Decision letter: Hawkmoths evaluate scenting flowers with the tip of their proboscis

Field: Agricultural and Biological Sciences (Life Sciences)

Full text Figures and data Side by side Abstract Introduction Results and discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Pollination by insects is essential to many ecosystems. Previously, we have shown that floral scent is important to mediate pollen transfer between plants (Kessler et al., 2015). Yet, the mechanisms by which pollinators evaluate volatiles of single flowers remained unclear. Here, Nicotiana attenuata plants, in which floral volatiles have been genetically silenced and its hawkmoth pollinator, Manduca sexta, were used in semi-natural tent and wind-tunnel assays to explore the function of floral scent. We found that floral scent functions to increase the fitness of individual flowers not only by increasing detectability but also by enhancing the pollinator's foraging efforts. Combining proboscis choice tests with neurophysiological, anatomical and molecular analyses we show that this effect is governed by newly discovered olfactory neurons on the tip of the moth's proboscis. With the tip of their tongue, pollinators assess the advertisement of individual flowers, an ability essential for maintaining this important ecosystem service. https://doi.org/10.7554/eLife.15039.001 Introduction Floral scent has been associated with insect pollination since the 18th century (Sprengler, 1793); however, the complex functions of floral volatiles have been only recently investigated in more detail, due to the availability of new molecular and analytical techniques (Raguso, 2008). Floral scent not only attracts pollinators (Klahre et al., 2011), but also manipulates them through chemical mimicry (Stökl et al., 2010) and repels herbivores (Junker and Blüthgen, 2010), altogether increasing plant fitness (Kessler et al., 2008). However, research studying the function of floral scent has been divided along two themes with little cross-fertilization: 1) studies examining the fitness effects of floral scent without the causal behavioral responses of pollinators (Kessler et al., 2008), or 2) studies examining the sensory physiology of pollinators, neglecting the ecological consequences for the plant (Raguso, 2008). Here, we meld these approaches and show that floral scent increases the fitness of individual flowers not only by increasing their detectability (Raguso and Willis, 2002), but also by enhancing the pollinator’s foraging motivation, and demonstrate that this is mediated by olfactory receptors on the tip of the moth’s proboscis which detect floral scent. The hawkmoth Manduca sexta (Figure 1A, Video 1) is a major pollinator of the wild tobacco Nicotiana attenuata in the Great Basin Desert (USA) (Kessler et al., 2008; 2010; 2015). N. attenuata emits a relatively simple floral scent dominated by a single compound: benzyl acetone (BA) (Euler and Baldwin, 1996). In spite of this simplicity, producing BA might come at considerable metabolic but especially ecological cost, as BA might attract not only nectar thieves and florivores, but also female hawkmoths in search of oviposition sites (Baldwin et al., 1997; Kessler et al., 2010). Plants might therefore reduce the amount of floral volatiles released as much as possible without losing their pollination services. Field experiments using plants in which the emissions of BA had been silenced by RNAi of the biosynthetic gene NaChal1 (CHAL) have shown that BA is required to maximize pollination success (Kessler et al., 2008; 2015). It was suggested that lacking this scent made plants in nature 'invisible' to hawkmoth pollinators. However, the precise mechanisms by which odors of single flowers influence pollinator behavior and thereby plant fitness have rarely been examined in the direct interaction between plant and pollinator (Klahre et al., 2011; Riffell et al., 2008). Hence, how floral scent emitted by individual flowers functions in this mutualistic interaction remained unknown, particularly as it is unclear how pollinators detect single volatile compounds within complex natural environments (Hansson and Stensmyr, 2011; Riffell et al., 2014). Figure 1 with 1 supplement see all Download asset Open asset Even though M. sexta visited the same number of benzyl acetone (BA)-emitting (EV) and non-emitting flowers (CHAL), BA-emitting flowers received superior pollination services, increasing seed production. (A) M. sexta feeding from N. attenuata flowers. (B) Number of EV and CHAL flowers visited per moth on each foraging flight when 10 randomly placed plants per line were presented in a two-choice, free-flight tent assay (Wilcoxon signed rank test). (C) Seeds matured per antherectomized flower after visitations by a moth experimentally loaded with pollen (Wilcoxon rank sum test). Extreme values are shown as numbers. https://doi.org/10.7554/eLife.15039.002 