Morphology and activities of cell populations of haemocytes in Oncomelania hupensis following Schistosoma japonicum infection.

Oncomelania hupensis is the only obligatory intermediate host of Schistosoma japonicum, the pathogen of zoonosis schistosomiasis. Haemocytes play a critical role in the cellular immune defence of O. hupensis against S. japonicum challenge. Here, the morphology and classification of haemocytes of O. hupensis were investigated by Giemsa staining and light microscopy, combining with the scanning and transmission electron microscopy and flow cytometry. Granulocytes and hyalinocytes were confirmed as two main types of haemocytes, account for ~ 10% and ~ 90% of all haemocytes, with size varying in 4.3-10.9 µm and 0.4-30.8 µm, respectively. Subpopulations can be identified further by granule feature, shape, size, and surface and inner structure of cells. The heterogeneity in morphology implied varied developmental process and function of haemocyte subpopulations. After the S. japonicum challenge, haemocytes of O. hupensis respond to S. japonicum invasion immediately. The dynamic change of haemocyte subpopulations indicates that the small hyalinocyte could differentiate into a larger one or granulocyte after S. japonicum challenge, and the granulocytes and larger hyalinocytes play leading roles in early defence reaction, but in different ways. Phagocytosis and apoptosis of haemocytes in O. hupensis were proved to be related to immune defence against S. japonicum, with the combined effect of granulocytes and larger hyalinocytes. However, the main pathway of each subpopulation to take effect in different periods need further investigation.

Mechanism by which the combination of SjCL3 and SjGAPDH protects against Schistosoma japonicum infection.

A vaccine is an important method to control schistosomiasis. Molecules related to lung-stage schistosomulum are considered potential vaccine candidates. We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cathepsin L3 (CL3) displayed differential expression in the lung-stage schistosomula of Schistosoma japonicum cocultured with host cells. In the present study, we prepared the two proteins and detected the protective effects of SjGAPDH by immunizing mice with this protein alone and in combination with SjCL3 with or without Freund's adjuvant. Then, we investigated the possible mechanisms underlying S. japonicum infection. The results showed that vaccination of adjuvanted SjGAPDH decreased the worm burden (37.8%) and egg load (38.1%), and the combination of adjuvanted SjGAPDH and SjCL3 further decreased the worm burden (65.6%) and egg load (70.9%) during Schistosoma japonicum infection. However, the immunization of a combination of adjuvant-free SjGAPDH and SjCL3 displayed a lower protective effect (< 15%) than those of the adjuvanted SjCL3, the adjuvanted SjGAPDH, and a combination of adjuvanted SjGAPDH and SjCL3. Flow cytometric results showed that the frequency of regulatory T cells (Tregs) was lower (P < 0.05) in the group with adjuvanted SjGAPDH and SjCL3 (2.61%) than the remaining groups. The enzyme-linked immunosorbent assay (ELISA) results indicated that except for the uninfected and infected control groups, the remaining groups displayed a Th1-type shift in immune responses. These results showed the immunization of SjGAPDH resulted in partial protection (approximately 38%); inoculation with a combination of SjCL3 and SjGAPDH in Freund's adjuvant resulted in a high immunoprotective effect (> 65%) against Schistosoma japonicum infection in mice, which was possibly caused by the reduced percentage of Tregs and a Th1-type shift in immune responses; and SjCL3 has no adjuvant-like effect, dissimilar to SmCL3.

[The Role of Virtual Reality Technology in Medical Education in the Context of Emerging Medical Discipline].

According to Healthy China, a national strategy of the Government of China, new requirements were put forward for high-quality medical education, high-level surgical research, and precise clinical diagnosis and treatment. In the context of Emerging Medical Discipline, a strategic blueprint of medical education in China, this paper reviews the concept and core value of virtual reality (VR) and its significant role in the medical industry. On that basis, we explore the role of VR technology in medical training against the background of Emerging Medicine Discipline. Furthermore, typical cases are presented to help analyze and illustrate in detail the important role of VR technology in the teaching and training of stomatological and clinical procedures, skills assessment, online self-directed training, and clinical thinking skills training. We herein summarize useful information from past experience so as to help build innovative models of medical education in the context of Emerging Medical Discipline.

Characteristics and function of cathepsin L3 from Schistosoma japonicum.

