DOI: 10.1111/j.1570-7458.2008.00669.x

Blackwell Publishing Ltd Odor-based recognition of nectar in cursorial

Joseph M. Patt* & Robert S. Pfannenstiel USDA–Agricultural Research Service, Subtropical Agricultural Research Center, Weslaco, TX 78596, USA Accepted: 18 December 2007

Key words: foraging, nectarivory, learning, olfaction, chemosensory, signals, local search, plant defense, biological control, futilis, Araneae,

Abstract Carnivorous are known to rely on non-prey foods, such as honeydew, pollen, and nectar. Consumption of plant-based nutrients by spiders also appears to be widespread, especially in cursorial species. This is not surprising, as studies have shown that these spiders’ activity levels, survivorship, and reproduction are increased when their diet includes plant-based nutrients, especially under conditions of prey scarcity. However, the sensory and behavioral means by which they recognize and locate non-prey food is unknown. Here we show that immatures of a nectarivorous [ Banks (Araneae: Anyphaenidae)] can recognize and remember particular chemical stimuli associated with nectar. Following ingestion of minute amounts of sugar, these spiders exhibited counterturning and other local searching behaviors that increased their chances of finding more nectar. When placed on test arenas, spiders that were naïve with respect to nectar aroma located artificial nectaries composed of diluted honey significantly faster than unscented nectaries composed of 1  sucrose solution. These results indicate that H. futilis is neurophysiologically and behaviorally adapted for recognizing olfactory stimuli. Interestingly, only spiders that ingested sugar and were engaged in local search responded to nectar aroma, suggesting that stimulation into local search is necessary to prime olfactory responses. We found that H. futilis could be conditioned to associate the presence of nectar with a novel aroma, in this case vanilla, and remember this aroma over the course of several hours. In arenas with vanilla-scented nectaries, spiders that had previous experience feeding on vanilla- scented sucrose droplets located the nectaries significantly faster than did vanilla-naïve spiders. The capacity to remember specific aromas could enhance the spiders’ ability to find nectar, either when moving between different parts of the same plant or among different plant species. The results here indicate that nectarivorous spiders possess the sensory capabilities and programed behaviors necessary for efficient detection, recognition, and location of nectar sources.

ghost spiders (Anyphaenidae), and sac spiders (Miturgidae Introduction and Clubionidae). Rather than relying on webs, these There is an increasing awareness of the central role that spiders actively search or lie in ambush for their prey non-prey foods, such as honeydew, pollen, and nectar, play (Pollard et al., 1995; Jackson et al., 2001). Cursorial in the lives of carnivorous arthropods (Wäckers et al., spiders, in general, use a variety of chemical stimuli to 2005). Their activity levels, development, survivorship, detect and recognize different resources, including mating success, and fecundity can be enhanced by prey, predators, mates, and hunting sites (Jackson & Pollard, consumption of non-prey food, especially during periods 1996; Persons & Rypstra, 2000; Jackson et al., 2002a,b,c, of prey scarcity (Smith & Mommsen, 1984; Vogelei & 2005; Heiling et al., 2004; Roberts et al., 2007). The cognitive Greissl, 1989; Pollard et al., 1995; Taylor & Foster, 1996; abilities of these spiders are considerable, extending to the Wäckers et al., 2005). Nectarivory is now known to be a ability to make decisions about prey-specific stalking common occurrence in at least five families of spiders routes (Jackson et al., 2001), selective attention to potential (Taylor & Foster, 1996; Jackson et al., 2001), including the prey occupied with their own foraging tasks (Jackson et al., jumping spiders (Salticidae), crab spiders (Thomisidae), 2002a,b,c), and multimodal (VanderSal & Hebets, 2007) and contextual learning (Skow & Jakob, 2006). This *Correspondence: E-mail: [email protected] suggested to us that they use chemical cues to detect and

© 2008 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 127:64–71, 2008 No claim to original US government works 64 Journal compilation © 2008 The Netherlands Entomological Society

