Olfactory learning in two Amblypygi species Paraphrynus laevifrons and Phrynus pseudoparvulus.

Olfactory learning in two Amblypygi species Paraphrynus laevifrons and Phrynus pseudoparvulus - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Anim Cogn . 2026 Apr 18;29(1):44. doi: 10.1007/s10071-026-02065-1 Olfactory learning in two Amblypygi species Paraphrynus laevifrons and Phrynus pseudoparvulus Laura Segura-Hernández 1, 2, ✉ , Kenna D S Lehmann 1, 3 , John Perez 1 , Daniel D Wiegmann 4, 6 , Verner P Bingman 5, 6 , Eileen A Hebets 1 Author information Article notes Copyright and License information 1 School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588 USA 2 Smithsonian Tropical Research Institute, Panamá City, Panamá 3 Human Biology Program, Michigan State University, East Lansing, MI 48824 USA 4 Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403 USA 5 Department of Psychology, Bowling Green State University, Bowling Green, OH 43403 USA 6 J. P. Scott Center for Neuroscience, Mind and Behavior, Bowling Green State University, LSH –, Bowling Green, OH 43403 USA ✉ Corresponding author. Received 2025 Oct 13; Revised 2026 Mar 29; Accepted 2026 Apr 2; Issue date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13233874  PMID: 41998460 Abstract A wide diversity of arthropod taxa have demonstrated the capacity for learning, but most of our current understanding comes from only a select subset of this highly diverse clade, with most studies focusing on various insect groups. Amblypygids (Order Amblypygi, Class Arachnida), however, are emerging as a model group for studying sensory integration and the neural substrates associated with learning and memory, especially as it relates to navigation. These nocturnal creatures possess specialized sensory appendages and one of the largest and most complex mushroom bodies - the part of the arthropod brain associated with learning and memory - of any arthropod. Prior field studies on multiple species demonstrate sophisticated homing abilities while laboratory-based behavioral assays in Phrynus marginemaculatus confirm olfactory-based learning associated with a refuge. In this study, we expand these laboratory-based assays to test the olfactory learning ability of two additional amblypygid species - Paraphrynus laevifrons and Phrynus pseudoparvulus - for which field data suggest complex navigation. We trained each species to associate an open refuge with a distinct chemical stimulus, using slightly different species-specific experimental details. We found evidence of olfactory learning in the training trials for both species, with an unexpected pattern of seeming avoidance learning of hexanol. Our test trials did not show significant associations with the trained stimulus, but this may have been in part due to our experimental design. Ultimately, we find some evidence of olfactory learning in additional amblypygid species, but our findings raise additional questions and highlight the need for species-specific study designs. Supplementary Information The online version contains supplementary material available at 10.1007/s10071-026-02065-1. Keywords: Arachnid, Comparative cognition, Mushroom body, Navigation, Olfaction, Whip spider Introduction Learning, the ability to “[update] system properties based on processing of new information,” affords organisms the opportunity to change their behavior and adapt to novel and changing environments within a single lifetime (Alcock 2005 ; Barron et al. 2015 ). Associative learning is particularly beneficial for navigation and homing, and research has demonstrated that arthropods are proficient learners capable of multiple kinds of associative learning, including classical and operant conditioning (Perry et al. 2013 ). Some of the most frequently studied learning abilities in arthropods focus on the eusocial Hymenoptera. This work has revealed that honeybees ( Apis melifera ) and bumblebees ( Bombus impatiens ) readily learn to discriminate flowers using odors, colors, and the presence of conspecifics (Leadbeater and Chittka 2009 ; Leonard et al. 2011 ; Lynn et al. 2005 ; Wright et al. 2005 ) and that ants ( Myrmica sabuleti ) are able to learn odors associated with a food reward (Cammaerts and Rachidi 2009 ). Fruit flies ( Drosophila melanogaster ) are also common subjects in learning studies and learn a number of body movements to avoid or prevent the application of negative stimuli (Booker and Quinn 1981 ; Brembs and Heisenberg 2000 ; Wustmann et al. 1996 ), and we observe similar abilities in the less-commonly studied weta ( Hemideina femorata , Hoyle and Field 1983 ), locust ( Schistocera americana , Forman 1984 ), and crayfish ( Procambarus clarkii , Kawai et al. 2004 ). Crabs ( Carcinus maenas , Abramson and Feinman 1990 ), and lobsters ( Homarus americanus , Tomina and Takahata 2010 ) can also learn to press or pull a button or lever to receive a food reward. Additional work has established that fruit flies (Brembs and Wiener 2006 ), bumblebees (Fauria et al. 2002 ), honeybees (Collett and Kelber 1988 ; Deisig et al. 2001 ; Gould 1987 ; Koltermann 1971 ; Mota et al. 2011 ; Schubert et al. 2002 ), cockroaches (Sato et al. 2006 ), crickets ( Gryllus bimaculatus , (Matsumoto and Mizunami 2004 ), and lobsters ( Panulirus argus , (Livermore et al. 1997 ) are capable of contextual learning (for additional details on learning in arthropods and invertebrates, see Perry et al. 2013 ) and the well-studied honeybee ( Apis mellifera ) is capable of conceptual learning (Bortot et al. 2019 ; Giurfa et al. 2001 ; Giurfa 2019a , b ; Muszynski and Couvillon 2015 , 2020 ). Learning appears to be widespread in arthropods, but one group in particular has emerged as a model group for studying sensory integration and the neural substrates associated with learning and memory related to navigation specifically (Wiegmann et al. 2016 ). This group, amblypygids, or ‘whip spiders’ (class Arachnida, order