Transcriptomic responses of Biomphalaria pfeifferi to Schistosoma mansoni: Investigation of a neglected African snail that supports more S. mansoni transmission than any other snail species

Abstract

Background: Biomphalaria pfeifferi is highly compatible with the widespread human-infecting blood fluke Schistosoma mansoni and transmits more cases of this parasite to people than any other snail species. For these reasons, B. pfeifferi is the world's most important vector snail for S. mansoni, yet we know relatively little at the molecular level regarding the interactions between B. pfeifferi and S. mansoni from early-stage sporocyst transformation to the development of cercariae.

Methodology/principal findings: We sought to capture a portrait of the response of B. pfeifferi to S. mansoni as it occurs in nature by undertaking Illumina dual RNA-Seq on uninfected control B. pfeifferi and three intramolluscan developmental stages (1- and 3-days post infection and patent, cercariae-producing infections) using field-derived west Kenyan specimens. A high-quality, well-annotated de novo B. pfeifferi transcriptome was assembled from over a half billion non-S. mansoni paired-end reads. Reads associated with potential symbionts were noted. Some infected snails yielded fewer normalized S. mansoni reads and showed different patterns of transcriptional response than others, an indication that the ability of field-derived snails to support and respond to infection is variable. Alterations in transcripts associated with reproduction were noted, including for the oviposition-related hormone ovipostatin and enzymes involved in metabolism of bioactive amines like dopamine or serotonin. Shedding snails exhibited responses consistent with the need for tissue repair. Both generalized stress and immune factors immune factors (VIgLs, PGRPs, BGBPs, complement C1q-like, chitinases) exhibited complex transcriptional responses in this compatible host-parasite system.

Significance: This study provides for the first time a large sequence data set to help in interpreting the important vector role of the neglected snail B. pfeifferi in transmission of S. mansoni, including with an emphasis on more natural, field-derived specimens. We have identified B. pfeifferi targets particularly responsive during infection that enable further dissection of the functional role of these candidate molecules.

Fig 1. Overview of novel bioinformatics pipeline developed to isolate and analyze B. pfeifferi transcriptomic expression from dual RNA-Seq data.

Fig 2. Identification of the innate immune recognition receptors TLRs in B. pfeifferi.

Partial CDS counts had a BLAST hit against a known TLR but all necessary domains could not be confidently determined by InterProScan5.

Fig 3. Identification of the innate immune recognition receptors VIgLs in B. pfeifferi with initial BLAST annotation and then verification of protein domains in InterProScan5.

Fig 4. Identification of all de novo assembled transcripts after S. mansoni read filtering.

Fig 5. Sum of non-B. pfeifferi de novo assembled CDS for each replicate. CDS were counted as present if read count >0.

Fig 6. Pie charts of unique CDS found to be differentially expressed in 3v3, 3v2, and 3v1 EBSeq analyses.

Fig 7. Multidimensional scaling (MDS) plots of pairwise comparisons of control versus 1d, 3d, and shedding replicates used for differential expression analyses.

Fig 8. Biomphalaria pfeifferi differential expression profiles in 1d, 3d, and shedding snails.

(A) Overall expression profiles for up- and down-regulated B. pfeifferi CDS in the 3v3 DE analysis with proportions shown for CDS with annotation known (white) and without annotation (gray) from one of the 5 databases searched (Table 3). Numbers by bars refer to numbers of up- and down-regulated features. (B) Heat map of differentially expressed B. pfeifferi CDS. (C) Up- and down-regulated B. pfeifferi CDS shared between 1d, 3d, and shedding snail groups in the 3v3 DE analysis are shown.

Fig 9. Biomphalaria pfeifferi CDS identified as neuropeptides, hormones, or involved in reproduction that are differentially expressed in 1d, 3d, and shedding snails.

Note that the 3v3 comparison includes all 3 infected snails within a time point, whereas 3v2 includes the two infected snails with the most S. mansoni reads and the 3v1 includes only the infected snail with the fewest S. mansoni reads.

Fig 10. Differential expression of Biomphalaria pfeifferi defense-related CDS in 1d, 3d, and shedding snails.

(A) Defense CDS in the 3v3 DE analysis. (B) Pie charts of proportions of CDS found to be DE in 3v3, 3v2, and 3v1 analyses. (C) Heat maps show expression levels from each of the three DE analyses highlighting the most relevant biological functional groups. Note that the 3v3 comparison includes all 3 infected snails within a time point, whereas 3v2 includes the two infected snails with the most S. mansoni reads and the 3v1 includes only the infected snail with the fewest S. mansoni reads.

Fig 11. qPCR results validate Illumina RNA-Seq differential expression results.

(A) Quantitative real-time PCR verifies Illumina trends among biological replicates in 1d, 3d, and shedding samples. (B) Corresponding Illumina DE results for the four genes tested. Asterisks indicate genes that are significantly DE.