A Screen for Genes Expressed in the Olfactory Organs of Drosophila melanogaster Identifies Genes Involved in Olfactory Behaviour

BACKGROUND: For insects the sense of smell and associated olfactory-driven behaviours are essential for survival. Insects detect odorants with families of olfactory receptor proteins that are very different to those of mammals, and there are likely to be other unique genes and genetic pathways involved in the function and development of the insect olfactory system. METHODOLOGY/PRINCIPAL FINDINGS: We have performed a genetic screen of a set of 505 Drosophila melanogaster gene trap insertion lines to identify novel genes expressed in the adult olfactory organs. We identified 16 lines with expression in the olfactory organs, many of which exhibited expression of the trapped genes in olfactory receptor neurons. Phenotypic analysis showed that six of the lines have decreased olfactory responses in a behavioural assay, and for one of these we showed that precise excision of the P element reverts the phenotype to wild type, confirming a role for the trapped gene in olfaction. To confirm the identity of the genes trapped in the lines we performed molecular analysis of some of the insertion sites. While for many lines the reported insertion sites were correct, we also demonstrated that for a number of lines the reported location of the element was incorrect, and in three lines there were in fact two pGT element insertions. CONCLUSIONS/SIGNIFICANCE: We identified 16 new genes expressed in the Drosophila olfactory organs, the majority in neurons, and for several of the gene trap lines demonstrated a defect in olfactory-driven behaviour. Further characterisation of these genes and their roles in olfactory system function and development will increase our understanding of how the insect olfactory system has evolved to perform the same essential function to that of mammals, but using very different molecular genetic mechanisms

Molecular analysis of pGT lines and predicted candidate genes.

<p><i>Note</i>.</p>a<p>Number of pGT inserts identified by Southern blot analysis.</p>b<p>BDGP prediction as taken from Flybase.</p>c<p>Cytological location of BDGP predicted gene.</p>d<p>Candidate gene identified from 3′RACE experiments.</p>e<p>Cytological location of gene identified by RACE.</p>f<p>Cytological location as determined by polytene chromosome <i>in situ</i> hybridisation with a <i>Gal4</i> probe.</p>g<p>As the Southern blot indicates two inserts there may be a second unidentified candidate gene for this line.</p>h<p>For BG02836 a Southern blot suggested one insert but polytene chromosome <i>in situ</i> hybridisation gave two signals, one at ∼18D1 and one at ∼67E thus there may be two inserts. N.D. – not determined.</p

GFP expression patterns observed in ‘olfactory positive’ pGT lines.

<p><i>Note</i>. Mouth - Mouthparts including proboscis, labellum and/or cibarial organs. Leg - Tips/distal parts or joints of legs. Wing - Wing margin or joints of wings. Brain - Majority of lines had staining in the mushroom bodies or uniformly in the brain, some lines also had staining in the optic lobes.</p>a<p>These lines showed inconsistent Elav co-localisation patterns and expression is also possibly in accessory cells.</p

Geotaxis Response Index is normal in pGT lines.

<p>Negative geotactic ability was tested to investigate CNS and locomotor function of the lines that exhibited abnormal olfactory behaviour. All lines tested showed negative geotactic behaviour to at least control levels, with one line (BG01746) showing a small increase (* ANOVA, t-test, p<0.007). The pGT lines are represented in numerical order. The error bars represent SEM; n = 5–19.</p

Precise excision of the pGT element in BG00076 and BG00973 restores olfactory behaviour.

<p>Comparison of response indices of wild type flies (wt), pGT insertion mutants (BG00076 or BG00973) and two precise excision lines for each (ex1, ex2). Asterisks for excision lines indicate significantly higher responses than pGT mutants (ANOVA, t-test, p<0.01). (A–B) For BG00076 mutant responses are rescued in both ex1 and ex2 in females (A) and males (B). (C–D) For BG00973 mutant responses are rescued in both ex1 and ex2 in females (C) but only in ex1 in males (D). The error bars represent SEM; n = 10 for all lines.</p

Olfactory Trap Response Index of pGT lines.

<p>Defects in olfactory behaviour were tested using an olfactory trap assay. Only seven pGT lines could be tested reproducibly for olfactory trap behaviour because of high mortality rates. The response index (RI) of flies entering traps was recorded at 20-hour intervals over 60 hours and the average at 60 hours is shown. A. Females. B. Males. The pGT lines are represented in numerical order. The error bars represent SEM; n = 10 for all lines. * p<0.05 t-test.</p