Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni

Abstract

Biomphalaria glabrata (B. glabrata) is an air-breathing aquatic mollusc found in freshwater habitats across the Western Hemisphere. It is most well-known for its recognized capacity to act as a major intermediate host for Schistosoma mansoni, the human blood fluke parasite. Ionotropic receptors (IRs), a variant family of the ionotropic glutamate receptors (iGluR), have an evolutionary ancient function in detecting odors to initiate chemosensory signaling. In this study, we applied an array of methods towards the goal of identifying IR-like family members in B. glabrata, ultimately revealing two types, the iGluR and IR. Sequence alignment showed that three ligand-binding residues are conserved in most Biomphalaria iGluR sequences, while the IRs did exhibit a variable pattern, lacking some or all known glutamate-interactingresidues, supporting their distinct classification from the iGluRs. We show that B. glabrata contains 7 putative IRs, some of which are expressed within its chemosensory organs. To further investigate a role for the more ancient IR25a type in chemoreception, we tested its spatial distribution pattern within the snail cephalic tentacle by in situ hybridization. The presence of IR25a within presumptive sensory neurons supports a role for this receptor in olfactory processing, contributing to our understanding of the molecular pathways that are involved in Biomphalaria olfactory processing.

Fig 1. Characterisation of Biomphalaria glabrata IRs and iGluRs.

(A) Molecular phylogeny for IR and iGluRs from B. blabrata (Bgla), A. californica (Acal), S. gregaria (Sgre), D. ponderosae (Dpon), P. argus (Parg) and D. melanogaster (Dmel). Bootstrap supports two IR subfamilies. The 7 newly identified Biomphalaria IRs are highlighted with red diamonds. Phylogenetic tree of nonIR8a/25a IRs is shown for 5 B. glabrata, 2 from P. argus and 9 from A. californica. Clades are indicated by different colours. All gene accession numbers can be found in S2 Table. (B) Alignment of predicted amino acid sequences of 5 candidate Biomphalaria IRs (BglaIR1-5), including regions encoding putative ligand-binding domains; S1 and S2 domains are shown by black asterisks below the sequences. Three key ligand-binding residues (R, T and D/E) are marked with red asterisks. Blue shading indicates identical or similar amino acids. Sequence logo conservation is presented above the sequence.

Fig 2. Analysis of ligand-binding domains in Biomphalaria glabrata IRs and iGluRs.

(A) Left: Protein domain structure of conventional iGluRs/IRs in schematic form [8]. Right: Illustration of the three Pfam domains present in iGluRs and IRs. Both IR8a and IR25a possess the Pfam domain corresponding to the iGluR ATD. All other IRs lack the same homology to the ATD. (B) Alignment of S1 and S2 ligand-binding domains from putative B. glabrata iGluRs and IRs with A.californica iGluRs. Biomphalaria and Aplysia S1 and S2 ligand-binding domains were manually aligned. Blue shading indicates identical or similar amino acids. Three key ligand-binding residues (R, T and D/E) are boxed. S1 and S2 domains are marked with coloured lines at the bottom. (C) Schematic representation of Biomphalaria iGluRs, showing conserved and invariable amino acids. Predicted ATD site is highlighted in red and the region of key ligand-binding residues is magnified and shown in yellow and green.

Fig 3. Tissue expression of Biomphalaria glabrata IRs.

Top: Schematic representation of B.glabrata showing tissues used for RT-PCR. Bottom: RT-PCR detection of 7 Biomphalaria IR genes in different tissues. Biomphalaria IRs can be detected in both olfactory and non-olfactory tissues. No expression could be detected from the lung or gonad. No amplification was detected in RNA samples in the absence of reverse transcription (data not shown) or template (-ve). Control RT-PCR products for comparative analysis of gene expression correspond to the β-actin.

Fig 4. Analysis of Biomphalaria glabrata IR25a.

(A) The protein domain organization of a typical IR25a is shown above a protein alignment of Biomphalaria (Bgla), Aplysia (Acal), Panulirus (Parg) and Drosophila (Dmel) IR25a. Conserved amino acid residues are highlighted in purple (≥80% conserved) and blue (≥50% conserved), and ligand-binding domain S1 and S2 domains are shown with red lines above the sequences. Three key ligand-binding residues (R, T and D/E) are marked with a black dot. (B) Schematic representation of Biomphalaria IRs, showing conserved and invariable amino acids. Predicted S1 and S2 region are highlighted in green and yellow, respectively. (C) Structure of BglaIR25a predicted by SWISS-MODEL in conjunction with MDS. Top: tertiary structure, purple-α helix, blue-3-10 helix, yellow-β sheet, cyan-turn and white-random coil. Bottom: space filling of predicted binding site, yellow-predicted ligand binding S1 region, green-predicted ligand binding S2 region, and blue-predicted TM region.

Fig 5. Expression of BglaIR25a as detected by in situ hybridization in Biomphalaria glabrata tentacle.

(A) Control whole-mount in situ hybridization on tentacle tissue with a DIG-labelled sense riboprobe for BglaIR25a. No signal is apparent. (B-D) Whole-mount tentacle probed with antisense riboprobe for BglaIR25a. (E-I) Cryostat sections showing cellular localization of IR25a within central and peripheral cells (arrows). d, distal; p, proximal.