Fig 1.
The relationship of Glossina morsitans to Drosophila melanogaster and its antennal sensilla.
(A) Phylogenetic tree showing the evolutionary relationship among four families within the order Diptera: Culicidae, Drosophilidae, Glossinidae, and Muscidae. Estimated divergence times are from Wiegmann et al., 2011 [4]. (B) Photograph of D. melanogaster and G. m. morsitans courtesy of Dr. Geoffrey Attardo (adapted from Sun et al. 2018 [27]). (C) Scanning electron micrograph of the antenna of G. morsitans. Arrows indicate the third antennal segment (Ant.), arista, and sensory pit (SP). Micrographs of trichoid (D, D’), basiconic (E, E’), coeloconic (F), and intermediate (G, G’) olfactory sensilla from G. morsitans. Arrows in D, E, and G indicate the sensilla that are shown at higher magnifications in the images to the right. Scale bars = 100 μm for C; 1 μm for D, E, G; 0.5 μm for D’, E’, G’; and 0.25 μm for F.
Fig 2.
The sensory pit and sacculus of the G. morsitans antenna.
(A) Scanning electron micrograph of a G. morsitans head with arrows indicating the openings of the sensory pit and sacculus. (B) Transmitted light image of an antennal cross section (coronal plane) showing the sensory pit and sacculus that open to the medial and lateral sides of the antenna, respectively. (C) Micrograph of a sectioned sensory pit with its opening to the antennal surface at the upper left. (D) Micrograph of the sensory pit, showing a top-down view of the olfactory sensilla that line the pit. (E, E’) The sensory pit is lined with basiconic Type II sensilla. The arrow in E indicates the sensillum shown in E’. (F) Dorsal chamber of the sacculus (medial is left, lateral is right) that is lined with basiconic Type III sensilla (G, G’). The arrow in G indicates the sensillum shown in G’. (H) Ventral chamber of the sacculus (medial is at left, lateral is at right) that is lined with coeloconic (I, J) and no-groove coeloconic sensilla (K). The asterisk in panel J marks an axial view of a coeloconic sensillum that had its tip cut off during cryosectioning, fortuitously revealing its inner structure. Scale bars = 200 μm for A; 5 μm for C, D, F and H; 1 μm for E, G; and 0.25 μm for E’, G’, I, J, and K.
Fig 3.
Expression of Odorant binding protein genes.
(A, B) In situ hybridization to GmmObp76a in (A) 40 μm and (B) 10 μm antennal cross sections. Arrow in panel B marks a trichoid sensillum in the same plane as a cell body marked by GmmObp76a. (C, D) GmmObp6 in (C) 40 μm and (D) 10 μm antennal cross sections. Arrow in D marks a basiconic sensillum in the same plane as a cell body marked by GmmObp6. (E, F) GmmObp84a in (E) 40 μm and (F) 10 μm antennal cross sections. Arrow in F marks a coeloconic sensillum in the ventral chamber of the sacculus in the same plane as a cell body marked by GmmObp84a. (G, H) GmmObp59a (red) in (G) 40 μm and (H) 10 μm antennal cross sections. Arrow in H marks a coeloconic sensillum in the same plane as a cell body marked by GmmObp84a.
Fig 4.
Expression of GmmOrco in the antenna.
(A) In situ hybridization to GmmOrco (red). (B) Immunostaining of pan-neuronal marker Elav (green). (C) Merged image of GmmOrco and anti-E lav. (D) Magnification of (C).
Fig 5.
Expression of GmmOrs in the antenna.
For all images dorsal is up and medial is to left. (A) GmmOrco. (B) GmmOr9. (C) GmmOr6. (D) GmmOr40. (E) GmmOr35. Some background fluorescence is visible that does not emanate from cell bodies; see sense strand control in S1H Fig. (F) GmmOr38. (G) GmmOr19. (H) GmmOr44. (I) GmmOr29. We note that in several of these images there is background fluorescence that emanates from the cuticle, which is thicker in the antenna of G. morsitans than in D. melanogaster. See S1 Fig for sense strand controls.
Fig 6.
GmmOr6 and GmmOr9 are transcribed in adjacent ORNs that encapsulate the sensory pit.
