Herbivore cues and plant damage-associated-compounds jointly alter seed germination and seedling herbivory

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These cues include chemicals released from damaged conspecifics and kairomones, non-attack-related substances emitted by an herbivore that plants can detect and use to their benefit. It is unknown, however, whether or how plants react to the interaction of these pre-attack cues. We measured germination, growth, and herbivore susceptibility of B. nigra seedlings in an experiment that crossed the presence/absence of crushed B. nigra leaves with the presence/absence of mucus of a generalist herbivore, A. subfuscus . Seeds exposed to both crushed leaves and slug mucus germinated 8% more quickly than control seeds; neither risk cue increased germination speed when tested individually. The same pattern was found in herbivore bioassays: Spodoptera exigua ate almost 10x more foliage from control seedlings than from from seedlings exposed to both crushed leaves and slug mucus. There was no difference in the final biomass of mature plants, suggesting that plants exposed to herbivore cues early in their development can increase defense without a measurable cost in size at maturity. Kairomone signal mollusk risk induced defense Figures Figure 1 Figure 2 Figure 3 Introduction Plants threatened by herbivores must balance the benefits of defense with its energetic costs. The modular design of plants, paired with the lack of a central vascular system and the highly plastic nature of meristem tissue, means that low levels of herbivory pose little risk to survival and fitness (Karban and Agrawal 2002 ). As a result, mature plants can generally afford to wait until attack begins to induce chemical and/or structural defenses to prevent further damage (Karban 2020 ). This risk-tolerant approach is less suitable for juvenile plants such as seeds and seedlings, that either lack the energetic reserves to recover from damage or are small enough to be eaten in their entirety before they have the chance to respond. As a result, the ability to detect and respond to pre-attack herbivore cues may be critically important to young plants. Because allocation to defense can reduce plant growth and lead to young plants losing out in competition for light and other resources, selection should favor individuals capable of balancing these competing demands and determining whether a given stimulus warrants the induction of pre-attack defense (Karban and Orrock 2018 ). Plants seeking to detect pre-attack herbivore cues may take advantage of kairomones, non-attack-related cues incidentally emitted by consumers that are detected by and benefit prey. Kairomones are often byproducts associated with daily life and include excrement, sex attractants, and fluids associated with locomotion or oviposition. Exposing Brassica nigra seeds and seedlings to snail ( Helix aspersa ) locomotion mucus, for example, decreased plant growth and herbivory relative to control plants (Orrock 2013 ); subsequent work found that the effect of H. aspersa mucus on tomato seedling growth and defense rivaled that of the plant defense elicitor methyl jasmonate (MeJA) (Orrock et al. 2018 ). Exposing Solidago altissima to sex attractants of the galling fly Eurosta solidaginis reduced herbivory and subsequent oviposition in both laboratory and field experiments (Helms et al. 2013 ). Insect frass from multiple herbivores has similarly been shown to induce defense in several host plant species (Ray et al. 2016 ). Plants may also assess risk of herbivore attack via ‘damaged self’ cues produced by nearby injured conspecific or heterospecific individuals. These damage-associated molecular patterns (DAMPs) are chemicals released by stressed or injured cells that alert nearby cells to a potential threat. Pattern recognition receptors (PRRs) located on the cell surface and spanning the plasma membrane detect damage and respond via increased production of reactive oxygen species, protein kinases, and hormones such as jasmonic acid (JA), salicylic acid (SA), and ethylene (Tanaka and Heil 2021 ). Because PRRs are most responsive to species-specific cues, defensive induction is greatest in response to DAMPs from the same or closely related species (Duran-Flores and Heil 2018). Bean ( Phaseolus vulgaris ) plants, for instance, increased reactive oxygen species production and JA induction in response to leaf homogenates from conspecific - but not heterogeneric - plants (Duran-Flores and Heil 2014 ). Although plant responses to non-damage herbivore kairomones and DAMPs have received considerable attention, the interaction between the two has only been explored in animal predator-prey systems. Studies of the separate and combined effects of both kairomones and alarm cues in several tadpole species found that while exposure to either alarm cues or kairomones induced prey defense, their combination elicited the most complete and effective set of antipredator responses (Schoeppner and Relyea 2005 ; Schoeppner and Relyea 2009a ). Prey responded more strongly to a kairomone-DAMP mix derived from predators fed conspecifics than those fed distantly related species, suggesting that the phylogenetic similarity of predator-consumed individuals is also important (Schoeppner and Relyea 2005 ). Similar results have been found in work exploring antipredator responses in the aquatic macroinvertebrates Caecidotea intermedi (Spivey et al. 2015 ) and Daphnia magna (Pestana et al. 2013 ; Ślusarczyk 1999 ; Wieski and Slusarczyk 2022 ), suggesting that kairomones and DAMPs together generally provide a reliable indication of predation risk. We describe the results of work examining how kairomones, DAMPs, and their interaction affect seed germination and seedling growth, chemistry, and herbivore palatability. Seeds and seedlings of our study species, Brassica nigra , have been shown to induce defense in response to locomotion mucus from the generalist molluscan herbivores Helix aspersa (Orrock 2013 ) and Arion subfuscus (Pellegrini et al. 2024 ). Our study crossed a kairomone treatment (control or locomotion mucus from A. subfuscus fed Brassica nigra ) with DAMP ( B. nigra leaf homogenate) presence/absence and measured various aspects of plant response. The results demonstrate that plants can utilize both signals and reveal an ecologically relevant story of growth and defense under exposure to pre-attack cues. Materials and Methods Study species Arion subfuscus is native to Europe and was introduced to North America in the early 1900s (Chichester and Getz 1973 ). It is an important molluscan herbivore and in the invaded range (Chichester and Getz 1969 ) and the most abundant species of slug in New England (French 2012). Adults were collected May-July 2024 from forested areas in South Kingstown, RI, USA. Slugs were maintained in six large soil- and detritus-filled terraria at 21–24°C on a diet of organic lettuces, carrots, and field-collected seedlings of various plant species. The terraria were misted daily in order to maintain high humidity levels, and decaying plant material was removed when necessary. Brassica nigra is a fast-growing plant native to the Mediterranean that is naturalized worldwide and grows wild in RI. Seeds were sourced from Outsidepride Seed Source, LLC (Independence, OR, USA); A. subfuscus is also found in Oregon (Burke 2013). Experimental design The 2x2 experimental design crossed two herbivore-risk treatments (Kairomone) with two damage-associated-molecular-pattern (DAMP) treatments for a total of six treatments. The herbivore-risk treatments were mucus from A. subfuscus fed B. nigra for three days (+ Kairomone) or a no-mucus control (-Kairomone). The two DAMP treatments were water mixed with ground B. nigra leaves (+ DAMP) or water alone (-DAMP). Each treatment was applied to B. nigra seeds and plants and their responses measured. Herbivore-risk treatment : Three days before preparing the risk treatments, our population of mature A. subfuscus was divided into multiple small plastic terraria lined with wet paper towels where they were fed lab-grown B. nigra seedlings. After three days, we flattened 4g of a soil-and-distilled-water mixture (1:4 ratio) into each of multiple 90 mm petri dishes. In dishes assigned to either the high-risk or control treatments, a mature A. subfuscus was allowed to crawl on the soil for 24 hours in a dark cabinet at 20–24°C. Control plates were held in similar conditions, but did not receive a slug. All slugs were then returned to the large dirt-filled terraria and the petri dishes frozen at -20°C freezer for 2–4 weeks until they were defrosted and used. We used frozen rather than ‘fresh’ plates for the work because it would have been extremely difficult to generate the > 1000 ‘fresh’ risk-treatment plates necessary for the work when the main experiment (detailed below) was running. To ensure that previously frozen slug mucus induced similar plant responses as freshly deposited mucus, we conducted a pilot experiment confirming that mucus frozen for up to six months accelerated B. nigra seed germination (Online Resource 