An angiotensin converting enzyme homolog is required for volatile pheromone detection, odorant binding protein secretion and normal courtship behavior in Drosophila melanogaster

Abstract

In many arthropods, including insects responsible for transmission of human diseases, behaviors that include mating, aggregation, and aggression are triggered by detection of pheromones. Extracellular odorant binding proteins are critical for pheromone detection in many insects and are secreted into the fluid bathing the olfactory neuron dendrites. In Drosophila melanogaster, the odorant binding protein LUSH is essential for normal sensitivity to the volatile sex pheromone, 11-cis vaccenyl acetate (cVA). Using a genetic screen for cVA pheromone insensitivity, we identified ANCE-3, a homolog of human angiotensin converting enzyme that is required for detection of cVA pheromone. The mutants have normal dose-response curves for food odors, although olfactory neuron amplitudes are reduced in all olfactory neurons examined. ance-3 mutants have profound delays in mating, and the courtship defects are primarily but not exclusively due to loss of ance-3 function in males. We demonstrate that ANCE-3 is required in the sensillae support cells for normal reproductive behavior, and that localization of odorant binding proteins to the sensillum lymph is blocked in the mutants. Expression of an ance-3 cDNA in sensillae support cells completely rescues the cVA responses, LUSH localization, and courtship defects. We show the courtship latency defects are not due to effects on olfactory neurons in the antenna nor mediated through ORCO receptors, but instead stem from ANCE-3-dependent effects on chemosensory sensillae in other body parts. These findings reveal an unexpected factor critical for pheromone detection with profound influence on reproductive behaviors.

Keywords: ACE; mating; olfaction; olfactory; reproduction.

Fig. 1.

ance-3 mutants are defective for cVA responses. a) Cartoon of a single at 1 sensillum showing the Or67d neuron (OSN) projecting a dendrite into the sensillum lymph within the shaft of the sensillum. Tormogen and trichogen support cells (To, Tr) secrete OBPs including LUSH into the sensillum lymph. The thecogen support cell (Th) acts as a glial sheath cell wrapping the sensory neuron. b) Cartoon of the single sensillum recording setup. A sharp glass electrode is used to puncture the sensillum and measure spontaneous and cVA-induced action potentials from the neuron. c) Single sensillum recordings (SSR) from Or67d neurons in at1 sensillae. Responses from wild type (WT), ance-3², ance-3³, and ance-3^RFP mutant alleles. d) Dose–response curves for wild type and three ance-3³ mutant alleles to various dilutions of cVA spotted on the stimulus filter paper. n = 28 for WT, n = 31 for ance-3³, n = 13 for ance-3², n = 11 for ance-3^RFP. No differences were observed between the alleles. e) ance-3³ mutants are defective for spontaneous activity in cVA-sensitive Or67d neurons. The genotypes are significantly different (P = 4.75 × 10⁻⁷, Student's t-test, n = 28 for WT, n = 31 for ance-3³, n = 13 for ance-3², n = 11 for ance-3^RFP). f) SSR amplitudes are significantly reduced in ance-3³ mutants compared to wild type (n = 10 for each genotype (P = 1.94 × 10⁻⁸, Student's t-test, n = 28 for WT, n = 31 for ance-3³, n = 13 for ance-3², n = 11 for ance-3^RFP).

Fig. 2.

The ance-3 gene, which is expressed in antennal support cells, is lesioned in the mutants. a) Genomic map of the ance-3 locus. The ance-3 coding sequence is distributed over 55 kb of genomic DNA. Location of the lesions is depicted. ance-3^RFP is a CRISPR Cas9 allele in which the first seven coding exons are replaced with 3xP3-dsRED. b) Anti-ANCE-3 antiserum reacted with wild-type antenna section detects protein in the antenna in a support cell pattern. Scale bar, 33 mm. c) Anti-ANCE-3 antiserum reacted with ance-3³ mutants detects no protein in the mutant. Scale bar, 33 mm. d) Anti-ANCE-3 antiserum on ance-3³ mutants expressing a wild-type ance-3 cDNA with lush GAL4 driver in the trichoid support cells. The strong lush promoter expresses high levels of ANCE-3. The brightness for panel c) is higher than b) and d), so the antenna is visible in the image. Scale bar, 8 mm.

Fig. 3.

ANCE-3 is required in support cells. a) SSR traces from wild type, ance-3³ mutants, and ance-3³ mutants expressing a wild-type ance-3 transgene in the support cells or Or67d neurons. Support cell expression rescues cVA sensitivity, while expression in the Or67d neuron does not. cVA responses from ance-3³ mutant flies expressing the transgene in both neurons and support cells are not different from expression in the support cells alone. ance-3 transgenic rescue with the tormogen socket cell-specific driver, ASE5 GAL4, rescues cVA responses from ance-3³, but expression in the thecogen sheath cell driver, nompA GAL4, does not rescue. b) Dose–response curves to air passed over various dilutions of cVA from wild type (WT) and ance-3³ mutants rescued with wild-type ance-3 cDNA expressed with different GAL4 drivers. (n = 28 for WT, n = 31 for ance-3³, n = 17 for Rescue in support cell, n = 15 for Rescue in neuron, n = 13 for Rescue in both, n = 11 for Rescue in thecogen, and n = 12 for Rescue in tormogen. c) Western blot of antennal extracts from wild type (+) and ance-3³ mutants (ance3). The mature LUSH protein with cleaved signal sequence is 14 kD. M, size markers.

