Group Title: BMC Biology
Title: Candidate chemoreceptor subfamilies differentially expressed in the chemosensory organs of the mollusc Aplysia
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Title: Candidate chemoreceptor subfamilies differentially expressed in the chemosensory organs of the mollusc Aplysia
Physical Description: Book
Language: English
Creator: Cummins, Scott
Erpenbeck, Dirk
Zou, Zhihua
Claudianos, Charles
Moroz, Leonid
Nagle, Gregg
Degnan, Bernard
Publisher: BMC Biology
Publication Date: 2009
 Notes
Abstract: BACKGROUND:Marine molluscs, as is the case with most aquatic animals, rely heavily on olfactory cues for survival. In the mollusc Aplysia californica, mate-attraction is mediated by a blend of water-borne protein pheromones that are detected by sensory structures called rhinophores. The expression of G protein and phospholipase C signaling molecules in this organ is consistent with chemosensory detection being via a G-protein-coupled signaling mechanism.RESULTS:Here we show that novel multi-transmembrane proteins with similarity to rhodopsin G-protein coupled receptors are expressed in sensory epithelia microdissected from the Aplysia rhinophore. Analysis of the A. californica genome reveals that these are part of larger multigene families that possess features found in metazoan chemosensory receptor families (that is, these families chiefly consist of single exon genes that are clustered in the genome). Phylogenetic analyses show that the novel Aplysia G-protein coupled receptor-like proteins represent three distinct monophyletic subfamilies. Representatives of each subfamily are restricted to or differentially expressed in the rhinophore and oral tentacles, suggesting that they encode functional chemoreceptors and that these olfactory organs sense different chemicals. Those expressed in rhinophores may sense water-borne pheromones. Secondary signaling component proteins Gaq, Gai, and Gao are also expressed in the rhinophore sensory epithelium.CONCLUSION:The novel rhodopsin G-protein coupled receptor-like gene subfamilies identified here do not have closely related identifiable orthologs in other metazoans, suggesting that they arose by a lineage-specific expansion as has been observed in chemosensory receptor families in other bilaterians. These candidate chemosensory receptors are expressed and often restricted to rhinophores and oral tentacles, lending support to the notion that water-borne chemical detection in Aplysia involves species- or lineage-specific families of chemosensory receptors.
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Research article

Candidate chemoreceptor subfamilies differentially expressed in
the chemosensory organs of the mollusc Aplysia
Scott F Cummins*', Dirk Erpenbeck2, Zhihua Zou3, Charles Claudianos4,
Leonid L Moroz5, Gregg T Nagle3 and Bernard M Degnan'


Address: 'School of Biological Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia, 2Department of Earth and
Environmental Sciences, Ludwig-Maximilians-University, Munich, Germany, 3Department of Neuroscience and Cell Biology, University of Texas
Medical Branch, Galveston, TX 77555, USA, 4Queensland Brain Institute, The University of Queensland, St. Lucia, Queensland, 4072, Australia
and 5Whitney Laboratory for Marine Science and Department of Neuroscience, University of Florida, St Augustine, Florida 32080, USA
Email: Scott F Cummins* s.cummins@uq.edu.au; Dirk Erpenbeck derpenb@gwdg.de; Zhihua Zou zhzou@utmb.edu;
Charles Claudianos c.claudianos@uq.edu.au; Leonid L Moroz moroz@whitney.ufl.edu; Gregg T Nagle gtnagle@utmb.edu;
Bernard M Degnan b.degnan@uq.edu.au
* Corresponding author


Published: 4 June 2009
BMC Biology 2009, 7:28 doi:10.1186/1741-7007-7-28


Received: 6 November 2008
Accepted: 4 June 2009


This article is available from: http://www.biomedcentral.com/1741-7007/7/28
2009 Cummins et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Marine molluscs, as is the case with most aquatic animals, rely heavily on olfactory
cues for survival. In the mollusc Aplysia californica, mate-attraction is mediated by a blend of water-
borne protein pheromones that are detected by sensory structures called rhinophores. The
expression of G protein and phospholipase C signaling molecules in this organ is consistent with
chemosensory detection being via a G-protein-coupled signaling mechanism.
Results: Here we show that novel multi-transmembrane proteins with similarity to rhodopsin G-
protein coupled receptors are expressed in sensory epithelia microdissected from the Aplysia
rhinophore. Analysis of the A. californica genome reveals that these are part of larger multigene
families that possess features found in metazoan chemosensory receptor families (that is, these
families chiefly consist of single exon genes that are clustered in the genome). Phylogenetic analyses
show that the novel Aplysia G-protein coupled receptor-like proteins represent three distinct
monophyletic subfamilies. Representatives of each subfamily are restricted to or differentially
expressed in the rhinophore and oral tentacles, suggesting that they encode functional
chemoreceptors and that these olfactory organs sense different chemicals. Those expressed in
rhinophores may sense water-borne pheromones. Secondary signaling component proteins Gaq,
Ga,, and Gao are also expressed in the rhinophore sensory epithelium.
Conclusion: The novel rhodopsin G-protein coupled receptor-like gene subfamilies identified
here do not have closely related identifiable orthologs in other metazoans, suggesting that they
arose by a lineage-specific expansion as has been observed in chemosensory receptor families in
other bilaterians. These candidate chemosensory receptors are expressed and often restricted to
rhinophores and oral tentacles, lending support to the notion that water-borne chemical detection
in Aplysia involves species- or lineage-specific families of chemosensory receptors.


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Background
All animals must recognize and respond to chemosensory
information in their environment. Although the marine
mollusc Aplysia has been a valuable model to investigate
the molecular basis of behavior [1,2] and reproduction
[3,4], our knowledge of how they recognize and respond
to environmental signals is limited. In particular, it is
unknown how they distinguish and bind water-soluble
molecules and transfer exogenous information intracellu-
larly. In contrast, the molecular components and mecha-
nisms of chemical detection in a range of vertebrates and
other invertebrates have been well studied.

Vertebrate chemoreception is made possible by six dis-
tinct classes of multi-transmembrane receptors: (i) olfac-
tory receptors (ORs) [5], (ii) trace amine-associated
receptors [6], vomeronasal receptors (iii) type 1 and (iv)
type 2 [7,8] and taste receptors (v) type 1 and (vi) type 2
[9,10]. Besides binding chemical molecules, all share the
common traits of seven transmembrane (7-TM) domains,
G-protein signaling and precise sensory cell expression. In
mammals, non-volatile pheromone perception is thought
to act primarily through the vomeronasal organ sensory
epithelium [11] and be mediated intracellular via the
interaction of chemical molecules with vomeronasal
receptors located on the dendrites ofvomeronasal sensory
neurons [12]. However, in teleost fishes who do not have
a vomeronasal organ, the vomeronasal receptors are
found in the main olfactory epithelium [13]. It appears
that genes involved in an animal's response to its environ-
ment are subject to extensive gene duplication, gene loss
and lineage-specific expansion over time, leading to large
gene families such as those observed in the OR and vome-
ronasal receptor repertoire. In fact, OR genes represent the
largest mammalian gene family [ 14].

Chemoreception through 7-TM domain receptors appears
to have evolved multiple times independently, as verte-
brate chemoreceptors are not closely related to those
known in insects and nematodes. Recognition of external
chemicals in Drosophila is accomplished by families of
130 genes encoding 7-TM domain receptors [15,16],
including OR (60) and gustatory receptors (70). Gusta-
tory receptors are greatly reduced in the honeybee [17].
Insect chemoreceptors do not belong to the G-protein
coupled receptor (GPCR) family due to a unique inverse
membrane topology [18]. Rather, they use an alternative,
non-G protein-based signaling pathway where receptors
not only detect chemicals but can also act as ion channels
[19]. In support of this, heterologous cells expressing silk-
moth, fruitfly or mosquito heteromeric OR complexes
showed G-protein independent extracellular calcium
influx and cation-non-selective ion conductance upon
stimulation with odorant [19]. Nevertheless, chemical


detection is still mediated by a large and divergent family
of 7-TM domain receptors.

