Olfactory responses of  Phortica variegata (Drosophilidae, Steganinae) the emerging vector of  Thelazia callipaeda (Rhabditida, Thelaziidae) to ecologically relevant volatiles

Olfactory responses of Phortica variegata (Drosophilidae, Steganinae) the emerging vector of Thelazia callipaeda (Rhabditida, Thelaziidae) to ecologically relevant volatiles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Olfactory responses of Phortica variegata (Drosophilidae, Steganinae) the emerging vector of Thelazia callipaeda (Rhabditida, Thelaziidae) to ecologically relevant volatiles Anna Laura Erdei, Magdolna Olívia Szelényi, Ferenc Deutsch, Balázs Kiss, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6128144/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jun, 2025 Read the published version in Parasites & Vectors → Version 1 posted 3 You are reading this latest preprint version Abstract Background: Phortica variegata (Drosophilidae: Steganinae), native to Europe, has emerged as a major vector of ocular nematosis caused by Thelazia callipaeda (Rhabditida: Thelaziidae), following the nematode's introduction into Europe from its original habitat in Asia. Males of P. variegata transmit these nematodes through feeding on tears of mammals including wild and domestic carnivorous mammals (foxes, beech martens, wild cats and dogs), lagomorphs, and humans. Insect vectors strongly rely on volatile cues to identify suitable hosts. Due to the increasing veterinary and medical concerns, there is a growing need for attractants. Understanding the olfactory responses of P. variegata is crucial, as insect vectors rely on volatile cues to locate hosts. The identification of key attractants could facilitate the development of vector surveillance and control strategies. However, the olfactory ecology of this species remains unexplored, limiting our ability to design effective attractant-based interventions. Methods: We used gas chromatography coupled electroantennography to measure antennal responses to synthetic and natural volatile blends. A comparative analysis was performed on the antennal responses of both sexes of P. variegata and its well-studied relative, Drosophila melanogaster . Components of the synthetic blends were selected based on the odorant receptor repertoire of D. melanogaster and established mosquito attractants, with the rationale that conserved olfactory receptors among dipterans may allow P. variegata to detect similar compounds. Volatile extracts collected using active carbon adsorbent traps were also tested on the antennae and analysed using gas chromatography coupled mass spectrometry. Results: Males of P. variegata showed higher antennal responses to phenol, 3-octanone, and sulcatone than females, indicating olfactory sexual dimorphism. Compared to D. melanogaster , the antennae of P. variegata did not respond to several common plant alcohols and terpenoids. Instead, they showed stronger responses to compounds such as anisole, ethyl propanoate, butyl propanoate, propyl acetate, 3-octanone, nonanal, and decanal, suggesting that peripheral olfaction in P. variegata may be more tuned to microbial volatiles. Conclusions: The antennal olfaction of P. variegata appears to be particularly tuned to microbial volatile emissions, suggesting that fungal and microbial substrates may play an important role in the life cycle of this species. Males show stronger relative responses to several compounds known to influence host-seeking behavior in other zoophilic dipterans, suggesting their potential as candidate attractants for future field studies. dipteran vector olfactory responses chemical ecology GC-EAD sexual olfactory dimorphism Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Drosophilid fruit flies are undoubtedly among the most studied organisms in the world. However, due to their lack of agricultural and veterinary importance, the fungivorous and zoophilic flies of the closely related subfamily Steganinae (Drosophilidae) have received little interest so far. This perspective began to change when the role of Phortica species in the transmission of ocular nematosis was recognised. Phortica variegata is native to Europe [1] and is known to feed on the tears of wild and domestic carnivores (foxes, beech martens, wild cats and dogs), lagomorphs, and humans [2]. Over the past decade, ocular nematosis caused by Thelazia callipaeda (also called oriental eyeworm, Rhabditida, Thelaziidiiae), an ocular nematode native to Southeast Asia [3], has sharply increased and has become a growing public health concern in Europe. P. variegata became an important vector of T. callipaeda when the nematode arrived in Europe. In its native habitat, T. callipaeda is mainly transmitted by other species of the genus Phortica, such as P. magna and P. okadaii . In Europe, however, P. variegata has emerged as the main vector [4]. Climate change and warmer winters have expanded the range and activity periods of this species by supporting overwintering and thereby facilitating the spread of T. callipaeda as well [5, 6]. Larvae of several Phortica species have been found to develop in fermenting tree sap [7, 8]. The distribution of P. variegata is associated with oak forests, and it has been shown that a chestnut-based rearing medium is suitable for larval development [9]. Studying dipteran guilts utilizing small-scale forestine food resources, Papp (2002) observed found that adult P. variegata were collectible around visited fox faeces and rotten fungi raising the possibility that such microhabitats are sites visited used for mating or, oviposition or feeding [10]. Additionally, larvae of other species in this subfamily were shown to develop on decaying plant matter and fungal substrates [8] that indicate P. variegata may also use similar substrates as breeding sites. Little is known about the vectoring behavior, apart from the fact that T. callipaeda is only found in males and that females were not observed to feed on tears [11]. This behavioral dimorphism is in contrast with all other known vector insects, where zoophagy and vectoring is either entirely female-linked or exhibited by both sexes [11]. Insects rely primarily on the sense of smell to find food, mates and oviposition sites in their complex environment. Their olfactory system is tuned to filter out “background noise” and detect volatile signals relevant for host recognition [12]. Chemical ecological research of vector insect olfaction supports control solutions by revealing the olfactory cues that modulate vector behavior. The identification of attractants and repellents can lead to new vector-control solutions such as repellent formulations, and baits for monitoring and mass trapping. P. variegata belongs to the Drosophilidae family, where adaptation to new food sources was shown to be reflected in corresponding changes in the olfactory system; Drosophila melanogaster , a species feeding and ovipositing on overripe fruits, has high sensitivity for fermentation volatiles [7, 13], herbivorous Scaptomyza flava has reduced sensitivity to those and increased sensitivity to leaf volatiles [14, 15]. and D. sechellia feeding on toxic Morinda fruits is attracted to short-chain fatty acids found in these fruits that repel other drosophilids [16] . Both sexes of P. variegata can be captured with fruit baits and vinegar and wine baits similar to D. melanogaster [17-19], although the majority of flies caught with these baits are females, while those caught around the eyes are exclusively males [4, 17]. It was recently shown that supplementing vinegar wine baits with carvacrol abolishes this attraction [19]. Although, currently there is limited information available on the olfactory repertoire of species belonging to the Steganinae subfamily it can be hypothesized that the zoophilic behavior exhibited by P. variegata and the indicated association with microbial substrates can be accompanied by adaptations of the olfactory system. While there are no other known drosophilids that exhibit attraction to mammalian hosts a useful parallel can be drawn with mosquitoes and tsetse flies, the most extensively studied insect vectors. The evolution of zoophily in mosquitoes has been directly linked to olfactory adaptations. Host-seeking female mosquitoes rely on a heightened sensitivity to mammalian body odours [20-24]. L-lactic acid attracts Aedes aegypti females and has been shown to be involved in human discrimination, as it is more abundant in human sweat than that of other mammals [20, 21]. It was also shown that an increased sensitivity to sulcatone is linked to human preference in A. aegypti where the increased expression of a sulcatone-sensitive odorant receptor, AaegOr4 supports host discrimination [24]. Additionally, the detection of short-chain saturated aldehydes in skin emissions [22, 23] and fluctuating CO₂ levels, which enhance A. aegypti 's sensitivity to human odors [25, 26] have been shown to play key roles in identifying mammalian hosts. These findings in mosquitoes and the well-described olfactory adaptation in other drosophilid species suggest that similar mechanisms may underlie the zoophilic behavior observed in P. variegata , where olfactory adaptations could facilitate host recognition. Currently, no efficient or species-specific attractants or repellents are available for Phortica variegata , posing a challenge for the development of targeted vector control strategies. To our knowledge, the chemical ecology of P. variegata , or any species within the Steganinae subfamily, remains entirely unexplored. However, the recent annotation of odorant receptor genes in the P. variegata genome [27] opens the possibility to explore olfactory mechanisms underlying tear feeding behavior. Given its attraction to mammals and fermentation-based baits, we hypothesized that P. variegata may rely on olfactory cues similar to those used by both mosquitoes and drosophilids to identify mammalian hosts and suitable microbial habitats. To investigate these hypotheses, we conducted olfactory investigations of P. variegata using gas chromatography coupled with electroantennographic detection (GC-EAD) comparing the response profile of both sexes to that of D. melanogaster using a panel of selected synthetic compounds and identified volatile components from host-related volatile sources and fermentation baits that are detected by the antennae of this species. Materials and methods Collection of experimental animals and taxonomic identification P. variegata m ales were collected by netting around the eyes of human collectors using hand-held aquarium nets in forest habitats around the outskirts of Budapest at Ördögárok (47.54254° N, 18.94572° E ), Iluska spring (47.64546° N, 18.86779° E) and Piliscsaba (47.63866° N, 18.85353°E) during the early afternoon, at temperatures around 19 °C ± 8°C. Females were captured using apple cider vinegar-baited live traps at the same locations. Animals were transferred into closed but not air-tight humidified vials using an aspirator. Species-level identification was based on the morphological taxonomic keys described by Bächli et al. 2005 in The Drosophilidae (Diptera) of Fennoscandia and Denmark [28] using methods identical to Kerezsi et al. 2019 [29] . Adults were sexed and placed individually in glass jars where access to apple fruit slices as food source was provided. The adults were kept on a 16/8 light cycle at room temperature (25 ±2°C) and at relative humidity of 50 ±5%. The wild type D. melanogaster used in the experiments were reared on a modified sugar-yeast-corn meal diet [30] under the same environmental conditions as described above. Preparation of synthetic mixtures A preliminary comparison of antennal sensitivities of the two fruit fly species was done by testing synthetic mixtures using electroantennography coupled gas chromatography. The 47 components included in the mixtures (Table S1) were selected based on known ligands of D. melanogaster odorant receptors listed in DoOR, the online database of D. melanogaster odorant receptor responses [31], as well as on reported attractants involved in host-seeking behavior of blood-feeding vector insects such as mosquitoes and tsetse flies [32-40]. All compounds were diluted in HPLC grade hexane to a concentration of 100 ng/µL and recordings with only solvent injected, were also conducted as control. The purity and manufacturers of synthetic compounds are listed in Table S1. Volatile collection methods Fresh faecal samples were collected from red fox ( Vulpes vulpes ), brown bear ( Ursus arctos ) and red deer ( Cervus elaphus ) in the Budakeszi Wildlife Park. Human body odour was sampled from two female volunteers (volunteers did not use scented soap or deodorant 24 hours prior to volatile sampling). The armpits and upper arms of the volunteers were rubbed with medical gauze for 5 minutes, which was later used to collect volatiles. For wine-apple cider vinegar (W+ACV) headspace sampling, 2 dL of apple cider vinegar and 1 dL of red wine were mixed in a glass beaker. The samples were placed in oven bags (35 x 43 cm, Hewa). Volatile sampling was performed in the laboratory at room temperature. The incoming air was filtered by a carbon air inlet. The air stream was drawn from the oven bag through an activated carbon volatile trap (5 mg cartridge, Brechbühler AG, Switzerland) at a flow rate of 500 mL/min for 4 hours. The volatile traps were eluted with 200 mL methylene chloride (Sigma Aldrich, HPLC grade etc.). Prior to volatile collection the active carbon airstream filters were cleaned at 200 °C and the volatile traps were cleaned in a series of 2 mL methanol, 2 mL acetone, 2 mL hexane and 2 mL methylene chloride and heated up to 100 ° C for 12 hours. The volatile sampling was launched within 40 minutes of collecting the faecal and human body odour samples. Gas chromatography coupled mass spectrometry The volatile extracts were analyzed by gas chromatography coupled with mass spectrometry (GC-MS, Agilent 6890 GC and 5975 MS, Agilent Technologies) equipped with HP-5 UI capillary column (30 m × 0.25 mm × 0.25 µm, J&W Scientific, Folsom, CA, USA). 