Recognition of nectar odor by spiders 65 locate nectaries and may learn to recognize specific cues associated with nectar. The purpose of this study was to determine whether nectarivorous spiders respond to gustatory and olfactory stimuli associated with nectar, and use these cues to detect and locate nectaries. We also wanted to determine if they could learn to recognize olfactory stimuli associated with nectar. The subjects of our study – immatures of a ghost spider, Hibana futilis Banks (Araneae: Anyphaenidae), forage for eggs and small prey while running across foliage (Pfannenstiel, 2005) – a searching strategy that may frequently bring them into proximity with flowers or extra-floral nectar glands. The addition of sucrose to their diet increases their survivorship and enables them to complete development when provided with only minimal prey (Taylor, 2004, Taylor & Pfannenstiel, unpubl.). Using the cold anthrone test, Taylor (2004) showed that 32% of female and 10% of male H. futilis were positive for nectar Figure 1 sugars in cotton fields. Given the benefits they receive from Diagram of the Hibana futilis test arena showing arrangement of artificial nectaries in the perimeter zone nectar consumption, we anticipated that immature H. futilis (perimeter nectaries; PN) and center zone (center nectaries; CN) possessed sensory and behavioral adaptations to enable (not to scale). Different combinations of scented and unscented them to efficiently detect and locate nectaries. artificial nectars were used to test for stimulation into local search and response to nectar aroma. Spiders were released in the Materials and methods perimeter zone halfway between two PN. We measured the behavioral responses of immature H. futilis to nectar stimuli on test arenas constructed from glass Petri dishes (Figure 1) in the laboratory. In the TX, USA. Spiders were reared individually in Petri dishes experiments described below, simulated ‘nectaries’ composed and kept in an incubator under an L15:D9 regime at of small droplets of artificial nectar were positioned in the 26 ± 1 °C. They were fed with moth [Helicoverpa zea arena center and perimeter (Figure 1). During pre-tests, (Boddie)] eggs ad libitum until they molted twice. Water spiders placed on an ordinary Petri dish displayed typical was provided continuously with a moistened cotton wick. thigmotaxis, that is, they showed a strong tendency to To improve their responsiveness during the experiments, crawl along the arena perimeter, and only occasionally spiders were starved prior to testing (J Patt, unpubl.). moved across the face of the dish. Therefore, on arenas When individuals began their molt to third instar, all with nectaries, departure from the perimeter to the remaining moth eggs were removed from their culture arena center was indicative of a response to nectar stimuli. dishes and then withheld following their emergence. To We conjectured that contact with the perimeter nectaries ensure that they were aroma-naïve, spiders were kept in the (PN) would stimulate local search, a behavioral state incubator and were never exposed to any of the stimuli (Dethier, 1976; Bell, 1991) in which frequently used in the experiments. Because H. futilis is nocturnal counterturn and reverse direction to increase their (Pfannenstiel, 2005), they were maintained on a reverse probability of locating a resource. Likewise, if the spiders photocycle to permit daytime testing. were stimulated by nectar aroma, then their movements Only spiders that were actively moving within their would direct them toward the arena center, the area with culture dishes when inspected at testing time were used in the highest aroma concentration. These behaviors, the experiments. Individuals that were hidden in their especially when expressed simultaneously, would enhance silken retreat tubes or otherwise quiescent in their culture their ability to recognize and locate the nectaries located in dishes were not selected. Because the spiders became very the arena center. agitated when handled, each individual was anesthetized by gentle chilling for 60 s just prior to testing. Chilling was Spiders accomplished by transferring a culture dish with its spider All tests used third instars of the ghost spider, H. futilis, from the incubator into a –12 °C freezer. Once anesthetized, that were F1 progeny of wild females collected in Weslaco, the spider was immediately transferred from its culture dish

66 Patt & Pfannenstiel

Table 1 Treatments and behaviors examined during experiments testing stimulation into local search, responses to nectar aroma, and effect of local search on responses to nectar aroma by Hibana futilis