Schistosomiasis is still prevalent and seriously endangering the health of people and livestock in many countries. There have been great efforts to develop vaccines against schistosomiasis for prolonged protection in epidemic areas. Molecules from lung-stage schistosomula have been regarded as potential vaccine candidates against schistosomiasis. Our previous work has shown that cathepsin L3 from Schistosoma japonicum (SjCL3) is expressed in lung-stage schistosomula, but its role is not well known. In the present study, we characterized SjCL3 and detected its effect as a possible vaccine in vivo and in vitro. From the results of quantitative PCR (qPCR) and western blot, SjCL3 was present throughout the lifecycle of the worm, and its relative expressed level was higher in the liver eggs and adult worms than other stages. Additionally, immunofluorescence assay showed that SjCL3 was mainly concentrated in the eggshell, alimentary canal, and musculature of worms. Compared with the adjuvant group, the immunization of SjCL3 in mice resulted in a 28.9% decrease in worm burden and a 29.2% reduction in egg number in the host liver. In antibody-dependent cell-mediated cytotoxicity (ADCC) insecticidal experiments in vitro, the existence of SjCL3 could in part suppress adherence between macrophages and worm. The above results indicated that the immunization of SjCL3 could induce limited immune protection against S. japonicum infection in mice, and this protease played a role in breaking the process of ADCC, which was beneficial to the survival of worms.

Morphological and enzymatical observations in Oncomelania hupensis after molluscicide treatment: implication for future molluscicide development.

A preparation of niclosamide named 50 % wettable powder of niclosamide ethanolamine salt (WPN), the only chemical molluscicide available in China, has been widely used for Oncomelania hupensis control over the past 20 years, but its molluscicidal mechanism has not been elucidated yet. Recently, a derivative of niclosamide, the salt of quinoid-2',5-dichloro-4'-nitro-salicylanilide (Liu Dai Shui Yang An, LDS), has been proven to have equivalent molluscicidal effects as WPN but with lower cost and significantly lower toxicity to fish than WPN. In our previous study, gene expression profiling of O. hupensis showed significantly effects after these two molluscicides had been applied. This study was designed to use morphological and enzymological analyses to further elucidate the mechanism by which these molluscicides cause snail death. After WPN or LDS treatment, the number of mitochondria of O. hupensis was reduced and their cristae appeared unclear, heterochromatin gathered to be polarized, ribosome numbers of the rough endoplasmic reticulums (rERs) decreased, myofilaments in muscle cells became disordered and loose, and cytoplasm in some liver cells was concentrated. Damage of cell structures and organelles suggested inhibited movement ability and effects on liver and energy metabolism following treatment. In parallel, activities of enzymes related with carbohydrate metabolism were inhibited except lactate dehydrogenase (LDH) increased in muscle tissue, and activities of enzymes related with stress response increased followed by decreasing to lower levels than those of the H2O-treated group. This shift of carbohydrate metabolism patterns led to insufficient energy supply and lactic acid accumulation, and variations of nitric oxide synthase (NOS), alanine aminotransferase (ALT), and superoxide dismutase (SOD) during process of molluscicide treatment suggested a stress response of snail to the molluscicides at early stages and later fatal damage in liver and nervous system. In general, effects of WPN and LDS were similar although LDS-treated snails showed more serious damage in the liver and a stronger inhibition of enzymes related with aerobic respiration and stress response. This was consistent with the transcriptome profile obtained previously. However, considering enzyme activities at post-transcriptional and protein levels, comprehensive identification and annotation of potential enzyme-related genes and regulation pattern would be necessary to provide great benefit for understanding of potential mechanism of these molluscicides and even for future molluscicide development.

Cloning and characterization of a bone morphogenetic protein homologue of Schistosoma japonicum.