2 µl of volatile extracts were autoinjected into the split/splitless injection port operated in splitless mode heated to 270 °C using a 1 min splitless time. Helium was used as a carrier gas with a flow rate of 1 mL/min. The initial oven temperature was held at 50 °C for 1 minute then increased by 10 °C/min to 270 °C. The final temperature was held for 10 minutes. The mass spectrometer source was operating at 250 °C in electron ionization mode 70 eV and the detector scanned in the 29-300 m/z range. The GC-MS results were analyzed using Agilent Mass Hunter B.08.00, the peaks were manually integrated. Compounds were tentatively identified by matching their mass spectra with those found in MS Libraries (NIST21 and Wiley12). Identifications were also verified by comparing calculated Kováts indices (KI) using C8-C20 alkane calibration standard to those found in NIST WebBook database, where only references using authentic standards were considered and key compounds were also identified using authentic synthetic standards (Table S2). Gas chromatography coupled electroantennography The biologically active components of volatile extracts were identified using gas chromatography coupled electroantennographic detection (GC-EAD). The Agilent 6890N gas chromatograph (GC) was equipped with an HP-5 UI capillary column (30 m × 0.32 mm × 0.25 µm, J&W Scientific, Folsom, CA, USA). 2 µl of volatile extracts were manually injected into the injection port of the gas chromatograph, operated in splitless mode and heated to 230 °C using a 1 min splitless time. The carrier gas was helium and the column flow was 4 mL/min. The initial oven temperature was held at 50 °C for 1 minute then in the first ramp increased by 10 °C/min to 270 °C in the second ramp by 30 °C/min to 230 °C. The final temperature was held for 5 minutes. The GC effluent was split in a low dead volume Graphpack 3D/2 four-way splitter. Two non-coated deactivated fused silica capillary columns (100 cm × 0.32 mm) were connected to the four-way splitter, one led to the flame ionization detector (FID) heated to 300 °C and the other line was fitted into a transfer line heated to 235 °C (Syntech, Kirchzarten, Germany). The capillary column protruded from the heated transfer line into an inert glass tube (10 mm I.D.) that had a charcoal-filtered and humidified airflow of 1 L/min transferring the effluent over the antennal preparation. Female and male adults of P. variegata and D. melanogaster were immobilized in 200 mL pipette tips. The tip of the pipette was cut and the animal was pushed forward until half of the eye was uncovered in the pipette tip but the proboscis was still covered. The silver/silver chloride electrodes were immersed in Ringer solution in two finely pulled glass capillaries. The glass capillary of the reference electrode was inserted into one of the eyes of the restrained fly and the glass capillary covering the recording electrode was pushed to hold firm contact with the dorsomedial region of the funiculus. The antennal signal was amplified 10 times, and the analog-digital conversion was done by IDAC-2 (Syntech). The recording was done simultaneously with the FID signal using GC-EAD software (GC-EAD 2014, vers. 1.2.5, Syntech). The eight synthetic mixtures were tested sequentially on the same individuals in a randomized order. Before each GC-EAD run, a glass Pasteur-pipette loaded with a 100 ng 1-hexanol in 10 µl mineral oil was used as a test stimulus to assess the quality of preparation and electric contact. The synthetic mixes were tested on at least 3 specimens of both species and both sexes. The volatile extracts were tested on male P. variegata specimens except for fox faeces, which was tested on both females and males. Data processing and statistical analysis Statistical analysis and data visualization, was performed using R (v. 4.2.0) in RStudio (RStudio Team (2023 v. 6.0.421). Similarities and dissimilarities between the antennal response profile of D . melanogaster and P . variegata individuals to synthetic compounds were investigated by Principal Coordinates Analysis (PCoA) using the capscale function and by Non-Metric Multidimensional Scaling (NMDS) of the vegan package (v. 2.6-4) [41] using Jaccard dissimilarity as a distance measure on non-binary data. The responses were standardized across individuals by dividing the responses by the average of responses for the individual. The responses were also standardized across each compound by Z-scoring: the average response to the compound is subtracted from the response of each individual and divided by the standard deviation of responses to the compound. Permutational multivariate analysis of variance (PERMANOVA) was performed on the Jaccard dissimilarity as a distance measure to compare groups using adonis2 function of the vegan package (v. 2.6-4) [41] and the significance values for multiple comparisons was adjusted by Benjamini-Hochberg correction (P-value adj). To identify differences between responses of species and between responses of sexes to individual compounds, we used multi-level pattern analysis by the multipatt function of indicspecies package [42] and adjusted P -values using Benjamini-Hochberg correction using the p.adjust function from package stats . All figures were visualized using geom_point (Fig. 1-4), geom_segment (Fig. 2.) geom_tile (Fig.3.) and geom_line (Fig. 4.) functions of the ggplot2 v.3.5.1 [43] package. Results We first assessed differences in antennal responses between P. variegata and D. melanogaster using synthetic compounds. These were selected based on their detection by olfactory receptors of D. melanogaster (as listed in the DoOR database [31]), as well as on their known roles in modulating host-seeking behavior in blood-feeding dipterans, such as mosquitoes and tsetse flies [32-40]. Both species were sensitive to aliphatic esters. However, P. variegata showed no antennal responses to the monoterpenoids and sesquiterpenoids tested, with the exception of linalool (Fig. 1, Fig. S1). PCoA (Fig. 2) and NMDS (Fig. S2) were applied using non-binary Jaccard dissimilarity to assess divergence in antennal response profiles. Individuals from the same species clustered closely together, whereas P. variegata and D. melanogaster separated along MDS1. The eigenvalues for MDS1 (0.58) and MDS2 (0.25) indicate that MDS1 explained the majority of the variation. PERMANOVA confirmed significant divergence in response profiles between species (F₁ = 7.386, P-value = 0.002). Pairwise comparisons with Benjamini-Hochberg correction also supported this difference (P-value adj = 0.004). The multi-level pattern comparison (Table S3) has shown that P. variegata had higher relative responses to , propyl acetate, ethyl propanoate, butyl propanoate, anisole, 3-octanone, nonanal and decanal than D. melanogaster . P. variegata males had higher responses to phenol, 3-octanone, and sulcatone (6-methyl-5-hepten-2-one) than females. Although males exhibited numerically higher responses to nonanal and decanal, this difference was not statistically significant (P-value adj > 0.05). Given that adult male P. variegata are captured in fermentation baits, associated with mammalian faeces, and attracted to mammals, further GC-EAD recordings were performed using ecologically relevant volatile samples, including W+ACV, and faeces from bear, deer, and fox, as well as human body odour. Electrophysiological recordings revealed antennal responses to 31 volatile components (Fig. 3). The detected components of mammalian samples closely resembled those of W+ACV (Table S4), as evidenced by similar antennal response profiles (Fig. 3). Ethyl lactate and isoamyl acetate were detected exclusively in W+ACV volatiles and elicited antennal responses. Phenol and dimethyl trisulfide were only present and were sensed in the bear and fox feces samples. Nonanal, sulcatone, and decanal were components of all samples (Table S4). Nonanal and sulcatone elicited antennal response in all volatile samples, while decanal did not elicit antennal responses in fox faeces volatiles, likely due to its low abundance (Fig. 3). Anisole and hexanoic acid elicited antennal responses but the former was only present in the volatile sample of fox faeces and the latter in human body odour samples. Since P. variegata has been observed to be present on fox faeces [10], we tested whether antennal responses of sexes differs to volatiles emitted from fox faeces (Fig. 4.). Sulcatone failed to elicit antennal responses in females, but response amplitudes to other volatiles did not differ significantly between sexes (Fig. 4). Discussion We compared antennal responses of P. variegata and D. melanogaster to a panel of synthetic odorants known to activate olfactory receptors in D. melanogaster and influence host-seeking in other mammal-attracted dipterans. While both species responded to aliphatic esters, P. variegata showed limited responses to monoterpenoids and sesquiterpenoids and multivariate analyses revealed clear species- and sex-related divergence in response profiles. The behavioral dimorphism, in which only male flies feed on tears, may also be reflected in sex-specific olfactory sensitivity, similar to A. aegypti where females express a higher number of ORs compared to males, likely linked to their need to recognize hosts for blood feeding [44]. To test this hypothesis, we compared the antennal response of female and male P. variegata using a panel of synthetic compounds (Fig. 1, Fig. 2). Multi-level pattern analysis revealed significant differences in response amplitudes between the sexes (Table S3.). The antenna of males exhibited significantly higher responses to synthetic phenol, 3-octanone, and sulcatone. Sulcatone was found in all tested relevant volatile samples (Fig. 3). Moreover, while sulcatone elicited responses in synthetic blends, it failed to trigger antennal responses in females when derived from fox faeces (Fig. 4). These results could be explained by the higher antennal sensitivity of male Phortica flies, as the amount of sulcatone in fox faeces was possibly below the detection threshold for females. As sulcatone is abundant in human skin and animal emissions [45, 46] a heightened sensitivity might allow males to detect and locate mammalian hosts more effectively than females. Sexual dimorphism in peripheral olfaction is a well-documented phenomenon in insects, often reflected in differences in antennal structure, the number of odorantreceptors, and sensitivity to specific compounds [47-49] . For instance, sexual dimorphism in olfaction of vector insects has been observed in A. aegypti [50], Culex pipiens quinquefasciatus [51] and Anopheles gambiae [47]. Notably, A. aegypti strains that prefer human hosts over cattle have been shown to overexpress the odorant receptor AaegOr4, which has a high affinity for sulcatone [24]. However, no significant sexual dimorphism was observed in the expression levels of this receptor [44] and the antennal sensitivity to sulcatone was not compared yet. To determine if male P. variegata files are more sensitive to sulcatone and if this compound is important in host-seeking, further studies involving dose-dependency measurements and behavioral experiments are needed. Differences observed for phenol in synthetic blends were not significant in fox faeces samples, likely due to dose dependency, as the fox faeces samples were more concentrated than the synthetic blends tested based on comparison FID traces. This observation again highlights the importance of considering dose dependency when evaluating olfactory responses and underscores the need for carefully controlled experiments to accurately assess sex-specific differences in chemosensation and behavior, as demonstrated in the case of both sulcatone and phenol with female Culicoides nubeculosus where attraction and repellence are strictly dose-dependent [46] . The observed sensory differences between sexes of P. variegata could explain the behavioral dimorphism, but further experiments are needed to establish a causal link between the detection of these compounds and their effect on behavior. Species specific differences were observed between the response profiles of D . melanogaster and P. variegata to synthetic compounds as they separated along the first axis of PCoA (Fig. 1). For several other compounds, a dose-dependent response was observed in the ecologically relevant volatiles samples (Fig. 3). Compared to D. melanogaster both sexes of P. variegata showed an increased sensitivity to anisole (methoxybenzene) which is main constituent of anise seed essential oil [52] and were described to be present in essential oils prepared from other plants [53] . Intriguingly, anisole is also emitted from decomposing leaf litter, such as that of poplar [54] and can be emitted by microbes such as Penicillium expansum during degradation of lignin [55]. It was reported that P. variegata adults are feeding on fermenting tree sap [8, 56], which might be a rich source of anisole and related methoxybenzenes. Surprisingly, anisole was also a minor component of fox faeces headspace, and both sexes of P. variegata responded to this component of the volatile extracts. The antennae of P. variegata were more sensitive than those of D. melanogaster to