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg M. sexta foraging on EV flowers emitting BA in a free-flight tent. https://doi.org/10.7554/eLife.15039.004 Results and discussion We investigated the function of floral scent in the context of individual flower-moth interactions, by offering individual male moths the choice between BA-emitting flowers (i.e. empty-vector transformed flowers (EV)) and non-emitting flowers (i.e. CHAL) in a free flight tent (24 m × 8 m × 4 m, 10 CHAL and 10 EV plants, spaced 50 cm apart). The flight tracking revealed that moths chose to visit the same number of emitting and non-emitting flowers in a random sequence (Figure 1B, Figure 1—figure supplement 1A, probability of changing between EV or CHAL flowers during consecutive visits: 0.47). In a second bioassay conducted in a wind tunnel (2.4 m × 0.9 m × 0.9 m, moonlight [0.5 lux of sunlight spectrum]), we presented plants with either emitting or non-emitting flowers to individual moths and analyzed their flight patterns, approaches and flower contacts. In none of these analyses, we found any difference between plants with emitting and non-emitting flowers (Figure 1—figure supplement 1B,C). These results suggest that visual cues and general vegetative plant odors provided sufficient information for the moths to locate flowers, consistent with previous work using artificial flowers (Raguso and Willis, 2002) and clearly showing that non-scenting flowers are not 'invisible' to moths. If non-scenting flowers are found by moths, why is plant fitness reduced? Does BA emission change the pollination probability? To test this, we loaded the moth’s proboscis with a standardized number of pollen grains using a fine brush. When such pollen-enhanced moths were allowed to forage freely on antherectomized N. attenuata flowers, seeds produced per flower of EV and CHAL plants differed significantly (Figure 1C). Scentless flowers matured very few seeds, reflecting the inferior pollination services provided by the moths despite a similar number of visits. This result highlights the importance of BA emission for the fitness of individual flowers and confirms the results of previous studies, which investigated the effects of BA emission on plant fitness at a population level (Kessler et al., 2008; 2015). But if the flowers were equally detectable by the moths, what behavioral mechanism was responsible for the plant fitness consequences? To analyze the effect of BA emission on moth behavior in greater detail, we quantified the time invested by a moth at individual flowers in a wind tunnel assay. The moths spent significantly more time at emitting than at non-emitting flowers (Figure 2A) particularly while trying to insert their proboscis, so even before tasting the floral nectar (Figure 2—figure supplement 1A). However, having successfully inserted their proboscis, the time of nectar uptake was similar between them (Figure 2—figure supplement 1B). This suggests that BA emission increased the motivation of moths to forage when individual flowers were evaluated at a close range, possibly because BA emissions, are closely linked to the physiological state and thereby also to the potential nectar amount of a flower (Bhattacharya and Baldwin, 2012; Yon et al., 2015; Kessler et al., 2015). By increasing the probing time in BA-emitting flowers, moths increased their success rate at their first as well as at consecutive flower visits and, therefore, collected more nectar per flower visit in tent (Figure 2B, Videos 1 and 2, Figure 2—figure supplement 1C) and wind tunnel assays (Figure 2—figure supplement 1D). These results agree with a study using different Petunia lines which found that although flower scent aided navigation, increased nectaring was the most consistent effect of floral scent (Klahre et al., 2011). Figure 2 with 1 supplement see all Download asset Open asset Moths spent more time and removed more nectar from BA-emitting (EV) flowers than from scentless (CHAL) flowers. (A) Time spent by moths at single flowers (Wilcoxon rank sum test) in a wind tunnel assay. (B) Nectar remaining in flowers after moths attempted to feed in a two choice tent assay (Wilcoxon signed rank test). https://doi.org/10.7554/eLife.15039.005 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg M. sexta attempting to forage on Chal flowers not emitting BA in a free-flight tent. https://doi.org/10.7554/eLife.15039.007 The large fitness consequences of floral volatiles for both moth and plant beg the question: how do moths evaluate the headspace of individual flowers? The wide spread of the antennae and their distance from the flower resulting from the moth’s long proboscis which is fully extended during nectaring suggests that the olfactory spatial resolution of the antennae might be too low to resolve individual flowers in an inflorescence or even between neighboring