Bone morphogenetic proteins (BMPs) are known to play an important role in the regulation of cell proliferation, survival, differentiation and apoptosis in many vertebrates and invertebrates through the TGF-ß signaling pathway. Although the TGF-ß signaling pathway exists in schistosomes, BMP homologue, a ligand of TGF-ß in Schistosoma japonicum, has not yet been identified. In this study, a BMP homologue of S. japonicum was cloned and characterized. The full length SjBMP cDNA is 3,020 bp and encodes 928 amino acids, which include a TGF-ß superfamily conserved domain at the C-terminus. BLAST analysis showed that, SjBMP has 68%, 51% and 43% homology with BMP from Schistosoma mansoni, Schmidtea mediterranea and Dugesia japonica at the amino acid level, respectively. According to data from real-time PCR, SjBMP was expressed in lung-stage schistosomula, 21-day liver-stage schistosomula, 50-day adult worms (the male and female), and eggs. The PCR data also indicated that, there was a ≈ 27- and ≈ 37-fold increase of SjBMP transcripts in the lung-stage schistosomula and eggs, respectively, and that there was relatively more SjBMP transcript in the adult male worm than in the adult female, in which the hepatic schistosomula was set as the calibrator for calculation. In situ hybridization based on FITC-labeled specific antisense oligonucleotide probes showed that SjBMP mRNA localized to the ovary of female worms and the integument and epithelium of female and male worms. After treatment with double-stranded RNA (dsRNA) at a concentration of 8 × 10(-2) µg/ml, which was added to the culture medium every other day for a week, the level of SjBMP mRNA in the cultured adult mixed-sex S. japonicum decreased at a range of ≈ 25-98% within 7 days compared with the level of SjBMP mRNA in the blank control group. On the 2nd day, the number of eggs produced per pair of worms decreased 28.7%, and the percent of normal eggs also decreased (12.7% vs. 4.3%) in the SjBMP dsRNA-treated group when compared with the eggs laid by the blank control group. No difference was detected between the two groups on the 7th day of treatment, because the eggs of the untreated worms were also mostly abnormal, similar to the eggs laid by the treated group. In addition, no significant difference in the morphological structure of the adult worms was observed. Thus, the preliminary in vitro experiment indicated that SjBMP may be involved in the oviposition behavior of S. japonicum, and further studies based on the recombinant virus vector-induced steady knockdown of SjBMP or in vivo experiments are required for more in-depth investigation.

Inhibition of alternative oxidase disrupts the development and oviposition of Biomphalaria glabrata snails.

BACKGROUND: Biomphalaria glabrata is one of the main intermediate hosts of Schistosoma mansoni, the most widespread species of Schistosoma. Our previous studies proved that alternative oxidase (AOX), the terminal oxidase in the mitochondrial respiratory chain, widely exists in several species of intermediate host snails of Schistosoma. Meanwhile, inhibition of AOX activity in Oncomelania hupensis snails could dramatically enhance the molluscicidal effect of niclosamide. As a hermaphroditic aquatic mollusc, the high fecundity and population density of B. glabrata increase the difficulty of snail control, which is one of the critical strategies for schistosomiasis elimination. The present study aimed to investigate the possible role of AOX in the development and fecundity of B. glabrata snail, which could be manipulated more manageable than other species of intermediate host snails of Schistosoma. METHODS: The dynamic expression of the AOX gene was investigated in different developmental stages and tissues of B. glabrata, with morphological change and oviposition behaviour observed from juvenile to adult snails. Furtherly, dsRNA-mediated knockdown of BgAOX mRNA and the AOX protein activity inhibiting was performed to investigate the effect of AOX on the development and oviposition of snails. RESULTS: The BgAOX gene expression profile is highly related to the development from late juveniles to adults, especially to the reproductive system of snails, with a positive correlation of 0.975 between egg production and BgAOX relative expression in ovotestis of snails. The inhibition of BgAOX at the transcriptional level and AOX activity could efficiently inhibit snail growth. However, the interference at the BgAOX protein activity level led to more severe tissue damage and more significant inhibition of oviposition than at the transcriptional level. This inhibition of growth and oviposition decreased gradually with the increase in the snail size. CONCLUSIONS: The inhibition of AOX could efficiently disrupt the development and oviposition of B. glabrata snails, and the intervention targeting AOX at the juvenile stage is more effective for snails. This investigation explored the role of AOX in the growth and development of snails. It would benefit snail control in the future by providing a potential target while using molluscicides more efficiently.

Identification of differentially expressed genes in Oncomelania hupensis chronically infected with Schistosoma japonicum.

