Divergent evolutionary pressures shape olfactory sensitivity of the maxillary palps in Tephritidae fruit flies

GC- EPD | maxillary palps | olfactory sensitivity | tephritids

Olfaction is a rapidly evolving sense. Given its diverse functions, from finding ecological niches to selection of mates, we hy - pothesized that olfaction is subjected to divergent evolutionary pressures. We compared the olfactory sensitivity of five species of Tephritidae fruit flies to two broad classes of volatiles: general niche- related volatiles (food and fruit odors) and volatiles used in sexual communication (pheromones and “parapheromones”). We then analyzed whether the differential sensitivities across species harbor “signals” of such contrasting evolutionary pressures. As recent studies highlight the maxillary palps as key aux - iliary olfactory organs for detecting both classes of volatiles, we focused our sensory analysis on this auxiliary olfactory organ. Using gas chromatography coupled to electropalpographic detection (GC- EPD), we recorded sensory responses from five species with a diverse phylogenetic and ecological separation. Detection overlapped considerably across taxa; however, the maxillary palp exhibited distinct sex and clade- specific patterns in sensitivity to pheromones and parapheromones. Cluster analysis of sen- sitivities to (para)pheromones aligned strongly with the species' phylogeny. In contrast, cluster analysis of sensitivities to general food and fruit odors clustered separately and showed a strong correlation with ecological niche rather than phylogeny. Clearly, the selection pressures that shape the evolutionary direction of olfactory sensitivity to (para)pheromones and niche- related odors are diametrical opposites, reminiscent of stabilizing versus directional selection. Understanding the detection and evolution of distinct volatile classes provides valuable insights into the evolutionary ecology of olfaction, studies on olfactory receptors, and sensory and preference coding, and supports the rational development of novel lures to manage these pest insects. 1 | Introduction Insets, which account for over 80% of all described species, represent the most diverse group of organisms (Stork  2018). Their short generation time and small size allow insects to rapidly invade new niches. Their evolutionary adaptability is paralleled by an equally rapidly evolving sense of smell, which detects key environmental cues, such as food sources and po - tential mates. This is for instance reflected at the protein level in olfactory receptors (ORs), with radiation events generally This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2025 The Author(s). Ecology and Evolution published by British Ecological Society and John Wiley & Sons Ltd. Chaymae Fennine and Sebastian Larsson Herrera shared first authorship. Wittko Francke passed away on 27 December 2020. 2 of 13 Ecology and Evolution, 2025 reflected in OR diversification (Missbach et al.  2014). However, whether the diversification of OR sequences equals functional radiation is largely unknown, partially due to the backlog of functional characterization of ORs. Similarly, it is largely un - known whether the evolutionary pressures acting on ORs are the same, irrespective of their function. It is, however, con - ceivable that the selection pressures acting on the detection of for example, food or oviposition site volatiles differ from the detection of sexual communication signals. We addressed this question by dissecting the odor sensitivity of the maxil - lary palps of tephritid fruit flies, which is sensitive to both fruit and food odors on the one hand, and sexual signals on the other. In insects, the antennae serve as the primary olfactory or - gans, but many species also possess auxiliary olfactory struc - tures such as the maxillary palps. Olfactory sensilla can also occur in other anatomical regions, including the ovipositor of Manduca sexta , where they detect odorants and may play a role in reproductive behaviors (Klinner et  al.  2016), and the labial pit of certain adult Lepidoptera, which houses CO 2- sensitive sensilla (Kent et al.  1986). The maxillary palps, located on the proximal part of the mouthparts, the labium, house a limited number of sensory neuron types compared to the antennae. In Drosophila, the maxillary palps contain only one morphological type of olfactory sensillum, the basiconic sensillum, with three subtypes, each harboring two olfactory sensory neuron (OSN) types (de Bruyne et  al.  1999; Dweck et  al.  2016). Similarly, in Tephritidae, the maxillary palps express three sensilla basicon - ica subtypes, each harboring two sensory neurons (Larsson- Herrera et al.  2024). Due to the proximity of feeding substrates, maxillary palps have been hypothesized to play a dispropor - tionate role in detecting food- related odors, complementing the broader olfactory functions of the antennae. Whereas this was not evident in Drosophila , in Tephritidae, the palps exhibited a preferential sensitivity to food odors over fruit odors (Larsson- Herrera et al. 2024 ). In addition to being sensitive to fruit and food odors, the palps in Tephritidae are also sensitive to spiroacetals (Noushini, Park, et al.  2020; Noushini, Perez, et al.  2020; Noushini et al.  2021). This group of compounds is produced in the rectal gland of Bactrocera species and, as they mediate sexual behaviors, are recognized pheromones of Tephritidae (Haniotakis  1974; Baker et al.  1980; Zhang et al.  1997; Booth et al.  2007; Noushini, Park, et al.  2020; Noushini, Perez, et al.  2020; Noushini et al.  2021). They have, however, not been reported from Ceratitis spp. Whereas spiroacetals are detected by the maxillary palps of Bactrocera bryoniae, B . kraussi , B. frauenfeldi , and B. oleae (Noushini, Park, et  al.  2020; Noushini, Perez, et  al.  2020; Noushini et  al.  2021), little is known about the detection in other species. Given the variety of spiroacetals and differences between sexes (Booth et al.  2009), comparative sensory studies would be helpful in understanding how these pheromones are detected, how their detection has evolved, and how olfactory input translates into behavior. Another group of compounds to which the palps are sensitive is phenylpropanoids (Chieng et al.  2018; Park et al.  2018; Verschut et al.  2018). Phenylpropanoids are of plant origin and typically attract males. Given their strong and sex- biased attractiveness, they are often referred to as parapheromones. Pheromones and parapheromones are extensively used in the monitoring and control of several Tephritidae species. Methyl eugenol (ME) is used for mass trapping, attract- and- kill, and monitoring of spe - cies; for example, B. dorsalis, raspberry ketone (RK), and its syn- thetic analog cuelure (CL) are employed for monitoring species, such as Z. cucurbitae (Clarke  2019; Biasazin et al.  2021). Ceratitis species do not appear to be attracted to phenylpropanoids, but two other male lures, terpinyl acetate and the synthetic chlori - nated trimedlure, fulfill a similar role for the monitoring and control of C. cosyra and C. capitata , respectively. Beyond de - tection, the palps are also essential for the orientation toward parapheromones in several tephritid species, as evidenced by ablation experiments in B. dorsalis (Chieng et al. 2018 ). In this study, we evaluated the responses of the maxillary palps of males and females from phylogenetically and ecologically diverse tephritid species using coupled gas chromatography electropalpographic detection (GC- EPD). We investigated the olfactory sensitivities of the maxillary palps in multiple tephritid species. These included B. dorsalis (Hendel), a polyphagous and severe horticultural pest that releases the spiroacetal (2 E,8E)- 2- ethyl- 8me- 1,7- dioxaspiro[5.5]undecane (Perkins et al.  1990) and is attracted to methyl eugenol (Kawano et al.  1968); Bactrocera latifrons (Hendel), a solanaceous specialist whose males are at - tracted to isophorone (Ishida et al.  2008) and produces the same spiroacetal as B. dorsalis , as well as (2S,6R,8S)- 2,8- dimethyl- 1,7- dioxaspiro[5.5]undecane (Zhang et  al.  1997); and the cucurbit specialist Zeugodacus cucurbitae (Coquillet), which is attracted to RK/CL (Kawashita et al.  2004) and is a known producer of spiroacetal (2 E,8E)/(2Z,8E)- 2- ethyl- 8me- 1,7- dioxaspiro[5.5]un- decane (Baker and Bacon  1985). We also included two polypha - gous species of Ceratitis: C. capitata (Wiedemann) and C. cosyra (Walker), which are attracted to trimedlure (Beroza et al.  1961) and terpinyl acetate (White and Elson- Harris  1992), respec - tively. However, neither of these Ceratitis species have been observed to produce spiroacetals. The list of stimuli further in - cluded compounds such as phenylpropanoids, esters, pyrazines, and phenols, which were selected based on literature (Biasazin et al.  2018; Chieng et al.  2018; Segura et al.  2018; Ono et al.  2021; Larsson- Herrera et al.  2024). Using synthetic compounds in con- junction with GC- EPD also removes false positives from impu - rities that may arise when employing non- GC approaches such as SSR and EAG/EPG with puffing (Schorkopf et al.  2019). The data provide insights into palpal detection and its evolutionary role in mediating sexually divergent behavioral responses to specific compounds. Finally, since these compounds are import- ant in pest control, understanding how they are detected may provide valuable insights for their use in pest management. 2 | Material and Methods 2.1 | Insects Lab colonies of the fly species were established from pupae ob - tained from the International Center of Insect Physiology and Ecology ( icipe, Kenya; B. dorsalis , B. latifrons , C. capitata , C. cosyra), and the International Atomic Energy Agency (IAEA, Vienna, Austria; Z. cucurbitae ). Emerging adult flies were kept in polyester netting Bugdorm cages (325 × 325 × 325 mm3) under 20457758, 2025, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ece3.72261 by Swedish University Of Agricultural Sciences, Wiley Online Library on [30/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3 of 13Ecology and Evolution, 2025 controlled conditions (25°C, 60% ± 5% RH and 12:12 LD), and provided with food (sugar and baker's yeast (Jästbolaget AB, Sollentuna, Sweden) mix, 3:1) and water (wet cotton). 2.2 | Chemicals Authentic chemical standards (> 95% purity) of compounds are summarized in Table  S1. Most of the compounds were pur - chased from Sigma, Aldrich, St. Louis, MO, USA, whereas others were from various sources and are available at the local chem - ical library of the Swedish University of Agricultural Sciences (SLU), Alnarp. The spiroacetals (except olean, provided by ISCA Technologies) were synthesized by Professor Wittcko Francke (Table  1). 