plants (Willis et al., 2013). Hence, we inferred that the moth’s proboscis might play a role in flower perception (Goyret and Raguso, 2006). Using reverse transcription PCR, we qualified the accumulation of transcripts of olfactory genes in the proboscis of M. sexta. Similar to the mosquito Aedes aegypti, but contrasting with predictions for nectar feeding insects (Jung et al., 2015), we found that the olfactory co-receptor Orco was expressed in the tip region of the proboscis along with the ionotropic co-receptor, IR25a (Figure 3A). Notably, Orco was only expressed in the first centimeter of the proboscis whereas the ionotropic co-receptor, IR8a, was only found in upper sections. This heterogeneous distribution of olfactory genes is consistent with the idea that the moth proboscis plays a more complex role in chemoreception than previously thought (Reiter et al., 2015). Screening the proboscis tip by scanning electron microscopy (Figure 3B, Figure 3—figure supplement 1A,B) we found a sensillum type that was not previously described for M. sexta (Reiter et al., 2015). This sensillum resembled the known sensillum styloconicum, but instead of a single tip pore, had a multiporous cone (Figure 3B4). Similar sensillum types have been described in other lepidopteran species (Faucheux, 2013), but their function remained unknown, although the presence of odorant-binding proteins suggested a role in olfaction (Nagnan-Le Meillour et al., 2000). We used an antibody raised against Orco (Nolte et al., unpublished), and found a single Orco- positive cell only in the first multiporous sensilla styloconica (mSt) at the tip of the proboscis (Figure 3C, Figure 3—figure supplement 1). Figure 3 with 1 supplement see all Download asset Open asset The M. sexta proboscis harbors sensilla, which house sensory neurons expressing olfactory genes. (A) Reverse transcription PCR using either the proboscis shaft (S), the first centimeter of the proboscis tip (T) or a water control (C) with primers for the three major olfactory co-receptors (IR8a, IR25a, Orco). The transcripts of the ribosomal gene RL131 and water were included as positive and negative control. (B1) Scanning electron microscopy images of the M. sexta proboscis tip show three types of potential chemosensory sensilla: sensilla styloconica (B2) with a uniporous (uSt (B3)) and multiporous (mSt (B4)) cone and uniporous sensilla basiconica (uBa (B5, Figure 3—figure supplement 1B)) as well as aporouse sensilla chaetica (aCh, Figure 3—figure supplement 1A). Asterisks mark tip pore, arrowheads indicate side pores. Neuronal labeling using anti-bodies against horseradish peroxidase (C1) and against Orco(C2) indicate three neurons close to the first mSt sensillum (arrows), of which one expresses the olfactory co-receptor Orco (C3, Figure 3—figure supplement 1). https://doi.org/10.7554/eLife.15039.008 Given the presence of potential olfactory sensilla on the proboscis of M. sexta, we wondered whether neurons housed in these sensilla play a role in the detection of BA and could thus help explain the pollination differences of EV and CHAL flowers. We performed single sensillum recordings and tested the response of neurons present in all sensillum types occurring at the tip of the proboscis to an air puff of BA at an ecologically relevant concentration (0.1 mM Kessler and Baldwin, 2007). Only neurons in the Orco-positive sensillum reacted to this compound (Figure 4B, Figure 4—figure supplement 1A). In a further test with 41 other ecologically relevant odorants, the first mSt was found to be more sensitive to BA and the structurally related benzylacetate (Figure 4C, Figure 4C—source data 1). Though these results show that neurons in the proboscis tip of M. sexta can detect volatile BA, it remained unclear whether the moth would also respond behaviorally to this compound based only on the input from neurons of the proboscis sensilla. Figure 4 with 1 supplement see all Download asset Open asset Olfactory sensory neurons housed in proboscis sensilla respond to BA and are sufficient for flower evaluation. (A) Single sensillum recordings from the first multiporous sensilla styloconica. Upper trace depicts a characteristic response to the water control; lower trace shows a response to BA from the neuron in the same sensillum. Red bar indicates time of stimulus. (B) Boxplot shows ∆ spikes per second recorded from the first mSt when stimulating with water control or BA (0.1 mM) for 0.5 s. Neurons responded with a significantly higher spike rate to BA than to the water control (Wilcoxon signed-rank test). (C) Response profile of the first mSt to 42 different odorants. Black bars indicate S.E.M. Names and spike rate of each odorant can be found in Figure 4C—source data 1. (D) Behavioral assay to test the response to either humidified air with BA (0.1 mM) or humidified air only. Exhaust excludes antennal olfactory