Oncomelania hupensis is the unique intermediate host of Schistosoma japonicum. The schistosome-snail interaction is biomedically important. To identify differentially expressed transcripts in O. hupensis chronically infected with S. japonicum, suppression subtractive hybridization (SSH) was used to construct a cDNA library in each direction for transcripts that are more abundantly enriched in head-foot part of the infected O. hupensis and for those that are more abundantly enriched in the uninfected, as head-foot part contains hemocytes and hemolymph which are associated with the snail internal defense system. After differential screening, 39 transcripts were identified, including nine and 30 transcripts enriched in infected and uninfected snails, respectively. Some of the transcripts have similar homology to available sequences in current databases, including transposase, caveolin-like protein, pancreatic trypsin inhibitor-like protein, prosaposin, glutathione s-transferase (GST), and several hypothetical proteins, while most of the transcripts do not match with any sequences in available databases. The identified transcripts were involved functionally in cell growth, metabolism, signal transduction, and immune responses. Two forward library transcripts and 11 reverse library transcripts were selected for real-time PCR, and 10 of them were confirmed to be consistent with the SSH results. It is intriguing to continue functional studies for some genes such as pancreatic trypsin inhibitor; a hypothetical protein (HS576367) related to calcium ion binding; GST; and several unknown proteins (HS576353 and HS576355). These identified differentially expressed genes may be key targets for understanding the molecular mechanism of co-existence during which the snail is unable to rid itself of the schistosome in chronic infection stage.

Effects of protein extract from head-foot tissue of Oncomelania hupensis on the growth and gene expression of mother sporocysts of Schistosoma japonicum.

Oncomelania hupensis is the intermediate host of Schistosoma japonicum. In the present study, we investigated the effects of protein extracts from head-foot or gland tissue of O. hupensis on mother sporocysts of S. japonicum cultured in vitro. In the presence of head-foot protein extract of snails from the native province Hunan, in-vitro-transformed mother sporocysts presented not only a longer survival time and stronger motility, but also a bigger size than parasites cultured with protein extracts of glands of the same snail or head-foot tissue of a non-native snail from the Hubei province. Using suppression subtractive hybridization, two subtractive libraries were constructed on the basis of RNA of sporocysts cultured with or without native snail head-foot protein extract. A number of 31 transcripts were found to be up-regulated. Sequence analyses revealed that they represented genes involved among others in metabolic process, electron transport chain, response to chemical stimulus, and oxidation-reduction processes. Opposite to that 20 down-regulated transcripts were among others related to pseudouridine synthesis, RNA processing, and ribosome biogenesis. The differential expression of three of these transcripts, encoding cytochrome c oxidase subunit 2 (Cox2), NADH-ubiquinone oxidoreductase (ND1), and dyskeratosis congenita 1 protein (DKC1), were confirmed by real-time PCR. The promoted development and the differential gene expression of cultured sporocysts under the influence of head-foot protein extract of native O. hupensis implied not only its ability to improve in vitro culture conditions for intramolluscan stages, it may also represent a priming result with respect to the identification and characterization of factors involved in the parasite-host interplay between S. japonicum and O. hupensis.

Studies on the establishment of a co-culture system of lung stage Schistosoma japonicum with host cells.

Due to their role in eliciting protective Th1 cell-mediated immune responses in definitive hosts lung stage schistosomula are in the focus of intensive research. In vitro culture approaches in the past exhibited significant differences in gene expression profiles between lung stage schistosomula isolated from hosts and those cultured conventionally. Therefore, new approaches to culture schistosomula are of broad interest. In the present study, co-culture systems of schistosomula of Schistosoma japonicum and different vertebrate host cells were tested. Among these, human hepatic venous endothelial cells (ED25) turned out to be very suitable and interesting feeder cells. Compared with controls cultured in vitro or co-cultured with other cells, schistosomula co-cultured with ED25 cells shared more similarities in morphology and tegumental structures with schistosomula directly obtained from infected mice as microscopically determined. According to results from a suppression subtractive hybridization approach to compare transcriptional differences of co-cultured and host group or control group parasites, four candidate transcripts encoding cathepsin L precursor, heat shock protein 70, glyceraldehyde 3-phosphate dehydrogenase, and programmed cell death protein 10 were shown to be differently expressed among the three groups by real-time PCR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis finally confirmed not only congruent protein patterns but also interesting differences among the compared schistosomula groups. The co-culture system between schistosomula of S. japonicum and ED25 cells established in the present study improved existing cultivation attempts. Although some differences to host-derived schistosomula were still observed, co-culture with ED25 cells positively influenced parasite morphology and gene expression in a more host-like manner.

