Báo cáo Y học: Expression pattern in the antennae of a newly isolated lepidopteran Gq protein a subunit cDNA potx

Fessard, CNRS, Gif-sur-Yvette, France; 4 Max-Planck-Institut fu¨r Verhaltensphysiologie, Seewiesen, Germany From the antennae of the moth Mamestra brassicae, we have identified a lepidopt

Expression pattern in the antennae of a newly isolated

lepidopteran Gq protein a subunit cDNA

Emmanuelle Jacquin-Joly1, Marie-Christine Franc¸ois1, Michael Burnet2, Philippe Lucas1,

Franck Bourrat3and Rosario Maida4

1

INRA, Unite´ de Phytopharmacie et Me´diateurs Chimiques, Route de Saint-Cyr, Versailles cedex, France;

2

Sympore GmbH, Reutlingen, Germany;3UPR 2197 DEPSN, Institut de Neurosciences A Fessard, CNRS, Gif-sur-Yvette, France;

4

Max-Planck-Institut fu¨r Verhaltensphysiologie, Seewiesen, Germany

From the antennae of the moth Mamestra brassicae, we have

identified a lepidopteran G protein a subunit belonging to

the Gq family, through immunological detection in crude

antennal extract and antennal primary cell cultures, followed

by molecular cloning.The complete cDNA sequence

(1540 bp) contains an open reading frame encoding a

protein of 353 amino acids.This deduced sequence possesses

all of the characteristics of the Gq family and shares a very

high degree of amino-acid sequence identity with vertebrate

(80% with mouse or human Gqa) and invertebrate subunits

(varying between 60 and 87% for Gqa from organisms as

diverse as sponge and Drosophila).The expression pattern of

the Gq subunit in adult antennae was associated with the olfactory sensilla suggesting a specific role in olfaction.These data provide molecular evidence for a component of the phosphoinositide signaling pathway in moth antennae: this

G protein a subunit may be involved in the olfaction trans-duction process through interaction with G-protein-coupled receptors, stimulating the phospholipase C mediated second messenger pathway

Keywords: G protein; a subunit; olfaction; Lepidoptera;

in situhybridization

For insects, olfaction plays an essential role in processing

chemical signals from the environment, leading to the

detection of food, reproductive partners, oviposition sites,

hosts, prey or predators.In particular, pheromone

percep-tion in moths has become a model for a growing number

of studies on the mechanisms of olfactory reception and

transduction.Although invertebrate chemosensory systems

show a great diversity across phyla, there are strong

similarities at the cellular level.The pheromone sensing

system of moths is morphologically very close to olfactory

systems from organisms as diverse as flies, nematodes or

lobsters.In moths, pheromone receptor cells are localized

in specialized sensory organs, the sensilla trichodea,

distributed on the antennae.Pheromone molecules, usually

emitted by the female, enter the sensilla of the male

antennae and are bound by specialized soluble proteins

that traffic through the extracellular lymph to the dendrite

membrane where they are recognized by specific olfactory

receptors.The transduction events following binding of the

receptor have been recently clarified by the discovery of the

first putative invertebrate odorant receptor genes in

Drosophila[1–3].The receptor proteins appear to belong

to the seven-transmembrane G protein coupled receptor

multigene family that also include vertebrate odorant

receptor molecules [4].These receptors relay signals from

cell surface to intracellular effectors through guanine nucleotide-binding proteins: the G proteins.G proteins play a central role in a wide variety of signal transduction pathways, mediating the perception of environmental cues

in all higher eukaryotic organisms.In particular, G pro-teins have been implicated in signal-transduction events underlying olfaction and vision (reviewed in [5]).They have been classified into different subtypes depending on which second messenger they predominantly control.Although these distinctions are not absolute, Gs frequently activates adenylate cyclase whereas Giinhibits it, Gq mediates the stimulation of phospholipase C and hence phosphoinosi-tide turnover, and G12 regulates Na+/K+exchanges [6] All G proteins consist of three subunits, a, b and c, with the nucleotide-binding and hydrolyzing a subunit defining the protein’s identity.The a subunit is believed to confer receptor and effector specificity on the heterotrimer.After its activation, different secondary pathways can occur: adenylate cyclase catalyses the formation of cAMP, whereas phospholipase C hydrolyses membrane phospha-tidylinositol, liberating inositol 1,4,5-triphosphate (InsP3) and diacylglycerol.Although cAMP and InsP3 cascades appear to be active as two alternative pathways in vertebrate olfaction [7], mechanisms of olfactory signal transduction in insects seem to involve the InsP3pathway Experiments on the rapid kinetics of second messengers in antennal homogenates of insects demonstrated an elevation