2.3 | Synthetic Blends Three blends were constructed of a total of 40 synthetic com - pounds (Table  1). Some of the compounds, such as spiroacetals, male lures, and others, were selected based on literature data from tephritids and drosophilids. Others were either known from our own unpublished work to elicit a response or shared similarities in their structure to either male lures or the reported spiroacetals. Additional compounds from fruit and fermentation TABLE 1 | Spiroacetals and male lures tested on the maxillary palps of five tephritids ( Bactrocera latifrons , Bactrocera dorsalis , Zeugodacus cucurbitae, Ceratitis capitata , and Ceratitis cosyra ). Class Trivial name IUPAC name Structure Spiroacetals Spiro A (2R)- 2- methyl- 1,7- dioxaspiro[5.5]undecane Spiro B 2- ethyl- 1,7- dioxaspiro[5.5] undecane (racemate) Spiro C (2S,6R,8S)- 2,8- dimethyl- 1,7- dioxaspiro[5.5] undecane Spiro D (2S,6S,8R)- 2,8- dimethyl- 1,7- dioxaspiro[5.5] undecane Spiro E 2- ethyl- 8- methyl- 1,7- dioxaspiro[5.5] undecane (racemate, fraction 1) Spiro F 2- ethyl- 8- methyl- 1,7- dioxaspiro[5.5] undecane (racemate, fraction 2) Spiro G 2- ethyl- 8- methyl- 1,7- dioxaspiro[5.5] undecane (racemate, fraction 3) Olean 1,7- dioxaspiro[5.5] undecane (racemate) Phenylpropanoids Methyl eugenol 1,2- dimethoxy- 4- prop- 2- enylbenzene Raspberry ketone 4- (4- hydroxyphenyl) butan- 2- one Cuelure [4- (3- oxobutyl)phenyl] acetate Zingerone 4- (4- hydroxy- 3- methoxyphenyl)butan- 2- one Terpenoids Trimedlure tert- butyl 4- chloro- 2- methylcyclohexane- 1- carboxylate Terpinyl acetate 2- (4- methylcyclohex- 3- en- 1- yl)propan- 2- yl acetate 20457758, 2025, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ece3.72261 by Swedish University Of Agricultural Sciences, Wiley Online Library on [30/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 of 13 Ecology and Evolution, 2025 that were observed to give robust responses in maxillary palps of tephritid species were also included to anchor the observations in previous work (Larsson- Herrera et al.  2024). The blends were thus diverse and contained esters, terpenoids, spiroacetals, pyr - azines, phenols, and phenylpropanoids (Table  S1). Chemical standards were analyzed prior to being combined into blends and injected at both 100 ng/μl and 10 ng/μl using a GC–MS (Agilent 6890 GC and 5975 MS, Agilent Technologies Inc., Santa Clara, CA, USA), using a polar DB- WAX column of 60 m × 0.25 mm × 0.25 μm film thickness, with helium as carrier gas. Injection was in splitless mode at 250°C. The oven tempera- ture program was as follows: 50°C for 1.5 min, ramping at 7°C/ min to 250°C, hold for 5 min. The lower concentration was used to separate compounds, and the higher concentration was used to parse out responses to synthetics from impurities. Major im - purities were tentatively identified, and main compounds were confirmed using the NIST 20 library in masshunter and NIST MS search v. 2.4 as well as published Kovats retention indices. Several synthetic compounds, such as farnesene, were excluded at this step and did not form part of the 40 final compounds due to containing large amounts of impurities. Three blends were constructed, assuring non- overlapping peaks of the synthetics, and again injected into the GC–MS for verification. An aliphatic alkane solution of C7- C30 was also injected to calculate Kovats retention indices. 2.4 | Electrophysiological Experiments Gas chromatography (GC) (Agilent Technologies 6890 GC (Santa Clara, CA, USA)) coupled with a flame ionization detector (FID) and an electropalpographic detector (EPD) was used to record olfactory responses from the palps of the five tephritid species. For the recording, the insect was immobilized in a 200 μL mi - cropipette tip with the palps exposed. Glass capillary electrodes filled with Beadle- Ephrussi Ringer's solution (7.5 g NaCl, 0.35 g KCl, 0.29 g CaCl2 dissolved in 1 L of distilled water) were used to record signals from the distal position of the palp against a

electrode on the head. Three blends were tested at 10 ng/μL, and signals were acquired using GC- EAD 2014 soft - ware (V.1.2.3, Syntech, Kirchzarten, Germany). The GC was equipped with a DB- WAX column (30 m × 0.25 mm × 0.25 μm, same method described above for the GC–MS), with hydrogen as the carrier gas. The effluent was split equally (1:1) between the FID and the EPD. Males and females of 10–20 days old were subjected to the three blends, with 2 μL injected per trial, and recordings across the blends were to a large extent performed on the same individual. For each blend, three to seven recordings were used, depending on the quality of recording throughout the run. GC- EPD active peaks were confirmed by comparing Kovats retention indices from the GC- EPD with GC–MS and published literature. 