Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni
Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail Biomphalaria glabrata, an Intermediate Host of Schistosoma mansoni
Di Liang 0 1
Tianfang Wang 0 1
Bronwyn A. Rotgans 0 1
Donald P. McManus 1
Scott F. Cummins 0 1
0 Faculty of Science , Health and Education , University of the Sunshine Coast , Maroochydore, Queensland 4558 , Australia , 2 Molecular Parasitology Laboratory , QIMR Berghofer Medical Research Institute , Brisbane, Queensland, 4006 , Australia
1 Editor: Matty Knight, George Washington University School of Medicine and Health Sciences , UNITED STATES
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 IRlike 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.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by the Australian
Research Council (Future Fellowship, FT110100990
to SFC). This research was undertaken with the
assistance of resources from the National
Computational Infrastructure (NCI), which is
supported by the Australian Government. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
Biomphalaria glabrata (B. glabrata) is an air-breathing aquatic pulmonate gastropod mollusc
in the family Planorbidae found in freshwater habitats across the Western Hemisphere. It is an
intermediate host for the Schistosoma mansoni parasite, causing one of the most prevalent
parasitic infections in humans, known as schistosomiasis (also called Bilharzia). Schistosomiasis is
a crippling disease found in 76 countries (primarily developing countries) [
] and causes
Competing Interests: The authors have declared
that no competing interests exist.
approximately 280,000 deaths per annum in sub-Saharan Africa alone [
]. It was believed
that preventive praziquantel chemotherapy and molluscicides were the most appropriate
means of eradicating this overwhelming disease burden . However, the fact that currently
available synthetic molluscicides tend to be generally biocidal, affecting many other animals
and/or plants in the snail habitat, along with the threat of praxiquantel drug resistance [
have spurred recognition of the pressing need for a practical and ideal supplementary approach
in addition to chemotherapy.
As an important intermediate snail host that is integral to the transmission of a significant
human pathogen, Biomphalaria presents itself as a powerful model organism for studying the
complexities of host-pathogen interactions. As is the case with most molluscs, Biomphalaria
snails have virtually no hearing and very limited vision so they obtain the vast majority of their
information about the environment by smell. Using this dependence, one may envisage the use
of a broad-spectrum of chemical cues to manipulate snail behavior, allowing for development
of environment-friendly control strategies. Towards realizing an olfactory-mediated control
strategy, it is important to be aware of the molecular biological makeup of the molusc’s
olfactory system, including the odor chemosensory receptors.
Ionotropic glutamate receptors (iGluRs) are a conserved family of ligand-gated ion channels
widespread across vertebrates [
] as well as invertebrates [
]. Ionotropic receptors (IRs), a
variant family of the iGluRs, were identified as a novel group of chemosensory receptors in
Drosophila melanogaster [
]. They were subsequently identified in several other species [
demonstrating that IRs had an evolutionary ancient function in detecting odors, likely playing
a general role in initiating chemosensory signaling.
By allowing neurons to communicate with each other in the brain in response to external
signals, iGluRs function in synaptic transmission as receptors for the excitatory
neurotransmitter glutamate and related ligands [
]. Based on their main agonist, traditional iGluRs can be
divided into three pharmacologically and molecularly distinct receptor subfamilies:
α-amino3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate
(NMDA). AMPA and kainate receptors are commonly grouped as non-NMDA receptors. Of
these, the AMPA receptors are best characterized for their function in mediating the fast
excitatory synaptic transmission while kainate receptors play a more subtle, modulatory role in this
]. NMDA receptors may have appeared after the non-NMDA receptors, and are well
known for their role in synaptic and neuronal plasticity, requiring two agonists (glutamate and
glycine) for activation [
]. The idea that iGluRs initiate metabotropic signaling has been
proposed for some types of mammals [
]. For example, GABA released in the supraoptic
nucleus of the hypothalamus is mediated by kainate iGluRs through an ionotropic mode of
Both iGluRs and IRs universally possess a conserved ligand-gated ion channel domain
encoded by Pfam domains PF10613 and PF00060 [
]. The ligand-gated ion channel domain
is made up of a combination of three transmembrane (TM) regions, an ion channel pore and a
large extracellular domain that contains a ligand binding domain (LBD), whose two
halfdomains (S1 and S2) combine to constitute a ‘‘Venus flytrap” that encloses glutamate and
related agonists [
]. Further, almost all iGluRs contain an extracellular amino-terminal