several common volatile compounds emitted from fermented substrates, including ethyl and butyl propanoate, propyl acetate, 3-octanone, nonanal and decanal. These compounds are found in a wide range of natural sources. Nonanal and decanal are also major components of human body odor and they were shown to be attractive to Culex mosquitoes [57] , while the high ratio of these compounds was shown to decrease the attraction of A. aegypti to human body emissions [23]. Several aliphatic esters from the synthetic blend, are often associated with fermenting plant materials, yeasts, and ripening fruits [58-60] , and similar to D. melanogaster antennae, those of P. variegata responded to isoamyl and isobutyl acetate and had a significantly higher sensitivity to ethyl- and butyl propanoate and propyl acetate. Interestingly, P. variegata exhibited a weaker response to ( E )-2-hexenal and ( E )-3-hexenol which are characteristic green leaf volatiles emitted upon mechanical damage of plant tissues [61]. These compounds are repellent for D. melanogaster and were hypothesized to be related to discrimination of ripe fruits from ripening ones that are unsuitable for oviposition [62]. Furthermore, P. variegata antennae did not respond to terpenoids selected from the DOOR, except males showing a weak response to linalool. β-caryophyllene, farnesol and α-humulene are common sesquiterpenoid compounds in plant volatile emissions [63]. β-caryophyllene, α-terpineol and α-humulene are major ligands of OR19a expressed in trichoid sensilla [64] and farnesol is major ligand of Or83c expressed in intermediate sensilla [65] on the antennae of D. melanogaster, several other Ors such as Or69a are also involved in the detection of terpenoids. Bastide et al. (2024) identified two orthologs of OR19a and one of Or83c in the genome of P. variegata , however, the functionality of these genes, their expression pattern as well as their main ligands are currently unknown [27]. Since terpenoids are detected by multiple odorant receptors in D. melanogaster , the lack of response in P. variegata to several terpenoids may reflect ecological differences between the species, suggesting that P. variegata relies less on the identification of plant-derived resources than D. melanogaster . The lower sensitivity of P. variegata to several ubiquitous plant volatile compounds compared to D. melanogaster and increased sensitivity to several volatile compounds common in microbial volatile emissions indicates that fungal and microbial substrates might be more important in the ecology of this species compared to D. melanogaster . According to our current knowledge, many species belonging to this group are associated with fungi or feeding on decaying plant material. Similarly to attraction of mosquitoes to their hosts [66, 67] , the attraction of P. variegata to mammalian hosts can be based on otherwise common microbial volatiles combined with carbon dioxide or visual cues. Based on this first report on the olfactory sensitivity of P. variegata , the behavioral significance of ethyl and butyl propanoate, propyl acetate, 3-octanone, nonanal, decanal and sulcatone for females and males should be further evaluated in laboratory and field behavioral bioassays. The identification of new attractants can provide a basis for developing both monitoring and mass trapping solutions for the future management of this vector species. Conclusions The olfaction of insects is shaped by their environment and ecology. Our study demonstrates that P . variegata exhibits sexual dimorphism in olfactory sensitivity, with males showing increased sensitivity to specific volatiles such as sulcatone, phenol, and 3-octanone which may help them locate mammalian hosts, aligning with their behavioral dimorphism in feeding on tears. Additionally, the comparative olfactory analysis with D. melanogaster revealed that P. variegata shows a stronger sensitivity to several microbial and yeast-related volatiles and a strongly reduced sensitivity to common plant volatile terpenoids, reinforcing the idea that its foraging ecology differs from that of D. melanogaster and fungal and microbial substrates might be more ecologically relevant for this species. The study highlights several antennally active volatiles that could be assessed in field and laboratory behavioral experiments to investigate their ecological roles and to potentially use them to develop monitoring and control strategies against this dipteran vector species. Abbreviations A. aegypti: Aedes aegypti, D. melanogaster: Drosophila melanogaster , GC-MS: Gas chromatography-mass spectrometry, GC-EAD: Gas chromatography-electroantennography, OR: odorant receptor, NMDS: Non-Metric Multidimensional Scaling, PCoA: Principal Coordinate Analysis, P. variegata: Phortica variegata, Declarations Acknowledgement We thank the Budakeszi Wildlife Park and its staff for providing the opportunity to collect samples from forestine mammals and for assisting us in the sampling procedure. We also acknowledge Dora Varga for illustrations of odour sources. Funding This research was supported by an NKFIH Research Proposal FK 137579 of the National Research, Development and Innovation Office. Availability of data and material All data utilized in this manuscript, along with the scripts to generate the figures, are available at https://figshare.com/s/5503fa00feca01ad50c Author’s contributions ALE, MOSz, and BPM were involved in the design and conception of the work. FD and BK collected and maintained the laboratory animals and FD performed the taxonomic identification. ALE completed the GC-MS and GC-EAD measurements and statistical analysis and ALE and MOSz prepared the figures. ALE and MOSz wrote the manuscript and all authors revised the text. Ethics approval and consent to participate: No ethical approval is necessary, for human odor collection the authors were sampled. Consent for publication: No consent is necessary for publication, no human experiments were done. Competing interests: The authors declare they have no competing interests. References Maca J. Revision of Palaearctic species of Amiota subge nus Photica (Diptera, Drosophilidae). Acta entomol bohemoslov. 1977;74:115-30. Bertos E, Sánchez-Cerdá M, Virgós E, Gil-Sánchez JM, Moleón M. Thelazia callipaeda as a potential new threat to european wildcats: insights from an eco-epidemiological study. Veterinary Research Communications. 2023;47(4):2153-60. Rossi L, Bertaglia PP. Presence of Thelazia callipaeda Railliet & Henry, 1910, in Piedmont, Italy. Parassitologia. 1989;31(2-3):167-72. Otranto D, Cantacessi C, Testini G, Lia RP. Phortica variegata as an intermediate host of Thelazia callipaeda under natural conditions: Evidence for pathogen transmission by a male arthropod vector. International Journal for Parasitology. 2006;36(10):1167-73. Palfreyman J, Graham-Brown J, Caminade C, Gilmore P, Otranto D, Williams DJL. Predicting the distribution of Phortica variegata and potential for Thelazia callipaeda transmission in Europe and the United Kingdom. Parasites & Vectors. 2018;11(1). Pombi M, Marino V, Jaenike J, Graham-Brown J, Bernardini I, Lia RP, et al. Temperature is a common climatic descriptor of lachryphagous activity period in Phortica variegata (Diptera: Drosophilidae) from multiple geographical locations. Parasites & Vectors. 2020;13(1). Markow TA, O’Grady P. Reproductive ecology of Drosophila. Functional Ecology. 2008;22(5):747-59. Máca J, Otranto D. Drosophilidae feeding on animals and the inherent mystery of their parasitism. Parasites & Vectors. 2014;7(1):516. Bernardini I, Poggi C, Manzi S, Bezerra-Santos MA, Beugnet F, Fourie J, et al. Laboratory breeding of two Phortica species (Diptera: Drosophilidae), vectors of the zoonotic eyeworm Thelazia callipaeda. Parasites & Vectors. 2022;15(1):200. Papp L. Dipterous guilds of small-sized feeding sources in forests of Hungary. Acta Zoologica Academiae Scientiarum Hungaricae. 2002;48(supplement 1):197-213. Otranto D, Stevens JR, Cantacessi C, Gasser RB. Parasite transmission by insects: a female affair? Trends Parasitol. 2008;24(3):116-20. Schröder R, Hilker M. The Relevance of Background Odor in Resource Location by Insects: A Behavioral Approach. BioScience. 2008;58(4):308-16. Becher PG, Flick G, Rozpędowska E, Schmidt A, Hagman A, Lebreton S, et al. Yeast, not fruit volatiles mediate rosophila melanogaster attraction, oviposition and development. Functional Ecology. 2012;26(4):822-8. Goldman-Huertas B, Mitchell RF, Lapoint RT, Faucher CP, Hildebrand JG, Whiteman NK. Evolution of herbivory in Drosophilidae linked to loss of behaviors, antennal responses, odorant receptors, and ancestral diet. Proc Natl Acad Sci U S A. 2015;112(10):3026-31. Whiteman NK, Groen SC, Chevasco D, Bear A, Beckwith N, Gregory TR, et al. Mining the plant-herbivore interface with a leafmining Drosophila of Arabidopsis. Mol Ecol. 2011;20(5):995-1014. Amlou M, Pla E, Moreteau B, David JR. Genetic analysis by interspecific crosses of the tolerance of Drosophila sechellia to major aliphatic acids of its host plant. Genetics Selection Evolution. 1997;29(4):511. Otranto D, Brianti E, Cantacessi C, Lia RP, Máca J. The zoophilic fruitfly Phortica variegata: morphology, ecology and biological niche. Medical and Veterinary Entomology. 2006;20(4):358-64. Roggero C, Schaffner F, Bächli G, Mathis A, Schnyder M. Survey of Phortica drosophilid flies within and outside of a recently identified transmission area of the eye worm Thelazia callipaeda in Switzerland. Veterinary parasitology. 2010;171(1-2):58-67. González MA, Bravo-Barriga D, Alarcón-Elbal P, Álvarez-Calero JM, Quero C, Ferraguti M, et al. Development of Novel Management Tools for Phortica variegata (Diptera: Drosophilidae), Vector of the Oriental Eyeworm, Thelazia callipaeda (Spirurida: Thelaziidae), in Europe. Journal of Medical Entomology. 2022;59(1):328-36. Acree Jr F, Turner R, Gouck H, Beroza M, Smith N. L-Lactic acid: a mosquito attractant isolated from humans. Science. 1968;161(3848):1346-7. Zwiebel L, Takken W. Olfactory regulation of mosquito–host interactions. Insect biochemistry and molecular biology. 2004;34(7):645-52. Irish S, Moore S, Bruce J, Cameron M. Preliminary evaluation of a nonanal lure for collection of gravid Culex quinquefasciatus. Journal of the American Mosquito Control Association. 2014;30(1):37-41. Logan JG, Birkett MA, Clark SJ, Powers S, Seal NJ, Wadhams LJ, et al. Identification of human-derived volatile chemicals that interfere with attraction of Aedes aegypti mosquitoes. Journal of chemical ecology. 2008;34:308-22. McBride CS, Baier F, Omondi AB, Spitzer SA, Lutomiah J, Sang R, et al. Evolution of mosquito preference for humans linked to an odorant receptor. Nature. 2014;515(7526):222-7. Dekker T, Geier M, Cardé RT. Carbon dioxide instantly sensitizes female yellow fever mosquitoes to human skin odours. J Exp Biol. 2005;208(Pt 15):2963-72. Majeed S, Hill SR, Ignell R. Impact of elevated CO2 background levels on the host-seeking behaviour of Aedes aegypti. Journal of Experimental Biology. 2014;217(4):598-604. Bastide H, Legout H, Dogbo N, Ogereau D, Prediger C, Carcaud J, et al. The genome of the blind bee louse fly reveals deep convergences with its social host and illuminates Drosophila origins. Current Biology. 2024;34(5):1122-32.e5. Bächli G, Viljoen F, Escher SA, Saura A. The drosophilidae (Diptera) of fennoscandia and Denmark: Brill; 2005. Kerezsi V, Kiss B, Deutsch F, Kontschán J. First record of Blattisocius mali (Oudemans, 1929) in Hungary associated with the drosopilid fly Phortica semivirgo (Máca, 1977). Redia. 2019;102:69-72. Ashburner M. Drosophila: A Laboratory Handbook. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. Münch D, Galizia CG. DoOR 2.0-comprehensive mapping of Drosophila melanogaster odorant responses. Scientific reports. 2016;6(1):21841. Melo N, Wolff GH, Costa-da-Silva AL, Arribas R, Triana MF, Gugger M, et al. Geosmin attracts Aedes aegypti mosquitoes to oviposition sites. Current Biology. 2020;30(1):127-34. e5. Owaga ML, Hassanali A, McDowell P. The role of 4-cresol and 3-n-propylphenol in the attraction of tsetse flies to buffalo urine. International Journal of Tropical Insect Science. 1988;9(1):95-100. Takken W, Kline D. Carbon dioxide and 1-octen-3-ol as mosquito attractants. Journal of the American Mosquito Control Association. 1989;5(3):311-6. Vale G, Hall D, Gough A. The olfactory responses of tsetse flies, Glossina spp.(Diptera: Glossinidae), to phenols and urine in the field. Bulletin of Entomological Research. 1988;78(2):293-300. Vale G, Hall D. The role of 1-octen-3-ol, acetone and carbon dioxide in the attraction of tsetse flies, Glossina spp.(Diptera: Glossinidae), to ox odour. Bulletin of Entomological Research. 1985;75(2):209-18. Magalhães-Junior JT, Barrouin-Melo SM, Corrêa AG, da Rocha Silva FB, Machado VE, Govone JS, et al. A laboratory evaluation of alcohols as attractants for the sandfly Lutzomyia longipalpis (Diptera: Psychodidae). Parasites & vectors. 2014;7:1-5. Dormont L, Mulatier M, Carrasco D, Cohuet A. Mosquito attractants. Journal of chemical ecology. 2021;47:351-93. Zhang H, Zhu Y, Liu Z, Peng Y, Peng W, Tong L, et al. A volatile from the skin microbiota of flavivirus-infected hosts promotes mosquito attractiveness. Cell. 2022;185(14):2510-22. e16. Harraca V, Syed Z, Guerin P. Olfactory and behavioural responses of tsetse flies, Glossina spp., to rumen metabolites. Journal of Comparative Physiology A. 2009;195:815-24. Oksanen J. Vegan: community ecology package. R package version. 2015;2:3. Cáceres MD, Legendre P. Associations between species and groups of sites: indices and statistical inference. Ecology. 