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Figure S1: Representative TEVC recordings of sugar-induced currents derived from Xenopus oocytes expressing different Gr-ensembles. The gustatory receptors AmGr1-3 have been transiently expressed in Xenopus oocytes either alone or in different Gr combinations. Oocytes were clamped at a holding membrane potential of -80 mV and tested for sugar response to sucrose (Suc), glucose (Gluc), fructose (Fruc), maltose (Malt), arabinose (Arab), mannose (Mann), galactose (Galac), raffinose (Raffi) and melezitose (Mele) (160 mM each; perfusion indicated by bars). For quantification of sugar specificities, sugar-induced steady-state currents (ISS) were recorded at a membrane potential of -140 mV and normalized to the currents in either sucrose (A, C, D and F) or fructose (B and E) solution (bar diagrams). A AmGr1 (mean of n = 16 oocytes ± SD); B AmGr3 (mean of n = 8 oocytes ± SD); C AmGr1 and AmGr2 co-expression (mean of n = 13 oocytes ± SD); D AmGr1 and AmGr3 co-expression (mean of n = 13 oocytes ± SD); E AmGr2 and AmGr3 co-expression (mean of n = 9 oocytes ± SD); F AmGr1, AmGr2 and AmGr3 co-expression (mean of n = 10 oocytes ± SD); G AmGr2, Inset: magnification of the current trace of AmGr2-expressing oocyte for sucrose, glucose and fructose application.
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Figure S3: Fluorescence-based studies of Xenopus oocytes expressing YFP-tagged AmGr1-3 constructs. Schematic models of YFP-tagged AmGrs and AmGrs tagged with YFP halves for BiFC experiments (A and B, upper panels); Pictures show a quarter of an optical slice of an oocyte, representing an overlay of brightfield and detection of fluorescence (depicted in yellow). Images were taken with a confocal laser scanning microscope (A and B, lower panels). (A) Representative images of AmGr1 3 tagged with YFP to the N-terminus (YFP::AmGr1; YFP::AmGr2; YFP::AmGr3) transiently expressed in Xenopus oocytes. (B) Interaction studies of AmGr1 and AmGr2 by bimolecular fluorescence complementation (BiFC). N- and C-terminal YFP halves fused to either AmGr1 or AmGr2 subunits complemented YFP fluorescence when co-expressed in oocytes, indicating physical interaction, i.e. via homomerization of AmGr1 subunits (YFPN::AmGr1 + YFPC::AmGr1) or AmGr2 subunits (YFPN::AmGr2 + YFPC::AmGr2). When corresponding YFP halves were fused to AmGr1 and AmGr2 (YFPN::AmGr1 + YFPC::AmGr2 / YFPN::AmGr2 + YFPC::AmGr1) co-expression in oocytes led to yellow fluorescence (YFP complementation), indicating physical interaction of AmGr1 and AmGr2 subunits that assemble to heterotetrameric receptors. N-terminal YFP half fused to AmGr1, co-expressed with C-terminal YFP half fused to AmGr3 (YFPN::AmGr1 + YFPC::AmGr3), yields no fluorescence, suggesting that heteromer formation does not occur. When corresponding YFP halves fused to AmGr2 and AmGr3 (YFPN::AmGr2 + YFPC::AmGr3) are co-expressed, fluorescence signals can be detected, indicative of heteromerization. (Scale bar = 200 µm)
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FIGURE 1. Functional analysis of A. mellifera gustatory receptor AmGr1 using a matched heterologous expression system and in vivo comparative approach. (A1–A3): two-electrode voltage clamp measurements: Current traces were recorded at a holding potential of −80 mV in response to perfusion with sucrose (Suc), glucose (Gluc) and fructose (Fruc) in standard solution (left panel). Sugar-induced steady-state currents (I
SS
) were recorded at a membrane potential of −140 mV (middle panel). (A1) Representative whole oocyte current trace of AmGr1-expressing oocyte (left panel); Quantification of sugar-induced I
SS
. Currents were normalized to sucrose-evoked I
SS
at −140 mV (mean of n = 16 oocytes ± SD; middle panel). (A2) Control: Inward whole oocyte currents from AmGr1/AmGr2/AmGr3-expressing oocyte (wild-type mimicry; left panel); Quantification of sugar-induced I
SS
that were normalized to sucrose-evoked I
SS
at −140 mV (mean of n = 10 oocytes ± SD, middle panel). These same values are displayed in all figures as consistent control. (A3) Whole oocyte currents from AmGr2/AmGr3 co-expressing oocyte (left panel); Quantification of sugar-induced I
SS
. Currents are normalized to the fructose-evoked I
SS
at −140 mV (mean of n = 9 oocytes ± SD; middle panel). (B1–3): behavioural evaluation through proboscis extension response (PER, in vivo). Wild-type (wt/wt, N = 20) and AmGr1 mutant bees (ns/ns; N = 19) were presented a series of sugar concentrations (16%, 20%, 25%, 32%, 40%, 50% and 63% (w/v); representing .47 M, .58 M, .73 M, .93 M, 1.17 M, 1.46 M and 1.84 M) of all three sugars sucrose (B1), glucose (B2) and fructose (B3). The sum of the responses (PERs) towards the concentrations of one of the sugars was recorded as a sugar-specific GRS (gustatory response score). The distribution of all GRS values of all measured bees is shown as data points and the resulting medians as lines. AmGr1 mutants were less responsive to sucrose when compared with wild-type bees and had significantly lower GRS (B1); Mann-Whitney-U, ns/ns vs. wt/wt, p = .0032, **). Glucose responsiveness in AmGr1 mutants was significantly lower than that in wild-types (B2); Mann-Whitney-U, ns/ns vs. wt/wt, p = .0125, *). Both groups did not differ in fructose GRS (B3); Mann-Whitney-U, ns/ns vs. wt/wt, p = .0779, n.s.).
