Drosophila Mutants in Phospholipid Signaling Have Reduced Olfactory

Responses as Adults and Larvae

Pinky Kain,1 Shanti Chandrashekaran,2 Veronica Rodrigues,1,3 and Gaiti Hasan1

1National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India2Indian Agricultural Research Institute, New Delhi, India

3Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India

Abstract: In this paper, we show that mutants in the gene stambhA (stmA), which encodes a putative phosphatidylinositol 4,5

bisphosphate-diacylglycerol lipase, exhibit a significant reduction in the amplitudes of odor-evoked responses recorded from the

antennal surface of adult Drosophila. This lends support to previously published findings that olfactory transduction in Drosophilarequires a phospholipid intermediate. Mutations in stmA also affect the olfactory behavior response of larvae. Moreover, there is a

requirement for Gqa and phospholipase Cb function in larval olfaction. The results suggest that larval olfactory transduction, like that of

the adult, utilizes a phospholipid second messenger, generated by the activation of Gqa and Plcb21c, and modulated by the stmA gene

product.

Keywords: stmA, antenna, dgq, plc21C, dorsal organ, olfactory sensory neurons

INTRODUCTION

Identification of genes encoding olfactory receptors has

helped understand the genetic basis of odor recognition

and chemotaxis in Drosophila. There are 62 members of

the odorant receptor (Or) family in Drosophila, each with

different odor specificities; these are located on the

dendritic surface of olfactory sensory neurons (OSNs).

Upon odor binding, OSNs change the rate of spontaneous

firing, which can be measured either directly by single-

unit recordings or indirectly through changes in the field

potential on the antennal surface. Recent studies, which

examined receptor function in heterologous systems,

suggest that Ors heterodimerize with a coreceptor

(Or83b) to form a ligand-gated ion inselective channel

(Sato et al., 2008; Wicher et al., 2008). In addition, there

is considerable previous evidence for both cAMP (Martin

et al., 2001; Gomez-Diaz et al., 2004) and inositol 1,4,

5-trisphosphate (IP3) signaling in insect olfactory trans-

duction (Krieger & Breer, 1999). We have recently shown

that olfactory responses from the adult antenna are

significantly reduced in Drosophila mutants for the

heterotrimeric G-protein, dgq; a phospholipase Cb ortho-

log, plcb21c, and a diacylglycerol kinase, rdgA. Genetic

interactions between mutants for these genes suggest that

a phospholipid second messenger is a major component in

Drosophila olfactory transduction (Kain et al., 2008). In

this paper, we provide further evidence for this model by

examining the role of another gene, stmA, in Drosophilaadult and larval olfaction. Mutants in stmA have been

shown to alter phospholipid levels and affect adult visual

transduction (Huang et al., 2004). At the systemic level,

odor perception requires sensory transduction in periph-

eral neurons, followed by integration of this information

in the brain, finally leading to a behavioral output. The

simplicity of the Drosophila larval olfactory system

makes it an attractive system to study the effects of genes

implicated in olfactory transduction on olfactory beha-

vior. Drosophila larvae are capable of sensing many, if

not all, odorants that stimulate adults. The basic organiza-

tion of the larval olfactory circuit is surprisingly similar to

that of adults and also to mammalian circuits; the system

is significantly simpler with just 21 OSNs that express

about 25 Ors, of which 11 are shared with the adult

(Stocker, 2006). An individual receptor appears to

transmit signals via a single OSN to a single glomerulus

in the larval antennal lobe (Kreher et al., 2005). Despite

this apparent simplicity, larval OSNs exhibit several

complex features also found in adult receptors (Hallem

et al., 2004; Kreher et al., 2005; Kreher et al., 2008). An

Received 17 May 2008; Accepted 27 July 2008.

Gaiti Hasan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore,

560065, India. E-mail: gaiti@ncbs.res.in

J. Neurogenetics, 23: 303�312

Copyright # 2009 Informa UK Ltd.

ISSN: 0167-7063 print/1563-5260 online

DOI: 10.1080/01677060802372494

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understanding of transduction events downstream of

larval receptors will help in understanding how multiple

broadly tuned receptors can aid the discrimination of

single odors.