1). DAMP treatment : Brassica nigra seeds were sown in potting soil and grown in trays on a sunny windowsill. To prepare the leaf homogenate for the DAMP + treatment, B. nigra seedlings (~ 3 cm in height) were cut at the base of the stem and blended with distilled water (1:30 ratio by weight, as per Duran-Flores and Heil 2014 ) in a blender (Waring Commercial, Torrington, CT, USA) and then allowed to sit for two hours before use as per Duran-Flores and Heil ( 2014 ). A water-only control solution was also prepared for use in the -DAMP treatment. Seed Responses Germination assay On 15 July, 16 petri dishes from each of the two herbivore-risk treatments were removed from the freezer. Once fully defrosted, a piece of 90mm white filter paper was placed on top of the soil inside each of the dishes. Half of the dishes in each herbivore-risk treatment were then wetted with either 1 mL of the leaf homogenate solution (+ DAMP) or 1 mL of distilled water (-DAMP). Each of the four treatments was thus replicated eight times for a total of 32 dishes. Immediately after the addition of the DAMP solution, 50 B. nigra seeds were placed on top of the filter paper in each dish. Dishes from all four treatments were then interspersed and placed in a dark cabinet at 21°C. Starting 15 hours after seed placement and continuing every three hours afterwards, the seeds in each petri dish were checked for germination. We chose to start germination checks at 15 hours on the basis of prior work that found virtually no seed germination before this point (Pellegrini et al. 2024 ). At each interval, each seed was carefully checked for radicle emergence. Seeds with a radicle were counted and then removed from their petri dish. The checks continued until hour 45, when at least 90 percent of seeds from each treatment group had germinated. Seedling Responses Planting, growth, and cue application Prior to the start of the experiment, 320 6 x 6 cm black plastic pots were filled with MiracleGro potting mix (Marysville, OH, USA). The pots were placed in fourteen square boxes (36 cm x 36 cm) with a mesh bottom. Each box contained six plants from each treatment, for a total of 36 pots per box in 13 of the 14 boxes; the fourteenth box had empty pots added to the ‘empty’ slots in it. The first 80 seeds to germinate in each of the four treatments (320 total pots) were removed and buried individually 1 cm under the soil in a pot that was then placed in a box. The boxes were placed along a windowsill where they received approximately seven hours of direct sunlight per day. Each box was rotated clockwise every other day, and every week each pot within each box was rotated within its box by moving it one slot down and one slot to the right. Each plant received 5mL of water daily, administered via pipette at the base of the plant. Starting one week after germination, one petri dish of soil from the appropriate herbivore-risk treatment was removed from the freezer, defrosted, and the soil within it added carefully to the base of a plant from the appropriate treatment. Immediately after the soil had been applied, each plant was watered with 5 mL of freshly prepared leaf homogenate (+ DAMP) or control (-DAMP) solution. Seedlings were subsequently watered every 2 days with 5mL of water. The herbivore-risk and DAMP treatments were applied weekly for a total of five weeks; each plant received the same cue combination for the duration of the experiment. Biomass and glucosinolate analysis On day 36 of the experiment (i.e., one day following final cue application), 20 plants from each treatment (80 total plants) were destructively harvested. We first cut the third-newest leaf from each plant at the petiole, weighed it, then rolled it into a 15mL falcon tube that was capped, flash frozen using liquid nitrogen, and placed in a -80°C freezer for later glucosinolate analysis. The rest of the plant was removed by hand from the soil and cut at the cotyledon scar. The aboveground material was immediately placed in a coin envelope; the belowground material was carefully rinsed in water and patted dry before doing so. All samples were then placed in a drying oven (Blue M Electric Company, Blue Island, IL, USA) for four days at 60°C before weighing the above- and below-ground portions of each plant. A separate set of leaves were cut from unused plants and weighed immediately before being dried and re-weighed; we used the resulting wet:dry weight regression to convert the wet weight of the leaf removed for glucosinolate analyses into dry weight for addition to the aboveground biomass. Glucosinolates were extracted and analyzed using standard methods (Pieck 2015) (Brown 2003). Briefly, frozen samples were ground under liquid nitrogen in a Retsch MM400 ball-mill grinder (Retsch GmbH, Haan, Germany). Once ground, 70–120 mg of frozen leaf tissue in 2mL microcentrifuge tubes was extracted in 1mL methanol. Glucosinolates were bound to 20 mg of a DEAE Sephadex A25 pellet. Sulfatase (4 mg/mL) was then used to cleave desulfoglucosinolates from the pellet, and the supernatant was filtered, 0.22 um, into HPLC vials. A sinigrin standard was processed using the same method; 90% of the glucosinolates in Brassica nigra are sinigrin (Chaplin-Kramer 2011; Feeny 1982). The concentration of sinigrin was analyzed by HPLC using a 150 x 4.6 mm C18 reverse phase column (Phenomenox, Torrance, CA) with a water acetonitrile gradient (Brown 2003) Each sample was injected twice and the concentration of sinigrin (mg/g fresh leaf mass) was averaged for each sample. Spodoptera exigua bioassays Five weeks after the start of the experiment, a series of paired-choice assays were performed in which late-instar Spodoptera exigua (Frontier Agricultural Services, Newark, DE, USA) were allowed to choose between a leaf from a control (-Kairomone/-DAMP) plant and another leaf from one of the three remaining treatment groups (-Kairomone/+DAMP, +Kairomone/-DAMP, +Kairomone/+DAMP). There were 15 replicates for each of the three bioassay combinations (45 total bioassays). For each paired-choice bioassay, a single B. nigra leaf from a control and one from a specific treatment plant was cut from a plant, weighed, and placed in a prepared petri dish for bioassays. Leaves of similar age and sizes were paired together whenever possible, and both plants were selected from the same box. For each bioassay, a 90mm petri dish was lined with 90mm white filter paper and sprayed twice with distilled water before the leaves were placed next to each other on opposite sides of the dish. One S. exigua was weighed and placed in the center of the dish, which was then closed and held in a dark cabinet for 24 hours. At the end of the bioassays, the S. exigua larvae and both leaves were reweighed. We also conducted no-herbivory bioassays (necessary to quantify ambient changes in leaf weight over time); these were identical to the choice assays except for the absence of S. exigua larvae. Statistical analysis We initially examined the germination data using failure-time analyses (Cox proportional hazards model) but ultimately opted to use a generalized linear mixed model (GLMM) with a binomial response distribution because model diagnostics revealed that the proportional hazards assumption was violated. The model specified kairomone and DAMP treatments as fixed effects, the hour since the experiment was initiated as a covariate, and also included a cubic term for hour. We modelled time as non-linear because preliminary analyses indicated a non-linear relationship between probability of germination and the duration of the experiment and a cubic relationship was a significantly better fit than linear or quadratic based on comparison of AICc and log-likelihood tests. We explored all possible interaction terms. The combined herbivore bioassay dataset contained a few anomalously high and low datapoints spread across multiple treatments. Because these likely reflected errors in weighing/data entry, we removed the highest and lowest 2.5% of values from the combined dataset before analyzing each of the choice bioassays separately. For each choice bioassay, data on leaf proportion remaining per treatment was analyzed using GLM (identity link function) with treatment and bioassay date as fixed effects and plate identity nested within date as a random effect. We examined plant growth and glucosinolate concentration using linear models that specified kairomone and DAMP as fixed effects. Analyses were conducted in R version 3.6.1 (R Core Development Team 2019) and JMP version 18.1.2 (SAS Institute Inc. 2025). Results Seed germination Patterns of germination in time were strongly cubic (X 2 = 579.40, 3 d.f., p < 0.001) with patterns of germination accelerating early in the experiment and then levelling off as time proceeded (Online Resource 2). Germination was affected by kairomones and DAMPs, as reflected by a highly significant kairomone*DAMP*hour interaction (Fig. 1; X 2 = 19.85, 3 d.f., p < 0.001), as well as a significant kairomone*hour interaction (X 2 = 34.89, 3 d.f., p < 0.00) and a significant DAMP*hour interaction (X 2 = 50.72, 3 d.f., p < 0.001). Averaged