Infection with Clonorchis sinensis (Cobbold, 1875) Metacercariae in Fish from the East Lake of Wuhan: Freshwater Fish in Urban Lakes May Act as Infection Sources of Liver Fluke.

The liver fluke disease caused by Clonorchis sinensis is one of the most serious food-borne parasitic diseases in China. Many freshwater fish and shrimps can be infected with C. sinensis metacercariae as the second intermediate hosts in endemic regions. Owing to the lack of infected humans and the good administration of pet dogs and cats in cities of non-endemic regions, few fish are expected to be infected with C. sinensis metacercariae in urban lakes. To determine the infection of C. sinensis metacercariae in freshwater fish and shrimps in urban lakes, a total of 18 fish species and one shrimp species were investigated in the East Lake of Wuhan City. Metacercariae were isolated by artificial digestive juice and identified using morphology and rDNA-ITS2 sequences. Five species of fish, Pseudorasbora parva, Ctenogobius giurinus, Squalidus argentatus, Hemiculter leuciclus, and Rhodeus spp., were infected with C. sinensis metacercariae. The overall prevalence of C. sinensis was 32.5%. The highest prevalence was found in P. parva with 57.9%, while S. argentatus exhibited the highest mean abundance (13.9). Apart from the C. sinensis metacercariae, four species of other trematode metacercariae were also identified across twelve fish species in total. Owing to the consumption of undercooked fish and feeding cats with small fish caught by anglers, there is a potential risk that the small fish infected with C. sinensis metacercariae may act as an infection source to spread liver fluke. Given the complete life cycle of C. sinensis, stray cats and rats were inferred to act as the important final hosts of C. sinensis in urban lakes in non-endemic areas.

Comprehensive analyses of prognostic biomarkers and immune infiltrates among histone lysine demethylases (KDMs) in hepatocellular carcinoma.

BACKGROUND: Histone lysine demethylases (KDMs) are closely related to the occurrence and development of different tumors through epigenetic mechanisms. However, the prognosis and immune infiltration of KDMs in hepatocellular carcinoma (HCC) remain undefined. METHODS: In the current study, we analyzed the expression of KDMs on HCC patients using the Oncomine, GEPIA, UALCAN, Kaplan-Meier Plotter, cBioPortal, GeneMANIA, STRING, Metascape, GSEA, and TIMER databases. Finally, we investigated KDM expression in HCC by qRT-PCR, Western blotting, and IHC. RESULTS: We found that KDM3A/3B/5A/5B and KDM6A were upregulated in HCC patients, while KDM6B and KDM8 were downregulated. The high expressions of KDM1A/2B/3B/5B/5C were markedly related to tumor stages and grades of HCC patients. The abnormal expression of KDM1A/1B/3A/4A/5A/5C/6A/6B/7A and KDM8 were associated with HCC patients' prognosis. Also, we found that HCC tissues presented higher expression levels of KDM1A/2A/5A/5B and lower expression levels of KDM6B. The function of KDMs was primarily related to the histone demethylase activity and cell cycle, p53 signaling pathway, pathways in cancer, transcriptional mis-regulation in cancer, viral carcinogenesis, and FoxO signaling pathway. Furthermore, we indicated that the pathways most involved were the mitotic spindle and DNA repair. Additionally, we found that the expression of KDM1A/1B/3A/4A/5B/5C and KDM6A were significantly correlated with HCC immune infiltration. CONCLUSIONS: Overall, our current results indicated that KDM1A/1B/3A/4A/5B/5C and KDM6A could be novel prognostic biomarkers and provide insights into potential immunotherapy targets to HCC patients.

Comparative characterization of microRNAs of Schistosoma japonicum from SCID mice and BALB/c mice: Clues to the regulation of parasite growth and development.

