Sattrachai Prasopdee

Temperature dependence of Opisthorchis viverrini infection in first intermediate host snail, Bithynia siamensis goniomphalos

Determining of the success of a parasites infectiveness in its snail host clearly depends on environmental conditions. Temperature, one of the most influential factors impinging on metabolism of cold-blooded animals, is believed to be an important factor in parasitic infection in snails. In order to elucidate the influence of temperature, sex and size of snails on infectivity of Opisthorchis viverrini to its first intermediate host, Bithynia siamensis goniomphalos, 960 snails were divided into 2 groups by sex. Each group was subdivided by their size into small and medium sub-groups. Each snail was fed with embryonated uterine-eggs of O. viverrini at different temperatures (16-37°C, 3°C intervals). Dissections were carried out 1, 7, 14, 28 and 56 days thereafter and detection of O. viverrini infection was undertaken by PCR using specific primers. Infection was strongly temperature-dependent, as temperature increases of 1°C resulted in increased odds of infection 5.4% (P<0.01). A temperature of 34°C gave the highest rate of infection of 44.14%. We also found that the odds of infection in small sized snails was 39.8% higher relative to medium sized snails (P<0.05). Relative to day 1, the decrease in the odds of infection was detected when the day post infection was longer (P<0.01). Proportion of infection in female was not different to male significantly.

Coming out of the shell: building the molecular infrastructure for research on parasite-harbouring snails.