A central issue that has not been adequately addressed is
how water-borne chemicals are detected at the molecular
level by the huge diversity of invertebrates that inhabit
marine environments. In marine invertebrates, chemo-
sensory abilities are essential for almost all aspects of their
life, from feeding to predator avoidance and reproduc-
tion. A recent bioinformatic survey of the sea urchin
genome resulted in the identification of a remarkable
diversity of chemoreceptors, expressed specifically and
differentially in adult sensory structures [20]. Meanwhile,
there have been important findings forthcoming from
research into the molluscan group. Olfactory studies of
squid have shown that both phospholipase C (PLC) and
cAMP-mediated pathways may be involved in olfactory
sensory neurons activation [21]. In support of this, immu-
nolocalization experiments revealed the presence of G
proteins involved in both cAMP (Gao) and PLC (Ga.q)
pathways which are clearly co-expressed in certain cell
types.

Aplysia possesses many advantages necessary for chemical
communication research, such as an extensive knowledge
of its anatomy, a detailed understanding of the molecular
and cellular basis of behavior, and now considerable
genomic and expressed sequence tag (EST) resources.
Moreover, we have found that in Aplysia, conspecific and
congener attraction is mediated by a remarkable cocktail
of water-borne protein pheromones [4,22]. In Aplysia,
freshly laid egg cordons are considered to be a source of
both water-borne and contact pheromones that attract
conspecifics and closely related species to the area and
induce them to mate and lay eggs. Egg laying results in the
release of at least four proteinaceous attraction pherom-
ones, including the 58-residue attraction [4,23,24]. T-maze
bioassays have demonstrated that binary blends of attrac-
tin with either enticin, temptin or seductin are sufficient
to attract potential mates [23].

At the anatomical level, Aplysia chemosensory detection is
achieved by the rhinophore [25], specialized anterior sen-
sory organs on the dorsal surface of the head. Rhinophore
are retractile and primarily used for distance chemorecep-
tion and rheoreception (response to water current),
whereas the oral tentacles, which are found more ven-
trally, are possibly involved in contact chemoreception
and mechanoreception [26]. The neuroanatomical organ-
ization of rhinophores includes a rhinophore groove
where most of the sensory cells appear to be concentrated.
Its sensory epithelium contains sensory neurons that
project axons back to rhinophore ganglia and dendrites
that end in either a surface-exposed cilium or a small pro-
tuberance [26-28]. Consistent with a potential role in


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chemical transduction, gene transcripts encoding G pro-
tein, PLC or inositol 1,4,5-trisphosphate receptor were
found to be expressed in Aplysia rhinophore sensory epi-
thelium [29]. The involvement of nitric oxide as a poten-
tial chemosensory processing component has also been
implicated in molluscan chemoreception based on cyto-
logical NADH-diaphorase histochemistry of the Aplysia
rhinophore [30]. Of significance, was the finding that
nitric oxide synthase is present in epithelial sensory-like
cells that had multiple apical ciliated processes exposed to
the environment. This is consistent with findings demon-
strating that inhibition of nitric oxide synthase disrupts
slime trail following, suggesting a role for nitric oxide in
neural processing of stimuli in snails [31].

The presence of G protein mRNAs in Aplysia sensory epi-
thelium suggested that multi-transmembrane GPCR-like
proteins could play an important role in chemosensory
detection. With the availability of a 2x genome coverage
for Aplysia californica, we expected that it would provide an
excellent and first opportunity to investigate the molecu-
lar basis of chemical detection in a mollusc. Here, we per-
formed iterative Basic Local Alignment Search Tool
(BLAST) searches to identify genes similar to rhodopsin
GPCR genes encoding 7-TM domains from the A. califor-
nica genome. We identified genes representing three
unique monophyletic families that show rhinophore, oral
tentacle and ovotestis expression. Based on their expres-
sion, these may encode chemosensory proteins, including
pheromone and gustatory receptors. Antisera directed
against a conserved region of a candidate chemosensory
receptor, as well as Aplysia Gcaq, Gai, and Gao, confirmed
their expression in sensory tissues, with localization to the
outer sensory epithelium.

Results
Identification of genes encoding rhodopsin G-protein
coupled receptor-like proteins in the Aplysia californica
genome
We performed iterative tBLASTn for closely related novel
genes and discovered a large number of genes encoding
rhodopsin GPCR-like proteins. Using this approach, we
successfully identified a total of 90 genes encoding pro-
teins belonging to the GPCR superfamily. Of these, 72
were predicted to contain 7-TM domains. It was not pos-
sible to annotate the full-length sequence of all genes,
especially in the 5'-regions, and 18 genes encoding six
transmembrane domains were considered partial-length.
Note that these numbers are the minimal estimates,
because the genome sequencing of A. californica had not
been fully completed. We expect that further rhodopsin
GPCR-like genes will be found beyond the ones we have
identified. Also, several of the multi-transmembrane gene
models appeared to be pseudogenes with various defects,
including the insertion of stop codons and frame-shifting


indels leading to premature termination of the coding
region. These were not included in the final data set.

Phylogenetic construction and analysis of identified
rhodopsin G-protein coupled receptor-like genes
We have performed phylogenetic analyses of the 90
selected rhodopsin GPCR-like genes ingroupp), together
with four non-Aplysia GPCR genes (outgroup). The 94
sequences (including outgroup taxa) comprised 264 to
444 characters. For the two phylogenetic analyses (with
and without outgroup, respectively) we had to restrict the
character sets to 96 and 166 alignable positions, respec-
tively, in order to maintain the conservative approach.
Both phylogenetic reconstructions, with and without out-
group, display a congruent picture regarding the phyloge-
netic relationship of the Aplysia GPCRs (Figures la and
ib). Branch lengths of groups A (subfamily a) and B (sub-
family b), especially in the former, are considerably
shorter than of group C (subfamily c). GPCR-like gene
group features are summarized in Additional file 1. The
phylogenetic analyses support a monophyly of the three
different subfamily groups, although sufficient phyloge-
netic signal for subfamily c monophyly is achievable only
when non-Aplysia taxa are not present.

Besides structural predictions that place these genes
within the 7-TM superfamily, which includes rhodopsin
GPCRs, these genes have little amino acid identity (<10%)
to any known genes, and do not appear in the published
Aplysia EST neuronal transcriptome [32]. They show only
distant similarity with known molluscan multi-trans-
membrane receptors, including well-characterized Aplysia
neurotransmitter GPCRs. GenBank tBLASTn searches also
reveal most amino acid identity with regions of orphan
GPCRs of the sea urchin, Strongylocentrotus purpuratus (E
value le-11), various ghrelin receptors (for example, Rat-
tus norvegicus, E value 2e-07) and a candidate GPCR
(Caenorhabditis elegans, E value 4e-04) for subfamilies a, b
and c, respectively. Based on the gene characteristics
described below and observed tissue distribution (see
Results, Tissue specificity of expression), we subsequently
called them candidate Aplysia californica chemosensory
receptors (AcCRs) subfamilies a to c.

Candidate Aplysia californica chemosensory receptors subfamily a
A total of 28 genes encoding rhodopsin GPCR-like pro-
teins were identified within the Aplysia genome that
grouped into the single monophyletic AcCRa. Of these, 10
appear to encode 7-TM domain proteins that range in size
from 355 to 368 amino acids (40.1 to 41.5 kDa). The
remaining 18 genes encoded 6-TM domain proteins; how-
ever, we were unable to identify an initiator methionine
suggesting that these represent partial length genes. Figure
2a is a comparative representation of the 7-TM proteins,
showing conservation of predicted amino acid sequences


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(a)






















(b)


















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Figure I
Phylogenetic reconstruction of identified Aplysia rhodopsin G-protein coupled receptor-like gene sequences.
Subfamily type a (red) type b (blue) and type c (green). The trees are based on the bayesian inference reconstruction combined
with the RAxML results. The numbers at the branches correspond to posterior probabilities (>75) and congruent bootstrap
probabilities (>60, with exceptions), respectively. (a) Unrooted tree without non-Aplysia sequences. (b) Phylogram with non-
Aplysia sequences included.