Amblypygi), is widespread, has unique sensory organs, and engages in complex behavior (e.g. navigation (Hebets et al. 2014b ; Ortega-Escobar 2020 ; Weygoldt 2000 ; Wiegmann et al. 2016 ) and agonistic interactions (Fowler-Finn and Hebets 2006 ). Their unique morphology and impressive homing abilities make them an excellent group in which to study olfactory learning (Hebets et al. 2014a ; Wiegmann et al. 2016 ). The order Amblypygi is relatively small compared to other arachnid orders. It is comprised of five families and 18 genera (Harvey 2007 ; updated from World Amblypygi Catalog 2026 ). Importantly, however, the 282 recognized species (World Amblypygi Catalog 2026 ) have been found nearly worldwide in a variety of habitats. All amblypygids are nocturnal and generally spend the day in moist microhabitats like hollow and rotting trees, under tree bark, in rock crevices, and under rocks (Chapin 2015 ; Fowler-Finn and Hebets 2006 ; Hebets and Chapman 2000a ; Quintero 1981 ). While most species are epigean, several species inhabit caves (Chapin 2015 ) and some are found only in ant and termite nests (Carvalho et al. 2012 ; Weygoldt 2000 ). This variety of microhabitats and associated lifestyles may underlie variation in the evolutionary history of navigation-related learning across amblypygid species. Amblypygids have impressive morphological adaptations and neural structures capable of supporting complex navigation and learning. In contrast with most other arachnids, amblypygids use only three pairs of legs for walking. The first, anterior pair of legs are elongated, slender, highly jointed, and covered in thousands of sensilla (reviewed in Santer and Hebets 2011 ). Amblypygids use these distinct legs like antennae to sense their environment in all directions (Weygoldt 2000 ). These unique sense organs and their accompanying neural structures are thought to be responsible for the ability of amblypygids to navigate through complex, three-dimensional habitats to relocate their home refuge (Beck and Görke 1974 ; Bingman et al. 2017 ; Hebets 2002 ; Hebets et al. 2014a , b ; Wiegmann et al. 2019 ). Olfactory and visual stimuli from the antenniform legs and eyes are integrated within the mushroom bodies, higher brain regions dense with dendrites, axons, and glial cells (Foelix et al. 2001 ; Santer and Hebets 2011 ; Sinakevitch et al. 2020 ; Spence and Hebets 2006 ; Strausfeld et al. 2009 ). Although mushroom bodies are found in other arthropods and their common ancestors (Kenyon 1896 ; Strausfeld et al. 2006 , 2009 ; Strausfeld 2012 ; Wolff et al. 2012 ), in comparison with other arthropods, amblypygid mushroom bodies are nearly the largest, relative to body size, and are more elaborately folded (Sinakevitch et al. 2020 ; Strausfeld et al. 1998 ; Wiegmann et al. 2016 ). Mushroom bodies are said to scale with olfactory navigational demands (Jacobs 2012 ), are involved in learning and memory in other taxa [honeybees: (Devaud et al. 2015 ; Erber et al. 1980 ; Menzel 2001 ; Menzel and Giurfa 2001 ), cockroaches: (Mizunami et al. 1998 , 2013 ), fruit flies: (Cognigni et al. 2018 ; de Belle and Heisenberg 1994 ; Guven-Ozkan and Davis 2014 ; Heisenberg 2003 ; Pascual and Préat 2001 ; Zars et al. 2000 )], and may even be homologous to the vertebrate center of spatial memory and navigation, the hippocampus (Jacobs 2012 ; Wolff and Strausfeld 2016 ). This combination of unique neuro- and sensory biology, wide range, and broad habitats makes amblypygids an excellent group for the comparative study of the evolution of learning and navigation. Sensory modality-specific learning has been extensively studied in one species of amblypygid, Phrynus marginemaculatus C.L. Kock 1840. Phrynus marginemaculatus is a medium-sized (18 mm body length, (Quintero 1981 ) amblypygid that lives in the pine rocklands and tropical hardwood hammocks of Southern Florida. Its range extends into the Caribbean islands as far south as Jamaica (Chapin et al. 2018 ; Quintero 1981 ). Phrynus marginemaculatus has demonstrated impressive abilities in associative learning. In the laboratory, they can locate a previously used shelter from self-derived scent cues (Casto et al. 2019 ) and they can learn to associate tactile (Santer and Hebets 2009 ), visual (Flanigan et al. 2021a ), and olfactory cues with a reward in the form of a shelter to hide from unwanted conditions (Wiegmann et al. 2019 ). Further, they retain their learning of olfactory cues for at least two weeks (Wiegmann et al. 2019 ), remember prior agonistic opponents up to three weeks (Fowler-Finn and Hebets 2006 ), and appear to integrate multimodal cues into a configural representation of their learned shelter (Flanigan et al. 2021b ). They may even be capable of higher-order conceptual learning by applying the concepts of ‘same’ and ‘different’ to novel stimuli (Lehmann et al. 2022 ). The navigation of additional amblypygid species has been studied in the field, but the sensory cues associated with their learning and navigation remain unstudied. This study explores the olfactory learning capacity of two additional amblypygid species – Paraphrynus laevifrons and Phrynus marginemaculatus . Paraphrynus laevifrons Pockock,1984 is a large amblypygid that inhabits vertical surfaces in the tropical wet forests of Costa Rica and Panama (Corey and Hebets 2017 ; Mullinex 1975 ). Field studies have shown that they can successfully navigate, presumably via the use of olfaction, through these complex forests when displaced up to ten meters from their home refuge (Bingman et al. 2017 ). In the laboratory, Paraphrynus laevifrons uses self-derived olfactory signals to locate previous refuges (Casto et al. 2020 ), much like Phrynus marginemaculatus . The only other learning experiment conducted on this species to date determined that Paraphrynus laevifrons does not learn to associate magnetic cues with a provided shelter, presumably because magnetic cues are not salient navigational