2.5 | Analysis Electrophysiological data were annotated using GC- EAD soft - ware and exported as CSV files. The data were then added to Google Sheets, with one workbook per mix and one work - sheet per species. The data were read into R (v. 4.2.2) using the package “googledrive” (D'Agostino McGowan and Bryan  2020). EPD responses were analyzed as relative values to account for inter- individual and inter- species differences in electrical prop - erties. Raw mV readings were log- transformed, and a baseline was computed for each experimental group. Absolute values were scaled to this baseline and further normalized by divid - ing by the mean scaled response, yielding a final relative metric. Species differences for each compound were tested using one- way ANOVA, followed by post hoc Tukey's HSD tests where appropriate. Sex differences within each species–compound combination were evaluated using independent- samples t- tests. All p- values were adjusted for multiple comparisons using the Benjamini–Hochberg method. Compounds were resolved against PubChem using webchem (Szöcs et al.  2020) and quality controlled for annotation discrepancies. Unknown compounds were given the name “unknown” plus their retention index. The biosynthetic pathway for each compound was resolved using SMILES through the API of NPClassifier (Kim et  al.  2021), with the exception of spiroacetals, which were manually la - beled. Linear models were constructed for each of the three major pathways of esters, shikimates, and phenylpropanoids, and spiroacetals across all pairwise combinations of insect species. Dendrograms of responses were constructed using a Jaccard dissimilarity index from the package “vegan” (Oksanen et al.  2022). A phylogenetic analysis was constructed by concate- nating the 16 s and COI gene sequences, using data from Virgilio et al. ( 2015), with Acanthiophilus helianthi as an outgroup; se - quences were aligned using MAFFT (Katoh and Standley  2013), and a consensus tree was constructed using “iqtree2” (Minh et al.  2020). All dendrograms were plotted using Yu et al. ( 2017) “ggtree” (), and all other plots and data manipulation were per - formed with “tidyverse” package (Wickham et al. 2019 ). 3 | Results The maxillary palps of B. latifrons , B. dorsalis , Z. cucurbitae , C. capitata, and C. cosyra detected 32 out of the 40 synthetic com - pounds in the synthetic mixes and consistently responded to an additional 36 impurity compounds, of which 34 could not be reliably identified (Figure  1, Figure  S1). Compounds were gen - erally detected by both sexes, with a detection overlap ranging from 87.5% in B. latifrons to 68% in Z. cucurbitae . Responses to compounds detected by only one sex were consistently weaker (< 0.2 mV). In all species, the three compounds that elicited the strongest re- sponses were either spiroacetals or phenylpropanoids. However, these differed between species: for B. dorsalis , the strongest re - sponses were elicited by spiro D, cue- lure, and olean, whereas in B. latifrons by cue- lure, zingerone, and raspberry ketone, and in Z. cucurbitae by spiro C, spiro A, and cue- lure. Both Ceratitis species responded most strongly to methyl eugenol and 4- ethyl guaiacol, followed by olean and dihydro methyl eugenol, an im - purity, for C. capitata and C. cosyra , respectively. All species detected the fatty acyl heptan- 2- one, and all, except B. latifrons , detected 1- octen- 3- ol. While Z. cucurbitae detected three fatty esters, all other species detected six. Pyrazines (2,3 and 2,5- dimethylpyrazines) were only detected by B. dorsalis , C. capitata , and C. cosyra . B. dorsalis and C. capitata detected 20457758, 2025, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ece3.72261 by Swedish University Of Agricultural Sciences, Wiley Online Library on [30/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 5 of 13 Ecology and Evolution, 2025 the most compounds from the shikimates and phenylpropanoids pathway, with 11 and 10 compounds, respectively, while all the other species detected 9. Only the two Bactrocera species de - tected caryophyllene, while Z. cucurbitae and both Ceratitis spp. detected isophorone. With the exception of Z. cucurbitae , all species detected beta- myrcene. In contrast, the following com - pounds were not detected by the palps of any species: ( E)- 4,8- dimethyl- 1,3,7- nonatriene (DMNT), 2- phenethyl propionate, 2- methylpropyl 3- methylbutanoate, 3- hydroxy- 2- butanone (acetoin), limonene, trimedlure, alpha- terpinyl acetate, and beta- ocimene. 3.1 | Sensitivity to Spiroacetals The five species showed both overlapping and differential palpal responses toward the eight spiroacetals in the panel (Figure  2). The two simplest forms of spiroacetals, olean and spiro A, were detected by all species and both sexes. Ceratitis cosyra detected the least number of spiros, only olean and spiro A. Zeugodacus cucurbitae, on the other hand, detected all spiroacetals except spiro F. B. dorsalis and B. latifrons detected the same six spiro - acetals, albeit with a differential response strength. Notably, spiro B elicited weak responses (< 0.25 mV) across all species, except C. cosyra , which did not detect this compound. Some spiroacetals were only detected by a few species, such as spiro C, which was detected exclusively by Z. cucurbitae and C. cap - itata, evoking strong (> 1 mV) and weak (< 0.25 mV) responses, respectively. In addition, the two Bactrocera species responded most strongly to spiro D, followed by olean. Spiro D and spiro G were detected by Z. cucurbitae , B. latifrons , and B. dorsalis . While Spiro F was detected solely by the two Bactrocera species, only Z. cucurbitae detected spiro E. Spiro E, spiro F, and spiro G are chiral isomers of 2- ethyl- 8- methyl- 1,7- dioxaspiro[5.5]un- decane racemate, for which the chirality could not be resolved. 