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Figure 2 (see previous page)
Analysis of candidate Aplysia californica chemosensory receptors subfamily a. (a) Conservation of predicted amino
acid sequences for 10 full-length genes is displayed as a consensus strength as color-coded histogram. In this representation,
the relative frequency with which an amino acid appears at a given position is reflected by the color, as depicted by the scale
bar. The seven transmembrane domains (based on the HMMTOP version 2.0 program) are indicated by a solid bar above the
sequence. (b) Schematic representation of the genome organization of clustered genes, partial AcCRI4a and full-length
AcCR2 7a genomicc contig AASC 01105652), including predicted start (ATG) and stop codons (TAG) and intron/exon structure
of AcCR27a leading to the mature multi-transmembrane protein. Met, methionine. (c) Comparative amino acid alignment of
AcCRI4a and AcCR27a. Identical amino acids are highlighted in black. Putative intracellular (IC), extracellular (EC), N-terminus
and C-terminus domains are shown. Arrow indicates the intron/exon boundary; asterisks show potential N-linked glycosyla-
tion sites; black circles represent highly conserved cysteines.


for the gene repertoire. Overall, amino acid sequence
identity among the subfamily ranges from 70% to 95%.
These genes are most distinct from other subfamily
sequences we identified due to the presence of a con-
served intron between coding regions ISLM95/GLAV
(based onAcCR27a, Figures 2b and 2c). This was later ver-
ified by reverse transcription-polymerase chain reaction
(RT-PCR) cloning and sequencing (See Results: laser cap-
ture microdissection (LCM)/RT-PCR gene identification).
Also, conservation of GLA/SV96._., Y120, ITAFITF150-156,
K178, G198, DRA/V271-273, MVT287-289, ET336-337, and
NSSVNI339_344 are distinct this subfamily. Most variability
is found within the N-terminal regions (also see align-
ments in the Additional file 2). Semi- and highly con-
served cysteine residues are located at C114, C159, C161,
C247, C248 and C298, while glycosylation sites can be found
at N18NS, N911S, N210A/VT and N339SS. Most share a sig-
nature motif with a FITAFITFERCLCIA amino acid
sequence in the third transmembrane domain and second
intracellular loop. We found that one genomic contig
AASC01105652 contained two genes, AcCR14a and
AcCR27a (Figure 2b). We predict that these are part of
larger clusters that may become apparent upon comple-
tion of the full genomic assembly. Within the Aplysia
genome (1.8 Gb), AcCR14a andAcCR27a are separated by
9031 bp, and are in the same transcriptional orientation.
A comparative amino acid alignment of the partial
AcCR14a and full-length AcCR27a proteins are shown in
Figure 2c.

Candidate Aplysia californica chemosensory receptors subfamily b
(AcCRb)
A total of 38 full-length intronless genes encoding pre-
dicted multi-transmembrane rhodopsin GPCR-like pro-
teins were identified belonging to AcCRb. Sizes ranged
from 319 to 364 amino acids in length (36.2 kDa to 40.8
kDa). Figure 3a is a comparative representation of these
proteins, showing conservation of predicted amino acid
sequence for the gene repertoire. Overall, amino acid
sequence identity among the subfamily members ranges
from 43% to 92%. Most variability is found at the N-ter-
minal region and most conservation is within the pre-


dicted transmembrane 3 (also see alignments in
Additional file 2). Most share a signature motif with a
WITAFVTFERCLCIA amino acid sequence in the putative
second intracellular loop. We found that at least some of
the genes are clustered in the genome, including genes
AcCR11b and AcCR12b (Figure 3b), as well as genes
AcCR13b and AcCR14b (Figure 3c). Within the Aplysia
genome, genes AcCR11b and AcCR12b are separated by
9173 bp while genes AcCR13b and AcCR14b are separated
by 13297 bp, which also includes a putative non-long ter-
minal repeat retrotransposon element in the reverse ori-
entation (10532 to 9078 bp) (Figure S1 in Additional file
3). Amino acid identity between AcCR1 lb and AcCR12b,
as well as AcCR13b and AcCR14b is high, 80.5% and
73.9% respectively (Figure S2 in Additional file 3). Con-
served cysteines (based on AcCR1 Ib) can be found at C88,
C133, C135, C221 and C269 and N-linked glycosylation sites
include NsES, Ns65T, N184KT, N310ST, and N320MS.

Candidate Aplysia californica chemosensory receptors subfamily c
(AcCRc)
A total of 24 full-length genes containing uninterrupted
coding regions were identified belonging to AcCRc. Sizes
ranged from 324 to 433 amino acids in length (37.7 kDa
to 49 kDa). Figure 4 is a comparative representation of
these proteins, showing conservation of predicted amino
acid sequence for the gene repertoire. Overall, amino acid
sequence gene identity ranges from 19% to 91%. Most
variability is found at the N-terminal region and within
the proposed third intracellular domain, which carried
length polymorphisms (also see alignments in Additional
file 2). Conserved cysteines (based on AcCR2c) are
located at C136 and C138, while semi- and highly conserved
N-linked glycosylation sites are located at Ns5ET, NIS,
N541T, N187TT, N2801S, N331TS.

Tissue specificity of expression
We next studied the expression of subfamily genes in
adult tissues using degenerate primers designed to con-
served codons specific to each of the AcCR subfamilies.
This approach was designed to detect if any members
within the three subfamilies were expressed in the target


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Figure 3
Analysis of candidate Aplysia californica chemosensory receptors subfamily b. (a) Conservation of predicted amino
acid sequences for 38 full-length genes is displayed as a consensus strength as color-coded histogram. In this representation,
the relative frequency with which an amino acid appears at a given position is reflected by the color, as depicted by the scale
bar. The seven transmembrane domains (based on the HMMTOP version 2.0 program) are indicated by a solid bar above the
sequence. (b) Schematic representation of the genome organization of clustered genes AcCRI Ib and AcCRI2b genomicc contig
AASCOI 159697), including predicted start (ATG) and stop codons (TAG). (c) Similar to (b) but clustered genes AcCRI3b and
AcCRI4b genomicc contig AASCO 1064969). A predicted retrotransposon element is identified in the reverse orientation
between the two gene sequences (see Figure SI in Additional file 3). Met, methionine.



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I


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370 380 390 400 410 420 430 440 450 460 470 480

Figure 4
Analysis of candidate Aplysia californica chemosensory receptors subfamily c. Conservation of predicted amino acid
sequences for 24 full-length genes is displayed as a consensus strength as color-coded histogram. In this representation, the rel-
ative frequency with which an amino acid appears at a given position is reflected by the color, as depicted by the scale bar. The
seven transmembrane domains (based on the HMMTOP version 2.0 program) are indicated by a black bar above the sequence.


0~ N

0 \0 C, j


Nf 0 CI


-711


-711

-404


Figure 5
Tissue-specific expression of candidate Aplysia chemoreceptors. Schematic representation of Aplysia californica show-
ing the location of selected sensory tissues (rhinophore, oral tentacle, skin), central nervous system (CNS) and reproductive
organs (albumen gland (AG), ovotestis, large hermaphroditic duct (LHD)) used for RNA isolation and RT-PCR. Amplification
products were 756 bp, 832 bp, 512 bp for AcCRa, AcCRb and AcCRc, respectively. The PCR control, using water instead of
cDNA, was negative (cont -ve). Actin cDNA was amplified from each tissue preparation (224 bp).



