Open Access

A next-generation microarray further reveals stage-enriched gene expression pattern in the blood fluke Schistosoma japonicum

Contributed equally
Parasites & Vectors201710:19

https://doi.org/10.1186/s13071-016-1947-x

Received: 5 November 2016

Accepted: 21 December 2016

Published: 10 January 2017

Abstract

Background

Schistosomiasis is caused by infection with blood flukes of the genus Schistosoma, and ranks, in terms of disability-adjusted life years (DALYs), as the third most important neglected tropical disease. Schistosomes have several discrete life stages involving dramatic morphological changes during their development, which require subtle gene expression modulations to complete the complex life-cycle.

Results

In the current study, we employed a second generation schistosome DNA chip printed with the most comprehensive probe array for studying the Schistosoma japonicum transcriptome, to explore stage-associated gene expression in different developmental phases of S. japonicum. A total of 328, 95, 268 and 532 mRNA transcripts were enriched in cercariae, hepatic schistosomula, adult worms and eggs, respectively. In general, genes associated with transcriptional regulation, cell signalling and motor activity were readily expressed in cercariae; the expression of genes involved in neuronal activities, apoptosis and renewal was modestly upregulated in hepatic schistosomula; transcripts involved in egg production, nutrition metabolism and glycosylation were enriched in adult worms; while genes involved in cell division, microtubule-associated mobility, and host-parasite interplay were relatively highly expressed in eggs.

Conclusions

The study further highlights the expressional features of stage-associated genes in schistosomes with high accuracy. The results provide a better perspective of the biological characteristics among different developmental stages, which may open new avenues for identification of novel vaccine candidates and the development of novel control interventions against schistosomiasis.

Keywords

Schistosoma japonicum Microarray Gene profiling Stage-enriched expression Developmental biology

Background

Schistosomiasis, a debilitating and chronic disease caused by infection with blood flukes (digenetic trematodes) of the genus Schistosoma, remains one of the most significant parasitic diseases worldwide, afflicting more than 230 million people, with about 800 million exposed to the risk of the infection [1, 2]. Schistosomiasis caused about 3.31 million DALYs in 2010, exceeded only by intestinal nematode infections and leishmaniasis, in the list of global neglected tropical diseases [3]. Schistosoma mansoni, S. haematobium and S. japonicum are the three main species of clinical relevance. Currently, there are no practical anti-schistosome vaccines available. The repeated use of a single effective drug, praziquantel, is required for schistosomiasis treatment, while a variety of morbidity management strategies have been adopted for control of the disease [4, 5].

The schistosome life-cycle involves an aquatic snail as an intermediate host and a mammal as definitive host [6]. Schistosome cercariae are shed from infected snails under a light stimulus and are released into water resources. The free-swimming cercariae infect a mammalian host by skin contact. After skin penetration, the larvae lose their tails and transform into schistosomula. Once entering into capillaries or lymphatic vessels, they are carried to the heart and lungs within 3–5 days depending on the species. The lung-stage schistosomula continue migration to the hepatic portal system at about 14-days post-infection, where the juveniles pair up and become sexually mature. Then the schistosomes in copula migrate to the mesenteric veins (S. mansoni and S. japonicum) or the pelvic venous plexus (S. haematobium), where the female worms lay eggs intravascularly, with varied patency periods among the species. Some eggs are lodged in tissues causing disease whereas others enter the intestine or bladder and are excreted from the host. The mature eggs hatch under favourable conditions to release miracidia which penetrate a snail host and develop asexually into mother and then daughter sporocysts, within which cercariae are produced, which are then released from the snail and continue the life-cycle.

The availability of schistosome transcriptome [7, 8] and genome sequences [911] for the three major Schistosoma spp., provides an invaluable resource to profile gene expression across different developmental stages and between the sexes. In this respect, high-throughput technologies, such as microarrays [1218], serial analysis of gene expression (SAGE) [1921], digital gene expression (DGE) [22], and, more recently, RNAseq [23, 24] have been employed in the analysis of gene profiling in schistosomes. These pioneering investigations have provided unique information on developmental-enriched, gender-biased, tissue-specific, strain-specific and host-associated gene expression features within schistosomes [12, 14, 2528], revealing critical insight on the biology of these parasites. With respect to using microarray platforms, the interpretation of microarray experiment depends on the quality of genetic information contained in the collection of DNA templates employed for probe design. The first-generation of schistosome cDNA chips were printed based on EST transcripts, so that the data obtained from these chip experiments resulted in a poor interpretation due to the problems in annotating these ESTs [1214]. We considered it essential to generate a second generation DNA microarray with a well-curated probe design, based on both transcriptomic and genomic sequences, in order to increase our understanding of schistosome biology.

We have constructed a second generation schistosome DNA chip printed with the most comprehensive coverage of probes, designed based on S. japonicum and S. mansoni genomic and transcriptomic sequences for transcriptomic studies [2931]. Here, we have identified stage-enriched transcripts in cercariae, hepatic schistosomula, adult worms and eggs using this next-generation DNA microarray. This study presents a comprehensive view of the expression features of stage-enriched genes for four developmental phases of S. japonicum, and provides novel insights on schistosome developmental biology.

Methods

Parasite materials

Schistosoma japonicum-infected snails (Oncomelania hupensis) were purchased from Hunan Institute of Parasitic Diseases, Yueyang, China. Cercariae were shed from these snails under light stimulation and were collected. Hepatic schistosomula at 14 days post-infection (p.i.) were perfused from S. japonicum-infected New Zealand rabbits via the vascular system. Mixed adult worms were perfused from S. japonicum-infected rabbits at 6 weeks p.i. Schistosome eggs were purified from liver tissues of infected rabbits (6 weeks p.i.) by enzyme digestion [32]. All parasite samples (except eggs) were soaked in RNAlater (Ambion, CA, USA), and stored at -80 °C until total RNA extraction. Total RNA from eggs was isolated immediately after purification.

Total RNA isolation

Total RNA samples were isolated from S. japonicum cercariae, hepatic schistosomula, adult worms and eggs using RNeasy Mini kits (QIAGEN, GmbH, Hilden, Germany) according to the manufacturer’s instructions. Potential contaminating genomic DNA was removed from RNA samples using a Turbo DNA-free kit (Ambion, CA, USA). The quantity of RNA in each sample was assessed by a NanoDropND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The integrity of total RNA in each sample was checked by denaturing agarose gel electrophoresis (Additional file 1: Figure S1).

Microarray construction and hybridization and subsequent data analysis

A schistosome genome-wide microarray was employed for profiling the gene expression in S. japonicum cercariae, hepatic schistosomula, adult worms and eggs. The details regarding the design and construction of the microarray, the hybridization method, and feature extraction have been reported [2933]. For each target sequence, 3 or 4 pairs of complementary oligonucleotide probes (forward and reverse, 60-mer) were designed (in total 145,000 probes). Probes with random sequences were printed as negative controls (background signal), while eight spike-RNA probes from the intergenic sequence of yeast were used as hybridization controls. Microarrays were printed in a 12 × 135 K feature format (Roche NimbleGen) representing 41,982 features. cDNA was labelled with a fluorescent dye (Cy3-dCTP) using a cRNA Amplification and Labelling Kit (CapitalBio, Beijing, China) [34]. Hybridization was performed using three biological replicates for all samples by CapitalBio, Beijing, China. Procedures for array hybridization, washing, scanning, and data acquisition were performed according to the NimbleGen Arrays User’s Guide. The arrays were scanned using a MS200 scanner (NimbleGen Systems) at 2-μm resolution, and NimbleScan software (NimbleGen) was employed to extract fluorescent intensity raw data from the scanned images. Normalized gene expression data were generated using the Robust Multichip Average (RMA) algorithm [35, 36]. Outlier probes were identified and their contribution was reduced at the reported gene expression level [36]. The expression value of a gene is a weighted average of all forward or reverse probe sets when both background correction and quantile normalization are performed.