input. (E) Moth inserted their proboscis significantly longer into the arm in which BA was present (Wilcoxon signed-rank test). (F) Moths chose equally often between Y-tube arms containing BA-scented air or solvent control at the first approach (Exact binominal test). https://doi.org/10.7554/eLife.15039.010 Figure 4—source data 1 Response spectrum of the first multpourus sensillum to ecological relevant odors. https://doi.org/10.7554/eLife.15039.011 Download elife-15039-fig4-data1-v2.xlsx To disentangle the proboscis input from other chemosensory organs, we devised a behavioral experiment in which the corolla tube of a flower was replaced by a Y-maze choice assay (Figure 4D). Each arm of the Y-maze was either connected to a source of humidified air or humidified air scented with BA (0.1 mM). By drawing air directly behind the entrance of the Y-maze, the experimental set-up excluded antenna-based olfaction. Hence, as soon as a moth entered the flower aperture during free hovering flight, only the proboscis experienced the air stream containing either a solvent control, or BA (Video 3). During their first and subsequent insertions, the moths chose both Y-tube arms with equal frequency (Figure 4F, Figure 4—figure supplement 1B), but inserted their proboscis for a significantly longer time into the arm containing the BA-scented air (Figure 4E), demonstrating that the moth was able to detect BA with only the proboscis. Moths seem to use the olfactory input from the proboscis not for orientation on the corolla, but rather to assess the specific quality of an individual flower, consistent with the notion that the close-range orientation of the proboscis on the flower can be informed by mechanical and visual cues (Goyret and Raguso, 2006; Sponberg et al., 2015). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Y-maze of the proboscis choice assay, BA-scented air is provided at the right arm and the solvent control in the left arm. Odors were removed by applying a vacuum at the entrance of the Y-maze. https://doi.org/10.7554/eLife.15039.013 Our findings show that hawkmoths are well adapted to visit and detect volatiles of single flowers. Floral volatiles, such as BA, not only function as navigational cues (Haverkamp et al., 2016), but also inform pollinators about the identity and the physiological state of individual flowers (Bhattacharya and Baldwin, 2012; Yon et al., 2015). Only BA-emitting flowers encourage the moth to visit a flower long enough to lead to successful pollination. Our results show that floral scent is an essential chemical feature for hawkmoths to gain nectar from, and pollinate, a single flower. Interestingly, many flowers require that their pollinators acquire particular handling skills on their first visits, before the insects are able to use the flower efficiently (Laverty, 1994). This energy investment by the pollinator not only helps ensure outcrossing for the plant, but provides the insect with a more exclusive nectar source (Heinrich, 1979). In a recent study, inexperienced M. sexta were found to sometimes expend more energy on handling flowers than they gained from the nectar; if additional experience increases foraging efficiency, this would compel the moth to visit additional flowers of the same species (Haverkamp et al., 2016). Notably, the ability to smell BA with the tip of the proboscis may not only increase the motivation of M. sexta to invest energy into BA emitting flowers, but also strengthen the moth’s learning of these flowers, as the nectar reward becomes associated with the presence of BA. Such a BA-conditioned learning rate might help to ensure a positive energy balance for the moth while at the same time ensuring cross-pollination for the plant (Heinrich and Raven, 1972). However, the question to what extent the interaction of moths and plants relies on the moths’ learning ability requires additional attention in future studies. Although both – metabolically costly and risky in terms of herbivory – thousands of plant species actively emit floral scent (Wright and Schiestl, 2009). These emissions might be a consequence of the physiological requirement for scent compounds by certain pollinator guilds when collecting nectar, even when visual cues would be sufficient to attract pollinators to a plant. The ongoing evolutionary interaction between plants and pollinators relies heavily on floral scent (Parachnowitsch et al., 2012; Schiestl and Johnson, 2013), and may explain the absence of scent-free plants in native N. attenuata populations (Kessler et al., 2015). To pollinators with the appropriate sensory system, floral scent provides a wealth of information, highlighting the importance of chemical communication in this mutualism, on which many of our crops rely (Radera et al., 2015). Materials and methods Plants Request a detailed protocol We used two transgenic Nicotiana attenuata