MATERIALS AND METHODS

Fly Stocks

The following stocks, dgq221c and dgq1370 alleles of dgq,

GqGAL4, RNAi construct for dgq (UASGq1F1), plc21cinsertion allele (plc21cP319), and the deficiency

Df(2L)p60A, which uncovers plc21C, RNAi line for

plc21C, Or83bGAL4, rdgA1 and rdgA3, norpAP24, have

been described earlier (Kain et al., 2008; Weinkove et al.,

1999; Vosshall et al., 2000; Inoue et al., 1989; Bloom-

quist et al., 1988). dgq14073 and FRT42B were obtained

from the Drosophila stock center Bloomington, Indiana,

USA. The isolation and characterization of stmA alleles,

which were generated in a CS genetic background, has

been described earlier (Shyngle & Sharma, 1985; Chan-

drashekaran & Sarla, 1993). The mutants have since been

introduced into the w1118 background. Hence, this strain

was used as the control genetic background for all

behavior tests with stmA. stmA1 was renamed rbots1,

and the rescue transgene, referred to as stmA-eGFP (or

rbo-eGFP), has been described in Huang et al. (2004). In

this work, the original nomenclature has been used. The

stmA-eGFP stock was obtained from Kendal Broadie

(Nashville, Tennesse, USA). All stocks were grown on

standard cornmeal medium at 258C.

Immunohistochemistry

Dissection and antibody staining of the larval AMC

(antenno-maxillary complex) and brain was according to

the procedure described as follows. Tissues were fixed in

4% paraformaldehyde in phosphate-buffered saline (PBS)

for 3 hours on ice, washed extensively, and blocked in

PBS containing 5% normal goat serum, 0.1% bovine

serum albumin, and 0.03% Triton-X-100 for 1 hour at

room temperature. Where mAbnc82 was to be used, the

protocol of Laissue et al. (1999) was followed. The

primary antibodies used were rabbit anti-GFP (1:10,000;

Invitrogen, Carlsbad, California, USA) and mAbnc82

(1:10; DSHB). Secondary antibodies were antimouse and

ant-rabbit IgG conjugated to either Alexa 488 or Alexa

568 (1:200; Invitrogen). Labeled samples were mounted

in 70% glycerol and examined in BioRad Radiance 2000

(Hemel Hempstead, Hertfordshire, UK) or an Olympus

FV1000 (Olympus, Tokyo, Japan) at 1 mm intervals; data

were processed using Image J (NIH, USA), Confocal

Assistant 4.2 (Biorad), and Adobe Photoshop 5.5, (San

Jose, California, USA).

For immunohistochemical staining of adult antenna,

the A3 segments were removed in chilled PBS and fixed

in 4% paraformadehyde with 3% Triton-X-100 on ice for

5 hours, followed by washes with PBS containing 2%

Triton-X-100, first at room temperature (3 times for 1

hour each), then at 48C for 2 days. Samples were

incubated for 3 days at 48C in anti-GFP (1:10,000).

Antennae were washed in PBS containing 0.1% Triton-X-

100 (3 times for 1 hour each) after primary antibody

incubation and treated with a 1:200 dilution of secondary

antibodies of antimouse and -rabbit IgG conjugated to

either Alexa 488 or Alexa 568 for 1 day at 48C. After

washes with PBS containing 0.1% Triton-X100, samples

were mounted in antifading agent (Vectashield; Vector

Labs, Burlingame, California, USA), and examined in an

Olympus FV1000 confocal microscope.

Electroantennogram Recordings

Electrophysiological recordings were carried out as

previously described (Ayer & Carlson, 1991). Two- to

6-day-old female flies were mounted into the tapering end

of a pipette tip, allowing the head to protrude. The head

was held against the thorax by myristic acid wax (melting

point, 58oC). Ag/AgCl2 wires were introduced into glass

capillaries (B1 mm in diameter) filled with 0.8% NaCl

and inserted into the head capsule for the reference

electrode, and the recording electrode was placed in the

basiconica-rich region of the third antennal segment and

connected to a high-impedance preamplifier (Electro 705;

WPI, Sarasota, Florida, USA). Signals were viewed on a

two-channel oscilloscope (Tektronix TDS320, Wilson-

ville, Oregon, USA). Odorants, ethyl acetate 10�4,

butanol 10�2, propionic acid 10�2, benzaldehyde

10�3, and iso amyl acetate 10�2, of the highest purity

available (Sigma, Saint Louis, Missouri, USA), diluted in

liquid paraffin, were the stimuli. Odor pulses were

delivered into a constant stream of air for 2 seconds,

followed by an interval of 5 minutes. When different

genotypes were being tested, the unpaired t-test was used

to calculate significance.