across these significant interactions with time, there was no main effect of DAMPs (X 2 = 2.18, 1 d.f., p = 0.14), kairomones (X 2 = 0.01, 1 d.f., p = 0.91) or interaction between DAMPs and kairomones (X 2 = 0.80, 1 d.f., p = 0.37). Seeds in the control treatment averaged 24.5 hours to germination, while those in the kairomone-only treatment averaged 24.4 hours (0.6% faster) and seeds in the DAMP-only treatment averaged 24.1 hours (1.7% faster). In contrast, seeds exposed to both kairomones and DAMPs germinated in 22.6 hours (7.9% faster than control seeds; Fig. 1). Seedling biomass Mean aboveground plant biomass (Fig. 2, green bars) was not affected by kairomones (F 1,82 = 0.013, p = 0.72), DAMPs (F 1,82 = 0.04, p = 0.97), or their interaction (F 1,82 = 0.07, p = 0.80). Mean belowground plant biomass (Fig. 2, brown bars) was similarly unaffected by kairomones (F 1,81 = 0.30, p = 0.59), DAMPs (F 1,81 = 0.18, p = 0.67), or their interaction (F 1,81 = 0.03, p = 0.86). Glucosinolate concentrations There was no effect of kairomones (F 1,76 = 1.62, p = 0.21), DAMPs (F 1,76 = 0.004, p = 0.99), or their interaction (F 1,76 = 1.06, p = 0.31) on leaf glucosinolate concentration. Herbivory bioassays : Spodoptera larvae did not discriminate between control leaves and those exposed to kairomones or DAMPs separately (all p < 0.10). In contrast, Spodoptera did exhibit a preference when choosing between control leaves and those from plants exposed to both kairomones and DAMPs (Fig. 3). Larve consumed 8.4% of controls versus > 1% of leaves from plants exposed to both risk cues (F 1,13 = 6.47, p = 0.025). Discussion Our results provide the first evidence that seeds and seedlings can integrate information from both herbivore kairomones and damage-associated molecular patterns (DAMPs) in their assessment of attack risk. Such induced responses to herbivory in plants provide essential defense against subsequent damage (Karban 2020 ). Because mature plants can generally wait until being attacked to induce defense (Sheriff et al. 2020 ), plant-herbivore research has focused on attack-associated cues such as tissue damage or volatile emissions. The ability to detect and respond to pre-attack cues like herbivore kairomones or DAMPs should be particularly beneficial for seeds and seedlings, life stages with limited resources for which herbivory is often lethal (Barton and Hanley 2013 ). Because defensive induction can be energetically costly, however, selection should also favor the use of multiple sources of pre-attack information to reduce undefended attacks while avoiding the cost of unnecessary defense (Orrock et al. 2015 ); our findings are consistent with this hypothesis. For B. nigra seeds and seedlings, our results demonstrate that pre-attack cues indicating the proximity of both herbivores and damaged conspecifics altered both seedling germination and herbivore susceptibility (i.e., heighten a plant’s awareness of danger in the vicinity). This finding parallels research in predator-prey systems showing that cues from predators that consume prey conspecifics (akin to an herbivore whose presence produces kairomones and whose feeding produces DAMPs) are likely more dangerous than those that do not (Scherer and Smee 2016 ). Terrestrial and aquatic studies confirm that prey respond more to cues from conspecific-fed predators than to either predator (in our study, kairomone) or crushed conspecific (in our study, DAMP) cues alone (Prada et al. 2018 ; Schoeppner and Relyea 2005 ). Cues from predators fed conspecific or congeneric prey consistently elicit stronger responses than cues produced by the consumption of distantly related species (Schoeppner and Relyea 2009b ; Shabani et al. 2008 ), arguing for the adaptive benefits of such gradated responses in both animal prey and plants. While we predicted that exposure to combined risk cues would induce defense, we were surprised that neither cue induced defense by themselves. This was unexpected because both herbivore kairomones and DAMPS have been shown singly to induce pre-attack plant defense. Helms (2013) reported that Solidago responds to the sex attractant of a gall-making fly, for instance, and Orrock ( 2013 ) found that snail mucus alters B. nigra growth and defense. We now know that cultivated and wild plants respond to slug and snail mucus (Falk et al. 2014 ; Kastner et al. 2014 ; Meldau et al. 2014 ; Orrock 2013 ; Orrock et al. 2018 ), and prior work found that B. nigra seeds and seedlings detect and respond to A. subfuscus (Pellegrini et al. 2024 ). Leaf homogenate from crushed conspecific plants similarly induces resistance in unwounded plants (Duran-Flores and Heil 2014 ); parallel responses to damaged-self cues occur in a range of taxa (Heil and Land 2014 ). Research demonstrating damaged-self responses to conspecific homogenates also found less induction in response to congeneric homogenates and none in response to crushed plants from different genera (Duran-Flores and Heil 2014 ). Although our work can be seen as contradictory to these findings, we suspect that aspects of our experimental design (detailed below) inadvertently reduced our ability to detect single-cue effects. Faster germination in response to risk cues has only recently been documented (Pellegrini 2024). We observed an increase in germination speed for seeds treated with both kairomone and DAMPs. This appears to be the first example of DAMPs acting as germination promoters in plants. Because risk cues changed germination speed but not the overall rate, detecting such shifts requires around-the-clock germination checks over a multi-day period; this may explain why our work is the first to identify this effect. While seedlings emerging from faster-germinating seeds typically tend to outcompete their slower-germinating counterparts (Orrock 2010 ), we found no difference in seedling biomass; Pellegrini et al ( 2024 ) actually found that faster-germinating seeds produced smaller seedlings. Both sets of results support the hypothesis that risk-induced changes in germination speed improve defense rather than competitive ability, an explanation consistent with the narrative that seeds must act quickly to avoid predation. While Pellegrini et al ( 2024 ) found that seeds with faster germination due to kairomones led to a smaller final biomass, we observed no difference in final biomass. Our studies do, however, parallel in linking faster germination to increased resistance to herbivory, but we observed generally low levels of herbivory across all treatments. There are several caveats to be considered in interpreting the results of our work. First, our finding that herbivore kairomones alone did not impact B. nigra seeds and seedlings contrasts with previously published research in this system (Pellegrini et al. 2024 ). The inability of A. subfuscus mucus alone to alter germination speed may result from our use of previously frozen mucus-covered soil rather than freshly collected material. While plant DAMPs do not appear to degrade after being frozen for a 16-week period (Duran-Flores and Heil 2014 ) and our pilot experiment found that the germination-accelerating effect of A. subfuscus slime persisted after nearly five months of freezing, freshly-produced mucus from B. nigra -fed slugs may have contained DAMPs or some other volatile cues absent from the frozen soil we used. Second, both Pellegrini et al ( 2024 ) and Orrock ( 2013 ) harvested their B. nigra seedlings at 21 days, too small a size to conduct glucosinolate analyses on individual plants. Because of our interest in glucosinolates as a mechanism for herbivore defense, we allowed plants in our experiment to grow to maturity (36 days) before harvesting them. While this ensured we had sufficient aboveground biomass for chemical analyses, it may also have allowed kairomone-and-DAMP-treated seedlings that started out smaller the time to ‘catch up’ in size to control seedlings. This difference in size may also explain both the absence of among-treatment differences in glucosinolate levels and the minimal herbivory in all treatments. Spodoptera consumed 5 ± 1.5% [SE] of leaf biomass in our work, while A. subfuscus herbivory in Pellegrini et al ( 2024 ) averaged 31 ± 11.2%. In addition to being larger, leaves from older plants also possessed trichomes and were significantly tougher to tear; while we know in most cases that seedlings are generally more palatable to mollusks than adults (Fenner et al. 1999 ), future research should address how physical defenses or non-glucosinolate chemical defenses might affect Spodoptera consumption. Additional work should explore the effect of individual and combined cues on the growth, chemical defense, and palatability on cotyledons and smaller true leaves, since both are produced when treated plants are likely more focused on defense. The fact that plants utilized multiple risk cues to alter defensive investment provides avenues for future research. For example, B. nigra and A. subfuscus co-occur in their native range; the substantial impact of Arion on endemic North American plants may reflect the lack of a coevolutionary history