3.2 | Olfactory Sensitivities to Phenylpropanoids (Male Lures) Significant interspecific differences in EPD responses were de - tected for some male- lure compounds (ANOVA: methyl eugenol, F(3,22) = 3.75, p = 0.026; zingerone, F(4,29) = 6.58, p = 0.0007), but no sex- based differences for any species- compound combi- nation (all p_adj ≥ 0.919). For ME, post hoc tests indicated that Z. cucurbitae responded significantly less than C. cosyra ( p_ adj = 0.019), with non- significant trends toward lower responses compared to B. dorsalis ( p = 0.305) and C. capitata ( p = 0.880). Consistent with this, the three species with strong ME responses (B. dorsalis , C. capitata , and C. cosyra ) were also sensitive to FIGURE 1 | Heatmap of maxillary palp olfactory sensitivities in Bactrocera dorsalis , Bactrocera latifrons , Zeugodacus cucurbitae , Ceratitis cap - itata and Ceratitis cosyra to synthetic compounds and two impurities (indicated with an *). From left to right: a) synthetic compounds of the three blends, b) their functional classes, c) olfactory sensitivities of each species to chemical compounds, d) chemical groups used for compound classifica - tion, and e) the normalized sensitivity of the fly responses ranging from light blue (0) to red (> 1 mv). The compounds are sorted from top to bottom in decreasing order of sharedness across tephritids species and within each cluster of males and females of a single species. 20457758, 2025, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ece3.72261 by Swedish University Of Agricultural Sciences, Wiley Online Library on [30/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 of 13 Ecology and Evolution, 2025 similar compounds such as eugenol and the impurity dihydro methyleugenol, whereas Z. cucurbitae and B. latifrons did not detect these ( Figure 3). In contrast, zingerone was detected by all species, though with significant variation in sensitivity. B. dorsalis and B. latifrons responded more strongly than Z. cucur- bitae and C. cosyra (all p _adj 0.17), which were only detected by the two Bactrocera spe- cies and Z. cucurbitae. Trimedlure and terpinyl acetate were not detected by any species. Moreover, only Z. cucurbitae and the two species of Ceratitis detected isophorone. 3.3 | Correlation Between Tephritids Palpal Olfactory Sensitivities to Spiroacetals, Phenylpropanoids and Esters Comparison between species showed that receptive ranges and strength of palpal responses were significantly correlated be - tween B. dorsalis and B. latifrons for esters ( p < 0.01, R2 = 0.86), phenylpropanoids ( p < 0.01, R2 = 0.52), and spiroacetals (p < 0.01, R2 = 0.84, Figure  4). In a similar way, C. capitata and C. cosyra were also significantly correlated for phenylpropanoids (p < 0.001, R2 = 0.83) and spiroacetals ( p < 0.001, R2 = 0.95). While many correlations were found between B. latifrons /B. dorsalis and C. cosyra/C. capitata for esters and spiroacetals, no correlation was observed between Z. cucurbitae and C. cosyra/C. capitata. Finally, a consensus tree of EPD responses of the five species to pheromones (spiroacetals) and parapheromones (male lures) using a dissimilarity index (Jaccard) closely aligned with a consensus phylogenetic tree derived from mitochondrial (COI) and ribosomal (16S) DNA, whereas EPD responses of the five species to general odors (which were primarily fatty acid and terpenoid derivatives) clustered separately, and this grouping correlated with their ecological niches (Figure 5 ). 4 | Discussion The maxillary palps of Tephritidae are increasingly studied, from OSN morphology (Zhang et  al.  2011; Chieng et  al.  2018; Liu et  al.  2020; Larsson- Herrera et  al.  2024) to functional studies using EPGs and EPDs (Chieng et  al.  2018; Verschut et al.  2018; Oh et al.  2019; Biswas et al.  2020; Noushini, Park, et  al.  2020; Noushini, Perez, et  al.  2020; Noushini et  al.  2021; Larsson- Herrera et al.  2024, this study). This growing attention is particularly due to the finding that the palps of tephritids mediate detection and attraction to pheromones and parapher - omones (Giannakakis and Fletcher  1981; Metcalf et  al.  1983), which are important in monitoring and control (Tan et al.  2014; Scolari et al.  2021). Yet, the receptive range of maxillary palps of Tephritidae is broader than just (para)pheromones and includes robust responses to fruit and food volatiles (Larsson- Herrera et al. 2024 ). Odor detection by the maxillary palps of tephritid flies, with a sensitivity to general odors and odors that induce sexually di - vergent behaviors, appears to serve multiple ecological func - tions that likely involve segregated neural processing pathways. While both odor classes activate palpal OSNs, (para)phero - mones may engage conserved lateral horn circuits driving hard - wired attraction behaviors, whereas food odors could integrate FIGURE 2 | Amplitude of olfactory responses of male and female maxillary palps of five tephritid species ( Bactrocera dorsalis , Bactrocera lati - frons, Zeugodacus cucurbitae , Ceratitis capitata , and Ceratitis cosyra ) toward spiroacetals, including olean, and synthesized spiro A, spiro B, spiro C, spiro D, spiro E, spiro F, spiro G, either emitted or shared between few tephritid species. 