Perimeter ‘nectaries’ Center ‘nectaries’ (CN) Behavior tested Treatment (PN) blank or present unscented or scented Stimulation into searching behavior Control Blank Unscented following sugar ingestion Experimental Present Unscented Responses to nectar aroma Control Present Unscented during local search Experimental Present Honey-scented Responses to nectar aroma Control Absent Unscented without a priori sugar ingestion Experimental Absent Honey-scented

to the arena with an artist’s paintbrush. Spiders that did a 1:1 (vol/vol) solution of distilled water mixed with honey not become active within 90 s after transfer to the arena (Harry’s wildflower honey, San Sabastian, TX, USA), or were returned to the incubator. Each individual was tested from vanilla-scented sucrose solution [15 µl vanilla extract with one particular treatment. (Alcirelli; Trade Markets LC, Pharr, TX, USA) dissolved in 0.5 ml of 0.002  aqueous sucrose solution]. Unscented General testing procedures CNs were made from 1  aqueous sucrose solution. Pair-wise tests were performed with different artificial Dispersal of nectar aroma was accomplished by dif- nectar combinations to compare spider response to various fusion from the CN droplets. Delivery of the aroma to the types of nectar stimuli (Table 1). The arena was constructed spiders from a directed airflow system was precluded, from a glass Petri dish, 10 cm in diameter (Figure 1) that because the spiders initiated ballooning when exposed to was positioned horizontally. A transparent acetate template an air current (J Patt, pers. obs.). To reduce the amount of was inserted beneath the arena (relative to the observer) air circulation around the arena, the observer wore a paper and used as a simple visual aid. The template was created mask and the tests were conducted inside a fume hood with PowerPoint (Microsoft Corporation, Redmond, WA, fitted with a black theatre curtain cloth to cover the opening. USA) and then printed onto an acetate sheet with an inkjet To discourage spiders from leaving, the arena was placed printer. An array of thin black lines and characters on the inside a dish filled approximately 5 mm deep with water. template permitted the observer to delineate the arena into Tests were conducted in darkness with the arena illuminated a perimeter zone (6 mm wide) and center zone (88 mm by a 150 W fiber optic lamp with red filters. Direct visual wide) (Figure 1). observations of spiderling behavior and position on the Combinations of gustatory and olfactory stimuli were arena were recorded with a digital voice-recorder. When presented to the spiders in the arena via two sets of necessary, behaviors were clarified by examination of the simulated nectaries that were made of droplets of artificial spiders through a binocular dissection microscope that nectar. Sucrose solution was used to provide gustatory was positioned directly over the arena with a boom arm. stimulation. Four droplets, each composed of 2 µl of Individual tests began when a spiderling resumed crawling 0.002  aqueous sucrose solution, were positioned at after anesthesia and ended either when it located a CN, left equidistant intervals along the arena perimeter, the location the arena, or surpassed an arbitrary time limit of 600 s. where the spiders were most likely to encounter it. The The key test parameters measured were: the amount of simulated nectaries positioned along the arena perimeter time a spiderling engaged either in crawling, local search, were referred to as PNs. To avoid possible effects of water or dispersal behavior, or feeding on PN, its position on the intake on the spiders’ response, the PN sucrose droplets arena, the latency period required for it to find a CN, and were reduced to a dried residue by evaporation. In experi- the percentage of spiders successfully locating a CN. ments requiring a control for ingestion of sugar, the arena Crawling was indicated by the absence of counterturning perimeter was left blank (blank perimeter). Olfactory and movement reversals while a spider was crawling, that stimuli were provided by four simulated nectaries placed is, its movement was primarily in a forward direction and in the center of the arena, the area least frequented by the it displayed little or no deviation from a straight-line spiders. The droplets were 8 µl in volume and positioned trajectory. Local search was indicated by counterturning 10 mm apart from each other. The simulated nectaries and movement reversals that occurred more or less con- positioned within the arena center were referred to as tinuously while crawling. Dispersal behavior was indicated center nectaries (CN). Scented CNs were made either from by ballooning postures (Turnbull, 1998) in which the