Larval Behavioral Assay

The response of larvae to odorant stimuli was measured,

as described in Ayyub et al. (1990). Briefly, approxi-

mately 150 third instar larvae were placed on 1% agar

9-cm petri plates containing two filter discs. Then, 25 mL

of odorant and the diluent were placed on the disc on

either side of the plate. Larvae were allowed to migrate

for 5 minutes, after which the numbers of larvae on the

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diluent (C) and stimulus (S) halves of the plate were

counted. Response index (RI) was calculated by subtract-

ing the number of animals on the control (C) side from the

stimulus (S) side of the dish as a fraction of the

participating larvae RI�(S�C)/(S�C). RI thus varies

from 1 (complete attraction) to 0 (no response). Often, a

small percentage of animals remained in the center of the

plate; these were not counted as C or as S and, therefore,

did not figure in the computation of the RI. A minimum

of 10 test plates were run for each experiment and the

mean and standard deviation were calculated. Statistical

significance was calculated by the Student’s t-test in all

cases.

RESULTS

Expression of a Membrane Lipase Encoded by the

stambhA Locus in the Adult Olfactory System

The product of the stambhA (stmA) gene (subsequently

also called rolling blackout or rbo by Huang et al. (2004),

is characterized as a putative phosphatidylinositol 4,5

bisphosphate- diacyl glycerol (PIP2-DAG) lipase. stmAmutants affect phototransduction by the misregulation of

phospholipid levels (Huang et al., 2004). We first tested

whether Stambh was expressed in the OSNs. This was

done by studying the expression of an eGFP transgene

driven by the stmA promoter, and hence expected to

replicate the endogenous stmA gene expression pattern

(Huang et al., 2004). In animals of the genotype stmA1;stmA-eGFP strong green fluorescent protein (GFP)

expression can be seen in sensory neurons of the third

antennal segment (Figure 1A). Their number is compar-

able with the number of GFP-expressing OSNs observed

in antennae of the genotype OR83bGAL4, UASGFPknown to express in a majority of OSNs (data not shown).

These data suggest that the stmA gene product could

affect phospholipid second-messenger levels in the

Drosophila antenna and, consequently, modify olfactory

responses.

Projections from these GFP-positive sensory neurons

extend to the adult antennal lobe (AL); a careful

examination of sections through the antennal lobe shows

expression in the entire glomeruli. Expression in the

OSNs alone would be expected to highlight only the

sensory terminals, which remain only on the periphery of

each glomerulus. The observation of complete glomerular

staining suggests that other neurons (possibly projection

neurons and local interneurons), which innervate the

antennal lobe, also express stm-eGFP. Expression was

also detected within all lobes of the mushroom body (MB;

Figure 1B) in the adult brain. The expression is strong,

and several cell bodies around the mushroom bodies are

also labeled (data not shown). Thus, stmA is expressed in

a large number of cells that project into the MB neuropi.

Reduced Odorant Responses Are Obtained from

Antennae of stmA Mutant Alleles

To understand the functional significance of stmAexpression in the adult antenna, we measured electro-

antennogram (EAG) responses to multiple odorants from

the antennae of stmA1 and stmA2 mutant alleles. Both

mutations were isolated in a screen for temperature-

sensitive paralytic mutants (Shyngle & Sharma, 1985;

Chandrashekaran & Sarla, 1993). Flies and larvae

paralyzed at nonpermissive temperature (35oC) and

recovered on return to lower permissive temperatures

Figure 1. stambhA expression in the adult olfactory sensory

system. (A) Confocal image of stmA1; stmA-eGFP transgenic

animal with GFP expression in OSNs of the third antennal

segment (A3). A single 1 mm slice of a confocal stack is shown

for clarity. (B) GFP in a stmA1; stmA-eGFP transgenic animal is

expressed in the adult antennal lobes (AL; dotted circle) and the

mushroom body (MB; white arrow head).