of InsP3 upon stimulation with pheromones [8–10] and nonpheromonal compounds [11] and it has been shown that G proteins are functionally active in signal transduc-tion of different sensory systems of invertebrates [12] Additionally, a phospholipase C b and a protein kinase C were recently identified in pheromone receptor neurons of the moth Antheraea polyphemus [13]

Correspondence to E.Jacquin-Joly, Phytopharmacie, INRA, Route

de Saint-Cyr, 78026 Versailles cedex, France.

Fax: + 33 1 30 83 31 19, Tel.: + 33 1 30 83 32 12,

E-mail: jacquin@versailles.inra.fr

Abbreviations: InsP 3 , inositol 1,4,5-triphosphate.

(Received 28 November 2001, revised 28 February 2002, accepted 4

March 2002)

G proteins from different families have been studied in

several invertebrate species including locust Go [14],

Dro-sophilaGq [15], the Lymnaea stagnalis Gq [16] or lobsters

Gq [17–19] and Gs [20].The presence of different G proteins

was reported in lepidopteran antennae in toxin sensitivity

studies [8,21,22].However, using antibodies raised against

different G proteins, Laue et al.[23] could detect positive

stain only with an antiserum raised against the a subunit of

a G protein belonging to the Gq/11 G protein family

So far, no G a subunit sequence is available in

Lepidop-tera, except a Go a cloned in the moth Manduca sexta [24]

In order to develop a better understanding of all the

elements of the olfactory signaling pathway in insects, we

report here characterization, molecular cloning and

expres-sion localization in the antennae of the first lepidopteran G

protein a subunit belonging to the Gq family

M A T E R I A L S A N D M E T H O D S

Insects

Animals were reared in Domaine du Magneraud (INRA,

France) on a semiartificial diet [25] at 20C, 60% relative

humidity, exposed to a 16-h/8-h light/dark photoperiod and

sexed as pupae.Antennae from 3-day-old adults were

dissected and stored at)80 C until use

Preparation of extracts, gel electrophoresis

and immunoblotting

Two hundred whole antennae from either male and female

adults were homogenized in 1 mL of 20 mM Tris/HCl,

pH 7.3, with a home-made moto-driven homogenizer, and

centrifuged at 10 000 g for 30 min The supernatant,

con-taining soluble proteins and membrane vesicles, was used in

the further experiments

PAGE was performed at a concentration of 10% of

polyacrylamide in the presence of 5% SDS, according to the

procedure of Laemmli [26].Protein bands were detected with

Coomassie Brilliant Blue R-250 (Serva).After

electropho-retic separation, proteins were electrotransferred onto

nitrocellulose membranes (Schleicher & Schuell, Germany)

and were treated with 2.5% BSA, 2.5% gelatin, 1% goat

serum and 0.05% Tween 20 in NaCl/Pifor 2 h in order to

prevent unspecific binding and incubated overnight with a

Gq/11 a antiserum (Calbiochem), at a dilution of 1 : 1000

Bound antibodies were detected with goat anti-rabbit

conjugated with alkaline phosphatase (dilution 1 : 10 000),

using 5-bromo-4-chloroindolyl phosphate/nitroblue

tetra-zolium as substrate.The affinity purified Gq/11 a antiserum

was raised against a synthetic decapeptide corresponding to

the C-terminal of a G protein a subunit and cross-reacts

with the a subunits of Gq and G11(Calbiochem)

Primary cultures of antennal neurons

Cultures were prepared as previously described [27].Briefly,

antennal flagella from 3-day-old male pupae were dissected

in 3 + 2 medium (three parts of Leibovitz’s L15 medium

and two parts of Grace’s medium supplemented with

lactalbumine hydrolysate and yeastolate).Flagella were

disrupted by incubation in L-cysteine-activated papain

(1 mgÆmL)1) followed by trituration with a fire-polished

Pasteur pipette.The resulting cell suspension was then plated onto uncoated Falcon Petri dishes.Two hours after plating the cells, the culture medium was replaced by a