that would otherwise allow them to employ risk-induced defense. Interplay between risk cues might also alter interactions with plants and other non-molluscan herbivores (Orrock et al. 2018 )or affect competition between conspecific or heterospecific seedlings that differ in their vulnerability to attack risk(Hanley M. E. 1995; Orians et al. 2013 ). Since herbivory in the parental generation can increase defense their offspring (Agrawal et al. 1999 ), it would also be of interest to see whether kairomone and DAMP exposure in the absence of actual herbivory would alter the cue receptivity or response in their offspring. Declarations Acknowledgements The authors would like to thank A. Gill, M. Miller, and L. Pintado for their assistance with methods development, and I. Perez Agudelo and V. Pleitez Aguilar for their assistance during the 2024 experiment. Funding: This study was funded by NSF DEB-2117367 to ELP, CMO, and JLO. Conflict of Interest: The authors declare that they have no conflict of interest. Ethical Approval: All applicable institutional and/or national guidelines for the care and use of animals were followed. Consent to participate: Not applicable. Consent for publication: Not applicable. Availability of data and material (data transparency): Should the paper be provisionally accepted, the authors pledge to archive all data on figshare prior to final acceptance. This will be the permanent repository. Code availability: Not applicable. Declaration of Authorship: ELP, CMO, and JLO originally formulated the idea. References Agrawal AA, Laforsch C, Tollrian R (1999) Transgenerational induction of defences in animals and plants. Nature 401:60–63. doi: 10.1038/43425 Barton KE, Hanley ME (2013) Seedling-herbivore interactions: insights into plant defence and regeneration patterns. Ann. Bot. 112:643–650. doi: 10.1093/aob/mct139 Brown PD, Tokuhisa, J.G., Reichelt, M., and Gershenzon, J. (2003) Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana . Phytochemistry 62:471–481. doi: 10.1016/s0031-9422(02)00549-6 Chaplin-Kramer R, Kliebenstein, D.J., Chiem, A., Morrill, E., Mills, N.J. and Kremen, C. 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ONE 9:e86500. doi: 10.1371/journal.pone.0086500 Meldau S, Kastner J, von Knorre D, Baldwin IT (2014) Salicylic acid-dependent gene expression is activated by locomotion mucus of different molluscan herbivores. Commun. Integr. Biol. 7:e28728. doi: 10.4161/cib.28728 Orians CM, Fritz RS, Hochwender CG, Albrectsen BR, Czesak ME (2013) How slug herbivory of juvenile hybrid willows alters chemistry, growth and subsequent susceptibility to diverse plant enemies. Ann. Bot. 112:757–765. doi: 10.1093/aob/mct002 Orrock JL (2013) Exposure of unwounded plants to chemical cues associated with herbivores leads to exposure-dependent changes in subsequent herbivore attack. PLoS. ONE 8:e79900. doi: 10.1371/journal.pone.0079900 Orrock JL, Connolly BM, Choi WG, Guiden PW, Swanson SJ, Gilroy S (2018) Plants eavesdrop on cues produced by snails and induce costly defenses that affect insect herbivores. Oecologia 186:703–710. doi: 10.1007/s00442-018-4070-1 Orrock JL et al. (2015) Error management in plant allocation to herbivore defense. Trends Ecol. Evol. 30:441–445. doi: 10.1016/j.tree.2015.06.005 Orrock JL, Christopher CC (2010) Density of intraspecific competitors determines the occurrence and benefits of accelerated germination. Am J Bot 97:694–699 Pellegrini BP, Pintado LS.; Sousa, P.; Bhavanam, S.; Orians, C.; Orrock, J.; & Preisser, E. (2024) Herbivore kairomones affect germination speed, seedling growth, and herbivory. Oecologia 206:215–223 Pestana JLT, Baird DJ, Soares AMVM (2013) Predator threat assessment in Daphnia magna : the role of kairomones versus conspecific alarm cues. Mar. Freshw. Res. 64. doi: 10.1071/mf13043 Pieck M, Yuan, Y., Godfrey, J., Fisher, C., Zolj, S., Vaughan, D., Thomas, N., Wu, C., Ramos, J., Lee, N., Normanly, J., Celenza, J.L. (2015) Auxin and tryptophan homeostasis are facilitated by the ISS1/VAS1 aromatic aminotransferase in Arabidopsis. Genetics 201:185–199. doi: 10.1534/genetics.115.180356 Prada LM, Guerrero-Casado J, Tortosa FS (2018) European rabbits recognise conspecifics in their predators’ diets. Acta Ethol. 21:163–168. doi: 10.1007/s10211-018-0295-6 Ray S et al. (2016) Lessons from the far end: caterpillar frass-induced defenses in maize, rice, cabbage, and tomato. J. Chem. Ecol. 42:1130–1141. doi: 10.1007/s10886-016-0776-x Scherer AE, Smee DL (2016) A review of predator diet effects on prey defensive responses. Chemoecology 26:83–100. doi: 10.1007/s00049-016-0208-y Schoeppner N, Relyea R (2005) Damage, digestion, and defence: the roles of alarm cues and kairomones for inducing prey defences. Ecol. Lett. 8:505–512. doi: 10.1111/j.1461-0248.2005.00744.x Schoeppner NM, Relyea RA (2009a) Interpreting the smells of predation: how alarm cues and kairomones induce different prey defences. Funct. Ecol. 23:1114–1121. doi: 10.1111/j.1365-2435.2009.01578.x Schoeppner NM, Relyea RA (2009b) When should prey respond to consumed heterospecifics? Testing hypotheses of perceived risk. Copeia 1:190–194. doi: 10.1643/ce-08-041 Shabani S, Kamio M, Derby CD (2008) Spiny lobsters detect conspecific blood-borne alarm cues exclusively through olfactory sensilla. J. Exp. Biol. 211:2600–2608. doi: 10.1242/jeb.016667 Sheriff MJ et al. (2020) Proportional fitness loss and the timing of defensive investment: a cohesive framework across animals and plants. Oecologia 193:273–283 Ślusarczyk M (1999) Predator-induced diapause in Daphnia magna may require two chemical cues. Oecologia 119:159–165. doi: 10.1007/s004420050772 Spivey KL, Chapman TL, Schmitz AL, Bast DE, Smith ALB, Gall BG (2015) The alarm cue obstruction hypothesis: isopods respond to alarm cues, but do not respond to dietary chemical cues from predatory bluegill. Behaviour 152:167–179 Tanaka K, Heil M (2021) Damage-associated molecular patterns (DAMPs) in plant innate immunity: applying the danger model and evolutionary perspectives. Annu. Rev. Phytopathol. 59:53–75. doi: 10.1146/annurev-phyto-082718-100146 Wieski K, Slusarczyk M (2022) On the different role of alarm substances and fish kairomones in diapause induction in a freshwater planktonic crustacean. J. Plankton Res. 44:278–287. doi: 10.1093/plankt/fbac004 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7041192","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487563206,"identity":"a8d3d753-44a6-4d80-9777-5ee10a785d8f","order_by":0,"name":"Katherine Overstrum","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIie3QMUvDQBjG8VcOLsvJre8RyWdIKdhufhVDoItWhI5CKDhkCXS9wQ9REZwCvhDoJHQtuFQCN1Wwm0MHc6mIlWtxdLj/kkvILw8EwOf7hyEA315+dLTcPjnfQ9gusScW/4HszAL/+oKbqHw+W67L/pUENsN1mWU9fTG4OS77IIPL2EVCkQYdbXCkxnygtKnwZDF8fBEGQRUrJ4kg5aEgTKYkul1BhIiWEEK8cK9EsubhpiFPlmwoa8nIkrM9JMRmBewKiE4NxFrC2hV0E6XrU1U0RFc8eS2oUlq8Pag7QoHP5tpFcJ4Y/KAsmeS3RM1BYjC8f19RFsk8nTr/8nfs1704/LrP5/P5DvUJ4tBY5Vskr3IAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0008-4215-5883","institution":"University of Rhode Island","correspondingAuthor":true,"prefix":"","firstName":"Katherine","middleName":"","lastName":"Overstrum","suffix":""},{"id":487563207,"identity":"7815073f-62d5-4cca-9672-4f6508429e11","order_by":1,"name":"Eirette Santiago","email":"","orcid":"","institution":"University of Rhode Island","correspondingAuthor":false,"prefix":"","firstName":"Eirette","middleName":"","lastName":"Santiago","suffix":""},{"id":487563208,"identity":"344b5c60-11fc-4819-9bac-58bc17bd73d0","order_by":2,"name":"Brooke Pellegrini","email":"","orcid":"","institution":"University of Rhode Island","correspondingAuthor":false,"prefix":"","firstName":"Brooke","middleName":"","lastName":"Pellegrini","suffix":""},{"id":487563209,"identity":"7986d352-1b69-4282-8044-73042d719acd","order_by":3,"name":"Kevin Headrick","email":"","orcid":"","institution":"Tufts University","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Headrick","suffix":""},{"id":487563210,"identity":"ee6e0663-7ed1-4609-82f0-da177920eff8","order_by":4,"name":"Colin Orians","email":"","orcid":"","institution":"Tufts University","correspondingAuthor":false,"prefix":"","firstName":"Colin","middleName":"","lastName":"Orians","suffix":""},{"id":487563211,"identity":"aca1fdc8-c1c7-46e7-b347-744882645f7f","order_by":5,"name":"John Orrock","email":"","orcid":"","institution":"University of Wisconsin-Madison","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Orrock","suffix":""},{"id":487563212,"identity":"847f03f0-cb63-4eb9-afb8-7cf02d674305","order_by":6,"name":"Evan Preisser","email":"","orcid":"","institution":"University of Rhode Island","correspondingAuthor":false,"prefix":"","firstName":"Evan","middleName":"","lastName":"Preisser","suffix":""}],"badges":[],"createdAt":"2025-07-03 20:12:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7041192/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7041192/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87304620,"identity":"67e65d53-4b59-444c-928c-532a4adcdc7c","added_by":"auto","created_at":"2025-07-22 14:01:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":325318,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of exposing \u003cem\u003eB. nigra \u003c/em\u003eseeds to control (soil and water only), kairomone only, DAMP only, or both kairomone and DAMP prior to and during germination. Seeds exposed to both kairomone and DAMP germinated significantly faster.