stimuli to P . laevifrons (Wiegmann et al. 2020 ). Phrynus pseudoparvulus Armas & Viquez 2001 is a medium-sized amblypygid that lives in the tropical rainforests of Costa Rica. Their diurnal refuges are often located in the crevices of large, buttressed trees. At night, they venture out from their home tree to forage and traverse impressive distances through the complex rainforest matrix, sometimes returning days or even weeks later (Hebets 2002 ). Displacement experiments in the wild revealed they can navigate back to their home tree from distances as far as 25 m (Hebets et al. 2014b ) and this navigation is dependent on olfaction and, to a lesser extent, vision (Hebets et al. 2014a ). While they demonstrate excellent homing in the wild, their olfaction-based learning ability has yet to be experimentally tested in the laboratory. This study directly tests olfactory-based learning associated with homing in the laboratory in Paraphrynus laevifrons and Phrynus pseudoparvulus . We used a modified learning assay developed in Phrynus marginemaculatus (Flanigan et al. 2021a , b ; Wiegmann et al. 2019 ) to test whether Paraphrynus laevifrons (Experiment 1, Fig. 1 a) and Phrynus pseudoparvulus (Experiment 2, Fig. 1 b) can learn to associate an olfactory stimulus with a shelter reward. Individuals were subjected to multiple days of training trials, followed by a final test trial. If these two species rely on olfaction-based learning in homing, as field studies suggest, we predicted (P1) a higher proportion of individuals to enter the shelter associated with the conditioned stimulus (C+) as training progressed and (P2) individuals would take less time to enter the C+ shelter. Across test trials, we also predicted that (P3) most individuals would choose to spend most of their time near the C+ stimulus (where “choice” was a categorical outcome defined as the side of the arena the amblypygid spent the highest proportion of time) and (P4) individuals would spend a higher proportion of time on the side of the arena with the C+ stimulus. Fig. 1. Open in a new tab Photos of Paraphrynus laevifrons ( a ) and Phrynus pseudoparvulus ( b ) in their natural habitats Methods and results General methods We placed amblypygids in a well-lit arena (Fig. 2 ) with two possible darkened shelters that contained the conditioned (C+) and unconditioned (C-) olfactory stimuli. Amblypygids are nocturnal and tend to hide from light, so bright lights above the arenas were meant to encourage them to find the open shelter associated with the C+ cue during training trials, while the C- shelter remained closed by a plastic mesh. Individuals were subjected to multiple days of training trials, followed by a final test trial, wherein entry to both shelters was blocked. Fig. 2. Open in a new tab Photo ( a ) and diagram ( b ) of the arena used in the training and test trials for both species. Black dotted lines indicate permanent plastic screening while dashed black lines indicate removable plastic screening. C+, C-, and neutral areas delineated in grey were used for scoring the amount of time the amblypygid spent near each stimulus. This is the same arena design used in (Flanigan et al. 2021a ; b ; Wiegmann et al. 2019 ) For the C + and C- stimuli, we used distilled water or two volatile chemicals (with different treatments depending on the species and experiment) commonly used in amblypygid learning experiments (Flanigan et al. 2021b ; Lehmann et al. 2022 ; Wiegmann et al. 2019 ). Our two chosen chemical stimuli, geraniol (C10H18O, SigmaAldrich, Product Number 163333) and 1-hexanol (C6H14O, SigmaAldrich, Product Number 471402), are naturally-occurring plant compounds and are discriminable and not aversive to Phrynus marginemaculatus (Wiegmann et al. 2019 ). Further, an electrophysiology study of the scent receptors on the antenniform legs of Phrynus pseudoparvulus determined this species can detect hexanol and citral, which is composed of two-thirds geraniol (Hebets and Chapman 2000b ). All training and testing trials were video recorded for later blind scoring. For training trials, we recorded whether the amblypygid entered the C+ shelter before the trial time ended (P1) and the latency to enter (P2). We divided the arena into quarters, with the middle 2 quarters designated as a “neutral” or “no choice” area and the remaining quarters next to each shelter designated as C + or C- (Fig. 2 ). We scored test trials for (i) which side of the arena area the amblypygid spent the most time in (conditioned, unconditioned, and ‘none’ if they spent the entire trial in the neutral zone) (P3) and (ii) how long the test animals spent in each designated C-, C+, or N area (P4). The walls of the arena (interior dimensions: 28 × 14 × 5 cm, Fig. 2 ) were constructed of an opaque acrylic plastic while the floor was a clear acrylic to allow us to visualize designations of C+, C-, and neutral zones on a piece of paper placed underneath the arena. The arena had three crevices (3 wide x 1 cm tall) centered at the base of three walls, two at either end of the arena and one placed at the center of the long wall. These crevices led to shelters constructed of opaque black acrylic (14 × 5 × 3 cm) with a removable lid. At the end opposite the crevice, each shelter contained a single clear acrylic cube (2.5 × 2.5 × 1 cm) with a small well drilled into the top of it. This well had 15 µL of the appropriate cue placed into it for each trial. Each cube was separated from the rest of the shelter by a hard plastic screen to prevent the animals from interacting with it directly. A similar screen was used to block the crevices as necessary. The amblypygids could not put their antenniform legs through the screen. We covered the floor of the arena and shelters with mesh screening for traction. In Experiment 1 ( Paraphrynus laevifrons ) one 60-watt spiral CFL light bulb (900 lumens) was attached via a clamp to the center of the lengthwise wall opposite the