3 + 2 medium supplemented with 5% fetal bovine serum The cultures were then inverted to form a hanging column and were maintained for 2–4 weeks at 22C in humid atmosphere

Antennal cells were grown in culture for 2–3 weeks prior

to harvesting in 20 mM Tris/HCl, pH 7.3 buffer, and extracting into Laemmli sample buffer

RNA extraction and cDNA synthesis Total RNA was extracted from 200 antennae with the Tri-Reagent (Euromedex).Single stranded cDNA was synthesized from 1 lg of total RNA with M-MLV (USB), using buffer and protocol supplied with the enzyme.The reaction mixture contained dNTP mix (Pharmacia), Rnasin (Promega), oligodT18with an anchor: CATGCATGCGGC CGCAAGCT18VN (synthesized by Isoprim, Toulouse, France), sterile water and template RNA to a final volume

of 50 lL.The mix was heated at 68C for 5 min and chilled

on ice before adding the M-MLV (600 U), then incubated 1

h at 37C and finally the reverse transcriptase was inactivated at 95C for 5 min.For the 3¢ RACE, reverse transcription was performed on 1 lg of total RNA accord-ing to the manufacturer’s instructions

(3¢-AmpliFIND-ERTM RACE Kit, Clontech), using a 20-lL reaction mixture.For the 5¢ RACE, cDNA was synthesized from

1 lg of male antennae total RNA at 42C for 1 5 h using the SMARTTMRACE cDNA Amplification Kit (Clontech) with 200 U of Superscript II (Gibco BRL), 5¢ CDS-primer and SMART II oligonucleotide, according to the manufac-turer’s instructions

Internal amplification Two degenerate primers were designed according to consensus regions of several G protein a subunit sequences from different species, including the mollusk Lymnaea stagnalissequence [16].The nucleotide sequence of the sense primer is based on the amino-acid motif FIKQMR (5¢-CG CGAATTCNTTYATHAARCARATGMG-3¢) and the antisense primer is based on the amino-acid sequence

TNGC-3¢) EcoRI and BamHI restriction sites (indicated

by underlining), respectively, have been included to facilitate subcloning.Approximately 1 ng of cDNA was used for polymerase chain reaction carried out with Taq polymerase (1 U) (Promega) in 10 mMTris/HCl, pH 9.0, 50 mMKCl, 0.1% Triton X-100, 1.5 mMMgCl2, 0 2 mMof each dNTP

A 900-bp PCR product was generated after 40 cycles consisting of 1 min at 94C, 1 min at 44 C and 1 min at

72C in a Hybaid thermocycler.Subcloning in pZERO (Invitrogen) using EcoRI and BamHI resulted in loss of a part of the amplified product due to a BamHI internal site

So, cloning was then performed using a TA vector from Invitrogen, pCRTMII, using the TOPO cloning kit 3¢ RACE-PCR

For the 3¢ RACE-PCR, two amplifications were con-ducted as described in the manufacturer’s instructions

(3¢-AmpliFINDERTMRACE Kit, Clontech).The first one

was conducted on 1 lL of the 3¢ reverse transcription

reaction, with a primary sense gene-specific primer deduced

from the sequence obtained after the internal amplification

(5¢-GCATTATAGAATACCCATTTGACCTG-3¢) and

with an antisense Anchor Primer (furnished in the kit).It

consisted of 30 cycles of 1 min at 94C, 1 min at 55 C and

1 min at 72C.The second amplification consisted of a

nested PCR and was carried out on 1 lL of the first

amplified product, using a second sense gene-specific primer

(5¢-GACCTGGAAGAAATACGATTTAGAATGG-3¢)