Recognition of nectar odor by spiders 67 abdomen and fore tarsi were upraised. Test parameters an arena with blank PN + vanilla-scented CN (15 µl vanilla were quantified during replay of the audio recordings with extract dissolved in 0.5 ml of 0.002  aqueous sucrose The Observer software (Noldus Corporation, Wageningen, solution). In the initial part of the experiment, spiders were The Netherlands). conditioned to vanilla-scented nectar (1 µl vanilla extract To prevent possible contamination from nectar residues dissolved in 0.5 ml of 0.002  aqueous sucrose solution). and silk lines, a clean arena with fresh PN and CN was used Conditioning was achieved by placing a spider on an arena for each individual spider. The arenas were cleaned by with four vanilla-scented PNs and allowing it to consume a soaking them in hot detergent water followed by rinsing in single vanilla-scented PN. Unconditioned individuals were hot tap water and distilled water and then drying with a generated by allowing a second group of spiders to consume clean auto glass-drying towel. an unscented PN composed of 0.002  sucrose solution. For the tests, the scented and unscented PNs were made Effect of sugar ingestion on local searching behavior from 2 µl droplets of artificial nectar that were air dried as To determine if ingestion of sugar stimulated local search, in the previous experiments. The vanilla-scented CNs were spider response was compared between arenas with and made of a solution of 15 µl of vanilla extract dissolved in without PN. In both arenas the CNs were unscented, thus 0.5 ml of 0.002  aqueous sucrose solution. To ensure a their discovery would be the result of a chance encounter uniform level of response during the tests, spiders that by the spiders. If sugar stimulated local search, then spiders did not locate a PN in ≤300 s or fed for a total of <60 s were tested on the arenas with PN were expected to locate the disqualified CN relatively quickly, because local search motions would The spiders’ response to vanilla aroma was tested 3–5 h cause them to move toward the arena center and encounter after conditioning on an arena with unscented PN + vanilla- the CN. On the other hand, spiders in the arena without scented CN. Spiders were given 300 s to locate the CN. PN were expected to remain along the perimeter, limiting Testing continued until 30 individuals from the vanilla- their opportunity to encounter the CN. conditioned and control groups successfully located a CN.

Responses of aroma-naïve spiders to honey aroma Statistical analysis To determine whether honey aroma stimulated aroma-naïve Paired comparisons of the mean time to locate CN and the spiders, their responses were compared between an arena percentage of total observation time spent inside the center with PN + unscented CN vs. one with PN + honey-scented zone were made with the Mann–Whitney U-test (Zar, 1999). CN. Because of the presence of PN, spiders in both arenas For these comparisons, only spiders that successfully were expected to be stimulated into local search. If honey located CN were included in the analysis, that is, spiders aroma was also stimulatory, then spiders tested in the arena that either left the arena or failed to encounter the CN with honey-scented CN were expected to locate the CN during the allotted time period were excluded. Paired more quickly than those in the arena with unscented CN. comparisons of the proportion of spiders that successfully To determine whether stimulation into local searching located CN were made with the G test (Zar, 1999). behavior was a necessary prerequisite for the spiders to respond to nectar aroma, spider response was compared Results on arenas without PN, but having either unscented- or honey-scented CN. If contact with PN was required to affect Responses of naïve spiders to gustatory and olfactory stimuli spider response to honey aroma, then the time required to When spiders fed at the PN, they lowered their bodies and locate the CN would be the same in both arenas. made forward and backward motions, which enabled them to consume the entire PN. Their feeding behavior Conditioned response to novel aromas resembled that of jumping spiders feeding on floral nectar The capacity of spiders to learn to recognize novel nectar (Jackson et al., 2001). Following ingestion of sucrose aromas was tested by presenting the spiders with sugar from the PN, the spiders initiated local search and made solution (unconditioned stimulus) scented with vanilla counterturns (~180°) and side-to-side turning (~30°–90°) (conditioned stimulus) and later testing their responses motions. The intensity of local search appeared to decay to vanilla aroma on the arena. Vanilla was chosen as the over time without additional sugar consumption. Spiders novel aroma, because it has been used successfully in that were not engaged in local search continued to move in other studies that examined conditioned responses in a forward direction and made few counterturns. nectarivorous carnivores, for example, Lewis & Takasu Spiders tested on arenas with blank perimeters spent (1990). To ensure that the vanilla-naïve spiders were most of their time attempting to disperse and displayed unresponsive to vanilla, we conducted preliminary tests on little or no local search. Spiders tested in the blank perimeter