Torr. (Solanaceae) lines derived from Agrobacterium tumefaciens (strain LBA 4404) transformation of wild type N. attenuata plants which were collected in a native population at the DI Ranch (Santa Clara, UT, USA) in 1988 and subsequently inbred for 22 generations (Krügel et al., 2002). Both lines have been described earlier, empty vector control plants (EV) transformed with pSOL3NC (line number A-04-266-3) (Bubner et al., 2006), as well and plants silenced by RNAi in the production of floral scent, CHAL line (N. attenuata chalcone synthase; pRESC5CHAL, line number (Kessler et al., 2008). Seeds were and on with as described in et with seeds were and 8 in a USA) for 10 and were to with in the 10 plants were to cm × cm for wind tunnel experiments or to 1 for tent The are described in et use in the wind plants were to a USA) with an time was from at and time were with a of Plants used in the tent experiment were in the for the of plants were to a second at the same in the plants were used for the tent Moths used in the wind tunnel experiments were from a at the in were as previously described et al., 2015). were collected from female M. sexta moths, which could freely on N. attenuata the tent from native M. sexta populations at the were collected and to were on artificial or on Nicotiana plants at an of and a of were into individual for before were and male were to a flight with and and were to and during and to and during the of was used between were used for experiments after tunnel Request a detailed protocol assays were performed in a wind tunnel cm × cm × air was through the tunnel at a of and was to and Plants were to the wind tunnel at 1 before the to plants were in a with an additional air before each a single plant was placed at the of the wind tunnel with the flower cm the tunnel cm from the tunnel and cm from each tunnel Moths were placed in cm × 1 before the experiment and to a to the same and as the wind each a single M. sexta moth was placed on a cm the tunnel cm from the tunnel and cm from each tunnel was to which not within this time were as and excluded from subsequent the behavior of each moth was recorded for 4 using a video tracking The tracking of at and a resolution of × Using a in the of the moth was at a rate of 10 on these tracking we analyzed the flight of the moth during the two before the flower in the wind tunnel using recorded flight were with video data and only recordings were used for further In to learning we only the first flower by each Moth behavior at the flower was recorded at a rate of by a which had been placed into the wind tunnel at a distance of about cm from the flower. were by a detection probing and time were by the individual video Similar to the flight analyses, we only the first flower in the Request a detailed protocol To a natural we conducted pollination experiments in a large tent 4 8 with an to from and for natural (Kessler et al., 2015). Plants were from the to the tent which was directly to the in the tent were conducted between and 8 plants of each of the two lines EV and CHAL were in at the of the tent. EV and CHAL plants were directly to each even The of the EV and CHAL plants were after each to potential In to use only flowers, all flowers were removed each before experiments were to male M. sexta moths were released per new moth was released only when the previous had and moth was only used Single flower visitations were the and time at a single flower was and after the the flowers were removed to the remaining Each moth had to 10 flower and for each moth the nectar gain all flower was Nectar and pollen Request a detailed protocol after each experimental wind tunnel both the moth and the plant were The remaining nectar in the flower was by the flower and the nectar with a The nectar amount was by the and the two In the tent nectar was quantified directly using a with a of by the corolla (Kessler et al., 2007). The pollen on the moth proboscis was by the proboscis three in 1 of 10 of were to each to the pollen The were and for 2 at the was without the pollen and water was to each The were and 10 were into a to the pollen number in each was and the was used for pollination experiment Request a detailed protocol To pollination in EV and CHAL plants by M. sexta in the wind fully flowers were in the previous to (Kessler et al., 2008). this, flowers per plant of each line and CHAL) were used in the wind one plant and one moth at each pollen was collected in the from plants not used for pollination. EV pollen which had been collected the previous was on the hawkmoth proboscis using a fine to its in the wind in to the pollen to experimental flowers. If moths not flight within 3 after placed in the wind tunnel they were excluded from the which flight were allowed to do so for in each wind tunnel The of matured as well as the seeds produced from each were after were collected before after the