In Thailand and Laos alone, approximately 10 million people are infected with the liver fluke Opisthorchis viverrini [1]. Chronic infection with this parasite is considered the leading cause of cholangiocarcinoma (CCA, or bile-duct cancer) in large areas of Southeast Asia [2]. In these regions, CCA caused by O. viverrini is typically diagnosed 30–40 years after infection, with death occurring within 3–6 months post diagnosis [3]. O. viverrini is characterised by a three-host life cycle, with prosobranch snails of the genus Bithynia and cyprinid fishes acting as first and second intermediate hosts, respectively, while piscivorous mammals, including dogs, cats, and humans, act as definitive hosts [2]. Over the last two decades, much attention has been paid to studies on the epidemiology, developmental biology, and diagnosis of O. viverrini [4], while recent biotechnological advances are contributing large-scale explorations of the fundamental molecular biology of this liver fluke, with a view toward identifying key molecules essential for its development, reproduction, and survival, as well as dissecting the molecular pathways leading to the development of CCA [5]–[8]. These advances provide a solid foundation for the development of novel strategies to fight this devastating disease. However, long-term control of O. viverrini–induced cancer strictly relies on the development of integrated approaches, targeting the parasite as well as its intermediate hosts. Recently, an article in PLOS Neglected Tropical Diseases by Adema and colleagues [9] served to highlight the substantial gap in knowledge of aspects of the fundamental molecular biology of molluscs harbouring parasites, as well as the extraordinary opportunities that modern research toolkits, including microarray platforms, RNA interference, and high-throughput sequencing, offer for investigations of snail-parasite interactions [9]. Indeed, despite the massive expansion in the demand for and access to low-cost, high-throughput sequencing, large-scale genomic analyses of snails are limited to the draft genome sequence of the pulmonate snail intermediate host of Schistosoma blood flukes (Biomphalaria glabrata Genome Initiative at http://biology.unm.edu/biomphalaria-genome/index.html). Until now, there has been no genomic or transcriptomic information available for the prosobranch snail intermediate hosts of carcinogenic liver flukes, such as O. viverrini and Clonorchis sinensis. In order to provide the research community with a solid resource for molecular studies of these organisms, we generated the first reference transcriptome of Bithynia siamensis goniomphalos (Gastropoda, Bithyniidae), the intermediate host of O. viverrini in areas of northeast Thailand, Lao PDR, Cambodia, and South Vietnam, where the incidence of CCA is highest [cf. 10] (cf. Figure 1). A cDNA library from adult snails [11] was constructed, sequenced using RNA-seq (Illumina), and annotated using an established bioinformatic workflow [12]. Briefly, snails were collected from a natural body of water in Muang District, Khon Kaen Province, northeast Thailand (Figure 1); the taxonomic identity of the specimens was confirmed based on characteristic morphological features of the shells [13]; RNA was extracted from whole adult, parasite-free B. siamensis goniomphalos (n = 5), reverse-transcribed to cDNA, adaptor-ligated, and paired-end sequenced on a Genome Analyzer II (Illumina). Almost 50 million high-quality (Phred score >28) reads were generated; the assembly, produced using the Trinity software [14], yielded 167,029 contigs >200 bp in length, with a GC content of 44.4% (Table 1). Using sequence homology–based searches [12], approximately 40% of the assembled contigs could be annotated (cf. Table 1). A total of 32,026 contigs could be annotated with Gene Ontology terms (via Blast2GO; [15]), according to the categories “biological process,” “cellular component,” and “molecular function.” Approximately 77,000 non-overlapping protein sequences (of which ∼15,000 were full-length) could be inferred from the transcriptome of B. siamensis goniomphalos via BLASTx alignments with protein sequences available in public databases. Of the B. siamensis goniomphalos transcripts encoding proteins that could be mapped to orthologues in the Clusters of Orthologous Groups (COGs) database [16] (Table 1), the largest set was assigned to “general function prediction only” (16%), followed by “translation, ribosomal structure and biogenesis” (10%), “replication, recombination and repair” (7%), and “transcription” (7%) (Table 1). Approximately 54,000 protein-coding transcripts had orthologues in one of the 29 known biological pathways in the KEGG database (Table 1), including “regulation of actin cytoskeleton” (4%), “focal adhesion” (4%), and “spliceosome” (3.9%) (Table 1). Among the major protein classes included in the B. siamensis goniomphalos transcriptome were peptidases (n = 1,944; 4%), kinases (n = 4,757; 9%), phosphatases (n = 2,151; 4%), GTPases (n = 2,533; 5%), receptors (n = 6,243; 11%), transcription factors (n = 1,478; 3%), and channels and transporters (n = 1,287; 2%). Annotation information linked to each transcript characterised in the present study, including top BLASTx hit, GO classification, KEGG pathway mapping, and COG orthologues, is available from Table S1. In the absence of a reference genome for Bithynia spp., the annotation of the sequence data analysed herein was based on comparison with data available in public databases. The relatively small proportion of B. siamensis goniomphalos annotated protein sequences (∼60%) is likely to reflect the paucity of genomic sequence information available for prosobranch molluscan species in these databases [17]; supported by the availability of this dataset, as well as of the draft genome sequence of B. glabrata, future sequencing efforts will provide the depth of coverage required for the determination of the genome of Bithynia spp., which, in turn, will pave the way for comparative genomic studies of prosobranch and pulmonate snails harbouring parasite infections. This knowledge will be pivotal to improve our understanding of the biology of snail-borne parasites, and their “choice” of distinct snail species as their intermediate hosts. Figure 1 Distribution and prevalence of Opisthorchis viverrini, cholangiocarcinoma, and Bithynia spp. snails in Thailand. Table 1 Summary of the RNA-seq data for Bythinia siamensis goniomphalos prior to and following assembly, and bioinformatics annotation and analyses. Both raw and assembled sequence data generated in the present study are freely available from the Sequence Read Archive (SRA) and the Transcriptome Shotgun Assembly Sequence Database at NCBI (http://www.ncbi.nlm.nih.gov) under accession numbers SRR768418 and GAGS0000000, respectively. To our knowledge, this sequence data represents the first large-scale transcriptomic resource for a prosobranch mollusc intermediate host of a human platyhelminth and represents a major contribution to future fundamental explorations of the developmental biology of O. viverrini in Bithynia spp. snails, as well as the molecular interactions occurring at the snail-parasite interface. We suggest that the extensive sequence data generated in this study will be of great value in future studies aimed at exploring the changes in gene transcription occurring in Bithynia spp. upon infection with O. viverrini eggs and at different time points following infection. Besides yielding a general picture of the modified biology of trematode-infected snails, this data will set a basis for the identification of key genes, gene products, biological pathways, and/or bacterial and viral symbionts [cf. 9] involved in the cascade of molecular events leading to the development of the parasite through the stages of miracidium, sporocyst, redia, and cercaria. In turn, this advance will ultimately result in the development of novel targeted strategies to control snail-borne NTDs. New molecular technologies have a tremendous potential to aid our efforts in controlling snail-borne diseases. However, despite the unanimous acknowledgment that an increased understanding of the fundamental molecular biology of parasite-harbouring snails will provide us with a range of new tools to aid current efforts aimed at controlling snail-borne infections, the progressive but steady decline of malacology expertise and funding for snail vector–related research [cf. 9] poses a serious obstacle to the application of such technologies to snail-oriented NTD control. With this first step toward the establishment of a reference database for genetic research of prosobranch snail vectors of parasitic helminths, we hope to stimulate integrated, interdisciplinary research across malacology, helminthology, genomics, and bioinformatics, in the bid to fight snail-borne infections.