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tissues. Transcripts from AcCRa and AcCRb were identi-
fied in rhinophore, as well as the oral tentacle; AcCRa
transcript was also present in the ovotestis (Figure 5).
AcCRc transcripts were detected in the oral tentacle, indi-
cating that each of the gene subfamilies are differentially
expressed in the sensory tissues. No transcripts were
detected in the skin, central nervous system, albumen
gland or large hermaphroditic duct using the method
described. We used the Aplysia housekeeping gene actin to
demonstrate the integrity of each RT sample (224 bp). An
alignment of deduced AcCRa amplicon sequences
obtained from rhinophore, oral tentacle and ovotestis
revealed that different members are present, which corre-
spond most closely to genes AcCR5a (97%), AcCR19a
(100%), and AcCR7a (90%), respectively (Figure S3 in
Additional file 3). A comparative analysis of proteins
encoded in AcCRb amplicons showed that the rhinophore
and oral tentacle express a common candidate chemore-
ceptor gene, corresponding to the gene AcCR 17b. Several
point mutations, however, were present within the rhino-
phore transcript, which led to a premature stop codon
(Figure S3 in Additional file 3). The AcCRc amplicon cor-
responded to the clone AcCR2c.

Scanning electron microscopy and molecular
identification of candidate chemoreceptors
Based on results from tissue-specific expression experi-
ments, we focused on the chemosensory organs. In repro-
ductively mature Aplysia adults, the rhinophore (about 1
cm in length) is round and tapered from the base to the
tip (Figure 6). Scanning electron microscopy (SEM) sup-
ports histological examination [27,28] and reveals that
rhinophore grooves comprise folds of sensory epithelia
bearing numerous cilia extending from a common pore
(Figures 6a to 6c). The tip and outside surface of rhino-
phores are largely devoid of obvious cilia. This rhino-
phore groove epithelium was previously isolated by LCM
and used to construct a cDNA library [12]. In a mature
adult, the oral tentacle extends laterally and anteriorly
from the ventral surface of the head, with an epithelium
containing numerous bunched cilia (Figures 6d to 60.
Although ciliated regions were most common, the oral
tentacle did contain regions of no obvious cilia. We next
performed PCR with the aim to identify full-length candi-
date chemoreceptors from the rhinophore epithelium
LCM library or prepared oral tentacle cDNA. Three clones
were selected for further analysis. Subsequent protein
sequence analyses using the Protein Families database of
alignments and Interpro database [33] revealed that the
deduced amino acid sequences have characteristics com-
mon to rhodopsin GPCRs (Figure S4 in Additional file 3).

AcCRa
PCR amplification of a rhinophore epithelium LCM
library using degenerate primers that were selective for


members of AcCRa sequences (primer combinations Al
to A5) generated amplification products of 757 bp. Sev-
eral amplicons were successfully cloned and sequenced,
revealing multiple partial-length AcCRa genes. Subse-
quently, the full length of one gene sequence was identi-
fied by 5'- and 3'-RACE, containing 1269 bp and encoding
a protein of 354 amino acids (Figure 7a corresponding
most closely, 91%, to partial gene AcCR8a). The sequence
data has been submitted to the GenBank database under
accession number EU935862. It possesses three protein
kinase C (PKC) phosphorylation sites (T203YK, S256DR,
S337SK,) and four N-linked glycosylation sites (Ni8ET,
N76IS, N19GAT and N325SS). The intron/exon boundary
exists between coding regions ISLM80/GLAV. Kyte-Doolit-
tie hydropathy profiles indicate the existence of seven
hydrophobic transmembrane segments that were com-
posed of between 20 and 25 residues, connected by extra-
cellular and cytoplasmic loops.

AcCRb
PCR amplification of a rhinophore epithelium LCM
library using degenerate primers that were selective for
AcCRb sequences (primer combinations B1 to B8) gener-
ated amplification products of 744 bp. Several amplicons
were successfully cloned and sequenced, revealing multi-
ple partial-length AcCRb genes. Subsequently, the full
length of one gene sequence was identified by 5'- and 3'-
RACE, containing 1483 bp and encoding a protein of 354
amino acids (Figure 7b corresponding most closely,
99.4%, to gene AcCR29b). The sequence data has been
submitted to the GenBank database under accession
number EI808014. It possesses potentially six PKC phos-
phorylation sites (S11SK, TisQK, T147PR, T249NR, S322SK,
S343ER) and four N-linked glycosylation sites (N5NS,
N65IT, N184VT and N310ST) within the predicted N-termi-
nal region and intracellular loop domains. Kyte-Doolittle
hydropathy profiles of the deduced amino acid sequence
showed that it contained seven hydrophobic transmem-
brane segments that were each composed of 25 residues.

AcCRc
AcCRc genes could not be PCR-amplified from a rhino-
phore LCM library. However, transcripts could be
obtained from oral tentacle cDNA preparations (Figure 7c
- corresponding most closely, 98%, to identified gene
AcCR2c). PCR amplification of oral tentacle cDNAs using
degenerate primers selective for AcCRc (gene combina-
tion Cl) generated an amplification product of 824 bp.
The amplicon was successfully cloned and sequenced.
Subsequently, the full-length gene sequence was identi-
fied by 5'- and 3'-RACE, containing 1752 bp and encoding
a protein of 398 amino acids. The sequence data has been
submitted to the GenBank database under accession
number EIJ808013. It possesses 10 PKC phosphorylation
sites (T7ER, T28LR, T150FK, T188TR, S195SK, S275RR, S295NK,


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Figure 6 (see previous page)
Scanning electron micrograph analysis of two Aplysia chemosensory organs. Aplysia species possess rhinophore
(rhino) and oral tentacles (ot) to detect chemical stimuli in their marine environment. (a to c) Scanning electron microscopy
(SEM) migrographs showing the surface of the Aplysia rhinophore. Scale bars: 300 rpm, 100 pm and 10 rpm, respectively. (a) A
low-power SEM micrograph showing the rhinophore tip. rg, rhinophore groove; tip, rhinophore tip. (b) A medium-power SEM
micrograph of the rhinophore tip showing the cilia-bearing epithelium within the rhinophore groove. f, folds. (c) Higher-power
SEM micrograph of groove epithelium (boxed region in b) showing numerous bunched cilia extending from a common pore.
Also evident are pores lacking obvious bunched cilia. ci, numerous long cilia. (d to f) SEM micrographs showing the surface of
the Aplysia oral tentacle. Scale bars I mm, 10 pm and I rpm, respectively. (d) A low-power SEM micrograph showing the oral
tentacle, ant, anterior. (e) A medium-power SEM micrograph of the oral tentacle showing a mat of cilia-bearing epithelium. (f)
Higher-power SEM micrograph of epithelium (boxed region in e) showing numerous bunched cilia extending from a common
pore.


S308AK, T332SR, S385YR, a cAMP phosphorylation site
(K234KSS) and six N-linked glycosylation sites (N5ET,
N16IS, N54IT, N18yTT, N280IS, N329TS) within the N-termi-
nal region and intracellular loop domains. Kyte-Doolittle
hydropathy profiles of the deduced amino acid sequence
showed that it contained seven hydrophobic transmem-
brane segments that were composed of between 20 and
25 residues.

Immunofluorescent localization of a candidate
chemoreceptor
We complemented our RT-PCR gene expression study by
analyzing the spatial distribution of AcCR29b protein
within rhinophore and oral tentacle. Figures 8a and 8b
shows representative sections of immunoreactivity within
similar cell types located at the epithelial surface of rhino-
phore and oral tentacle, respectively. In both, antisera
strongly label cell bodies and processes that extend to the
surface, containing no apparent cilia. Rhinophore immu-
nolabeling was most prominent in cells located in epithe-
lia at the tip and outer surfaces, while not obvious within
epithelium of the rhinophore groove at the magnification
tested. Controls in which the primary antibody was prea-
bsorbed against its antigenic peptide showed greatly
reduced staining at the same exposure (Figure 8b, inset).