Bioinformatics analysis on stage-enriched mRNA and EST transcripts

mRNA and EST transcripts highly enriched in cercariae, hepatic schistosomula, adult worms and eggs of S. japonicum were retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/sites/batchentrez) based on fold-change (FC = the mean intensity/the median of the mean intensity values of the four developmental stages) values. (FC ≥ 2 for both forward and reverse probe sets, and three biological replicates were used for each stage). Student’s t-test was used to determine differentially expressed genes between one particular stage and any of the other three stages [28, 30] (P < 0.05). Heat maps were constructed based on the transformed log2FC values (forward probe sets) using HemI 1.0 software [37]. Blast2GO was used to annotate the four gene sets functionally [38]. A comprehensive re-annotation was performed against these gene sets using the BLASTx algorithm, with the annotation of S. mansoni, S. haematobium and Clonorchis sinensis homologues as a reference. For possible improved annotation, potential conserved protein domains were searched against genes annotated as hypothetical protein or unknown in the NCBI CDD database (v3.14) [39].

Quantitative real-time PCR

A total of 20 stage-enriched genes were selected for validation using qRT-PCR as described [29]. One microgram total RNA samples were reverse transcribed into first-strand cDNA using a SuperScript III Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) with oligo (dT) 15 primer. The cDNA products were diluted 20-fold with nuclease-free water before undertaking the qPCR. Each 25 μl PCR reaction contained 12.5 μl of 2 × Brilliant II SYBR Green QPCR Master Mix (Agilent, USA), 1 μl cDNA, 1 μl of the forward and reverse primer pair (Additional file 2: Table S1), and 10.5 μl of sterile water. PCR cycling conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 30 s denaturation at 95 °C and 1 min annealing and extension at 60 °C. A dissociation step (95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s and 60 °C for 15 s) was performed to confirm the amplification specificity for each gene. 26S proteasome non-ATPase regulatory subunit 4 (PSMD4) [29, 40] was employed as a house-keeping gene in the assays. PCR reactions were performed in technical triplicates on the 7300 Real-Time PCR system (Applied Biosystems). The relative expression level of each gene was analysed using SDS 1.4 software (Applied Biosystems). Correlations between the microarray and qPCR results for 20 stage-enriched genes were analysed with the Spearman’s rho.

Results and discussion

Global view of stage-enriched mRNA transcripts in S. japonicum

By employing a microarray with the most comprehensive probe coverage design to date, signal intensities from 3571, 1014, 1728 and 3381 sequences were found to be enriched (FC of mean of intensity value to the median of the mean of intensity values of the four stages ≥ 2) in cercariae, hepatic schistosomula, adult worms and eggs, respectively. Based on the initial screening, we further retrieved a total of 1768 potential mRNA transcripts and 470 expressed sequence tags (ESTs) associated with developmental stages from the NCBI database (Additional file 3: Table S2). The gene collection was further filtered by requiring FC values from both forward and reverse probe sets ≥ 2. This filtration finally retained 328, 95, 268 and 532 mRNA transcripts highly enriched in cercariae, hepatic schistosomula, adult worms and eggs, respectively (Additional files 4, 5, 6 and 7: Tables S3–S6), which contrasted with 128, 31, 83 and 84 ESTs, respectively, highly enriched in these four stages (Additional files 8, 9, 10 and 11: Tables S7–S10). However, the percentage of genes that were annotated as hypothetical protein or unknown (23.57% in the mRNA data in contrast to 69.01% in the EST data), highlights the utility of the second generation S. japonicum DNA chip in profiling gene expression in this parasite.

We observed that more mRNA transcripts were enriched in the egg stage than in the other stages, with a stronger biased expression (higher FC value) (Figs 1 and 2a-d). For example, 46.1% egg-enriched mRNA transcripts showed a strong biased expression (FC > 10); this number decreased to 22.0% in adult worms, and further dropped to only 3.0 and 1.1% in cercariae and hepatic schistosomula, respectively. A similar tendency was observed when analysing the stage-enriched EST transcripts (Additional file 12: Figure S2). In regards to fluorescence intensity, 13.4, 8.42, 25.0 and 27.5% mRNA transcripts enriched in cercariae, hepatic schistosomula, adult worms and eggs, respectively showed an average intensity value > 10,000 (Fig. 2e-h).
Fig. 1

Heatmap for mRNA transcripts enriched in cercariae, hepatic schistosomula, adult worms and eggs. A total of 328, 95, 268 and 532 mRNA transcripts were enriched in cercariae, hepatic schistosomula, adult worms and eggs, respectively. The heatmap was created by HemI 1.0 based on the transformed data of log2 FC values. The data are based on the mean of weighted signal intensity values of forward probe sets (three biological replicates)

Fig. 2

Bias ratio and signal intensity analysis of stage-associated genes. Scatter plot showing the distribution of the bias ratio and fluorescence intensity for mRNA transcripts enriched in cercariae (a), hepatic schistosomula (b), adult worms (c), and eggs (d). The y-axis corresponds to bias ratios (FC value) and the x-axis corresponds to the fluorescence intensities, both of which are log10-transformed. Pie diagrams representing the percentage of stage-enriched mRNA transcripts in cercariae (e), hepatic schistosomula (f), adult worms (g), and eggs (h) showed by different fluorescence intensities

Comparing the results with previous transcriptome data

A complete and accurate comparison of the results obtained in the current study with data from previous reports is hindered due to the following reasons. Firstly, the annotation of stage-enriched genes was not ideal in previous reports due to the fact that EST sequences were used for probe design coupled with less sequence homology information from other trematode species being available. Secondly, the annotation for the same gene may not have been unique. Thirdly, the screening criteria for stage-enriched genes may have varied among different studies. Nevertheless, we compared our data with these from previous Schistosoma transcriptome data [7, 13, 14, 23, 28] by manual checking. Globally, about 4.57, 10.07 and 12.97% genes enriched in cercariae, adult worms and eggs, respectively, were reported in previous studies (Additional files 4, 5 and 7: Tables S3, S4, S6). With respect to hepatic schistosomula (14 days p.i.), to our knowledge the only other relevant investigation on this particular stage was carried out on S. mansoni by Fitzpatrick et al. [28], but no enriched gene clustering was evident in that study. This was probably due to the fact a large number (15) of distinct stages were analysed [28], and this has made comparison with our data for hepatic schistosomula difficult.

qPCR validation of the expression pattern of stage-enriched genes

A subset of 20 representative stage-enriched genes was selected for qRT-PCR validation (Figs 3 and 4a-d). Most genes were associated with important biological functions in each of the parasite forms. The expression of these genes at the four developmental stages validated by qRT-PCR analysis significantly correlated with the results obtained by microarray: for cercariae-enriched genes selected, r (30) = 0.8959, P < 0.0001 (Fig. 4e); for hepatic schistosomula-enriched genes selected, r (30) = 0.7375, P < 0.0001 (Fig. 4f); for adult-enriched genes selected, r (20) = 0.9082, P < 0.0001 (Fig. 4g); for egg-enriched genes selected, r (21) = 0.8983, P < 0.0001 (Fig. 4h).
Fig. 3

Twenty stage-enriched genes selected for qPCR validation. The heat map illustrates the hierarchical clustering of 20 stage-enriched genes based on the transformed data of log2 FC value of the three biological replicates. Abbreviations: C, cercariae; S, hepatic schistosomula; A, adult worms; E, eggs

Fig. 4

qPCR validation of stage-enriched genes. The expression of 5 selected genes enriched in cercariae (a), hepatic schistosomula (b), adult worms (c), and eggs (d), respectively, was quantified by qRT-PCR analysis. The PSMD4 gene was used for internal normalization among the four developmental stages. The highest expression level in one particular stage was set as 1. The error bars represent the standard deviation for three technical replicates. Correlations between the microarray and qPCR results for the selected genes enriched in cercariae (e), hepatic schistosomula (f), adult worms (g), and eggs (h), were analysed using Spearman’s rho

