{"paper_id":"408017fa-0c3c-4080-a5ba-8105adf58f8e","body_text":"The Chemosensory Toolkit of a Cursorial Spider | 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 The Chemosensory Toolkit of a Cursorial Spider Mohammad Belal Talukder, Carsten H. G. Müller, Andreas Fischer, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6228127/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Chemosensation is vital for organisms to detect food, avoid predators, and find mates. Spiders, like many arthropods, rely on chemosensory input, but the modes and structures for detecting chemicals are not well understood. Cursorial spiders use chemical information for orientation and mate-finding. Behavioral studies suggest gustation plays a major role in male spiders following silken draglines to locate females, but the role of olfaction and tools involved in chemosensing are not known, despite the important ecological role of spiders. Results High-resolution electron microscopy revealed two types of chemosensory sensilla in the cursorial spider Pisaura mirabilis . Tip-pore sensilla, on the walking legs and pedipalps of both sexes, support contact chemoreception. Wall-pore sensilla, only occurring on adult male walking legs, are associated with olfaction. Behavioral assays confirmed that males detect airborne chemical signals from females, supporting these sensilla's role in mate search. The distribution of both sensilla types on walking legs is nearly complementary, with tip-pore sensilla towards leg tips and wall-pore sensilla towards leg bases supporting their respective contact and olfactory functions. Conclusions This study links the morphology, location, and function of chemosensory organs in cursorial spiders, providing insights into their chemosensory world. The findings advance the understanding of chemical sensing evolution in spiders and have implications for broader arthropod research. The identification of specialized olfactory sensilla in male spiders highlights the significance of airborne chemical detection in mate-finding, while contact chemoreceptors in both sexes suggest a general role in environmental chemical sensing. Evolutionary Biology Animal Behavior Cellular & Molecular Neuroscience Chemical ecology Araneae tip-pore sensilla wall-pore sensilla behavior gustation olfaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Chemical sensing is a crucial sensory modality across all animal phyla (1), supporting a wide range of behaviors essential to ecological success and reproductive fitness. Land-living arthropods, such as insects, utilize highly specialized chemosensory systems to detect and interpret environmental chemical information from, e.g., food sources, predators, and conspecifics in the form of surface-bound compounds upon contact and airborne molecules. In insects, contact chemoreception (gustation/taste) is mediated by sensory hairs (sensilla) featuring a terminal or sub terminal pore. These tip-pore sensilla are located primarily on the mouthparts, antennae, and distal segments of the legs (2). In contrast, the perception of airborne chemicals (olfaction/smelling) is facilitated by sensilla with wall pores distributed along their shafts, which are predominantly found on the insect antennae (3). In both types of sensilla, chemicals enter their pore(s) and bind to receptor cell dendrites within the sensillum (4,5). Similarly, in crustaceans, contact chemosensilla (gustatory sensilla) with a terminal pore are distributed unevenly across the body, with dense concentrations on mouthparts and walking legs. In contrast, the olfactory sensilla of crustaceans, known as aesthetascs, are thin-walled pegs that lack pores but contain numerous olfactory neurons and are located primarily on the first and second antennae (6). Compared to insects and crustaceans, limited knowledge exists regarding the chemical senses of other arthropods, limiting our comprehension of their sensory structures and the evolutionary history of these structures within this clade. For spiders, many behavioral studies support the use of both gustatory and olfactory information (7–9). For example, males are attracted to female-produced airborne sex pheromones (10–13), and upon contact, gustatory female sex pheromones elicit courtship behavior in males (12,14,15). Other than in the mating context, there is behavioural evidence that spiders use contact chemoreception and olfaction for habitat selection (16,17), prey detection (18,19), and predator avoidance (20,21). The cuticular chemosensory structures currently identified in spiders are tip-pore sensilla (22–24), which are found in large numbers on all walking legs and pedipalps of both sexes. These sensilla have been shown to function as contact chemoreceptors in an electrophysiological study on the spider Cupiennius salei (25). Prior to recent findings, reports of wall-pore sensilla in spiders were lacking (but see (22). However, wall-pore sensilla have now been found in the orb-weaver spider Argiope bruennichi , where they occur in large numbers on all walking legs and exclusively in males (26). Using single sensillum electrophysiological recordings, it was demonstrated that the male-specific wall-pore sensilla respond to the female-produced airborne sex pheromone (26). Adult females release the pheromone from their webs and attract males from a distance (10). As typical for web-building spiders, A. bruennichi females are stationary in their webs, whereas adult males actively search for females. However, many spider species are cursorial and do not reside in silken webs; instead, they roam freely and deposit dragline silk as they walk. Behavioral observations have demonstrated that in cursorial spiders such as lycosids, males follow female silk trails to locate potential mating partners (27,28). These findings suggest that cursorial spider rely primarily on silk-bound chemicals (gustation) for mate finding (but see (29) for evidence of olfaction). An examination of the sensilla apparatus of a cursorial spider, in conjunction with a behavioral assessment of the ability to perform olfaction during mate search, will help to contribute to the understanding of the chemosensory world of non-web-building spiders. The European nursery web spider Pisaura mirabilis (Clerck, 1757), also known as the nuptial-gift-giving spider, is a compelling model for exploring chemical communication in cursorial spiders. The lifestyle of P. mirabilis combined with well-studied knowledge of its mating behavior (30–33), which relies on silk-bound chemical information (34–37), makes it particularly valuable for investigating chemosensory mechanisms. Furthermore, choice experiments have shown that P. mirabilis utilizes chemical information from plants for habitat selection (38) and avoids the cuticular hydrocarbons of an ant predator (21). However, most experiments have not differentiated between the sensory modes of gustation and olfaction. Although mating-related contact chemoreception (gustation) has been supported in several studies (35,39), whether olfaction is a relevant sensory mode remains to be investigated. Recent identification of putative wall-pore sensilla in adult P. mirabilis that are male-specific (26) suggests that olfaction may play a significant role during mate search and possibly other contexts in this species. To shed light on the chemical communication system of cursorial spiders, we investigated the chemosensory structures of P. mirabilis and hypothesized that P. mirabilis possesses diverse types of chemosensilla if they are capable of performing contact-chemoreception and olfaction. Using high-resolution electron microscopy, we examined legs and pedipalps for candidate sensilla in both sexes and subadults and characterized the morphology and distribution of these sensilla. To address the knowledge gap regarding olfaction, we tested the hypothesis that males detect and respond to female-emitted airborne signals. Results Tip-pore sensilla External morphology of the tip-pore sensilla Sensilla with a blunt tip and a slightly oval terminal pore (Figs. 1 A, B), measuring 400–700 nm in diameter (N = 10 pores for each sex), were found on all walking legs and pedipalps of both males and females (Fig. 2 , Additional file 1: Figure S1). They are hair like, appear slightly S shaped, and feature spirally oriented microtrichia (spine-like protuberances) along the shaft (Fig. 1 A). The spination begins approximately 30 µm from the finely ribbed shaft base and extends up to 10–15 µm below the tip. The tip region contains many shallow longitudinal grooves (Figs. 1 B, C). The shafts of tip-pore sensilla emerge from a round, slightly elevated ‘crater-like’ socket (Fig. 1 D) and project from it at a steep angle (50–65°) to the leg surface cuticle, in contrast to the longer, branched, striated, and sharp-pointed tactile setae. In females, the sensillum shafts are approximately 188.14 ± 40.80 µm [arithmetic mean ± standard deviation] long (N = 30), whereas in males, they are slightly shorter (175.15 ± 37.39 µm, N = 30). The shaft diameter is very similar between the sexes, with values of 6.58 ± 0.71 µm (N = 30) in females and 6.24 ± 0.65 µm (N = 30) in males (Additional file 1: Table S1). Internal anatomy of the tip-pore sensilla We examined the internal anatomy of the tip-pore sensilla from the femur of the 1st walking leg of both males and females of P. mirabilis . Ultrathin cross-sections of the middle region of the sensillum shaft reveal a star-shaped profile and a distinctly corrugated surface of the shaft wall cuticle (Fig. 1 E), corresponding to the spirally oriented, ribbed microtrichia observed with SEM (Fig. 1 A), whereas in the proximal region, the shaft possesses a circular profile along with a finely ribbed surface (Figs. 1 C, D). The thick shaft wall cuticle features a system of thin longitudinal lymph canals running inside its length, giving the shaft a double-wall appearance in a cross section (Figs. 1 C, E-F). None of the cross-sections examined revealed that these longitudinal shaft canals are connected to the lymph space or to the outside. Cross-sections show a hollow sensillum shaft filled with lymph (Figs. 1 E, F). The lymph space is divided into two compartments. These compartments are separated by a thick cuticular tube called the peridendritic shaft cylinder (Figs. 1 C, E-G) (according to Müller et al.(24). The inner compartment, called the inner sensillum lymph space, contains 15–18 nonbranching dendrites (Figs. 1 E-G), which are likely chemoreceptive. Each dendrite is characterized by a set of microtubules. All dendrites running through the shaft terminate below the terminal pore. The outer compartment, called the outer sensillum lymph space, surrounds the inner compartment (Figs. 1 E-G). The outer sensillum lymph space often contains cytoplasmic processes of accessory sheath cells located beneath the leg cuticle (Figs. 1 F, G). A mechanoreceptive portion, typical for tip-pore sensilla in spiders, is present in the socket region of this sensillum, and the somata of both chemoreceptive and mechanoreceptive neurons are located beneath the socket of the sensillum, as shown in A. bruennichi (24). Distribution of tip-pore sensilla In P. mirabilis , adult males possess approximately 4,100 tip-pore sensilla distributed across all walking legs and pedipalps, whereas females have approximately 2,900 (Fig. 2 , Additional file 1: Figures S1, S2A-C, and S4). On all walking legs of both males and females, these sensilla are most abundant on the tarsus, metatarsus, and tibia; occur in lower numbers on the patella and femur; and are absent on the trochanter (Fig. 2 B, Additional file 1: Figure S1). In both sexes, there are few (3–9) tip-pore sensilla on the coxa of all walking legs. On the tarsus, metatarsus, and tibia of all walking legs in both males and females, the tip-pore sensilla are arranged in seven to eight rows along the segment axes. The number of tip-pore sensilla was greater on the 1st and 2nd walking legs than on the 3rd and 4th walking legs in both sexes (Additional file 1: Figure S2C). On all walking legs, the tip-pore sensilla are predominantly located on the lateral and ventral sides, with fewer on the dorsal side (Fig. 2 B, Additional file 1: Figure S1). On the pedipalps, the density of tip-pore sensilla is high on the dorsal and prolateral sides of the tarsus of the female and the dorsal side of the cymbium (the outer part of the modified palpal tarsus) of the male (Fig. 2 A). In both sexes, toward the body, there are only a few tip-pore sensilla on the tibia, patella, and femur and none on the trochanter or coxa of the pedipalp. Wall-pore sensilla External morphology of wall-pore sensilla The wall-pore sensilla are thin, trichoid hairs with round, slightly elevated \"crater-like\" sockets (Figs. 3 A-D). These sensilla occur exclusively on all walking legs of adult males and are not found in females or subadult males (Additional file 1: Figure S3). The sensilla have a curved shape with a tapered tip. The insertion angles of the wall-pore sensilla are slightly steeper than those of the mechanoreceptive sensilla (tactile hairs) found on all leg and pedipalp segments in high abundance. On average, the wall-pore sensilla of P. mirabilis are 141.78 ± 11.41 µm [mean ± standard deviation] long (N = 15) (Additional file 1: Table S2). The sensilla diameter is 2.12 ± 0.16 µm, as measured approximately 20 µm distal to the hair base (N = 15). The surface of the sensillum shaft shows fine diagonal ridges and grooves (Fig. 3 B), which are longitudinally oriented toward the tip. Numerous pore-like invaginations (Fig. 3 B), with an average diameter of 41.93 ± 3.26 nm (N = 45), are distributed inside the groves over almost the entire length of the shaft, except for the sensilla base region (Fig. 3 D). No marked differences in size or shape were observed in the wall-pore sensilla across different walking legs and their segments in male P. mirabilis (Additional file 1: Table S2). Internal anatomy of wall-pore sensilla In our TEM analysis, we focused on wall-pore sensilla located on the femur of the 1st walking leg of male P. mirabilis . Cross-sections taken from different regions of the shaft reveal that the wall-pore sensilla possess two lymph spaces (Figs. 3 E-I). There is a voluminous sensillum lymph space inside the shaft. This so-called outer lymph space contains an inner sensillum lymph space that is surrounded by a dendritic sheath (Figs. 3 E-I). The inner lymph space hosts 2–4 (mostly 4) dendrites characterized by a set of microtubules (Figs. 3 F-I). The dendrites are tightly packed inside the dendritic sheath, which is present throughout the shaft. Consecutive ultrathin cross-sections revealed that there are pores in the shaft cuticle (Figs. 3 E, G). These pores are connected to thin canal-like structures (spoke canals, approximately 100 nm in diameter; Fig. 3 F) that traverse the shaft wall cuticle and open into the outer sensillum lymph space. As these spoke canals run obliquely through the shaft wall, we did not capture their full course in our cross sections. Pore tubules, such as those found in single-walled sensilla of ticks ( Amblyomma variegatum ) (40), were not observed in the spoke canals of the wall-pore sensilla of P. mirabilis . The shaft wall cuticle exhibits narrow and distinct longitudinal lymph canals in a ring-like formation, giving the shaft wall a double-walled appearance (Figs. 3 E-H). At the basal shaft level, most of the longitudinal shaft canals fuse to form a ring (Fig. 3 G). The longitudinal shaft canals do not interconnect with the radial spoke canals. In some sections, the outer sensillum lymph space contains cytoplasmic extensions of the accessory sheath cells (Fig. 3 I) that are located beneath the leg cuticle. Like the wall-pore sensilla of A. bruennichi (26), we did not detect tubular bodies, a key component of mechanoreceptors, in the socket region, indicating that these sensilla are not multimodal, as is characteristic of tip-pore sensilla. Distribution of male-specific wall-pore sensilla The male-specific wall-pore sensilla of P. mirabilis are abundant (approx. 