and the Anchor Primer from the kit, and consisted of 30

cycles of 1 min at 94C, 1 min at 55 C and 1 min at 72 C

A 600-bp amplification product was obtained

5¢ RACE-PCR

Amplification was performed on 2.5 lL of 5¢-RACE-ready

cDNA using Universal Primer Mix (Clontech) as a sense

primer and an antisense gene-specific primer, designed

according to the cDNA sequence obtained from the

internal amplification (5¢-TCGCCTGCCGTCGTAGCAC

TCCTG -3¢).The 50-lL amplification mix was prepared

according to the SMARTTMRACE cDNA Amplification

kit instructions using the Advantage 2 Polymerase mix

(Clontech).Touchdown PCR was performed using

hot-start as follows: after 1 min at 94C, 5 cycles of 30 s at

94C and 3 min at 72 C, then 5 cycles of 30 s at 94 C,

30 s at 70C and 3 min at 72 C, then 25 cycles of 30 s at

94C, 30 s at 68 C and 3 min at 72 C, then 5 min at

72C

Cloning and sequencing

The amplified cDNAs were ligated into the plasmid

pCRTM-II using the TOPO cloning kit from Invitrogen

(the Netherlands).Recombinant plasmids were isolated

using Plasmid Midi kit from Qiagen and both strands were

subjected to automated sequencing by ESGS (Evry,

France).Database searches were performed with theBLAST

program (NCBI) and sequence alignment with the

CLUSTALW(NPS @IBCP)

In situ hybridization

RNA sense and antisense probes (900 bp long) were in vitro

transcribed from linearized pCRII-cDNA plasmid,

result-ing from the clonresult-ing of the internal amplification, usresult-ing T7

and SP6 RNA polymerase (Promega) following

recom-mended protocol and in the presence of 1.5 U of Rnasin

(Promega).Probe quality was confirmed under denaturing

conditions by formaldehyde agarose gel electrophoresis and

the probes stored at)80 C until use

For hybridization, antennae were removed from adult

head, cut into pieces and fixed overnight at 4C in 4%

paraformaldehyde in NaCl/Pi.Fixed tissues were

dehydra-ted in 100% methanol and stored at)20 C.The

hybrid-ization protocol was performed on whole-mount pieces of

antennae as previously described [28].Hybridization was

detected using alkaline-phosphatase-conjugated

anti-digoxygenin Ig (1 : 4000) and stained with Nitro blue

tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate,

toluidine salt (Boehringer Mannheim).After sufficient

staining, specimens were washed in NaCl/Piand fixed in 4% paraformaldehyde for 20 min, then dehydrated through

a graded series of ethanol and wax-embeded.Six-micro-meter longitudinal sections were cut and counter-stained with acridine orange.Sections were photographed, then pictures were digitized and processed usingADOBE PHOTO-SHOP5.0

R E S U L T S

Immunodetection of the Gq/11 a subunit

Proteins extracted from male and female antennae and from primary antennal cell culture of M brassicae were separated by SDS/PAGE and analysed by Western-blot using a Gq/11 a antiserum (Fig.1).Crude homogenates of male and female antennae contained an immunoreactive band with an apparent molecular mass of 40 kDa (Fig.1, left, A,B).In the sample of primary cell culture of

M brassicae, a band with the same apparent molecular weight was labeled by the antiserum, indicating that the protein is also present in the in vitro cell cultures (Fig.1, left, C)

Cloning and cDNA sequencing

A 900-bp cDNA product was amplified with RT-PCR using degenerate oligonucleotide primers.After cloning and sequencing, this product was translated and the deduce amino-acid sequence was compared with sequences

in the GenBank database.This product appeared to be very similar to a subunits from G proteins belonging to the Gq family.It was then extended to the 5¢ and the 3¢ untranslated regions by 5¢ and 3¢ RACE, respectively This allowed us to obtain the sequence of a full length

Fig 1 Biochemical detection of Gq/11 a M, molecular markers.Left (A,B,C) Coomassie stain after 10% SDS/PAGE of antennal and cell culture homogenates.(A) Male M brassicae (4 antennae equivalent), (B) female M brassicae (6 antennae equivalent), (C) primary cell cul-tures of M brassicae male antennae (15 Petri dishes equivalent to 15 antennae).Right (A,B,C) Western-blot after SDS/PAGE of antennal and cell culture homogenates using Gq/11 a antiserum (dilution

1 : 1000).The antiserum cross-reacted only with a single band of about

40 kDa in both male (A) and female (B) antennal extracts as well as in the primary cell culture extracts (C).

cDNA of 1541 bp (Fig.2) This sequence has been

deposited in the GenBank database with accession

number AF448447.Nucleotide sequence analysis revealed

that the cDNA contains a putative coding region of

1059 bp, encoding a 353 amino-acid protein with a

theoretical molecular mass of 41 400 Da and an isoelectric

point of 5.35, as determined using MWCALC (Infobiogen)

