The main PMRs in females can be separated.

A, B) Schematic depiction of head and trunk expression in Drosophila elav FRTstopFRT GAL4; otdflp (A) and in tshGAL4 (B) visualized by UAS GFP (green).

C, D) Receptivity (C) and oviposition (D) of wild type control virgin (red) and mated (orange) females, and virgin females expressing UAS mSP (green) pan-neuronally with nsybGAL4 or in head and trunk patterns shown as means with standard error from three repeats for receptivity (21 females per repeat) by counting the number of females mating within a 1 h period or for oviposition by counting the eggs laid within 18 hours from 30 females. Statistically significant differences from ANOVA post-hoc comparison are indicated by different letters (p<0.0001).

E-H) Representative adult female genital tract showing tshGAL4 UAS H2BYFP (green) and elavLexA LexAop NLStomato (red) nuclear expression. The magnification (F-H) shows sensory genital tract neurons. Scale bar shown in E and H are 100 μm and 20 μm, respectively.

Distinct regulatory regions in SPR, fru and dsx genes induce PMRs from mSP expression.

A-C) Schematic representation of SPR, fru, and dsx chromosomal regions depicting coding and non-coding exons as black or white boxes, respectively, and splicing patterns in solid lines. Vertical lines below the gene model depict enhancer GAL4 lines with names and those in red showed PMRs by expression of mSP.

D, E) Receptivity (D) and oviposition (E) of wild type control virgin (red) and mated (orange) females, and virgin females expressing UAS mSP (green) under the control of GAL4 pan-neuronally in nsyb or in SPR8, SPR12, fru11, fru12, and dsx24 patterns shown as means with standard error from three repeats for receptivity (21 females per repeat) by counting the number of females mating within a 1 h period or for oviposition by counting the eggs laid within 18 hours from 30 females. Statistically significant differences from ANOVA post-hoc comparison are indicated by different letters (p≤0.0001).

F-O) Representative adult female brains (F-J) and ventral nerve cords (VNC, K-O) expressing UAS CD8GFP under the control of SPR8, SPR12, fru11, fru12 and dsx24 GAL4. Scale bars shown in J and O are 50 µm and 100 µm, respectively.

Expression of mSP in secondary ascending abdominal ganglion neurons induces PMRs.

A, B) Receptivity (A) and oviposition (B) of wild type control virgin (red) and mated (orange) females, and virgin females expressing UAS mSP (green) under the control of GAL4 pan-neuronally in nsyb or in FD1, FD2, FD3, FD4, FD5, and FD6 patterns shown as means with standard error from three repeats for receptivity (21 females per repeat) by counting the number of females mating within a 1 h period or for oviposition by counting the eggs laid within 18 hours from 30 females. Statistically significant differences from ANOVA post-hoc comparison are indicated by different letters (p<0.0001).

Distinct circuits from intersection of SPR, fru, dsx and FD6 patterns in the brain and VNC induce PMRs from mSP expression.

A) Schematic showing the intersectional gene expression approach: GAL4 activation (AD, orange) and DNA binding domains (DBD, blue) are expressed in different, but overlapping patterns. Leucine zipper dimerization reconstitutes a functional split-GAL4 in the intersection (pink) to express UAS reporters.

B, C) Receptivity (B) and oviposition (C) of wild type control virgin (red) and mated (orange) females, and virgin females expressing UAS mSP (green) under the control of split-GAL4 intersecting SPR8 ∩ fru11/12, SPR8 ∩ dsx, SPR8 ∩ FD6, fru11/12 ∩ dsx and fru11/12 ∩ FD6 patterns shown as means with standard error from three repeats for receptivity (21 females per repeat) by counting the number of females mating within a 1 h period or for oviposition by counting the eggs laid within 18 hours from 30 females. Statistically significant differences from ANOVA post-hoc comparison are indicated by different letters (p<0.0001).

D-M) Representative adult female brains and ventral nerve cords (VNC) expressing UAS CD8GFP under the control of SPR8 ∩ fru11/12, SPR8 ∩ dsx, SPR8 ∩ FD6, fru11/12 ∩ dsx and fru11/12 ∩ FD6. Scale bars shown in H and M are 50 µm and 100 µm, respectively.

Distinct neuronal circuitries from intersection of SPR, fru and dsx sense SP after mating to induce PMRs.

A, B) Receptivity (A) and oviposition (B) of wild type control virgin (red) and mated (orange) females, and virgin females expressing UAS mSP (green) under the control of split-Gal4 intersecting SPR8 ∩ dsx, fru11/12 ∩ dsx, and SPR8 ∩ fru11/12 patterns in SPR/Df mutant females or SPR RNAi knock-down shown as means with standard error from three repeats for receptivity (21 females per repeat) by counting the number of females mating within a 1 h period or for oviposition by counting the eggs laid within 18 hours from 30 females. Statistically significant differences from ANOVA post-hoc comparison are indicated by different letters (p<0.0001 except p=0.002 and p=0.006 for c and d in A, and p=0.004 for c in B).

Distinct neuronal circuitries in the brain senses SP to induce PMRs.

A, B) Schematic depiction of UAS GFP (green) expression in the head of Drosophila (A) combining split-GAL4 intersectional expression (AD-GAL4 and GAL4-DBD) with brain-expressed otdflp mediated recombination of UAS FRTGFPstopFRTmSP (B).

C, D) Receptivity (C) and oviposition (D) of wild type control virgin (red) and mated (orange) females, and virgin females expressing UAS FRTGFPstopFRTmSP (grey), UAS FRTGFPstopFRTTrpA1 (purple) and UAS FRTGFPstopFRTTNT (pink) under the control of split-GAL4 intersecting SPR8 ∩ dsx, fru11/12 ∩ dsx and SPR8 ∩ fru11/12 patterns with brain-specific FRT-mediated recombination by otdflp shown as means with standard error from three repeats for receptivity (21 females per repeat) by counting the number of females mating within a 1 h period or for oviposition by counting the eggs laid within 18 hours from 30 females. Statistically significant differences from ANOVA post-hoc comparison are indicated by different letters (p<0.0001 except p<0.0004 for c in C, p<0.007 for c in D).

retro- and trans-Tango identification of pre- and post-synaptic neurons of SP target neurons reveals higher order neuronal input canalized into shared output circuitries.

A-O) Representative adult female brains expressing QUAST tomato3xHA retro-Tango (left, A-E), UAS myrGFP (middle, F-J) and QUAST tomato3xHA trans-Tango (right, K-O) in SPR8 ∩ dsx, fru11/12 ∩ dsx, SPR8 ∩ fru11/12, SPR8 ∩ FD6 and fru11/12 ∩ FD6 split-GAL4s. The presynaptic (A-E, left), split-GAL4 (F-J, middle) and postsynaptic (K-O, right) neuronal circuitries are shown in an inverted grey background. Arrows (magenta) indicate neurons and their corresponding projections in different regions in female brain. The scale bar shown in O is 50 μm.

P) Model for the SP induced post-mating response. SP interferes with interpretation of sensory cues, e.g. vison, hearing, smell, taste, and touch at distinct sites in the brain indicated by higher order projections revealed by intersectional expression in the following patterns: SPR8 ∩ dsx (blue), fru11/12 ∩ dsx (black), SPR8 ∩ fru11/12 (yellow), SPR8 ∩ FD6 (pink) and fru11/12 ∩ FD6 (olive). and VNC (fru11/12 ∩ dsx) during higher order neuronal processing.