Putative functions predicted by GO analysis

We analysed the potential biological functions of the stage-enriched genes in S. japonicum using GO classification [41] (Fig. 5, Additional files 13, 14, 15 and 16: Tables S11–S14). Of the biological process categories, the most highly enriched GO terms were organic substance metabolic process, single-organism cellular process, primary metabolic process and cellular metabolic process for cercariae, adult worms and eggs; the first three of these GO terms and regulation of cellular process were the most highly enriched GO terms for hepatic schistosomula. The percentages of genes involved in regulation of cellular process, cellular response to stimulus, and single organism signaling were higher in cercariae and schistosomula than those in adults and eggs. Of the molecular function categories, the percentages of genes involved in ion, heterocyclic compound and organic cyclic compound, small molecule and carbohydrate derivative binding were higher in cercariae and schistosomula than in adults and eggs. A higher percentage of genes related to protein binding, signaling receptor activity and receptor activity were observed in schistosomula, while the GO term extracellular matrix structural constituent was only evident for this stage. In addition, a higher percentage of genes involved in hydrolase activity were assigned to adult worms. In the cellular component categories, gene products localised to intracellular, intracellular part and intracellular organelle were more abundant in cercariae, while gene products localised to intrinsic component of membrane were more enriched in the other three stages. Further, genes with GO terms of protein complex, cell periphery, plasma membrane, plasma membrane part and proteinaceous extracellular matrix were relatively enriched in hepatic schistosomula. In addition, the GO term cilium was present only in the egg stage.
Fig. 5

GO analysis of mRNA transcripts enriched in the four developmental stages of S. japonicum. The Blast2Go program defined the GO terms into three categories: biological processes (a), molecular functions (b) and cellular component (c). The y-axis shows the ratio of the number of mapped genes versus total number of genes in each cognate stage identified as a function of all available GO terms. The x-axis shows GO terms at the 3rd level

The top 25 genes enriched in S. japonicum cercariae, hepatic schistosomula, adult worms and eggs

The top 25 highly stage-associated genes in cercariae, hepatic schistosomula, adult worms and eggs were analysed (Table 1). Collectively, the upregulated expression of these genes in cercariae indicates that signal transduction (ribosomal protein S6 kinase beta-2 [42]), vesicular trafficking (calcium-dependent secretion activator [43] and small GTPase Rab-protein 11 [44]) and energy metabolism (AMP deaminase [45] and 5′-AMP-activated protein kinase [46]) and transcriptional regulation (krueppel-like factor 11, homeobox protein SMOX-1, and retinoid X receptor RXR-2) are active processes in this stage.
Table 1

The top 25 genes enriched in S. japonicum cercariae, hepatic schistosomula, mixed adult worms and eggs

NCBI Nucleotide

NCBI Protein

Annotation

FC

Enriched in cercariae

 AY811679.1

AAX27568.2

Tegumental antigen

94.004

 AY812964.1

AAW24696.1

Lysophosphatidic acid phosphatase type 6

89.015

 AY808793.1

AAX24682.2

Krueppel-like factor 11

20.463

 AY814888.1

AAP06195.1

Hypothetical protein

20.323

 AY915869.1

AAX31090.1

UPF0506 domain containing protein

15.144

 AY811006.1

AAX26895.2

Putative sodium-dependent transporter

14.884

 FN319257.1

CAX74986.1

Ribosomal protein S6 kinase beta-2

13.668

 AY813254.1

CAX83692.1

Gag-Pol polyprotein

11.090

 AY812158.1

AAX28047.2

Calcium-dependent secretion activator

10.898

 FN327240.1

CAX82964.1

UPF0364 protein

10.005

 FN319112.1

CAX74840.1

Anti-inflammatory protein 16

9.750

 AY809199.1

AAX25088.2

Dynein light intermediate chain 1 cytosolic

9.060

 AY815066.1

AAW26798.1

Calpain

8.200

 FN314407.1

CAX70140.1

Rab-protein 11

8.118

 AY813232.1

AAW24964.1

DM9 domain-containing protein

7.327

 AY915497.1

AAX30718.2

Homeobox protein SMOX-1

7.320

 AY813605

AAW25337.1

Hypothetical protein

7.234

 FN319705.1

CAX75429.1

THO complex subunit 1

6.827

 AY813585.1

AAW25317.1

Hypothetical protein

6.756

 AY811834.1

AAX27723.2

AMP deaminase

6.524

 AY813088.1

AAW24820.1

Hypothetical protein

6.357

 FN314484

CAX70217.1

Hypothetical protein

6.196

 AY811464.1

ABA40369.1

5′-AMP-activated protein kinase subunit gamma-1

6.165

 EU046089.1

AAW25910.1

Cercarial stage-specific protein Sj20H8

6.075

 AY808884.1

AF129816_1

Retinoid X receptor RXR-2

6.011

Enriched in hepatic schistosomula

 AY809629.1

AAX25518.2

Hypothetical protein

33.897

 AY810683

AAX26572.2

Putative collagen alpha-1(V) chain precursor

9.200

 AY815366.1

AAW27592.1

Alpha-ketoglutarate-dependent dioxygenase alkB 6

6.931

 AY813429.1

AAW25161.1

Hypothetical protein

5.290

 AY810949.1

AAX26838.2

Homeobox protein engrailed-like SMOX-2

5.057

 EZ000055.1

ACE06835.1

Vacuolar protein sorting-associated protein 29

5.021

 AY810397.1

AAX26286.2

Protocadherin Fat 4

4.839

 AY811075.1

AAX26964.2

Hypothetical protein

4.831

 AY815532.1

AAW27264.1

Hypothetical protein

4.727

 AY814356

AAW26088.1

RhoGAP domain containing protein

4.610

 AY811025.1

AAX26914.2

Serine/threonine-protein kinase Sgk1

4.342

 AY809477.1

AAX25366.2

SAM and SH3 domain-containing protein 1

4.248

 FN314446.1

CAX70179.1

Annexin A3 (Annexin III)

4.037

 AY814048.1

AAW25780.1

Basic proline-rich protein-like isoform

3.967

 AY808501.1

AAR28090.2

Nuclear receptor subfamily 4 group A

3.956

 AY809584.1

AAX25473.2

Hypothetical protein

3.894

 AY812287.1

AAX28176.2

Run domain Beclin-1 interacting and cysteine-rich containing protein

3.806

 AY813648.1

AAW25380.1

Hypothetical protein

3.439

 AY915540.1

ABA40872.1

Leishmanolysin-like peptidase

3.419

 AY812557.1

AAX28446.2

Aromatic-L-amino-acid decarboxylase

3.335

 AY808377.1

AAX24266.2

Regulator of G-protein signaling 3

3.250

 FN313634.1

CAX69368.1

Collagen alpha-2(I) chain

3.244

 AY813683.1

AAW25415.1

Delphilin

3.240

 AY812144.1

AAX28033.2

Hypothetical protein

3.212

 AY813563

AAW25295.1

Hypothetical protein

3.203

Enriched in mixed adult worms

 FN314868.1

CAX70600.1

Asparagine-rich antigen Pfa35-2

1651.245

 EZ000096

ACE06876.1

Putative eggshell protein precursor

934.084

 FN314999

CAX70731.1

TES domain containing protein

704.455

 AY813556.1

AAW25288.1

Hypothetical protein

692.180

 AY814029

AAW25761.2

Stress protein DDR48 (DNA damage-responsive protein 48)

678.514

 FN313935.1

CAX69669.1

Stress protein DDR48 (DNA damage-responsive protein 48)

665.581

 FN317103

CAX72834.1

Stress protein DDR48 (DNA damage-responsive protein 48)

645.627

 FN313912

CAX69646.1

TES domain containing protein

604.574

 FN313715.1

CAX69449.1

TES domain containing protein

561.444

 AY812810.1

AAW24542.1

Histidine-rich glycoprotein precursor

526.698

 FN315504.1

CAX71236.1

TES domain containing protein

517.929

 AY815518

AAW27250.1

TES domain containing protein

489.519

 FN314997

CAX70729.1

TES domain containing protein

422.784

 AY813405

AAW25137.1

TES domain containing protein

407.588

 AY815264.1

AAW26996.1

Tyrosinase 1

346.094

 AY812315.1

AAX28204.2

Hypothetical protein

330.410

 FN330801

CAX83018.1

Stress protein DDR48 (DNA damage-responsive protein 48)