Schistosomiasis, caused by a parasite with a wide range of mammalian hosts, remains one of the most prevailing parasitic diseases in the world. While numerous studies have reported that the growth and reproduction of schistosomes in immunodeficient mice was significantly retarded, the underlying molecular mechanisms have yet to be revealed. In this study, we comparatively analyzed the microRNA expression of Schistosoma japonicum derived from SCID and BALB/c mice on the 35th day post-infection by high-throughput RNA sequencing as prominent morphological abnormalities had been observed in schistosomes from SCID mice when compared with those from BALB/c mice. The results revealed that more than 72% and 61% of clean reads in the small RNA libraries of female and male schistosomes, respectively, could be mapped to the selected miRs in the miRBase or the sequences of species-specific genomes. Further analysis identified 122 miRNAs using TPM >0.01 as the threshold value, including 75 known and 47 novel miRNAs, 96 of which were commonly expressed across all the four tested schistosome libraries. Comparative analysis of the libraries of schistosomes from SCID and BALB/c mice identified 15 differentially expressed miRNAs (5 up-regulated and 10 down-regulated) among females and 16 among males (9 up-regulated and 7 down-regulated). Integrated analysis of the two sets of differentially expressed miRNAs of female and male worms identified 2 miRNAs (sja-miR-3488 and sja-miR-novel_29) that overlapped between female and male datasets. Prediction of miRNA targets and Gene Ontology (GO) term enrichment analysis of the predicted target genes revealed that these genes were involved in some important biological processes, such as nucleic acid metabolic process, macromolecule modification, and cellular aromatic compound metabolic process. The predicted target genes were further matched to the differentially expressed genes in male and female schistosomes from the above two hosts, obtaining 7 genes that may be responsible for regulating the growth, development and sex maturation of schistosomes. Taken together, this study provides the first identification of differentially expressed miRNAs in schistosomes from SCID and BALB/c mice. These miRNAs and their predicted target mRNAs are probably involved in the regulation of development, growth, and maturation of schistosomes. Therefore, this study expands our understanding of schistosome development regulation and host-parasite relationship, and also provides a valuable set of potential anti-schistosomal targets for prevention and control of schistosomiasis.

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.

Prohibitin 1 (PHB1) controls growth and development and regulates proliferation and apoptosis in Schistosoma japonicum.

Schistosomiasis is a zoonotic parasitic disease caused by the trematode blood flukes of the genus Schistosoma. The prodigious egg output of females is the main cause of the disease in definitive hosts, while the female worm relies on continuous pairing with the male worm to fuel the growth and maturation of the reproductive organs and egg production. Prohibitin, which contains the functionally interdependent PHB1 and PHB2 subunits in human and some other species, has been proposed to participate in the cell proliferation and apoptosis regulation in mammals. However, little is known about the function of PHB homolog in the growth and reproductive development of schistosomes. Here, we reported the Phb1 gene that was structurally and evolutionarily conserved in Schistosoma japonicum when compared with that of other species from Caenorhabditis elegans to human. Real-time PCR detected that SjPhb1 was highly transcribed in the vitellaria of female worms. SjPhb1 knockdown achieved through the dsRNA-mediated RNAi in vivo resulted in retarded growth, decreased pairing, and fecundity in adult worms, as well as attenuated pathogenicity or virulence of worms to their hosts. Cell proliferation and apoptosis examination found decreased cell proliferation and increased cell apoptosis in SjPhb1 dsRNA-treated worms. Therefore, our study provides the first characterization of S. japonicum PHB1 and reveals its fundamental role in the regulation of growth and development of S. japonicum by specific dsRNA-mediated RNAi in vivo. Our findings prompt for a promising molecular of schistosomes that can be targeted to effectively retard the growth and development of the schistosomes.

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.

Comparative Transcriptome Analyses of Schistosoma japonicum Derived From SCID Mice and BALB/c Mice: Clues to the Abnormality in Parasite Growth and Development.

Schistosomiasis, caused by the parasitic flatworms called schistosomes, remains one of the most prevailing parasitic diseases in the world. The prodigious oviposition of female worms after maturity is the main driver of pathology due to infection, yet our understanding about the regulation of development and reproduction of schistosomes is limited. Here, we comparatively profiled the transcriptome of Schistosoma japonicum recovered from SCID and BALB/c mice, which were collected 35 days post-infection, when prominent morphological abnormalities could be observed in schistosomes from SCID mice, by performing RNA-seq analysis. Of the 11,183 identified genes, 62 differentially expressed genes (DEGs) with 39 upregulated and 23 downregulated messenger RNAs (mRNAs) were found in male worms from SCID mice (S_M) vs. male worms from BALB/c mice (B_M), and 240 DEGs with 152 upregulated and 88 downregulated mRNAs were found in female worms from SCID mice (S_F) vs. female worms from BALB/c mice (B_F). We also tested nine DEGs with a relatively higher expression abundance in the gonads of the worms (ovary, vitellaria, or testis), suggesting their potential biological significance in the development and reproduction of the parasites. Gene ontology (GO) enrichment analysis revealed that GO terms such as "microtubule-based process," "multicellular organismal development," and "Rho protein signal transduction" were significantly enriched in the DEGs in S_F vs. B_F, whereas GO terms such as "oxidation-reduction process," "response to stress," and "response to DNA damage stimulus" were significantly enriched in the DEGs in S_M vs. B_M. These results revealed that the differential expression of some important genes might contribute to the morphological abnormalities of worms in SCID mice. Furthermore, we selected one DEG, the mitochondrial prohibitin complex protein 1 (Phb1), to perform double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) in vivo targeting the worms in BALB/c mice, and we found that it was essential for the growth and reproductive development of both male and female S. japonicum worms. Taken together, these results provided a wealth of information on the differential gene expression profiles of schistosomes from SCID mice when compared with those from BALB/c mice, which were potentially involved in regulating the growth and development of schistosomes. These findings contributed to an understanding of parasite biology and provided a rich resource for the exploitation of antischistosomal intervention targets.