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in the PN + unscented CN treatment spent 52 ± 5% of their total observation time in local search, while spiders tested in the PN + honey-scented CN treatment spent 57 ± 6% of their time in local search. Local search movements increased encounters with unscented CN. When compared to the blank perimeter + unscented PN treatment, spiders tested on the PN + unscented CN treatment located the CN on average 93 s more quickly (U = 306, P = 0.114; Figure 2B). A significantly greater percentage of spiders provided with PN located the CN (88% in the PN + unscented CN treatment, 73% in the blank perimeter + unscented CN treatment; G = 2.47, P = 0.091, and α = 0.1). Lastly, when compared to the blank perimeter + unscented PN treatment, spiders tested on the PN + unscented CN treatment spent significantly more time in the arena center (U = 384, P<0.001; Figure 3). When engaged in local search, aroma-naïve spiders were strongly stimulated by honey aroma. The decrease in time required to locate CN in the PN + honey-scented CN relative to the PN + unscented CN treatment was highly significant (U = 433, P = 0.002; Figure 4). As well, the Figure 2 Mean time (+ SEM) to locate center nectaries (CN) by percentage of spiders that located CN in the PN + honey- aroma-naïve spiders; n = 30 spiders tested per treatment; number in each treatment that located CN shown in parenthesis. (A) scented CN treatment was significantly greater than in the Responses to honey-scented and unscented CN by spiders blank perimeter + honey-scented CN treatment (96% in without access to sugar: blank perimeter + unscented CN the blank perimeter + honey-scented CN treatment, 70% (n = 22) vs. blank perimeter + honey-scented CN (n = 20) in the blank perimeter + unscented CN treatment; G = 5.27, (P = 0.166). (B) Effect of sugar consumption on stimulation into P = 0.026). local search in the absence of honey aroma: blank perimeter + unscented CN (n = 22) vs. PN + unscented CN (n = 22) Conditioned response to novel aromas (P = 0.114). Bars with different letters are significantly different A conditioned response was observed in H. futilis following (Mann–Whitney U-test). the pairing of vanilla odor as a conditional stimulus with

+ unscented CN treatment displayed dispersal behavior for 73 ± 10% (mean ± SEM) of the total observation time, while spiders tested in the blank perimeter + honey-scented CN spent 61 ± 8% of their time displaying dispersal behavior. In the absence of PN, spider response to honey aroma was weak. The mean location time of CN for spiders on the blank PN + unscented CN treatment was 224 ± 44 s (mean ± SEM), while that of spiders on the blank PN + honey- scented CN decreased to 160 ± 37 s. The time required to locate the CN was similar on arenas with blank perimeters, regardless of whether the CNs were honey-scented or unscented (U = 275, P = 0.166; Figure 2A). Likewise, there was no significant difference in the percentage of spiders that located CN in these arenas (70% in the blank perimeter unscented CN treatment, 73% in the blank perimeter + Figure 3 Mean percentage (+ SEM) of total test time spent by spiders in the center zone of the test arena; n = 30 spiders tested honey-scented CN treatment; G = 0.082, P = 0.603). per treatment. Number in each treatment that located center Contact with PN had a strong effect on searching nectaries (CN): 21 in blank perimeter + unscented CN, and 22 in behavior. Spiders tested on arenas with PN typically spent PN + unscented CN. Bars with different letters are significantly >50% of their time engaged in local search. Spiders tested different (Mann–Whitney U-test).

Recognition of nectar odor by spiders 69

(G = 9.949, P<0.001). This difference suggests that the concentration of aroma of vanilla extract used in the CN may be slightly repellent.