The identification of alternative oxidase in intermediate host snails of Schistosoma and its potential role in protecting Oncomelania hupensis against niclosamide-induced stress.

BACKGROUND: Snail intermediate hosts are mandatory for the transmission of schistosomiasis, which has to date infected more than 200 million people worldwide. Our previous studies showed that niclosamide treatment caused the inhibition of aerobic respiration and oxidative phosphorylation, and the disruption of energy supply, in one of the intermediate hosts of schistosomiasis, Oncomelania hupensis, which eventually led to the death of the snails. Meanwhile, the terminal oxidase in the mitochondrial respiratory chain, alternative oxidase (AOX), was significantly up-regulated, which was thought to counterbalance the oxidative stress and maintain metabolic homeostasis in the snails. The aims of the present study are to identify the AOXs in several species of snails and investigate the potential activation of O. hupensis AOX (OhAOX) under niclosamide-induced stress, leading to enhanced survival of the snail when exposed to this molluscicide. METHODS: The complete complementary DNA was amplified from the AOXs of O. hupensis and three species of Biomphalaria; the sequence characteristics were analysed and the phylogenetics investigated. The dynamic expression and localisation of the AOX gene and protein in O. hupensis under niclosamide-induced stress were examined. In addition, the expression pattern of genes in the mitochondrial respiratory complex was determined and the production of reactive oxygen species (ROS) calculated. Finally, the molluscicidal effect of niclosamide was compared between snails with and without inhibition of AOX activity. RESULTS: AOXs containing the invertebrate AOX-specific motif NP-[YF]-XPG-[KQE] were identified from four species of snail, which phylogenetically clustered together into Gastropoda AOXs and further into Mollusca AOXs. After niclosamide treatment, the levels of OhAOX messenger RNA (mRNA) and OhAOX protein in the whole snail were 14.8 and 2.6 times those in untreated snails, respectively, but varied widely among tissues. Meanwhile, the level of cytochrome C reductase mRNA showed a significant decrease in the whole snail, and ROS production showed a significant decrease in the liver plus gonad (liver-gonad) of the snails. At 24 h post-treatment, the mortality of snails treated with 0.06-0.1 mg/L niclosamide and AOX inhibitor was 56.31-76.12% higher than that of snails treated with 0.1 mg/L niclosamide alone. CONCLUSIONS: AOX was found in all the snail intermediate hosts of Schistosoma examined here. AOX was significantly activated in O. hupensis under niclosamide-induced stress, which led to a reduction in oxidative stress in the snail. The inhibition of AOX activity in snails can dramatically enhance the molluscicidal effect of niclosamide. A potential target for the development of an environmentally safe snail control method, which acts by inhibiting the activity of AOX, was identified in this study.

Conservation and variation in mitochondrial genomes of gastropods Oncomelania hupensis and Tricula hortensis, intermediate host snails of Schistosoma in China.

The complete mitochondrial genomes of intermediate host snails for Schistosoma in China were sequenced, including the sub-species Oncomelania hupensis hupensis in two types, and O. hupensis robertsoni, intermediate hosts for S. japonicum, and Tricula hortensis, the intermediate host of S. sinensium. Four genomes have completely the same gene order as in other caenogastropods, containing 13 protein-coding genes and 22 transfer RNAs. The gene size, start codon and termination codon are mostly the same for all protein-coding genes. However, pairwise sequence alignments revealed quite different degrees of variation. The ribbed-shelled O. hupensis hupensis and the smooth-shelled but with varix O. hupensis hupensis had a lower level of genetic distance (3.1% for protein-coding genes), but the coden usages differed obviously in the mitochondrial genomes of these two types of snails, implying that their genetic difference may be larger than previously recognized. The mean genetic distance between O. hupensis hupensis and O. hupensis robertsoni was 12% for protein-coding genes, indicating a higher degree of genetic difference. In consideration of the difference in morphology and distribution, we considered that O. hupensis hupensis and O. hupensis robertsoni can be considered as separate species. The ribbed-shelled O. hupensishupensis and smooth-shelled O. hupensis robertsoni were phylogenetically clustered together within a same clade, which was then clustered with T. hortensis, confirming their close relationship. However, species or sub-species in the Oncomelania from southeastern Asian countries should be included in future study in order to resolve the phylogenetic relationship and origination of all snails in the genus.