Comparative serum metabolomics between SCID mice and BALB/c mice with or without Schistosoma japonicum infection: Clues to the abnormal growth and development of schistosome in SCID mice.

The small blood flukes of genus Schistosoma, which cause one of the most prevalent and serious parasitic zoonosis schistosomiasis, are dependent on immune-related factors of their mammalian host to facilitate their growth and development, and the formation of granulomatous pathology caused by eggs deposited in host's liver and intestinal wall. Schistosome development is hampered in the mice lacking just T cells, and is even more heavily retarded in the severe combined immunodeficient (SCID) mice lacking both T and B lymphocytes. Nevertheless, it's still not clear about the underlying regulatory molecular mechanisms of schistosome growth and development by host's immune system. This study, therefore, detected and compared the serum metabolic profiles between the immunodeficient mice and immunocompetent mice (SCID mice vs. BALB/c mice) before and after S. japonicum infection (on the thirty-fifth day post infection using liquid chromatography-mass spectrometry (LC-MS). Totally, 705 ion features in electrospray ionization in positive-ion mode (ESI+) and 242 ion features in ESI- mode were identified, respectively. First, distinct serum metabolic profiles were identified between SCID mice and BALB/c mice without S. japonicum worms infection. Second, uniquely perturbed serum metabolites and their enriched pathways were also obtained between SCID mice and BALB/c mice after S. japonicum infection, which included differential metabolites due to both species differences and differential responses to S. japonicum infection. The metabolic pathways analysis revealed that arachidonic acid metabolism, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, alpha-linolenic acid metabolism, glycerophospholipid metabolism, sphingolipid metabolism and purine metabolism were enriched based on the differential serum metabolites between SCID mice and BALB/c mice after S. japonicum infection, which was addressed to be related to the retarded growth and development of S. japonicum in SCID mice. These findings provide new clues to the underlying molecular events of host's systemic metabolic changes on the growth and development of S. japonicum worms, and also provide quite promising candidates for exploitation of drugs or vaccines against schistosome and schistosomiasis.

Comparative Metabonomic Investigations of Schistosoma japonicum From SCID Mice and BALB/c Mice: Clues to Developmental Abnormality of Schistosome in the Immunodeficient Host.

The growth and development of schistosome has been affected in the immunodeficient hosts. But it remains unresolved about the molecular mechanisms involved in the development and reproduction regulation of schistosomes. This study tested and compared the metabolic profiles of the male and female Schistosoma japonicum worms collected from SCID mice and BALB/c mice at 5 weeks post infection using liquid chromatography tandem mass spectrometry (LC-MS/MS) platform, in which the worms from SCID mice were the investigated organisms and the worms from BALB/c mice were used as the controls. There were 1015 ion features in ESI+ mode and 342 ion features in ESI- mode were identified after filtration by false discovery rate. Distinct metabolic profiles were found to clearly differentiate both male and female worms in SCID mice from those in BALB/c mice using multivariate modeling methods including the Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA). There were more differential metabolites in female worms than in male worms between SCID mice and BALB/c mice. And common and uniquely perturbed metabolites and pathways were identified among male and female worms from SCID mice when compared with BALB/c mice. The enriched metabolite sets of the differential metabolites in male worms between SCID mice and BALB/c mice included bile acid biosynthesis, taurine and hypotaurine metabolism, sphingolipid metabolism, retinol metabolism, purine metabolism, etc. And the enriched metabolite sets of differential metabolites in female worms included retinol metabolism, alpha linolenic acid and linoleic acid metabolism, purine metabolism, sphingolipid metabolism, glutamate metabolism, etc. Further detection and comparison in transcript abundance of genes of the perturbed retinol metabolism and its associated meiosis process in worms identified clues suggesting accumulated retinyl ester and perturbed meiotic process. These findings suggested an association between the schistosome with retarded growth and development in SCID mice and their perturbed metabolites and metabolic pathways, and provided a new insight into the growth and development regulation of S. japonicum worms from the metabolic level, which indicated great clues for discovery of drugs or vaccines against the parasites and disease with more researches.