start shelter opening. In Experiment 2 ( Phrynus pseudoparvulus ), two 60-watt spiral CFL light bulbs (900 lumens) were attached to either end of the arena. Differences in lighting were due to the desire for enhanced negative stimuli (i.e. light) after Experiment 1. The bulbs were placed approximately 15–20 cm from the floor of the arena. To ensure amblypygids were not learning to go to a single side of the arena, a coin was flipped to randomly determine which side should contain the C + cue. In between trials, all parts of the arena and experimental shelters were cleaned and dried with 95% ethanol to remove any remaining olfactory stimulus. In each trial, an amblypygid was placed in the darkened start shelter with a clear acrylic cube containing distilled (Experiment 1, Paraphrynus laevifrons ) or tap water (Experiment 2, Phrynus pseudoparvulus ) and a hard-plastic mesh screen on either side prevented the animal from entering the arena or accessing the acrylic cube. Distilled water was not available for Phrynus pseudoparvulus experiments, as this work was conducted at a tropical field station. After an acclimation period, the opaque lid was removed from the start shelter to let in light and encourage the amblypygid to enter the area through the now-open crevice. Each trial began once the animal entered the arena and the crevice was covered behind it, preventing it from re-entering the start shelter. Statistical analyses To test whether the individuals were more likely to enter the C+ shelter as training progressed (P1), we built generalized binomial models with the likelihood of entering the C+ shelter as the response variable and individual identity as a random effect. We then compared the fit of models with and without the inclusion of trial number as a fixed effect. To test whether amblypygid learning was affected by the training chemical, we included chemical identity and an interaction between chemical identity and trial number in the first model. This interaction was significant, so we split the datasets by treatment and analyzed these data separately in subsequent analyses. Comparative model fit was assessed via likelihood ratio test and AIC comparison. We used a Cox proportional-hazards model to determine if amblypygids entered the C+ shelter more quickly as training progressed (P2). We included individual identity as a random effect to control for repeated measures. To test our final two predictions, we calculated the proportion of time the amblypygid spent in the C+, C-, and neutral areas during the final test trial. We then compared the time spent in the C + and C- areas. Amblypygids that did not leave the neutral area were determined to have made no choice and the remaining individuals were coded as choosing the area (C + or C-) they spent the most time in (P3). We used a Chi-squared goodness of fit test to compare counts of conditioned and unconditioned choices to determine if amblypygids that made a choice were more likely to make the conditioned choice. We also used a beta regression model to determine if amblypygids spent a larger proportion of time on the conditioned side of the arena (note: this analysis excluded the amount of time spent in the neutral area) (P4). Experiment 1: Paraphrynus laevifrons Paraphrynus laevifrons (Fig. 1 a) were collected around Las Cruces Biological Station in Coto Brus, Puntarenas, Costa Rica by Tyler Corey in January and June 2016 and transported to the laboratory at the University of Nebraska—Lincoln (Corey and Hebets 2020 ), where they were housed until the experiments were conducted from April to August 2016. They were housed separately in plastic terraria (18.3 × 11.2 × 14 cm) in a temperature- (21–26 °C) and humidity- (20–60%) controlled room with a 12-hour day/night cycle. Each terrarium contained 2.5 cm of a moist coconut coir substrate (Zoo Med), water dish, and a rectangle (10.2 × 16.2 × 0.64 cm) of cork bark for shelter and molting. Study animals were fed an appropriately sized cricket each week. S ixteen Paraphrynus laevifrons were evenly divided between four treatments: (1) geraniol -, (2) hexanol -, (3) geraniol +, and (4) hexanol + (Table 2 ). For the training trials, the amblypygid was presented with a chemical-containing shelter (open in positive treatments and closed in negative treatments) opposite a shelter with distilled water (closed in positive treatments and open in negative treatments). Thus, each trial had one chemical (geraniol or hexanol) versus water. In the negative treatments, we expected the amblypygids to learn to associate a chemical with a closed shelter while in the positive treatments, we expected the amblypygids to learn to associate a chemical with an open shelter. Training was conducted over three days, with five training trials on the first two days, and four training trials followed by a test trial on the third day. Table 2. Model Results for Experiment 1 Training trials - Paraphrynus laevifrons and water vs. chemical training trials. Likelihood ratio test p-values compare generalized linear models with individual identity as a random effect and trial count included and excluded. ∆AIC is the AIC value of the model without trial count minus the AIC of the model with trial count included. Positive ∆AIC indicates the model listed was not a better fit than the model without trial count. Negative ∆AIC indicate the model listed is a better fit than the model without trial count and trial count was a significant predictor of whether the amblypygids enter the shelter Dataset Predictors Log-Odds SE p geraniol - N = 56 (Intercept) -3.08 2.95 0.298 Trial Count 0.67 0.27 0.012 Likelihood ratio test p < 0.0001 ∆AIC -15.122 hexanol - N = 56 (Intercept) -3.16 0.01 < 0.001 Trial Count 0.28 0.01 < 0.001 Likelihood ratio test p = 0.001718 ∆AIC -7.829 geraniol + N = 56 (Intercept) -1.69 0.01 < 0.001 Trial Count 0.37 0.01 < 0.001 Likelihood ratio test p < 0.0001 ∆AIC -14.33 hexanol + N = 56 (Intercept) -0.01 