domain (ATD, Pfam domain PF01094) involved in the assembly of subunits into heteromeric
complexes, which are discernible only in well-characterized Drosophila melanogaster IR25a
and IR8a [
], but not in other known IR. In order to respond fast to the binding of extracellular
ligands through action potential generation, both iGluRs and IRs depolarize these domains by
permitting TM ion conduction [
]. Given that IRs possess a similar structure to iGluRs, it is
not surprising that IRs evolved from an animal iGluR ancestor without drastic functional
modifications, simply transitting in expression from an interneuron (where it modifies synaptic
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transmission in response to external amino acid) to a sensory neuron (where it may detect
chemical signals from the external molecules). Similar to iGluR, IRs are situated in distal
membrane regions of neuronal dendrites, but on cilia instead of post-synaptic membranes [
Integrating all publicly available data, we found that there is substantial variation in the size
of the IR repertoire throughout Protostomes, from three in C. elegans to eighty-five in the
crustacean Daphnia pulex and studies [
] point out IRs as the only known putative
chemosensory receptors expressed in crustacean antennules. In the study by Croset et al. , the
gastropod Aplysia expresses ionotropic receptors (IRs) in chemosensory organs. However, the
picture is far from complete, as besides Aplysia, very little attention has been given to the
genetic basis of chemoreception in other molluscs. Whether nor not IRs also play a role in the
olfactory system of Biomphalaria has remained elusive.
A significant breakthrough will be the annotation of the 931-Mb genome sequence of the B.
glabrata, the third complete molluscan genome available to date after oyster and octopus [
]. Its completion has provided an excellent opportunity to characterize the chemoreceptor
repertoire of Biomphalaria. In this study, a total of 19 candidate iGluR and 7 IR genes have
been obtained by utilizing the resources from this recently available B. glabrata genome. Of
these, 14 sequences are predicted to house a 3TM domain with full-length open reading frames
(ORF). We characterized the phylogenetic clustering and carried out an extensive tissue
expression profile for all Biomphalaria IRs that showed a widespread expression of BglaIRs in
non-antennal tissues, except for the IR25a which are found predominantly expressed in the
tentacle and central nervous system (CNS), implying that in this species the IRs have a more
complex function. We also complemented our PCR experiments by analyzing the expression
and spatial distribution pattern of IR25a with RNA by situ hybridization.
Materials and Methods
The conduct and procedures involving animal experimentation were approved by the Animal
Ethics Committee of the QIMR Berghofer Medical Research Institute (project number P242).
This study was performed in accordance with the recommendations in the Guide for the Care
and Use of Laboratory Animals of the National Institutes of Health.
Animal rearing and tissue collection
B. glabrata (BB02 strain) were maintained in flow-through aquarium tanks at Queensland
Institute of Medical Research (QIMR) during January, 2014, in a constant temperature room
set to 25°C, and fed to satiety on lettuce. For collection of tissues, animals were removed from
the aquarium and relevant tissues dissected out and either (1) embedded in optimal cutting
temperature compound for cryostat sectioning to perform in situ hybridization, or (2) snap
frozen in liquid nitrogen for RNA and protein isolation.
Gene identification and functional annotation
The B. glabrata genome and genome protein annotation files were downloaded from the
following resources: Bioinformatics Resource for Invertebrate Vectors of Human Pathogens
(https://www.vectorbase.org/organisms/biomphalaria-glabrata), NCBI contigs file (http://
www.ncbi.nlm.nih.gov/Traces/wgs/?val=APKA01#contigs) and NCBI scaffolds file (ftp://ftp.
ncbi.nlm.nih.gov/genbank/genomes/ Eukaryotes/ invertebrates/Biomphalaria_glabrata).
To identify target sequences, the B. glabrata genome was imported into the CLC Genomics
Workbench (v6.0; Finlandsgade, Dk). In this framework, previously identified putative Aplysia
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IRs were used to query (tBLASTn and BLASTx) the databases to help guide receptor
identification efforts. Annotated genomic regions retrieved from the databases were translated and
screened for the presence of recurrent transmembrane motifs using TMHMM Server v2.0
(http://www.cbs.dtu.dk/services/TMHMM/). The proteins that survived this filter were loaded
into the Pfam database (http://hmmer.janelia.org/search/phmmer and http://pfam.xfam.org/
search) and searched against the set of profile TMHMMs. Multiple sequence alignments for
IRs were performed using the Muscle algorithm, with phylogenetic trees constructed using the
neighbor-joining method with a minimum 1000 bootstrap replicates for node support. MikTex
Texshade software was used to generate multiple sequence alignments and schematics showing
amino acid conservation for the presented figures.