Distribution of Ga q Ga, and Gao immunoreactivity in
rhinophore
Sections were taken from the sites of pheromone detec-
tion, the rhinophore. Aplysia G proteins encoded by previ-
ously isolated transcripts from rhinophore sensory
epithelium [29] are shown schematically in Figure 9a.
Commercial antibodies used for this study were directed
to the C-terminus which shares 100% identity with Aplysia
Ga proteins. Immunofluorescence studies confirmed the
immunoblot expression results [29] and demonstrated
localization of immunoreactive Aplysia Goiq in rhinophore
sections. Numerous Goiq immunoreactive fibers were
observed proximal to the rhinophore epithelium and
within the distal layer (Figure 9b); Guqimmunoreactivity
appeared to be present in the outer sensory surface, con-


sistent with a potential role in pheromone signal trans-
duction. Gai-labeled cells were identified throughout
rhinophore sections, where immunoreactivity was partic-
ularly concentrated in the distal regions of sensory epithe-
lia. Inspection of these sections at high magnification
revealed that the majority of cells in the epithelium were
labeled, including cells presumed to be supporting cells
and sensory neurons (Figure 9c). Immunoreactive fibers
could also be found spreading into the cortex of the rhi-
nophore, although this was less prominent. In contrast,
immunoreactive Aplysia GCa had a more restricted distri-
bution, in that each section contained several immunore-
active fibers that were observed primarily within
presumptive olfactory neuron dendrites (Figure 9d); fewer
immunoreactive presumptive sensory neuron cell bodies
were observed. No immunoreactivity was observed when
primary antibody to Goiq, Gai or Gao was omitted (data
not shown).

Discussion
In this study, we provide an important step towards
understanding the molecular and cellular basis of chemo-
sensory recognition in molluscs. Mining of the incom-
plete A. californica genome revealed a novel and diverse set
of genes encoding 90 (72 considered full-length intact)
rhodopsin GPCR-like proteins that are likely to mediate
chemosensory responses in Aplysia. To initially identify
genes encoding multi-transmembrane GPCR-like proteins
that may play a role in chemosensory detection, a number
of assumptions were made that have proved important for
the successful isolation of chemoreceptors in other meta-
zoans. First, receptor genes would encode 7-TM domains
and be clustered in the genome. Second, receptors would
be relatively rapidly evolving and thus have limited amino
acid sequence identity to members of known conserved
GPCR families. Finally, receptors would be encoded by
unique families of related genes, as has been observed in
a range of other bilaterians [5,15,34]. Phylogenetic analy-
sis of the identified Aplysia genes revealed the existence of
three monophyletic subfamilies, which we have named
candidate Aplysia californica chemosensory receptor


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(a)
MPLSRLWTNQEVYSPDGNETALMATTTGSSL 31
SSTDILSDEINS CLLLVLNVI
IVVFAKQGFQD SMNISLGLASLSLVTM 93
III YKPLFYLSELPFDPRDI
TF LCIAVPLKVK 155
TIITPGRTIKT R
LEWVFDFRTNATVLKATYKAKREILEAITFL: 17
AKLNSKTKW
RKATAAKSDRATDGVGVKDQKVVK 279
TTDFRIDGVYRNE
-- KMSSKYR 341
AVFMKTFLNKQER

(b) C...
MGPGNNSQARSSKSTQKGLLDDYTLT L 31
LNIIVFCKQGFKD
TFNPLF 93
IEADLPMVYQDI
LCTITVPLKVKMILTPRR) 155
~GPRHFPERNVT
IIGSVYNENGPFYEG 217
VITIHNFLLKSKWRQSASSATRQEFL DYC
TNRDKKVV 279
SSEYRTGGRYQNVY
SSSKYRKILDEMLKRKEGNRG 341
PSERKAVRCIQSL Ir-llT

(c)
MSLVNETERELKGSHNISEHGGLIDDQTLR 31
NITVFLTLG
AKD CDI 93
LDAYGSADFYVDPRGLYYQ
T I CVALPFRFKELFTEKPAV 155
LRVQWDPRT
NTTRVLLWSSKDMPAITAFLDLWNH 217
SLKKSSQFQRRGAARPS
EPDGPNNFKTSNEVGEGEENVLRDPDSRRDN 279
NISTPYCPTNVEKESSNKIKVEKSPQTLSAK
NRRRRVV 341
RAEPDINFGHRYH
PKLNPSYRKTFSQIFGIGQTK

Figure 7
Molecular identification of candidate Aplysia californica chemoreceptor genes. Deduced amino acid sequence and
schematic model of (a) AcCRa cDNA isolated from rhinophore laser capture microdissection (LCM) RNA (GenBank:
EU935862), (b) AcCRb cDNA isolated from rhinophore LCM RNA (GenBank: EU808014) and, (c) AcCRc cDNA isolated
from oral tentacle cDNA (GenBank: EU808013). Arrowhead, indicates position of intronlexon boundary and boxed grey areas
indicate predicted transmembrane domains (based on HMMTOP version 2.0 program). Unfilled boxes show relative positions
of the conserved motif Glu-Arg-Cys (ERC) at the proximal part of the cytoplasmic 2 domain.



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Figure 8
Immunofluorescence localization of AcCR29b. An antibody designed to the N-terminal region of AcCR29b was used to
localize corresponding protein within epithelial cells of (a) rhinophore and (b) oral tentacle, shown in green. Arrowheads
show immunopositive cell bodies and arrows point to processes exposed to the surface. Sections were counterstained with
DAPI (blue) to show nuclear staining. A control in which the primary antibody was preabsorbed against its antigenic peptide
showed greatly reduced staining at the same exposure (b, inset). Scale bars = 50 pnm.


(AcCR) subfamilies a to c; their primary features are sum-
marized in Table 1. The gene expansion observed could
provide the diversity of receptors required to enable the
animal to recognize diverse water-soluble molecules, as
well as complex pheromone blends during the coordina-
tion of attraction and reproduction.

Consistent with known chemosensory receptors (for
example, insect, rodent, fish), all selected genes that were
considered full length encoded 7-TM regions and semi-
conserved glycosylation sites, as well as several common
cysteine residues and amino acid sequence motifs. Con-
served amino acids and post-translational modifications
would likely contribute to the correct folding and func-
tioning within the plasma membrane so that they may
bind chemical stimuli and couple to appropriate second-
ary signaling molecules. For example, Katada and col-
leagues [35] demonstrated that in rodents, N-terminal
glycosylation is critical for proper targeting of ORs to the
plasma membrane. While proteins encoded by AcCRa
and AcCRb genes share notably high sequence identity,
comparative analysis within AcCRc shows that they share
as little as 19% amino acid identity. As a consequence,
there are very few defining sequence motifs which are
retained throughout. However, it is not uncommon for
chemoreceptors, and in particular gustatory receptors, to


be divergent; similarity between most insect and mamma-
lian gustatory receptor pairs is only 15% and 25% or less
at the amino acid sequence level, respectively [10,36].