235.455

 AY814142.1

AAW25874.1

Putative FAM75 family member

224.325

 AY812904

AAW24636.1

Tyrosinase 2

209.523

 FN315510.1

CAX71242.1

Hypothetical protein

164.941

 AY814814

AAW26546.1

Cadherin

145.264

 AY815418

AAW27150.1

Female-specific protein 800

135.097

 FN316955

CAX72686.1

Prostatic spermine-binding protein precursor

132.448

 AY222885

AAP05897.1

Stress protein DDR48 (DNA damage-responsive protein 48)

127.238

 FN314903.1

CAX70635.1

Hypothetical protein

107.908

Enriched in eggs

 FN317800

CAX73529.1

Glutenin high molecular weight subunit DX5

1794.846

 FN319280

CAX75008.1

Tetraspanin 22

1769.270

 FN322023.1

CAX77751.1

Histidine-rich glycoprotein

1656.913

 FN324495.1

CAX80219.1

Hypothetical protein

1549.720

 FN326817

CAX82541.1

Histidine-rich glycoprotein

1523.735

 FN317759.1

CAX73488.1

Similar to venom allergen-like (VAL) 25 protein

1062.695

 FN324480.1

CAX80126.1

Hypothetical protein

938.553

 FN321785

CAX77509.1

Ribonuclease T2

850.487

 FN321171.1

CAX76897.1

Hypothetical protein

831.194

 FN324498.1

CAX80222.1

Hypothetical protein

776.801

 FN319117.1

CAX74843.1

CIA30 domain containing protein

665.147

 FN317754

CAX73483.1

Tetraspanin

663.055

 FN322724.1

CAX78439.1

Peptidase inhibitor 16

651.579

 FN319142

CAX74870.1

Hypothetical protein

628.202

 FN320551

CAX76277.1

Egg protein CP1531

592.491

 FN326664

CAX82388.1

Hypothetical protein

577.505

 AY816014.1

AAW27746.1

Ribonuclease S-4

534.668

 FN321764.1

CAX77484.1

Cell wall integrity and stress response component 1

488.342

 FN326758

CAX82480.1

Hypothetical protein

484.608

 FN317167

CAX72898.1

Hypothetical protein

481.352

 FN319216.1

CAX74944.1

Hypothetical protein

453.890

 FN320451

CAX76177.1

GLIPR1-like protein 1/venom allergen-like protein 5

422.820

 FN317231

CAX72962.1

GLIPR1-like protein 1/venom allergen-like protein 5

417.438

 FN326877

CAX82601.1

Hypothetical protein

416.455

 FN330952.1

CAX83183.1

Ribonuclease Oy

414.347

The over-expression of the top 25 genes in hepatic schistosomula appears to reflect a diversity of physiological activities, including transcriptional (homeobox protein engrailed-like SMOX-2 [47, 48], serum and glucocorticoid-regulated kinase 1 (SGK1) [49] and nuclear receptor subfamily 4 group A [50, 51]) and neuronal (protocadherin FAT4 [52], Aromatic-L-amino-acid decarboxylase [53] and delphilin [54]) activities, together with tegumental integrity (annexin A3 [55, 56]), skeletal morphogenesis (protocadherin FAT4 [57]) and endosome-to-Golgi retrieval (vacuolar protein sorting-associated protein 29 [58]).

In mixed adult worms, genes encoding a number of trematode eggshell synthesis (TES) domain-containing proteins, DDR48 stress proteins, an asparagine-rich antigen Pfa35-2, two distinct tyrosinase homologues, cadherin, female-specific protein 800 and a prostatic spermine-binding protein are listed in the top 25 enriched mRNA transcripts (Table 1). Most of these genes are female-biased expressed genes [59] with potential molecular functions in egg production [60].

In the egg stage, genes encoding a glutenin high molecular weight subunit DX5, egg protein CP1531, two histidine-rich glycoproteins, three ribonucleases, two tetraspanins, three venom allergen-like (VAL) proteins and cell wall integrity and stress response component 1 are present in the top 25 upregulated mRNA transcripts (Table 1). Notably, it has been shown that T2 ribonuclease omega-1 in soluble egg antigen is a major Th2 polarizing component, which is capable of regulating inflammasome activity [61]. It has been shown previously that VAL-5 is mainly present in the egg, miracidium and sporocyst developmental stages [62].

Genes enriched in cercariae

Interestingly, a group of genes encoding transcription factors, i.e. homeobox protein SMOX-1 (AY915497), bhlhzip transcription factor max/bigmax (FN314500), pre-B-cell leukemia transcription factor 2 (AY809282), transcription factor 25 (AY808969), 20 (AY813668), BTF3 (EZ000130), TFIID subunit 3 (AY812404) and 7 (FN317813), IIIB subunit (AY812330), LIM/homeobox protein (AY915618) and transcriptional repressor NF-X1(AY813973) were actively transcribed in cercariae (Additional file 4: Table S3), indicating gene transcription may not be as silent as previously suggested in this stage. It has been shown that the highest ratio of miRNAs, the critical post-transcriptional regulators, in the total small RNA population was observed in cercariae compared with other different developmental stages of S. japonicum [32, 63], leading us to hypothesise that a specific group of genes may be actively transcribed in this aquatic stage. In addition, miRNAs may inhibit the translation of a subset of these transcripts, forming a repertoire of genes that make schistosomula ready to adapt to subsequent intra-mammalian life. Further, there is epigenetic control of gene expression in S. mansoni cercariae [64]. Overall, these observations indicate that active transcriptional regulation occurs at different layers in cercariae to subtly control gene expression in this stage.

We also observed that an extensive gene panel involved in cellular signalling transduction, i.e. F-box protein 25/32 (EZ000162), dual specificity mitogen-activated protein kinase 2 (AY815572), Serine/threonine kinase NLK (FN317434), Rho GTPase-activating protein 39 (FN317833), GDP/GTP exchange factor Sec2p domain containing protein (FN317362), Rho-associated protein kinase 1 (FN330915), mitogen-activated protein kinase 3 (EZ000180), Ran binding protein 9-related protein (AY812647), GTP-binding protein 2 (FN317377), NF-kappa-B inhibitor-interacting Ras-like protein 1 (AY812481), son of sevenless (AY915633), MAP kinase (AY594257), C-Jun-amino-terminal kinase-interacting protein 4 (AY808598), and regulator of G-protein signaling 7 (AY810841), were over-expressed in cercariae (Additional file 4: Table S3). These results support recent finding that three signaling pathways, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and protein kinase C (PKC), are modulated in cercariae in response to light and temperature cues as well as the skin fatty acid linoleic acid (LA) and are important in host penetration mechanisms [65].

In line with, and expanding on, previous transcriptional studies on schistosomes [13, 14, 66], genes encoding an array of cytoskeleton motor proteins, including dynein light intermediate chain 1, cytosolic (AY809199), troponins (FN317001 and AY809606), tensin-1 (AY809674), villin (AY808977), myosin light chain kinase, actin-related protein 5 (FN326677), dynamin (AY809889), catenin beta (AY814842), coronin (AY814365), dynein light chain Tctex-type 1 (AY811669) and alpha-actinin (FN326862) (Additional file 4: Table S3) were more highly expressed in cercariae than the other stages evaluated. Transcripts encoding LIM or PDZ domain-containing proteins, which contribute to cytoskeletal organisation, such as LIM/homeobox protein (AY915618), actin binding LIM protein 1 (AY813306), four and a half LIM domains protein 2 (FN317368), and PDZ and LIM domain protein 7 (FN317962) (Additional file 4: Table S3), were also enriched in cercariae. Proteomic studies also revealed that cytoskeleton-related proteins are abundant in schistosome cercariae [67]. Together, these data indicate modulated signalling and motor activities and rigid transcriptional regulation are the most important biological events in cercariae, which enable them to seek, invade and adapt to a suitable definitive host.

Genes enriched in hepatic schistosomula

On invading a mammalian host, schistosomes have evolved several mechanisms to adapt to, and survive in, the hostile host environment; in particular, they develop a unique syncytial tegument, as well as mechanisms of antigenic mimicry [33], immune modulation [68] and evasion [69, 70]. In this study, we found extracellular matrix constituents, that are located in the tegumental protein assemblage, were enriched in hepatic schistosomula. These collagen components included, for example, collagen alpha-1(V) chain (AY810683, AY811988, and AY815998), alpha-1(IV) chain (AY809845), alpha-1(XXIV) chain (AY814344), alpha-2(I) chain (AY810097, FN313634) and alpha-2(V) chain (AY813923) (Additional file 5: Table S4). This observation raises the possibility that collagen components may form a protective barrier on the worm surface, which may help the schistosomula evade host attack.