0.84 0.994 Trial Count 0.02 0.07 0.763 Likelihood ratio test p = 0.7645 ∆AIC 1.91 Open in a new tab Each individual was given five minutes to acclimate in the start shelter before exposing the amblypygid to light. If the amblypygid had not left the start shelter after five minutes, it was encouraged into the arena with a blunt probe. The amblypygid was given fifteen minutes to locate and enter the open C+ shelter. After this time, if the subject still had not entered the open C+ shelter, it was gently coaxed to enter with a blunt probe. To reinforce learning, the subject was held in the darkened shelter for five minutes before being returned to their cage. In the 15-minute test trial, both experimental shelters were closed so that one side had geraniol and the other had hexanol. Notably, this was a different design than all training trials, which had a chemical vs. water. This paired chemical design was meant to test whether Paraphrynus laevifrons had learned attraction/avoidance of their assigned chemical and eliminated the possibility that they had learned attraction/avoidance of a strong scent in general. We expected individuals that experienced geraniol or hexanol with the open shelter (geraniol + and hexanol +) would move toward those chemicals in the test trials, while individuals that were exposed to an open shelter with water (geraniol - and hexanol -) would more away from the test chemical (Table 1 ). Table 1. Experiment 2 training and expected C+ stimulus for each treatment Treatments C+ training cues C- training cues Expected Test Trial C+ geraniol - water geraniol hexanol hexanol - water hexanol geraniol geraniol + geraniol water geraniol hexanol + hexanol water hexanol Open in a new tab Training trial results In support of our first prediction (P1), the likelihood that amblypygids entered the C+ shelter increased over training trials for those individuals trained to move away from geraniol (geraniol -), toward geraniol (geraniol +), or away from hexanol (hexanol -). The likelihood that subjects trained to move toward hexanol (hexanol +), however, remained constant (Fig. 3 ; Table 2 ). Overall, these data suggest Paraphrynus laevifrons have the capacity to learn an olfactory cue and associate it with shelter, supporting our first prediction. Fig. 3. Open in a new tab Training trials for Experiment 1. Paraphrynus laevifrons trained to associate geraniol and hexanol to a closed shelter (geraniol - and hexanol -, respectively) and geraniol with an open shelter (geraniol +) were more likely to enter the open, C+ shelter as training progressed. Individuals trained to associate hexanol with an open shelter (hexanol +) were not more likely to enter the open, C+ shelter as training progressed We also found support for our second prediction (P2). In the Cox proportional-hazards model, trial number was a significant predictor of time to enter the C+ shelter for all treatments (geraniol -: Fig. 4 a, Trial count β = 0.09028, SE = 0.04248, p = 0.0336; geraniol +: Fig. 4 b, Trial count β = 0.1533, SE = 0.04345, p = < 0.0005; hexanol -: Fig. 4 c, Trial count β = 0.1680, SE = 0.0634, p = < 0.00806), except hexanol + (Fig. 4 d, Trial count β = -0.0009, SE = 0.0422, p = 0.983). Paraphrynus laevifrons reduced the time to enter the C+ shelter over the course of training in all but the hexanol + treatment. Fig. 4. Open in a new tab Survival curves for time to enter C+ shelter during training trials in Experiment 1 ( Paraphrynus laevifrons ). Individuals entered the C+ shelter more quickly as training progressed in all treatments ( a - c ) except the hexanol + treatment ( d ) Test trial results We found mixed results for our third and fourth predictions (P3 and 4). Across all trials, Paraphrynus laevifrons individuals did not choose the C + cue more often than expected by chance (P3, Fig. 5 a) (All trials: X 2 = 1.9231, df = 1, p = 0.1655; geraniol - trials: X 2 =0, df = 1, p = 1; hexanol - trials: X 2 = 0.333, df = 1, p = 0.5637; geraniol + trials: = X 2 = 0.333, df = 1, p = 0.5637; hexanol + trials: X 2 = 3, df = 1, p = 0.08326), but individuals did spend a larger proportion of time on the C+ side of the arena overall (P4), although this effect varied by treatment (Fig. 5 b). Fig. 5. Open in a new tab Experiment 1 - Paraphrynus laevifrons test trials: geraniol vs. hexanol. Bar plot of the number of trials in which amblypygids made each choice ( a ). Choice was defined as the side of the arena the amblypygid spent the highest proportion of time in. Choice was coded as “none” if the amblypygid never left the neutral area. The beta regression model estimates and 95% confidence intervals of the proportion of time spent on the C+ side of the arena out of the total time spent on either side ( b ) Experiment 2: Phrynus pseudoparvulus Phrynus pseudoparvulus (Fig. 1 b) were collected and experiments were conducted in La Tirimbina Biological Reserve, Sarapiquí, Heredia, Costa Rica in April and October of 2017. Between trials, Phrynus pseudoparvulus were housed in small plastic containers (15 × 15 × 6 cm) with a moistened paper towel. They were released at the completion of their trials. Similarly to Experiment 1, we aimed to test whether Phrynus pseudoparvulus can learn to associate a chemical stimulus with a darkened shelter. Because we were doing this experiment in the field and needed to conduct experiments on a very shortened timeline (due to limited time at the field station), we trained 18 individuals over two days instead of three. Day one consisted of seven (as opposed to five in Experiment 1) 15-minute training trials and day two consisted of six 15-minute training trials followed by a single 20-minute (April collection) or 30-minute (October collection) test trial. The October trials were extended to ensure we provided the animals enough time to express a choice. Given our limited sample sizes, we also altered the study design such that every individual was trained with both geraniol