Discussion Hibana futilis responded to chemical cues associated with nectar and exhibited a number of behaviors that, collectively, would enhance their ability to recognize and locate nectar. Specifically, we found that they initiated local search after ingesting sugar, Figure 4 Mean time (+ SEM) to locate honey-scented center were stimulated by honey aroma while engaged in local nectaries (CN) by aroma-naïve spiders when engaged in local search, and could learn to recognize novel olfactory cues search; n = 30 spiders tested per treatment; number in each associated with nectar. The propensity of the spiders to treatment that located CN shown in parenthesis. PN + unscented CN (n = 22) vs. PN + honey-scented CN (n = 23) (P = 0.002). encounter CN was influenced by the presence or absence Bars with different letters are significantly different (Mann– of discrete gustatory and olfactory stimuli on the arena. Whitney U-test). The importance of local search in resource-finding has long been established (Dethier, 1976; Bell, 1991), and secretions from extra-floral nectaries stimulate local search in other carnivorous arthropods (Heil & McKey, 2003). sucrose solution as an unconditional stimulus. When tested Thus, the observation that sugar-feeding stimulated local on an arena with PN + vanilla-scented CN, spiders from search in H. futilis was not unexpected. The counter turning the vanilla-conditioned group located the CN significantly inherent in local search caused the spiders to move toward faster than did unconditioned spiders (U = 652, P = 0.003; the arena center. This resulted in longer residence times in Figure 5). Also, more vanilla-conditioned spiders located the arena center and quicker location times of unscented the CN (88% in the vanilla-conditioned treatment, 73% in CN in arenas with PN’s vs. arenas with blank perimeters the unconditioned treatment; G = 5.27, P = 0.026). (Figures 2B and 4). In the salticid Portia fimbriata, detection In pre-study tests, spiders were not responsive to vanilla- of contact and olfactory kairomones increased movements scented CN on an arena with a blank perimeter. Of the 20 toward unseen prey and enhanced prey location (Jackson spiders tested, 15 (75%) did not locate the CN within a set et al., 2002a,b,c). This searching strategy may correlate limit of 360 s. This percentage is significantly different with the quicker discovery time of unscented CN by from the 28% of spiders that failed to locate a CN within H. futilis engaged in local search. 360 s, when tested on the blank perimeter + unscented CN Nectar aroma appears to be a primary proximate cue to foraging H. futilis (Figure 2C). Further evidence that nectar aromas function as proximate cues to nectarivorous spiders is provided by studies that showed that jumping spiders remained longer on nectaried vs. nectary-less plants (Ruhren & Handel, 1999), and that crab spiders preferred scented vs. unscented flowers (Heiling et al., 2004). That H. futilis can learn to recognize novel aromas associated with nectar and retain this information over the course of several hours is in keeping with current ideas about the relative importance of associative learning in locating resources that are spatially – and temporally unpredictable (Lewis & Takasu, 1990; Vet & Dicke, 1992; Turlings et al., 1993). Our test arena was adequate for demonstrating the Figure 5 Mean time (+ SEM) to locate vanilla-scented center nectaries (CN). Bars with different letters are significantly general responses of immature H. futilis to nectar stimuli. different (Mann–Whitney U-test: P = 0.002). Number of tests The design of the arena took advantage of the spiders’ required for 30 individuals from each treatment to locate CN: thigmotaxic tendencies and their responses to the various n = 34 in vanilla-conditioned group and n = 41 in unconditioned treatment combinations on the gross level were suitably group. obvious and quantifiable. The open design of the arena to

70 Patt & Pfannenstiel a certain extent mimicked a more natural arrangement specific information and does not imply recommendation than other types of olfactometers, and may have been less or endorsement by the USDA for its use. problematic with respect to certain behaviors, that is, ballooning in response to air currents. To decrease stimulation into dispersal behavior, we used References diffusion to disperse the nectar aroma from the CN rather Bell WJ (1991) Searching Behavior: The Behavioral Ecology of than a directed airflow with a well-defined odor zone. As Finding Resources. Chapman & Hall, London, UK. we could not determine the concentration gradient and Dethier VG (1976) The Hungry Fly. Harvard University Press, boundaries of the aroma plume, the manner in which Cambridge, MA, USA. olfactory cues were utilized by the spiders following sugar Heil M & McKey D (2003) Protective ant-plant interactions as ingestion could not be ascertained. 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