2009;90(12):3566-74. Wickham H, Sievert C. ggplot2: elegant graphics for data analysis: springer New York; 2009. Tallon AK, Hill SR, Ignell R. Sex and age modulate antennal chemosensory-related genes linked to the onset of host seeking in the yellow-fever mosquito, Aedes aegypti. Scientific Reports. 2019;9(1):43. Hinze A, Pelletier J, Ghaninia M, Marois E, Hill SR, Ignell R. Knockout of OR39 reveals redundancy in the olfactory pathway regulating the acquisition of host seeking in Anopheles coluzzii. Proceedings of the Royal Society B. 2023;290(2011):20232092. Isberg E, Bray DP, Birgersson G, Hillbur Y, Ignell R. Identification of cattle-derived volatiles that modulate the behavioral response of the biting midge Culicoides nubeculosus. Journal of chemical ecology. 2016;42:24-32. Pitts RJ, Rinker DC, Jones PL, Rokas A, Zwiebel LJ. Transcriptome profiling of chemosensory appendages in the malaria vector Anopheles gambiae reveals tissue-and sex-specific signatures of odor coding. BMC genomics. 2011;12:1-17. Zhou S, Stone EA, Mackay TF, Anholt RR. Plasticity of the chemoreceptor repertoire in Drosophila melanogaster. PLoS genetics. 2009;5(10):e1000681. Nyanjom SG, Tare C, Wamunyokoli F, Obiero GF. Expression levels of odorant receptor genes in the savanna tsetse fly, Glossina morsitans morsitans. Journal of Medical Entomology. 2018;55(4):855-61. Basrur NS, De Obaldia ME, Morita T, Herre M, von Heynitz RK, Tsitohay YN, et al. Fruitless mutant male mosquitoes gain attraction to human odor. eLife. 2020;9:e63982. Pelletier J, Leal WS. Genome Analysis and Expression Patterns of Odorant-Binding Proteins from the Southern House Mosquito Culex pipiens quinquefasciatus. PLOS ONE. 2009;4(7):e6237. Kubo I, Fujita KI, Nihei KI. Antimicrobial activity of anethole and related compounds from aniseed. Journal of the Science of Food and Agriculture. 2008;88(2):242-7. MacLeod AJ, Pieris NM, de Troconis NG. Aroma volatiles of Cynara scolymus and Helianthus tuberosus. Phytochemistry. 1982;21(7):1647-51. Isidorov V, Jdanova M. Volatile organic compounds from leaves litter. Chemosphere. 2002;48(9):975-9. Azeem M, Rajarao GK, Nordenhem H, Nordlander G, Borg-Karlson AK. Penicillium expansum Volatiles Reduce Pine Weevil Attraction to Host Plants. Journal of Chemical Ecology. 2013;39(1):120-8. Okada T. Systematic study of the early stages of Drosophilidae: Bunka Zugeisha; 1968. Leal HM, Hwang JK, Tan K, Leal WS. Attraction of Culex mosquitoes to aldehydes from human emanations. Sci Rep. 2017;7(1):17965. Plata C, Millán C, Mauricio JC, Ortega JM. Formation of ethyl acetate and isoamyl acetate by various species of wine yeasts. Food Microbiology. 2003;20(2):217-24. Castellan I, Duménil C, Rehermann G, Eisenstecken D, Bianchi F, Robatscher P, et al. Chemical and Electrophysiological Characterisation of Headspace Volatiles from Yeasts Attractive to Drosophila suzukii. Journal of Chemical Ecology. 2024;50(11):830-46. Erdei AL, Szelényi MO, Deutsch F, Rikk P, Molnár BP. Lure design for Drosophila suzukii based on liquid culture of fruit epiphytic yeasts: Comparing the attractivity of fermentation volatiles for seasonal morphs. Journal of Applied Entomology. 2022;146(6):773-85. Ameye M, Allmann S, Verwaeren J, Smagghe G, Haesaert G, Schuurink RC, et al. Green leaf volatile production by plants: a meta-analysis. New Phytologist. 2018;220(3):666-83. Gao XJ, Clandinin TR, Luo L. Extremely Sparse Olfactory Inputs Are Sufficient to Mediate Innate Aversion in Drosophila. PLOS ONE. 2015;10(4):e0125986. Yu F, Utsumi R. Diversity, regulation, and genetic manipulation of plant mono- and sesquiterpenoid biosynthesis. Cell Mol Life Sci. 2009;66(18):3043-52. Dweck HK, Ebrahim SA, Kromann S, Bown D, Hillbur Y, Sachse S, et al. Olfactory preference for egg laying on citrus substrates in Drosophila. Curr Biol. 2013;23(24):2472-80. Ronderos DS, Lin CC, Potter CJ, Smith DP. Farnesol-detecting olfactory neurons in Drosophila. J Neurosci. 2014;34(11):3959-68. McMeniman CJ, Corfas RA, Matthews BJ, Ritchie SA, Vosshall LB. Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans. Cell. 2014;156(5):1060-71. Vinauger C, Van Breugel F, Locke LT, Tobin KKS, Dickinson MH, Fairhall AL, et al. Visual-Olfactory Integration in the Human Disease Vector Mosquito Aedes aegypti. Curr Biol. 2019;29(15):2509-16.e5. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Additionalfile2.docx Additionalfile3.docx Additionalfile4.docx Additionalfile5.docx Additionalfile6.docx FigS1.pdf FigS2.pdf Graphicalabstarct.png Cite Share Download PDF Status: Published Journal Publication published 02 Jun, 2025 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 27 Apr, 2025 Submission checks completed at journal 17 Apr, 2025 First submitted to journal 13 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6128144","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":442329491,"identity":"0f9a51e5-443a-4452-b384-6e1fb620b410","order_by":0,"name":"Anna Laura Erdei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBADxgZm5gMMD0jUwpbAkECaFgYeA+K08M/uPfiBoeKebD87z+cPiW0M9vyEtEjcOZcswXCm2HhmM+82CaCWxJkNhPTcyDGQYGxLSNxwmHcbA1BLgsEBAjrkb+QY/2D8l5C4/zDPY7DD7AlpMbiRYybB2AC0hZmHAeQwxg2E3GUI1GKRcCzBeMZhNjOJhHMSiTMI2SIHdNiNDzUJsv39hx9/+FBmY8/fQMgaEEhAMCWIUT8KRsEoGAWjgBAAAByMPm8fdbftAAAAAElFTkSuQmCC","orcid":"","institution":"Swedish University of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"Laura","lastName":"Erdei","suffix":""},{"id":442329492,"identity":"ea916382-cb09-492a-b31a-5590ad4f58e9","order_by":1,"name":"Magdolna Olívia Szelényi","email":"","orcid":"","institution":"HUN-REN Centre for Agricultural Research","correspondingAuthor":false,"prefix":"","firstName":"Magdolna","middleName":"Olívia","lastName":"Szelényi","suffix":""},{"id":442329493,"identity":"399ebaef-ef11-45ed-8ea6-ff3b0c4a5060","order_by":2,"name":"Ferenc Deutsch","email":"","orcid":"","institution":"HUN-REN Centre for Agricultural Research","correspondingAuthor":false,"prefix":"","firstName":"Ferenc","middleName":"","lastName":"Deutsch","suffix":""},{"id":442329494,"identity":"a2d8da11-517e-4bca-b534-ff7d381ff125","order_by":3,"name":"Balázs Kiss","email":"","orcid":"","institution":"HUN-REN Centre for Agricultural Research","correspondingAuthor":false,"prefix":"","firstName":"Balázs","middleName":"","lastName":"Kiss","suffix":""},{"id":442329495,"identity":"81547ebb-91d6-46e7-bacf-c7a5c22eea42","order_by":4,"name":"Béla Péter Molnár","email":"","orcid":"","institution":"HUN-REN Centre for Agricultural Research","correspondingAuthor":false,"prefix":"","firstName":"Béla","middleName":"Péter","lastName":"Molnár","suffix":""}],"badges":[],"createdAt":"2025-02-28 11:23:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6128144/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6128144/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06850-8","type":"published","date":"2025-06-02T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80691460,"identity":"6f78bf11-48bf-495d-84ef-1185b751dfc5","added_by":"auto","created_at":"2025-04-16 05:35:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":359840,"visible":true,"origin":"","legend":"\u003cp\u003eThe antennal sensitivity of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e and \u003cem\u003ePhortica variegata\u003c/em\u003e to a panel of synthetic compounds was measured using GC-EAD. The responses were normalized by individuals by dividing each antennal response with the average of responses for the individual. The radius of symbols represents the relative response size. Color codes indicate the chemical classes of the selected synthetic compounds.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/f7a053d37071402a78bdb40b.png"},{"id":80691461,"identity":"5e8af1f7-81e9-4210-8118-4edac3f13871","added_by":"auto","created_at":"2025-04-16 05:35:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":137720,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal coordinate analysis (PCoA) based on the relative amplitude of antennal responses of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e and \u003cem\u003ePhortica variegata \u003c/em\u003eindividuals to synthetic volatiles. a) Clustering of individuals along MDS1 and MDS2. The relative corrected eigenvalues denoting the percentage contribution of each axis to the total variation is 0.58 for MDS1 and 0.25 for MDS2. b) The length and direction of vectors on the biplot shows the contribution of individual responses to the separation along MDS1 and MDS 2. Only compounds with eigenvalues lower than -0.3 or higher than 0.3 are plotted.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/d537d4876d88abde67b508cc.png"},{"id":80691467,"identity":"a7f02865-de48-4772-b98d-f5fa43d922b9","added_by":"auto","created_at":"2025-04-16 05:35:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":193116,"visible":true,"origin":"","legend":"\u003cp\u003eThe antennal responses of male \u003cem\u003ePhortica variegata \u003c/em\u003eto components of volatile sample of red wine-apple cider vinegar bottle traps (W+ACV), and ecologically relevant volatile blends (human body odour, deer, bear and fox faeces). The relative peak areas of compounds are shown using a color scale whereas response sizes are shown as the radius of circles.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/69d0802c8770292fddb9c664.png"},{"id":80691466,"identity":"6a7938da-570c-467f-950c-4373cf761f26","added_by":"auto","created_at":"2025-04-16 05:35:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116819,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of female and male antennal responses of \u003cem\u003ePhortica variegata \u003c/em\u003eto fox feces volatiles. Section of the FID chromatogram of the fox feces volatile sample is plotted against the average responses of \u003cem\u003eP. variegata\u003c/em\u003e individuals. The numbers represent the following active components: (1) RI738, (2) RI748, (3) 2-hexanone, (4) butyl acetate, (5) 3-methyl butanoic acid, (6) 3-heptanone, (7) RI900, (8) anisole, (9) RI948, (10) 6-methyl-2-heptanone, (11) dimethyl trisulfide, (12) phenol, (13) sulcatone, (14) nonanal. The standardized amplitude of average antennal response is visualized by the radius of yellow dots; the gray dots show the standard deviation of standardized antennal responses.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/935867acd1a0111c9e02f092.png"},{"id":84242693,"identity":"e06bffd1-850f-493d-bdde-2b50e5731ccc","added_by":"auto","created_at":"2025-06-09 16:11:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1460267,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/bc9881ea-7227-48d0-924c-d86b4f39f86d.pdf"},{"id":80692048,"identity":"6e759fa3-070a-4504-aaa2-025293511ee1","added_by":"auto","created_at":"2025-04-16 05:43:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":135180,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/07c91a8d93168a4027d9c023.docx"},{"id":80691459,"identity":"cb9d9326-b287-47fb-9247-741679b20c72","added_by":"auto","created_at":"2025-04-16 05:35:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21376,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/7c2ab9a4773eb23b1e052833.docx"},{"id":80692564,"identity":"a8c0f0d2-d589-4c30-87fe-ef9bd7760165","added_by":"auto","created_at":"2025-04-16 05:59:52","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13874,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/4d7ad8d43c4e843efb6bb238.docx"},{"id":80692050,"identity":"9b88caa2-6b53-4f70-8a02-5664477ea9dd","added_by":"auto","created_at":"2025-04-16 05:43:52","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":27484,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/f5c50608ac280cab3fc4a3ac.docx"},{"id":80691469,"identity":"f0debfb3-a322-4d71-9249-017c525b848e","added_by":"auto","created_at":"2025-04-16 05:35:52","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10935,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile5.docx","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/62bb75f27e5b39bfb5bb74c8.docx"},{"id":80691471,"identity":"0a5b98ba-3adc-4026-bf2f-4abc622e0e56","added_by":"auto","created_at":"2025-04-16 05:35:52","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":20918,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile6.docx","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/73b64f53725303631b9eabd0.docx"},{"id":80691142,"identity":"d3693c80-71ee-4614-8512-5e53343d42ad","added_by":"auto","created_at":"2025-04-16 05:27:52","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":21660,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/a3822524f269420e4ecbd67f.pdf"},{"id":80691464,"identity":"6dadc35f-fcc4-4337-9d2c-aaefa519fc1e","added_by":"auto","created_at":"2025-04-16 05:35:52","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":21480,"visible":true,"origin":"","legend":"","description":"","filename":"FigS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/18df8537da2660d6b8accc4c.pdf"},{"id":80692051,"identity":"3844e58c-3074-41dc-9c0f-518f0aa28c0b","added_by":"auto","created_at":"2025-04-16 05:43:52","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":128067,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstarct.png","url":"https://assets-eu.researchsquare.com/files/rs-6128144/v1/96da2f26c7891b46380e981c.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Olfactory responses of Phortica variegata (Drosophilidae, Steganinae) the emerging vector of Thelazia callipaeda (Rhabditida, Thelaziidae) to ecologically relevant volatiles","fulltext":[{"header":"Background","content":"\u003cp\u003eDrosophilid fruit flies are undoubtedly among the most studied organisms in the world. However, due to their lack of agricultural and veterinary importance, the fungivorous and zoophilic flies of the closely related subfamily Steganinae\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Drosophilidae) have received little interest so far. This perspective began to change when the role of \u003cem\u003ePhortica\u0026nbsp;\u003c/em\u003especies in the transmission of ocular nematosis was recognised.