3,000 sensilla) and somewhat irregularly arranged on the proximal leg segments (femur, patella, and tibia) of all walking legs of the males (Fig. 2 B, Additional file 1: Figure S1). They are most abundant in the middle to distal regions of the femur and patella and in the proximal region of the tibia in the first, second, and fourth legs. In contrast, wall-pore sensilla were not found on the distal segments (metatarsus and tarsus) of any leg or on the male pedipalps. Compared with the dorsal and ventral sides, the wall-pore sensilla are more densely distributed along the lateral sides of the legs (Fig. 2 B, Additional file 1: Figure S1). The first and second legs each possess approximately 460–470 wall-pore sensilla, whereas the fourth leg possesses approximately 340, and the shorter third leg has approximately 200 wall-pore sensilla (Additional file 1: Figure S2D). Olfactometer test Olfactory attraction of males to females was observed (binomial test, p = 0.012; Fig. 4 ), with 16 (70%) out of 23 males choosing the female-equipped branch, four (17%) choosing the control branch, and three males (13%) not leaving the holding tube. In the control experiment, no side preference was observed, as four males (18%) chose the left branch, six males (27%) chose the right branch (binomial test, p = 0.754; Fig. 4 ), 12 males (55%) exhibited no behavioral response (Fig. 4 ), and one male died prior to their second trial. Males were more likely to make a choice when exposed to a female scent (87%) than when they were not exposed to a female scent (45%) (Fisher’s exact test, p = 0.005; Fig. 4 ). Contact area versus non-contact area of body appendages We conducted an analysis of the contact and non-contact regions of the body appendages of male and female P. mirabilis during locomotion on various surfaces, silk probing, prey capture, and mating. The contact and non-contact areas of the walking legs and pedipalps of P. mirabilis were delineated on the basis of the contact probability across all pooled contexts: locomotion, silk probing, prey capture, and mating (Fig. 5 , Additional file 1: Tables S3 and S4). Upon pooling all contexts (Additional file 1: Table S4), the ventral and lateral sides of the tarsus of all walking legs exhibited frequent substrate contact in both sexes (55%-100%). The metatarsus in males demonstrated frequent substrate contact on the first two walking legs (52%-53%), whereas in females, frequent contact was observed solely for the metatarsus of the first walking legs (53%). In both sexes. The tibia‒patella segments of all walking legs exhibited infrequent substrate contact across all contexts (1%-11%), and the femur did not establish contact with the substrate or mating partner in any context of our experimental trials. With respect to pedipalps, the cymbium in males and the tarsus in females frequently probed substrates, nuptial gifts, and prey, with contact frequencies ranging from 86%-100%. Conversely, the remaining pedipalp segments in both sexes rarely establish contact in any context. Detailed observations of all the behavioral contexts are provided in the supplemental material (Additional file 1: Supplement text, Tables S3 and S4). Discussion Chemosensory sensilla in Pisaura mirabilis We detected two distinct types of chemosensory sensilla in the cursorial spider P. mirabilis : tip-pore sensilla and wall-pore sensilla. The first type, tip-pore sensilla (Fig. 1 ), is associated with contact chemoreception (gustation), a well-documented function in insects (5), crustaceans (6), and one spider species (25). In P. mirabilis , tip-pore sensilla are present in both sexes, as well as in subadults, and are distributed across all walking legs and pedipalps (Fig. 2 ). They are predominantly located on the distal segments of the appendages, which frequently contact substrates (Fig. 5 ), suggesting their role in detecting surface-bound chemical information. The second type, wall-pore sensilla (Fig. 3 ), functions as olfactory sensilla, a role demonstrated in insects (4), crustaceans (6), and more recently in a spider, (26). Our Y-tube olfactometer assays demonstrated that P. mirabilis males respond to airborne signals emitted by females (Fig. 4 ). In P. mirabilis , wall-pore sensilla are exclusive to adult males and absent in females and subadult males (Additional file 1: Figure S3). These sensilla are located on the proximal segments of all walking legs (Fig. 2 B, Additional file 1: Figure S1), areas that rarely contact substrate, prey, or mating partners (Fig. 5 ), which is consistent with findings in the orb-weaving spider A. bruennichi (26), corroborating their specialization for detecting airborne signals emitted by females. Tip-pore sensilla Tip-pore sensilla are present on all walking legs and pedipalps of both sexes and subadult P. mirabilis . These sensilla feature chemoreceptive dendrites housed within a trichoid shaft, with the dendrites terminating at a blunt terminal pore (Fig. 1 ). This pore provides the only connection between the dendrites and the external environment, supporting the role of tip-pore sensilla in contact chemoreception, as confirmed in, e.g., insects (5), crustaceans (6), and arachnids such as opilionids and ricinulei (41,42). The gustatory role of tip-pore sensilla has also been demonstrated in the cursorial spider Cupiennius salei through electrophysiological studies (25). Although the outer morphology of the tip-pore sensilla can differ between species, the internal ultrastructures of the sensilla shafts in P. mirabilis and C. salei are highly similar to those of other cursorial spiders and web-building spiders (24,43). In all cases, tip-pore sensilla also entail a mechanoreceptive component (24,25,43). In P. mirabilis , tip-pore sensilla are positioned at steeper angles (Additional file 1: Table S1) than pure mechanoreceptive sensilla are and are more densely distributed on distal segments (tarsus and metatarsus) of the walking legs and pedipalps, with fewer proximal segments (tibia, patella, and femur) (Fig. 2 , Additional file 1: Figure S1). A similar distal abundance of tip-pore sensilla with steeper angles has been observed in both web-building (23) and cursorial spiders (43). In various insect orders, the presence of tip-pore sensilla on the mouthparts and distal segments of walking legs is common (2). For example, in leaf beetles (Coleoptera, Chrysomelidae), the tip-pore sensilla present on the tarsi of legs play a crucial role in host selection (44). This distal placement and steeper angles of tip-pore sensilla increase the probability of encountering substrate-bound chemical information, facilitating gustation during navigation and mating. Our video observations further support this, demonstrating that distal segments of the body appendages of P. mirabilis frequently contact substrates during locomotion, silk probing, prey capture, and mating, whereas proximal segments rarely do (Fig. 5 , Additional file 1: Tables S3 and S4). Additionally, there are fewer tip-pore sensilla on the dorsal sides of all walking legs, where contact with the substrate is rare (Fig. 2 B, Additional file 1: Figure S1), which further corroborates the functional role of tip-pore sensilla as gustatory organs. There is a marked difference between the sexes in the number of tip-pore sensilla in P. mirabilis , with males possessing approximately 4,100 sensilla compared with 2,900 in females (Additional file 1: Figure S4), despite their similar body sizes. In males, the highest densities occur on the distal segments of the first and second walking legs and on the cymbium of the pedipalps. When encountering the dragline silk of a female, males touch the silk with these segments, and their tip-pore sensilla likely play a role in the detection of the reproductive status of a female and trail-following decisions (34,36,39,45). Consequently, mate detection and assessment are likely necessary for a greater number of tip-pore sensilla in males. In more ancient spiders, such as liphistiids, gustation-based mate search is likely also accomplished through the available tip-pore sensilla (46), but male-specific scopulate hairs have also been suggested to perform this function (47). Male-specific gustatory sensilla are also known from the lady beetle Semiadalia undecimnotata (Coleoptera), where they occur on the antennae and have been proposed to play a role in mating-related functions (48). The greater quantity of tip-pore sensilla in the cursorial spider P. mirabilis than in the orb-weaver A. bruennichi , which has 1,000 sensilla in males and 2,000 in females (23), likely reflects the necessity for cursorial spiders to perceive more detailed information on prey presence, habitat quality, and predation risk while roaming in the undergrowth. In contrast, most stationary web-building spiders predominantly remain on their web—an extended perceptual system through which they can even decipher prey types (49). In the web, physical contact occurs solely with intercepted prey items (with the exception of contact with a mating partner), which the spiders examine with their gustatory sensilla prior to wrapping or during prey consumption (50). Wall-pore sensilla Wall-pore sensilla are found exclusively in adult male P. mirabilis . These sensilla contain multiple pores connecting to a central lymph space within the sensillum shaft via radial spoke canals, forming a direct pathway from the external environment to the chemoreceptive dendrites in the lymph space (Fig. 3 ). The presence of longitudinal shaft wall canals indicates that these multiporous sensilla belong to the class of double-walled sensilla in insects; however, they lack slit-like grooves on the shaft surface and vase-shaped chambers below the pore (40). Distal processes of accessory sheath cells within the shaft (Fig. 3 I) have been observed in single-walled sensilla with pore openings (40), similar to those found on the tips of the sensory legs of amblypygid spiders (22). The wall-pore sensilla of male P. mirabilis closely resemble those of A. bruennichi , for which the perception of the airborne female sex pheromone was corroborated through electrophysiological tests (26). The wall-pore sensilla of both species, however, differ in ultrastructure from the single wall-pore sensilla described for a gradungulid species, a basally branching spider taxon (22), and whether wall-pore sensilla evolved once within spiders or several times convergently remains to be investigated. P. mirabilis males possess approximately 3,000 wall-pore sensilla on all walking legs, predominantly in the proximal leg segments (Fig. 2 B, Additional file 1: Figure S1), which are non-contact areas (Fig. 5 ). This distribution suggests that their ability is to detect airborne signals released by females, similar to A. bruennichi males (26). A. bruennichi males possess approximately 7,000 male-specific wall-pore sensilla, which are likewise concentrated in non-contact areas of their walking legs and are finely tuned to airborne female-produced sex pheromone (26). The greater number of wall-pore sensilla in A. bruennichi males may indicate a greater reliance on olfaction for mate search. Male A. bruennichi are attracted from substantial distances to stationary females by signals released from the web (10), whereas P. mirabilis males may utilize both olfactory and gustatory information from female dragline silk in their environment. In arthropods, male-specific olfactory sensilla are abundant and typically specialize in detecting sex pheromones. For example, in a moth species, males possess approximately 50,000 specialized antennal trichoid sensilla that are dedicated to detecting female sex pheromones, whereas other olfactory sensilla in both males and females respond to general odors, as reviewed previously (4,51). Male-specific olfactory sensilla have also been found in other moth species (52–54). Similarly, in aquatic mysids (crustaceans), approximately 2,000 male-specific olfactory sensilla located basally on the first antennae have been proposed to function as pheromone receptors (55). These observations support the hypothesis that male-specific wall-pore sensilla in P. mirabilis likewise have evolved to enhance the detection of female sex pheromones and that these sensilla must be airborne. Our behavioral assays indeed demonstrated that P. mirabilis males respond to airborne signals emitted by females (Fig. 4 ). This finding aligns with observations in the lycosid spider Pardosa milvina , where males are attracted to airborne signals from adult virgin females but not from subadult females or males (29). The sexual pheromone of P. mirabilis that resulted in remote attraction in our behavioral trials remains unidentified. No pheromone candidates were detected via gas chromatography‒mass spectrometry (GC‒MS) (unpublished data; G. Uhl, S. Schulz), possibly because these signals involve polar compounds that are undetectable via GC‒MS (7). Recently, liquid chromatography coupled with tandem mass spectrometry was used to identify polar olfactory and gustatory sex pheromones of widow spiders (11,12,56), which has been elusive in GC‒MS analyses. We propose similar investigations for P. mirabilis to explore the compound responsible for mate attraction in a cursorial spider. Olfaction of adult female and immature cursorial spiders. The olfactory capabilities of subadult and female spiders remain uncertain, as wall pore sensilla have been found exclusively in male spiders (26). Nevertheless, studies indicate that female and juvenile cursorial spiders utilize olfactory information for purposes such as habitat selection (16) and prey detection (19). Consequently, it can be postulated that tip-pore sensilla potentially serve dual gustatory (taste) and olfactory (smell) functions, analogous to certain tip-pore chemoreceptors identified in insects (5,57,58). Apart from this \"dual-function hypothesis\", a “functional zone hypothesis” warrants investigation to determine whether the tip-pore sensilla located on the proximal segments of the legs, where direct contact with surfaces is infrequent, would engage in olfaction, and those in the distal part of the leg might be limited to gustation. Consequently, the function of the tip-pore sensilla may vary depending on their location, as has been reviewed in insects (59). Conclusion We set out to explore the chemosensory toolkit of arthropods other than insects as a step to unravel the evolution of chemosensing within arthropods. We elucidated the chemosensory apparatus and mate-finding mode of the cursorial spider P. mirabilis . Tip-pore sensilla, present in both sexes and associated with contact chemoreception, and male-specific wall-pore sensilla linked to olfaction were discovered and described on the basis of their outer and inner anatomical features. The significance of olfaction in mate search was demonstrated by male attraction to female scents. Morphological and behavioral findings suggest that male-specific sensilla function in female pheromone detection, as observed in insects and crustaceans. Male P. mirabilis possess wall-pore sensilla on all walking legs in regions close to the body that normally