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7041192/v1/daaca4adde3a93751816daa8.png"},{"id":87304619,"identity":"618f9169-8692-4514-a695-e8c2549e83c0","added_by":"auto","created_at":"2025-07-22 14:01:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122054,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of exposing \u003cem\u003eB. nigra \u003c/em\u003eseeds and seedlings to control (soil and water only), kairomone only, DAMP only, or both kairomone and DAMP. There was no significant difference in aboveground, belowground, or total biomass for seedlings at time of harvest.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7041192/v1/fb97edc9a66dcb4d0324f26e.png"},{"id":87306238,"identity":"0cc9673c-a519-4e7f-816f-29c5636246cb","added_by":"auto","created_at":"2025-07-22 14:09:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":119242,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of plants eaten by \u003cem\u003eS. exigua \u003c/em\u003elarvae when allowed to choose between leaves from the four treatments. \u003cstrong\u003e3A\u003c/strong\u003e: leaves from control versus DAMP-only plants, \u003cstrong\u003e3B\u003c/strong\u003e: leaves from control versus kairomone-only, \u003cstrong\u003e3C\u003c/strong\u003e: leaves from control versus kairomone+DAMP plants. \u003cem\u003eS. exigua \u003c/em\u003edid not differentiate between control plants and single treatment plants, but did prefer control plants over those treated with both pre-attack cues. *p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7041192/v1/40b48ad8a4d1d20892177bc7.png"},{"id":89956589,"identity":"4641c0e3-4d9d-4531-a3d0-2a48c72b43c5","added_by":"auto","created_at":"2025-08-26 22:17:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1186165,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7041192/v1/102e23fd-f727-4e85-882d-ec463c9a3298.pdf"}],"financialInterests":"","formattedTitle":"Herbivore cues and plant damage-associated-compounds jointly alter seed germination and seedling herbivory","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants threatened by herbivores must balance the benefits of defense with its energetic costs. The modular design of plants, paired with the lack of a central vascular system and the highly plastic nature of meristem tissue, means that low levels of herbivory pose little risk to survival and fitness (Karban and Agrawal \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). As a result, mature plants can generally afford to wait until attack begins to induce chemical and/or structural defenses to prevent further damage (Karban \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This risk-tolerant approach is less suitable for juvenile plants such as seeds and seedlings, that either lack the energetic reserves to recover from damage or are small enough to be eaten in their entirety before they have the chance to respond. As a result, the ability to detect and respond to pre-attack herbivore cues may be critically important to young plants. Because allocation to defense can reduce plant growth and lead to young plants losing out in competition for light and other resources, selection should favor individuals capable of balancing these competing demands and determining whether a given stimulus warrants the induction of pre-attack defense (Karban and Orrock \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePlants seeking to detect pre-attack herbivore cues may take advantage of kairomones, non-attack-related cues incidentally emitted by consumers that are detected by and benefit prey. Kairomones are often byproducts associated with daily life and include excrement, sex attractants, and fluids associated with locomotion or oviposition. Exposing \u003cem\u003eBrassica nigra\u003c/em\u003e seeds and seedlings to snail (\u003cem\u003eHelix aspersa\u003c/em\u003e) locomotion mucus, for example, decreased plant growth and herbivory relative to control plants (Orrock \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e); subsequent work found that the effect of \u003cem\u003eH. aspersa\u003c/em\u003e mucus on tomato seedling growth and defense rivaled that of the plant defense elicitor methyl jasmonate (MeJA) (Orrock et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Exposing \u003cem\u003eSolidago altissima\u003c/em\u003e to sex attractants of the galling fly \u003cem\u003eEurosta solidaginis\u003c/em\u003e reduced herbivory and subsequent oviposition in both laboratory and field experiments (Helms et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Insect frass from multiple herbivores has similarly been shown to induce defense in several host plant species (Ray et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePlants may also assess risk of herbivore attack via \u0026lsquo;damaged self\u0026rsquo; cues produced by nearby injured conspecific or heterospecific individuals. These damage-associated molecular patterns (DAMPs) are chemicals released by stressed or injured cells that alert nearby cells to a potential threat. Pattern recognition receptors (PRRs) located on the cell surface and spanning the plasma membrane detect damage and respond via increased production of reactive oxygen species, protein kinases, and hormones such as jasmonic acid (JA), salicylic acid (SA), and ethylene (Tanaka and Heil \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Because PRRs are most responsive to species-specific cues, defensive induction is greatest in response to DAMPs from the same or closely related species (Duran-Flores and Heil 2018). Bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e) plants, for instance, increased reactive oxygen species production and JA induction in response to leaf homogenates from conspecific - but not heterogeneric - plants (Duran-Flores and Heil \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough plant responses to non-damage herbivore kairomones and DAMPs have received considerable attention, the interaction between the two has only been explored in animal predator-prey systems. Studies of the separate and combined effects of both kairomones and alarm cues in several tadpole species found that while exposure to either alarm cues or kairomones induced prey defense, their combination elicited the most complete and effective set of antipredator responses (Schoeppner and Relyea \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Schoeppner and Relyea \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009a\u003c/span\u003e). Prey responded more strongly to a kairomone-DAMP mix derived from predators fed conspecifics than those fed distantly related species, suggesting that the phylogenetic similarity of predator-consumed individuals is also important (Schoeppner and Relyea \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Similar results have been found in work exploring antipredator responses in the aquatic macroinvertebrates \u003cem\u003eCaecidotea intermedi\u003c/em\u003e (Spivey et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and \u003cem\u003eDaphnia magna\u003c/em\u003e (Pestana et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ślusarczyk \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Wieski and Slusarczyk \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), suggesting that kairomones and DAMPs together generally provide a reliable indication of predation risk.\u003c/p\u003e\u003cp\u003eWe describe the results of work examining how kairomones, DAMPs, and their interaction affect seed germination and seedling growth, chemistry, and herbivore palatability. Seeds and seedlings of our study species, \u003cem\u003eBrassica nigra\u003c/em\u003e, have been shown to induce defense in response to locomotion mucus from the generalist molluscan herbivores \u003cem\u003eHelix aspersa\u003c/em\u003e (Orrock \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and \u003cem\u003eArion subfuscus\u003c/em\u003e (Pellegrini et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our study crossed a kairomone treatment (control or locomotion mucus from \u003cem\u003eA. subfuscus\u003c/em\u003e fed \u003cem\u003eBrassica nigra\u003c/em\u003e) with DAMP (\u003cem\u003eB. nigra\u003c/em\u003e leaf homogenate) presence/absence and measured various aspects of plant response. The results demonstrate that plants can utilize both signals and reveal an ecologically relevant story of growth and defense under exposure to pre-attack cues.