Schistosomula undertake a lengthy migration in the mammalian host to the portal venous system, where they mature into adult worms and pair. This migration is closely associated with locomotion activity controlled by the neuronal system. The data presented here show that neuronal activities may be particularly active in hepatic schistosomula, which could be linked to the fact that responses to environmental cues from the host and the subsequent control of mobility are required to guarantee that they reach their destination [22]. A cohort of genes involved in neuronal activities in this stage includes netrin receptor unc5B (AY915275), nephrin (AY809045), caskin 2 (AY812623), spondin-1 (AY812421), as well as the previously described genes protocadherin FAT4, aromatic-L-amino-acid decarboxylase and delphilin. Although the precise functions of these genes in schistosomes remain unknown, there is evidence from other studies that at least three are involved in axon guidance. In mammals, it has been shown that the unc5B receptor, interacting with netrin-1, activates the downstream signal transduction pathway that mediates axon guidance [71]. A caskin ortholog in Drosophila is a cytoplasmic adaptor protein, which has been shown to mediate Lar signal transduction motor axon guidance [72]. Similarly, spondin-1 is an extracellular matrix protein, and previous research showed that its C. elegans ortholog functions in axon guidance and fasciculation in motoneurons [73]. Also, the expression of nephrin homologues has been observed in the central nervous system of mammals, and nephrin may potentially interact with glutamate receptors [74, 75].

In multicellular organisms, apoptosis is a highly controlled cellular process of programmed cell death which plays a key role in maintaining cell populations during an organism’s life-cycle. The apoptosis pathway has been suggested as a potential intervention target in schistosomes [76]. The activities of two central proteolytic enzymes involved in the apoptosis process, caspase-3 and -7, were shown to peak in S. japonicum schistosomula (14 days p.i.) [77]. The upregulated expression of caspase 7 (AY813428) in hepatic schistosomula was confirmed in this study (Additional file 5: Table S4). It is of note that a cohort of planarian neoblast-like cells with self-renewal function has been identified in S. mansoni, with a potential role in renewal of the tegument [78]. In this respect, fibroblast growth factor receptor 2, a crucial gene for the maintenance of neoblast-like cell population in schistosomes [79], was enriched in hepatic schistosomula (Additional file 5: Table S4), emphasising the requirement for rapid tegumental renewal during this period of fast-growth.

Genes enriched in adult worms

One of the major biological roles of adult worms is to produce a large number of eggs, a key process in the schistosome life-cycle. As earlier mentioned, within the top 25 adult-enriched genes, most are associated with egg production. However, two pre-requisites for egg production are mating and nutrient acquisition. In fulfilment of the former process, the gene encoding gynecophoral canal protein has been shown upregulated in adults, with a dramatic bias towards male worms [59]. In regards to nutrient uptake, and consistent with a previous study [18], over-expression of a number of ‘blood processing’ proteases in adult worms was also revealed here. For instance, cathepsin family members, i.e. cathepsin C (FN315267), cathepsin D2-like (AY812817), cathepsin B-like (AY814095), cathepsin L (FN313884) and cathepsin L-like isoforms (AY222874, FN314782, and FN314778), and aminopeptidase N (FN317672) were readily identified as adult worm-enriched genes (Additional file 6: Table S5). In addition, saposin B domain-containing proteins (FN314931, FN315898 and FN314355), which have been proposed as being involved in nutrient acquisition by disrupting the membrane of red blood cells to release haemoglobin [80], were highly expressed in adult worms.

In schistosomes, glycosylation is a complex process which plays a crucial role in their biology, particularly in terms of immune modulation [81]. A subset of transcripts involved glycosylation in was enriched in adult worms of S. japonicum. These genes included beta-1,4-galactosyltransferase 4 (AY813412), glycosyltransferase 1 domain-containing protein 1 (FN319898), GDP-fucose protein O-fucosyltransferase 2 (AY810860), beta-1,3-galactosyltransferase 5 (AY814132), glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1 (AY809881), glycoprotein 3-alpha-L-fucosyltransferase A (FN317387), alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (AY812621), and alpha-L-fucosidase-like protein (FN317475) (Additional file 6: Table S5). However, given the inherent complexity of glycosylation and that multiple glycosyltransferases responsible for similar molecular functions are present in the Schistosoma genomes [81, 82], it is difficult to conclude that the global level of glycosylation or the expression of specific glycans is higher in adults than in the other stages examined here.

Genes enriched in eggs

Globally, genes associated with the egg stage are involved in a diversity of biological functions, which may be the result of using samples for analysis that comprise a mixture of immature and mature eggs. In addition to anticipated genes encoding egg proteins, immunogenic miracidial antigens and major egg antigens, a number of genes involved in the cell cycle and proliferation, including meiosis expressed protein 1 (FN317540), meiosis-specific nuclear structural protein 1 (AY810474), mitogen-activated protein kinase 15 (FN317209), putative chromosome segregation protein SMC (AY812773), different isoforms of leishmanolysin-like peptidase (AY811259, FN317512, AY810562 and FN319863) and probably protein VHS3 (FN330961), placenta-specific gene 8 protein (FN317134), placental protein 25 homolog (FN317187) and centrosomal protein of 162 kDa (AY810094), were upregulated in eggs (Additional file 7: Table S6). These transcripts may be enriched in immature eggs, hinting that active cell division is essential for embryonic development.

Further, a group of transcripts encoding tubulin and microtubule-associated motor proteins, i.e. tubulin alpha (FN317215), tubulin beta (FN320386), tubulin beta-2C chain (FN320061), cytoplasmic dynein light chain 1 (FN317588) and 2 (AY914882), dynein light chain 1, axonemal (FN317727), inner dynein arm light chain, axonemal (FN317915), outer dynein arm protein 1 (AY813443), dynein heavy chain 5, axonemal (AY810177), as well as the ciliary and flagellar microtubule components, i.e. tektins (AY814061, AY914954, FN317819 and FN314465), dynein intermediate chain 3 (AY810742) and outer dense fibre protein 3-B (FN318315) were over-expressed in eggs (Additional file 7: Table S6). These transcriptional differences may reflect the fact that a miracidium is enclosed in the eggshell of the mature egg, and once the egg is released into the external environment and contacts freshwater, a high level of movement is required for the larva to hatch and escape from the eggs [83], and to seek the snail intermediate host in order to establish an infection.

Though the miracidium is enclosed by an eggshell, an active parasite-host interplay takes place via pores in the egg [83]. On one hand, nutrients (e.g. iron, amino acid and lipid) are acquired by eggs from the host, a process supported by the upregulation of genes involved in transport and exchange activities, such as putative sodium-dependent transporter (FN318875), sodium/hydrogen exchanger (AY815720), sodium/calcium exchanger (FN318247), large neutral amino acids transporter small subunit 2 (FN327074), Y + L amino acid transporter 2 (FN313722), high-affinity choline transporter 1 (FN317430), iron channels (i.e. voltage-gated hydrogen channel (FN318209), two pore calcium channel protein 2 (FN326741), and TWiK family of potassium channels protein (AY813707), and lipid metabolism (i.e. fatty acid-binding protein (FN318753) (Additional file 7: Table S6). On the other hand, it has been shown that major egg products from S. mansoni such as ribonuclease omega-1, kappa 5 (FN329842) and IPSE/alpha-1 are released into host tissues and modulate host immune responses [8487]. In this study, S. japonicum homologues of ribonuclease omega-1 (FN330952) and kappa 5 (FN321248) were also enriched in the egg stage, although as yet, no homologue of IPSE/alpha-1 has been identified in this schistosome species.

Conclusions

In this study, we present the most comprehensive transcriptomic profile to date of four stage-associated genes in S. japonicum based on a next-generation DNA chip. The study has revealed the key biological and physiological features of the four development stages: cercariae, hepatic schistosomula, adult parasites and eggs. Overall, this study adds new insights on the developmental biology of S. japonicum which further the discovery of novel intervention targets against this persistent parasite and the disease it causes.