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePhortica variegata\u0026nbsp;\u003c/em\u003eis native to Europe [1] and is known to feed on the tears of wild and domestic carnivores (foxes, beech martens, wild cats and dogs), lagomorphs, and humans [2]. Over the past decade, ocular nematosis caused by \u003cem\u003eThelazia callipaeda\u0026nbsp;\u003c/em\u003e(also called oriental eyeworm, Rhabditida, Thelaziidiiae), an ocular nematode native to Southeast Asia [3], has sharply increased and has become a growing public health concern in Europe.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003ebecame an important vector of \u003cem\u003eT. callipaeda\u0026nbsp;\u003c/em\u003ewhen the nematode arrived in Europe. In its native habitat, \u003cem\u003eT. callipaeda\u003c/em\u003e is mainly transmitted by other species of the genus Phortica, such as \u003cem\u003eP. magna\u003c/em\u003e and \u003cem\u003eP. okadaii\u003c/em\u003e. In Europe, however, \u003cem\u003eP. variegata\u003c/em\u003e has emerged as the main vector [4]. Climate change and warmer winters have expanded the range and activity periods of this species by supporting overwintering and thereby facilitating the spread of \u003cem\u003eT. callipaeda\u003c/em\u003e as well \u0026nbsp;[5, 6]. Larvae of several Phortica species have been found to develop in fermenting tree sap [7, 8]. The distribution of \u003cem\u003eP. variegata\u003c/em\u003e is associated with oak forests, and it has been shown that a chestnut-based rearing medium is suitable for larval development [9]. Studying dipteran guilts utilizing small-scale forestine food resources, Papp (2002) observed found that adult \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003e were collectible around visited fox faeces and rotten fungi raising the possibility that such microhabitats are sites visited used for mating or, oviposition or feeding [10]. Additionally, larvae of other species in this subfamily were shown to develop on decaying plant matter and fungal substrates [8] that indicate \u003cem\u003eP. variegata\u003c/em\u003e may also use similar substrates as breeding sites. Little is known about the vectoring behavior, apart from the fact that \u003cem\u003eT. callipaeda\u003c/em\u003e is only found in males and that females were not observed to feed on tears \u0026nbsp; [11]. This behavioral dimorphism is in contrast with all other known vector insects, where zoophagy and vectoring is either entirely female-linked or exhibited by both sexes [11]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInsects rely primarily on the sense of smell to find food, mates and oviposition sites in their complex environment. Their olfactory system is tuned to filter out \u0026ldquo;background noise\u0026rdquo; and detect volatile signals relevant for host recognition [12]. Chemical ecological research of vector insect olfaction supports control solutions by revealing the olfactory cues that modulate vector behavior. The identification of attractants and repellents can lead to new vector-control solutions such as repellent formulations, and baits for monitoring and mass trapping. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. variegata\u003c/em\u003e belongs to the Drosophilidae family, where adaptation to new food sources was shown to be reflected in corresponding changes in the olfactory system; \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, a species feeding and ovipositing on overripe fruits, has high sensitivity for fermentation volatiles [7, 13], herbivorous \u003cem\u003eScaptomyza flava\u003c/em\u003e has reduced sensitivity to those and increased sensitivity to leaf volatiles [14, 15]. and \u003cem\u003eD. sechellia\u003c/em\u003e feeding on toxic Morinda fruits is attracted to short-chain fatty acids found in these fruits that repel other drosophilids [16] .\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBoth sexes of \u003cem\u003eP. variegata\u003c/em\u003e can be captured with fruit baits and vinegar and wine baits similar to \u003cem\u003eD. melanogaster\u003c/em\u003e [17-19], although the majority of flies caught with these baits are females, while those caught around the eyes are exclusively males [4, 17]. It was recently shown that supplementing vinegar wine baits with carvacrol abolishes this attraction [19]. Although, currently there is limited information available on the olfactory repertoire of species belonging to the Steganinae subfamily it can be hypothesized that the zoophilic behavior exhibited by \u003cem\u003eP. variegata\u003c/em\u003e and the indicated association with microbial substrates can be accompanied by adaptations of the olfactory system.\u003c/p\u003e\n\u003cp\u003eWhile there are no other known drosophilids that exhibit attraction to mammalian hosts a useful parallel can be drawn with mosquitoes and tsetse flies, the most extensively studied insect vectors. The evolution of zoophily in mosquitoes has been directly linked to olfactory adaptations. Host-seeking female mosquitoes rely on a heightened sensitivity to mammalian body odours [20-24]. L-lactic acid attracts \u003cem\u003eAedes aegypti\u003c/em\u003e females and has been shown to be involved in human discrimination, as it is more abundant in human sweat than that of other mammals [20, 21]. It was also shown that an increased sensitivity to sulcatone is linked to human preference in \u003cem\u003eA. aegypti\u003c/em\u003e where the increased expression of a sulcatone-sensitive odorant receptor, AaegOr4 supports host discrimination \u0026nbsp;[24].\u003c/p\u003e\n\u003cp\u003eAdditionally, the detection of short-chain saturated aldehydes in skin emissions [22, 23] and fluctuating CO₂ levels, which enhance \u003cem\u003eA. aegypti\u003c/em\u003e\u0026apos;s sensitivity to human odors [25, 26] have been shown to play key roles in identifying mammalian hosts. These findings in mosquitoes and the well-described olfactory adaptation in other drosophilid species suggest that similar mechanisms may underlie the zoophilic behavior observed in \u003cem\u003eP. variegata\u003c/em\u003e, where olfactory adaptations could facilitate host recognition.\u003c/p\u003e\n\u003cp\u003eCurrently, no efficient or species-specific attractants or repellents are available for \u003cem\u003ePhortica variegata\u003c/em\u003e, posing a challenge for the development of targeted vector control strategies. To our knowledge, the chemical ecology of \u003cem\u003eP. variegata\u003c/em\u003e, or any species within the Steganinae subfamily, remains entirely unexplored. \u0026nbsp;However, the recent annotation of odorant receptor genes in the \u003cem\u003eP. variegata\u003c/em\u003e genome [27] opens the possibility to explore olfactory mechanisms underlying tear feeding behavior. Given its attraction to mammals and fermentation-based baits, we hypothesized that \u003cem\u003eP. variegata\u003c/em\u003e may rely on olfactory cues similar to those used by both mosquitoes and drosophilids to identify mammalian hosts and suitable microbial habitats. To investigate these hypotheses, we conducted olfactory investigations of \u003cem\u003eP. variegata\u003c/em\u003e using gas chromatography coupled with electroantennographic detection (GC-EAD) comparing the response profile of both sexes to that of \u003cem\u003eD. melanogaster\u003c/em\u003e using a panel of selected synthetic compounds and identified volatile components from host-related volatile sources and fermentation baits that are detected by the antennae of this species.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eCollection of experimental animals and taxonomic identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. variegata m\u003c/em\u003eales were collected by netting around the eyes of human collectors using hand-held aquarium nets in forest habitats around the outskirts of Budapest at \u0026Ouml;rd\u0026ouml;g\u0026aacute;rok (47.54254\u0026deg; N, 18.94572\u0026deg; E ), Iluska spring (47.64546\u0026deg; N, 18.86779\u0026deg; E) \u0026nbsp;and Piliscsaba (47.63866\u0026deg; N, 18.85353\u0026deg;E) during the early afternoon, at temperatures around 19 \u0026deg;C \u0026plusmn; 8\u0026deg;C. Females were captured using apple cider vinegar-baited live traps at the same locations. Animals were transferred into closed but not air-tight humidified vials using an aspirator. Species-level identification was based on the morphological taxonomic keys described by B\u0026auml;chli et al. 2005 in The Drosophilidae (Diptera) of Fennoscandia and Denmark [28] using methods identical to Kerezsi et al. 2019 [29] . Adults were sexed and placed individually in glass jars where access to apple fruit slices as food source was provided. The adults were kept on a 16/8 light cycle at room temperature (25 \u0026plusmn;2\u0026deg;C) and at relative humidity of 50 \u0026plusmn;5%. The wild type \u003cem\u003eD. melanogaster\u003c/em\u003e used in the experiments were reared on a modified sugar-yeast-corn meal diet [30] under the same environmental conditions as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of synthetic mixtures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA preliminary comparison of antennal sensitivities of the two fruit fly species was done by testing synthetic mixtures using electroantennography coupled gas chromatography. The 47 components included in the mixtures (Table S1) were selected based on known ligands of D. melanogaster odorant receptors listed in DoOR, the online database of D. melanogaster odorant receptor responses [31], as well as on reported attractants involved in host-seeking behavior of blood-feeding vector insects such as mosquitoes and tsetse flies [32-40]. All compounds were diluted in HPLC grade hexane to a concentration of 100 ng/\u0026micro;L\u0026nbsp;and recordings with only solvent injected, were also conducted as control. The purity and manufacturers of synthetic compounds are listed in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVolatile collection methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh faecal samples were collected from red fox (\u003cem\u003eVulpes vulpes\u003c/em\u003e), brown bear (\u003cem\u003eUrsus arctos\u003c/em\u003e) and red deer (\u003cem\u003eCervus elaphus\u003c/em\u003e) in the Budakeszi Wildlife Park. Human body odour was sampled from two female volunteers (volunteers did not use scented soap or deodorant 24 hours prior to volatile sampling). The armpits and upper arms of the volunteers were rubbed with medical gauze for 5 minutes, which was later used to collect volatiles.\u003c/p\u003e\n\u003cp\u003eFor wine-apple cider vinegar (W+ACV) headspace sampling, 2 dL of apple cider vinegar and 1 dL of red wine were mixed in a glass beaker. The samples were placed in oven bags (35 x 43 cm, Hewa). Volatile sampling was performed in the laboratory at room temperature. The incoming air was filtered by a carbon air inlet. The air stream was drawn from the oven bag through an activated carbon volatile trap (5 mg cartridge, Brechb\u0026uuml;hler AG, Switzerland) at a flow rate of 500 mL/min for 4 hours.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe volatile traps were eluted with 200 mL methylene chloride (Sigma Aldrich, HPLC grade etc.). Prior to volatile collection the active carbon airstream filters were cleaned at 200 \u0026deg;C and the volatile traps were cleaned in a series of 2 mL methanol, 2 mL acetone, 2 mL hexane and 2 mL methylene chloride and heated up to 100 \u003cem\u003e\u0026deg;\u003c/em\u003eC for 12 hours. The volatile sampling was launched within 40 minutes of collecting the faecal and human body odour samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGas chromatography coupled mass spectrometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe volatile extracts were analyzed by gas chromatography coupled with mass spectrometry (GC-MS, Agilent 6890 GC and 5975 MS, Agilent Technologies) equipped with HP-5 UI capillary column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m, J\u0026amp;W Scientific, Folsom, CA, USA). 2 \u0026micro;l of volatile extracts were autoinjected into the split/splitless injection port operated in splitless mode heated to 270 \u0026deg;C using a 1 min splitless time. \u0026nbsp;Helium was used as a carrier gas with a flow rate of 1 mL/min. The initial oven temperature was held at 50 \u0026deg;C for 1 minute then increased by 10 \u0026deg;C/min to 270 \u0026deg;C. The final temperature was held for 10 minutes. The mass spectrometer source was operating at 250 \u0026deg;C in electron ionization mode 70 eV and the detector scanned in the 29-300 m/z range.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe GC-MS results were analyzed using Agilent Mass Hunter B.08.00, the peaks were manually integrated. Compounds were tentatively identified by matching their mass spectra with those found in MS Libraries (NIST21 and Wiley12). Identifications were also verified by comparing calculated Kov\u0026aacute;ts indices (KI) using C8-C20 alkane calibration standard to those found in NIST WebBook database, where only references using authentic standards were considered and key compounds were also identified using authentic synthetic standards (Table S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGas chromatography coupled electroantennography\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biologically active components of volatile extracts were identified using gas chromatography coupled electroantennographic detection (GC-EAD). The Agilent 6890N gas chromatograph (GC) was equipped with an HP-5 UI capillary column (30 m \u0026times; 0.32 mm \u0026times; 0.25 \u0026micro;m, J\u0026amp;W Scientific, Folsom, CA, USA). 