do not contact the substrate, which corroborates their function in olfaction. The absence of wall-pore sensilla in females and immature spiders raises novel questions regarding the sensilla involved in olfaction, other than mate search. Candidates are tip-pore sensilla that may form functional zones depending on their location on the legs. More broadly, this study contributes to our understanding of the ecological and evolutionary drivers of chemosensory diversity in arthropods, offering insights into how organisms detect and respond to chemical information in their environment and providing material for comparative analyses of sensory organs and their evolution across arthropods. Methods Study species Juvenile, subadult (one molt prior to adulthood), and adult P. mirabilis (Clerck, 1757) individuals of both sexes were collected each year from 2021–2024 from grasslands in Greifswald, Germany (54° 05' 49.91''N 13° 23' 16.58'' E) and brought to the laboratory. The spiders were individually housed in Drosophila culture tubes measuring 5 cm in diameter and 10 cm in height, with a netted top and a sponge lid at the bottom (35). Inside each vial, a substrate of artificial aquarium plants was provided. To maintain high humidity levels, vials with sponges at the bottom were placed in trays filled with water. The spiders were kept at a constant temperature of 22°C (± 5°C) under a 12:12 h light‒12 h dark cycle. Adult and subadult spiders were fed one housefly ( Musca domestica ) or blowfly ( Lucilia caesar ) twice a week, whereas juveniles were provided with several fruit flies ( Drosophila hydei ) on the same schedule. Juveniles and subadults were monitored regularly for the presence of exuviae to monitor their development. Scanning electron microscopy: examination and mapping of chemosensilla To investigate and map chemosensilla, freshly molted adult males and females as well as subadults of P. mirabilis were anesthetized with CO 2 . They were then mounted on a polyethylene board (Plastazote) with their legs straightened and secured with wire clamps. The mounted samples were submerged and preserved in 80% ethanol. For SEM analysis, the straightened walking legs and pedipalps on the right side were detached and dehydrated through a graded ethanol series up to 99% and then dried using a Leica EM CPD300 critical point dryer. The straightened legs and pedipalps were vertically mounted on metal SEM stubs. The samples were then coated with gold-palladium for 2.5 min using a Polaron SC7640 sputter coater (Fisons Instruments). Then, the stubs were placed horizontally into a custom-made stub holder that allowed the samples to be rotated at various angles. The samples were examined using a Zeiss EVO LS 10 scanning electron microscope (SEM) operated at 10 kV. The mounted samples were oriented perpendicular to the electron beam and rotated at 0°, 90°, 180°, and 270° to capture images from the dorsal, prolateral, ventral, and retrolateral sides of the body appendages. Owing to variation in the diameter of the segments of the body appendages (coxa to tarsus), we used a range of magnifications (130× to 220×), but each segment was imaged at a consistent magnification for all males and females. The images were stitched together to reconstruct the relative positions of the sensilla on outline drawings of the spider’s legs, derived from multifocus stereomicroscopic photographs (Zeiss SteREO Discovery V20). Sensilla near the drawings´ borders appear on several maps but were excluded from the total count of sensilla. During mapping, the sensilla account for individual variation, and three males and three females were examined. All the sensillum parameters (length, diameter, insertion angle, and pore diameter) were measured via SEM built-in software. For a higher resolution of the properties of the sensillum shafts, a Zeiss SUPRA 40VP field emission SEM was used. The samples were cleaned with a 5% KOH solution and glacial acetic acid, following the methods described by Schneeberg et al. (60), before they were subjected to critical point drying. The legs were then mounted onto SEM stubs and sputter-coated with an Au-Pd (80:20) mixture at 5 mA (Q150T ES, QUORUM, UK), resulting in a coating thickness of 10 nm. Transmission electron microscopy: investigation of the internal anatomy of chemosensilla To prepare the samples for transmission electron microscopy, freshly molted samples—two males and two females of P. mirabilis —were anesthetized with CO₂. The walking legs were then dissected using micro-scissors while immersed in an ice-cold prefixative solution modified from Karnovsky's protocol (61), consisting of 2.0% glutaraldehyde, 2.5% paraformaldehyde, and 1.5% sucrose in 0.1 mol/L sodium phosphate buffer at pH 7.4. To improve tissue preservation, each leg segment was cut into 2–5 pieces, each measuring 1–3 mm in length, and incubated in the same prefixative solution. To further enhance fixation, the samples were subjected to three sets of 2 min microwave pulses at a power of 120 W using a PELCO BioWave Pro instrument equipped with a PELCO SteadyTemp Pro Thermo Cube for solid-state cooling. Throughout the BioWave application, the sample temperature was monitored, remaining between 8–10°C prior to treatment and 9–15°C afterward. The leg pieces were stored in the same prefixative solution at 4°C for at least one night. The samples were then washed three times in 0.1 M sodium phosphate buffer for 45 min in total. The samples were subsequently fixed with 2% osmium tetroxide for 3 to 4 h at room temperature (RT) and then washed three times with ddH₂O for a total of 30 min. The samples were dehydrated through a series of ethanol solutions (50%, 60%, 70%, 80%, 90%, 100%), with dehydration at each concentration up to 90% carried out for 2 × 10 min at RT. For the 100% ethanol stage, the samples were washed over 3 × 10 min. Subsequent infiltration of the samples with epoxy resin (Embed812, an Epon substitute) was conducted at RT using the solvent propylene oxide (PO) over several stages of dilution: 2 × 100% PO for 15 min in total, 66% for 2 h, 50% for 12 h, and 33% for 24 h. The samples were placed on a shaker throughout infiltration to support efficient infiltration. For preembedding, the samples were incubated in 100% epoxy resin and again placed on a shaker for 2 h. To further increase infiltration speed and efficiency, the samples were then transferred to a Heraeus VT-6025 vacuum heating cabinet for a total of 2 h at 40°C. During that time span, the samples were exposed to high vacuum conditions (3 × 30 min at 150 mbar, each phase separated by 10-min breaks under normal atmospheric pressure). For final embedding, the samples were placed in silicon molds containing fresh epoxy resin. Each piece of the femur, tibia, patella, metatarsus, or tarsus was oriented in specific resin blocks (transverse, oblique, or longitudinal to the block face). The resin blocks were polymerized in a heating cabinet at 60°C for 48 hours. Transverse, horizontal, and longitudinal ultrathin sections (70–90 nm thick) of the tibia and femur were produced using a Leica UCT ultramicrotome. In areas from which ultrathin sections were obtained (with trimming gaps of 2–5 µm), semithin sections (700 nm thick) were also taken and stained with toluidine blue (dissolved in 1% sodium tetraborate) for examination under a light microscope. The ultrathin sections were placed on Formvar-coated slot grids (PLANO), stained with uranyl acetate and lead citrate for 4 min each, and then examined with a JEOL JEM-1011 transmission electron microscope operated at 80 kV. Digital photomicrographs were captured using a mid-mount camera (MegaView III, Soft Imaging System) with iTEM imaging software (Olympus, Soft Imaging Solution). Olfactometer test Olfactory attraction of males to unmated female P. mirabilis was tested in December 2024 using Y-tube olfactometers (Additional file 1: Figure S5) following established protocols (62). Females were placed in stimulus chambers (26 × 2.5 cm glass tube with mesh on both sides) for two hours to allow for acclimatization and silk deposition. The stimulus chamber was then connected by tubing to one arm of the Y-olfactometer (main stem: 24 cm long, side arms: 21 cm long, diameter: 2.5 cm), while a control chamber, without a female, was connected to the other arm. To initiate the assay, a male was placed into a glass tube (26 × 2.5 cm, with mesh on both sides) for two minutes to acclimatize, while a pump drew 0.4 L/min air. The acclimatization tube was then connected to the stem of the Y-tube. The male was subsequently given 15 min to reach the end of either arm, which was considered a choice. Upon first choice, the experiment was ended. To provide a grip for the spiders, all glass tubes held bamboo skewers (20 × 0.4 cm, Exnima, Damiel, Spain). Tissue paper, with a hole, lined the three terminal stoppers connected to tubing. The presentation sides were altered 50:50 across replicates. Bamboo skewers and tissues were discarded upon use, while the glassware was rinsed with water and baked for 2 h at 250°C to remove any stimuli. The experiment was replicated 23 times with randomly paired males and females (N = 23 each). To test for any side bias, each male was tested again one week later in the same setup but without any female stimuli. Male olfactory choice data were statistically compared against binomial or Fisher’s exact tests in R (63). Contact and non-contact areas of body appendages To identify the contact and non-contact areas on the body appendages (legs and pedipalps) of male and female P. mirabilis , we observed their legs and pedipalps movements via high-speed camera recordings in 2021. The following scenarios were recorded in a laboratory: a) locomotion, b) silk probing by a male, c) capturing of prey, and d) mating. For the locomotion trials, the spiders were placed on four different substrates that they might encounter in their natural habitat: a flat surface, natural common nettles (a plant on which we frequently find the species in the field), a 20 cm long (2 mm in diameter) horizontally placed skewer, and a vertically placed bundle of natural grass (approximately 30 cm long). The substrates were cleaned with 70% ethanol and air-dried after each trial to eliminate any residual silk left by spiders. For the trials of silk probing by the male, we analyzed video footage from a previous study (34) in which males were also allowed to walk on and around dragline silk left by females in a transparent plastic box (17 cm × 8 cm × 6 cm) to observe their silk probing behavior. During prey capture, we observed how the spiders interacted with different-sized prey, including comparatively small houseflies ( Musca domestica ) and larger house crickets ( Acheta domesticus ). For each prey capture trial, an individual spider was placed in a transparent plastic box (17 cm × 8 cm × 6 cm), and a live prey item was released into the box. In our prey capture analysis, we included video footage from an earlier study (64). For mating observations, an unmated female was placed in an open arena (40 cm × 30 cm × 4 cm) for 5 min to deposit dragline silk. Females typically leave dragline silk behind when they roam (35,37). A male, carrying a nuptial gift (prey item wrapped in silk) in his chelicerae, was then introduced at the periphery of the arena. Upon contact with the female's dragline silk, the male followed it and approached the female, tapped the female with his legs and offered a nuptial gift. If the female accepted the gift and started feeding on it, the male inserted his pedipalps (sperm transfer organ) into her genital opening (epigyne) for mating. We recorded movements of all the above-mentioned behavioral contexts using a high-speed camera (Miro LC320S, AMETEK Vision Research) at 500 frames per second for a maximum of 8 seconds and analyzed the footage in slow motion with ImageJ software (public domain, https://imagej.net/ij/ ). We noted which segments (tarsus, metatarsus, tibia, patella, and femur) of the leg or pedipalp made contact in these different contexts and which did not. The segments that contacted the substrates or partner at least once during a trial were considered contact areas for the specific appendages in that trial. We compared the number of trials in which contact occurred to the total number of trials (6–24 trials, depending on the context; see Additional file 1: Table S3) for all contexts, resulting in a contact probability ranging from 0–100%. The leg and pedipalp segments that made contact in more than 50% of the cases were labeled contact areas, whereas those below 50% were classified as infrequent contact areas. Those segments that did not contact the substrates during our trials were defined as non-contact areas. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was funded by the German Science Foundation (DFG, UH 87/14-1, Grant No. 451487045). Author contributions Conceptualization, G.B.U., C.H.G.M.; Methodology, M.B.T., C.H.G.M., G.B.U., J.O.W., A.F.; Investigation, M.B.T., C.H.G.M., A. F., and V.M.; Writing-Original draft, M.B.T., G.B.U., and A.F.; Writing-Review & Editing, G.B.U., C.H.G.M., J.O.W., and A.F.; Visualization, M.B.T., A.F.; Supervision, G.B.U. and C.H.G.M.; Funding acquisition, G.B.U. All the authors have read and approved the final manuscript . Acknowledgments We appreciate the technical support for the SEM analyses provided by Rabea Schlüter and Stefan Bock from the Imaging Center at the University of Greifswald. We also thank Carmen Noske (Greifswald) for her assistance in filming the high-speed videos and rearing the spiders. 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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-6228127\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":428958635,\"identity\":\"7094582a-7b5b-4005-9342-86a294fb41fe\",\"order_by\":0,\"name\":\"Mohammad Belal Talukder\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Greifswald\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mohammad\",\"middleName\":\"Belal\",\"lastName\":\"Talukder\",\"suffix\":\"\"},{\"id\":428958613,\"identity\":\"ba093503-c010-4701-bde3-aec10ea82cd8\",\"order_by\":1,\"name\":\"Carsten H. G. 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Uhl\",\"email\":\"data:image/png;base64,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\",\"orcid\":\"https://orcid.org/0000-0001-8758-7913\",\"institution\":\"University of Greifswald\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Gabriele\",\"middleName\":\"B.