(Fig.2) There are several upstream ATG codons but

soon followed by stop codons.The ATG at position 199

has a favorable sequence context for translation initiation

[29] and could be proposed to be the start of the protein

coding domain.Sequence analysis of the 3¢ end cDNA

revealed that there is a polyadenylation signal upstream of

the poly(A)

Analysis of the primary structure ofM brassicae Gqa The putative protein product encoded by the cloned cDNA was aligned with different G proteins from invertebrates and vertebrates retrieved from blast search (Fig.3).This putative protein showed a high degree of identity to other known Gqa proteins from invertebrates (Drosophila, 87%; Limulus, 83%; lobster, 85%) but also from vertebrates (mouse, 80%; human, 80%), and is less similar to other Ga types (47.5% with Go of the Lepidoptera M sexta, for example) (Table 1)

Furthermore, the M brassicae Gqa subunit exhibits important characteristics of other Gqa proteins, namely: the amino-acid sequence G40TGESGKSTFI typical of the

Fig 2 cDNA and deduced amino-acid sequence of the M brassicae Gq a subunit (GenBankaccession number no AF448447) The suggested start ATG and stop TGA codons are in bold italics.Positions of the primers for the internal amplification are underlined (solid line), as are gene specific primers and nested primer for the 3¢ RACE amplification (dashed lines) and the gene specific primer used for the 5¢RACE amplifi-cation (dotted line).Palmitoylation sites C3C4, G40TGES box and putative cholera toxin site Arg177 are in boxes.

A domain, with the characteristic residues underlined [30], a

N-terminal cysteine doublet (Cys3, Cys4) in a MXCC motif

that represent putative sites for palmitoylation [31], a

putative cholera toxin ADP-ribosylation site (Arg177) and a

G40TGES GAG box sequence that is present in the

GTP-binding domain of other Gqa proteins (Fig.2)

Expression pattern in male antennae

In situ hybridization experiments were performed using

digoxigenin incorporated antisense and sense RNA probes

against adult male antennae.The M brassicae antenna is

filiform, 1 cm long and comprises about 72 segments [32]

Each segment exhibits the same general organization: the

dorsal side is covered with two rows of scales and the

olfactory hairs (the sensilla) are located on the ventral side

as can be seen using scanning electron microscopy

(Fig.4A).In males, the olfactory hairs are distributed in

two classes according to their length.The long ones (long sensilla trichodea) are located on the lateral part of the ventral area and are arrayed in four to five parallel rows [32] (Fig.4A, white arrows).Short sensilla trichodea are located medio-ventrally and are not arranged in rows

Sense strand controls gave no signals (not shown) whereas antisense probe hybridization is restricted to the sensilla (ventral) side of the antennae (Fig.4B,E) Close examination revealed hybridization in cells at the bases of the sensilla hairs (Fig.4C,D) and sometimes two labeled somata can be seen at the base of one sensillum (Fig.4E)

On longitudinal sections through the antennae, it is difficult

to distinguish between long and short sensilla as only parts

of the sensilla are visible (Fig.4B,E) However, sections through the cuticle permitted the observation of labeled spots distributed in the ventro-lateral region with a row pattern consistent with the distribution of the long sensilla trichodea (Fig.4F, white arrows) Typical structures of

Fig 3 Alignment using CLUSTAL W of G protein a subunit of the q family from different species, including invertebrates and vertebrates Amino-acid identities are in bold.Sequences compared to M brassicae Gqa sequence are from Drosophila melanogaster (GenBank accession numbers M58016; M30152; U31092), Homarus americanus (U89139), Panulirus argus (AF201328), the mouse Mus musculus (P21279), the dog Canis familiaris (Q28294) and human (P50148).Several motifs indicative of this Ga family are conserved: N-terminal cysteines, arginine177, and a GAG box.

