Deborah A. Kimbrell

Pheromone reception in fruit flies expressing a moth's odorant receptor

We have expressed a male-specific, pheromone-sensitive odorant receptor (OR), BmorOR1, from the silkworm moth Bombyx mori in an “empty neuron” housed in the ab3 sensilla of a Drosophila Δhalo mutant. Single-sensillum recordings showed that the BmorOR1-expressing neurons in the transgenic flies responded to the B. mori pheromone bombykol, albeit with low sensitivity. These transgenic flies responded to lower doses of bombykol in an altered stimulation method with direct delivery of pheromone into the sensillum milieu. We also expressed a B. mori pheromone-binding protein, BmorPBP, in the BmorOR1-expressing ab3 sensilla. Despite the low levels of BmorPBP expression, flies carrying both BmorOR1 and BmorPBP showed significantly higher electrophysiological responses than BmorOR1 flies. Both types of BmorOR1-expressing flies responded to bombykol, and to a lesser extent to a second compound, bombykal, even without the addition of organic solvents to the recording electrode buffer. When the semiochemicals were delivered by the conventional puffing of stimulus on the antennae, the receptor responded to bombykol but not to bombykal. The onset of response was remarkably slow, and neural activity extended for an unusually long time (>1 min) after the end of stimulus delivery. We hypothesize that BmorOR1-expressing ab3 sensilla lack a pheromone-degrading enzyme to rapidly inactivate bombykol and terminate the signal. We also found an endogenous receptor in one of the sensillum types on Drosophila antenna that responds to bombykol and bombykal with sensitivity comparable to the pheromone-detecting sensilla on B. mori male antennae.

Bombykol receptors in the silkworm moth and the fruit fly

Male moths are endowed with odorant receptors (ORs) to detect species-specific sex pheromones with remarkable sensitivity and selectivity. We serendipitously discovered that an endogenous OR in the fruit fly, Drosophila melanogaster, is highly sensitive to the sex pheromone of the silkworm moth, bombykol. Intriguingly, the fruit fly detectors are more sensitive than the receptors of the silkworm moth, although its ecological significance is unknown. By expression in the “empty neuron” system, we identified the fruit fly bombykol-sensitive OR as DmelOR7a (= DmOR7a). The profiles of this receptor in response to bombykol in the native sensilla (ab4) or expressed in the empty neuron system (ab3 sensilla) are indistinguishable. Both WT and transgenic flies responded with high sensitivity, in a dose-dependent manner, and with rapid signal termination. In contrast, the same empty neuron expressing the moth bombykol receptor, BmorOR1, demonstrated low sensitivity and slow signal inactivation. When expressed in the trichoid sensilla T1 of the fruit fly, the neuron housing BmorOR1 responded with sensitivity comparable to that of the native trichoid sensilla in the silkworm moth. By challenging the native bombykol receptor in the fruit fly with high doses of another odorant to which the receptor responds with the highest sensitivity, we demonstrate that slow signal termination is induced by overdose of a stimulus. As opposed to the empty neuron system in the basiconic sensilla, the structural, biochemical, and/or biophysical features of the sensilla make the T1 trichoid system of the fly a better surrogate for the moth receptor.

Host Immune Response and Differential Survival of the Sexes in Drosophila

Innate immunity is essential for the survival of organisms across the evolutionary spectrum. Drosophila is well studied as a model of innate immunity and has been instrumental in establishing principles of defense and gene signaling pathways that are shared with humans. Previous studies in Drosophila have not focused on differences between the sexes, and in this report we present evidence that it is essential to include differences between the sexes. Survival rates post-infection, after a fungal or bacterial infection, varied according to the combination of signaling pathway (Toll and Imd) and sex tested. We also found that antimicrobial protein gene mRNA levels for Drosomycin and Metchnikowin showed both similarities and differences between the sexes. These studies highlight the need to include both sexes in studies of immune function as well as the associated opportunities for advancing our understanding of immunity.

Cuticle protein gene expression during the third instar of Drosophila melanogaster

Abstract Genes that encode cuticle proteins of Drosophila melanogaster are useful for the study of coordinate gene regulation. During the development of Drosophila , five cuticles are made: one for each of the three larval instars, one for the pupa, and one for the adult. The production of each cuticle is controlled by the steroid moulting hormone, 20-hydroxyecdysone. In this study we investigated the basis for the sequential appearance of third instar larval cuticle proteins. Among five major proteins, larval cuticle proteins (LCPs) 3–5 accumulate before LCPs 1 and 2. Metabolic labelling of the proteins suggests that LCPs 3–5 are synthesized before LCPs 1 and 2. The genes that encode LCPs 1–4 are clustered within 8 kilobases on chromosome 2. Using probes specific for these genes, we find that transcripts of genes 3 and 4 accumulate before those of genes 1 and 2. Thus, the late accumulation of transcripts from genes 1 and 2 during the 3rd instar appears to be the basis for the delayed accumulation of larval cuticle proteins 1 and 2.

Facile functional analysis of insect odorant receptors expressed in the fruit fly: validation with receptors from taxonomically distant and closely related species

With the advent of genomic sequences and next-generation sequencing technologies (RNA-Seq), multiple repertoires of olfactory proteins in various insect species are being unraveled. However, functional analyses are lagging behind due in part to the lack of simple and reliable methods for heterologous expression of odorant receptors (ORs). While the Xenopus oocyte recording system fulfills some of this lacuna, this system is devoid of other olfactory proteins, thus testing only the “naked” ORs. Recently, a moth OR was expressed in the majority of neurons in the antennae of the fruit fly using Orco-GAL4 to drive expression of the moth OR. Electroantennogram (EAG) was used to de-orphanize the moth OR, but generic application of this approach was brought to question. Here, we describe that this system works with ORs not only from taxonomically distant insect species (moth), but also closely related species (mosquito), even when the fruit fly has highly sensitive innate ORs for the odorant being tested. We demonstrate that Orco-GAL4 flies expressing the silkworm pheromone receptor, BmorOR1, showed significantly higher responses to the sex pheromone bombykol than the control lines used to drive expression. Additionally, we show that flies expressing an OR from the Southern house mosquito, CquiOR2, gave significantly stronger responses to the cognate odorants indole and 2-methylphenol than the “background noise” recorder from control lines. In summary, we validate the use of Orco-GAL4 driven UAS-OR lines along with EAG analysis as a simple alternative for de-orphanization and functional studies of insect ORs in an intact olfactory system.

Regulation of Hemocytes in Drosophila Requires dappled Cytochrome b5

A major category of mutant hematopoietic phenotypes in Drosophila is melanotic tumors or nodules, which consist of abnormal and overproliferated blood cells, similar to granulomas. Our analyses of the melanotic mutant dappled have revealed a novel type of gene involved in blood cell regulation. The dappled gene is an essential gene that encodes cytochrome b5, a conserved hemoprotein that participates in electron transfer in multiple biochemical reactions and pathways. Viable mutations of dappled cause melanotic nodules and hemocyte misregulation during both hematopoietic waves of development. The sexes are similarly affected, but hemocyte number is different in females and males of both mutants and wild type. Additionally, initial tests show that curcumin enhances the dappled melanotic phenotype and establish screening of endogenous and xenobiotic compounds as a route for analysis of cytochrome b5 function. Overall, dappled provides a tractable genetic model for cytochrome b5, which has been difficult to study in higher organisms.