i. but not at 3 and 4 weeks p.i. The results of this study suggest that the host diet influences the lipid content of E. caproni adults.

The expulsion of Echinostoma trivolvis (Trematoda) from ICR mice: extension/retraction mechanisms and ultrastructure of the collar spines.

Ultrastructural observations of collar spines and surrounding tissues associated with extension/retraction mechanisms of the spines were made on Echinostoma trivolvis recovered from ICR mice or golden hamsters. The spine consisted mainly of a homogeneous, crystalline structure of moderate to heavy electron density. The spine was surrounded by an outer layer of tegument-like material. Interstitial material was located between this outer layer and the surrounding tegument. Muscle bundles associated with spine movements were anchored by hemidesmosomes to interstitial material. Contraction of these muscles caused an invagination of the tegument surrounding a spine, resulting in spinal protrusion. Relaxation of these muscles resulted in spinal retraction. An immunocytochemistry technique using colloidal gold confirmed the presence of actin in the collar spines. Most of the collar spines of excysted metacercariae and juveniles maintained in a defined medium supplemented with fresh hamster serum were extended, whereas worms maintained in mouse serum mainly showed retracted spines. Apparently, factors in mouse serum are involved in spinal retraction.

Fatty acid composition of Echinostoma trivolvis (Trematoda) rediae and adults and of the digestive gland-gonad complex of Helisoma trivolvis (Gastropoda) infected with the intramolluscan stages of this echinostome.

Gas-liquid chromatographic studies were done to determine the fatty acid composition of the digestive gland-gonad (DGG) complex of Helisoma trivolvis snails infected with the intramolluscan stages of Echinostoma trivolvis, of rediae freed from the DGG, of uninfected DGG, and of 41-day-old adult worms grown in golden hamsters. The DGG of infected snails showed significantly higher levels of stearic acid (18:0), hexatrienoic acid (16:3n-4), and docosahexanoic acid (22:6n-3) than that of uninfected snails. However, the DGG of uninfected snails showed significantly higher levels of 20:2 non-methylene-interrupted diene (NMID) and adrenic acid (22:4n-6) than that of infected snails. The profiles of other fatty acids were remarkably similar in both infected and uninfected snails. Adult worms showed significantly higher amounts of numerous saturated fatty acids and dienes as compared with the rediae. However, the rediae showed significantly higher amounts of certain monoenes and trienes as compared with the adults. Fatty acid differences between rediae and adults probably reflect differences in either the available lipid pools in the immediate host sites or the metabolic activity of each stage of this echinostome.