Abbreviations

CDS: 

Coding DNA sequences

DALYs: 

Disability adjusted life years

DGE: 

Digital gene expression

ERK: 

Extracellular signal-regulated kinase

ESTs: 

Expressed sequence tags

FC: 

Fold-changes

LAPs: 

Hydrolysis of lysophosphatidic acids

MAPK: 

Mitogen-activated protein kinase

PKC: 

Protein kinase C

SAGE: 

Serial analysis of gene expression

TES: 

Trematode eggshell synthesis

UTRs: 

Untranslated regions

VAL: 

Venom allergen-like

Declarations

Acknowledgements

We thank the Chinese National Genome Center at Shanghai for making S. japonicum genome publicly available.

Funding

This study was supported by the National Natural Science Foundation of China (Grant No. 81270026), the National S & T Major Program (Grant No. 2012ZX10004-220), the Special Fund for Health Research in the Public Interest (Grant No. 201202019), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT13007). DPM is a NHMRC Senior Principal Research Fellow and Senior Scientist at QIMR Berghofer Medical Research Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

Raw data and the normalized data have been deposited at the public domain Gene Expression Omnibus under the accession number for the platform GPL18617 and series GSE57143.

Authors’ contributions

PC and QC conceived the project and designed the strategy. PC, SL, XP and NH carried out the experiments. PC, SL, DPM and QC analysed the data. PC, HY, DPM and QC wrote the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

All procedures performed on animals within this study were conducted following animal husbandry guidelines of the Chinese Academy of Medical Sciences and with permission from the Experimental Animal Committee (Institute of Pathogen Biology, CAMS) with Ethical Clearance Number IPB-2011-6.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College
(2)
Molecular Parasitology Laboratory, QIMR Berghofer Medical Research Institute
(3)
Key Laboratory of Zoonosis, Shenyang Agriculture University