2 \u0026micro;l of volatile extracts were manually injected into the injection port of the gas chromatograph, operated in splitless mode and heated to 230 \u0026deg;C using a 1 min splitless time. The carrier gas was helium and the column flow was 4 mL/min. The initial oven temperature was held at 50 \u0026deg;C for 1 minute then in the first ramp increased by 10 \u0026deg;C/min to 270 \u0026deg;C in the second ramp by 30 \u0026deg;C/min to 230 \u0026deg;C. The final temperature was held for 5 minutes.\u003c/p\u003e\n\u003cp\u003eThe GC effluent was split in a low dead volume Graphpack 3D/2 four-way splitter. Two non-coated deactivated fused silica capillary columns (100 cm\u0026thinsp;\u0026times;\u0026thinsp;0.32 mm) were connected to the four-way splitter, one led to the flame ionization detector (FID) heated to 300 \u0026deg;C and the other line was fitted into a transfer line heated to 235 \u0026deg;C (Syntech, Kirchzarten, Germany). The capillary column protruded from the heated transfer line into an inert glass tube (10 mm I.D.) that had a charcoal-filtered and humidified airflow of 1 L/min transferring the effluent over the antennal preparation.\u003c/p\u003e\n\u003cp\u003eFemale and male adults of \u003cem\u003eP. variegata\u003c/em\u003e and \u003cem\u003eD. melanogaster\u003c/em\u003e were immobilized in 200 mL pipette tips. The tip of the pipette was cut and the animal was pushed forward until half of the eye was uncovered in the pipette tip but the proboscis was still covered. The silver/silver chloride electrodes were immersed in Ringer solution in two finely pulled glass capillaries. The glass capillary of the reference electrode was inserted into one of the eyes of the restrained fly and the glass capillary covering the recording electrode was pushed to hold firm contact with the dorsomedial region of the funiculus. The antennal signal was amplified 10 times, and the analog-digital conversion was done by \u0026nbsp;IDAC-2 (Syntech). The recording was done simultaneously with the FID signal using GC-EAD software (GC-EAD 2014, vers. 1.2.5, Syntech). The eight synthetic mixtures were tested sequentially on the same individuals in a randomized order. Before each GC-EAD run, a glass Pasteur-pipette loaded with a 100 ng 1-hexanol in 10 \u0026micro;l mineral oil was used as a test stimulus to assess the quality of preparation and electric contact. The synthetic mixes were tested on at least 3 specimens of both species and both sexes. The volatile extracts were tested on male \u003cem\u003eP. variegata\u003c/em\u003e specimens except for fox faeces, which was tested on both females and males.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData processing and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis and data visualization, was performed using R (v. 4.2.0) in RStudio (RStudio Team (2023 v. 6.0.421). Similarities and dissimilarities between the antennal response profile of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003emelanogaster\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP\u003c/em\u003e. \u003cem\u003evariegata\u0026nbsp;\u003c/em\u003eindividuals to synthetic compounds were investigated by Principal Coordinates Analysis (PCoA) using the \u003cem\u003ecapscale\u003c/em\u003e function and by Non-Metric Multidimensional Scaling (NMDS) of the \u003cem\u003evegan\u0026nbsp;\u003c/em\u003epackage (v. 2.6-4) [41] \u0026nbsp;using Jaccard dissimilarity as a distance measure on non-binary data. The responses were standardized across individuals by dividing the responses by the average of responses for the individual. The responses were also standardized across each compound by Z-scoring: the average response to the compound is subtracted from the response of each individual and divided by the standard deviation of responses to the compound. Permutational multivariate analysis of variance (PERMANOVA) was performed on the Jaccard dissimilarity as a distance measure to compare groups using \u003cem\u003eadonis2\u003c/em\u003e function of the \u003cem\u003evegan\u0026nbsp;\u003c/em\u003epackage (v. 2.6-4) [41] \u0026nbsp;and the significance values for multiple comparisons was adjusted by Benjamini-Hochberg correction (P-value adj).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo identify differences between responses of species and between responses of sexes to individual compounds, we used multi-level pattern analysis by the \u003cem\u003emultipatt\u003c/em\u003e function of \u003cem\u003eindicspecies\u003c/em\u003e package [42] \u0026nbsp;and adjusted \u003cem\u003eP\u003c/em\u003e-values using Benjamini-Hochberg correction using the \u003cem\u003ep.adjust\u0026nbsp;\u003c/em\u003efunction from package \u003cem\u003estats\u003c/em\u003e. All figures were visualized using geom_point (Fig. 1-4), geom_segment (Fig. 2.) geom_tile (Fig.3.) and geom_line (Fig. 4.) functions of the \u0026nbsp;\u003cem\u003eggplot2\u0026nbsp;\u003c/em\u003ev.3.5.1 [43] \u0026nbsp;package.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe first assessed differences in antennal responses between \u003cem\u003eP. variegata\u003c/em\u003e and \u003cem\u003eD. melanogaster\u003c/em\u003e using synthetic compounds. These were selected based on their detection by olfactory receptors of \u003cem\u003eD. melanogaster\u003c/em\u003e (as listed in the DoOR database [31]), as well as on their known roles in modulating host-seeking behavior in blood-feeding dipterans, such as mosquitoes and tsetse flies [32-40]. Both species were sensitive to aliphatic esters. However, \u003cem\u003eP. variegata\u003c/em\u003e showed no antennal responses to the monoterpenoids and sesquiterpenoids tested, with the exception of linalool (Fig. 1, Fig. S1).\u003c/p\u003e\n\u003cp\u003ePCoA (Fig. 2) and NMDS (Fig. S2) were applied using non-binary Jaccard dissimilarity to assess divergence in antennal response profiles. Individuals from the same species clustered closely together, whereas\u003cem\u003e\u0026nbsp;P. variegata\u0026nbsp;\u003c/em\u003eand \u003cem\u003eD. melanogaster\u003c/em\u003e separated along MDS1. The eigenvalues for MDS1 (0.58) and MDS2 (0.25) indicate that MDS1 explained the majority of the variation. PERMANOVA confirmed significant divergence in response profiles between species (F₁ = 7.386, P-value = 0.002). Pairwise comparisons with Benjamini-Hochberg correction also supported this difference (P-value adj = 0.004).\u003c/p\u003e\n\u003cp\u003eThe multi-level pattern comparison (Table S3) has shown that \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003ehad higher relative responses to , propyl acetate, ethyl propanoate, butyl propanoate, anisole, 3-octanone, nonanal and decanal than \u003cem\u003eD. melanogaster\u003c/em\u003e. \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003emales had higher responses to phenol, 3-octanone, and sulcatone (6-methyl-5-hepten-2-one) than females. Although males exhibited numerically higher responses to nonanal and decanal, this difference was not statistically significant (P-value adj \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003eGiven that adult male \u003cem\u003eP. variegata\u003c/em\u003e are captured in fermentation baits, associated with mammalian faeces, and attracted to mammals, further GC-EAD recordings were performed using ecologically relevant volatile samples, including W+ACV, and faeces from bear, deer, and fox, as well as human body odour. Electrophysiological recordings revealed antennal responses to 31 volatile components (Fig. 3).\u003c/p\u003e\n\u003cp\u003eThe detected components of mammalian samples closely resembled those of W+ACV (Table S4), as evidenced by similar antennal response profiles (Fig. 3). Ethyl lactate and isoamyl acetate were detected exclusively in W+ACV volatiles and elicited antennal responses. Phenol and dimethyl trisulfide were only present and were sensed in the bear and fox feces samples. Nonanal, sulcatone, and decanal were components of all samples (Table S4). Nonanal and sulcatone elicited antennal response in all volatile samples, while decanal did not elicit antennal responses in fox faeces volatiles, likely due to its low abundance (Fig. 3). Anisole and hexanoic acid elicited antennal responses but the former was only present in the volatile sample of fox faeces and the latter in human body odour samples.\u003c/p\u003e\n\u003cp\u003eSince \u003cem\u003eP. variegata\u003c/em\u003e has been observed to be present on fox faeces [10], we tested whether antennal responses of sexes differs to volatiles emitted from fox faeces (Fig. 4.). \u0026nbsp;Sulcatone failed to elicit antennal responses in females, but response amplitudes to other volatiles did not differ significantly between sexes (Fig. 4).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe compared antennal responses of \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003eand \u003cem\u003eD. melanogaster\u003c/em\u003e to a panel of synthetic odorants known to activate olfactory receptors in \u003cem\u003eD. melanogaster\u003c/em\u003e and influence host-seeking in other mammal-attracted dipterans. While both species responded to aliphatic esters, \u003cem\u003eP. variegata\u003c/em\u003e showed limited responses to monoterpenoids and sesquiterpenoids and multivariate analyses revealed clear species- and sex-related divergence in response profiles.\u003c/p\u003e\n\u003cp\u003eThe behavioral dimorphism, in which only male flies feed on tears, may also be reflected in sex-specific olfactory sensitivity, similar to \u003cem\u003eA. aegypti\u0026nbsp;\u003c/em\u003ewhere females express a higher number of ORs compared to males, likely linked to their need to recognize hosts for blood feeding [44]. To test this hypothesis, we compared the antennal response of female and male \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003eusing a panel of synthetic compounds (Fig. 1, Fig. 2). Multi-level pattern analysis revealed significant differences in response amplitudes between the sexes (Table S3.).\u003c/p\u003e\n\u003cp\u003eThe antenna of males exhibited significantly higher responses to synthetic phenol, 3-octanone, and sulcatone. Sulcatone was found in all tested relevant volatile samples (Fig. 3). Moreover, while sulcatone elicited responses in synthetic blends, it failed to trigger antennal responses in females when derived from fox faeces (Fig. 4). These results could be explained by the higher antennal sensitivity of male \u003cem\u003ePhortica\u0026nbsp;\u003c/em\u003eflies, as the amount of sulcatone in fox faeces was possibly below the detection threshold for females. As sulcatone is abundant in human skin and animal emissions [45, 46] \u0026nbsp;a heightened sensitivity might allow males to detect and locate mammalian hosts more effectively than females. Sexual dimorphism in peripheral olfaction is a well-documented phenomenon in insects, often reflected in differences in antennal structure, the number of odorantreceptors, and sensitivity to specific compounds [47-49] . For instance, sexual dimorphism in olfaction of vector insects has been observed in \u003cem\u003eA.\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e [50], \u003cem\u003eCulex pipiens quinquefasciatus\u0026nbsp;\u003c/em\u003e[51] \u003cem\u003e\u0026nbsp;\u003c/em\u003e and \u003cem\u003eAnopheles gambiae\u003c/em\u003e [47]. Notably, \u003cem\u003eA. aegypti\u003c/em\u003e strains that prefer human hosts over cattle have been shown to overexpress the odorant receptor AaegOr4, which has a high affinity for sulcatone [24]. However, no significant sexual dimorphism was observed in the expression levels of this receptor [44] and the antennal sensitivity to sulcatone was not compared yet. To determine if male \u003cem\u003eP. variegata\u003c/em\u003e files are more sensitive to sulcatone and if this compound is important in host-seeking, further studies involving dose-dependency measurements and behavioral experiments are needed.\u003c/p\u003e\n\u003cp\u003eDifferences observed for phenol in synthetic blends were not significant in fox faeces samples, likely due to dose dependency, as the fox faeces samples were more concentrated than the synthetic blends tested based on comparison FID traces. This observation again highlights the importance of considering dose dependency when evaluating olfactory responses and underscores the need for carefully controlled experiments to accurately assess sex-specific differences in chemosensation and behavior, as demonstrated in the case of both sulcatone and phenol with female\u003cem\u003e\u0026nbsp;Culicoides nubeculosus\u0026nbsp;\u003c/em\u003ewhere attraction and repellence are strictly dose-dependent\u003cem\u003e\u0026nbsp;[46]\u003c/em\u003e. The observed sensory differences between sexes of \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003ecould explain the behavioral dimorphism, but further experiments are needed to establish a causal link between the detection of these compounds and their effect on behavior.