and hexanol (as opposed to one chemical vs. water as we did with Paraphrynus laevifrons ). This design also allowed us to test whether the individuals had learned to distinguish chemicals with fewer treatments than Experiment 1 (two treatments versus four). In each training trial, half ( n = 9) of the individuals were taught to associate geraniol and the other half ( n = 9) were taught to associate hexanol with the open shelter. The opposing closed shelter contained the unconditioned stimulus (C-), which was the opposite chemical. After acclimating to the dark, start shelter for three minutes, the amblypygids were encouraged to enter the arena with a paintbrush and the start shelter was blocked behind them. The individuals were then allowed to explore the arena for approximately 15 min. Similar to Experiment 1, if they did not go into the retreat by that time, they were gently encouraged to enter the open shelter with a paintbrush. Amblypygids were rewarded with a few minutes in the darkened C+ shelter before being returned to their darkened cage for approximately 60 min, while other individuals underwent trials. Test trials were identical to the training trials, except all crevices were closed and individuals were given either 20-minutes (April collection) or 30-minutes (October collection) to explore the arena. Training trial results We failed to find support for our first prediction (P1). Phrynus pseudoparvulus individuals that were trained with geraniol did not increase the likelihood of entering the C+ shelter over training trials (Table 3 ; Fig. 6 a). In contrast to our prediction, those trained with hexanol were less likely to enter the C+ shelter over the course of training (Table 3 ; Fig. 6 c). Table 3. Model Results for Experiment 2 - Phrynus pseudoparvulus training trials. Likelihood ratio test p-values compare generalized linear models with individual identity as a random effect and trial count included and excluded. ∆AIC is the AIC value of the model without trial count minus the AIC of the model with trial count included. Positive ∆AIC indicates the model listed was not a better fit than the model without trial count. Negative ∆AIC indicate the model listed is a better fit than the model without trial count and trial count was a significant predictor of whether the amblypygids enter the C+ shelter Dataset Predictors Log-Odds SE p geraniol N = 117 (Intercept) 2.89 1.65 0.081 Trial count -0.07 0.08 0.399 Likelihood ratio test p = 0.3961 ∆AIC 1.28 hexanol N = 117 (Intercept) 3.43 1.04 0.001 Trial count -0.37 0.09 < 0.001 Likelihood ratio test p < 0.0001 ∆AIC -24.37 Open in a new tab Fig. 6. Open in a new tab Training trials for Experiment 2. Phrynus pseudoparvulus trained to associate geraniol with an open shelter were not more likely to enter the open, C+ shelter ( a ) and they did not enter the C+ shelter more quickly ( b ) as training progressed. Individuals trained to associate hexanol with an open shelter were less likely ( c ) and took longer ( d ) to enter the C+ shelter as training progressed We also did not find general support for our second prediction (P2). In the Cox proportional-hazards model, trial number was not a significant predictor of time to enter the C+ shelter for the geraniol-trained animals. Similar to results for P1, however, our hexanol-trained animals show the exact opposite pattern to what we expected for P2: trial number was significant, but negative, in the model of time to enter the C+ shelter. Similarly, amblypygids trained to associate geraniol with a shelter did not reduce the time to enter the shelter over the course of training (Fig. 6 b, Trial count β = -0.044, SE = 0.957, p = 0.14) while those trained with hexanol took longer to enter the shelter as training progressed (Fig. 6 d, Trial count β = -0.176, SE = 0.839, p = < 0.0001). Overall, Phrynus pseudoparvulus appeared to learn to avoid hexanol. Test trial results We did not find support for our third prediction (P3). Phrynus pseudoparvulus did not choose the conditioned C+ scent more often than expected by chance (Fig. 7 a- All trials: X 2 = 0.28571, df = 1, p = 0.593; geraniol trials: X 2 = 0, df = 1, p = 1; hexanol trials: X 2 = 0.66667, df = 1, p = 0.4142) and they did not spend a larger proportion of time on the C+ side of the arena (Fig. 7 b). Fig. 7. Open in a new tab Test trials for Experiment 2 - Phrynus pseudoparvulus . Bar plot of the number of trials in which amblypygids made each choice ( a ). Choice was defined as the side of the arena the amblypygid spent the highest proportion of time in. Choice was coded as “none” if the amblypygid never left the neutral area. The beta regression model estimates and 95% confidence intervals of the proportion of time spent on the conditioned side of the arena out of the total time spent on either side ( b ) Post experimental procedures During trials with Phrynus pseudoparvulus and prior to any data analysis, a worry arose that this species finds hexanol inherently aversive, because individuals were taking long periods of time to enter the open shelter during training trials. Thus, after the completion of these experiments, we tested whether Phrynus pseudoparvulus found geraniol or hexanol aversive without any prior training. To test this, we conducted an aversion test on 18 individuals in October 2017. We found that Phrynus pseudoparvulus is not inherently avoidant of geraniol or hexanol (see Supplementary Material for full methods and results), suggesting that Phrynus pseudoparvulus’s change in behavior in the learning trials was not the result of an innate aversion to conditioned odors, but instead a learned aversion. Discussion Both of our study species demonstrated changes in behavior, especially during training trials, that indicate that they could use olfactory cues to find, or avoid, shelters as they navigate their natural environment. For instance, in support of our first two predictions, Paraphrynus laevifrons were more