Proteomic profile of Bithynia siamensis goniomphalos snails upon infection with the carcinogenic liver fluke Opisthorchis viverrini

UNLABELLED The snail Bithynia siamensis goniomphalos acts as the first intermediate host for the human liver fluke Opisthorchis viverrini, the major cause of cholangiocarcinoma (CCA) in Northeast Thailand. The undisputed link between CCA and O. viverrini infection has precipitated efforts to understand the molecular basis of host-parasite interactions with a view to ultimately developing new control strategies to combat this carcinogenic infection. To date most effort has focused on the interactions between the parasite and its human host, and little is known about the molecular relationships between the liver fluke and its snail intermediate host. In the present study we analyse the protein expression changes in different tissues of B. siamensis goniomphalos induced by infection with larval O. viverrini using iTRAQ labelling technology. We show that O. viverrini infection downregulates the expression of oxidoreductases and catalytic enzymes, while stress-related and motor proteins are upregulated. The present work could serve as a basis for future studies on the proteins implicated in the susceptibility/resistance of B. siamensis goniomphalos to O. viverrini, as well as studies on other pulmonate snail intermediate hosts of various parasitic flukes that infect humans. BIOLOGICAL SIGNIFICANCE Despite the importance and high prevalence of opisthorchiasis in some regions of Southeast Asia and the direct relationship between infection by Opisthorchis viverrini and the incidence of cholangiocarcinoma, little is known of the modifications induced by this parasite in its snail intermediate hosts. This time-course study provides the first in-depth quantitative proteomic analysis of experimentally infected Bithynia siamensis goniomphalos. We show how motor and stress-related proteins are upregulated in infected snails, while O. viverrini infection downregulates the expression of oxidoreductases and catalytic enzymes. This work serves as a basis for the development of new strategies, focused on the invertebrate intermediate hosts, to control parasite transmission.

Effects of Bayluscide on Bithynia siamensis goniomphalos, the first intermediate host of the human liver fluke, Opisthorchis viverrini, in laboratory and field trials

The molluscicidal effects of Bayluscide (niclosamide) were investigated on Bithynia siamensis goniomphalos, the first intermediate host of human liver fluke, Opisthorchis viverrini. Lethal concentrations of 50% (LC(50)) and 95% (LC(95)) against young and adult males were 0.38 and 0.80, 0.42 and 0.86 ppm, respectively. The LC(50) and LC(95) against young and adult females were 0.42 and 0.86, 0.46 and 0.97 ppm, respectively. No significant differences in mortality rate between sexes or snail size (p > 0.05) was detected. Bayluscide-related tissue damage in B. siamensis goniomphalos included detachment of cilia of the epithelial layer of the digestive tract and decreased number of calcium cells. In tests of lethal concentrations of Bayluscide on non-target animals, no lethal effect was observed on Filopaludina martensi martensi (Viviparous snail) but high mortality rates were recorded in Puntius gonionotus fingerling, Ricefish (Oryzias mekongensis) and shrimp (Macrobrachium lanchesteri), but lower in guppy fish (Poecilia reticulata) after 24h exposure. For field trials, sufficient Bayluscide was sprayed in 3 roadside ditches to result in final concentrations of 5, 10 or 20 ppm, with mortality rates on B. siamensis goniomphalos of 10.94, 20.00 and 31.25%, respectively. Non-target snails died in small numbers but no effect was observed in other aquatic vertebrate animals. Field trials of Bayluscide on B. siamensis goniomphalos revealed low mortality rates, suggesting the need for application methods of higher efficacy or that Bayluscide is not suitable for application to operculate snails or snails which are able to escape by burying in mud.