B. glabrata gene nomenclature was based upon a four-letter species abbreviation consisting
of an uppercase initial letter of the genus name and three lower case initial letters of the species
name (e.g. Aplysia californica = Acal; Biomphalaria glabrata = Bgla). iGluRs genes were
represented according to the subtype of the receptor (GluN for NMDA and iGluR for non-NMDA),
and named based on similarities with previously annotated A. californica iGluRs, or a logical
variant where no corresponding gene was identified. An additional number suffix after a point
was appended to the ends of these labels, where necessary, to distinguish them from multiple
gene models associated with a single contig or scaffold (e.g. BglaGluR8.1).
Molecular dynamics simulation
The initial conformations of the receptors were built using SWISS-MODEL by sequence
alignment with proteins with known 3D structures (template proteins) [
]. The structure with the
highest quality estimation (QMEAN score) was chosen, and subjected to the molecular
dynamics simulation (MDS) using AMBER version 14 [
]. The structure was imported using the
LEAP module of AMBER; the sequence segment(s) that was miss-represented (normally at
Nor C- terminus) due to different sequence length of the template proteins, was built as a linear
structure using LEAP and linked back to the corresponding positions. The MDS was fully
unrestrained and carried out in the canonical ensemble using the SANDER module. The
ff14SB force field [
] was employed. Energy minimisation with 2500 steps was first performed
to remove unfavourable contacts. The AMBER structure was then heated to 325K over 50 ps to
avoid being kinetically trapped in local minima, then subjected to unrestrained MD
simulations at 325K for the purpose of peptide equilibration. The structural information was sampled
every 1 ps (i.e., 10,000 structures were calculated for 10 ns MD simulation). This MD
simulation was continued until the root mean square deviation (RMSD) of structures within a
reasonable long time range was stable at/less than 3~4Å. Then, a lowest energy structure was
determined, and considered as the representative of the conformations simulated over this
period. Visualisation of the systems was effected using VMD software [
Total RNAs were prepared from each tissue [central nervous system (comprising pooled
cerebral, pleural, buccal, pedal and abdominal ganglia), tentacle, foot, heart, lung, blood vessel (in
close proximity to the heart), gonad, digestive system and cerebral ganglia using Trizol reagent
(Invitrogen, CA, USA), and NanoDrop measured the purity and quantity of each RNA sample.
First-strand cDNA was generated using random primers and the Superscript Preamplification
System for First-strand Synthesis (Invitrogen). PCR amplification was performed using
REDTaq DNA polymerase (Sigma) per the manufacturer's instructions with specific primer
combinations that were specific to the target gene (S2 File). PCR products were visualized by 2.0%
agarose gel electrophoresis to confirm transcript expression. Reactions were run in triplicate
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with tissues obtained from at least five different animals. Controls included no reverse
transcriptase. Biomphalaria β-actin (487 bp) was used as a control for all cDNA templates.
In situ hybridization
After identification of putative IR genes, one candidate, IR25a, was selected for tissue
expression localisation using whole-mount in situ hybridisation on B. glabrata cephalic tentacles.
Total RNA was extracted from B. glabrata cephalic tentacles using TriZol reagent (Life
Technologies) following the manufacturer’s instructions. First-strand cDNA was synthesised from
1 μg total RNA using random hexamers and the TaqMan Reverse Transcription kit (Applied
Biosystems). A fragment of IR25a was amplified from cephalic tentacle cDNA using specific
nested primers. The PCR product was then purified using a QIAquick Gel Purification kit
(Qiagen) and ligated into a pGEM-T Easy vector (Promega) according to the manufacturer’s
instructions, followed by transformation into JM109 competent cells (Promega). Blue-white
screening was used to choose colonies for PCR using T7 and SP6 primers (Promega) and
plasmid purification was performed using the QIAprep Spin Miniprep kit (Qiagen). Purified
plasmid was then amplified using M13 primers before gel purification of bands in the correct size
range using the QIAquick Gel Purification kit (Qiagen). Sense and antisense RNA probes were
prepared using a Digoxygenin RNA labelling kit (Roche) with T7 and SP6 polymerase. Spatial
localization of IR25a within B. glabrata cephalic tentacles was performed essentially as
described (Cummins et al., 2009). Colour was developed with NBT/BCIP (Roche) before
tissues were cleared in BB:BA for observation. Tissues were photographed using an Olympus
BX60 with Nomarski optics and a Nikon Digital Sight DS-U1 camera.