\",\"lastName\":\"Uhl\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-03-14 16:41:39\",\"currentVersionCode\":1,\"declarations\":{\"humanSubjects\":false,\"vertebrateSubjects\":false,\"conflictsOfInterestStatement\":false,\"humanSubjectEthicalGuidelines\":false,\"humanSubjectConsent\":false,\"humanSubjectClinicalTrial\":false,\"humanSubjectCaseReport\":false,\"vertebrateSubjectEthicalGuidelines\":false},\"doi\":\"10.21203/rs.3.rs-6228127/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6228127/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":78720585,\"identity\":\"a6cb48a7-d95a-4082-9a4d-e56790677593\",\"added_by\":\"auto\",\"created_at\":\"2025-03-18 04:25:51\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":822874,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eExternal appearance and internal anatomy of tip-pore sensilla in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003ePisaura mirabilis\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e. SEM (A-D), TEM (E-G). A \\u003c/strong\\u003eAn overview of a tip‒pore sensillum showing a spiny, tapered shaft with a blunt tip. Note that the basal region of the shaft is not spiny but finely ribbed. \\u003cstrong\\u003eB\\u003c/strong\\u003eClose-up image of the tip region, highlighting the terminal pore. \\u003cstrong\\u003eC\\u003c/strong\\u003e A broken sensillum shaft revealing the hollow lymph space surrounded by the cuticular peridendritic shaft cylinder (pdc) and a longitudinal canal (lca) within the shaft wall. \\u003cstrong\\u003eD\\u003c/strong\\u003e Round, elevated socket of the sensillum with a nonporous, finely ribbed base. \\u003cstrong\\u003eE\\u003c/strong\\u003e Cross-section of the distal mid-region of the sensillum shaft, showing a strongly corrugated, star-shaped cross profile of the shaft wall cuticle (swc). A ring-like formation of thin, partly fused longitudinal canals (lca) is present within the shaft wall. The sensillum shaft contains two distinct lymph spaces, the inner (isl) and outer (osl) spaces, which are separated by the thick peridendritic shaft cylinder (pdc). The inner lymph space (isl) houses 17 dendrites (de) of chemoreceptive cells. \\u003cstrong\\u003eF\\u003c/strong\\u003e Cross-section of the basal shaft region, showing a finely ribbed shaft wall cuticle (swc) that is crossed by a system of thin longitudinal canals (lca) in a ring-like formation. The outer sensillum lymph (osl) space contains cytoplasmic projections of accessory sheath cells (pasc). \\u003cstrong\\u003eG\\u003c/strong\\u003e Close-up view of the inner sensillum lymph space (isl), surrounded by the thick peridendritic shaft cylinder (pdc). The isl contains 16 tightly packed chemoreceptive dendrites (de). See also Additional file 1: Table S1.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6228127/v1/da3ca13ca53f5a1ae3018e05.png\"},{\"id\":78720586,\"identity\":\"9bd9a8ac-9495-4933-bd25-d869cd398686\",\"added_by\":\"auto\",\"created_at\":\"2025-03-18 04:25:51\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":220365,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eDistribution of chemosensilla in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003ePisaura mirabilis.\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e A \\u003c/strong\\u003ePedipalp of a male (left panels) and a female (right panels).\\u003cstrong\\u003e B \\u003c/strong\\u003eFirst walking leg of a male (upper panels) and a female (lower panels). Each panel shows four perspectives from top to bottom: dorsal, prolateral, ventral, and retrolateral. Notably, wall-pore sensilla are present only on the walking leg of males. All scale bars: 1 mm. See also Additional file 1: Figure S1\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6228127/v1/0cd679a99c375c19d36f6a4f.png\"},{\"id\":78720599,\"identity\":\"e488a508-4c74-441d-89cf-c674372b70ba\",\"added_by\":\"auto\",\"created_at\":\"2025-03-18 04:25:51\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":722546,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eExternal appearance and internal anatomy of wall-pore sensilla in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003ePisaura mirabilis\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e. \\u003c/strong\\u003eSEM (A-D) and TEM (E-I). \\u003cstrong\\u003eA\\u003c/strong\\u003e An overview of a wall-pore sensillum with a curved-tapered shaft and round socket. \\u003cstrong\\u003eB\\u003c/strong\\u003e Close-up image of the middle region of the sensillum shaft showing a grooved and ridged surface with multiple pore-like depressions (some are indicated by white arrows) located within the grooves. \\u003cstrong\\u003eC\\u003c/strong\\u003e A broken sensillum shaft revealing the hollow center (lymph space). \\u003cstrong\\u003eD\\u003c/strong\\u003eSocket of the sensillum with a nonporous, finely ribbed base. \\u003cstrong\\u003eE\\u003c/strong\\u003e Cross-section of the tip region of the sensillum shaft, showing wall pores (black arrows) in the shaft wall cuticle (swc). The outer lymph space (osl) and inner lymph space (isl) are separated by a dendritic sheath (ds), and dendrites are not clearly discernible. Longitudinal canals (lca) are present in the shaft wall cuticle. \\u003cstrong\\u003eF\\u003c/strong\\u003e Spoke canals (sc) (marked with white arrows) in the shaft wall cuticle (swc) connect the outer environment to the lymph spaces. Some partly disintegrated dendrites (de) of receptor cells are visible in the inner lymph space (isl). A longitudinal canal (lc) is shown in the shaft wall cuticle (swc). \\u003cstrong\\u003eG\\u003c/strong\\u003eCross-section of the mid-region of the sensillum shaft, showing pores connected to the lymph space via spoke canals. Two chemoreceptive dendrites (de), one of which arcuces, are visible in the inner lymph space (isl). \\u003cstrong\\u003eH\\u003c/strong\\u003e Cross-section of the basal region of the sensillum shaft, showing four dendrites (de) enclosed by the dendritic sheath (ds). The longitudinal canals (lca) are mostly fused and form a compact ring-like structure. \\u003cstrong\\u003eI\\u003c/strong\\u003e Close-up of the basal region of the sensillum shaft in cross-section, highlighting four dendrites (de) in the inner lymph space (isl). The outer lymph space (osl) contains cytoplasmic projections of accessory sheath cells (pasc). See also Additional file 1: Table S2\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6228127/v1/c1c840a3721d1e744b6d1667.png\"},{\"id\":78721009,\"identity\":\"a798168c-2610-41ce-9fd1-7bdcbd0d6d36\",\"added_by\":\"auto\",\"created_at\":\"2025-03-18 04:41:51\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":31083,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eResponses of male \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003ePisaura mirabilis\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003eto the scent of a female or a stimulus-free control.\\u003c/strong\\u003e The numbers on the bars represent responders, whereas the\\u003cstrong\\u003e \\u003c/strong\\u003ecentral gray boxes represent nonresponders.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6228127/v1/8ba1e2785a59fe7155121059.png\"},{\"id\":78720596,\"identity\":\"92cd5ce5-676c-41f4-b91c-f3670e237c4f\",\"added_by\":\"auto\",\"created_at\":\"2025-03-18 04:25:51\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":97301,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSchematic representation of contact and non-contact areas of walking legs and pedipalps in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003ePisaura mirabilis\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e males and females. \\u003c/strong\\u003eThese areas were categorized on the basis of substrate contact during locomotion, prey capture, and interactions with mating partners via high-speed video recordings. Segments with a contact probability of more than 50% across all trials were classified as contact areas. Segments with a contact probability of 1% to less than 50% were categorized as infrequent contact areas. The segments that never contacted the substrate during the trials were classified as non-contact areas. The segments from tip to body are the tarsus, metatarsus, tibia, patella, femur, trochanter, and coxa. See also Additional file 1: supplemental text, Tables S3 and S4.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6228127/v1/2582c3668b5dbb0433ac0f2e.png\"},{\"id\":78721319,\"identity\":\"331c7606-20b8-442c-964e-e279b8ded8f3\",\"added_by\":\"auto\",\"created_at\":\"2025-03-18 04:49:52\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3034725,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6228127/v1/f560cc62-bb3c-4955-a2d4-be9d2637baf6.pdf\"},{\"id\":78720584,\"identity\":\"b77f6674-37fb-451e-a3ff-8784a40457a1\",\"added_by\":\"auto\",\"created_at\":\"2025-03-18 04:25:50\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1675411,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003e\\u003cstrong\\u003eSupplemental information\\u003c/strong\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAdditional file 1. Figures S1–S5 and Tables S1-S4\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"PisauraBMCAdditionalfile.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6228127/v1/cd6043679bc41c432009cdf0.docx\"}],\"financialInterests\":\"The authors declare no competing interests.\",\"formattedTitle\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe Chemosensory Toolkit of a Cursorial Spider\\u003c/strong\\u003e\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eChemical sensing is a crucial sensory modality across all animal phyla (1), supporting a wide range of behaviors essential to ecological success and reproductive fitness. Land-living arthropods, such as insects, utilize highly specialized chemosensory systems to detect and interpret environmental chemical information from, e.g., food sources, predators, and conspecifics in the form of surface-bound compounds upon contact and airborne molecules. In insects, contact chemoreception (gustation/taste) is mediated by sensory hairs (sensilla) featuring a terminal or sub terminal pore. These tip-pore sensilla are located primarily on the mouthparts, antennae, and distal segments of the legs (2). In contrast, the perception of airborne chemicals (olfaction/smelling) is facilitated by sensilla with wall pores distributed along their shafts, which are predominantly found on the insect antennae (3). In both types of sensilla, chemicals enter their pore(s) and bind to receptor cell dendrites within the sensillum (4,5). Similarly, in crustaceans, contact chemosensilla (gustatory sensilla) with a terminal pore are distributed unevenly across the body, with dense concentrations on mouthparts and walking legs. In contrast, the olfactory sensilla of crustaceans, known as aesthetascs, are thin-walled pegs that lack pores but contain numerous olfactory neurons and are located primarily on the first and second antennae (6).\\u003c/p\\u003e \\u003cp\\u003eCompared to insects and crustaceans, limited knowledge exists regarding the chemical senses of other arthropods, limiting our comprehension of their sensory structures and the evolutionary history of these structures within this clade. For spiders, many behavioral studies support the use of both gustatory and olfactory information (7\\u0026ndash;9). For example, males are attracted to female-produced airborne sex pheromones (10\\u0026ndash;13), and upon contact, gustatory female sex pheromones elicit courtship behavior in males (12,14,15). Other than in the mating context, there is behavioural evidence that spiders use contact chemoreception and olfaction for habitat selection (16,17), prey detection (18,19), and predator avoidance (20,21).\\u003c/p\\u003e \\u003cp\\u003eThe cuticular chemosensory structures currently identified in spiders are tip-pore sensilla (22\\u0026ndash;24), which are found in large numbers on all walking legs and pedipalps of both sexes. These sensilla have been shown to function as contact chemoreceptors in an electrophysiological study on the spider \\u003cem\\u003eCupiennius salei\\u003c/em\\u003e (25). Prior to recent findings, reports of wall-pore sensilla in spiders were lacking (but see (22). However, wall-pore sensilla have now been found in the orb-weaver spider \\u003cem\\u003eArgiope bruennichi\\u003c/em\\u003e, where they occur in large numbers on all walking legs and exclusively in males (26). Using single sensillum electrophysiological recordings, it was demonstrated that the male-specific wall-pore sensilla respond to the female-produced airborne sex pheromone (26). Adult females release the pheromone from their webs and attract males from a distance (10). As typical for web-building spiders, \\u003cem\\u003eA. bruennichi\\u003c/em\\u003e females are stationary in their webs, whereas adult males actively search for females. However, many spider species are cursorial and do not reside in silken webs; instead, they roam freely and deposit dragline silk as they walk. Behavioral observations have demonstrated that in cursorial spiders such as lycosids, males follow female silk trails to locate potential mating partners (27,28). These findings suggest that cursorial spider rely primarily on silk-bound chemicals (gustation) for mate finding (but see (29) for evidence of olfaction). An examination of the sensilla apparatus of a cursorial spider, in conjunction with a behavioral assessment of the ability to perform olfaction during mate search, will help to contribute to the understanding of the chemosensory world of non-web-building spiders.\\u003c/p\\u003e \\u003cp\\u003eThe European nursery web spider \\u003cem\\u003ePisaura mirabilis\\u003c/em\\u003e (Clerck, 1757), also known as the nuptial-gift-giving spider, is a compelling model for exploring chemical communication in cursorial spiders. The lifestyle of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e combined with well-studied knowledge of its mating behavior (30\\u0026ndash;33), which relies on silk-bound chemical information (34\\u0026ndash;37), makes it particularly valuable for investigating chemosensory mechanisms. Furthermore, choice experiments have shown that \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e utilizes chemical information from plants for habitat selection (38) and avoids the cuticular hydrocarbons of an ant predator (21). However, most experiments have not differentiated between the sensory modes of gustation and olfaction. Although mating-related contact chemoreception (gustation) has been supported in several studies (35,39), whether olfaction is a relevant sensory mode remains to be investigated. Recent identification of putative wall-pore sensilla in adult \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e that are male-specific (26) suggests that olfaction may play a significant role during mate search and possibly other contexts in this species.\\u003c/p\\u003e \\u003cp\\u003eTo shed light on the chemical communication system of cursorial spiders, we investigated the chemosensory structures of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e and hypothesized that \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e possesses diverse types of chemosensilla if they are capable of performing contact-chemoreception and olfaction. Using high-resolution electron microscopy, we examined legs and pedipalps for candidate sensilla in both sexes and subadults and characterized the morphology and distribution of these sensilla. To address the knowledge gap regarding olfaction, we tested the hypothesis that males detect and respond to female-emitted airborne signals.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTip-pore sensilla\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eExternal morphology of the tip-pore sensilla\\u003c/h2\\u003e \\u003cp\\u003eSensilla with a blunt tip and a slightly oval terminal pore (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA, B), measuring 400\\u0026ndash;700 nm in diameter (N\\u0026thinsp;=\\u0026thinsp;10 pores for each sex), were found on all walking legs and pedipalps of both males and females (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, Additional file 1: Figure S1). They are hair like, appear slightly S shaped, and feature spirally oriented microtrichia (spine-like protuberances) along the shaft (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). The spination begins approximately 30 \\u0026micro;m from the finely ribbed shaft base and extends up to 10\\u0026ndash;15 \\u0026micro;m below the tip. The tip region contains many shallow longitudinal grooves (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB, C). The shafts of tip-pore sensilla emerge from a round, slightly elevated \\u0026lsquo;crater-like\\u0026rsquo; socket (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD) and project from it at a steep angle (50\\u0026ndash;65\\u0026deg;) to the leg surface cuticle, in contrast to the longer, branched, striated, and sharp-pointed tactile setae. In females, the sensillum shafts are approximately 188.14\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;40.80 \\u0026micro;m [arithmetic mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation] long (N\\u0026thinsp;=\\u0026thinsp;30), whereas in males, they are slightly shorter (175.15\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;37.39 \\u0026micro;m, N\\u0026thinsp;=\\u0026thinsp;30). The shaft diameter is very similar between the sexes, with values of 6.