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy species\u003c/strong\u003e\u003cp\u003e\u003cem\u003eArion subfuscus\u003c/em\u003e is native to Europe and was introduced to North America in the early 1900s (Chichester and Getz \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). It is an important molluscan herbivore and in the invaded range (Chichester and Getz \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1969\u003c/span\u003e) and the most abundant species of slug in New England (French 2012). Adults were collected May-July 2024 from forested areas in South Kingstown, RI, USA. Slugs were maintained in six large soil- and detritus-filled terraria at 21\u0026ndash;24\u0026deg;C on a diet of organic lettuces, carrots, and field-collected seedlings of various plant species. The terraria were misted daily in order to maintain high humidity levels, and decaying plant material was removed when necessary.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eBrassica nigra\u003c/em\u003e is a fast-growing plant native to the Mediterranean that is naturalized worldwide and grows wild in RI. Seeds were sourced from Outsidepride Seed Source, LLC (Independence, OR, USA); \u003cem\u003eA. subfuscus\u003c/em\u003e is also found in Oregon (Burke 2013).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eExperimental design\u003c/strong\u003e\u003cp\u003eThe 2x2 experimental design crossed two herbivore-risk treatments (Kairomone) with two damage-associated-molecular-pattern (DAMP) treatments for a total of six treatments. The herbivore-risk treatments were mucus from \u003cem\u003eA. subfuscus\u003c/em\u003e fed \u003cem\u003eB. nigra\u003c/em\u003e for three days (+\u0026thinsp;Kairomone) or a no-mucus control (-Kairomone). The two DAMP treatments were water mixed with ground \u003cem\u003eB. nigra\u003c/em\u003e leaves (+\u0026thinsp;DAMP) or water alone (-DAMP). Each treatment was applied to \u003cem\u003eB. nigra\u003c/em\u003e seeds and plants and their responses measured.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHerbivore-risk treatment\u003c/b\u003e: Three days before preparing the risk treatments, our population of mature \u003cem\u003eA. subfuscus\u003c/em\u003e was divided into multiple small plastic terraria lined with wet paper towels where they were fed lab-grown \u003cem\u003eB. nigra\u003c/em\u003e seedlings. After three days, we flattened 4g of a soil-and-distilled-water mixture (1:4 ratio) into each of multiple 90 mm petri dishes. In dishes assigned to either the high-risk or control treatments, a mature \u003cem\u003eA. subfuscus\u003c/em\u003e was allowed to crawl on the soil for 24 hours in a dark cabinet at 20\u0026ndash;24\u0026deg;C. Control plates were held in similar conditions, but did not receive a slug. All slugs were then returned to the large dirt-filled terraria and the petri dishes frozen at -20\u0026deg;C freezer for 2\u0026ndash;4 weeks until they were defrosted and used. We used frozen rather than \u0026lsquo;fresh\u0026rsquo; plates for the work because it would have been extremely difficult to generate the \u0026gt;\u0026thinsp;1000 \u0026lsquo;fresh\u0026rsquo; risk-treatment plates necessary for the work when the main experiment (detailed below) was running. To ensure that previously frozen slug mucus induced similar plant responses as freshly deposited mucus, we conducted a pilot experiment confirming that mucus frozen for up to six months accelerated \u003cem\u003eB. nigra\u003c/em\u003e seed germination (Online Resource 1).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDAMP treatment\u003c/b\u003e: \u003cem\u003eBrassica nigra\u003c/em\u003e seeds were sown in potting soil and grown in trays on a sunny windowsill. To prepare the leaf homogenate for the DAMP\u0026thinsp;+\u0026thinsp;treatment, \u003cem\u003eB. nigra\u003c/em\u003e seedlings (~\u0026thinsp;3 cm in height) were cut at the base of the stem and blended with distilled water (1:30 ratio by weight, as per Duran-Flores and Heil \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) in a blender (Waring Commercial, Torrington, CT, USA) and then allowed to sit for two hours before use as per Duran-Flores and Heil (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). A water-only control solution was also prepared for use in the -DAMP treatment.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eSeed Responses\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eGermination assay\u003c/strong\u003e\u003cp\u003eOn 15 July, 16 petri dishes from each of the two herbivore-risk treatments were removed from the freezer. Once fully defrosted, a piece of 90mm white filter paper was placed on top of the soil inside each of the dishes. Half of the dishes in each herbivore-risk treatment were then wetted with either 1 mL of the leaf homogenate solution (+\u0026thinsp;DAMP) or 1 mL of distilled water (-DAMP). Each of the four treatments was thus replicated eight times for a total of 32 dishes.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eImmediately after the addition of the DAMP solution, 50 \u003cem\u003eB. nigra\u003c/em\u003e seeds were placed on top of the filter paper in each dish. Dishes from all four treatments were then interspersed and placed in a dark cabinet at 21\u0026deg;C. Starting 15 hours after seed placement and continuing every three hours afterwards, the seeds in each petri dish were checked for germination. We chose to start germination checks at 15 hours on the basis of prior work that found virtually no seed germination before this point (Pellegrini et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). At each interval, each seed was carefully checked for radicle emergence. Seeds with a radicle were counted and then removed from their petri dish. The checks continued until hour 45, when at least 90 percent of seeds from each treatment group had germinated.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eSeedling Responses\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePlanting, growth, and cue application\u003c/strong\u003e\u003cp\u003ePrior to the start of the experiment, 320 6 x 6 cm black plastic pots were filled with MiracleGro potting mix (Marysville, OH, USA). The pots were placed in fourteen square boxes (36 cm x 36 cm) with a mesh bottom. Each box contained six plants from each treatment, for a total of 36 pots per box in 13 of the 14 boxes; the fourteenth box had empty pots added to the \u0026lsquo;empty\u0026rsquo; slots in it.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eThe first 80 seeds to germinate in each of the four treatments (320 total pots) were removed and buried individually 1 cm under the soil in a pot that was then placed in a box. The boxes were placed along a windowsill where they received approximately seven hours of direct sunlight per day. Each box was rotated clockwise every other day, and every week each pot within each box was rotated within its box by moving it one slot down and one slot to the right. Each plant received 5mL of water daily, administered via pipette at the base of the plant.\u003c/p\u003e\u003cp\u003eStarting one week after germination, one petri dish of soil from the appropriate herbivore-risk treatment was removed from the freezer, defrosted, and the soil within it added carefully to the base of a plant from the appropriate treatment. Immediately after the soil had been applied, each plant was watered with 5 mL of freshly prepared leaf homogenate (+\u0026thinsp;DAMP) or control (-DAMP) solution. Seedlings were subsequently watered every 2 days with 5mL of water. The herbivore-risk and DAMP treatments were applied weekly for a total of five weeks; each plant received the same cue combination for the duration of the experiment.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eBiomass and glucosinolate analysis\u003c/strong\u003e\u003cp\u003eOn day 36 of the experiment (i.e., one day following final cue application), 20 plants from each treatment (80 total plants) were destructively harvested. We first cut the third-newest leaf from each plant at the petiole, weighed it, then rolled it into a 15mL falcon tube that was capped, flash frozen using liquid nitrogen, and placed in a -80\u0026deg;C freezer for later glucosinolate analysis.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eThe rest of the plant was removed by hand from the soil and cut at the cotyledon scar. The aboveground material was immediately placed in a coin envelope; the belowground material was carefully rinsed in water and patted dry before doing so. All samples were then placed in a drying oven (Blue M Electric Company, Blue Island, IL, USA) for four days at 60\u0026deg;C before weighing the above- and below-ground portions of each plant. A separate set of leaves were cut from unused plants and weighed immediately before being dried and re-weighed; we used the resulting wet:dry weight regression to convert the wet weight of the leaf removed for glucosinolate analyses into dry weight for addition to the aboveground biomass.