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(their name arises from the Hindi word, Stambh, meaning

motionless). EAG responses were reduced to a range of

chemicals when tested at both 25 and 29oC; this lack of a

temperature-sensitive effect for the olfactory response

prompted us to carry out all subsequent experiments at

25oC. Homozygous stmA1, stmA2 and the heteroallelic

combination stmA1/ stmA2 were tested with multiple

odorants. Their EAG responses were significantly lower

(PB0.0001, calculated by Students t-test) than the

response of heterozygous controls, stmA1/� and stmA2/�(Figure 2A�2C). Moreover, on introduction of the stmA-eGFP transgene in the stmA1 mutant background, EAG

responses were rescued to a significant extent (PB

0.0005, when compared with stmA1 homozygotes). The

stmA-eGFP transgene has also been shown to rescue

temperature-sensitive blindness and embryonic lethality

in stmA mutant allelic combinations (Huang et al., 2004).

Genetic Interactions among Heteroallelic Mutant

Combinations of stmA, dgq, and plcb21c

The reduced EAG responses of stmA mutants support the

idea that this gene product alters levels of a phospholipid

second messenger in adult olfactory transduction. This

was tested further by measuring the response of stmA1

heterozygotes in combination with a single copy of either

dgq mutants (dgq221c and dgq1370) or plc21c mutants

(P319 and p60A) predicted to reduce the function of their

cognate gene products. Both EAG and single olfactory

neuron responses are sensitive to a reduction in levels of

Gqa and Plc21c (Kain et al., 2008). Trans-heterozygotes

of the genotype dgq221c/stmA1, dgq1370/stmA1, dgq1370/stmA2, and dgq221c/stmA2 had significantly lower EAG

responses (Figure 2D; 4�5 mV), as compared with

heterozygous controls (9�10 mV; PB0.0001). Similarly,

a strong genetic interaction was observed among the

heteroallelic combinations of stmA and plc21c mutants

(e.g., plc21cp319/stmA1 and plc21cp60A/stmA1; 4�5 mV;

Figure 2D). The reduction observed in the heteroallelic

combinations is significantly greater than the value

expected from the mere additive effect of the reduction

observed in each heterozygote. All heterozygotes and

double mutant combinations were tested in parallel. The

response of plc21c and dgq heterozygotes has been

published earlier (Kain et al., 2008) and is shown here

for comparison. Our data suggest that dgq, plc21c, and

stmA function in the same signaling pathway and together

help odor transduction in adult olfactory sensory neurons.

These observations, together with published data

(Kain et al., 2008), suggests that odor transduction in

the Drosophila antenna includes signaling through phos-

pholipid intermediates generated by activation of the

Gqa protein. Is this signaling system operational during

olfactory transduction in larvae?

GqGAL4 Expression in Peripheral and Central

Olfactory System of Drosophila Larvae

The robust chemotactic responses of third instar larvae

to odorants are mediated by 21 OSNs that project to

glomeruli in the larval antennal lobe (Couto et al., 2005;

Kreher et al., 2005). In order to explore the putative role

of G-protein and phospholipid signaling downstream of

larval ORs, we began by examining the expression of dgqin the larval olfactory system (Figure 3A). Expression of

DGq was monitored by using a GqGAL4 (3.9 kb of the

genomic region upstream of the dgq gene fused to

GAL4), which drives the expression of UASGFP. GFP

expression was observed in the larval chemosensory

organ, that is, the dorsal organ (white box in Figure 3B,

magnified in Figure 3C�3D). Dendritic projections of

larval OSNs are labeled by GFP driven by GqGAL4 in the

dorsal organ (DO, shown by a dotted circle; Figure 3C

and also the cell bodies, which are located in the dorsal

organ ganglion; DOG; Figure 3D). Analysis of all the

confocal sections shows that a majority (at least 19 of 21)

of larval OSNs express GqGAL4 driven GFP. Axonal

projections from the DOG traverse in the antennal nerve

(AN, thick white arrow in Figure 3D) to the larval

antennal lobe (demarcated by dotted lines in Figure 3B).