The genetic variation of different developmental stages of Schistosoma japonicum: do the distribution in snails and pairing preference benefit the transmission?

BACKGROUND: Schistosoma japonicum is a waterborne parasite that causes schistosomiasis in humans and in more than 40 animal species. Schistosoma japonicum shows distinct genetic differentiation among geographical populations and multiple hosts, but the genetic diversity of different developmental stages of S. japonicum from is less studied. Such studies could elucidate ecological mechanisms in disease transmission by analysing feedbacks in individual physiology and population state. METHODS: After infection using cercariae from a pool of snails shedding together (Method I) and infection using mixed equal numbers of cercariae from individually shed snails (Method II), different developmental stages of S. japonicum were genotyped with microsatellite loci, including 346 cercariae, 701 adult worms and 393 miracidia. Genetic diversity and molecular variation were calculated at different population levels. Kinships (I') among cercariae at intra-snail and inter-snail levels were evaluated. Genetic distance (Dsw) was compared between paired and unpaired worms, and partner changing was investigated through paternity identification for miracidia. RESULTS: The cercaria clones in individual snails varied from 1 to 8 and the kinship of cercariae within individual snails was significant higher (P < 0.001) than that among different snails after deleting near-identical multi-locus genotypes (niMLGs). The allelic diversity of worms in Method I was lower (P < 0.001) than that in Method II, and allele frequency among mice in Method I was also less consistent. The parents of some miracidia were worms that were not paired when collected. The Dsw between each female of paired and unpaired males was much larger (P < 0.001) than that between the female and male in each pair. CONCLUSIONS: Most of the infected snails contained multiple miracidia clones. The aggregation of genetically similar S. japonicum miracidia in individual snails and the unbalanced distribution of miracidia among snails suggests a non-uniform genetic distribution of cercariae among snails in the field. This further influenced the genetic structure of adult worms from infections with different cercariae sampling methods. Schistosoma japonicum in mice can change paired partner, preferring to mate with genetically similar worms. These characteristics provide implications for understanding the balance in genetic diversity of S. japonicum related to the transmission of schistosomiasis.

Metabolic profiles of Oncomelania hupensis after molluscicidal treatment: Carbohydrate metabolism targeted and energy deficiency.

Oncomelania hupensis is the intermediate host of Schistosoma japonicum, one of the Schistosoma species that can cause human schistosomiasis. Molluscicidal treatment remains the primary means to control snail. Niclosamide is the only molluscicide recommended by the World Health Organization, and it has been used throughout schistosomiasis-endemic areas in China for almost 30 years. In our previous studies on transcriptomics, morphology, and enzymology of snails after molluscicidal treatment, two effective molluscicides were used, 50% wettable powder of niclosamide ethanolamine salt (WPN) and a new molluscicide derived from niclosamide, the salt of quinoid-2', 5-dichloro-4'-nitro-salicylanilide (LDS, simplified for Liu Dai Shui Yang An). Genes involved in cell structure mintenance, inhibition of neurohumoral transmission, and energy metabolism showed significant differential expression after molluscicide treatments. Damages in the structure of liver and muscle cells were accompanied by inhibited activities of enzymes related to carbohydrate metabolism and energy supply. This study was designed to clarify the dynamic metabolic process by metabonomics, together with the previous transcriptomic and enzymological profiles, to identify potential metabolite markers and metabolism pathways that related to the toxic mechanism of the molluscicide. In total, 56 metabolites were identified for O. hupensis, and 75% of these metabolites consisted of amino acids and derivatives, organic acids, and nucleic acid components. The concentration of glucose, maltose, succinate, choline, and alanine changed significantly after molluscicide treatments. These changes in metabolites mainly occurred in the process of carbohydrate metabolism, energy metabolism, and amino acid metabolism, primarily related to glycolysis/gluconeogenesis, oxidative phosphorylation, and transamination by KEGG pathway identification. Most of the identified pathways were also related to those differentially expressed unigenes and observed enzymes from our previous studies. Inhibited aerobic respiration and oxidative phosphorylation, and energy deficiency were implied further to be the leading causes of the final death of snails after molluscicide treatments. The hypothesised mathematical model in this study identified the rational hysteresis to explain the inconsistency of responses of unigenes, enzymes, and metabolites to molluscicide treatments. This study contributes to the comprehensive understanding of the molluscicidal mechanism in the metabolic process and this could assist in improving existing molluscicide formulations or development of new molluscicides.