Of the major GPCR superfamily groups, the identified
Aplysia genes categorize most closely to the rhodopsin
GPCR family (based on Interpro database [33]). As is the
case with many other rhodopsin family GPCRs, these
genes largely lack introns. Moreover, all encoded proteins
have a short N-terminus and a highly conserved arginine
(R) residue located at the cytosolic end of the third trans-
membrane domain. This residue is typically associated
with the DRY (Asp-Arg-Tyr) motif, crucial for controlling
agonist-dependent receptor activation. Of the three resi-
dues constituting DRY, arginine is the most conserved res-
idue, and appears to be essential for forming
intramolecular interactions that constrain receptors in
either the inactive or activated conformation [37]. Con-
sistent with this, receptors lacking the arginine side chain
fail to activate G-protein signaling [38,39]. In the novel
Aplysia proteins, however, this has been replaced by an
ERC motif, a feature also observed in the human prostag-
landin F2a receptor and most other prostanoid receptors
[40]. Studies show that substitution of the glutamic acid
to a threonine residue leads to full constitutive activation
and implicates the region in agonist-dependent G-protein


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Figure 9
Analysis of Aplysia Guq, Gui, and Gao proteins in rhinophore. (a) Schematic diagrams of Gaq (GenBank: D0397515),
Gui (GenBank: D06561 I 1 ) and Ga. (GenBank: D06561 12) showing the location of N-terminal cysteines that may be sites for
palmitoylation (wavy lines), a putative cholera toxin ADP-ribosylation site (0), and the binding site of Ga-specific antibodies
used for immunolocalization analyses. (b to d) Immunofluorescent (green) localization of Guq (b), Ga, (c) and Gao (d) in the
rhinophore sensory epithelium (SE), including presumptive sensory neuron cell fibers (arrows) and outer epithelia (arrow-
heads). Sections were counterstained with propidium iodide (red). Scale bars = 100 rpm. Higher magnifications are shown in
insets.



Table I: Summary of candidate Aplysia chemoreceptor genes selected by genome mining

Subfamily Number of intact genes Size range (aa's) Predicted TM domains Intron number Sites of expression**

a 10* 355-368 7 I rhino, ot, ovo
a 38 319-364 7 0 rhino, ot
c 24 324-433 7 0 ot

* Not including those considered partial-length (non seven transmembrane domain).
** Based on degenerate reverse transcription-polymerase chain reaction strategy outlined in this study (Methods)
rhino, rhinophore; ot, oral tentacle; ovo, ovotestis.


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coupling control [40]. We predict that this may also be
essential to receptor activation in the identified Aplysia
receptors.

The canonical model of GPCR activation is via an interac-
tion with intracellular heterotrimeric G-protein signaling
components. The genes identified in this study show little
amino acid identity to GPCRs found in the Metazoa and
we have not shown that they directly interact with G-pro-
teins. Despite this, our study indicates that Ga proteins
are present in the rhinophore sensory epithelium, possi-
bly in close association with transmembrane GPCRs. The
presence of sensory tissue G-protein immunoreactivity
adds further support to studies of other marine inverte-
brate olfactory systems implicating G-proteins in sensory
transduction [21,41]. Moreover, the existence of multiple
G-type proteins in sensory epithelium suggests that multi-
ple signal transduction pathways may be activated follow-
ing ligand stimulus. In squid, for example, the pattern of
immunolabeling implies that a G protein coupled to a
PLC pathway (Gatq and Gao) may be present in similar
cells as those coupled to a cAMP pathway (Gai). As sug-
gested by Mobley and colleagues [21], overlapping G-pro-
tein pathways could facilitate discrimination between
odorants detected by the same neuron. This contrasts
rodent models where the role of G proteins in olfactory
transmembrane signaling at the dendrites has been stud-
ied extensively. Researchers have demonstrated spatially
restricted patterns of expression of respective G proteins
[42-44]. Gao and Gai2 are highly expressed by separate
subsets of neurons that are located in different regions of
the vomeronasal neuroepithelium [44].

Of particular relevance to Aplysia chemosensory studies is
the rhinophore epithelium, where water-borne molecules
such as pheromones presumably bind and initiate activa-
tion of pheromone-receptive neurons. In the rhinophore
groove, receptor cells with a suspected chemosensory role
have been described previously in molluscs [26-28] and
their presence was further supported by our SEM analysis.
The rhinophore groove ciliary aggregations are likely nec-
essary in the separation and circulation of fluid through-
out the groove, and may also be directly involved in
detection of external chemical stimuli. It is from this pre-
cise location that we isolated cDNAs encoding identified
novel GPCR-like proteins. Their presence raised the ques-
tion as to whether their expression was specific to sensory
tissues. Subsequently, representatives of each subfamily
were found to be restricted to or differentially expressed in
the rhinophore, oral tentacles and ovotestis, suggesting
that they encode functional receptors and that these olfac-
tory organs sense different chemicals. In the rhinophore,
spatial expression of cells immunoreactive to candidate
chemoreceptor AcCR29b was most prominent in the tip
and outer epithelium, peripheral to the groove. Chemo-


sensory detection could likely benefit from this broad dis-
tribution, whereby stimulation may activate sensory fibers
that extend to higher brain centers. Although this finding
clearly indicates a sensory role, a more extensive study of
Aplysia sensory organs at higher magnification is required
to delineate the precise distribution of this receptor, as
well as other receptor subfamily members.

Interestingly, we found gene expression within the ovotes-
tis, and our preliminary analysis of various Aplysia neuro-
nal EST databases indicate that a relatively small fraction
of these genes may be expressed in the central nervous sys-
tem. Deep sequencing of neuronal transcripts has resulted
in identification of tags for 13 different genes in the cen-
tral ganglia of A. californica (that is, AcCRIa, AcCR5a,
AcCR15a, AcCR32b, AcCR5c, AcCR9c, AcCR1 Ic, AcCR15c,
AcCR16c and AcCR20c, see Additional file 1) as well as
several orphan receptors similar to vertebrate ghrelin and
histamine receptors (L Moroz, unpublished). Some of
those are associated with centrally located sensory neu-
rons that send neuronal processes to the periphery and
therefore may be involved in chemoreception. Other neu-
ronal cell types were previously described as motomeu-
rons. Taken together, these findings imply that external
chemical detection may not be their sole function, which
is consistent with that described of chemoreceptors in
mammalian olfactory bulb [45], cardiac muscle [46] and
vertebrate germ tissues [47-49]. The functional signifi-
cance of such expression is currently unknown. We specu-
late that there may be a role for Aplysia chemoreceptors in
oocyte recognition, possibly because both are activated by
identical or structurally similar hormones and pherom-
ones. In addition to the exocrine albumen gland, the Aply-
sia pheromone seductin is known to be expressed in
ovotestis tissue [24].

It will be of interest to perform a comparative gene analy-
sis to determine whether A. californica candidate chemore-
ceptors are also present in other Aplysia species. This
finding would suggest that these genes are highly similar
throughout Aplysia species and strongly imply a selective
pressure for conservation. We have already established
that several Aplysia species share a comparable attraction
pheromone blend [4,23], and therefore cognate receptor
binding sites are likely to be similar. The identification of
ligands for chemosensory receptors is often problematic
and therefore it is an advantage that we have these attrac-
tion pheromones for use in future functional studies to
definitively link identified genes to chemosensory detec-
tion. As these are the first such novel GPCRs identified in
molluscs, it will also be of interest to see if analogous
receptors are found in evolutionarily distant molluscs.
However, apart from the highly conserved insect receptor
Or83b [50], it is generally accepted that chemoreceptors



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seem to be very divergent with little sequence conserva-
tion within and across orders [51,52].

Our analysis of the Aplysia genome noted that AcCRa
genes as well as various AcCRb genes are clustered, a com-
mon feature of fast-evolving genes such as chemoreceptor
genes [15,53-55]. We also found that, although the assem-
bly of the genome used was incomplete, some genes con-
tain mutations that introduce stop codons to encode
truncated proteins, one of which appears to be expressed
in the A. californica rhinophore. Hominoids, in particular,
are known to possess a high pseudogene content (50%)
among their ORs, whereas only 20% of OR genes are
pseudogenes in the mouse [34] and less in the Drosophila
melanogaster genome [56]. Upon genome completion, a
more comprehensive analysis of GPCR gene families in
Aplysia will be necessary to determine the precise pseudo-
gene number. Indeed, some of the pseudogenes identified
may in fact be 'flatliners', that is, genes whose functional
versus pseudogene status is unclear [57]. As demonstrated
in C. elegans, many of these genes have apparently func-
tional alleles in one or more wild isolates and therefore
are not pseudogenes. Evidence for this has also been
shown for some Drosophila ORs [58] and gustatory recep-
tors [59], as well as Anopheles gambiae gustatory receptors
[51]. Although pseudogenes are generally accepted as
nonfunctional and therefore not transcribed, occasionally
it has been shown that such pseudogenes can be tran-
scribed [60]; however, there is no evidence of the func-
tional relevance.