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSpecies specific differences were observed between the response profiles of \u003cem\u003eD\u003c/em\u003e. \u003cem\u003emelanogaster\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003eto synthetic compounds as they separated along the first axis of PCoA (Fig. 1). For several other compounds, a dose-dependent response was observed in the ecologically relevant volatiles samples (Fig. 3). Compared to \u003cem\u003eD. melanogaster\u003c/em\u003e both sexes of \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003eshowed an increased sensitivity to anisole (methoxybenzene) which is main constituent of anise seed essential oil [52] \u0026nbsp;and were described to be present in essential oils prepared from other plants [53] . Intriguingly, anisole is also emitted from decomposing leaf litter, such as that of poplar [54] \u0026nbsp;and can be emitted by microbes such as \u003cem\u003ePenicillium expansum\u003c/em\u003e during degradation of lignin [55]. It was reported that \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003eadults are feeding on fermenting tree sap [8, 56], which might be a rich source of anisole and related methoxybenzenes. Surprisingly, anisole was also a minor component of fox faeces headspace, and both sexes of \u003cem\u003eP. variegata\u003c/em\u003e responded to this component of the volatile extracts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe antennae of \u003cem\u003eP. variegata\u003c/em\u003e were more sensitive than those of \u003cem\u003eD. melanogaster\u003c/em\u003e to several common volatile compounds emitted from fermented substrates, including ethyl and butyl propanoate, propyl acetate, 3-octanone, nonanal and decanal. These compounds are found in a wide range of natural sources. Nonanal and decanal are also major components of human body odor and they were shown to be attractive to \u003cem\u003eCulex\u0026nbsp;\u003c/em\u003emosquitoes [57] , while the high ratio of these compounds was shown to decrease the attraction of \u003cem\u003eA. aegypti\u003c/em\u003e to human body emissions [23]. Several aliphatic esters from the synthetic blend, are often associated with fermenting plant materials, yeasts, and ripening fruits [58-60] , and similar to \u003cem\u003eD. melanogaster\u0026nbsp;\u003c/em\u003eantennae, those of \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003eresponded to isoamyl and isobutyl acetate and had a significantly higher sensitivity to ethyl- and butyl propanoate and propyl acetate.\u003c/p\u003e\n\u003cp\u003eInterestingly,\u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003eexhibited a weaker response to (\u003cem\u003eE\u003c/em\u003e)-2-hexenal and (\u003cem\u003eE\u003c/em\u003e)-3-hexenol which are characteristic green leaf volatiles emitted upon mechanical damage of plant tissues [61]. These compounds are repellent for \u003cem\u003eD. melanogaster\u003c/em\u003e and were hypothesized to be related to discrimination of ripe fruits from ripening ones that are unsuitable for oviposition [62]. Furthermore, \u003cem\u003eP. variegata\u0026nbsp;\u003c/em\u003eantennae did not respond to terpenoids selected from the DOOR, except males showing a weak response to linalool.\u0026nbsp;\u0026beta;-caryophyllene, farnesol and \u0026alpha;-humulene are common sesquiterpenoid compounds in plant volatile emissions [63]. \u0026beta;-caryophyllene, \u0026alpha;-terpineol and \u0026alpha;-humulene are major ligands of OR19a expressed in trichoid sensilla [64] and farnesol is major ligand of Or83c expressed in intermediate sensilla [65] \u0026nbsp; on the antennae of \u003cem\u003eD. melanogaster,\u003c/em\u003e several other Ors such as Or69a are also involved in the detection of terpenoids. Bastide et al. (2024) identified two orthologs of OR19a and one of Or83c in the genome of \u003cem\u003eP. variegata\u003c/em\u003e, however, the functionality of these genes, their expression pattern as well as their main ligands are currently unknown [27].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince terpenoids are detected by multiple odorant receptors in \u003cem\u003eD. melanogaster\u003c/em\u003e, the lack of response in \u003cem\u003eP. variegata\u003c/em\u003e to several terpenoids may reflect ecological differences between the species, suggesting that \u003cem\u003eP. variegata\u003c/em\u003e relies less on the identification of plant-derived resources than \u003cem\u003eD. melanogaster\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe lower sensitivity of \u003cem\u003eP. variegata\u003c/em\u003e to several ubiquitous plant volatile compounds compared to \u003cem\u003eD. melanogaster\u003c/em\u003e and increased sensitivity to several volatile compounds common in microbial volatile emissions indicates that fungal and microbial substrates might be more important in the ecology of this species compared to \u003cem\u003eD. melanogaster\u003c/em\u003e. According to our current knowledge, many species belonging to this group are associated with fungi or feeding on decaying plant material. \u0026nbsp; Similarly to attraction of mosquitoes to their hosts [66, 67] , the attraction of \u003cem\u003eP. variegata\u003c/em\u003e to mammalian hosts can be based on otherwise common microbial volatiles combined with carbon dioxide or visual cues.\u003c/p\u003e\n\u003cp\u003eBased on this first report on the olfactory sensitivity of \u003cem\u003eP. variegata\u003c/em\u003e, the behavioral significance of ethyl and butyl propanoate, propyl acetate, 3-octanone, nonanal, decanal and sulcatone for females and males should be further evaluated in laboratory and field behavioral bioassays. The identification of new attractants can provide a basis for developing both monitoring and mass trapping solutions for the future management of this vector species.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe olfaction of insects is shaped by their environment and ecology. Our study demonstrates that \u003cem\u003eP\u003c/em\u003e. \u003cem\u003evariegata\u0026nbsp;\u003c/em\u003eexhibits sexual dimorphism in olfactory sensitivity, with males showing increased sensitivity to specific volatiles such as sulcatone, phenol, and 3-octanone which may help them locate mammalian hosts, aligning with their behavioral dimorphism in feeding on tears. Additionally, the comparative olfactory analysis with \u003cem\u003eD. melanogaster\u003c/em\u003e revealed that \u003cem\u003eP. variegata\u003c/em\u003e shows a stronger sensitivity to several microbial and yeast-related volatiles and a strongly reduced sensitivity to common plant volatile terpenoids, reinforcing the idea that its foraging ecology differs from that of \u003cem\u003eD. melanogaster\u0026nbsp;\u003c/em\u003eand fungal and microbial substrates might be more ecologically relevant for this species. The study highlights several antennally active volatiles that could be assessed in field and laboratory behavioral experiments to investigate their ecological roles and to potentially use them to develop monitoring and control strategies against this dipteran vector species.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eA. aegypti: Aedes aegypti, D. melanogaster: Drosophila melanogaster\u003c/em\u003e, GC-MS: Gas chromatography-mass spectrometry, GC-EAD: Gas chromatography-electroantennography, OR: odorant receptor, NMDS: Non-Metric Multidimensional Scaling, PCoA: Principal Coordinate Analysis, \u003cem\u003eP. variegata: Phortica variegata,\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Budakeszi Wildlife Park and its staff for providing the opportunity to collect samples from forestine mammals and for assisting us in the sampling procedure. \u0026nbsp;We also acknowledge Dora Varga for illustrations of odour sources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by an NKFIH Research Proposal FK 137579 of the National Research, Development and Innovation Office.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data utilized in this manuscript, along with the scripts to generate the figures, are available at https://figshare.com/s/5503fa00feca01ad50c\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eALE, MOSz, and BPM were involved in the design and conception of the work. FD and BK collected and maintained the laboratory animals and FD performed the taxonomic identification. ALE completed the GC-MS and GC-EAD measurements and statistical analysis and ALE and MOSz prepared the figures. ALE and MOSz wrote the manuscript and all authors revised the text.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo ethical approval is necessary, for human odor collection the authors were sampled.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo consent is necessary for publication, no human experiments were done.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMaca J. Revision of Palaearctic species of Amiota subge nus Photica (Diptera, Drosophilidae). Acta entomol bohemoslov. 1977;74:115-30.\u003c/li\u003e\n\u003cli\u003eBertos E, S\u0026aacute;nchez-Cerd\u0026aacute; M, Virg\u0026oacute;s E, Gil-S\u0026aacute;nchez JM, Mole\u0026oacute;n M. Thelazia callipaeda as a potential new threat to european wildcats: insights from an eco-epidemiological study. Veterinary Research Communications. 2023;47(4):2153-60.\u003c/li\u003e\n\u003cli\u003eRossi L, Bertaglia PP. Presence of Thelazia callipaeda Railliet \u0026amp; Henry, 1910, in Piedmont, Italy. Parassitologia. 1989;31(2-3):167-72.\u003c/li\u003e\n\u003cli\u003eOtranto D, Cantacessi C, Testini G, Lia RP. Phortica variegata as an intermediate host of Thelazia callipaeda under natural conditions: Evidence for pathogen transmission by a male arthropod vector. International Journal for Parasitology. 2006;36(10):1167-73.\u003c/li\u003e\n\u003cli\u003ePalfreyman J, Graham-Brown J, Caminade C, Gilmore P, Otranto D, Williams DJL. Predicting the distribution of Phortica variegata and potential for Thelazia callipaeda transmission in Europe and the United Kingdom. Parasites \u0026amp;amp; Vectors. 2018;11(1).\u003c/li\u003e\n\u003cli\u003ePombi M, Marino V, Jaenike J, Graham-Brown J, Bernardini I, Lia RP, et al. Temperature is a common climatic descriptor of lachryphagous activity period in Phortica variegata (Diptera: Drosophilidae) from multiple geographical locations. Parasites \u0026amp;amp; Vectors. 2020;13(1).\u003c/li\u003e\n\u003cli\u003eMarkow TA, O\u0026rsquo;Grady P. Reproductive ecology of Drosophila. Functional Ecology. 2008;22(5):747-59.\u003c/li\u003e\n\u003cli\u003eM\u0026aacute;ca J, Otranto D. Drosophilidae feeding on animals and the inherent mystery of their parasitism. Parasites \u0026amp; Vectors. 2014;7(1):516.\u003c/li\u003e\n\u003cli\u003eBernardini I, Poggi C, Manzi S, Bezerra-Santos MA, Beugnet F, Fourie J, et al. Laboratory breeding of two Phortica species (Diptera: Drosophilidae), vectors of the zoonotic eyeworm Thelazia callipaeda. Parasites \u0026amp; Vectors. 2022;15(1):200.\u003c/li\u003e\n\u003cli\u003ePapp L. Dipterous guilds of small-sized feeding sources in forests of Hungary. Acta Zoologica Academiae Scientiarum Hungaricae. 2002;48(supplement 1):197-213.\u003c/li\u003e\n\u003cli\u003eOtranto D, Stevens JR, Cantacessi C, Gasser RB. Parasite transmission by insects: a female affair? Trends Parasitol. 2008;24(3):116-20.\u003c/li\u003e\n\u003cli\u003eSchr\u0026ouml;der R, Hilker M. The Relevance of Background Odor in Resource Location by Insects: A Behavioral Approach. BioScience. 2008;58(4):308-16.\u003c/li\u003e\n\u003cli\u003eBecher PG, Flick G, Rozpędowska E, Schmidt A, Hagman A, Lebreton S, et al. Yeast, not fruit volatiles mediate rosophila melanogaster attraction, oviposition and development. Functional Ecology. 2012;26(4):822-8.\u003c/li\u003e\n\u003cli\u003eGoldman-Huertas B, Mitchell RF, Lapoint RT, Faucher CP, Hildebrand JG, Whiteman NK. Evolution of herbivory in Drosophilidae linked to loss of behaviors, antennal responses, odorant receptors, and ancestral diet. Proc Natl Acad Sci U S A. 2015;112(10):3026-31.\u003c/li\u003e\n\u003cli\u003eWhiteman NK, Groen SC, Chevasco D, Bear A, Beckwith N, Gregory TR, et al. Mining the plant-herbivore interface with a leafmining Drosophila of Arabidopsis. Mol Ecol. 2011;20(5):995-1014.\u003c/li\u003e\n\u003cli\u003eAmlou M, Pla E, Moreteau B, David JR. Genetic analysis by interspecific crosses of the tolerance of Drosophila sechellia to major aliphatic acids of its host plant. Genetics Selection Evolution. 1997;29(4):511.\u003c/li\u003e\n\u003cli\u003eOtranto D, Brianti E, Cantacessi C, Lia RP, M\u0026aacute;ca J. The zoophilic fruitfly Phortica variegata: morphology, ecology and biological niche. Medical and Veterinary Entomology. 2006;20(4):358-64.\u003c/li\u003e\n\u003cli\u003eRoggero C, Schaffner F, B\u0026auml;chli G, Mathis A, Schnyder M. Survey of Phortica drosophilid flies within and outside of a recently identified transmission area of the eye worm Thelazia callipaeda in Switzerland. Veterinary parasitology. 2010;171(1-2):58-67.\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez MA, Bravo-Barriga D, Alarc\u0026oacute;n-Elbal P, \u0026Aacute;lvarez-Calero JM, Quero C, Ferraguti M, et al. Development of Novel Management Tools for Phortica variegata (Diptera: Drosophilidae), Vector of the Oriental Eyeworm, Thelazia callipaeda (Spirurida: Thelaziidae), in Europe. Journal of Medical Entomology. 2022;59(1):328-36.\u003c/li\u003e\n\u003cli\u003eAcree Jr F, Turner R, Gouck H, Beroza M, Smith N. L-Lactic acid: a mosquito attractant isolated from humans. Science. 1968;161(3848):1346-7.\u003c/li\u003e\n\u003cli\u003eZwiebel L, Takken W. Olfactory regulation of mosquito\u0026ndash;host interactions. Insect biochemistry and molecular biology. 2004;34(7):645-52.\u003c/li\u003e\n\u003cli\u003eIrish S, Moore S, Bruce J, Cameron M. Preliminary evaluation of a nonanal lure for collection of gravid Culex quinquefasciatus. Journal of the American Mosquito Control Association. 2014;30(1):37-41.