GFP-labeled OSN terminals can be visualized within the

larval antennal lobe (Figure 3E); the lobe architecture is

highlighted by staining by using antibodies against the

presynaptic protein Bruchpilot (mAbnc82; Figure 3F�3G;

Laissue et al., 1999). Gqa is thus expressed in sensory

neurons of the larval olfactory system. Molecules likely to

function downstream of dgq such as plc21c (Hasan, 2003)

and stmA (Huang et al., 2004) have been documented in

parts of the embryonic nervous system.

Dgq Mutant Alleles and RNAi in Sensory Neurons

Strongly Impairs Larval Olfactory Responses

The requirement for dgq in larval olfaction was tested by

measuring behavioral responses of third instar larvae in the

larval chemotaxis assay described earlier (Monte et al.,

1989; Ayyub et al., 1990). Larvae are offered a choice

between odorant stimuli and controls on an agar plate and

the distribution after 5 minutes is quantified (see repre-

sentative plate of wild-type in Figure 4A). The responses

of dgq1370/� and dgq221c/� heterozygotes were tested

against the strongly attractant odor ethyl acetate; these

genotypes also showed reduced EAG responses in the

adult (Kain et al., 2008). At a dilution of 10�6 of the

odorant the response index of dgqnull/� larvae was

markedly reduced (0.4�0.07) in comparison with wild-

type (0.68�0.07, PB0.0001; Students t-test). The

P-insertion used to generate dgq221c causes no behavioral

defects (dgq14073 in Figure 4B). Animals of the original

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FRT42B genetic background, used for creating dgq1370,also show normal responses (similar to wild-type) toward

all concentrations of ethyl acetate tested (Figure 4B).

Homozygous dgq221c and dgq1370 larvae die as early first

or early second instar larvae and hence could not be tested

in the olfactory plate assay.

Behavioral changes, such as those measured in the

olfactory plate test, could arise from mutant effects at

multiple levels of the olfactory pathway. To test the

function of Gqa directly in larval OSNs, Gqa levels were

downregulated in all the larval sensory neurons with the

help of a GqRNAi strain (UASGq1F1; Banerjee et al.,

2006) driven by the OR83bGAL4 driver, which is known

to express in all 21 larval sensory neurons (Fishilevich

et al., 2005). The UASGq1F1 strain has been shown to

reduce Gqa?levels? but not eliminate expression (Bane-

rjee et al., 2006). The responses of UASGq1F1/�;

OR83bGAL4/� animals at all four dilutions was sig-

nificantly reduced, as compared to control animals

UASGq1F1/� and OR83bGAL4/� (Figure 4C; PB

0.005). Olfactory responses of larvae in which Gqa levels

were reduced by expression of UASGq1F1 in the GqGAL4

Figure 2. stmA mutants exhibit reduced sensitivity to different classes of odorants. (A) EAG responses of flies of the indicated

genotypes, to five different stimuli, were tested. Homozygotes of stmA1 and stmA2 showed significantly lower responses than the

heterozygotes stmA1/� and stmA2/� (PB0.0001). The stmA1/stmA2 transheterozygote combination also had reduced EAG responses

(PB0.0001). The phenotype could be rescued by the stmA genomic transgene (stmA1; stmA-eGFP; PB0.0005, compared to stmA1).

N�10 or more flies for each genotype tested. (B�C) Representative EAG traces from stmA1/� and stmA1/ stmA1 animals. Scale bar 5

mV/1sec. (D) Genetic interaction between stmA and dgq or plc21c. Transheterozygote combinations of dgq221c/stmA1, dgq1370/stmA1,dgq1370/stmA2, and dgq221c/stmA2 have reduced EAG responses, as compared to heterozygous controls dgq/� stmA1/� and stmA2/�(PB0.0001). Similarly, a strong genetic interaction was observed for the heteroallelic combinations of plc21cp319/stmA1 and plc21cp60A/stmA1.

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expression domain were also tested and found to be

significantly lower at ethyl acetate dilutions of 10�4,

10�5, and 10�6, as compared with control strains

GqGAL4/� and UASGq1F1/� (Figure 4C; PB0.001).

The response of UASGq1F1/GqGAL4 animals compared

to UASGq1F1/�; OR83bGAL4/� was significantly lower

(Figure 4C; PB0.005), reflecting the fact that GqGAL4more faithfully recapitulates the endogenous expression

of the dgq gene. Our genetic data, together with the

expression patterns described earlier, provide strong

evidence for implicating Gqa signaling in olfactory

transduction to ethyl acetate in the larva.