\u003c/p\u003e\u003cp\u003eGlucosinolates were extracted and analyzed using standard methods (Pieck 2015) (Brown 2003). Briefly, frozen samples were ground under liquid nitrogen in a Retsch MM400 ball-mill grinder (Retsch GmbH, Haan, Germany). Once ground, 70\u0026ndash;120 mg of frozen leaf tissue in 2mL microcentrifuge tubes was extracted in 1mL methanol. Glucosinolates were bound to 20 mg of a DEAE Sephadex A25 pellet. Sulfatase (4 mg/mL) was then used to cleave desulfoglucosinolates from the pellet, and the supernatant was filtered, 0.22 um, into HPLC vials. A sinigrin standard was processed using the same method; 90% of the glucosinolates in \u003cem\u003eBrassica nigra\u003c/em\u003e are sinigrin (Chaplin-Kramer 2011; Feeny 1982). The concentration of sinigrin was analyzed by HPLC using a 150 x 4.6 mm C18 reverse phase column (Phenomenox, Torrance, CA) with a water acetonitrile gradient (Brown 2003) Each sample was injected twice and the concentration of sinigrin (mg/g fresh leaf mass) was averaged for each sample.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSpodoptera exigua bioassays\u003c/em\u003e\u003c/strong\u003e\u003cp\u003eFive weeks after the start of the experiment, a series of paired-choice assays were performed in which late-instar \u003cem\u003eSpodoptera exigua\u003c/em\u003e (Frontier Agricultural Services, Newark, DE, USA) were allowed to choose between a leaf from a control (-Kairomone/-DAMP) plant and another leaf from one of the three remaining treatment groups (-Kairomone/+DAMP, +Kairomone/-DAMP, +Kairomone/+DAMP). There were 15 replicates for each of the three bioassay combinations (45 total bioassays).\u003c/p\u003e\u003c/p\u003e\u003cp\u003eFor each paired-choice bioassay, a single \u003cem\u003eB. nigra\u003c/em\u003e leaf from a control and one from a specific treatment plant was cut from a plant, weighed, and placed in a prepared petri dish for bioassays. Leaves of similar age and sizes were paired together whenever possible, and both plants were selected from the same box. For each bioassay, a 90mm petri dish was lined with 90mm white filter paper and sprayed twice with distilled water before the leaves were placed next to each other on opposite sides of the dish. One \u003cem\u003eS. exigua\u003c/em\u003e was weighed and placed in the center of the dish, which was then closed and held in a dark cabinet for 24 hours. At the end of the bioassays, the \u003cem\u003eS. exigua\u003c/em\u003e larvae and both leaves were reweighed. We also conducted no-herbivory bioassays (necessary to quantify ambient changes in leaf weight over time); these were identical to the choice assays except for the absence of \u003cem\u003eS. exigua\u003c/em\u003e larvae.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003cp\u003eWe initially examined the germination data using failure-time analyses (Cox proportional hazards model) but ultimately opted to use a generalized linear mixed model (GLMM) with a binomial response distribution because model diagnostics revealed that the proportional hazards assumption was violated. The model specified kairomone and DAMP treatments as fixed effects, the hour since the experiment was initiated as a covariate, and also included a cubic term for hour. We modelled time as non-linear because preliminary analyses indicated a non-linear relationship between probability of germination and the duration of the experiment and a cubic relationship was a significantly better fit than linear or quadratic based on comparison of AICc and log-likelihood tests. We explored all possible interaction terms. The combined herbivore bioassay dataset contained a few anomalously high and low datapoints spread across multiple treatments. Because these likely reflected errors in weighing/data entry, we removed the highest and lowest 2.5% of values from the combined dataset before analyzing each of the choice bioassays separately. For each choice bioassay, data on leaf proportion remaining per treatment was analyzed using GLM (identity link function) with treatment and bioassay date as fixed effects and plate identity nested within date as a random effect. We examined plant growth and glucosinolate concentration using linear models that specified kairomone and DAMP as fixed effects. Analyses were conducted in R version 3.6.1 (R Core Development Team 2019) and JMP version 18.1.2 (SAS Institute Inc. 2025).\u003c/p\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSeed germination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatterns of germination in time were strongly cubic (X\u003csup\u003e2\u003c/sup\u003e = 579.40, 3 d.f., p \u0026lt; 0.001) with patterns of germination accelerating early in the experiment and then levelling off as time proceeded (Online Resource 2). Germination was affected by kairomones and DAMPs, as reflected by a highly significant kairomone*DAMP*hour interaction (Fig.\u0026nbsp;1; X\u003csup\u003e2\u003c/sup\u003e = 19.85, 3 d.f., p \u0026lt; 0.001), as well as a significant kairomone*hour interaction (X\u003csup\u003e2\u003c/sup\u003e = 34.89, 3 d.f., p \u0026lt; 0.00) and a significant DAMP*hour interaction (X\u003csup\u003e2\u003c/sup\u003e = 50.72, 3 d.f., p \u0026lt; 0.001). Averaged across these significant interactions with time, there was no main effect of DAMPs (X\u003csup\u003e2\u003c/sup\u003e = 2.18, 1 d.f., p = 0.14), kairomones (X\u003csup\u003e2\u003c/sup\u003e = 0.01, 1 d.f., p = 0.91) or interaction between DAMPs and kairomones (X\u003csup\u003e2\u003c/sup\u003e = 0.80, 1 d.f., p = 0.37). Seeds in the control treatment averaged 24.5 hours to germination, while those in the kairomone-only treatment averaged 24.4 hours (0.6% faster) and seeds in the DAMP-only treatment averaged 24.1 hours (1.7% faster). In contrast, seeds exposed to both kairomones and DAMPs germinated in 22.6 hours (7.9% faster than control seeds; Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeedling biomass\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMean aboveground plant biomass (Fig.\u0026nbsp;2, green bars) was not affected by kairomones (F\u003csub\u003e1,82\u003c/sub\u003e = 0.013, p = 0.72), DAMPs (F\u003csub\u003e1,82\u003c/sub\u003e = 0.04, p = 0.97), or their interaction (F\u003csub\u003e1,82\u003c/sub\u003e = 0.07, p = 0.80). Mean belowground plant biomass (Fig.\u0026nbsp;2, brown bars) was similarly unaffected by kairomones (F\u003csub\u003e1,81\u003c/sub\u003e = 0.30, p = 0.59), DAMPs (F\u003csub\u003e1,81\u003c/sub\u003e = 0.18, p = 0.67), or their interaction (F\u003csub\u003e1,81\u003c/sub\u003e = 0.03, p = 0.86).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlucosinolate concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no effect of kairomones (F\u003csub\u003e1,76\u003c/sub\u003e = 1.62, p = 0.21), DAMPs (F\u003csub\u003e1,76\u003c/sub\u003e = 0.004, p = 0.99), or their interaction (F\u003csub\u003e1,76\u003c/sub\u003e = 1.06, p = 0.31) on leaf glucosinolate concentration.\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eHerbivory bioassays\u003c/b\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSpodoptera\u003c/em\u003e larvae did not discriminate between control leaves and those exposed to kairomones or DAMPs separately (all p \u0026lt; 0.10). In contrast, \u003cem\u003eSpodoptera\u003c/em\u003e did exhibit a preference when choosing between control leaves and those from plants exposed to both kairomones and DAMPs (Fig.\u0026nbsp;3). Larve consumed 8.4% of controls versus \u0026gt; 1% of leaves from plants exposed to both risk cues (F\u003csub\u003e1,13\u003c/sub\u003e = 6.47, p = 0.025).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results provide the first evidence that seeds and seedlings can integrate information from both herbivore kairomones and damage-associated molecular patterns (DAMPs) in their assessment of attack risk. Such induced responses to herbivory in plants provide essential defense against subsequent damage (Karban \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Because mature plants can generally wait until being attacked to induce defense (Sheriff et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), plant-herbivore research has focused on attack-associated cues such as tissue damage or volatile emissions. The ability to detect and respond to pre-attack cues like herbivore kairomones or DAMPs should be particularly beneficial for seeds and seedlings, life stages with limited resources for which herbivory is often lethal (Barton and Hanley \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Because defensive induction can be energetically costly, however, selection should also favor the use of multiple sources of pre-attack information to reduce undefended attacks while avoiding the cost of unnecessary defense (Orrock et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); our findings are consistent with this hypothesis.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003eB. nigra\u003c/em\u003e seeds and seedlings, our results demonstrate that pre-attack cues indicating the proximity of both herbivores and damaged conspecifics altered both seedling germination and herbivore susceptibility (i.e., heighten a plant\u0026rsquo;s awareness of danger in the vicinity). This finding parallels research in predator-prey systems showing that cues from predators that consume prey conspecifics (akin to an herbivore whose presence produces kairomones and whose feeding produces DAMPs) are likely more dangerous than those that do not (Scherer and Smee \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Terrestrial and aquatic studies confirm that prey respond more to cues from conspecific-fed predators than to either predator (in our study, kairomone) or crushed conspecific (in our study, DAMP) cues alone (Prada et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Schoeppner and Relyea \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Cues from predators fed conspecific or congeneric prey consistently elicit stronger responses than cues produced by the consumption of distantly related species (Schoeppner and Relyea \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009b\u003c/span\u003e; Shabani et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), arguing for the adaptive benefits of such gradated responses in both animal prey and plants.