\u003c/li\u003e\n\u003cli\u003eLogan JG, Birkett MA, Clark SJ, Powers S, Seal NJ, Wadhams LJ, et al. Identification of human-derived volatile chemicals that interfere with attraction of Aedes aegypti mosquitoes. Journal of chemical ecology. 2008;34:308-22.\u003c/li\u003e\n\u003cli\u003eMcBride CS, Baier F, Omondi AB, Spitzer SA, Lutomiah J, Sang R, et al. Evolution of mosquito preference for humans linked to an odorant receptor. Nature. 2014;515(7526):222-7.\u003c/li\u003e\n\u003cli\u003eDekker T, Geier M, Card\u0026eacute; RT. Carbon dioxide instantly sensitizes female yellow fever mosquitoes to human skin odours. J Exp Biol. 2005;208(Pt 15):2963-72.\u003c/li\u003e\n\u003cli\u003eMajeed S, Hill SR, Ignell R. Impact of elevated CO2 background levels on the host-seeking behaviour of Aedes aegypti. Journal of Experimental Biology. 2014;217(4):598-604.\u003c/li\u003e\n\u003cli\u003eBastide H, Legout H, Dogbo N, Ogereau D, Prediger C, Carcaud J, et al. The genome of the blind bee louse fly reveals deep convergences with its social host and illuminates Drosophila origins. Current Biology. 2024;34(5):1122-32.e5.\u003c/li\u003e\n\u003cli\u003eB\u0026auml;chli G, Viljoen F, Escher SA, Saura A. The drosophilidae (Diptera) of fennoscandia and Denmark: Brill; 2005.\u003c/li\u003e\n\u003cli\u003eKerezsi V, Kiss B, Deutsch F, Kontsch\u0026aacute;n J. First record of Blattisocius mali (Oudemans, 1929) in Hungary associated with the drosopilid fly Phortica semivirgo (M\u0026aacute;ca, 1977). Redia. 2019;102:69-72.\u003c/li\u003e\n\u003cli\u003eAshburner M. Drosophila: A Laboratory Handbook. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;nch D, Galizia CG. DoOR 2.0-comprehensive mapping of Drosophila melanogaster odorant responses. Scientific reports. 2016;6(1):21841.\u003c/li\u003e\n\u003cli\u003eMelo N, Wolff GH, Costa-da-Silva AL, Arribas R, Triana MF, Gugger M, et al. Geosmin attracts Aedes aegypti mosquitoes to oviposition sites. Current Biology. 2020;30(1):127-34. e5.\u003c/li\u003e\n\u003cli\u003eOwaga ML, Hassanali A, McDowell P. The role of 4-cresol and 3-n-propylphenol in the attraction of tsetse flies to buffalo urine. International Journal of Tropical Insect Science. 1988;9(1):95-100.\u003c/li\u003e\n\u003cli\u003eTakken W, Kline D. Carbon dioxide and 1-octen-3-ol as mosquito attractants. Journal of the American Mosquito Control Association. 1989;5(3):311-6.\u003c/li\u003e\n\u003cli\u003eVale G, Hall D, Gough A. The olfactory responses of tsetse flies, Glossina spp.(Diptera: Glossinidae), to phenols and urine in the field. Bulletin of Entomological Research. 1988;78(2):293-300.\u003c/li\u003e\n\u003cli\u003eVale G, Hall D. The role of 1-octen-3-ol, acetone and carbon dioxide in the attraction of tsetse flies, Glossina spp.(Diptera: Glossinidae), to ox odour. Bulletin of Entomological Research. 1985;75(2):209-18.\u003c/li\u003e\n\u003cli\u003eMagalh\u0026atilde;es-Junior JT, Barrouin-Melo SM, Corr\u0026ecirc;a AG, da Rocha Silva FB, Machado VE, Govone JS, et al. A laboratory evaluation of alcohols as attractants for the sandfly Lutzomyia longipalpis (Diptera: Psychodidae). Parasites \u0026amp; vectors. 2014;7:1-5.\u003c/li\u003e\n\u003cli\u003eDormont L, Mulatier M, Carrasco D, Cohuet A. Mosquito attractants. Journal of chemical ecology. 2021;47:351-93.\u003c/li\u003e\n\u003cli\u003eZhang H, Zhu Y, Liu Z, Peng Y, Peng W, Tong L, et al. A volatile from the skin microbiota of flavivirus-infected hosts promotes mosquito attractiveness. Cell. 2022;185(14):2510-22. e16.\u003c/li\u003e\n\u003cli\u003eHarraca V, Syed Z, Guerin P. Olfactory and behavioural responses of tsetse flies, Glossina spp., to rumen metabolites. Journal of Comparative Physiology A. 2009;195:815-24.\u003c/li\u003e\n\u003cli\u003eOksanen J. Vegan: community ecology package. R package version. 2015;2:3.\u003c/li\u003e\n\u003cli\u003eC\u0026aacute;ceres MD, Legendre P. Associations between species and groups of sites: indices and statistical inference. Ecology. 2009;90(12):3566-74.\u003c/li\u003e\n\u003cli\u003eWickham H, Sievert C. ggplot2: elegant graphics for data analysis: springer New York; 2009.\u003c/li\u003e\n\u003cli\u003eTallon AK, Hill SR, Ignell R. Sex and age modulate antennal chemosensory-related genes linked to the onset of host seeking in the yellow-fever mosquito, Aedes aegypti. Scientific Reports. 2019;9(1):43.\u003c/li\u003e\n\u003cli\u003eHinze A, Pelletier J, Ghaninia M, Marois E, Hill SR, Ignell R. Knockout of OR39 reveals redundancy in the olfactory pathway regulating the acquisition of host seeking in Anopheles coluzzii. Proceedings of the Royal Society B. 2023;290(2011):20232092.\u003c/li\u003e\n\u003cli\u003eIsberg E, Bray DP, Birgersson G, Hillbur Y, Ignell R. Identification of cattle-derived volatiles that modulate the behavioral response of the biting midge Culicoides nubeculosus. Journal of chemical ecology. 2016;42:24-32.\u003c/li\u003e\n\u003cli\u003ePitts RJ, Rinker DC, Jones PL, Rokas A, Zwiebel LJ. Transcriptome profiling of chemosensory appendages in the malaria vector Anopheles gambiae reveals tissue-and sex-specific signatures of odor coding. BMC genomics. 2011;12:1-17.\u003c/li\u003e\n\u003cli\u003eZhou S, Stone EA, Mackay TF, Anholt RR. Plasticity of the chemoreceptor repertoire in Drosophila melanogaster. PLoS genetics. 2009;5(10):e1000681.\u003c/li\u003e\n\u003cli\u003eNyanjom SG, Tare C, Wamunyokoli F, Obiero GF. Expression levels of odorant receptor genes in the savanna tsetse fly, Glossina morsitans morsitans. Journal of Medical Entomology. 2018;55(4):855-61.\u003c/li\u003e\n\u003cli\u003eBasrur NS, De Obaldia ME, Morita T, Herre M, von Heynitz RK, Tsitohay YN, et al. Fruitless mutant male mosquitoes gain attraction to human odor. eLife. 2020;9:e63982.\u003c/li\u003e\n\u003cli\u003ePelletier J, Leal WS. Genome Analysis and Expression Patterns of Odorant-Binding Proteins from the Southern House Mosquito Culex pipiens quinquefasciatus. PLOS ONE. 2009;4(7):e6237.\u003c/li\u003e\n\u003cli\u003eKubo I, Fujita KI, Nihei KI. Antimicrobial activity of anethole and related compounds from aniseed. Journal of the Science of Food and Agriculture. 2008;88(2):242-7.\u003c/li\u003e\n\u003cli\u003eMacLeod AJ, Pieris NM, de Troconis NG. Aroma volatiles of Cynara scolymus and Helianthus tuberosus. Phytochemistry. 1982;21(7):1647-51.\u003c/li\u003e\n\u003cli\u003eIsidorov V, Jdanova M. Volatile organic compounds from leaves litter. Chemosphere. 2002;48(9):975-9.\u003c/li\u003e\n\u003cli\u003eAzeem M, Rajarao GK, Nordenhem H, Nordlander G, Borg-Karlson AK. Penicillium expansum Volatiles Reduce Pine Weevil Attraction to Host Plants. Journal of Chemical Ecology. 2013;39(1):120-8.\u003c/li\u003e\n\u003cli\u003eOkada T. Systematic study of the early stages of Drosophilidae: Bunka Zugeisha; 1968.\u003c/li\u003e\n\u003cli\u003eLeal HM, Hwang JK, Tan K, Leal WS. Attraction of Culex mosquitoes to aldehydes from human emanations. Sci Rep. 2017;7(1):17965.\u003c/li\u003e\n\u003cli\u003ePlata C, Mill\u0026aacute;n C, Mauricio JC, Ortega JM. Formation of ethyl acetate and isoamyl acetate by various species of wine yeasts. Food Microbiology. 2003;20(2):217-24.\u003c/li\u003e\n\u003cli\u003eCastellan I, Dum\u0026eacute;nil C, Rehermann G, Eisenstecken D, Bianchi F, Robatscher P, et al. Chemical and Electrophysiological Characterisation of Headspace Volatiles from Yeasts Attractive to Drosophila suzukii. Journal of Chemical Ecology. 2024;50(11):830-46.\u003c/li\u003e\n\u003cli\u003eErdei AL, Szel\u0026eacute;nyi MO, Deutsch F, Rikk P, Moln\u0026aacute;r BP. Lure design for Drosophila suzukii based on liquid culture of fruit epiphytic yeasts: Comparing the attractivity of fermentation volatiles for seasonal morphs. Journal of Applied Entomology. 2022;146(6):773-85.\u003c/li\u003e\n\u003cli\u003eAmeye M, Allmann S, Verwaeren J, Smagghe G, Haesaert G, Schuurink RC, et al. Green leaf volatile production by plants: a meta-analysis. New Phytologist. 2018;220(3):666-83.\u003c/li\u003e\n\u003cli\u003eGao XJ, Clandinin TR, Luo L. Extremely Sparse Olfactory Inputs Are Sufficient to Mediate Innate Aversion in Drosophila. PLOS ONE. 2015;10(4):e0125986.\u003c/li\u003e\n\u003cli\u003eYu F, Utsumi R. Diversity, regulation, and genetic manipulation of plant mono- and sesquiterpenoid biosynthesis. Cell Mol Life Sci. 2009;66(18):3043-52.\u003c/li\u003e\n\u003cli\u003eDweck HK, Ebrahim SA, Kromann S, Bown D, Hillbur Y, Sachse S, et al. Olfactory preference for egg laying on citrus substrates in Drosophila. Curr Biol. 2013;23(24):2472-80.\u003c/li\u003e\n\u003cli\u003eRonderos DS, Lin CC, Potter CJ, Smith DP. Farnesol-detecting olfactory neurons in Drosophila. J Neurosci. 2014;34(11):3959-68.\u003c/li\u003e\n\u003cli\u003eMcMeniman CJ, Corfas RA, Matthews BJ, Ritchie SA, Vosshall LB. Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans. Cell. 2014;156(5):1060-71.\u003c/li\u003e\n\u003cli\u003eVinauger C, Van Breugel F, Locke LT, Tobin KKS, Dickinson MH, Fairhall AL, et al. Visual-Olfactory Integration in the Human Disease Vector Mosquito Aedes aegypti. Curr Biol. 2019;29(15):2509-16.e5.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"dipteran vector, olfactory responses, chemical ecology, GC-EAD, sexual olfactory dimorphism","lastPublishedDoi":"10.21203/rs.3.rs-6128144/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6128144/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003ePhortica variegata\u003c/em\u003e (Drosophilidae: Steganinae), native to Europe, has emerged as a major vector of ocular nematosis caused by \u003cem\u003eThelazia callipaeda \u003c/em\u003e(Rhabditida: Thelaziidae), following the nematode's introduction into Europe from its original habitat in Asia.\u003c/p\u003e\n\u003cp\u003eMales of \u003cem\u003eP. variegata\u003c/em\u003e transmit these nematodes through feeding on tears of mammals including wild and domestic carnivorous mammals (foxes, beech martens, wild cats and dogs), lagomorphs, and humans. Insect vectors strongly rely on volatile cues to identify suitable hosts. Due to the increasing veterinary and medical concerns, there is a growing need for attractants. Understanding the olfactory responses of \u003cem\u003eP. variegata\u003c/em\u003eis crucial, as insect vectors rely on volatile cues to locate hosts. The identification of key attractants could facilitate the development of vector surveillance and control strategies. However, the olfactory ecology of this species remains unexplored, limiting our ability to design effective attractant-based interventions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe used gas chromatography coupled electroantennography to measure antennal responses to synthetic and natural volatile blends. A comparative analysis was performed on the antennal responses of both sexes of \u003cem\u003eP. variegata\u003c/em\u003e and its well-studied relative, \u003cem\u003eDrosophila\u003c/em\u003e \u003cem\u003emelanogaster\u003c/em\u003e. Components of the synthetic blends were selected based on the odorant receptor repertoire of \u003cem\u003eD. melanogaster \u003c/em\u003eand established mosquito attractants, with the rationale that conserved olfactory receptors among dipterans may allow \u003cem\u003eP. variegata\u003c/em\u003e to detect similar compounds. Volatile extracts collected using active carbon adsorbent traps were also tested on the antennae and analysed using gas chromatography coupled mass spectrometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eMales of \u003cem\u003eP. variegata\u003c/em\u003eshowed higher antennal responses to phenol, 3-octanone, and sulcatone than females, indicating olfactory sexual dimorphism. Compared to \u003cem\u003eD. melanogaster\u003c/em\u003e, the antennae of \u003cem\u003eP. variegata\u003c/em\u003e did not respond to several common plant alcohols and terpenoids. Instead, they showed stronger responses to compounds such as anisole, ethyl propanoate, butyl propanoate, propyl acetate, 3-octanone, nonanal, and decanal, suggesting that peripheral olfaction in \u003cem\u003eP. variegata\u003c/em\u003e may be more tuned to microbial volatiles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe antennal olfaction of \u003cem\u003eP. variegata\u003c/em\u003e appears to be particularly tuned to microbial volatile emissions, suggesting that fungal and microbial substrates may play an important role in the life cycle of this species. Males show stronger relative responses to several compounds known to influence host-seeking behavior in other zoophilic dipterans, suggesting their potential as candidate attractants for future field studies.\u003c/p\u003e","manuscriptTitle":"Olfactory responses of Phortica variegata (Drosophilidae, Steganinae) the emerging vector of Thelazia callipaeda (Rhabditida, Thelaziidae) to ecologically relevant volatiles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 05:27:47","doi":"10.21203/rs.3.rs-6128144/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-27T11:49:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-17T10:35:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-04-13T18:15:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"324334a6-dc3a-4964-b883-4851a44df2fe","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:06:04+00:00","versionOfRecord":{"articleIdentity":"rs-6128144","link":"https://doi.org/10.1186/s13071-025-06850-8","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-06-02 15:56:58","publishedOnDateReadable":"June 2nd, 2025"},"versionCreatedAt":"2025-04-16 05:27:47","video":"","vorDoi":"10.1186/s13071-025-06850-8","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06850-8","workflowStages":[]},"version":"v1","identity":"rs-6128144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6128144","identity":"rs-6128144","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}