Transcriptome sequencing and differential gene expression analysis of the schistosome-transmitting snail Oncomelania hupensis inhabiting hilly and marshland regions

Abstract

The freshwater snail Oncomelania hupensis is the unique intermediate host of the blood fluke Schistosoma japonicum, which is the major cause of schistosomiasis. The snail inhabits two contrasting environments: the hilly and marshland regions. The hilly snails are smaller in size and have the typical smooth shell, whereas the marshland snails are larger and possess the ribbed shell. To reveal the differences in gene expression between the hilly and marshland snails, a total of six snails, three per environment, were individually examined by RNA sequencing technology. All paired-end reads were assembled into contigs from which 34,760 unigenes were predicted. Based on single nucleotide polymorphisms, principal component analysis and neighbor-joining clustering revealed two distinct clusters of hilly and marshland snails. Analysis of expression changes between environments showed that upregulated genes relating to immunity and development were enriched in hilly snails, while those associated with reproduction were over-represented in marshland snails. Eight differentially expressed genes between the two types of snails were validated by qRT-PCR. Our study identified candidate genes that could be targets for future functional studies, and provided a link between expression profiling and ecological adaptation of the snail that may have implications for schistosomiasis control.

Figure 1

Geographical locations of the sampling regions. (a) Location of Anhui province in China; The map was generated using ArcGIS version 10.2 (ESRI, Redlands, CA, USA); (b) Sampling sites in Wuhu City and Nanling County within Anhui province. The satellite images were taken from Google Earth http://earth.google.com/.

Figure 2

The gene ontology categories in the transcriptome of Oncomelania hupensis. Each of three major categories was shown in bold, while all subcategories within each major category were shown in plain on the y-axis. The bar length represents the log10 transformation of unigene number, and the actual number of unigenes is shown on the x-axis. The percentage of unigene counts is presented on the right.

Figure 3

Population clustering analysis. (a) Principal component analysis (PCA) of the snails. Black squares (H1-H3) refer to three individuals of hilly snails; Red triangles (M1–M3) indicate three marshland snails. (b) Neighbor-joining (NJ) tree based on all single nucleotide polymorphisms (SNPs). Black, hilly snails; Red, marshland snails.

Figure 4

Analysis of differentially expressed unigenes. (a) Comparison between the hilly and marshland snails via RNA-seq measured in fragments per kilobase per million fragments mapped (FPKM). Each dot represents a unigene, and two green lines bracket unigenes having less than 4-fold differences in expression between environments; and the unigenes with ≥ 4-fold differences in expression were considered to be differentially expressed in this study. A total of 1,064 genes were upwardly expressed and 2,392 were downwardly expressed in hilly snails relative to marshland snails; (b) Heatmap of all 3,456 differentially expressed unigenes between the hilly and marshland snails.

Figure 5

Gene ontology (GO) enrichment analysis of differentially expressed unigenes between the hilly and marshland snails. Gene number is provided adjacent to each GO term.

Figure 6

Expression profiles of eight differentially expressed genes. Expression levels in marshland snails were calculated as the control for each gene. Red bars represent the relative expression levels in hilly snails by RT-PCRs and grey bars indicate the relative expression levels in hilly snails by transcriptome sequencing. All relative expression values were shown with log2 (fold change) on the Y axis.