Identification of Toll-like receptor family members in Oncomelania hupensis and their role in defense against Schistosoma japonicum.

The amphibious snail, Oncomelania hupensis, primarily distributed in the Far East, is the only intermediate host of Schistosoma japonicum, which causes the most virulent form of schistosomiasis. Obligatory parasitism of snails is the main vehicle for human and livestock infection and depends primarily on parasite infectivity, snail defense capacity and specificity, and parasite-snail compatibility. Therefore, the schistosome-snail interaction is biomedically significant, particularly the molecular mechanisms involved in the innate immune response against S. japonicum. Several immune effectors and signaling pathways have been successfully identified in mollusks, especially in Biomphalaria glabrata, the intermediate snail host of S. mansoni; however, limited information is available for O. hupensis. Here, we identified 16 Toll-like receptors (TLRs) in O. hupensis. These O. hupensis TLRs (OhTLRs) are highly expressed in haemocytes, the primary immune cell of mollusks. Most of the OhTLRs were more highly expressed in female gonads than in other tissues, which may suggest maternal immune transfer in O. hupensis. After S. japonicum challenge, the expression levels of all of the OhTLRs were significantly up-regulated at 6 h post-challenge; many of the OhTLR expression levels were inhibited at later time points in haemocytes, while they were inhibited and fluctuated to varying degrees in other tissues. Additionally, we further determined the tissue-specific expression and dynamic response against S. japonicum of one of the TLR signaling adaptors, myeloid differentiation factor 88 (MyD88), from O. hupensis. Three OhMyD88 genes were highly expressed in haemocytes, and were up-regulated in haemocytes and inhibited in the head-foot muscle at the early time-point after S. japonicum challenge; however, these had slower changes and longer durations compared to OhTLRs. These results provide evidence suggesting that immune effectors are involved in innate immune responses of O. hupensis against S. japonicum and may play a role in the activation of different haemocytes, and not limited for the early response to S. japonicum invasion. Further investigation into the varied expression of OhTLRs in other tissues after S. japonicum challenge will improve our understanding of TLR function in innate immunity of O. hupensis.

Influence of Schistosoma japonicum programmed cell death protein 10 on the growth and development of schistosomula.

BACKGROUND: Schistosomiasis caused by Schistosoma japonicum is among the most serious endemic zoonoses in China. To study interactions between schistosomula, the pre-adult juvenile stage, and hosts, it is important to study the functions of key genes involved in schistosomula growth and development. Programmed cell death protein 10 (pcdp10) is an important apoptosis-related gene with various biological functions. This study described the molecular characterization of S. japonicum PCDP10 (SjPCDP10) and evaluated its functions in schistosomula. METHODS: Real-time quantitative polymerase chain reaction (qPCR) and western blot were used to detect Sjpcdp10 mRNA and protein levels, respectively, at different developmental stages. Immunolocalization was performed to determine SjPCDP10 expression in the parasite. RNA interference (RNAi) experiments were used to assess gene functions associated with SjPCDP10 in schistosomula growth and development. RESULTS: Real-time qPCR revealed that Sjpcdp10 was expressed during all investigated developmental stages and upregulated during schistosomula growth and development. Histochemical localization showed that SjPCDP10 was mainly distributed in the teguments of schistosomula in all investigated stages and part of the parenchymal area of 14-, 18-, and 21-day-old schistosomula. Following Sjpcdp10 knockdown by RNAi, the lengths, widths, areas, and volumes of schistosomula were significantly lower than those in the control group. Scanning electron microscopy showed that the body surfaces of schistosomula subjected to RNAi were seriously damaged, with few tegumental spines and sensory papillae. Transmission electron microscopy indicated that the teguments of Sjpcdp10-knockdown schistosomula were incomplete, the number of layers was reduced, and the thickness decreased significantly as compared with those in the control group. Furthermore, terminal deoxynucleotidyl transferase dUTP nick-end labelling results showed that the rate of apoptosis in Sjpcdp10-knockdown schistosomula was significantly higher than that in the control group. CONCLUSIONS: Sjpcdp10-knockdown influenced the growth and development of schistosomula. Therefore, our results indicated that SjPCDP10 contributes to the regulation of cell apoptosis and is essential for schistosomula growth and development.