Larval Olfactory Behavior Deficits in plcb21c and

stmA Gene Mutants

Activation of Gqa is known to lead to activation of

PhospholipaseCb; this enzyme is encoded by two genes,

norpA (Bloomquist et al., 1988) and plc21c (Shortridge

et al., 1991), in the Drosophila genome. The norpA gene

product is essential for Drosophila visual transduction

(Bloomquist et al., 1988) and for olfactory transduction

in the OSNs of the maxillary palps (Riesgo-Escovar

et al., 1995). plcb21C has been recently implicated in

the response to multiple odorants in the adult antenna

Figure 3. dgq expression in the Drosophila larval chemosensory system. (A) Schematic of the larval olfactory system. There are 21

olfactory sensory neurons (OSNs) with dendrites in the dorsal organ (DO) and cell bodies in the dorsal organ ganglion (DOG). Axons

from the cell bodies of the dorsal organ OSNs (AN; antennal nerve) project to the larval antennal lobe (LAL) in the brain. The position

of the mushroom bodies (MB) and ventral nerve cord (VG) are indicated. (B) Third instar larva of GqGAL4/UAS2XEGFP stained with

anti-GFP (green) and mAbnc82 (red). The region of the dorsal organ (DO) is indicated in the white box and enlarged in C and D. The

animal is oriented with anterior toward the top and the posterior below. (C) The dendritic projections of larval olfactory sensory neurons

are present in the DO (white dotted circle). (D) Cell bodies of larval OSNs (white arrows) are present in the dorsal organ ganglion

(DOG). OSN axons originating from the DO exit the periphery in the AN (thick white arrow). (E�F) Larval antennal lobe in

GqGAL4/UAS2XEGFP larvae shows GFP expression (D, white dotted circle). The neuropil is labeled with nc82 (red). (G) Merge of E

and F.

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downstream of Gqa (Kain et al., 2008). To identify which

effector molecule functions downstream of Gqa during

larval olfactory transduction, mutants in the two Plcbgenes were tested for larval olfactory behavior. As

homozygotes, a hypomorphic mutant allele of plc21c(plc21cP319 or P319) gave reduced responses at 10�5 and

10�6 dilutions of ethyl acetate in the larval plate assay

(Figure 5A; PB0.0001). Lower responses, compared to

controls, were also observed in animals with a single

copy of a small deficiency uncovering plc21c(plc21cp60A/� or p60A/�; PB0.001; Weinkove et al.,

1999). Attraction toward the odorant was further reduced

in the heteroallelic mutant combination of p60A/P319(PB0.0001). The response curves for p60A/P319, p60A/�and P319/P319 are significantly different from P319/�control animals (PB0.001 for all genotypes). In contrast,

behavioral responses of a homozygous null allele of

norpA (norpAP24) appeared normal (Figure 5A). Our data

suggest that plc21c, and not norpA, functions directly

downstream of Gqa in larval olfaction.

We confirmed a role for the plc21c gene in larval

olfactory sensory neurons by expressing an RNAi

transgene for plc21c (referred to as plc21cRNAi557) under

regulation of the OR83bGAL4 driver. UASplc21C557 is a

recently generated UASRNAi strain reported as specific

for plc21C (http://stockcenter.vdrc.at). Reduced responses

were observed at 10�6, 10�5, and 10�4 concentrations

of ethyl acetate as compared to wild-type, OR83bGAL4/�, and plc21cRNAi557/� control animals (PB0.001).

When both plc21c and dgq levels were reduced in the

larval sensory neurons, by generating a double RNAi

combination of plc21cRNAi557/UASGq1F1; OR83bGAL4/,� the responses were further reduced at all concentra-

tions of ethyl acetate, as compared to plc21cRNAi557/�;

OR83bGAL4/� and UASGq1F1/�; OR83bGAL4/� ani-

mals (Figure 5B; PB0.05). These data are consistent with

the proposed role of these genes in adult olfactory

transduction (Kain et al., 2008) and support the idea

that both larval and adult olfactory receptors stimulate the

heterotrimeric G-protein Gqa upon odor binding, followed

by activation of Plcb21c. This enzyme cleaves the

membrane-bound lipid phosphoinositol bis-phosphate or

PIP2 to generate diacyglycerol (DAG) and inositol 1,4,

5-trisphosphate (InsP3).