sensilla coeloconica, that resemble flowers, can be observed

on sections through the cuticle, without any associated

labeling (Fig.4G, black arrows), whereas on the same

section other sensilla without any particular distribution are

labeled, that correspond to short sensilla trichodea

D I S C U S S I O N

Several studies have previously suggested that

G-protein-mediated signal transduction pathway may occur in

pheromone-sensitive receptor cells in insects.For example,

it has been shown that nerve impulse activity of

phero-mone receptor cells increased significantly after G

protein-activating natrium fluoride application to their outer

dendrite in single sensilla trichodea of the moth Bombyx

mori [23].Additionally, using a Gq/11 antiserum, the

same authors revealed the presence of a protein that is

likely to belong to the Gq family in antennae of both

B moriand Antheraea pernyi.In this context, we report

here the immunodetection of a protein with the same

characteristics, the molecular cloning of the corresponding

cDNA to get the total amino-acid sequence of the protein,

and the expression pattern of the corresponding mRNA

as a first step to clarify the role of G protein a subunit in

olfaction

Immunodetection of Gq/11 a subunit in antennae homogenate and in neuron primary culture The molecular mass of the immunoreactive band observed

is consistent with the molecular mass of other G protein a subunits, and may represent the a subunit(s) of one or several proteins belonging to the Gq/11 family

The visualization of such proteins in our olfactory cell culture is consistent with the occurrence of a Gq protein in antennal primary cell culture already observed in lobster [17], which mediates excitatory odor transduction in olfac-tory receptor neurons in this species

Molecular cloning of a cDNA coding for a Gqa subunit

in male antennae Because of the strong conservation of the G protein a subunit throughout evolution, we decided to use the PCR technique to identify a cDNA encoding M brass-icae Gqa-like protein.We then identified a putative transcript encoding a G protein a subunit homologous to invertebrate and vertebrate Gqa, suggesting the presence

of a specific Gqa gene in M brassicae.The molecular mass of the predicted protein is consistent with that determined by Western blot after SDS/PAGE (Fig.1)

Table 1 G protein a subunit characterized in insects, including the Gq of M brassicae described here, and in some other invertebrate groups Databank accession numbers, references and expression pattern/putative role are also given.ORN, olfactory receptor neurons.

Species

G protein a subunit class

Accession no.Ref.

% identity with Gq

of M brassicae Possible function Insecta

Lepidoptera Mamestra brassicae Gq AF448447 This paper 100 Expression in ORN

embryonic neurons Diptera Drosophila

melanogaster

system and ovaries DGq3

G

U31092

M23094

[15]

[56]

87

48.6

Expression in chemosensory cells and central nervous system Expression in embryos and pupae

Calliphora vicina G AJ250443 [57] 81.5 Visual protein of the

compound eyes

nervous tissues Crustacea Panulirus argus Gq/11 AF201328 [19] 85 Expression in ORN

Homarus

americanus

olfactory organs and brain Chelicerata Limulus polyphemus Gq U88586 [58] 83 Expression in eyes Mollusca Patinopecten

yessoensis

photoreceptor cells

subunit

Demospongiae Geodia cydonium Gq Y14248 [63] 50.1 Oocyte maturation

The antibody used for the Western-blot is directed to the

decapeptide QSALKEFNLA that corresponds to a

defined C-terminal sequence found in both Gqa and

G11a (Calbiochem).The deduced amino-acid sequence

shares high C-terminal identity with this sequence

(QLNLKEYNLV) and thus should have been detected

with such antibodies.Although proteins of this class are

highly conserved in sequence and molecular mass, the

observation of only a single band on the Western blot,

combined with the molecular cloning data, suggests that

the cloned cDNA probably encodes the protein detected

using commercial antibodies

The predicted protein we obtained shares high identities

with other already known Gq protein a subunits and

therefore can be placed within this family.Indeed,

M brassicaeGqa possesses all the characteristics observed

in Gqa-like proteins.In particular, the cysteine doublet at

the N-terminal part probably serves as a site for

post-translational attachment of a palmitoyl group (16-carbon,

saturated fatty acid) through a labile, reversible thioester

linkage [33,34], and which could serve as a membrane

anchor

It is noteworthy that proteins from Gq family are

highly conserved throughout evolution: the putative

M brassicae Gq sequence is 80% identical to mouse,

dog and human Gq; however, it shares only 47%

identity with a Go sequence from M sexta, another

lepidopteran (Table 1).In particular, the M brassicae G

subunit is 87% identical to the dGqa-3 of Drosophila

[15].It differs only in two domains: 70–130 and the

C-terminal region that is important for receptor

interac-tions [35,36]