Conclusion
Aplysia is an excellent model animal for studying the
molecular mechanism of chemical communication in the
marine environment. In this study we have isolated a
novel group of genes encoding multi-transmembrane
rhodopsin GPCR-like proteins that show expression in
chemosensory tissues. This expression pattern and
observed genomic clustering provide strong evidence that
these have arisen via gene duplication and may be used to
discriminate the large diversity of water-soluble mole-
cules. The expression of some of these in rhinophore sug-
gests that they are excellent candidates to be involved in
pheromone detection. Further knowledge of the receptor
gene genome organization, characterization of their
developmental and spatial expression profile, secondary
signaling and their evolutionary relationship to other
molluscan species would be the next significant steps
towards defining the logic behind how chemical commu-
nication in molluscs, and potentially other marine ani-
mals, operates.


Methods
Database mining for multi-transmembrane rhodopsin G-
protein coupled receptor-like genes
A. californica genome contig sequences were procured
from the NCBI trace database
http:www.ncbi.nlm.nih.gov/sutils!
genom table.cgi?organ ism=euk[61]. This was a prelimi-
nary genome assembly from 2x coverage of the genome
by the Broad Institute at MIT http://www.genome.goP-
ages/Research/Sequencing/SeqProposals/Aply
siaSeq.pdf[62]. An iterative tBLASTn strategy was adopted
to identify multi-transmembrane rhodopsin GPCR-like
genes in the Aplysia genome. Selection criteria included
that receptors would be encoded by a family of related
genes; at least some receptor genes would be clustered at
the same genetic loci; receptors would have limited amino
acid identity to members of known GPCR superfamilies;
and a full-length coding region would encode multiple
transmembrane domains. A search was initially per-
formed using molluscan GPCR protein sequences already
submitted to GenBank, as queries; a non-stringent expec-
tation value cutoff of le-4 was employed. During this
search we retrieved two sequences on contig
AASC01105652 with genes encoding hydrophobic multi-
transmembrane domains with no significant amino acid
identity to other proteins. Putative Aplysia transmembrane
receptors were in turn employed in searches to find more
genes in an iterative tBLASTn process. A candidate rho-
dopsin GPCR-like gene having a complete open reading
frame (methionine, 7-TM domains, three extracellular
domains, three intracellular domains, and a stop codon)
was considered intact and probably functional. To be con-
servative, genes that were >98% identical in amino acid
sequence were considered allelic variants. As the Aplysia
genome has not yet been fully assembled and consists of
only contigs, this method does not identify splice-variants
or provide a comprehensive analysis of gene clustering.
Pseudogenes were identified by premature stop codons
and we have arbitrarily chosen to disregard those genes
that encode less than six transmembrane domains. Com-
puter analyses of sequences were performed using BLAST
and CLUSTALW for nucleotide alignment. Transmem-
brane helix domain and topology of predicted receptors
was performed using HMMTOP version 2.0 program;
http://www.enzim.hu/hmmtop/index.html[63].
Hydrophilicity plots were generated using the TMHMM
program at http://www.enzim.hu/hmmtop/html/sub
mit.html[64]. In order to categorize identified genes, we
used http://pfam.janelia.org/search[65] and the Interpro
database [33].

Phylogenetic and gene analysis
Selection of outgroup sequences was performed by Gen-
Bank tBLASTn searches. The approach provided homolo-
gous counterparts from C. elegans for the Aplysia


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rhodopsin GPCR-like genes representing subfamilies a
and b. We did not find any significant counterparts for
subfamily c. The sequences were aligned with t-coffee [66]
under default settings. Alignments have subsequently
been improved by eye. Character positions, which could
not be aligned unambiguously, were not considered for
the phylogenetic analyses in order to avoid conflicting
phylogenetic signal. The inclusion of outgroup sequences
of C. elegans and Strongylocentrotus purpuratus resulted in a
large proportion of only ambiguously alignable positions
and consequently required deletion of a relatively high
number of characters. Therefore we performed two sepa-
rate analyses: first, we aligned and analyzed the ingroup
sequences only in order to reconstruct their evolution
under a maximal number of informative characters. Sec-
ond, we aligned and analyzed the dataset with the non-
Aplysia sequences included in order to infer the polarity to
the phylogeny.

Phylogenetic analyses were conducted on a multi-proces-
sor Linux-Cluster under the likelihood criterion using
RAxML v. 7.0 [67] for Maximum Likelihood and MrBayes
v. 3.1.2 [68] for the Bayesian Inference. MrBayes analyses
were performed in two runs of eight MCMCMC chains
and under the GTR+G+I Model [69]. Chains ran for
10,000,000 generations or were stopped when the stand-
ard deviation of split frequencies between both runs fell
below 0.01. RAxML bootstrap analyses on 1,000 replicates
have been performed under the PROTMIX algorithm with
the WAG amino acid substitution model [70]. In PROT-
MIX the tree inference is performed under the PROTCAT
model followed by the final tree evaluation under the
PROTGAMMA model in order to obtain stable likelihood
values (see the RAxML manual for further details).

Animal and sample preparation
Adult A. californica (100 to 500 g) were obtained from
Marine Research and Educational Products (Escondido,
CA, USA). Animals were anesthetized in isotonic MgCl2
(337 mM) equivalent to 50% of their weight, relevant tis-
sues dissected out and either (1) embedded in optimal
cutting temperature (OCT) compound for LCM or sec-
tioning, (2) snap frozen in liquid nitrogen for RNA and
protein isolation, or (3) prepared for SEM.

Reverse transcription-polymerase chain reaction
Total RNA was extracted from rhinophore, oral tentacle,
skin, pooled central nervous system, albumen gland, large
hermaphroditic duct, and ovotestis tissues of A. californica
using a Tripure Isolation Reagent. Any contaminating
genomic DNA was removed by treatment with DNase I
followed by lithium chloride/ethanol precipitation. First-
strand cDNA synthesis was performed in a 20 il reverse
transcription mixture containing oligo d(T)12_18 and 200
U Superscript III RNase H- reverse transcriptase, following


the manufacturer's instructions. PCR was performed using
1 il of prepared cDNA using subfamily-specific primers
(see Table S1 of Additional file 3: primer combinations
A3, B3 and C1). Each reaction was performed in a final
concentration of lx PCR Buffer, 1.5 mM MgCl2, 200 tiM
of each dNTP, 0.5 jiM of sense and antisense primer, 1.25
units of Red Taq polymerase and ddH20. Negative con-
trols contained no template cDNA. PCR using actin-spe-
cific primers (sense, 5'-GCTTCACCACCACTGCCGAGAG-
3' and antisense, 5'-ACCAGCAGATTCCATACCCAGG-3')
were used to ensure the integrity of each tissue cDNA sam-
ple. Reactions were heated at 94 C for 2 min and ampli-
fied for 36 cycles (94C, 60 s; 50 to 55 C, 30 s; 72C, 60
s). Following PCR, 15 pl of reaction mix was fractionated
on 2% agarose gels and visualized by ethidium bromide
staining. Based on the primer design, the expected ampli-
fication sizes were 756 bp (AcCRa), 832 bp (AcCRb), 512
bp (AcCRc) and 224 bp actinn). PCR products were cloned
into pGEM-T vector and sequenced.

Scanning electron microscopy of rhinophore and oral
tentacle
Adult Aplysia rhinophore and oral tentacle were fixed with
2.5% glutaraldehyde in phosphate buffer (pH 7.2 to 7.4)
for 3 days at 40C. Secondary fixation was in 1% OsO4
(osmium tetroxide) in 0.1M sodium cacodylate. This
material was dehydrated in a graded series of ethanol
(20% to 100%). The samples were dried using hexameth-
yldisilazane and platinum-coated using Eiko IB-5 Sputter
Coater. The specimens were viewed using a Jeol 6300
Field Emission Scanning Electron Microscope.