DNA Barcode Identification of Freshwater Snails in the Family Bithyniidae from Thailand

Freshwater snails in the family Bithyniidae are the first intermediate host for Southeast Asian liver fluke (Opisthorchis viverrini), the causative agent of opisthorchiasis. Unfortunately, the subtle morphological characters that differentiate species in this group are not easily discerned by non-specialists. This is a serious matter because the identification of bithyniid species is a fundamental prerequisite for better understanding of the epidemiology of this disease. Because DNA barcoding, the analysis of sequence diversity in the 5’ region of the mitochondrial COI gene, has shown strong performance in other taxonomic groups, we decided to test its capacity to resolve 10 species/ subspecies of bithyniids from Thailand. Our analysis of 217 specimens indicated that COI sequences delivered species-level identification for 9 of 10 currently recognized species. The mean intraspecific divergence of COI was 2.3% (range 0-9.2 %), whereas sequence divergences between congeneric species averaged 8.7% (range 0-22.2 %). Although our results indicate that DNA barcoding can differentiate species of these medically-important snails, we also detected evidence for the presence of one overlooked species and one possible case of synonymy.

RNA-Seq Reveals Infection-Induced Gene Expression Changes in the Snail Intermediate Host of the Carcinogenic Liver Fluke, Opisthorchis viverrini

Background Bithynia siamensis goniomphalos is the snail intermediate host of the liver fluke, Opisthorchis viverrini, the leading cause of cholangiocarcinoma (CCA) in the Greater Mekong sub-region of Thailand. Despite the severe public health impact of Opisthorchis-induced CCA, knowledge of the molecular interactions occurring between the parasite and its snail intermediate host is scant. The examination of differences in gene expression profiling between uninfected and O. viverrini-infected B. siamensis goniomphalos could provide clues on fundamental pathways involved in the regulation of snail-parasite interplay. Methodology/Principal Findings Using high-throughput (Illumina) sequencing and extensive bioinformatic analyses, we characterized the transcriptomes of uninfected and O. viverrini-infected B. siamensis goniomphalos. Comparative analyses of gene expression profiling allowed the identification of 7,655 differentially expressed genes (DEGs), associated to 43 distinct biological pathways, including pathways associated with immune defense mechanisms against parasites. Amongst the DEGs with immune functions, transcripts encoding distinct proteases displayed the highest down-regulation in Bithynia specimens infected by O. viverrini; conversely, transcription of genes encoding heat-shock proteins and actins was significantly up-regulated in parasite-infected snails when compared to the uninfected counterparts. Conclusions/Significance The present study lays the foundation for functional studies of genes and gene products potentially involved in immune-molecular mechanisms implicated in the ability of the parasite to successfully colonize its snail intermediate host. The annotated dataset provided herein represents a ready-to-use molecular resource for the discovery of molecular pathways underlying susceptibility and resistance mechanisms of B. siamensis goniomphalos to O. viverrini and for comparative analyses with pulmonate snail intermediate hosts of other platyhelminths including schistosomes.