20457758, 2025, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ece3.72261 by Swedish University Of Agricultural Sciences, Wiley Online Library on [30/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 7 of 13 Ecology and Evolution, 2025 into mushroom body networks for context- dependent valuation (Dweck et al.  2016; Giurfa  2013). These functional differences may subject palpal detection systems to divergent selection pres- sures. For food odors, directional selection may optimize sen - sitivity to dominant host volatiles (e.g., fruit terpenes), while stabilizing selection may preserve high- affinity receptors for trace (para)pheromones due to their reproductive significance (Gonzalez et al.  2020). Temporal niche partitioning (e.g., crepus- cular pheromone release vs. diurnal feeding) likely minimizes masking effects between these odor classes. The current study shows that, while the palps indeed respond to fruit and food odors, they are particularly sensitive to (para)pher- omones, with a surprising breadth and cross sensitivity across taxa that do not reflect behavioral sensitivity. In addition, a cluster analysis showed that the responses to parapheromones closely followed phylogeny rather than ecology, and thereby differed from fruit and food odor sensitivities, which followed ecology rather than phylogeny (Biasazin et  al.  2019; Larsson- Herrera et al.  2024, this study). Below, the results are discussed in the context of (para)pheromone production, known behav - ioral responses, and the evolutionary ecology of Tephritidae. 4.1 | Sensitivity to Male Lures or Parapheromones Bactrocera and Zeugodacus spp. are generally classified into three categories based on their behavioral responses to phen - ylpropanoids: ME responsive, RK/CL responsive, and non- lure responsive (Clarke  2019; Royer et  al.  2017; Drew et  al.  1982; Metcalf and Metcalf  1992; Tan et  al.  2010). How the olfactory circuitry regulates these, often very strong, male- specific behav- ioral responses is not understood, and neither is the evolutionary ecology of the sensitivity to these compounds. Whereas ablation experiments show the importance of maxil - lary palps, the receptive ranges of the maxillary palps to these compounds across species are generally not known. Our data provide some important correlates that warrant further study. As the palpal minicircuitry consists of only six OSN types, sev - eral of which mediate responses to a wide range of chemically diverse fruit and food odors (including esters, terpenes, ketones, pyrazines, as well as spiroacetals, Larsson- Herrera et al.  2024, this study), the number of OSNs types mediating phenylpro - panoid responses would have to be very limited. Possibly, only one OSN type detects phenylpropanoids and responses may thus reflect the tuning breadth of a single OSN that is critical in me - diating male attraction. In the maxillary palps of D. melanogas - ter, a single OSN class (pb1b) also displays a strong sensitivity to phenylpropanoids, with two other neurons being sensitive to other phenolics (de Bruyne et  al.  1999; Dweck et  al.  2016). However, no particular role of phenylpropanoids or other phe - nolics has been described in the ecology of drosophilids. Surprisingly, however, palpal sensitivity to phenylpropanoids was not always congruent with behavioral sensitivity. For in - stance, whereas the palps of B. dorsalis are essential for induc - ing the male behavioral response to ME (Chieng et al.  2018), they were also broadly sensitive to other phenylpropanoids that are not male lures for this species. Conversely, the palps FIGURE 3 | Amplitude of olfactory responses of male and female maxillary palps of five tephritid species ( Bactrocera dorsalis , Bactrocera lati - frons, Zeugodacus cucurbitae , Ceratitis capitata , and Ceratitis cosyra ) toward phenylpropanoids (zingerone, methyl eugenol (ME), cuelure (CL), rasp- berry ketone (RK)), known as male lures for many Bactrocera species. 20457758, 2025, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ece3.72261 by Swedish University Of Agricultural Sciences, Wiley Online Library on [30/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 of 13 Ecology and Evolution, 2025 of B. latifrons , a “non- lure responsive” species (Clarke  2019), while not sensitive to ME, responded strongly to RK and CL, even stronger than in Z. cucurbitae , a species routinely mon - itored using RK and CL (Kawashita et  al.  2004 ). Similarly, despite not being behaviorally responsive to ME, both Ceratitis species exhibited strong responses to this compound, while displaying no palpal sensitivity to their own male lures, trimedlure or α- terpinyl acetate (Ripley and Hepburn  1935; FIGURE 4 | Linear models for olfactory responses of maxillary palps of the five tephritid species ( Bactrocera dorsalis , Bactrocera latifrons , Zeugodacus cucurbitae , Ceratitis capitata , and Ceratitis cosyra ) to esters, shikimates, and phenylpropanoids and spiroacetals, as well as across all pairwise combinations. Stars highlight the level of significance * < 0.05, ** < 0.01, *** < 0.001. 20457758, 2025, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ece3.72261 by Swedish University Of Agricultural Sciences, Wiley Online Library on [30/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 9 of 13 Ecology and Evolution, 2025 Khan et al.  2021). Finally, whereas zingerone, RK and CL are known attractants for Zeugodacus species (Inskeep et al.  2018; Tan and Nishida  2024), the maxillary palps of Z. cucurbitae were relatively insensitive to these phenylpropanoids com - pared to both Bactrocera species, for which these compounds are likely behaviorally insignificant. These apparent mismatches should be interpreted with caution. Our electrophysiological assays tested compounds in isolation, whereas natural (para)pheromonal signals typically occur as multi- component blends in precise ratios that determine be - havioral valence. Peripheral neuronal firing to an individual