[Proteomics and its application in parasitology].

Proteomics is an important high throughout method in modern life science. In this paper, the definition, background and methods used in proteomics were introduced, and the last part was focused on its application in parasitology.

Molecular cloning and characterization of copper/zinc-superoxide dismutase of Paragonimus westermani.

Superoxide dismutases (SODs; EC 1.15.1.1) play important roles in the protection of the parasites against cellular oxygen-mediated killing of the hosts. A copper/zinc-containing SOD (Cu/Zn-SOD) was identified previously from lung fluke, Paragonimus westermani. To expand our understanding of P. westermani SOD, we isolated a complementary DNA encoding a Cu/Zn-SOD, expressed the active enzyme in Escherichia coli, and characterized its biochemical properties. The deduced amino acid (aa) sequence of the gene shared up to 73.7% identities with Cu/Zn-SODs of other helminths and shared well-conserved characteristic motifs and essential aa residues involved in coordinating copper and zinc enzymatic functions. Recombinant Cu/ Zn-SOD exhibited comparable biochemical properties with that of the native enzyme, including pH optima and potassium cyanide-and hydrogen peroxide-sensitive inhibition profiles. The active enzyme consisted of 2 identical subunits covalently linked by disulfide bonds. The enzyme was constitutively expressed throughout various developmental stages of the parasite. The levels increased as P. westermani matured and plateaued in adult stage. Our result suggests the enzyme might play an important role for parasites to survive in the hosts through its superoxide anion-detoxifying function.

[Progress of pattern recognition receptors of molluscs].

Molluscs have established complete innate immunity to defense against pathogens. The pattern recognition receptors (PRRs) are the sensory receptors of molluscs to resist outside invaders, as the first reactor to initiate the innate immune response. Some PRRs have been identified in several molluscs, including Toll-like receptors (TLRs) , C-type lectins, galectins, lipopolysaccharide-ß-1,3-glucan binding protein (LGBP), Clq domain-containing protein (ClqDC), and peptidoglycan recognition protein (PGRP). PRRs have various biological activities and play important roles in the defense system of molluscs. This paper reviews the research progress of PRRs in molluscs.

De Novo transcriptome analysis of Oncomelania hupensis after molluscicide treatment by next-generation sequencing: implications for biology and future snail interventions.

The freshwater snail Oncomelania hupensis is the only intermediate host of Schistosoma japonicum, which causes schistosomiasis. This disease is endemic in the Far East, especially in mainland China. Because niclosamide is the only molluscicide recommended by the World Health Organization, 50% wettable powder of niclosamide ethanolamine salt (WPN), the only chemical molluscicide available in China, has been widely used as the main snail control method for over two decades. Recently, a novel molluscicide derived from niclosamide, the salt of quinoid-2',5-dichloro-4'-nitro-salicylanilide (Liu Dai Shui Yang An, LDS), has been developed and proven to have the same molluscicidal effect as WPN, with lower cost and significantly lower toxicity to fish than WPN. The mechanism by which these molluscicides cause snail death is not known. Here, we report the next-generation transcriptome sequencing of O. hupensis; 145,008,667 clean reads were generated and assembled into 254,286 unigenes. Using GO and KEGG databases, 14,860 unigenes were assigned GO annotations and 4,686 unigenes were mapped to 250 KEGG pathways. Many sequences involved in key processes associated with biological regulation and innate immunity have been identified. After the snails were exposed to LDS and WPN, 254 unigenes showed significant differential expression. These genes were shown to be involved in cell structure defects and the inhibition of neurohumoral transmission and energy metabolism, which may cause snail death. Gene expression patterns differed after exposure to LDS and WPN, and these differences must be elucidated by the identification and annotation of these unknown unigenes. We believe that this first large-scale transcriptome dataset for O. hupensis will provide an opportunity for the in-depth analysis of this biomedically important freshwater snail at the molecular level and accelerate studies of the O. hupensis genome. The data elucidating the molluscicidal mechanism will be of great benefit in future snail control efforts.