Humidity response depends on the small soluble protein Obp59a in Drosophila
- Yale University, United States
Figures
Figure 1
Obp59a maps to the second chamber of the sacculus.
(A) In situ hybridization of Obp59a to the third segment of the Drosophila antenna. Transcript (red) is observed around the second chamber of the sacculus. (B,C) Obp59a-GAL4 drives expression of UAS-GFP (green) in the same location as the RNA probe. Scale bar = 12 μm. (D) The tsetse fly Glossina morsitans morsitans (right); the third antennal segment is indicated by the arrowhead. Drosophila melanogaster (left) is shown to illustrate the relative sizes of these flies. Photo courtesy of Dr. Geoffrey Attardo. (E) In situ hybridization with the G. morsitans morsitans ortholog of Obp59a to the tsetse antenna shows localization to the sacculus. ‘S’ indicates the sacculus; ‘P’ designates a distinct sensory pit observed in the tsetse antenna. Scale bar = 50 μm.
Figure 2 with 2 supplements
Obp59a maps to hygrosensitive sensilla.
Antennal IRX-GAL4; UAS-GFP drivers (green) were used in double-label experiments with an Obp59a probe (red). Arrowheads indicate neuronal dendrites. Obp59a localizes to cells adjacent to hygrosensitive neurons that express drivers of IR93a, IR25a, IR40a, and IR68a. The 12 fluorescent images are all from the region of the antenna containing the sacculus (rectangle in black and white image above). The dendrite labeled by the IR68a-GAL4 driver can be visualized more clearly by examining multiple focal planes. Scale bar of fluorescent images = 6 μm.
Figure 2—figure supplement 1
Obp59a does not map to sensilla expressing IR8a-GAL4.
. IR8a-GAL4; UAS-GFP driver (green) was used in a double-label experiment with an Obp59a probe (red). The arrowhead indicates a neuronal dendrite. Obp59a does not label cells adjacent to IR8a-GAL4-expressing neurons, which are not hygrosensory (Enjin et al., 2016). Scale bar = 6 μm.
Figure 2—figure supplement 2
Hygrosensitive neurons are present in Obp59a1.
An anti-IR93a antibody, an anti-IR25a antibody, and an IR68a-GAL4 driver all labeled neurons in the second chamber of the sacculus of Obp59a1, in a pattern comparable to that observed in controls (+). Scale bar = 6 um.
Figure 3 with 1 supplement
Obp59a protein localizes within the sensillum shaft.
(A) αOBP59a antibody labels the second chamber of the sacculus, in a pattern that overlaps with that of the Obp59a RNA probe (B,C). (D) The αOBP59a antibody does not label Obp59a1. Scale bar = 18 μm. (E) Ultrastructure of a hygrosensory sensillum in the second chamber of the sacculus. Scale bar = 100 nm. (F) Antennal section through the second chamber, showing hygrosensitive sensilla (arrowheads). (G–J) Double-labeling with αOBP59a antibody and the Obp59a RNA probe, showing that Obp59a protein is localized within the shafts of hygrosensitive sensilla, which in (J) are outlined by thin solid lines within the dotted regions of interest; immunolabeling is also observed in non-neuronal cells that express the Obp59a RNA, presumably representing nascent protein before secretion. Scale bar = 2 μm.
Figure 3—figure supplement 1
Gross morphology of the sacculus is normal in Obp59a1.
. Z-stack of the sacculus from control (+) and Obp59a1 flies observed via confocal microscopy. Scale bar = 5 μm.
Figure 4 with 2 supplements
An Obp59a mutant is defective in a fast hygrotaxis behavior.
(A) Hygrotaxis paradigm in a Petri dish. (B–D) Hygrotaxis response of Obp59a1 (red) is lower than control (black) in all cases in which flies are given a choice between two different humidities. (E) Neither genotype shows a response when given a choice between two identical humidities. (F–H) Expression of a UAS-Obp59a rescue construct (dark blue) increases humidity response above each parental control value. (I) Under the condition of identical humidities, no genotype shows a response. ****p<0.0001, n.s. = not significant, Friedman test with Dunn’s multiple comparisons test, n = 30 replicates.
Figure 4—figure supplement 1
Desiccated Obp59a1 flies are defective in a fast hygrotaxis behavior.
(A–C) When desiccated prior to experimentation, Obp59a1 shows reduced hygrotaxis responses in all cases in which flies are given a choice between two different humidities. (D) Neither genotype shows a response when given a choice between two identical humidities. ****p<0.0001, n.s. = not significant, Friedman test with Dunn’s multiple comparisons test, n = 14–25 replicates.
Figure 4—figure supplement 2
The hygrotaxis phenotype maps to Obp59a.
. Obp59a2 and an Obp28a mutant described previously (Larter et al., 2016) were evaluated in the Petri dish hygrotaxis assay. Both mutations are on the second chromosome. (A–D) The hygrotaxis response of Obp59a2 (red) is lower than control (black) in all cases in which flies are given a choice between two different humidities. This experiment was carried out in parallel with that of Figure 4A–D and the control values are the same. (E–H) Obp28a- does not show an altered humidity response. (I–P) Following desiccation, Obp59a2 flies show a phenotype but Obp28a mutant flies do not. The data shown in panels I-L were collected in parallel with those in Figure 4—figure supplement 1A–D and the control values are the same. ****p<0.0001, n.s. = not significant, Friedman test with Dunn’s multiple comparisons test. n = 14–30 replicates.
Figure 5
Obp59a is defective in humidity preference, but not in other behaviors.