likely to enter the shelters (P1) and entered more quickly over the course of training (P2) in all treatments except hexanol+ (i.e., geraniol+, geraniol- and hexanol-). This increased likelihood to enter and decreased latency to enter are consistent with our predictions of olfactory-based associative learning in regard to navigation. In the test trials, however, they did not choose the conditioned side more often than expected by chance (P3) but they did spend a higher proportion of time on the C+ side of the arena with the conditioned stimulus (P4). Notably, this is despite the fact that our test trials provided P. laevifrons with an additional challenge, as each individual was presented with a novel odor in the test trial (i.e. the opposite chemical that they received training on). In hindsight, this design in which individuals were trained to one chemical vs. water but tested with two chemicals complicates our data interpretation, as the presence of a novel odor could, in principle, override prior learning. Paraphyrnus laevifrons individuals, for example, may have been motivated to explore the novel stimulus. A combination of associative learning and neophilia, or exploratory behavior, could help explain our inability to detect a clear signature of choice in the test trials of this species. Regardless, our data are consistent with three of our four predictions in Paraphyrnus laevifrons , providing support for the overarching hypothesis that, like Phrynus marginemaculatus , this species can use olfactory learning in navigation. Phrynus pseudoparvulus also changed their behavior throughout training trials, suggesting that they could learn olfactory cues to find shelters. In contrast to Paraphrynus laevifrons , however, the patterns were opposite from our predictions (P1 and 2). Phrynus pseudoparvulus were less likely (P1) and took longer (P2) to enter the C+ shelter only in the hexanol+ treatment. Again, this pattern is exactly opposite our predicted results and suggests that they learned avoidance versus attraction to hexanol. We did not see this pattern in the test trials, as Phrynus pseudoparvulus did not associate more with the C + or C- sides of the arena (P3 and 4). Our test trial analyses, however, included individuals trained to associate the open crevice with both geraniol and hexanol, which appeared to have different impacts on Phrynus pseudoparvulus behavior. Thus, it is perhaps not surprising that we observed no pattern of learning in these test trials. Overall, most (but not all) of our results suggest that both Paraphrynus laevifrons and Phrynus pseudoparvulus are capable of associative olfactory learning in regards to locating shelters and indicate intriguing similarities and differences concerning their responses to particular chemical stimuli. The hexanol stimulus had a surprising effect on both species. Despite no evidence of innate aversion (see Supplementary Material for aversion trial details), Paraphrynus laevifrons did not enter the C+ shelter with hexanol more quickly over the training trials, as was observed in the other treatments. Similarly, Phrynus pseudoparvulus demonstrated what looks like a learned aversion to hexanol. To reiterate, we chose geraniol and hexanol for several reasons: (i) they are plant-derived compounds that have comparable volatilities and are readily discriminated by many terrestrial arthropods (Hansson and Stensmyr 2011 ; Leonard and Masek 2014 ); (ii) a prior electrophysiology study using Phrynus pseudoparvulus showed a capacity for detecting monoterpenoids structurally similar to geraniol and a strong response to hexanol (Hebets and Chapman 2000b ), and (iii) a behavioral learning study (using a similar design as our study) demonstrated that Phrynus marginemaculatus showed no innate attraction or aversion to either chemical when tested (Wiegmann et al. 2019 ). It is also relevant to note that geraniol has been shown to affect the sensing ability in distant arachnids (Gaudet et al. 2024 ). Ultimately, we had no a priori reason to expect distinct behavioral responses to geraniol and hexanol. We are left wondering if the aversion was the result of the chemical itself, a random occurrence in our small sample size, or another untested factor of the experimental design. Future research is necessary to explore this unanticipated behavioral response to hexanol. It is important to note the limitations of our comparative data as they relate to the differences in protocols used for each species. As highlighted in the methods, these differences stemmed from constraints associated with different locations of behavioral assays (field station versus university laboratory) and different limitations in time and sample size. As the trials for Phrynus pseudoparvulus were done after those of Paraphrynus laevifrons , we simplified the design to increase sample size with the limited number of amblypygids collected. This design meant that we trained all individuals with both chemicals present and we shortened the number of days for training trials (from three to two). These differences in protocols make it harder to draw direct comparisons between the species (hence why they were analyzed separately) and should be considered when addressing these results. Nonetheless, both experiments still demonstrate interesting patterns that provide insights in the potential use of learning olfactory cues to guide navigation in amblypygids. Though our training trials provide evidence that Paraphrynus laevifrons and Phrynus pseudoparvulus are capable of olfactory learning, they also appear to be less proficient learners than Phrynus marginemaculatus. Phrynus marginemaculatus learned to associate tactile, olfactory, and visual stimuli with a shelter reward within 10, 14, and 10 training trials, respectively (Flanigan et al. 2021a ; Santer and Hebets 2009 ; Wiegmann et al. 2019 ). In contrast, the 13–14 training trials conducted here were not enough to demonstrate