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.

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.

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.

[The Effect of Lipopolysaccharide-Induced Mouse B Cell Activation on Schistosoma japonicum Development].

OBJECTIVE: To investigate the effect of lipopolysaccharide (LPS)-induced B cell activation on the development of Schistosoma japonicum. METHODS: Eighteen BALB/c nude mice deficient in T cells and 23 BALB/c SCID mice deficient in T and B cells were used in this study. Each was infected with 30 ± 1 S. japonicum cercariae. The nude (n=9, NL group) and SCID (n=12, SL group) mice then received 2-3 (every two weeks) intraperitoneal injections with LPS (100 µg/mL, 0.2 mL for each mouse). The remaining nude(n=9, N group) and SCID (n=11, S group) mice received PBS injection as control. The mice were sacrificed on days 28 and 36 after infection (n=4/5, 4/5, 5/6, 6/6 for N, NL, S and SL groups, respectively), and adult worms were collected by hepatic portal vein perfusion. The collecting rate of the adult worms was calculated, the body-length measured, and pairs of worms recorded. The liver tissue was collected and digested with 5% KOH, and the number of eggs per gram of liver tissue was calculated. The levels of TGF-ß, IFN-γ and IL-10 in peripheral blood were evaluated. Spleen cell suspension was prepared for detecting the proportion of regulatory B cells (Bregs) in splenic lymphocytes. RESULTS: On day 28 after infection, the body-lengths of male worms in NL and N groups were (7.65±2.85) mm and (5.28±1.64) mm (P<0.01), and those of female worms were (9.64±1.99) mm and (7.49±1.63) mm (P<0.01), respectively. On day 36 after infection, the number of eggs per gram of liver tissue was significantly higher in the NL group than in the N group (1 088±297 vs 715±404, P<0.05), and significantly lower in the SL group than in the S group (217±33 vs 573±160, P<0.01). The proportions of CD(hi)CD5(+)CD19(+) Bregs in N group on days 28 and 36 after infection were (12.73±0.96)% and (37.15±3.04)% (P<0.05), respectively, with no significant difference with that of NL group. The serum levels of TGF-ß and IFN-y on day 28 after infection were significantly different between N and NL groups (TGF-ß, 101.75±46.72 vs 260.90±45.34 pg/mL; IFN-y, 7.91±1.62 vs 14.11±3.72 pg/mL, both P<0.01). Similarly, significant difference was found for the plasma level of IL-10 on day 36 after infection between the S and N groups (41.85±3.14 vs 66.25±4.16 pg/mL, P<0.01), and between the SL and NL groups (44.48±3.87 vs 72.22±17.76 pg/mL, P<0.01), but not between the LPS groups and the control groups. CONCLUSION: LPS can induce the release of cytokines (e.g. TGF-ß) from B cells of mice infected with S. japonium, to facilitate the early development of adult female and male worms.

[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.

[Research progress of immunoregulation of B cells to schistosome infection].

Recent studies found that B cell subsets and their factors have double effects on anti- and aiding schistosome infection. This article summarizes the research progress of positive and negative immunoregulation of schistosome infection involving B lymphocytes, antibody and regulatory B cells (Bregs) relating cytokines (IL-10, IL-7 and TGF-ß).

Effect of cytotoxic T-lymphocyte-associated protein 4 on CD4(+)CD25(+) regulatory T cells in murine schistosomiasis japonica.

In a previous study we demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) contributed to the escape of Schistosoma japonicum (S. japonicum) from the host's immune responses. In this paper, we studied the effect of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on CD4(+)CD25(+) Tregs in murine Schistosomiasis japonica and its corresponding role in the immune evasion of S. japonicum in mice. The results showed substantial reductions of worm burden and egg production in worm groups treated with anti-CD25 or anti-CTLA-4 monoclonal antibodies (mAb) compared to an infected but untreated control. The reduction effect was even enhanced in an experimental group co-treated with both mAbs. Compared to the control group, the percentage of CD4(+)CD25(+) Tregs was very much lower in the anti-CD25 mAb group as determined by FACS analyses and higher in the anti-CTLA-4 mAb group. ELISA analyses showed that both the anti-CTLA-4 mAb and the co-treated groups had higher levels of cytokines compared to the control group as well as larger egg granuloma sizes as determined by microscopical analyses of liver sections of infected mice. These results suggest that treatment with an anti-CTLA-4 mAb allows the host to clear S. japonicum, but at the cost of elevated pathological damage. The latter indicated a role of CTLA-4 in granuloma formation. Moreover, CD4(+)CD25(+) Tregs and CTLA-4 may exert synergistic effects during immune evasion processes by enhancing Th1-type immune response.