Identification of B. glabrata ionotropic receptors (IRs) and ionotropic
glutamate receptors (iGluRs)
Aplysia IR sequences were used to mine the B. glabrata genome, leading to the identification of
40 IR-like sequences. Subsequent filtering removed false positives and a primary cut-off
(Evalue = 1.0E-40) was selected. These BLAST-based annotations underwent manual inspection
of gene structure. In total, 26 IR-like sequences were inspected manually for homology to the
target query and the corresponding gene models were edited where necessary (S1 Table). Of
the 26 IR-like sequences, 19 contained typical motifs for iGluRs while the remaining 7 were
designated as IRs (Table 1). Twenty-five sequences did span at least two of the three
characteristic transmembrane domains, ranging in size from 229 to 1092 amino acids. Unigene
reference, length, and the number of predicted transmembrane domains for the final sequence
dataset are shown in Table 1.
Given that 11 of the 19 putative iGluRs had at least 67% identity with the corresponding
iGluRs of Aplysia (Biomphalaria orthologues), we therefore name these following their
orthologous genes. BglaGluR6.2 was named due to multiple copies of an orthologue of a B. glabrata
gene which exist and its relatively low similarity to AcalGluR6. The four novel NMDA type
iGluRs were named BglaGluN11 through BglaGluN14 to avoid confusion with the names of
non-NMDA type iGluRs, which number up to BglaGluR10. A previous report by Croset et al.
] did indicate that iGluRs contain the Pfam domain corresponding to the ATD, similarly
observed within most of the novel Biomphalaria iGluRs.
Regarding the IRs, two candidate IR subunits correspond to IR8a and IR25a, which share
31% and 56% amino acid identities with the spiny lobster Panulirus argus IR8a and 25a,
respectively. Also, there exists 29% and 53% identity at the amino acid level to the fruitfly
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Drosophila melanogaster, IR8a and 25a, respectively. Further, BglaIR25a showed a higher
amino acid identity (69%) with the A. californica candidate IR25a sequence, a potential
orthologue identified in B. glabrata. The remaining five putative IRs do not display considerable
conservation to any reported IRs, particularly in the key functional domains, but retained their
characteristic features, and thus these were named using the Arabic numerals 1–5 based on the
order of their identification in the B. glabrata transcriptomes.
Molecular phylogeny and structure of B. glabrata IR and iGluRs
Comparative alignment between all B.glabrata iGluRs and IRs was performed (S1 File), then
aligned with other known and novel candidates and reference sequences of IRs and iGluRs
retrieved from NCBI, including Panulirus argus, Aplysia californica, Biomphalaria glabrata
and Drosophila melanogaster (Fig 1A). It is immediately obvious that there are four primary
phylogenetic groupings with the presence of two different phylum-specific IR lineages. All
putative B. glabrata iGluRs have been dispersed over two groups (NMDA and non-NMDA)
and clustered with their corresponding orthologous genes into a group, in congruence with the
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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.
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BLAST results. The non-NMDAR (AMPAR/KainateR) group is the largest, containing 11 and
15 members from A. californica and B. glabrata, respectively, while the NMDAR group
includes AcalGluN and 4 NMDA type iGluRs of B. glabrata.
The second largest cluster next to the NMDAR group, composed of nine Aplysia IRs and two
Panulirus IRs (IR4 and 7), along with five B. glabrata IRs (IR1-5), was labeled as an IR group. In
addition to the phylogenetic clustering of these sequences into the primary IR group, the
Neighbor-joining tree further clustered these IRs into three clear species-specific subfamilies; homology
ranged from 19% to 26% between Panulirus and Biomphalaria IRs and 21% to 35% between
Aplysia and Biomphalaria IRs. Even though they grouped together and largely clustered into a
separate monophyletic clade, the five B. glabrata IRs (includes 3 partial and 2 full-length)
exhibited weak similarities when compared together, and the relevant phylogenetic separation is
mirrored by noticeable structural differences shown in their alignment analyses (Fig 1B).