Expression pattern in the adult male antennae

In situhybridization revealed that this G protein subunit is expressed in both long and short sensilla trichodea (Fig.4F,G) in cells that could be neurons because of several observations.On Fig.4C, for instance, the labeled cell is located at the base of the cuticular hair and protrusions emanating from the soma that could corres-pond to the dendrite are seen entering the base of sensillum hair (Fig.4C) Such protrusions have already been observed after in situ hybridization in labeled neurons of

M sextaantennae [37].Furthermore, two somata can be seen that are labeled at the base of the same sensilla (Fig.4E), possibly corresponding to the two receptor neurons observed in all sensilla.The shape, size and position of the stained cells also suggest their identity as olfactory neurons

The Gqa appeared to be associated only with sensilla trichodea, devoted to pheromone reception [32], with no expression in sensilla coeloconica.These latter structures have been shown to be involved in plant-related volatile detection, at least in B mori [38].We can then suppose that although both sensilla types are implicated in olfaction, they

do not express same G protein a subunits, maybe according

to the ligands they are tuned to.Such a phenomenon has already been observed in the vertebrate vomeronasal organ,

an organ responsible for detecting pheromones.Two

G protein subtypes are selectively activated by different classes of compounds [39]: some neurons express receptors encoded by one multigene family and the G protein a subunit ai, whereas some others express receptors encoded

by another multigene family and the G protein a subunit a

Fig 4 Expression pattern of M brassicae G

protein a subunit revealed by in situ

hybridiza-tion to mRNA in longitudinal sechybridiza-tions of male

antennae (A) Scanning electron microscopy of

a male antennae.The ventral surface is

cov-ered by short and long sensilla, the last being

arranged in parallel rows (white arrows).(B,E)

expression of G protein a subunit on the

sen-silla side of the antennae.(C,D) sensen-silla

trichodea at higher magnification.(F) Section

through the cuticule in the ventro-lateral

region of the antennae showing G protein a

subunit expression in row pattern (white

arrows) consistent with the distribution of the

long sensilla trichodea devoted to pheromone

reception.(G) Sensilla coeloconica are not

labeled (black arrows) whereas the surrounded

sensilla (short sensilla) are labeled.Scale:

(A,B,E,F) 50 lm; (C,D,G) 10 lm.

Recently, the a subunit of a G protein of the Gq family

has been immunolocalized in olfactory sensilla preparations

of the silkmoth Antherea pernyi [23].Using

immunocytol-ogy, the authors were able to show that all types of olfactory

sensilla are labeled.However, labeling is not restricted to

sensillar cells and can be observed in auxiliary cells,

epidermal cells and subcuticular extracellular space.This

is not in contradiction with our observations given that the

tools and the organisms used in the two studies are different:

using antibodies they visualized the localization of the

protein whereas by using in situ hybridization to mRNA we

revealed only the expressing cells that are likely to be

olfactory neurons.The immunological study [23] suggests

that Gq plays a role in olfactory signal transduction as long

as the protein predominates in the dendrites of olfactory

receptor cells

Implication of Gq in lepidoptera olfaction

Gqa have been frequently presumed to play a role in

olfaction in invertebrates.For example, the protein dGqa-3

of Drosophila was detected in the third antennal segment,

maxillary palps, the tip of the proboscis and in the brain

[15].Some of the immunoreactive cells have been identified

as antennal olfactory neurons, non-neuronal accessory cells,

or gustatory neurons, suggesting that this protein is involved

in olfactory and gustatory responses in Drosophila.Several

studies on lobsters support Gq involvement in odor

transduction in olfactory receptor neurons.An anti-Gq/11

Ig has been shown to selectively block odor-evoked inward

current in voltage-clamped cultured neurons and

immuno-labeled a band of 45-kDa in Western-blot analyses [17]