Results described above on the adult olfactory

neurons suggest that the putative PIP2-DAG lipase

encoded by stmA participates in the same pathway as

Gqa and Plcb21c (Figure 2). To test this in the context of

larval olfactory behavior, the response of stmA1 homo-

zygotes and heterozygotes was measured in the larval

plate assay. Both stmA1/� and stmA1/stmA1 larvae show

significantly reduced attraction toward ethyl acetate at

all dilutions, as compared with control larvae of

the genotypes w1118/� and w1118/w1118 (Figure 5C;

Figure 4. Reduced dgq levels in larval OSNs results in reduced

olfactory behavior. (A) A representative larval plate test with

stimulus (S) and control (C) containing filter paper discs. Larvae

show biased movement toward S. (B) Null alleles of dgq(dgq221c and dgq1370) as heterozygotes (dgq221c/� and dgq1370/�) have reduced responses to ethyl acetate at 10�6 dilution,

compared to heterozygous controls of the appropriate genetic

backgrounds, dgq14073/� and FRT42B/� (PB0.0001). (C) Gqa

levels were downregulated in larval sensory neurons by

expressing a GqRNAi strain (UASGq1F1) driven by the

OR83bGAL4 driver. Olfactory behavior responses at concentra-

tions ranging from 10�4 to 10�6 were significantly reduced,

compared to the control animals, UASGq1F1/� and OR83b-GAL4/� (PB0.005). Reducing Gqa levels in the GqGAL4expression domain (GqGAL4/UASGq1F1) also led to signifi-

cantly lower responses, as compared with the control strains

GqGAL4/� and UASGq1F1/� (PB0.001). N�10 plates for

each genotype tested.

Phospholipid Signaling in Drosophila Olfaction 309

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PB0.001). However, transheterozygote combinations,

such as dgq221c/ plc21Cp60A, dgq221c/ plc21Cp319, stmA1/dgq221c, and stmA1/ plc21Cp319, did not appear different

from the heterozygous controls (Figure 5D).

DISCUSSION

We have shown that adult olfactory responses are reduced

in mutants for Gqa? which signals through Phospholipase

Cb. Genetic as well as electrophysiological data favor the

possibility that a phospholipid intermediate, rather than

IP3, acts in olfactory signaling (Kain et al., 2008). In this

paper, we have provided further support for this conclu-

sion. The product of the stmA gene, which can function as

a PIP2-DAG lipase, is strongly expressed in adult OSNs.

Mutants, which presumably compromise phospholipid

levels, also compromise the ability of OSNs to evoke a

response to several different odors and affect the

behavioral response to one of these odorants (e.g., ethyl

acetate) in larvae. Recent data from studies in cell-culture

systems provide support to a body of literature suggesting

a role for cAMP in Drosophila olfactory reception

(Wicher et al., 2008). A further complexity lies in the

Figure 5. plc21c acts as a downstream effector in larval olfactory signaling. (A) Response indexes for different concentrations of ethyl

acetate are shown for the indicated genotypes. plc21Cp319/� (P319/�) is the genetic background control strain for the deficiency

plc21Cp60A/� (p60A/�). Their responses are significantly different (PB0.001). plc21Cp60A/ plc21Cp319 transheterozygotes and

plc21Cp319 homozygous animals show a reduction in responses, as compared to the plc21Cp319/� animals (PB0.001). norpAP24

homozygotes showed normal responses. (B) An RNAi-producing transgene of plc21c ( plc21cRNAi557) driven by OR83bGAL4 in all the

larval olfactory sensory neurons, reduces responses at 10�6, 10�5, and 10�4 concentrations of ethyl acetate, as compared to wild-type,

OR83bGAL4/�, and plc21cRNAi557/� control animals (PB0.001). A knockdown of plc21c and Gqa in the larval sensory neurons by

double RNAi combination, plc21cRNAi557/UASGq1F1 ; OR83bGAL4/� further reduces the responses significantly at all concentrations

of ethyl acetate, as compared to plc21cRNAi557/�; OR83bGAL4/� and UASGq1F1/� ; OR83bGAL4/� animals (shown in Figure 2D;