The newly identified candidate Biomphalaria IR25a and 8a formed a distinct cluster
together with their counterparts from the other species (two PargIRs, SgreIR8a, DponIR8a,
antennal DmelIR25a and IR25a of A. californica,) and, apart from the existing IR lineages,
formed a separated clade next to the existing IR25a/8a lineages. BglaIR25a bears a relatively
high similarity to the other broadly expressed olfactory IR25a, regardless whether molluscan
(69% with A. californica) or with non-molluscan (56% with P. argus, 55% with M. mediator,
54% with S. gregaria, 53% with both D. busckii and H. assulta). Although its strongest level of
similarity is with PargIR8a, the novel BglaIR8a is only 31% similar to Panulirus sequence at the
amino acid level. Yet it can be located clearly together with the IR family in the IR25a/8a clade
as shown in Fig 1A. BglaIR8a shows a less clear relationship with its selected counterparts but
appears to fall in the phylogenetic vicinity of the major IR25a/8a probably because of the lack
of overall homology (currently there is no IR8a identified in Aplysia or any other mollusc).
Structural features of the ligand-binding domain
The interaction of glutamate receptors with their ligand is supposed to occur within a “Venus
flytrap” that is formed by an extracellular two-lobed ligand-binding domain and three
ligandbinding residues (R, arginine; T, threonine; and either D, aspartate or E, glutamate) that align
to form salt bridges with the glutamate ligand [
]. Fig 2A illustrates the orientation and
protein domain structure of conventional iGluRs/IRs and three Pfam domains present in the
iGluRs and IRs.
Sequence alignments of the LBD, which is specific to this protein family, based on conserved
residues in S1 and S2, were used to help make a final decision with respect to the potential
nomenclature of the iGluRs or IRs. As shown in Fig 2A, the amino acid sequences of 19
candidate Biomphalaria iGluRs and 7 IRs were aligned with A. californica iGluRs. A conserved amino
acid profile in the three key glutamate binding residues was observed in all Biomphalaria iGluRs,
except in the case of BglaGluR6, 9 and BglaGluN11, 14 which are predicted to contain one or
two. However, the profile was not conserved for the candidate Biomphalaria IRs, which lack one
or more residues, confirming their membership of the IR sub-family rather than the iGluR
subfamily. As indicated by the alignment, these IRs contain variable key glutamate binding residues.
Protein structural analysis demonstrated that all candidate B. glabrata iGluRs contain three
key residues at relative positions (Fig 2B shows R518, T669 and D717 based on BglaBluR6.2).
Additionally, ATD sites are found preceding the LBD S1 domain.
Tissue-specific expression of B. glabrata IRs
Expression profiles of all BglaIRs were performed to compare expression in defined tissues
with gene-specific primers by RT-PCR as shown in Fig 3. Tissues investigated included the
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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 [
]. 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.
sensory regions of adult olfactory organs, as well as non-sensory tissues such as the central
nervous system (comprising pooled cerebral, pleural, buccal, pedal and abdominal ganglia),
reproductive tissues and various tissues of the visceral mass. All IRs showed widespread expression
patterns in tentacle, foot, CNS, cerebral ganglia, and heart and blood vessels, except for IR25a
that appeared to be exclusive to the tentacle and CNS, including the cerebral ganglia. No
expression was detected in lung, gonad and digestive system.
Characterization and spatial expression pattern of B. glabrata IR25a
A comparative multiple amino acid sequence alignment of candidate Biomphalaria IRs and
reference sequences of IR25a retrieved from NCBI, including P. argus, A. californica and D.
melanogaster is shown in Fig 4A. The five sequences comprised 327 to 947 amino acids so we
chose to restrict the character sets to 410 alignable positions, in order to maintain a
conservative approach. All the IR25a receptors selected displayed remnants of classical IR motifs at
corresponding positions and the predicted domains that are critical structural regions responsible
for detecting odorous ligands and contributing to ligand specificity, are highlighted by lines
above the alignment. On one hand, the putative glutamate-interacting key residues (R,T,D/E)
are completely conserved only in lobster IR25a and two of these three residues are conserved in
Aplysia and Drosophila IR25a. In contrast, with Biomphalaria, only one key residue is
conserved in the predicted amino acid sequences of IR25a. On the other hand, all these IR25a
retain the R residue in S1 that interacts with glutamate in the iGluRs. However, the glutamate
binding residues in the S2 sequences are not conserved other than in PargIR25a, suggesting the
S2 domain has a much more variable sequence. Furthermore, when considering just the S2, the
unequal distribution of variable amino acids and, in particular, their strongest variability in
overall length (varies between 84 and 93 amino acids), displays significant variation.