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.71 \\u0026micro;m (N\\u0026thinsp;=\\u0026thinsp;30) in females and 6.24\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.65 \\u0026micro;m (N\\u0026thinsp;=\\u0026thinsp;30) in males (Additional file 1: Table S1).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eInternal anatomy of the tip-pore sensilla\\u003c/h3\\u003e\\n\\u003cp\\u003eWe examined the internal anatomy of the tip-pore sensilla from the femur of the 1st walking leg of both males and females of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e. Ultrathin cross-sections of the middle region of the sensillum shaft reveal a star-shaped profile and a distinctly corrugated surface of the shaft wall cuticle (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE), corresponding to the spirally oriented, ribbed microtrichia observed with SEM (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA), whereas in the proximal region, the shaft possesses a circular profile along with a finely ribbed surface (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC, D). The thick shaft wall cuticle features a system of thin longitudinal lymph canals running inside its length, giving the shaft a double-wall appearance in a cross section (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC, E-F). None of the cross-sections examined revealed that these longitudinal shaft canals are connected to the lymph space or to the outside. Cross-sections show a hollow sensillum shaft filled with lymph (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE, F). The lymph space is divided into two compartments. These compartments are separated by a thick cuticular tube called the peridendritic shaft cylinder (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC, E-G) (according to M\\u0026uuml;ller et al.(24). The inner compartment, called the inner sensillum lymph space, contains 15\\u0026ndash;18 nonbranching dendrites (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE-G), which are likely chemoreceptive. Each dendrite is characterized by a set of microtubules. All dendrites running through the shaft terminate below the terminal pore. The outer compartment, called the outer sensillum lymph space, surrounds the inner compartment (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE-G). The outer sensillum lymph space often contains cytoplasmic processes of accessory sheath cells located beneath the leg cuticle (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eF, G). A mechanoreceptive portion, typical for tip-pore sensilla in spiders, is present in the socket region of this sensillum, and the somata of both chemoreceptive and mechanoreceptive neurons are located beneath the socket of the sensillum, as shown in \\u003cem\\u003eA. bruennichi\\u003c/em\\u003e (24).\\u003c/p\\u003e\\n\\u003ch3\\u003eDistribution of tip-pore sensilla\\u003c/h3\\u003e\\n\\u003cp\\u003eIn \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e, adult males possess approximately 4,100 tip-pore sensilla distributed across all walking legs and pedipalps, whereas females have approximately 2,900 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, Additional file 1: Figures S1, S2A-C, and S4). On all walking legs of both males and females, these sensilla are most abundant on the tarsus, metatarsus, and tibia; occur in lower numbers on the patella and femur; and are absent on the trochanter (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, Additional file 1: Figure S1). In both sexes, there are few (3\\u0026ndash;9) tip-pore sensilla on the coxa of all walking legs. On the tarsus, metatarsus, and tibia of all walking legs in both males and females, the tip-pore sensilla are arranged in seven to eight rows along the segment axes. The number of tip-pore sensilla was greater on the 1st and 2nd walking legs than on the 3rd and 4th walking legs in both sexes (Additional file 1: Figure S2C). On all walking legs, the tip-pore sensilla are predominantly located on the lateral and ventral sides, with fewer on the dorsal side (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, Additional file 1: Figure S1). On the pedipalps, the density of tip-pore sensilla is high on the dorsal and prolateral sides of the tarsus of the female and the dorsal side of the cymbium (the outer part of the modified palpal tarsus) of the male (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). In both sexes, toward the body, there are only a few tip-pore sensilla on the tibia, patella, and femur and none on the trochanter or coxa of the pedipalp.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eWall-pore sensilla\\u003c/h3\\u003e\\n\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eExternal morphology of wall-pore sensilla\\u003c/h2\\u003e \\u003cp\\u003eThe wall-pore sensilla are thin, trichoid hairs with round, slightly elevated \\\"crater-like\\\" sockets (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA-D). These sensilla occur exclusively on all walking legs of adult males and are not found in females or subadult males (Additional file 1: Figure S3). The sensilla have a curved shape with a tapered tip. The insertion angles of the wall-pore sensilla are slightly steeper than those of the mechanoreceptive sensilla (tactile hairs) found on all leg and pedipalp segments in high abundance. On average, the wall-pore sensilla of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e are 141.78\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;11.41 \\u0026micro;m [mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation] long (N\\u0026thinsp;=\\u0026thinsp;15) (Additional file 1: Table S2). The sensilla diameter is 2.12\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.16 \\u0026micro;m, as measured approximately 20 \\u0026micro;m distal to the hair base (N\\u0026thinsp;=\\u0026thinsp;15). The surface of the sensillum shaft shows fine diagonal ridges and grooves (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB), which are longitudinally oriented toward the tip. Numerous pore-like invaginations (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB), with an average diameter of 41.93\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.26 nm (N\\u0026thinsp;=\\u0026thinsp;45), are distributed inside the groves over almost the entire length of the shaft, except for the sensilla base region (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD). No marked differences in size or shape were observed in the wall-pore sensilla across different walking legs and their segments in male \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e (Additional file 1: Table S2).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eInternal anatomy of wall-pore sensilla\\u003c/h3\\u003e\\n\\u003cp\\u003eIn our TEM analysis, we focused on wall-pore sensilla located on the femur of the 1st walking leg of male \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e. Cross-sections taken from different regions of the shaft reveal that the wall-pore sensilla possess two lymph spaces (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE-I). There is a voluminous sensillum lymph space inside the shaft. This so-called outer lymph space contains an inner sensillum lymph space that is surrounded by a dendritic sheath (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE-I). The inner lymph space hosts 2\\u0026ndash;4 (mostly 4) dendrites characterized by a set of microtubules (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF-I). The dendrites are tightly packed inside the dendritic sheath, which is present throughout the shaft. Consecutive ultrathin cross-sections revealed that there are pores in the shaft cuticle (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE, G). These pores are connected to thin canal-like structures (spoke canals, approximately 100 nm in diameter; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF) that traverse the shaft wall cuticle and open into the outer sensillum lymph space. As these spoke canals run obliquely through the shaft wall, we did not capture their full course in our cross sections. Pore tubules, such as those found in single-walled sensilla of ticks (\\u003cem\\u003eAmblyomma variegatum\\u003c/em\\u003e) (40), were not observed in the spoke canals of the wall-pore sensilla of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e. The shaft wall cuticle exhibits narrow and distinct longitudinal lymph canals in a ring-like formation, giving the shaft wall a double-walled appearance (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE-H). At the basal shaft level, most of the longitudinal shaft canals fuse to form a ring (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eG). The longitudinal shaft canals do not interconnect with the radial spoke canals. In some sections, the outer sensillum lymph space contains cytoplasmic extensions of the accessory sheath cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eI) that are located beneath the leg cuticle. Like the wall-pore sensilla of \\u003cem\\u003eA. bruennichi\\u003c/em\\u003e (26), we did not detect tubular bodies, a key component of mechanoreceptors, in the socket region, indicating that these sensilla are not multimodal, as is characteristic of tip-pore sensilla.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eDistribution\\u003c/b\\u003e \\u003cb\\u003eof male-specific\\u003c/b\\u003e \\u003cb\\u003ewall-pore sensilla\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe male-specific wall-pore sensilla of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e are abundant (approx. 3,000 sensilla) and somewhat irregularly arranged on the proximal leg segments (femur, patella, and tibia) of all walking legs of the males (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, Additional file 1: Figure S1). They are most abundant in the middle to distal regions of the femur and patella and in the proximal region of the tibia in the first, second, and fourth legs. In contrast, wall-pore sensilla were not found on the distal segments (metatarsus and tarsus) of any leg or on the male pedipalps. Compared with the dorsal and ventral sides, the wall-pore sensilla are more densely distributed along the lateral sides of the legs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, Additional file 1: Figure S1). The first and second legs each possess approximately 460\\u0026ndash;470 wall-pore sensilla, whereas the fourth leg possesses approximately 340, and the shorter third leg has approximately 200 wall-pore sensilla (Additional file 1: Figure S2D).\\u003c/p\\u003e\\n\\u003ch3\\u003eOlfactometer test\\u003c/h3\\u003e\\n\\u003cp\\u003eOlfactory attraction of males to females was observed (binomial test, p\\u0026thinsp;=\\u0026thinsp;0.012; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e), with 16 (70%) out of 23 males choosing the female-equipped branch, four (17%) choosing the control branch, and three males (13%) not leaving the holding tube. In the control experiment, no side preference was observed, as four males (18%) chose the left branch, six males (27%) chose the right branch (binomial test, p\\u0026thinsp;=\\u0026thinsp;0.754; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e), 12 males (55%) exhibited no behavioral response (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e), and one male died prior to their second trial. Males were more likely to make a choice when exposed to a female scent (87%) than when they were not exposed to a female scent (45%) (Fisher\\u0026rsquo;s exact test, p\\u0026thinsp;=\\u0026thinsp;0.005; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eContact area versus non-contact area of body appendages\\u003c/h2\\u003e \\u003cp\\u003eWe conducted an analysis of the contact and non-contact regions of the body appendages of male and female \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e during locomotion on various surfaces, silk probing, prey capture, and mating. The contact and non-contact areas of the walking legs and pedipalps of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e were delineated on the basis of the contact probability across all pooled contexts: locomotion, silk probing, prey capture, and mating (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e, Additional file 1: Tables S3 and S4). Upon pooling all contexts (Additional file 1: Table S4), the ventral and lateral sides of the tarsus of all walking legs exhibited frequent substrate contact in both sexes (55%-100%). The metatarsus in males demonstrated frequent substrate contact on the first two walking legs (52%-53%), whereas in females, frequent contact was observed solely for the metatarsus of the first walking legs (53%). In both sexes. The tibia‒patella segments of all walking legs exhibited infrequent substrate contact across all contexts (1%-11%), and the femur did not establish contact with the substrate or mating partner in any context of our experimental trials. With respect to pedipalps, the cymbium in males and the tarsus in females frequently probed substrates, nuptial gifts, and prey, with contact frequencies ranging from 86%-100%. Conversely, the remaining pedipalp segments in both sexes rarely establish contact in any context. Detailed observations of all the behavioral contexts are provided in the supplemental material (Additional file 1: Supplement text, Tables S3 and S4).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eChemosensory sensilla in Pisaura mirabilis\\u003c/h2\\u003e \\u003cp\\u003eWe detected two distinct types of chemosensory sensilla in the cursorial spider \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e: tip-pore sensilla and wall-pore sensilla. The first type, tip-pore sensilla (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), is associated with contact chemoreception (gustation), a well-documented function in insects (5), crustaceans (6), and one spider species (25). In \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e, tip-pore sensilla are present in both sexes, as well as in subadults, and are distributed across all walking legs and pedipalps (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). They are predominantly located on the distal segments of the appendages, which frequently contact substrates (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e), suggesting their role in detecting surface-bound chemical information. The second type, wall-pore sensilla (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e), functions as olfactory sensilla, a role demonstrated in insects (4), crustaceans (6), and more recently in a spider, (26). Our Y-tube olfactometer assays demonstrated that \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e males respond to airborne signals emitted by females (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). In \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e, wall-pore sensilla are exclusive to adult males and absent in females and subadult males (Additional file 1: Figure S3). These sensilla are located on the proximal segments of all walking legs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, Additional file 1: Figure S1), areas that rarely contact substrate, prey, or mating partners (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e), which is consistent with findings in the orb-weaving spider \\u003cem\\u003eA. bruennichi\\u003c/em\\u003e (26), corroborating their specialization for detecting airborne signals emitted by females.