References

  1. Steinmann P, Keiser J, Bos R, Tanner M, Utzinger J. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect Dis. 2006;6:411–25.View ArticlePubMedGoogle Scholar
  2. Weerakoon KG, Gobert GN, Cai P, McManus DP. Advances in the diagnosis of human schistosomiasis. Clin Microbiol Rev. 2015;28:939–67.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Hotez PJ, Alvarado M, Basanez MG, Bolliger I, Bourne R, Boussinesq M, et al. The global burden of disease study 2010: interpretation and implications for the neglected tropical diseases. PLoS Negl Trop Dis. 2014;8:e2865.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Mutapi F, Rujeni N, Bourke C, Mitchell K, Appleby L, Nausch N, et al. Schistosoma haematobium treatment in 1-5 year old children: safety and efficacy of the antihelminthic drug praziquantel. PLoS Negl Trop Dis. 2011;5:e1143.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Chen MG. Assessment of morbidity due to Schistosoma japonicum infection in China. Infect Dis Poverty. 2014;3:6.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Cai P, Gobert GN, You H, McManus DP. The Tao survivorship of schistosomes: implications for schistosomiasis control. Int J Parasitol. 2016;46:453–63.View ArticlePubMedGoogle Scholar
  7. Hu W, Yan Q, Shen DK, Liu F, Zhu ZD, Song HD, et al. Evolutionary and biomedical implications of a Schistosoma japonicum complementary DNA resource. Nat Genet. 2003;35:139–47.View ArticlePubMedGoogle Scholar
  8. Verjovski-Almeida S, DeMarco R, Martins EA, Guimaraes PE, Ojopi EP, Paquola AC, et al. Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni. Nat Genet. 2003;35:148–57.View ArticlePubMedGoogle Scholar
  9. Berriman M, Haas BJ, LoVerde PT, Wilson RA, Dillon GP, Cerqueira GC, et al. The genome of the blood fluke Schistosoma mansoni. Nature. 2009;460:352–8.View ArticlePubMedPubMed CentralGoogle Scholar
  10. The Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium. The Schistosoma japonicum genome reveals features of host-parasite interplay. Nature. 2009;460:345–51.View ArticleGoogle Scholar
  11. Young ND, Jex AR, Li B, Liu S, Yang L, Xiong Z, et al. Whole-genome sequence of Schistosoma haematobium. Nat Genet. 2012;44:221–5.View ArticlePubMedGoogle Scholar
  12. Waisberg M, Lobo FP, Cerqueira GC, Passos LK, Carvalho OS, Franco GR, et al. Microarray analysis of gene expression induced by sexual contact in Schistosoma mansoni. BMC Genomics. 2007;8:181.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Jolly ER, Chin CS, Miller S, Bahgat MM, Lim KC, DeRisi J, et al. Gene expression patterns during adaptation of a helminth parasite to different environmental niches. Genome Biol. 2007;8:R65.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Gobert GN, Moertel L, Brindley PJ, McManus DP. Developmental gene expression profiles of the human pathogen Schistosoma japonicum. BMC Genomics. 2009;10:128.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Chai M, McManus DP, McInnes R, Moertel L, Tran M, Loukas A, et al. Transcriptome profiling of lung schistosomula, in vitro cultured schistosomula and adult Schistosoma japonicum. Cell Mol Life Sci. 2006;63:919–29.Google Scholar
  16. Moertel L, McManus DP, Piva TJ, Young L, McInnes RL, Gobert GN. Oligonucleotide microarray analysis of strain- and gender-associated gene expression in the human blood fluke, Schistosoma japonicum. Mol Cell Probes. 2006;20:280–9.View ArticlePubMedGoogle Scholar
  17. Fitzpatrick JM, Johansen MV, Johnston DA, Dunne DW, Hoffmann KF. Gender-associated gene expression in two related strains of Schistosoma japonicum. Mol Biochem Parasitol. 2004;136:191–209.View ArticlePubMedGoogle Scholar
  18. Parker-Manuel SJ, Ivens AC, Dillon GP, Wilson RA. Gene expression patterns in larval Schistosoma mansoni associated with infection of the mammalian host. PLoS Negl Trop Dis. 2011;5:e1274.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Ojopi EP, Oliveira PS, Nunes DN, Paquola A, DeMarco R, Gregorio SP, et al. A quantitative view of the transcriptome of Schistosoma mansoni adult-worms using SAGE. BMC Genomics. 2007;8:186.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Williams DL, Sayed AA, Bernier J, Birkeland SR, Cipriano MJ, Papa AR, et al. Profiling Schistosoma mansoni development using serial analysis of gene expression (SAGE). Exp Parasitol. 2007;117:246–58.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Cogswell AA, Kommer VP, Williams DL. Transcriptional analysis of a unique set of genes involved in Schistosoma mansoni female reproductive biology. PLoS Negl Trop Dis. 2012;6:e1907.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Piao X, Cai P, Liu S, Hou N, Hao L, Yang F, et al. Global expression analysis revealed novel gender-specific gene expression features in the blood fluke parasite Schistosoma japonicum. PLoS One. 2011;6:e18267.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Anderson L, Amaral MS, Beckedorff F, Silva LF, Dazzani B, Oliveira KC, et al. Schistosoma mansoni egg, adult male and female comparative gene expression analysis and identification of novel genes by RNA-Seq. PLoS Negl Trop Dis. 2015;9:e0004334.View ArticlePubMedPubMed CentralGoogle Scholar
  24. Picard MA, Boissier J, Roquis D, Grunau C, Allienne JF, Duval D, et al. Sex-biased transcriptome of Schistosoma mansoni: host-parasite interaction, genetic determinants and epigenetic regulators are associated with sexual differentiation. PLoS Negl Trop Dis. 2016;10:e0004930.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Jones MK, Higgins T, Stenzel DJ, Gobert GN. Towards tissue specific transcriptomics and expression pattern analysis in schistosomes using laser microdissection microscopy. Exp Parasitol. 2007;117:259–66.View ArticlePubMedGoogle Scholar
  26. Gobert GN, McManus DP, Nawaratna S, Moertel L, Mulvenna J, Jones MK. Tissue specific profiling of females of Schistosoma japonicum by integrated laser microdissection microscopy and microarray analysis. PLoS Negl Trop Dis. 2009;3:e469.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Nawaratna SS, McManus DP, Moertel L, Gobert GN, Jones MK. Gene Atlasing of digestive and reproductive tissues in Schistosoma mansoni. PLoS Negl Trop Dis. 2011;5:e1043.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Fitzpatrick JM, Peak E, Perally S, Chalmers IW, Barrett J, Yoshino TP, et al. Anti-schistosomal intervention targets identified by life-cycle transcriptomic analyses. PLoS Negl Trop Dis. 2009;3:e543.Google Scholar
  29. Liu S, Cai P, Hou N, Piao X, Wang H, Hung T, et al. Genome-wide identification and characterization of a panel of house-keeping genes in Schistosoma japonicum. Mol Biochem Parasitol. 2012;182:75–82.View ArticlePubMedGoogle Scholar
  30. Liu S, Cai P, Piao X, Hou N, Zhou X, Wu C, et al. Expression profile of the Schistosoma japonicum degradome reveals differential protease expression patterns and potential anti-schistosomal intervention targets. PLoS Comput Biol. 2014;10:e1003856.View ArticlePubMedPubMed CentralGoogle Scholar
  31. Liu S, Zhou X, Piao X, Wu C, Hou N, Chen Q. Comparative analysis of transcriptional profiles of adult Schistosoma japonicum from different laboratory animals and the natural host, water buffalo. PLoS Negl Trop Dis. 2015;9:e0003993.View ArticlePubMedPubMed CentralGoogle Scholar
  32. Cai P, Piao X, Hao L, Liu S, Hou N, Wang H, et al. A deep analysis of the small non-coding RNA population in Schistosoma japonicum eggs. PLoS One. 2013;8:e64003.View ArticlePubMedPubMed CentralGoogle Scholar
  33. Wu C, Hou N, Piao X, Liu S, Cai P, Xiao Y, et al. Non-immune immunoglobulins shield Schistosoma japonicum from host immunorecognition. Sci Rep. 2015;5:13434.View ArticlePubMedPubMed CentralGoogle Scholar
  34. Li DD, Zhao LX, Mylonakis E, Hu GH, Zou Y, Huang TK, et al. In vitro and in vivo activities of pterostilbene against Candida albicans biofilms. Antimicrob Agents Chemother. 2014;58:2344–55.Google Scholar
  35. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003;4:249–64.View ArticlePubMedGoogle Scholar
  36. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003;31:e15.View ArticlePubMedPubMed CentralGoogle Scholar
  37. Deng W, Wang Y, Liu Z, Cheng H, Xue Y. HemI: a toolkit for illustrating heatmaps. PLoS One. 2014;9:e111988.View ArticlePubMedPubMed CentralGoogle Scholar
  38. Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, et al. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res. 2008;36:3420–35.View ArticlePubMedPubMed CentralGoogle Scholar
  39. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, et al. CDD: NCBI’s conserved domain database. Nucleic Acids Res. 2015;43:D222–6.View ArticlePubMedGoogle Scholar
  40. Cai P, Piao X, Hou N, Liu S, Wang H, Chen Q. Identification and characterization of argonaute protein, Ago2 and its associated small RNAs in Schistosoma japonicum. PLoS Negl Trop Dis. 2012;6:e1745.View ArticlePubMedPubMed CentralGoogle Scholar
  41. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25–9.View ArticlePubMedPubMed CentralGoogle Scholar
  42. Valovka T, Verdier F, Cramer R, Zhyvoloup A, Fenton T, Rebholz H, et al. Protein kinase C phosphorylates ribosomal protein S6 kinase betaII and regulates its subcellular localization. Mol Cell Biol. 2003;23:852–63.View ArticlePubMedPubMed CentralGoogle Scholar
  43. Daily NJ, Boswell KL, James DJ, Martin TF. Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion. J Biol Chem. 2010;285:35320–9.View ArticlePubMedPubMed CentralGoogle Scholar
  44. Ligasova A, Bulantova J, Sebesta O, Kasny M, Koberna K, Mikes L. Secretory glands in cercaria of the neuropathogenic schistosome Trichobilharzia regenti - ultrastructural characterization, 3-D modelling, volume and pH estimations. Parasit Vectors. 2011;4:162.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Morisaki T, Gross M, Morisaki H, Pongratz D, Zollner N, Holmes EW. Molecular basis of AMP deaminase deficiency in skeletal muscle. Proc Natl Acad Sci U S A. 1992;89:6457–61.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Neumann D, Woods A, Carling D, Wallimann T, Schlattner U. Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli. Protein Expr Purif. 2003;30:230–7.View ArticlePubMedGoogle Scholar