compound, therefore, does not necessarily predict attraction, as central processing in higher olfactory centers ultimately deter - mines behavioral output. Thus, whereas the maxillary palps are important in mediat - ing the behavioral response to male lures (Chieng et  al.  2018; Verschut et  al.  2018), palpal tuning did not match behavioral sensitivity to (para)pheromones. As palpal sensitivity overlaps with antennal sensitivity, it would seem logical that, to elicit the full sequence of behaviors that lead to for example, trap catches, input from both organs is required (Biasazin et  al.  2025), and that this combination of input also renders behavioral speci - ficity. Indeed, the combination of the maxillary palps and the antennae appears critical for the strong behavioral responses to male lures of B. dorsalis and Z. cucurbitae (Chieng et al.  2018; Verschut et al.  2018). Along the same lines, the palps of B. lati - frons did not detect isophorone, a reported male attractant that is not nearly as attractive for B. latifrons as ME for B. dorsalis (Ishida et al.  2008). Similarly, trimedlure and alpha- terpinyl ac- etate were not detected by the palps and are not as effective lures for Ceratitis species as ME for B. dorsalis (Vargas et al.  2012). To fully understand how palpal responses to various compounds lead to behaviors, a solid understanding of the antennal circuitry would seem important. 4.2 | Spiroacetals Although species differ in the spiroacetals they produce, the palpal sensitivities overlapped seemingly independent of that. For instance, the simplest spiroacetal tested here, olean (ra - cemic 1,7- dioxaspiro[5.5]undecane, the major pheromone of the olive fruit fly), was detected by all species in this study, even though olean is not reported from any of these spe - cies (Baker et  al.  1980). Similarly, spiro A and spiro B were also detected by all or almost all species, respectively, while spiroacetals have not been reported from Tephritidae (Booth et  al.  2009). Spiro C, a widespread spiroacetal that is com - monly found in Bactrocera and Zeugodacus species (Baker and Bacon  1985; Zhang et al.  1997; Francke and Kitching  2001), was detected only by Z. cucurbitae and slightly by C. capitata , while its stereoisomer and uncommon spiro D (Francke and Kitching  2001) was detected by B. dorsalis and B. latifrons . Spiro E, F, and G, a racemic mixture of 2- ethyl- 8- methyl- 1,7- d ioxaspiro[5.5]undecane and identified from several Bactrocera species (Booth et al.  2009 ; El- Sayed et al.  2019; Noushini, Park, et al.  2020 ; Noushini, Perez, et al.  2020 ; Noushini et al.  2021), induced a mosaic sensitivity pattern across the five species, indicating a differential effect of chirality on receptor sensi - tivity. In this context, it is important to note that spiroacetals are well- known pheromones not only of Tephritidae but also widely reported from across insect orders (Booth et al.  2009). This may indicate a deeply rooted, ancient OSN and OR sys - tem for spiroacetals in insects and, given the abundance and overlap in production across insect lineages, a lower behav - ioral specificity than what perhaps the connotation of the word “pheromone” might suggest. Further research should look at the OSNs, their ORs underlying spiroacetal responses, as well as the cross- sensitivity of the OSNs and ORs to various spiroacetals and phenolics. In addition, given the sensitivity of Ceratitis species, the potential production of spiroacetals in this and related genera would seem warranted. FIGURE 5 | On the left: A tree based on EPD responses of five tephritid species ( Bactrocera dorsalis , Bactrocera latifrons , Zeugodacus cucurbitae , Ceratitis capitata , and Ceratitis cosyra ) to spiroacetals and phenylpropanoids. Since the responses did not consistently differ between sexes, the data were combined. On the right: A tree based on collapsed EPD responses to general odors (esters, fatty acyls, and terpenes). In the center: A phyloge - netic tree of the five tephritids plotted using phylogenetic data concatenated from COI and 16 s, based on a subset of data from Virgilio et al. ( 2015). Whereas responses to compounds from the spiroacetal and phenylpropanoid pathways followed phylogeny, responses to compounds from fatty acid and terpenoid pathways formed a cluster that correlates with ecological niche. 20457758, 2025, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ece3.72261 by Swedish University Of Agricultural Sciences, Wiley Online Library on [30/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 10 of 13 Ecology and Evolution, 2025 4.3 | EPD Responses to Pheromones and Phenylpropanoids Align With Phylogeny Previous studies demonstrated that ecology is of overriding importance in the overall olfactome tuning of the antennae (Biasazin et al.  2019) and the maxillary palps (Larsson- Herrera et al.  2024) to general fruit and food odors. This study provides evidence that, in spite of phylogenetic distance, the sensory responses to these general odors were highly similar between species with a similar ecological niche, even though phyloge - netically distant. However, a diametrically opposite pattern emerged for responses to pheromones and parapheromones, which instead closely matched phylogenetic relatedness. Apparently, opposing selection pressures shape the olfactory circuitry to either niche odors, resulting in directional selection that shows a pattern correlating with