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTip-pore sensilla\\u003c/h2\\u003e \\u003cp\\u003eTip-pore sensilla are present on all walking legs and pedipalps of both sexes and subadult \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e. These sensilla feature chemoreceptive dendrites housed within a trichoid shaft, with the dendrites terminating at a blunt terminal pore (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). This pore provides the only connection between the dendrites and the external environment, supporting the role of tip-pore sensilla in contact chemoreception, as confirmed in, e.g., insects (5), crustaceans (6), and arachnids such as opilionids and ricinulei (41,42). The gustatory role of tip-pore sensilla has also been demonstrated in the cursorial spider \\u003cem\\u003eCupiennius salei\\u003c/em\\u003e through electrophysiological studies (25). Although the outer morphology of the tip-pore sensilla can differ between species, the internal ultrastructures of the sensilla shafts in \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e and \\u003cem\\u003eC. salei\\u003c/em\\u003e are highly similar to those of other cursorial spiders and web-building spiders (24,43). In all cases, tip-pore sensilla also entail a mechanoreceptive component (24,25,43).\\u003c/p\\u003e \\u003cp\\u003eIn \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e, tip-pore sensilla are positioned at steeper angles (Additional file 1: Table S1) than pure mechanoreceptive sensilla are and are more densely distributed on distal segments (tarsus and metatarsus) of the walking legs and pedipalps, with fewer proximal segments (tibia, patella, and femur) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, Additional file 1: Figure S1). A similar distal abundance of tip-pore sensilla with steeper angles has been observed in both web-building (23) and cursorial spiders (43). In various insect orders, the presence of tip-pore sensilla on the mouthparts and distal segments of walking legs is common (2). For example, in leaf beetles (Coleoptera, Chrysomelidae), the tip-pore sensilla present on the tarsi of legs play a crucial role in host selection (44). This distal placement and steeper angles of tip-pore sensilla increase the probability of encountering substrate-bound chemical information, facilitating gustation during navigation and mating. Our video observations further support this, demonstrating that distal segments of the body appendages of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e frequently contact substrates during locomotion, silk probing, prey capture, and mating, whereas proximal segments rarely do (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e, Additional file 1: Tables S3 and S4). Additionally, there are fewer tip-pore sensilla on the dorsal sides of all walking legs, where contact with the substrate is rare (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, Additional file 1: Figure S1), which further corroborates the functional role of tip-pore sensilla as gustatory organs.\\u003c/p\\u003e \\u003cp\\u003eThere is a marked difference between the sexes in the number of tip-pore sensilla in \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e, with males possessing approximately 4,100 sensilla compared with 2,900 in females (Additional file 1: Figure S4), despite their similar body sizes. In males, the highest densities occur on the distal segments of the first and second walking legs and on the cymbium of the pedipalps. When encountering the dragline silk of a female, males touch the silk with these segments, and their tip-pore sensilla likely play a role in the detection of the reproductive status of a female and trail-following decisions (34,36,39,45). Consequently, mate detection and assessment are likely necessary for a greater number of tip-pore sensilla in males. In more ancient spiders, such as liphistiids, gustation-based mate search is likely also accomplished through the available tip-pore sensilla (46), but male-specific scopulate hairs have also been suggested to perform this function (47). Male-specific gustatory sensilla are also known from the lady beetle \\u003cem\\u003eSemiadalia undecimnotata\\u003c/em\\u003e (Coleoptera), where they occur on the antennae and have been proposed to play a role in mating-related functions (48). The greater quantity of tip-pore sensilla in the cursorial spider \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e than in the orb-weaver \\u003cem\\u003eA. bruennichi\\u003c/em\\u003e, which has 1,000 sensilla in males and 2,000 in females (23), likely reflects the necessity for cursorial spiders to perceive more detailed information on prey presence, habitat quality, and predation risk while roaming in the undergrowth. In contrast, most stationary web-building spiders predominantly remain on their web\\u0026mdash;an extended perceptual system through which they can even decipher prey types (49). In the web, physical contact occurs solely with intercepted prey items (with the exception of contact with a mating partner), which the spiders examine with their gustatory sensilla prior to wrapping or during prey consumption (50).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWall-pore sensilla\\u003c/h2\\u003e \\u003cp\\u003eWall-pore sensilla are found exclusively in adult male \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e. These sensilla contain multiple pores connecting to a central lymph space within the sensillum shaft via radial spoke canals, forming a direct pathway from the external environment to the chemoreceptive dendrites in the lymph space (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). The presence of longitudinal shaft wall canals indicates that these multiporous sensilla belong to the class of double-walled sensilla in insects; however, they lack slit-like grooves on the shaft surface and vase-shaped chambers below the pore (40). Distal processes of accessory sheath cells within the shaft (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eI) have been observed in single-walled sensilla with pore openings (40), similar to those found on the tips of the sensory legs of amblypygid spiders (22). The wall-pore sensilla of male \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e closely resemble those of \\u003cem\\u003eA. bruennichi\\u003c/em\\u003e, for which the perception of the airborne female sex pheromone was corroborated through electrophysiological tests (26). The wall-pore sensilla of both species, however, differ in ultrastructure from the single wall-pore sensilla described for a gradungulid species, a basally branching spider taxon (22), and whether wall-pore sensilla evolved once within spiders or several times convergently remains to be investigated.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e males possess approximately 3,000 wall-pore sensilla on all walking legs, predominantly in the proximal leg segments (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, Additional file 1: Figure S1), which are non-contact areas (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). This distribution suggests that their ability is to detect airborne signals released by females, similar to \\u003cem\\u003eA. bruennichi\\u003c/em\\u003e males (26). \\u003cem\\u003eA. bruennichi\\u003c/em\\u003e males possess approximately 7,000 male-specific wall-pore sensilla, which are likewise concentrated in non-contact areas of their walking legs and are finely tuned to airborne female-produced sex pheromone (26). The greater number of wall-pore sensilla in \\u003cem\\u003eA. bruennichi\\u003c/em\\u003e males may indicate a greater reliance on olfaction for mate search. Male \\u003cem\\u003eA. bruennichi\\u003c/em\\u003e are attracted from substantial distances to stationary females by signals released from the web (10), whereas \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e males may utilize both olfactory and gustatory information from female dragline silk in their environment.\\u003c/p\\u003e \\u003cp\\u003eIn arthropods, male-specific olfactory sensilla are abundant and typically specialize in detecting sex pheromones. For example, in a moth species, males possess approximately 50,000 specialized antennal trichoid sensilla that are dedicated to detecting female sex pheromones, whereas other olfactory sensilla in both males and females respond to general odors, as reviewed previously (4,51). Male-specific olfactory sensilla have also been found in other moth species (52\\u0026ndash;54). Similarly, in aquatic mysids (crustaceans), approximately 2,000 male-specific olfactory sensilla located basally on the first antennae have been proposed to function as pheromone receptors (55). These observations support the hypothesis that male-specific wall-pore sensilla in \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e likewise have evolved to enhance the detection of female sex pheromones and that these sensilla must be airborne.\\u003c/p\\u003e \\u003cp\\u003eOur behavioral assays indeed demonstrated that \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e males respond to airborne signals emitted by females (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). This finding aligns with observations in the lycosid spider \\u003cem\\u003ePardosa milvina\\u003c/em\\u003e, where males are attracted to airborne signals from adult virgin females but not from subadult females or males (29). The sexual pheromone of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e that resulted in remote attraction in our behavioral trials remains unidentified. No pheromone candidates were detected via gas chromatography‒mass spectrometry (GC‒MS) (unpublished data; G. Uhl, S. Schulz), possibly because these signals involve polar compounds that are undetectable via GC‒MS (7). Recently, liquid chromatography coupled with tandem mass spectrometry was used to identify polar olfactory and gustatory sex pheromones of widow spiders (11,12,56), which has been elusive in GC‒MS analyses. We propose similar investigations for \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e to explore the compound responsible for mate attraction in a cursorial spider.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eOlfaction of adult female and immature cursorial spiders.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe olfactory capabilities of subadult and female spiders remain uncertain, as wall pore sensilla have been found exclusively in male spiders (26). Nevertheless, studies indicate that female and juvenile cursorial spiders utilize olfactory information for purposes such as habitat selection (16) and prey detection (19). Consequently, it can be postulated that tip-pore sensilla potentially serve dual gustatory (taste) and olfactory (smell) functions, analogous to certain tip-pore chemoreceptors identified in insects (5,57,58). Apart from this \\\"dual-function hypothesis\\\", a \\u0026ldquo;functional zone hypothesis\\u0026rdquo; warrants investigation to determine whether the tip-pore sensilla located on the proximal segments of the legs, where direct contact with surfaces is infrequent, would engage in olfaction, and those in the distal part of the leg might be limited to gustation. Consequently, the function of the tip-pore sensilla may vary depending on their location, as has been reviewed in insects (59).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eWe set out to explore the chemosensory toolkit of arthropods other than insects as a step to unravel the evolution of chemosensing within arthropods. We elucidated the chemosensory apparatus and mate-finding mode of the cursorial spider \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e. Tip-pore sensilla, present in both sexes and associated with contact chemoreception, and male-specific wall-pore sensilla linked to olfaction were discovered and described on the basis of their outer and inner anatomical features. The significance of olfaction in mate search was demonstrated by male attraction to female scents. Morphological and behavioral findings suggest that male-specific sensilla function in female pheromone detection, as observed in insects and crustaceans. Male \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e possess wall-pore sensilla on all walking legs in regions close to the body that normally do not contact the substrate, which corroborates their function in olfaction. The absence of wall-pore sensilla in females and immature spiders raises novel questions regarding the sensilla involved in olfaction, other than mate search. Candidates are tip-pore sensilla that may form functional zones depending on their location on the legs. More broadly, this study contributes to our understanding of the ecological and evolutionary drivers of chemosensory diversity in arthropods, offering insights into how organisms detect and respond to chemical information in their environment and providing material for comparative analyses of sensory organs and their evolution across arthropods.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section3\\\"\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \"},{\"header\":\"Methods\",\"content\":\"\\u003ch2\\u003eStudy species\\u003c/h2\\u003e\\u003cp\\u003eJuvenile, subadult (one molt prior to adulthood), and adult \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e (Clerck, 1757) individuals of both sexes were collected each year from 2021–2024 from grasslands in Greifswald, Germany (54° 05' 49.91''N 13° 23' 16.58'' E) and brought to the laboratory. The spiders were individually housed in Drosophila culture tubes measuring 5 cm in diameter and 10 cm in height, with a netted top and a sponge lid at the bottom (35). Inside each vial, a substrate of artificial aquarium plants was provided. To maintain high humidity levels, vials with sponges at the bottom were placed in trays filled with water. The spiders were kept at a constant temperature of 22°C (± 5°C) under a 12:12 h light‒12 h dark cycle. Adult and subadult spiders were fed one housefly (\\u003cem\\u003eMusca domestica\\u003c/em\\u003e) or blowfly (\\u003cem\\u003eLucilia caesar\\u003c/em\\u003e) twice a week, whereas juveniles were provided with several fruit flies (\\u003cem\\u003eDrosophila hydei\\u003c/em\\u003e) on the same schedule. Juveniles and subadults were monitored regularly for the presence of exuviae to monitor their development.\\u003c/p\\u003e\\u003ch2\\u003eScanning electron microscopy: examination and mapping of chemosensilla\\u003c/h2\\u003e\\u003cp\\u003eTo investigate and map chemosensilla, freshly molted adult males and females as well as subadults of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e were anesthetized with CO\\u003csub\\u003e2\\u003c/sub\\u003e. They were then mounted on a polyethylene board (Plastazote) with their legs straightened and secured with wire clamps. The mounted samples were submerged and preserved in 80% ethanol. For SEM analysis, the straightened walking legs and pedipalps on the right side were detached and dehydrated through a graded ethanol series up to 99% and then dried using a Leica EM CPD300 critical point dryer. The straightened legs and pedipalps were vertically mounted on metal SEM stubs. The samples were then coated with gold-palladium for 2.5 min using a Polaron SC7640 sputter coater (Fisons Instruments). Then, the stubs were placed horizontally into a custom-made stub holder that allowed the samples to be rotated at various angles.