  47. Morgan R. Engrailed: complexity and economy of a multi-functional transcription factor. FEBS Lett. 2006;580:2531–3.View ArticlePubMedGoogle Scholar
  48. Webster PJ, Mansour TE. Conserved classes of homeodomains in Schistosoma mansoni, an early bilateral metazoan. Mech Dev. 1992;38:25–32.View ArticlePubMedGoogle Scholar
  49. Mansley MK, Watt GB, Francis SL, Walker DJ, Land SC, Bailey MA, et al. Dexamethasone and insulin activate serum and glucocorticoid-inducible kinase 1 (SGK1) via different molecular mechanisms in cortical collecting duct cells. Physiol Rep. 2016;4:e12792.Google Scholar
  50. Wu W, LoVerde PT. Nuclear hormone receptors in parasitic helminths. Mol Cell Endocrinol. 2011;334:56–66.View ArticlePubMedGoogle Scholar
  51. Wu W, Loverde PT. Schistosoma mansoni: identification of SmNR4A, a member of nuclear receptor subfamily 4. Exp Parasitol. 2008;120:208–13.View ArticlePubMedPubMed CentralGoogle Scholar
  52. Badouel C, Zander MA, Liscio N, Bagherie-Lachidan M, Sopko R, Coyaud E, et al. Fat1 interacts with Fat4 to regulate neural tube closure, neural progenitor proliferation and apical constriction during mouse brain development. Development. 2015;142:2781–91.View ArticlePubMedPubMed CentralGoogle Scholar
  53. Brun L, Ngu LH, Keng WT, Ch’ng GS, Choy YS, Hwu WL, et al. Clinical and biochemical features of aromatic L-amino acid decarboxylase deficiency. Neurology. 2010;75:64–71.View ArticlePubMedGoogle Scholar
  54. Miyagi Y, Yamashita T, Fukaya M, Sonoda T, Okuno T, Yamada K, et al. Delphilin: a novel PDZ and formin homology domain-containing protein that synaptically colocalizes and interacts with glutamate receptor delta 2 subunit. J Neurosci. 2002;22:803–14.PubMedGoogle Scholar
  55. Tararam CA, Farias LP, Wilson RA, Leite LC. Schistosoma mansoni Annexin 2: molecular characterization and immunolocalization. Exp Parasitol. 2010;126:146–55.View ArticlePubMedGoogle Scholar
  56. Leow CY, Willis C, Osman A, Mason L, Simon A, Smith BJ, et al. Crystal structure and immunological properties of the first annexin from Schistosoma mansoni: insights into the structural integrity of the schistosomal tegument. FEBS J. 2014;281:1209–25.View ArticlePubMedGoogle Scholar
  57. Mao Y, Kuta A, Crespo-Enriquez I, Whiting D, Martin T, Mulvaney J, et al. Dchs1-Fat4 regulation of polarized cell behaviours during skeletal morphogenesis. Nat Commun. 2016;7:11469.View ArticlePubMedPubMed CentralGoogle Scholar
  58. Fuse A, Furuya N, Kakuta S, Inose A, Sato M, Koike M, et al. VPS29-VPS35 intermediate of retromer is stable and may be involved in the retromer complex assembly process. FEBS Lett. 2015;589:1430–6.View ArticlePubMedGoogle Scholar
  59. Cai P, Liu S, Piao X, Hou N, Gobert GN, McManus DP, et al. Comprehensive transcriptome analysis of sex-biased expressed genes reveals discrete biological and physiological features of male and female Schistosoma japonicum. PLoS Negl Trop Dis. 2016;10:e0004684.View ArticlePubMedPubMed CentralGoogle Scholar
  60. Fitzpatrick JM, Hirai Y, Hirai H, Hoffmann KF. Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases. FASEB J. 2007;21:823–35.View ArticlePubMedGoogle Scholar
  61. Ferguson BJ, Newland SA, Gibbs SE. Tourlomousis P, Fernandes dos Santos P, Patel MN, et al. The Schistosoma mansoni T2 ribonuclease omega-1 modulates inflammasome-dependent IL-1beta secretion in macrophages. Int J Parasitol. 2015;45:809–13.View ArticlePubMedGoogle Scholar
  62. Chalmers IW, McArdle AJ, Coulson RM, Wagner MA, Schmid R, Hirai H, et al. Developmentally regulated expression, alternative splicing and distinct sub-groupings in members of the Schistosoma mansoni venom allergen-like (SmVAL) gene family. BMC Genomics. 2008;9:89.View ArticlePubMedPubMed CentralGoogle Scholar
  63. Cai P, Hou N, Piao X, Liu S, Liu H, Yang F, et al. Profiles of small non-coding RNAs in Schistosoma japonicum during development. PLoS Negl Trop Dis. 2011;5:e1256.View ArticlePubMedPubMed CentralGoogle Scholar
  64. Roquis D, Lepesant JM, Picard MA, Freitag M, Parrinello H, Groth M, et al. The epigenome of Schistosoma mansoni provides insight about how cercariae poise transcription until infection. PLoS Negl Trop Dis. 2015;9:e0003853.View ArticlePubMedPubMed CentralGoogle Scholar
  65. Ressurreicao M, Kirk RS, Rollinson D, Emery AM, Page NM, Walker AJ. Sensory protein kinase signaling in Schistosoma mansoni cercariae: host location and invasion. J Infect Dis. 2015;212:1787–97.View ArticlePubMedPubMed CentralGoogle Scholar
  66. Han ZG, Brindley PJ, Wang SY, Chen Z. Schistosoma genomics: new perspectives on schistosome biology and host-parasite interaction. Annu Rev Genomics Hum Genet. 2009;10:211–40.View ArticlePubMedGoogle Scholar
  67. Liu M, Ju C, Du XF, Shen HM, Wang JP, Li J, et al. Proteomic analysis on cercariae and schistosomula in reference to potential proteases involved in host invasion of Schistosoma japonicum larvae. J Proteome Res. 2015;14:4623–34.View ArticlePubMedGoogle Scholar
  68. Jenkins SJ, Hewitson JP, Jenkins GR, Mountford AP. Modulation of the host’s immune response by schistosome larvae. Parasite Immunol. 2005;27:385–93.View ArticlePubMedPubMed CentralGoogle Scholar
  69. Cai P, Bu L, Wang J, Wang Z, Zhong X, Wang H. Molecular characterization of Schistosoma japonicum tegument protein tetraspanin-2: sequence variation and possible implications for immune evasion. Biochem Biophys Res Commun. 2008;372:197–202.View ArticlePubMedGoogle Scholar
  70. Zhang W, Li J, Duke M, Jones MK, Kuang L, Zhang J, et al. Inconsistent protective efficacy and marked polymorphism limits the value of Schistosoma japonicum tetraspanin-2 as a vaccine target. PLoS Negl Trop Dis. 2011;5:e1166.View ArticlePubMedPubMed CentralGoogle Scholar
  71. Bradford D, Cole SJ, Cooper HM. Netrin-1: diversity in development. Int J Biochem Cell Biol. 2009;41:487–93.View ArticlePubMedGoogle Scholar
  72. Weng YL, Liu N, DiAntonio A, Broihier HT. The cytoplasmic adaptor protein Caskin mediates Lar signal transduction during Drosophila motor axon guidance. J Neurosci. 2011;31:4421–33.View ArticlePubMedPubMed CentralGoogle Scholar
  73. Woo WM, Berry EC, Hudson ML, Swale RE, Goncharov A, Chisholm AD. The C. elegans F-spondin family protein SPON-1 maintains cell adhesion in neural and non-neural tissues. Development. 2008;135:2747–56.View ArticlePubMedPubMed CentralGoogle Scholar
  74. Li M, Armelloni S, Ikehata M, Corbelli A, Pesaresi M, Calvaresi N, et al. Nephrin expression in adult rodent central nervous system and its interaction with glutamate receptors. J Pathol. 2011;225:118–28.View ArticlePubMedGoogle Scholar
  75. Putaala H, Soininen R, Kilpelainen P, Wartiovaara J, Tryggvason K. The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death. Hum Mol Genet. 2001;10:1–8.View ArticlePubMedGoogle Scholar
  76. Lee EF, Young ND, Lim NT, Gasser RB, Fairlie WD. Apoptosis in schistosomes: toward novel targets for the treatment of schistosomiasis. Trends Parasitol. 2014;30:75–84.View ArticlePubMedGoogle Scholar
  77. Han H, Peng J, Gobert GN, Hong Y, Zhang M, Han Y, et al. Apoptosis phenomenon in the schistosomulum and adult worm life cycle stages of Schistosoma japonicum. Parasitol Int. 2013;62:100–8.View ArticlePubMedGoogle Scholar
  78. Pearson MS, Loukas A. The parasite’s new clothes. Elife. 2016;5:e12473.Google Scholar
  79. Collins 3rd JJ, Wang B, Lambrus BG, Tharp ME, Iyer H, Newmark PA. Adult somatic stem cells in the human parasite Schistosoma mansoni. Nature. 2013;494:476–9.View ArticlePubMedPubMed CentralGoogle Scholar
  80. Don TA, Bethony JM, Loukas A. Saposin-like proteins are expressed in the gastrodermis of Schistosoma mansoni and are immunogenic in natural infections. Int J Infect Dis. 2008;12:e39–47.View ArticlePubMedGoogle Scholar
  81. Mickum ML, Prasanphanich NS, Heimburg-Molinaro J, Leon KE, Cummings RD. Deciphering the glycogenome of schistosomes. Front Genet. 2014;5:262.View ArticlePubMedPubMed CentralGoogle Scholar
  82. Smit CH, van Diepen A, Nguyen DL, Wuhrer M, Hoffmann KF, Deelder AM, et al. Glycomic analysis of life stages of the human parasite Schistosoma mansoni reveals developmental expression profiles of functional and antigenic glycan motifs. Mol Cell Proteomics. 2015;14:1750–69.View ArticlePubMedPubMed CentralGoogle Scholar
  83. Jones MK, Bong SH, Green KM, Holmes P, Duke M, Loukas A, et al. Correlative and dynamic imaging of the hatching biology of Schistosoma japonicum from eggs prepared by high pressure freezing. PLoS Negl Trop Dis. 2008;2:e334.View ArticlePubMedPubMed CentralGoogle Scholar
  84. Steinfelder S, Andersen JF, Cannons JL, Feng CG, Joshi M, Dwyer D, et al. The major component in schistosome eggs responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease (omega-1). J Exp Med. 2009;206:1681–90.View ArticlePubMedPubMed CentralGoogle Scholar
  85. Meyer NH, Mayerhofer H, Tripsianes K, Blindow S, Barths D, Mewes A, et al. A Crystallin fold in the Interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE/alpha-1) mediates IgE binding for antigen-independent basophil activation. J Biol Chem. 2015;290:22111–26.View ArticlePubMedPubMed CentralGoogle Scholar
  86. Schramm G, Mohrs K, Wodrich M, Doenhoff MJ, Pearce EJ, Haas H, et al. Cutting edge: IPSE/alpha-1, a glycoprotein from Schistosoma mansoni eggs, induces IgE-dependent, antigen-independent IL-4 production by murine basophils in vivo. J Immunol. 2007;178:6023–7.Google Scholar
  87. Schramm G, Hamilton JV, Balog CI, Wuhrer M, Gronow A, Beckmann S, et al. Molecular characterisation of kappa-5, a major antigenic glycoprotein from Schistosoma mansoni eggs. Mol Biochem Parasitol. 2009;166:4–14.View ArticlePubMedGoogle Scholar

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