Fig. 4.

ance-3 mutants fail to secrete odorant binding proteins into the sensillum lymph. Frozen tissue sections from wild type (a, c, and e) or ance-3³ mutant (b, d, f, g, and h) individuals reacted with anti-LUSH (a, b, g, and h), anti-Os-E (c and d), or anti-Os-F (e and f). Arrows indicate trichoid sensillae shafts. g) ance-3³ mutants expressing a wild-type ance-3 cDNA with lush GAL4 reacted with anti-LUSH antiserum reveals restoration of LUSH secretion. h) ance-3³ mutant antenna expressing ance-3^QE with the lush GAL4 driver reacted with anti-LUSH antiserum partially restores LUSH secretion to ance-3 mutants. Scale bar, 10 mm.

Fig. 5.

ANCE, ANCE-3^His, and ANCE-3^QE transgenic individuals for rescue of cVA sensitivity. a) SSR traces recorded from wild-type at1 sensillae, or sensillae from ance-3³ mutants expressing ANCE-3^His lacking the zinc coordinating histidines (Histidine mutant), or expressing ANCE, the closest paralog to ANCE-3 in the Drosophila genome (ance rescue). b) cVA dose–response curves for wild type (WT, black squares), and ance-3³ mutants expressing the ance-3^His mutant (open red circles) or the ance transgene rescue (ance rescue, green triangles). n = 28 for WT, n = 31 for ance-3³, n = 9 for Histidine mutant, n = 12 for Ance rescue. c) Sample SSR traces induced by air passed over filters with 1%, 10%, or 100% cVA dilutions recorded from at1 sensillae from ance-3³ mutants rescued with the ance-3^QE construct. The ance-3^QE rescue consistently restores cVA responses, even at low cVA concentrations, but has little effect on the reduced spike amplitude phenotype. d) cVA dose–response curves for wild type (WT, black squares) and ance-3³ mutants expressing ance-3^QE with the lush GAL4 driver (ance-3^QE, red pentagons). ANCE-3^QE significantly rescues ance-3 loss of function on cVA sensitivity, but the genotypes are significantly different at cVA applications above 10% (P < 0.05 for 10, 30, and 100%). n = 28 for WT, n = 8 for Ance QE. e–h) Anti-ANCE-3 antiserum reacted on ance-3³ mutants expressing transgenic rescue constructs. e) ance-3³ mutants. f) ance-3³ mutants expressing wild type ance-3 cDNA with lush GAL4. g) ance-3³ mutants expressing ance-3^His with lush GAL4. h) ance-3³ mutants expressing ance-3^QE with lush GAL4. Scale bar, 7 mm.

Fig. 6.

ance-3 mutants have defective courtship behaviors. a) Time to copulation (Mating Latency) for single pairs of wild type, ance-3³ mutants, ance-3³ mutants expressing a wild-type ance-3 transgene with lush GAL4 (ance-3³ res), lush mutants, or double mutants defective for expression of Os-E, Os-F odorant binding proteins (Scheuermann and Smith 2019), and triple mutants lacking LUSH, Os-E, and Os-F odorant binding proteins (Os-E/Os-F⁻, lush⁻). n = 10 for each genotype. **** genotypes different at P < 0.0001, one-way ANOVA with Tukey test. If no bar with asterixis, not significantly different between genotypes. b) Time to copulation (Mating Latency) for virgin females of different genotypes with wild-type males. n = 10 for each genotype. **P < 0.01, ***P < 0.001, ****P < 0.0001, by Kruskal–Wallis test with Dunn's correction. If no bar with asterixis, not significantly different between genotypes. c) Time to copulation (Mating Latency) for wild-type virgin females crossed to males of different genotypes. n = 10 for each genotype. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by Kruskal–Wallis test with Dunn's correction. If no bar with asterixis, not significantly different between genotypes. d) Male–male courtship index. n = 10 for each genotype. Genotypes are not significantly different by Kruskal–Wallis test with Dunn's correction. Hypersexual behavior in w¹¹¹⁸ has been observed previously (Krstic et al. 2013). e) Locomotor behavior is not significantly different for wild type and ance-3³ mutants by two-tailed Student's t-test. n = 16 for each genotype.

Fig. 7.

Trichoid neuron receptor mutant mating latencies are less severe than ance-3 mutants. a) Time to copulation (Mating Latency) for wild type (w¹¹¹⁸), ance-3 cDNA rescue of ance-3³ mutants with lush GAL4 (ance-3³ res), nompA GAL4 (nompA res), and ASE5 GAL4 (ASE5 res). All rescue copulation latency. n = 10 for each genotype. No differences between genotypes using one-way ANOVA with Tukey test. b) Time to copulation (Mating Latency) for lush GAL4 expression of ANCE-3^QE partially rescues copulation latency in ance-3³ males from 19 to 4 h. n = 15 for each genotype. Genotypes are different by two-tailed Student's t-test. c) Time to copulation (Mating Latency) for single pairs of wild type, Or47b receptor mutants (Or47b⁻), Or88a receptor mutants (DOr88a), Or65abc deletion mutants (DOr65abc), Or65abc; Or67d^GAL4 double mutants (DOr65abc; Or67d^GAL4), Orco mutants (Or83b²), Or67d receptor mutants (Or67d^GAL4), and ance-3³ mutants. n = 10 for each genotype. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, significantly different by Kruskal–Wallis test with Dunn's correction. If no bar with asterixis, not significantly different between genotypes.