(A) A humidity preference paradigm based on that of Knecht et al. (2016). (B–D) Control flies (black) show a sustained preference for higher humidities; Obp59a1 flies show reduced responses during most of the 24 hr test period. We note that the first time point was taken at 5 min, by which time a number of flies had migrated to the region of high humidity. ****p<0.0001, Friedman test with Dunn’s multiple comparisons test, n = 24 replicates. (E) Modified Proboscis Extension Response paradigm. (F) Mutants have reduced PER responses to water vapor. **p<0.01, Mann-Whitney test, n = 22 replicates. (G) RING test of climbing behavior. n.s. = not significant, Mann-Whitney test, n = 10 replicates. (H) Mutants show no phenotype in an olfactory trap assay. Apple cider vinegar was diluted 10−3, and trans-2-hexenal was diluted 10−2, both in paraffin oil. n.s. = not significant, Mann-Whitney test, n = 15 replicates.
Figure 6 with 1 supplement
Obp59a mutations confer increased desiccation resistance.
Mutant flies survive longer under desiccating conditions than controls. (A), Obp59a1 males. (B), Obp59a2 males. (C), Obp59a1 females. (D), Obp59a2 females. The control data in (A) and (B) are the same because the two mutant alleles were tested in parallel; likewise for (C) and (D). ****p<0.0001, log-rank (Mantel-Cox) test, n = 20–28 replicates. Supplemental Information Legends.
Figure 6—figure supplement 1
Normal survival of Obp59a at 70% humidity and normal survival of Obp28a.
(A–D) Obp59a1 (red), Obp59a2 (pink), and control (black) flies show similar survival curves when exposed to 70% RH. (E–H) Obp28a shows normal survival when exposed to 0% or 70% RH. The control data in (E) are the same as in Figure 6A and B because the mutants were tested in parallel; likewise for (F) and Figure 6C and D. n.s., not significant by log-rank (Mantel-Cox) test, n = 20–28 replicates.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (D. melanogaster) | Obp59a | NA | FLYB:FBgn0034766 | |
| Gene (G. morsitans morsitans) | Obp59a | NA | Vectorbase: GMOY006081 | |
| Genetic reagent (D. melanogaster) | wCS | DOI: 10.1016/j. neuron.2014.07.012 | ||
| Genetic reagent (D. melanogaster) | Obp28a- | DOI: 10.7554/eLife.20242 | ||
| Genetic reagent (D. melanogaster) | Obp59a1 and Obp59a2 | this paper | ||
| Genetic reagent (D. melanogaster) | IR40a-GAL4 | Bloomington Drosophila Stock Center | BDSC:41727 | |
| Genetic reagent (D. melanogaster) | IR25a-GAL4 | Bloomington Drosophila Stock Center | BDSC:41728 | |
| Genetic reagent (D. melanogaster) | IR93a-GAL4 | DOI: 10.1016/j. cub.2016.03.049 | FLYB:FBtp0102896 | |
| Genetic reagent (D. melanogaster) | IR68a-GAL4 | DOI: 10.7554/ eLife.26654 | ||
| Genetic reagent (D. melanogaster) | IR8a-GAL4 | Bloomington Drosophila Stock Center | BDSC:41731 | |
| Genetic reagent (D. melanogaster) | Obp59a-GAL4 | this paper | Created using 5' and 3' fragments cloned into pBGRY1. Injected into attP2 site. | |
| Genetic reagent (D. melanogaster) | attP2 | Bloomington Drosophila Stock Center | BDSC:8622 | |
| Genetic reagent (D. melanogaster) | IR8a-GAL4 | Bloomington Drosophila Stock Center | BDSC:51324 | |
| Genetic reagent (D. melanogaster) | UAS-GFP | DOI: 10.1016/S0896- 6273 (00)80701–1 | ||
| Protein | Obp59a | this paper | Novoprotein:SBP5A | Created using insect cell-optimized cDNA cloned into pFastbac1. |
| Antibody | mouse αObp59a | this paper | 1:250, mouse polyclonal. | |
| Antibody | rabbit αIR93a | DOI: 10.7554/eLife.17879 | 1:250 | |
| Antibody | guinea pig αIR25a | DOI: 10.7554/eLife.17879 | 1:250 | |
| Antibody | goat αmouse Alexa 488 | ThermoFisher | ThermoFisher:A-32723 | 1:500 |
| Antibody | goat αrabbit Alexa 488 | ThermoFisher | ThermoFisher:A-11034 | 1:500 |
| Antibody | goat αguinea pig Alexa 488 | ThermoFisher | ThermoFisher:A-11073 | 1:500 |
| Recombinant DNA reagent | pU6-BbsI-chiRNA (plasmid) | Addgene | Addgene:45946 | pBS-SK(+) vector backbone |
| Recombinant DNA reagent | pHD-DsRed-attP (plasmid) | Addgene | Addgene:51019 | pJ204 vector backbone |
| Recombinant DNA reagent | pBGRY1 (plasmid) | GenBank | GenBank:KM016698 |
Additional files
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Supplementary file 1
The Obp59a deletion.
Cut sites were located 1-nt downstream (first large capital letter) of the 5’ end of the coding region, and 71-nt downstream (last large capital letter) of the 3’ end of the coding region, thereby removing essentially all of the coding sequence (blue letters) of Obp59a. Homology arms were used to replace the coding region with a DsRed marker.
- https://doi.org/10.7554/eLife.39249.015
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Transparent reporting form
- https://doi.org/10.7554/eLife.39249.016
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Humidity response depends on the small soluble protein Obp59a in Drosophila
eLife 7:e39249.
https://doi.org/10.7554/eLife.39249