Protein structure analysis and alignment for the B. glabrata IRs reveals that they share a
conserved ligand-gated ion channel structure closely resembling that of conventional iGluRs
and IRs with the 2 Pfam domains (PF10613 and PF00060,) formed by an extracellular
twolobed LBD, an ion channel pore and three TM regions. We selected BglaIR5 as a representative
to demonstrate the conserved and variable amino acids among these Biomphalaria IRs,
displaying the predicated ligand-binding S1 domain (D75-K102) and S2 (F306-R342) domain
regions (Fig 4B). The structure model of the Venus flytrap domain of Biomphalaria IR25a is
shown in Fig 4C, while the respective putative ligand binding site is indicted with an arrow.
To further explore the expression pattern of BglabIR25a in olfactory tissues, we analyzed its
cellular spatial distribution in the B. glabrata anterior tentacle by whole-mount in situ
hybridization. No tentacle staining was observed using a sense riboprobe for BglabIR25a (Fig 5A),
while localization was clearly visible within the distal and proximal tentacle regions using an
antisense riboprobe (Fig 5B–5D). Expression within the epithelium, as well as that of the
neuropil was determined following cryostat sectioning (Fig 5E–5I). This location is typical of
sensory neurons, although we lack an unambiguous neuronal marker to confirm this
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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.
The availability of the B. glabrata whole genome sequence has provided a basis for the in silico
identification and analysis of undiscovered and novel receptors. Application of the
homologybased searches against the original B. glabrata predicted proteome assemblies led to the
identification and examination of 7 putative IRs that possess regions homologous to the LBD site of
Aplysia IRs, as well as 19 iGluRs.
Identification of receptors was initially focused on the most highly-conserved structural
features that extend throughout the iGluR and IR families; then sequences were manually
reannotated to allow for a more refined phylogenetic analysis. For instance, it had been
established that the LBD exhibits 3 key residues (R, T, D/E) at fixed positions within S1 and S2,
forming a “Venus flytrap” structure [
]. Sequence alignment showed that these highly
conserved residues are present in most Biomphalaria iGluR sequences, while the IR exhibits a
variable pattern, lacking some or all known glutamate-interacting residues, supporting their
distinct classification from iGluRs.
Phylogenetic analysis confirmed the division of the 26 receptor sequences into two distinct
types (iGluRs and IRs) where 7 novel IR sequences could be categorized into two groups, that
is the IR25a/8a and other IRs. Our phylogenetic analysis is congruent with previous findings
with respect to the more ancient IR25a/8a lineages neighboring the non-NMDA group.
BglabIR1-5 are clearly distinct from the BglabIR25a/8a receptors and show only distant similarity
to the Aplysia IR sequences, and therefore represent a newly defined IRs cluster, probably
reflecting the snail’s very different ecological niche (freshwater versus marine). The lack of
obvious orthologs between these two molluscan species suggests expansion or contraction of
these receptors occurred after the splitting of the Gastropoda lineage.
A likely role for these BglabIRs as chemosensory receptors may be inferred based on their
noted expansion as well as spatial expression that includes the animal’s chemosensory organs
by RT-PCR. Furthermore, our whole mount in situ hybridization experiments enabled
visualization of IR25a expression in the tentacle of Biomphalaria, including the proximal and distal
regions, supporting a functional role in the detection of olfactory stimuli in all regions of the
organ. By comparison, in rhinophore sections of Aplysia dactylomela, IR25a has been
demonstrated to be expressed in small clusters of cells of a characteristic neuronal morphology close
to the sensory epithelial surface [
]. Also, the IR25a-related gene (OET-07) from Homarus
americanus, the American lobster, is expressed in topographically defined subpopulations of
mature olfactory sensory neurons [
]. Similarly, the presence of IR25a has been documented
in almost all of the lobster antennules, in a similar fashion, specifically within putative
chemosensory cells [
10, 27, 28
]. Together, these results are consistent with at least some of the
Biomphalaria IRs having a chemosensory function.