\\u003c/p\\u003e\\u003cp\\u003eThe samples were examined using a Zeiss EVO LS 10 scanning electron microscope (SEM) operated at 10 kV. The mounted samples were oriented perpendicular to the electron beam and rotated at 0°, 90°, 180°, and 270° to capture images from the dorsal, prolateral, ventral, and retrolateral sides of the body appendages. Owing to variation in the diameter of the segments of the body appendages (coxa to tarsus), we used a range of magnifications (130× to 220×), but each segment was imaged at a consistent magnification for all males and females. The images were stitched together to reconstruct the relative positions of the sensilla on outline drawings of the spider’s legs, derived from multifocus stereomicroscopic photographs (Zeiss SteREO Discovery V20). Sensilla near the drawings´ borders appear on several maps but were excluded from the total count of sensilla. During mapping, the sensilla account for individual variation, and three males and three females were examined. All the sensillum parameters (length, diameter, insertion angle, and pore diameter) were measured via SEM built-in software. For a higher resolution of the properties of the sensillum shafts, a Zeiss SUPRA 40VP field emission SEM was used. The samples were cleaned with a 5% KOH solution and glacial acetic acid, following the methods described by Schneeberg et al. (60), before they were subjected to critical point drying. The legs were then mounted onto SEM stubs and sputter-coated with an Au-Pd (80:20) mixture at 5 mA (Q150T ES, QUORUM, UK), resulting in a coating thickness of 10 nm.\\u003c/p\\u003e\\u003ch2\\u003eTransmission electron microscopy: investigation of the internal anatomy of chemosensilla\\u003c/h2\\u003e\\u003cp\\u003eTo prepare the samples for transmission electron microscopy, freshly molted samples—two males and two females of \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e—were anesthetized with CO₂. The walking legs were then dissected using micro-scissors while immersed in an ice-cold prefixative solution modified from Karnovsky's protocol (61), consisting of 2.0% glutaraldehyde, 2.5% paraformaldehyde, and 1.5% sucrose in 0.1 mol/L sodium phosphate buffer at pH 7.4. To improve tissue preservation, each leg segment was cut into 2–5 pieces, each measuring 1–3 mm in length, and incubated in the same prefixative solution. To further enhance fixation, the samples were subjected to three sets of 2 min microwave pulses at a power of 120 W using a PELCO BioWave Pro instrument equipped with a PELCO SteadyTemp Pro Thermo Cube for solid-state cooling. Throughout the BioWave application, the sample temperature was monitored, remaining between 8–10°C prior to treatment and 9–15°C afterward. The leg pieces were stored in the same prefixative solution at 4°C for at least one night. The samples were then washed three times in 0.1 M sodium phosphate buffer for 45 min in total. The samples were subsequently fixed with 2% osmium tetroxide for 3 to 4 h at room temperature (RT) and then washed three times with ddH₂O for a total of 30 min. The samples were dehydrated through a series of ethanol solutions (50%, 60%, 70%, 80%, 90%, 100%), with dehydration at each concentration up to 90% carried out for 2 × 10 min at RT. For the 100% ethanol stage, the samples were washed over 3 × 10 min. Subsequent infiltration of the samples with epoxy resin (Embed812, an Epon substitute) was conducted at RT using the solvent propylene oxide (PO) over several stages of dilution: 2 × 100% PO for 15 min in total, 66% for 2 h, 50% for 12 h, and 33% for 24 h. The samples were placed on a shaker throughout infiltration to support efficient infiltration. For preembedding, the samples were incubated in 100% epoxy resin and again placed on a shaker for 2 h. To further increase infiltration speed and efficiency, the samples were then transferred to a Heraeus VT-6025 vacuum heating cabinet for a total of 2 h at 40°C. During that time span, the samples were exposed to high vacuum conditions (3 × 30 min at 150 mbar, each phase separated by 10-min breaks under normal atmospheric pressure). For final embedding, the samples were placed in silicon molds containing fresh epoxy resin. Each piece of the femur, tibia, patella, metatarsus, or tarsus was oriented in specific resin blocks (transverse, oblique, or longitudinal to the block face). The resin blocks were polymerized in a heating cabinet at 60°C for 48 hours. Transverse, horizontal, and longitudinal ultrathin sections (70–90 nm thick) of the tibia and femur were produced using a Leica UCT ultramicrotome. In areas from which ultrathin sections were obtained (with trimming gaps of 2–5 µm), semithin sections (700 nm thick) were also taken and stained with toluidine blue (dissolved in 1% sodium tetraborate) for examination under a light microscope. The ultrathin sections were placed on Formvar-coated slot grids (PLANO), stained with uranyl acetate and lead citrate for 4 min each, and then examined with a JEOL JEM-1011 transmission electron microscope operated at 80 kV. Digital photomicrographs were captured using a mid-mount camera (MegaView III, Soft Imaging System) with iTEM imaging software (Olympus, Soft Imaging Solution).\\u003c/p\\u003e\\u003ch2\\u003eOlfactometer test\\u003c/h2\\u003e\\u003cp\\u003eOlfactory attraction of males to unmated female \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e was tested in December 2024 using Y-tube olfactometers (Additional file 1: Figure S5) following established protocols (62). Females were placed in stimulus chambers (26 × 2.5 cm glass tube with mesh on both sides) for two hours to allow for acclimatization and silk deposition. The stimulus chamber was then connected by tubing to one arm of the Y-olfactometer (main stem: 24 cm long, side arms: 21 cm long, diameter: 2.5 cm), while a control chamber, without a female, was connected to the other arm. To initiate the assay, a male was placed into a glass tube (26 × 2.5 cm, with mesh on both sides) for two minutes to acclimatize, while a pump drew 0.4 L/min air. The acclimatization tube was then connected to the stem of the Y-tube. The male was subsequently given 15 min to reach the end of either arm, which was considered a choice. Upon first choice, the experiment was ended. To provide a grip for the spiders, all glass tubes held bamboo skewers (20 × 0.4 cm, Exnima, Damiel, Spain). Tissue paper, with a hole, lined the three terminal stoppers connected to tubing. The presentation sides were altered 50:50 across replicates. Bamboo skewers and tissues were discarded upon use, while the glassware was rinsed with water and baked for 2 h at 250°C to remove any stimuli. The experiment was replicated 23 times with randomly paired males and females (N = 23 each). To test for any side bias, each male was tested again one week later in the same setup but without any female stimuli. Male olfactory choice data were statistically compared against binomial or Fisher’s exact tests in R (63).\\u003c/p\\u003e\\u003ch2\\u003eContact and non-contact areas of body appendages\\u003c/h2\\u003e\\u003cp\\u003eTo identify the contact and non-contact areas on the body appendages (legs and pedipalps) of male and female \\u003cem\\u003eP. mirabilis\\u003c/em\\u003e, we observed their legs and pedipalps movements via high-speed camera recordings in 2021. The following scenarios were recorded in a laboratory: a) locomotion, b) silk probing by a male, c) capturing of prey, and d) mating. For the locomotion trials, the spiders were placed on four different substrates that they might encounter in their natural habitat: a flat surface, natural common nettles (a plant on which we frequently find the species in the field), a 20 cm long (2 mm in diameter) horizontally placed skewer, and a vertically placed bundle of natural grass (approximately 30 cm long). The substrates were cleaned with 70% ethanol and air-dried after each trial to eliminate any residual silk left by spiders. For the trials of silk probing by the male, we analyzed video footage from a previous study (34) in which males were also allowed to walk on and around dragline silk left by females in a transparent plastic box (17 cm × 8 cm × 6 cm) to observe their silk probing behavior. During prey capture, we observed how the spiders interacted with different-sized prey, including comparatively small houseflies (\\u003cem\\u003eMusca domestica\\u003c/em\\u003e) and larger house crickets (\\u003cem\\u003eAcheta domesticus\\u003c/em\\u003e). For each prey capture trial, an individual spider was placed in a transparent plastic box (17 cm × 8 cm × 6 cm), and a live prey item was released into the box. In our prey capture analysis, we included video footage from an earlier study (64). For mating observations, an unmated female was placed in an open arena (40 cm × 30 cm × 4 cm) for 5 min to deposit dragline silk. Females typically leave dragline silk behind when they roam (35,37). A male, carrying a nuptial gift (prey item wrapped in silk) in his chelicerae, was then introduced at the periphery of the arena. Upon contact with the female's dragline silk, the male followed it and approached the female, tapped the female with his legs and offered a nuptial gift. If the female accepted the gift and started feeding on it, the male inserted his pedipalps (sperm transfer organ) into her genital opening (epigyne) for mating. We recorded movements of all the above-mentioned behavioral contexts using a high-speed camera (Miro LC320S, AMETEK Vision Research) at 500 frames per second for a maximum of 8 seconds and analyzed the footage in slow motion with \\u003cem\\u003eImageJ\\u003c/em\\u003e software (public domain, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://imagej.net/ij/\\u003c/span\\u003e\\u003cspan address=\\\"https://imagej.net/ij/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). We noted which segments (tarsus, metatarsus, tibia, patella, and femur) of the leg or pedipalp made contact in these different contexts and which did not. The segments that contacted the substrates or partner at least once during a trial were considered contact areas for the specific appendages in that trial. We compared the number of trials in which contact occurred to the total number of trials (6–24 trials, depending on the context; see Additional file 1: Table S3) for all contexts, resulting in a contact probability ranging from 0–100%. The leg and pedipalp segments that made contact in more than 50% of the cases were labeled contact areas, whereas those below 50% were classified as infrequent contact areas. Those segments that did not contact the substrates during our trials were defined as non-contact areas.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eEthics approval and consent to participate\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eConsent for publication\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAvailability of data and materials\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eCompeting interests\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eFunding\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was funded by the German Science Foundation (DFG, UH 87/14-1, Grant No. 451487045).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAuthor contributions\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization, G.B.U., C.H.G.M.; Methodology, M.B.T., C.H.G.M., G.B.U., J.O.W., A.F.; Investigation, M.B.T., C.H.G.M., A. F., and V.M.; Writing-Original draft, M.B.T., G.B.U., and A.F.; Writing-Review \\u0026amp; Editing, G.B.U., C.H.G.M., J.O.W., and A.F.; Visualization, M.B.T., A.F.; Supervision, G.B.U. and C.H.G.M.; Funding acquisition, G.B.U. All the authors have read and approved the final manuscript\\u003cstrong\\u003e\\u003cem\\u003e.\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAcknowledgments\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe appreciate the technical support for the SEM analyses provided by Rabea Schl\\u0026uuml;ter and Stefan Bock from the Imaging Center at the University of Greifswald. We also thank Carmen Noske (Greifswald) for her assistance in filming the high-speed videos and rearing the spiders. Some high-speed video recordings of prey capture and silk probing were taken by Benjamin Eggs and Michelle Beyer together with Jonas Wolff. We are grateful to Hong-Lei Wang (Lund) for valuable literature suggestions. Additionally, we thank Heidi Land (Greifswald) for illustrating the sketch in Fig. 5.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eBuchinger TJ, Li W. Chemical communication and its role in sexual selection across Animalia. Commun Biol. 2023 Nov 20;6(1):1178.\\u003c/li\\u003e\\n\\u003cli\\u003eKing BH, Gunathunga PB. Gustation across the class Insecta: body locations. Ann Entomol Soc Am. 2023 Mar 13;116(2):76\\u0026ndash;82.\\u003c/li\\u003e\\n\\u003cli\\u003eFleischer J, Pregitzer P, Breer H, Krieger J. Access to the odor world: olfactory receptors and their role for signal transduction in insects. Cell Mol Life Sci. 2018 Feb;75(3):485\\u0026ndash;508.\\u003c/li\\u003e\\n\\u003cli\\u003eSteinbrecht RA. Olfactory receptors. In: Eguchi E, Tominaga Y, editors. Atlas of arthropod sensory receptors: Dynamic morphology in relation to function. Tokyo: Springer; 1999. p. 155\\u0026ndash;76.\\u003c/li\\u003e\\n\\u003cli\\u003eOzaki M, Tominaga Y. Contact chemoreceptors. In: Eguchi E, Tominaga Y, editors. Atlas of arthropod sensory receptors: Dynamic morphology in relation to function. Springer; 1999. p. 143\\u0026ndash;54.\\u003c/li\\u003e\\n\\u003cli\\u003eHallberg E, Skog M. Chemosensory Sensilla in Crustaceans. In: Breithaupt T, Thiel M, editors. Chemical Communication in Crustaceans. New York Dordrecht Heidelberg London: Springer; 2011. p. 103\\u0026ndash;22.\\u003c/li\\u003e\\n\\u003cli\\u003eFischer A. Chemical communication in spiders \\u0026ndash; a methodological review. J Arachnol. 2019 Apr 27;47(1):1.\\u003c/li\\u003e\\n\\u003cli\\u003eUhl G. Spider Olfaction: Attracting, Detecting, Luring and Avoiding. In: Nentwig W, editor. Spider Ecophysiology. Berlin, Heidelberg: Springer Berlin Heidelberg: Springer; 2013. p. 141\\u0026ndash;57.\\u003c/li\\u003e\\n\\u003cli\\u003eUhl G, Elias DO. Communication. In: Herberstein ME, editor. Spider behaviour, flexibility and versatility. Cambridge, England: Cambridge University Press; 2011. p. 127\\u0026ndash;88.\\u003c/li\\u003e\\n\\u003cli\\u003eChinta SP, Goller S, Lux J, Funke S, Uhl G, Schulz S. The sex pheromone of the wasp spider\\u003cem\\u003e Argiope bruennichi\\u003c/em\\u003e. Angew Chem Int Ed. 2010 Mar 8;49(11):2033\\u0026ndash;6.\\u003c/li\\u003e\\n\\u003cli\\u003eFischer A, Roman-Torres AC, Vurdela J, Lee Y, Bahar N, Gries R, et al. Non-targeted metabolomics aids in sex pheromone identification: a proof-of-concept study with the triangulate cobweb spider, \\u003cem\\u003eSteatoda triangulosa\\u003c/em\\u003e. Sci Rep. 2023 Oct 27;13(1):18426.\\u003c/li\\u003e\\n\\u003cli\\u003eFischer A, Gries R, Alamsetti SK, Hung E, Roman Torres AC, Fernando Y, et al. Origin, structure and functional transition of sex pheromone components in a false widow spider. Commun Biol. 2022 Oct 30;5(1):1156.