PB0.05). (C) stmA mutant alleles exhibit reduced larval olfactory responses to ethyl acetate. stmA1, showed reduced behavior toward

different concentrations of ethyl acetate. Heterozygous stmA1/� larvae also showed significant reduction at all odor concentrations

tested, as compared to w1118/� controls (PB0.001). (D) Transheterozygotic combinations of dgq221c/ plc21Cp60A, dgq221c/ plc21Cp319,

stmA1/ dgq221c, and stmA1/ plc21Cp319 do not show a significant alteration in response to ethyl acetate tested, as compared to controls.

N�10 plates for each genotype.

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finding that odorant receptors, in combination with

coreceptor Or83b, form ligand-gated ion channels, which

presumably would trigger neuronal activity directly upon

stimulus binding (Sato et al., 2008). These mechanisms,

which could well exist in the same neuron, would operate

at different stimulus concentrations, thus providing a large

dynamic range for odor detection in insects.

Barring a few exceptions, larval as well as adult

OSNs generally express a single odorant receptor together

with Or83b. The response capabilities of different

receptors have been elegantly studied by ectopically

expressing them in an ‘‘empty’’ adult neuron (Hallem

et al., 2004). Most of the larval Ors are not only broadly

tuned, but show complex responses; and several Ors can

trigger either excitatory or inhibitory responses from two

different stimuli. Moreover, certain adult and larval

receptors are also capable of delayed responses to one

odorant while responding at normal time scales to others

(Hallem et al., 2004; Kreher et al., 2005). Explanations

for these phenomena may well come from modeling

differential activation of multiple signaling pathways

downstream of odorant receptors.

Increasingly, the close similarity between signaling

intermediates and transduction mechanisms in Drosophilavision and olfaction is becoming apparent (Kain et al.,

2008; Woodard et al., 1992). In this paper, we compare

the contribution of stmA gene in olfactory transduction

and its published role in vision (Huang et al., 2004).

Interestingly, some differences in the effects of stmA on

vision and olfaction are apparent. In stmA mutants, the

levels of DAG are elevated by �50% higher at 25oC; at

this ‘‘permissive’’ temperature, there is no effect seen on

electroretinograms (ERGs). In contrast, we find that

stmA1 homozygotes have significant defects in EAG

responses and larval olfactory behavior at 25oC. These

observations are consistent with the idea that the EAG

response requires a DAG metabolite, which is generated

by the product of the stmA gene. Levels of PIP and PIP2

are also altered in stmA1 mutants at nonpermissive

temperatures (37oC), and based on these observations it

has been proposed that the temperature-sensitive ERG

defect arises from a defect in light-activated PLC

function. It is possible that the stmA gene product has

differential effects on norpA encoded PLCb? as compared

with Plcb21c. The putative role of DAG or a DAG

metabolite as a second messenger in olfaction has been

tested directly by measuring EAGs from mutant alleles for

a DAG kinase gene, rdgA (Kain et al., 2008). The rdgAgene product is adult specific, and hence, rdgA mutants

have no effect on larval olfactory behavior (data not

shown). There are other DAG kinase homologs present in

Drosophila mutants, which need to be tested for their

effect on larval olfaction. Since similar mechanisms

appear to operate in larval as well as adult neurons (at

least as far as Gqa signaling is concerned), the larval

system provides a useful model to test the possibility of a

multiplicity of signaling mechanisms and to link this to its

consequence in behavior.

ACKNOWLEDGMENTS

This work was supported by funding from the National

Centre for Biological Sciences (NCBS) and the Depart-

ment of Biotechnology (GH), the National Institute on

Drug Abuse�associated Supplement for International

Collaboration Grant DA15495, and the Indian Council

for Medical Research (VR). The authors acknowledge the

Centre for Nanotechnology (Department of Science and

Technology No. SR/S5/NM-36/2005) for the Olympus

confocal microscope and are indebted to Prabhat Kumar

Tiwari, H. Krishnamurthy, and the NCBS imaging facility

for help with confocal imaging.

Declaration of interest: The authors report no conflicts of

interest. The authors alone are responsible for the content

and writing of this paper.

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