In vitro cultivation of Schistosoma japonicum-parasites and cells.

Schistosomiasis is a serious parasitic zoonosis caused by blood-dwelling flukes of the genus Schistosoma. Understanding functions of genes and proteins of this parasite is important for uncovering this pathogen's complex biology, which will provide valuable information to design new strategies for schistosomiasis control. Effective applications of molecular tools reported to investigate schistosome gene function, such as inhibitor studies and transgenesis, rely on the developments of in vitro cultivation system of this parasite and cells. Besides the in vitro culture studies dealing with Schistosoma mansoni, there are also numerous excellent studies about the in vitro cultivation of Schistosoma japonicum, which were performed by Chinese researchers and published in Chinese journals. Nearly every stage of the life-cycle of S. japonicum, including miracidia, mother sporocysts, cercariae, schistosomula, and egg-laying adult worms, was employed for developing in vitro cultivation methods, being accompanied by the introduction of several media and supplements that helped to improve culture conditions. It was not only possible to generate mother sporocysts from miracidia in vitro, but also to obtain adult worms from cercariae through in vitro cultivation. The main obstacles to complete the life cycle of S. japonicum in the lab are the transition from mother sporocysts to cercariae, and the production of fertilized and completely developed eggs by adult worms generated in vitro. With regard to cells from S. japonicum, besides established isolation protocols and morphological observations, media optimizations were conducted by using different chemical reagents, biological supplements and physical treatment. Among these, mutagens like N-methyl-N-nitro-N-nitrosoguanidine and the addition of extracellular matrix were found to be able to induce mitogenic activities. Although enzyme activities or the level of silver-stained nucleolar region associated protein in cultured cells indicated still suboptimal conditions, the achievements made point to the possibility of reaching the aim of establishing cell lines for S. japonicum. Both the improvements of the in vitro culture of larval and adult worms of S. japonicum as well as the access of cells of this parasite provide excellent advances for research on this important parasite in the future.

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.

The new national integrated strategy emphasizing infection sources control for schistosomiasis control in China has made remarkable achievements.

Schistosomiasis japonica remains one important public health concern that cause great loss of humans' health and social-economic development in the Peoples' Republic of China. At the end of 1990s and the beginning of 2000s, there were still about 0.8 million patients and nearly 85 million people living in the epidemic areas around China. We undertook full analysis of the epidemiological data of schistosomiasis taken from the report of schistosomiasis status in People's Republic of China from 1999 to 2010 for effectiveness assessment of China's new strategy for schistosomiasis control nationwide after its implementation since the beginning the 21st century. The schistosomiasis-endemic uncontrolled counties or towns decreased in number from 1,149 in 2002 to 643 in 2010 at a rate of 44%. The number of schistosomiasis patients decreased from nearly 800,000 to less than 326,000 in 2010 at a decrease rate of more than 50%. The number of acute schistosomiasis patients also decreased significantly, and only 43 cases were reported in 2010. The infection rates of cattle in the endemic uncontrolled provinces decreased greatly though the number of cattle and the actual snail habitat areas remained large with no obvious decline. The schistosome infection rates of human and cattle both decreased significantly by more than 64% and 75%. However, most of the uncontrolled schistosomiasis-endemic areas, schistosomiasis patients, and acute cases are generally located in the four provinces (Hunan, Hubei, Jiangxi, and Anhui) of the lake regions in the middle and lower reach of the Yangtze River, and the egg-positive rates in diagnosed human in endemic Hunan and Hubei remained higher than 10%. Therefore, the new strategy of schistosomiasis control via integrated measures emphasizing infection source control is scientific and successful around China, though it is essential to explore an effective and sustainable strategy for schistosomiasis control in the tough lake and marshland regions of China. The four provinces (Hunan, Hubei, Jiangxi, and Anhui) of the lake regions in China are the main battlefield of China's schistosomiasis control in the present and future.