(A-D) In situ hybridization to GmmOr6 (red) combined with anti-Elav immunostaining (green). (A) GmmOr6, (B) anti-Elav, and (C) merged image of the whole antenna. (D) Magnified merged image of the sensory pit without DIC. The circular structure in the center of the image that is uniformly green is autofluorescent cuticle of the pit. Likewise, the green fluorescence in the upper right corner is cuticular autofluorescence from the sacculus. (E-H) In situ hybridization to GmmOr9 (red) combined with anti-Elav immunostaining (green). (E) GmmOr9, (F) anti-Elav, and (G) merged image of the whole antenna. (H) Magnified merged image of the sensory pit without DIC. (I-L) Double in situ hybridization to GmmOr6 and GmmOr9 (both red) combined with anti-Elav immunostaining (green). (I) GmmOr6 and GmmOr9, (J) anti-Elav, and (K) merged image of the whole antenna. (L) Magnified merged image of the sensory pit without DIC.
Fig 7.
CRISPR/Cas9 knock-in of the Gal4 gene into the Or22a and Or22b coding regions.
(A) Genomic region of Or22a and Or22b before and after it is targeted for CRISPR/Cas9-mediated homology-directed repair to knock-in the Gal4 transcription factor and DsRed eye marker genes. The formal designation of this stock is Df(2L)Or22ab, TI{GAL4}Or22ab but for convenience it is indicated as Or22abGAL4. (B) GFP signal from antennal cross sections from D. melanogaster carrying the Gal4 knock-in (Or22abGAL4), the UAS-mcd8:GFP transgene on the second chromosome, or both. (C, D) Odorant response profiles from (C) wild type control ab3A and (D) homozygous Or22abGAL4 ab3A ORNs. (E, F) Odorant response profiles from D. melanogaster UAS-Or transgenes previously shown [15] to confer strong responses to E2-hexenal (Or7a) (E) or methyl salicylate (Or10a) (F), expressed in homozygous Or22abGAL4 ab3A olfactory receptor neurons. In panels C-F neuronal responses are reported in spikes/second +/- S.E.M., using individual odorants diluted at 10−2 and presented for 0.5 seconds. n≥5 for all odorants tested. Spontaneous firing frequencies have been subtracted from all responses; responses to the paraffin oil diluent have been subtracted from the responses to all odorants.
Fig 8.
GmmOr35 responds strongly to 1-hexen-3-ol and moderately to alpha-pinene.
(A) Odorant response profile of GmmOr35 expressed in homozygous Or22abGAL4 ab3A ORNs. Neuronal responses are reported in spikes/second +/- S.E.M., to individual odorants diluted 10−2. (B,C) Sample traces from ab3A empty neurons expressing the GmmOr35 transgene in response to the paraffin oil diluent (B) and 1-hexen-3-ol (C), each presented for 0.5 seconds (black bars). (D) Dose response curve for GmmOr35 and 1-hexen-3-ol. (E) Response profile of GmmOr19. In panels A, D, E, spontaneous firing frequencies have been subtracted from all responses; responses to the paraffin oil diluent have been subtracted from the responses to all odorants. n≥5 for all stimuli tested.
Fig 9.
GmmOr9 responds strongly to 2-propanol, 2-butanone, and acetone.
(A) Odorant response profile of GmmOr9 expressed in homozygous Or22abGAL4 ab3A. (B-E) Sample traces. Black bars represent 0.5 second stimuli. (F) Dose response curves for GmmOr9. Error bars indicate +/- S.E.M. n≥5 for all stimuli tested.
Fig 10.
Responses of tsetse to acetone, 2-butanone, and 2-propanol.
A) Scanning electron micrograph of the G. morsitans antenna. The white square is centered on the opening of the sensory pit. B) Magnified image of the opening to the sensory pit. The arrow indicates the approximate position of a recording electrode, which is inserted through the opening of the sensory pit in order to pierce a basiconic Type II sensillum for electrophysiological recording. Scale bar is 5 μm. (C) Odorant response profile of sensory pit B neurons. Odorants were diluted 10−2 in paraffin oil. n≥5 for all stimuli tested. (D,E) Sample traces from sensory pit sensilla in response to paraffin oil diluent (D) and 2-propanol (E) presented for 0.5 seconds (black bars). The B neuron, represented by the smaller spikes, shows a weak response to paraffin oil, a response that is also observed in some Drosophila neurons [46], and a strong response to 2-propanol. The A neuron, represented by large spikes, may be inhibited by 2-propanol. (F) The T-maze paradigm. (G) Behavioral responses of G. morsitans. Means were compared using one-way ANOVA, followed by Tukey’s test for pairwise comparison against paraffin oil for all odorants. **, p<0.01. n = 10 replicates for all odorants; n = 5 for the paraffin oil diluent. (H) Responses of G. fuscipes. t-test; p<0.05. n = 14 replicates.