clear, unambiguous olfactory learning in the test trials of our study species. There are multiple potential reasons why Phrynus marginemaculatus showed clearer results regarding olfactory associative learning to find shelter. For instance, the experimental design, modified from a study on Phrynus marginemaculatus , may have favored the natural history and behavior of Phrynus marginemaculatus. Phrynus marginemaculatus from Florida is primarily a horizontally dwelling species that moves along the ground and enters crevices underneath limestone rocks (Hebets and Chapman 2000a ; Chapin and Hebets 2016 ). In contrast, Paraphrynus laevifrons and Phrynus pseudoparvulus are found on vertical surfaces and both species reside in refuges with a variety of entrance orientations (Corey and Hebets 2017 ; Hebets 2002 ). Thus, the horizontal experimental design may be more compatible with the natural behavior of Phrynus marginemaculatus than that of the two species we tested here. In the future, it would be good to test the learning capacity of our two ‘vertical dwellers’ with a vertical testing arena like those used by Casto et al. 2020 or Lehmann et al. 2022 . In addition to the orientation of the experimental arena, the size of the arena relative to the size of the amblypygids also varied across the species. Our arena was the same dimensions as those used to test learning in Phrynus marginemaculatus. But, both Paraphrynus laevifrons and Phrynus pseudoparvulus are significantly larger than Phrynus marginemaculatus and may have benefited from training in a larger arena proportionate to their body size. Future studies should consider that learning should evolve from the challenges regularly faced by a species in the habitats and at scales (and with chemical stimuli) they regularly interact with. Finally, other aspects of the natural history of these species, such as their natural daily movement patterns, might help us interpret the differences we observed in apparent learning capacity. Preliminary field experiments with Phrynus marginemaculatus , for example, suggest that these animals leave their home refuge (e.g., a limestone rock) nightly (Hebets personal observation), likely to forage. In contrast, both Paraphrynus laevifrons and Phrynus pseudoparvulus have fairly high site fidelity within and across nights. In subsequent surveys, 30–55% of sighted Paraphrynus laevifrons were resighted in the same or adjacent grid and Phrynus pseudoparvulus were found on the same tree in 50–77% of resightings (Corey and Hebets 2017 ; Hebets 2002 ). Differences in foraging patterns and associated navigation may play a large role in selection for learning capabilities. In conclusion, to fully test how widespread the role olfactory associative learning is in the navigation abilities of amblypygids, we need to appropriately and thoroughly test a wider range of species using species-relevant designs. Amblypygids are fairly challenging to work with in the laboratory in terms of designing experiments that capture the animal’s natural behavior. So little is known about the natural behavior and life histories of different amblypygid species that designing species-appropriate assays is nearly impossible. Most studies have focused on new-world amblypygids in a single family, Phrynidae, and we know little about the behavior and habitats of the remaining families (Chapin and Hebets 2016 ). Nonetheless, we suggest that future learning experiments should (i) begin with a thorough investigation of the species’ natural history so experiments can be designed with the species’ habitat and behavior in mind, (ii) attempt to obtain larger sample sizes (though we fully recognize the challenge of this) with consistent experimental design, and (iii) potentially incorporate longer training periods or overnight training, as utilized in Flanigan et al. 2021b . Understanding the natural histories of more amblypygid species, coupled with appropriate laboratory experiments, will help us understand how olfactory learning can be used in navigation and if some species can solve complex navigation without olfactory learning. Supplementary Information Below is the link to the electronic supplementary material. Supplementary Material 1 (86.8KB, docx) Acknowledgements We would like to thank Tyler Corey for collecting Paraphrynus laevifrons and La Tirimbina Biological Reserve for making collection possible. We would also like to thank Tyler Corey and Neil Losin for photos of both species. Finally, we would like to thank two reviewers as well as the editor who provided feedback that greatly improved the manuscript. Author contributions Conceptualization: LSH, JP, EAH. Data curation: LSH, JP. Formal Analysis: LSH and KDSL. Funding acquisition: EAH, DDW, VPB. Investigation: LSH, JP. Methodology: LSH, JP, EAH. Project administration: EAH. Resources: EAH, DDW, VPB. Supervision: EAH. Validation: LSH, JP, KDSL. Visualization: LSH, KDSL. Writing – original draft: KDSL. Writing – review & editing: KDSL, LSH, EAH, JP, DDW, VPB. Funding This research was funded by NSF IOS—Collaborative Research—Navigation and the Neural Integration of Multimodal Sensory Information in the Brain of an Arthropod (PI-EAH) 2015–2019 (IOS-1638825; DEB-1456817). Funding from the University of Nebraska-Lincoln’s Undergraduate Creative Activities and Research Experience (UCARE) program also funded John Perez’s participation in this project. LSH was supported by the NOMIS Fellowship program at the Smithsonian Tropical Research Institute during the submission, review and publication of this work. Data availability The experimental data described can be accessed on Figshare with the identifier 10.6084/m9.figshare.30341929. Declarations Competing interests The authors declare no competing interests. Footnotes Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Abramson CI, Feinman RD (1990) Lever-press conditioning in the crab. 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