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FIGURE 2. Functional analysis of A. mellifera gustatory receptor AmGr2 using a matched heterologous expression system and in vivo comparative approach. (A1–A3): two-electrode voltage clamp measurements: Current traces were recorded at a holding potential of −80 mV in response to perfusion with sucrose (Suc), glucose (Gluc) and fructose (Fruc) in standard solution (left panel). Sugar-induced I
SS
were recorded at a membrane potential of −140 mV and normalized to the currents in sucrose solution (middle panel). (A1) Representative whole oocyte current trace of AmGr2-expressing oocyte. Inset: Magnification of the current trace reveals microscopic sustained or transient inward currents upon sugar application. (A2) Control (for clarification displayed again): The inward whole oocyte currents from AmGr1/AmGr2/AmGr3-expressing oocyte (wild type mimicry, left panel); Quantification of sugar-induced I
SS
that were normalized to sucrose-evoked I
SS
at −140 mV (mean of n = 10 oocytes ± SD; middle panel). These same values are displayed in all figures as consistent control. (A3) Whole oocyte currents from AmGr1/AmGr3 co-expressing oocyte (left panel); Currents are normalized to the sucrose-evoked I
SS
at −140 mV (mean of n = 13 oocytes ± SD; middle panel). (B1–B3): behavioural evaluation through proboscis extension response (PER, in vivo). Wild-type (wt/wt, N = 14) and AmGr2 mutant bees (ns/ns; N = 13) were presented a series of sugar concentrations (16%, 20%, 25%, 32%, 40%, 50% and 63% (w/v); representing .47 M, .58 M, .73 M, .93 M, 1.17 M, 1.46 M and 1.84 M) of all three sugars sucrose (B1), glucose (B2) and fructose (B3). The sum of the responses (PERs) towards the concentrations of one of the sugars was recorded as a sugar-specific GRS (gustatory response score) of each respective bee. The distribution of all GRS values of all measured bees is shown as data points and the resulting medians as lines. AmGr2 mutants (ns/ns) did not show any significant differences in their responsiveness towards all three sugars when compared to wild-type (wt/wt) bees, neither to sucrose (B1); Mann-Whitney-U, ns/ns vs. wt/wt, p = .5351, n.s.), to glucose (B2); Mann-Whitney-U, ns/ns vs. wt/wt, p = .0909, n.s.) or to fructose (B3); Mann-Whitney-U, ns/ns vs. wt/wt, p = .2536, n.s.).
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FIGURE 3. Functional analysis of A. mellifera gustatory receptor AmGr3 using a matched heterologous expression system and in vivo comparative approach. (A1–A3): two-electrode voltage clamp measurements: Current traces were recorded at a holding potential of −80 mV in response to perfusion with sucrose (Suc), glucose (Gluc) and fructose (Fruc) in standard solution (left panels). Sugar-induced I
SS
were recorded at a membrane potential of −140 mV (middle panels). (A1) Representative whole oocyte current trace of AmGr3-expressing oocyte (left panel); Currents are normalized to the fructose-evoked I
SS
at −140 mV (mean of n = 8 oocytes ± SD; middle panel). (A2) Control (for clarification displayed again): Inward whole oocyte currents from AmGr1/AmGr2/AmGr3-expressing oocyte (wild-type mimicry, left panel); currents were normalized to sucrose-induced I
SS
at −140 mV (mean of n = 10 oocytes ± SD; middle panel). These same values are displayed in all figures as consistent control. (A3) Whole oocyte currents from AmGr1/AmGr2 co-expressing oocyte (, left panel); currents were normalized to sucrose-induced I
SS
at −140 mV (mean of n = 13 oocytes ± SD; middle panel). (B1–B2) (as published previously in Degirmenci et al., 2020): behavioural evaluation through proboscis extension response (PER, in vivo). Wild-type (wt/wt, N = 26) and AmGr3 mutant bees (ns/ns; N = 31) were presented a series of sugar concentrations (16%, 20%, 25%, 32%, 40%, 50% and 63% (w/v); representing .47 M, .58 M, .73 M, .93 M, 1.17 M, 1.46 M and 1.84 M) of the sugars sucrose (B1) and fructose (B2). The sum of the responses (PERs) towards one of the sugars was recorded as a sugar-specific GRS (gustatory response score). The distribution of all GRS values of all measured bees is shown as data points and the resulting medians as lines. AmGr3 mutants did not show any difference in GRS when compared with wild-type bees (B1); Mann-Whitney-U, ns/ns vs. wt/wt, p = .4279, n.s.). Fructose GRS of AmGr3 mutants were significantly lower than those of wild-types (B2); Mann-Whitney-U, ns/ns vs. wt/wt, p = .0062, **). Because of experimental limitations (such as short survival of the AmGr3 mutants), glucose measurements were not implemented and cannot be pursued retrospectively.
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