Experimental infection of Opisthorchis viverrini cercariae to the cyprinid fish, Barbonymus gonionotus

Opisthorchis viverrini is an important public health problem, a major cause of cholangiocarcinoma in the Greater Mekong subregion including Lao PDR, Cambodia, Vietnam and Thailand. Humans acquire the infection by consumption of raw, fermented or partially cooked freshwater cyprinid fish containing infective metacercariae. This study examined the effect of cercarial infection dosage (25-200 cercariae), age (1-60 day) and size (1-24mm) of Barbonymus gonionotus fish on infection success of O. viverrini cercariae. Additionally, the site of cyst formation of O. viverrini cercariae and subsequent development to the metacercariae was examined. The result showed that cercarial infection dose (F4,95=8.52, P<0.001) age (P<0.001, ƛLR(2)=954.72) and size (P<0.001, ƛLR(2)=1204.84) were significantly associated with number of O. viverrini metacercariae recovery with in every additional fish age, the O. viverrini metacercarial recovery rate increased by 5.4% (P<0.001). Most metacercariae were found in the body of the fish (81.4%). We concluded that O. viverrini cercarial infection dosage, and age and size of fish were important determinants for a successful infection to fish.

Species-Specific Primers Designed from RAPD Products for Bithynia funiculata, the First Intermediate Host of Liver Fluke, Opisthorchis viverrini, in North Thailand

Abstract: Bithynia funiculata is the first intermediate host of the human liver fluke Opisthorchis viverrini in northern Thailand but its identification through morphological analysis is often problematic due to the shortage of gastropod taxonomists. As a consequence, we focused on the development of species-specific primers for use as an identification tool. Our work involved recovery of a 502-base pair (bp) amplicon of unknown function through species-specific primers whose effectiveness was tested by analyzing specimens of B. funiculata from 3 locations in northern Thailand. This primer set did not amplify other species in the Bithyniidae or in other gastropod families. By providing a tool to confirm morphological identifications of B. funiculata, and by enabling the identification of juvenile specimens and those with damaged shells, these primers will improve estimates of the prevalence of parasitic infections in this snail.

Data set from the proteomic analysis of Bithynia siamensis goniomphalos snails upon infection with the carcinogenic liver fluke Opisthorchis viverrini

The snail Bithynia siamensis goniomphalos acts as the first intermediate host for the human liver fluke Opisthorchis viverrini, the major cause of cholangiocarcinoma (CCA) in Northeast Thailand. This data article contains the results obtained from the analysis of the proteins differentially expressed in the snail B. siamensis goniomphalos upon infection with O. viverrini. It contains the data generated from iQuantitator software including a pdf of each sample with a protein׳s relative expression summary and a per-protein detailed analysis of all time points studied and an excel file for each sample containing the raw data from iQuantitator analysis, including ID, mean, standard deviation, credible interval, log2 and description for every protein identified in each of the samples.

Egg-Hatching Mechanism of Human Liver Fluke, Opisthorchis viverrini: A Role For Leucine Aminopeptidases From the Snail Host, Bithynia siamensis goniomphalos

Abstract The human liver fluke Opisthorchis viverrini (Platyhelminthes, Trematoda, Digenea) uses snails of the genus Bithynia as first intermediate host. Peculiarly among trematodes, the eggs of O. viverrini hatch within the digestive tract of its snail host. It remains uncertain whether hatching in this species is mediated through mechanical fracture of the eggshell or by digestion with specific digestive enzymes. This study aimed to characterize enzymes with specific inhibitors and factors involved in the hatching activity of O. viverrini eggs. For measuring egg hatching in vivo, 50 O. viverrini mature eggs were fed to individual Bithynia siamensis goniomphalos snails at various temperature conditions for 24 hr. Ex vivo, mature eggs were incubated with crude snail extract and commercial leucine aminopeptidase (LAP). Egg-hatching of O. viverrini was temperature dependent, with optimal hatching occurring at 24–28 C, with a peak of hatching of 93.54% in vivo and 30.55% ex vivo occurring at these temperatures. Ex vivo hatching rates increased to 45.87% under anaerobic conditions at 28 C. Some 22.70% and 16.21% of heat-killed eggs also hatched within the snail digestive tract and snail extract, respectively, indicating that host molecules are involved in the hatching response. Most eggs hatch in the anterior regions of the digestive tract. Hatching was completely inhibited in the presence of bestatin, an inhibitor of LAP, but not in the presence of phosphatase inhibitors. Bestatin inhibition of hatching was reversible. Finally, egg hatching could be induced by addition of a porcine LAP. The results indicate that this digenean utilizes both LAP of the snail host and movement of miracidia for hatching.