Previous studies, coupled with the results presented here confirm that IR25a is likely the
oldest IR family member, present in the early protostomian lineage, more than 600 Mya [
]. A representation of the IR25a from 4 species of Protostomia was used to unify protein
structure predictions across species, exhibiting a modified LBD. For example, Biomphalaria
and Aplysia receptor sequences show a deletion of 5 amino acid residues in the S2 domain
region compared with Drosophila and Panulirus (Fig 4). It is therefore tempting to speculate
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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
13 / 18
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
bluepredicted 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;
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that the length and structure of the LBD may play a functional role in recognizing various odor
Our tissue-specific expression studies show that BglaIR25a is exclusive to the tentacle and
CNS (of those tissues tested), while other IRs were detected in other tissues, including the
muscular foot, which may also contain chemoreceptor cells. IR expression determined in
non-sensory tissues supports the idea that IRs may play a more general role in Biomphalaria
chemosensation, also described in Aplysia [
]. The expression of IR genes in the blood
circulatory system (heart and blood vessel) is particularly intriguing, suggesting a possible role in
endocrine-mediated signaling. We anticipate that the knowledge gained from studies on the
chemical responses of the foot and blood circulatory system will help us determine whether IRs
are possibly involved in sensing or reacting to particular cues in these tissues. This should, in
turn, also be informative for determining the role of IR members in the tentacles and CNS of
Biomphalaria, and to establish the exact cues that each receptor is detecting. Another
important point pending further investigations is how the odors transfer to the receptor exposed on
the dendrite. In lobsters and other crustaceans, water-soluble odors are believed to dissolve in
the mucus covering the aesthetascs and diffuse through the cuticle into the receptor lymph
space where they contact the dendrite [
]. Whether this principle applies to molluscs or
whether a yet unknown mechanism is in operation remains to be tested.
It has been found that both excitatory and inhibitory olfactory signaling in gastropod
olfactory sensory neurons are mediated via G-protein-coupled second messenger pathways, which
are the largest superfamily of transmembrane proteins involved in cell signaling. For example,
water-borne chemical and pheromone detection in Aplysia may involve Gaq and can be
blocked by antisera specific for phospholipase C (PLC) and Ins(1,4,5)P3R [
]. Together with
these previous findings, it appears that the GPCRs are not the only known putative
chemosensory receptors expressed, which is entirely consistent with the findings of others [
chemosensory neurons of Aplysia represent an olfactory hybrid and utilize both classes of
olfactory receptors. Since GPCRs have been established as key receptors in olfaction for other
species, it raises the interesting possibility that IRs may also act in concert with GPCRs in B.
glabrata, where both pathways may contribute to the output of gastropod olfactory sensory
neurons. Furthermore, IRs have been detected in the rhinophore and oral tentacle of two
Aplysia genera, A. dactylomela and A. californica, coupled with 19 candidate iGluR and 7 IR genes
identified in this study. IR subunits have been found present in the olfactory tissue of two
divergent gastropoda subclasses, pulmonates (Biomphalaria) and opisthobranchs (Aplysia),
hinting at a general role of this ion channel family in initiating chemosensory signaling in the
Gastropoda. Indeed, we additionally found other IR genes with similarity to B. glabrata IR25a
within publically accessible databases of other aquatic and terrestrial molluscs such as oyster
(e.g. Crassostrea gigas), Lottia gigantea and Lymnaea stagnalis. Therefore, our study also allows
for a more thorough understanding of evolutionary relationships between the locotrophozoans
and the more popular model organisms that belong to other metazoan clades (e.g. the
ecdysozoa and deuterostomes).
S1 File. Comparative alignment between all Biomphalaria glabrata iGluRs and IRs.
S2 File. Gene-specific primers used for RT-PCR of IR genes. IR protein sequence alignments
are annotated with primer regions (underlined) and amplified regions highlighted. IR
nucleotide sequences have been annotated with primer regions highlighted.
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S1 Table. Information about all 26 B.glabrata IR-like sequences.
S2 Table. Accession numbers for Fig 1.
DPM is a National Health and Medical Research Council Senior Principal Research Fellow and
Senior Scientist at QIMR Berghofer. We acknowledge the Biomphalaria genome consortium
which provided a valuable resource for the gene sequences obtained in this study.
Conceived and designed the experiments: DL SFC. Performed the experiments: DL TW.
Analyzed the data: DL TW. Contributed reagents/materials/analysis tools: SFC DPM BAR. Wrote
the paper: DL TW SFC DPM.
16 / 18
stimulation of the mitogen-activated protein kinase signaling cascade in neurons. The Journal of
neuroscience: the official journal of the Society for Neuroscience. 1999; 19(14):5861–74. PMID: 10407026.
17 / 18
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