\\u003c/li\\u003e\\n\\u003cli\\u003eFischer A, Schulz S, Ayasse M, Uhl G. Pheromone communication among sexes of the garden cross spider \\u003cem\\u003eAraneus diadematus\\u003c/em\\u003e. Sci Nat. 2021 Oct;108(5):38.\\u003c/li\\u003e\\n\\u003cli\\u003eBaruffaldi L, Costa FG, Rodr\\u0026iacute;guez A, Gonz\\u0026aacute;lez A. Chemical communication in \\u003cem\\u003eSchizocosa malitiosa\\u003c/em\\u003e: evidence of a female contact sex pheromone and persistence in the field. J Chem Ecol. 2010 Jul;36(7):759\\u0026ndash;67.\\u003c/li\\u003e\\n\\u003cli\\u003eScott C, Gerak C, McCann S, Gries G. The role of silk in courtship and chemical communication of the false widow spider, \\u003cem\\u003eSteatoda grossa\\u003c/em\\u003e (Araneae: Theridiidae). J Ethol. 2018 May;36(2):191\\u0026ndash;7.\\u003c/li\\u003e\\n\\u003cli\\u003eDodson GN, Lang PL, Jones RN, Versprille AN. Specificity of attraction to floral chemistry in \\u003cem\\u003eMisumenoides\\u003c/em\\u003e \\u003cem\\u003eformosipes\\u003c/em\\u003e crab spiders. J Arachnol. 2013 Apr;41(1):36\\u0026ndash;42.\\u003c/li\\u003e\\n\\u003cli\\u003eFischer A, Hung E, Gries G. Female false black widow spiders, \\u003cem\\u003eSteatoda grossa,\\u003c/em\\u003e recognize webs based on physical and chemical cues. Entomol Exp Appl. 2019 Sep;167(9):803\\u0026ndash;10.\\u003c/li\\u003e\\n\\u003cli\\u003eC\\u0026aacute;rdenas M, Jiro\\u0026scaron; P, Pek\\u0026aacute;r S. Selective olfactory attention of a specialised predator to intraspecific chemical signals of its prey. Naturwissenschaften. 2012 Aug;99(8):597\\u0026ndash;605.\\u003c/li\\u003e\\n\\u003cli\\u003eJackson RR, Nelson XJ, Sune GO. A spider that feeds indirectly on vertebrate blood by choosing female mosquitoes as prey. Proc Natl Acad Sci. 2005 Oct 18;102(42):15155\\u0026ndash;60.\\u003c/li\\u003e\\n\\u003cli\\u003eFischer A, Lee Y, Dong T, Gries G. Know your foe: synanthropic spiders are deterred by semiochemicals of European fire ants. R Soc Open Sci. 2021 May;8(5):210279.\\u003c/li\\u003e\\n\\u003cli\\u003eNarimanov N, Heuschele JM, Entling MH, Menzel F, Mestre L. Differential effects of ephemeral and stable predator chemical cues on spider antipredator behaviour. J Chem Ecol. 2024 Nov;50(11):714\\u0026ndash;24.\\u003c/li\\u003e\\n\\u003cli\\u003eFoelix RF. Mechano- and chemoreceptive sensilla. In: Barth FG, editor. Neurobiology of arachnids. Berlin: Springer; 1985. p. 118\\u0026ndash;37.\\u003c/li\\u003e\\n\\u003cli\\u003eGanske AS, Uhl G. The sensory equipment of a spider \\u0026ndash; A morphological survey of different types of sensillum in both sexes of \\u003cem\\u003eArgiope bruennichi\\u003c/em\\u003e (Araneae, Araneidae). Arthropod Struct Dev. 2018 Mar;47(2):144\\u0026ndash;61.\\u003c/li\\u003e\\n\\u003cli\\u003eM\\u0026uuml;ller CHG, Ganske A, Uhl G. Ultrastructure of chemosensory tarsal tip‐pore sensilla of \\u003cem\\u003eArgiope spp.\\u003c/em\\u003e Audouin, 1826 (Chelicerata: Araneae: Araneidae). J Morphol. 2020 Dec;281(12):1634\\u0026ndash;59.\\u003c/li\\u003e\\n\\u003cli\\u003eTichy H, Gingl E, Ehn R, Papke M, Schulz S. Female sex pheromone of a wandering spider (\\u003cem\\u003eCupiennius salei\\u003c/em\\u003e): identification and sensory reception. J Comp Physiol A. 2001 Mar 8;187(1):75\\u0026ndash;8.\\u003c/li\\u003e\\n\\u003cli\\u003eTalukder MB, M\\u0026uuml;ller CHG, Zhang DD, Schulz S, L\\u0026ouml;fstedt C, Wang HL, et al. Olfaction with legs\\u0026mdash;Spiders use wall-pore sensilla for pheromone detection. Proc Natl Acad Sci. 2025 Jan 21;122(3):e2415468121.\\u003c/li\\u003e\\n\\u003cli\\u003eTietjen WJ. Dragline‐following by male Lycosid spiders. Psyche J Entomol. 1977 Jan;84(2):165\\u0026ndash;78.\\u003c/li\\u003e\\n\\u003cli\\u003eTietjen WJ, Rovner JS. Trail-following behavioour in two species of wolf spiders: sensory and etho-ecological concomitants. Anim Behav. 1980;(28):735\\u0026ndash;41.\\u003c/li\\u003e\\n\\u003cli\\u003eSearcy LE, Rypstra AL, Persons MH. Airborne chemical communication in the wolf spider \\u003cem\\u003ePardosa milvina\\u003c/em\\u003e. J Chem Ecol. 1999;25(11):2527\\u0026ndash;33.\\u003c/li\\u003e\\n\\u003cli\\u003eAlbo MJ, Toft S, Bilde T. Condition dependence of male nuptial gift construction in the spider \\u003cem\\u003ePisaura mirabilis\\u003c/em\\u003e (Pisauridae). J Ethol. 2011 Sep;29(3):473\\u0026ndash;9.\\u003c/li\\u003e\\n\\u003cli\\u003eBeyer M, Uludağ K\\u0026Ouml;, Tuni C. Female state and condition-dependent chemical signaling revealed by male choice of silk trails. Behav Ecol. 2023 Nov 8;34(6):919\\u0026ndash;29.\\u003c/li\\u003e\\n\\u003cli\\u003eGhislandi PG, Beyer M, Velado P, Tuni C. Silk wrapping of nuptial gifts aids cheating behavioour in male spiders. Behav Ecol. 2017 May 1;28(3):744\\u0026ndash;9.\\u003c/li\\u003e\\n\\u003cli\\u003eProkop P, Semelbauer M. Biometrical and behavioural associations with offering nuptial gifts by males in the spider \\u003cem\\u003ePisaura mirabilis\\u003c/em\\u003e. Anim Behav. 2017 Jul;129:189\\u0026ndash;96.\\u003c/li\\u003e\\n\\u003cli\\u003eBeyer M, Uludag K\\u0026Ouml;, Lailler M, Wolff JO, Eberhard MJB, Czaczkes TJ, et al. Testing presence of directionality information in female spider silk trails through male trail-following behavior. Behav Ecol Sociobiol. 2023 Dec;77(12):139.\\u003c/li\\u003e\\n\\u003cli\\u003eEberhard MJB, Machnis A, Uhl G. Condition-dependent differences in male vibratory pre-copulatory and copulatory courtship in a nuptial gift-giving spider. Behav Ecol Sociobiol. 2020 Nov;74(11):138.\\u003c/li\\u003e\\n\\u003cli\\u003eJežov\\u0026aacute; Z, Prokop P, Zvar\\u0026iacute;kov\\u0026aacute; M, Zvar\\u0026iacute;k M. Unraveling the significance of draglines: female sexual signalization in the nursery-web spider, \\u003cem\\u003ePisaura mirabilis\\u003c/em\\u003e. Insects. 2023 Sep 13;14(9):765.\\u003c/li\\u003e\\n\\u003cli\\u003eNitzsche ROM. Courtship, mating and agonistic behaviour in \\u003cem\\u003ePisaura mirabilis\\u003c/em\\u003e (Clerck, 1757). Arachnology. 2011 Mar;15(4):93\\u0026ndash;120.\\u003c/li\\u003e\\n\\u003cli\\u003eJunker RR, Bretscher S, D\\u0026ouml;tterl S, Bl\\u0026uuml;thgen N. Phytochemical cues affect hunting-site choices of a nursery web spider (\\u003cem\\u003ePisaura mirabilis\\u003c/em\\u003e) but not a crab spider (\\u003cem\\u003eMisumena vatia\\u003c/em\\u003e). J Arachnol. 2011 Apr;39(1):113\\u0026ndash;7.\\u003c/li\\u003e\\n\\u003cli\\u003eBeyer M, Czaczkes TJ, Tuni C. Does silk mediate chemical communication between the sexes in a nuptial feeding spider? Behav Ecol Sociobiol. 2018 Mar;72(3):49.\\u003c/li\\u003e\\n\\u003cli\\u003eTichy H, Barth FG. Fine structure of olfactory sensilla in myriapods and arachnids. Microsc Res Tech. 1992 Sep;22(4):372\\u0026ndash;91.\\u003c/li\\u003e\\n\\u003cli\\u003eGainett G, Michalik P, M\\u0026uuml;ller CHG, Giribet G, Talarico G, Willemart RH. Ultrastructure of chemoreceptive tarsal sensilla in an armored harvestman and evidence of olfaction across Laniatores (Arachnida, Opiliones). Arthropod Struct Dev. 2017 Mar;46(2):178\\u0026ndash;95.\\u003c/li\\u003e\\n\\u003cli\\u003eTalarico G, Palacios‐Vargas JG, Fuentes Silva M, Alberti G. Ultrastructure of tarsal sensilla and other integument structures of two \\u003cem\\u003ePseudocellus\\u003c/em\\u003e species (Ricinulei, Arachnida). J Morphol. 2006 Apr;267(4):441\\u0026ndash;63.\\u003c/li\\u003e\\n\\u003cli\\u003eFoelix RF, Chu-Wang IW. The morphology of spider sensilla II. chemoreceptors. Tissue Cell. 1973 Jan;5(3):461\\u0026ndash;78.\\u003c/li\\u003e\\n\\u003cli\\u003eYosano S, Kutsuwada Y, Akatsu M, Masuta S, Kakazu R, Masuoka N, et al. Taste recognition through tarsal gustatory sensilla potentially important for host selection in leaf beetles (Coleoptera: Chrysomelidae). Sci Rep. 2020 Mar 18;10(1):4931.\\u003c/li\\u003e\\n\\u003cli\\u003eEberhard MJB, M\\u0026ouml;ller TA, Uhl G. Dragline silk reveals female developmental stage and mediates male vibratory courtship in the nuptial gift‐giving spider \\u003cem\\u003ePisaura mirabilis\\u003c/em\\u003e. Ethology. 2021 Mar;127(3):267\\u0026ndash;77.\\u003c/li\\u003e\\n\\u003cli\\u003eHaupt J. The Mesothelae - a Monograph of an exceptional group of spiders (Araneae: Mesothelae). Stuttgart: E. Schweizerbart\\u0026rsquo;sche Verlagsbuchhandlung; 2003. (Zoologica).\\u003c/li\\u003e\\n\\u003cli\\u003eFoelix R, Erb B, Michalik P. Scopulate hairs in male \\u003cem\\u003eLiphistius\\u003c/em\\u003e spiders: probable contact chemoreceptors. J Arachnol. 2010 Dec;38(3):599\\u0026ndash;603.\\u003c/li\\u003e\\n\\u003cli\\u003eJourdan H, Barbier R, Bernard J, Ferran A. Antennal sensilla and sexual dimorphism of the adult ladybird beetle \\u003cem\\u003eSemiadalia undecimnotata\\u003c/em\\u003e Schn. (Coleoptera: Coccinellidae). Int J Insect Morphol Embryol. 1995;24(3):307\\u0026ndash;22.\\u003c/li\\u003e\\n\\u003cli\\u003eJapyass\\u0026uacute; HF, Laland KN. Extended spider cognition. Anim Cogn. 2017 May;20(3):375\\u0026ndash;95.\\u003c/li\\u003e\\n\\u003cli\\u003eRobinson MH, Mirick H, Turner O. The predatory behavior of some araneid spiders and the origin of immobilization wrapping. Psyche J Entomol. 1969 Jan;76(4):487\\u0026ndash;501.\\u003c/li\\u003e\\n\\u003cli\\u003eSteinbrecht RA. Structure and function of insect olfactory sensilla. In: Bock GR, Cardew G, editors. Novartis Foundation Symposia. 1st ed. Wiley; 1996. p. 158\\u0026ndash;83.\\u003c/li\\u003e\\n\\u003cli\\u003eMochizuki F, Shibuya T. Antennal single sensillum responses to sex pheromone in male beet armyworm, \\u003cem\\u003eSpodoptera exigua\\u003c/em\\u003e, Hubner (Lepidoptera: Noctuidae). Appl Entomol Zool. 1991;26(3):409\\u0026ndash;11.\\u003c/li\\u003e\\n\\u003cli\\u003eMochizuki F, Sugi N, Shibuya T. Pheromone sensilla of the beet armyworm, \\u003cem\\u003eSpodoptera exigua \\u003c/em\\u003e(Hubner) (Lepidoptera:Noctuidae). Appl Entomol Zool. 1992;27(4):547\\u0026ndash;56.\\u003c/li\\u003e\\n\\u003cli\\u003eAlmaas TJ, Mustaparta H. Pheromone reception in tobacco budworm moth \\u003cem\\u003eHeliothis virescens\\u003c/em\\u003e. J Chem Ecol. 1990 Apr;16(4):1331\\u0026ndash;47.\\u003c/li\\u003e\\n\\u003cli\\u003eJohansson KUI, Hallberg E. Male-specific structures in the olfactory system of mysids (Mysidacea; Crustacea). Cell Tissue Res. 1992 May;268(2):359\\u0026ndash;68.\\u003c/li\\u003e\\n\\u003cli\\u003eFischer A, Fischer AJ, Gries R, Hung E, Lau K, Monfared A, et al. Identification and seasonal abundance of web- and air-borne sex pheromone components of western black widow spiders, \\u003cem\\u003eLatrodectus hesperus\\u003c/em\\u003e. J Chem Ecol. 2025 Jan 29;51:36.\\u003c/li\\u003e\\n\\u003cli\\u003eKwon HW, Lu T, R\\u0026uuml;tzler M, Zwiebel LJ. Olfactory responses in a gustatory organ of the malaria vector mosquito \\u003cem\\u003eAnopheles gambiae\\u003c/em\\u003e. Proc Natl Acad Sci. 2006 Sep 5;103(36):13526\\u0026ndash;31.\\u003c/li\\u003e\\n\\u003cli\\u003eSt\\u0026auml;dler E, Hanson FE. Olfactory capabilities of the \\u0026ldquo;gustatory\\u0026rdquo; chemoreceptors of the tobacco hornworm larvae. J Comp Physiol A. 1975;104:97\\u0026ndash;102.\\u003c/li\\u003e\\n\\u003cli\\u003eKing BH, Gunathunga PB. Gustation in insects: taste qualities and types of evidence used to show taste function of specific body parts. J Insect Sci. 2023 Mar 1;23(2):11.\\u003c/li\\u003e\\n\\u003cli\\u003eSchneeberg K, Bauernfeind R, Pohl H. Comparison of cleaning methods for delicate insect specimens for scanning electron microscopy. Microsc Res Tech. 2017 Nov;80(11):1199\\u0026ndash;204.\\u003c/li\\u003e\\n\\u003cli\\u003eKarnovsky MJ. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol. 1965;27(2):137\\u0026ndash;8.\\u003c/li\\u003e\\n\\u003cli\\u003eFischer A, Goh XH, Varney JLS, Blake AJ, Tak\\u0026aacute;cs S, Gries G. Multimodal and multifunctional signaling? \\u0026ndash; Web reduction courtship behavior in a North American population of the false black widow spider. PLOS ONE. 2020 Feb 26;15(2):e0228988.\\u003c/li\\u003e\\n\\u003cli\\u003eR Core Team. R. R Foundation for Statistical Computing, Vienna, Austria; 2024. (A language and environment for statistical computing.).\\u003c/li\\u003e\\n\\u003cli\\u003eEggs B, Wolff JO, Kuhn‐Nentwig L, Gorb SN, Nentwig W. Hunting without a web: how Lycosoid spiders subdue their prey. Ethology. 2015 Dec;121(12):1166\\u0026ndash;77.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[{\"identity\":\"c2cfab71-0595-418d-9506-3eb57527dc53\",\"identifier\":\"10.13039/501100001659\",\"name\":\"Deutsche Forschungsgemeinschaft\",\"awardNumber\":\"451487045\",\"order_by\":0}],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"Universtiy of Greifswald\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Chemical ecology, Araneae, tip-pore sensilla, wall-pore sensilla, behavior, gustation, olfaction\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6228127/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6228127/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eChemosensation is vital for organisms to detect food, avoid predators, and find mates. Spiders, like many arthropods, rely on chemosensory input, but the modes and structures for detecting chemicals are not well understood. Cursorial spiders use chemical information for orientation and mate-finding. Behavioral studies suggest gustation plays a major role in male spiders following silken draglines to locate females, but the role of olfaction and tools involved in chemosensing are not known, despite the important ecological role of spiders.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eHigh-resolution electron microscopy revealed two types of chemosensory sensilla in the cursorial spider \\u003cem\\u003ePisaura mirabilis\\u003c/em\\u003e. Tip-pore sensilla, on the walking legs and pedipalps of both sexes, support contact chemoreception. Wall-pore sensilla, only occurring on adult male walking legs, are associated with olfaction. Behavioral assays confirmed that males detect airborne chemical signals from females, supporting these sensilla's role in mate search. The distribution of both sensilla types on walking legs is nearly complementary, with tip-pore sensilla towards leg tips and wall-pore sensilla towards leg bases supporting their respective contact and olfactory functions.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e \\u003cp\\u003eThis study links the morphology, location, and function of chemosensory organs in cursorial spiders, providing insights into their chemosensory world. The findings advance the understanding of chemical sensing evolution in spiders and have implications for broader arthropod research. The identification of specialized olfactory sensilla in male spiders highlights the significance of airborne chemical detection in mate-finding, while contact chemoreceptors in both sexes suggest a general role in environmental chemical sensing.\\u003c/p\\u003e\",\"manuscriptTitle\":\"The Chemosensory Toolkit of a Cursorial Spider\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-03-18 04:25:46\",\"doi\":\"10.21203/rs.3.rs-6228127/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"a7b0bef4-1015-4e2d-82af-c2abdca7dbce\",\"owner\":[],\"postedDate\":\"March 18th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":45701320,\"name\":\"Evolutionary Biology\"},{\"id\":45701321,\"name\":\"Animal Behavior\"},{\"id\":45701322,\"name\":\"Cellular \\u0026 Molecular Neuroscience\"}],\"tags\":[],\"updatedAt\":\"2025-03-18T04:25:46+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-03-18 04:25:46\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6228127\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6228127\",\"identity\":\"rs-6228127\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}