Similarly, a Gqa protein has been cloned in two lobster

species, Homarus americanus [18] and Panulirus argus [19],

that is expressed in olfactory receptor neurons, suggesting

that one function of Gqa is to mediate olfactory

transduc-tion.In our study, expression of proteins in olfactory

sensilla trichodea, apparently in neurons, leads us to

hypothesize that, in M brassicae, this G protein subunit is

involved in pheromone reception.In addition, our data

demonstrated that the olfactory organ of this species

expresses a gene that is critical for the phosphoinositide

signaling pathway: the fact that this protein belongs to the

Gqa subunits suggests that a phospholipase C second

messenger pathway may be implicated in transduction of

olfactory signals in lepidoptera.Such a hypothesis has

already been proposed for insects in a variety of species

using kinetics based methodology (reviewed in [40]) and

immunological detection of Gq/11a subunits in antennae

[23].Additionally, a phospholipase C b and a protein kinase

C, two enzymes involved in the InsP3transduction pathway,

were identified by using specific antibodies directed against

molecules involved in intracellular olfactory signalling [13]

The two enzymes were detected after Western blot with

homogenates of isolated pheromone-sensitive sensilla

trichodea, containing no other cellular elements than the

outer dendrites of pheromone receptor neurons.In lobsters,

several recent studies showed that phospholipase C b

mediates olfactory transduction as well [41].Molecular

evidence for two components of the phosphoinositide

signaling pathway in lobster olfactory receptor neurons

has been provided [19]: a G protein a subunit of the Gq

family and an InsP-gated channel or an InsP receptor.In

addition, the authors showed that the InsP3 receptor is associated with the plasma membrane, suggesting a novel mechanism for regulating intracellular ions within restricted cellular compartments of neurons [19].Interestingly, InsP3 receptors have also been immunolocalized within the dendritic membrane of olfactory sensilla of moths [42] Elevation of InsP3and InsP3-gated-Ca2+influx in phero-mone-stimulated cell cultured olfactory neurons has also been shown [43]

Here, we provide molecular evidence that support the previous findings and the first lepidopteran sequence of a Gqa subunit

The Caenorhabditis elegans genome project has revealed

20 genes encoding a-subunits of G proteins, 14 of which are expressed almost exclusively in subsets of chemosensory neurons [44,45].Then it seems likely that this nematode uses multiple Ga subunits per cell, leading us to hypothesize that neurons mediating more than one sensory modality can do

so via distinct intracellular pathways [46], each mediating a particular response to a specific class of chemical stimuli [47].However, C elegans expresses multiple chemosensory receptors per olfactory neurons, which is not the case in Drosophilawhere neurons are likely to express only a single olfactory receptor gene, although sometimes along with a broadly expressed receptor of unknown function [48].In moths, the lack of any information on putative olfactory receptors does not permit such considerations.However, it cannot be excluded that different types of Ga subunits may

be involved in olfactory transduction in Lepidoptera Different G proteins are found in specialized tissue and they have there different functions, although they all share structural properties such as the heterotrimeric composition with a, b, c subunits.However, a subunits are distinct whereas b subunits are quite similar [49].The similarity of our a subunit sequence with others implicated in olfactory transduction further supports our hypothesis that this subunit is involved in odor transduction cascade in moth antennae

Our identification of a Gqa subunit expressed in olfactory sensilla supports the hypothesis that G-protein-coupled olfactory receptors are functional in insects.In insects, seven transmembrane domain proteins coupled to G-protein-mediated second messenger cascades have been found to date only in Drosophila [1–3] and Anopheles gambie [50] and attempts to find similar receptor proteins in other insects have failed.An olfactory-specific protein (SNMP for sensory neuron membrane protein) of two transmembrane domains uniquely expressed in olfactory receptor neurons has been characterized in the silkmoth A polyphemus [51,52] as well as in the moths B mori, Heliothis virescens and M sexta [37].In this latter species, a second SNMP homologue was also identified [37,53].These proteins are homologous with the CD36 receptor family, which pre-dominately recognizes proteinaceous ligands.One could then not exclude a possible role as olfactory receptor, considering the probable interaction with odorant binding proteins carrying the odorant molecule to the receptor Although no olfactory receptor has been identified in Lepidoptera, the discovery of a Gqa subunit expressed in olfactory neurons and sharing high identities with the olfactory/gustatory Drosophila Gqa subunits suggests that seven transmembrane domain receptor proteins should exist

in moth antennae and are involved in olfaction

A C K N O W L E D G E M E N T S

This study was supported by founds from Institut National de la

Recherche Agronomique, including Rosario Maida fellowship during

his stay in Versailles.

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