ecology, or odors that me - diate (para)pheromone communication, resulting in some form of stabilizing selection which consequently follows phylogeny. This contrasting pattern spans tens of millions of years of olfac - tory evolution of Tephritidae (Zhang et al.  2023) and emerges by virtue of GC- coupled olfactome measurements, which provide the sums of olfactory sensitivity for a large number of odorants. Indeed, such correlations have not previously been reported from other functional studies on OSNs or ORs. As single sensil - lum studies generally focus on single or small subsets of OSNs (de Bruyne et al.  2009; Stensmyr et al.  2003; Prelic et al.  2022), overall sensitivities cannot easily be extracted. Similarly, recep - tor studies that describe the tuning curves of individual ORs are difficult to translate into ensemble in  vivo responses (Reisert and Restrepo  2009) and are difficult to understand in terms of selection regimes to which they are subjected. Besides surfacing evo- eco patterns of ORs, these evolutionary correlates can be used to direct OR studies. Given the limited number of ORs expressed in the palps, one could use the oppos - ing selection pressures on (sets of) ORs to tease out which palpal ORs putatively respond to general odors versus those that re - spond to (para)pheromones. Based on the differential selection pressures, one would expect sequences of (para)pheromone- sensitive ORs to more strictly follow phylogeny than ORs sensi - tive to general odors. Additionally, this may provide insights into the amino acid sequence(s) that are involved in ligand binding and give rise to these differential responses, particularly for ORs under directional selection amidst an abundance of sequence in- formation following phylogeny (Saad et al. 2018 ). 5 | Conclusions and Further Research The functional characterization of the maxillary palps of Tephritidae provides important insights into the circuitry un - derlying (para)pheromones, and how detection may, or may not, steer behavior toward these compounds. It appears that the detection of (para)pheromones by the maxillary palps is not an indication of behavioral sensitivity. While this supports the idea that behavioral responses rely on a synergy between palpal and antennal input in mediating the full range of behaviors, it also raises the question of how such synergy is mediated and which OSN/OR combinations are responsible for this integration. Further questions of interest include, for instance, whether such (sub)circuits exist in non- responsive species or non- responsive sexes, and whether they can be activated through hitherto unde- scribed odor combinations that induce responses in OSN types that are part of this circuitry. The data also provide novel in - sights of how differential selection pressures shape the evolution of olfactory sensitivities and follow either ecology or phylogeny. The emerging patterns may be useful in further unraveling which ORs underlie responses to which odors, as well as guide future studies deciphering ORs' function by indicating puta - tive sequences that may be involved in ligand binding. Finally, knowing the sensitivities of the palps could be used in designing novel odor combinations for testing in field trials. Author Contributions Chaymae Fennine: conceptualization (equal), data curation (equal), formal analysis (equal), methodology (equal), visualization (equal), writing – original draft (equal), writing – review and editing (equal). Sebastian Larsson Herrera: conceptualization (equal), data curation (equal), formal analysis (equal), methodology (equal), visualization (equal), writing – original draft (equal), writing – review and editing (equal). Tibebe Dejene Biasazin: conceptualization (equal), funding acquisition (equal), investigation (equal), methodology (equal), valida - tion (equal), writing – review and editing (equal). Wittko Francke: conceptualization (equal), investigation (equal), methodology (equal). Sergio Angeli: conceptualization (equal), supervision (equal), writing – review and editing (equal). Teun Dekker: conceptualization (equal), data curation (equal), formal analysis (equal), funding acquisition (equal), methodology (equal), project administration (equal), resources (equal), supervision (equal), validation (equal), visualization (equal), writing – original draft (equal), writing – review and editing (equal). Acknowledgments We acknowledge Prof. Wittko Francke for the synthesis of all spiroac - etals used in this study. This research was supported by three grants from the Swedish Research Council (VR): 2016- 05660 (T.D., S.L.H.), 2019- 04421 (T.D.B.), 2020- 05344 (network grant), as well as FORMAS grant: 2020- 01616, S.L.H., and T.D. Conflicts of Interest The authors declare no conflicts of interest. Data Availability Statement All data utilized in this manuscript, along with the scripts to generate the figures, are available for review at https:// zenodo. org/ recor ds/ 14893 742? previ ew= 1& token = eyJhb GciOi JIUzU xMiIs Imlhd CI6MT czOTk 4MDAy OSwiZ XhwIj oxNzY 3MTM5 MTk5fQ. eyJpZ CI6Ij M2NGQ zNjQy LTVhN TItNG Y3Mi1 hNmI1 LWE4M GY0Zm RkM2V hMSIs ImRhd GEiOn t9LCJ yYW5k b20iO iJkOW FlZjg 3N2Vh ZDIyN TE0MW NlOGJ hYzVh MDllZ DY1YS J9. oAILI UqJGF NsLJm 4B1Bl OIyX3 q7Nzzq_ fLJde LzARH Mg0jn ODB45 Qdu3_ muD25 BWzel iHQN2 EHygm Taf8W W6eA.

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See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License