Laser capture microdissection and molecular identification
of candidate A. californica chemosensory receptors
The location of the rhinophore groove, glomeruli under-
lying the sensory epithelium, and rhinophore ganglia in
Aplysia have been described previously [26-28]. To exam-
ine whether selected genes are expressed in rhinophore
sensory epithelial cells, a combination of LCM, total RNA
isolation, library construction and RT-PCR were per-
formed. Rhinophore tissue that had been embedded in
OCT compound was sectioned (10 jim) onto slides and
dehydrated. LCM was performed using a PixCell II laser
capture microscope with an infrared diode laser (Arcturus
Engineering Inc., Mountain View, CA, USA) and a laser
spot size of 15 jim. Cells were marked, captured on Cap-
Sure HS caps (Arcturus), and total RNA was isolated using
the Picopure Isolation Kit (Arcturus) including DNase I
incubation. RNA quality was determined by measurement
of absorbance ratio at 260 nm/280 nm.

First-strand cDNA was synthesized according to the
SMART cDNA Library Construction Kit protocol (Clon-
tech, Palo Alto, CA, USA), with the minor modification of
incorporated EcoRI restriction sites during cDNA synthesis


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(5'-AAGCAGTGGTATCAACGCAGAGTGAAT-
TCACGCGGG-3' and 5'-
AAGCAGTGGTATCAACGCAGAGTGAATTCT3oVN-3').
Amplification of cDNA was performed by PCR using 5'
and 3' PCR Primer mix (5'-CTAATACGACTCACTATAG-
GGCAAGCAGTGGTATCAACGCAGAGT-3' and 5'-
AAGCAGTGGTATCAACGCAGAGTGAATICT3oVN-3');
samples were heated at 95 C for 1 min and amplified for
30 cycles (95C, 1 min; 500C, 30 s; 680C, 4 min). Reac-
tion volumes of 50 il were treated with 2 il of proteinase
K (20 pig/pil) at 45 C for 20 min, and PCR products were
precipitated. Dried samples were resuspended in 80 il of
deionized water. EcoRI digestion was performed and size
fractionation was achieved using a CHROMA SPIN-400
column (Stratagene, La Jolla, CA, USA). Products were
purified by precipitation and the dried pellet resuspended
in 7 Atl deionized water. EcoRI cDNA was cloned into the
EcoRI sites of Lambda ZAP II vector, and the cDNA library
(complexity 1 x 106) amplified once.

The sequences of oligonucleotide primers (Sigma-Geno-
sys, Australia) used for library PCR are located in Table S1
of Additional file 3. For AcCRa and AcCRb genes, PCR was
performed using degenerate sense and antisense primer
combinations Al to A5 (AcCRa) and BI to B8 (AcCRb).
Each reaction was performed using Red Taq polymerase
(Sigma) following the manufacturer's instructions. Sam-
ples were heated at 94C for 3 min and amplified for 36
cycles (940C, 60 s; 450C, 30 s; 720C, 60 s), followed by a
7-min extension at 72 o C. PCR products were cloned into
the TA vector pGEM-T (Promega) and sequenced as previ-
ously described [23]. To obtain 5' and 3' sequences, PCR
was performed using gene-specific primers. For 3'-RACE,
gene-specific sense primers (A3' and B3') were used in
combination with vector primer T3. For 5'-RACE, gene-
specific antisense primers (A5' and B5') were used in com-
bination with vector primer T7. Samples were heated at
94 C for 2 min and amplified for 36 cycles (94 C, 60 s;
50 C, 30 s; 720 C, 60 s), followed by a 7-min extension at
72 C. PCR products were cloned into pGEM-T vector and
sequenced.

Molecular identification of candidate Aplysia
chemosensory receptors within subfamily c
Total RNA was extracted from oral tentacle tissue of A. cal-
ifornica using a Tripure Isolation Reagent (Roche), and
any contaminating genomic DNA was removed by treat-
ment with DNase I (Invitrogen). First strand cDNA syn-
thesis was performed using 1 |ig of total RNA in a 20 1il
reverse transcription mixture containing oligo d(T)12_18
and 200 U SuperscriptM III RNase H- reverse transcriptase,
following the manufacturer's instructions. The sequences
of oligonucleotide primers used for PCR are located in
Table S1 of Additional file 3. PCR was performed using
degenerate sense and antisense primer combinations C1-


C3. 3'- and 5'-RACE was performed with the SMART RACE
amplification kit (BD Biosciences) and using gene-specific
primers sense and antisense primers (C3' and C5'). Sam-
ples were heated at 94C for 2 min and amplified for 36
cycles (94C, 60 s; 50C, 30 s; 72C, 60 s), followed by a
7-min extension at 72C. PCR products were cloned into
a pGEM-T vector and sequenced.

Immunohistochemical localization
A rabbit polyclonal antibody was generated to the N-ter-
minal region of candidate chemoreceptor 29b, corre-
sponding to N6SQARSSKSTQKGL (GenScript
Corporation). This region was chosen due to its lack of
significant amino acid identity to other receptors. Details
of the immunohistochemical protocol have been
described [23,24]. Briefly, tissue cryostat sections of rhi-
nophore were incubated overnight at 4C in either affin-
ity-purified 29b antibody (0.6 mg/ml, 1:500), Goiq, Gai or
Gao antisera (Chemicon, 1:500 dilution), rinsed in phos-
phate buffered saline (PBS), incubated in fluorescein iso-
thiocyanate (FITC)-conjugated goat anti-rabbit Ig (Sigma-
Aldrich, St. Louis, MO, USA) for 1 h at 22 C, rinsed in
PBS, and then mounted in FITC mounting media (90%
glycerol/100 mM Tris pH 8.0). Preparations were exam-
ined using an Olympus FluoView confocal microscope
(Leeds Precision Instruments, Inc., Minneapolis, USA),
and the images captured on a spot-cooled charged cou-
pled device camera. Sections were counterstained with
4',6-diamidino-2-phenylindole or propidium iodide at 1
ig/ml in water. As a control, the primary antiserum was
replaced with no primary antibody. For 29b, a control
also included using the primary antibody that had been
preabsorbed against its antigenic peptide (20 ig/ml).

Abbreviations
7-TM: seven transmembrane; AcCR: Aplysia californica
chemosensory receptor; BLAST: basic local alignment
search tool; EST: expressed sequence tag; FITC: fluorescein
isothiocyanate; GPCR: G-protein coupled receptor; LCM:
laser capture microdissection; OCT: optimal cutting tem-
perature; PBS: phosphate-buffered saline; PLC: phosphol-
ipase C; RT-PCR: reverse transcription-polymerase chain
reaction; OR: olfactory receptor.

Authors' contributions
SFC, DE, GTN and BMD designed research. SFC and DE
performed research. LLM and BMD analyzed data. SFC,
ZZ, CC, GTN and BMD wrote the paper. All authors read
and approved the final manuscript.









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Additional material


Acknowledgements
We acknowledge the Broad Institute Aplysia Genome Initiative for making
the partial genome sequence of A. californica available. We thank Dr Darren
Boehning for comments on an earlier version of the manuscript. We
acknowledge the assistance of the UTMB Protein Chemistry Lab, Steve
LePage (MREP) and Erica Lovus (Institute of Molecular Biology). SFC is sup-
ported by a University of Queensland Fellowship. This research was sup-
ported by grants to BMD from the Australian Research Council, LLM from
NIH and NSF, and to GTN from the NSF (grant IBN-0314377).

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Additional file 1
A. californica rhodopsin G-protein coupled receptor-like genes. This
file summarizes details for all identified rhodopsin G-protein coupled
receptor-like genes.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1741-
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Additional file 2
Candidate A. californica chemosensory receptor alignments. The com-
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