\u003c/p\u003e\u003cp\u003eWhile we predicted that exposure to combined risk cues would induce defense, we were surprised that neither cue induced defense by themselves. This was unexpected because both herbivore kairomones and DAMPS have been shown singly to induce pre-attack plant defense. Helms (2013) reported that \u003cem\u003eSolidago\u003c/em\u003e responds to the sex attractant of a gall-making fly, for instance, and Orrock (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) found that snail mucus alters \u003cem\u003eB. nigra\u003c/em\u003e growth and defense. We now know that cultivated and wild plants respond to slug and snail mucus (Falk et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kastner et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Meldau et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Orrock \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Orrock et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and prior work found that \u003cem\u003eB. nigra\u003c/em\u003e seeds and seedlings detect and respond to \u003cem\u003eA. subfuscus\u003c/em\u003e (Pellegrini et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Leaf homogenate from crushed conspecific plants similarly induces resistance in unwounded plants (Duran-Flores and Heil \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e); parallel responses to damaged-self cues occur in a range of taxa (Heil and Land \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Research demonstrating damaged-self responses to conspecific homogenates also found less induction in response to congeneric homogenates and none in response to crushed plants from different genera (Duran-Flores and Heil \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although our work can be seen as contradictory to these findings, we suspect that aspects of our experimental design (detailed below) inadvertently reduced our ability to detect single-cue effects.\u003c/p\u003e\u003cp\u003eFaster germination in response to risk cues has only recently been documented (Pellegrini 2024). We observed an increase in germination speed for seeds treated with both kairomone and DAMPs. This appears to be the first example of DAMPs acting as germination promoters in plants. Because risk cues changed germination speed but not the overall rate, detecting such shifts requires around-the-clock germination checks over a multi-day period; this may explain why our work is the first to identify this effect. While seedlings emerging from faster-germinating seeds typically tend to outcompete their slower-germinating counterparts (Orrock \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), we found no difference in seedling biomass; Pellegrini et al (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) actually found that faster-germinating seeds produced smaller seedlings. Both sets of results support the hypothesis that risk-induced changes in germination speed improve defense rather than competitive ability, an explanation consistent with the narrative that seeds must act quickly to avoid predation. While Pellegrini et al (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found that seeds with faster germination due to kairomones led to a smaller final biomass, we observed no difference in final biomass. Our studies do, however, parallel in linking faster germination to increased resistance to herbivory, but we observed generally low levels of herbivory across all treatments.\u003c/p\u003e\u003cp\u003eThere are several caveats to be considered in interpreting the results of our work. First, our finding that herbivore kairomones alone did not impact \u003cem\u003eB. nigra\u003c/em\u003e seeds and seedlings contrasts with previously published research in this system (Pellegrini et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The inability of \u003cem\u003eA. subfuscus\u003c/em\u003e mucus alone to alter germination speed may result from our use of previously frozen mucus-covered soil rather than freshly collected material. While plant DAMPs do not appear to degrade after being frozen for a 16-week period (Duran-Flores and Heil \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and our pilot experiment found that the germination-accelerating effect of \u003cem\u003eA. subfuscus\u003c/em\u003e slime persisted after nearly five months of freezing, freshly-produced mucus from \u003cem\u003eB. nigra\u003c/em\u003e-fed slugs may have contained DAMPs or some other volatile cues absent from the frozen soil we used. Second, both Pellegrini et al (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and Orrock (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) harvested their \u003cem\u003eB. nigra\u003c/em\u003e seedlings at 21 days, too small a size to conduct glucosinolate analyses on individual plants. Because of our interest in glucosinolates as a mechanism for herbivore defense, we allowed plants in our experiment to grow to maturity (36 days) before harvesting them. While this ensured we had sufficient aboveground biomass for chemical analyses, it may also have allowed kairomone-and-DAMP-treated seedlings that started out smaller the time to \u0026lsquo;catch up\u0026rsquo; in size to control seedlings. This difference in size may also explain both the absence of among-treatment differences in glucosinolate levels and the minimal herbivory in all treatments. \u003cem\u003eSpodoptera\u003c/em\u003e consumed 5\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.5% [SE] of leaf biomass in our work, while \u003cem\u003eA. subfuscus\u003c/em\u003e herbivory in Pellegrini et al (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) averaged 31\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;11.2%. In addition to being larger, leaves from older plants also possessed trichomes and were significantly tougher to tear; while we know in most cases that seedlings are generally more palatable to mollusks than adults (Fenner et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), future research should address how physical defenses or non-glucosinolate chemical defenses might affect \u003cem\u003eSpodoptera\u003c/em\u003e consumption. Additional work should explore the effect of individual and combined cues on the growth, chemical defense, and palatability on cotyledons and smaller true leaves, since both are produced when treated plants are likely more focused on defense.\u003c/p\u003e\u003cp\u003eThe fact that plants utilized multiple risk cues to alter defensive investment provides avenues for future research. For example, \u003cem\u003eB. nigra\u003c/em\u003e and \u003cem\u003eA. subfuscus\u003c/em\u003e co-occur in their native range; the substantial impact of \u003cem\u003eArion\u003c/em\u003e on endemic North American plants may reflect the lack of a coevolutionary history that would otherwise allow them to employ risk-induced defense. Interplay between risk cues might also alter interactions with plants and other non-molluscan herbivores (Orrock et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)or affect competition between conspecific or heterospecific seedlings that differ in their vulnerability to attack risk(Hanley M. E. 1995; Orians et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Since herbivory in the parental generation can increase defense their offspring (Agrawal et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), it would also be of interest to see whether kairomone and DAMP exposure in the absence of actual herbivory would alter the cue receptivity or response in their offspring.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank A. Gill, M. Miller, and L. Pintado for their assistance with methods development, and I. Perez Agudelo and V. Pleitez Aguilar for their assistance during the 2024 experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was funded by NSF DEB-2117367 to ELP, CMO, and JLO.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eAll applicable institutional and/or national guidelines for the care and use of animals were followed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material (data transparency):\u0026nbsp;\u003c/strong\u003eShould the paper be provisionally accepted, the authors pledge to archive all data on figshare prior to final acceptance. This will be the permanent repository.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\u003cp\u003eDeclaration of Authorship: ELP, CMO, and JLO originally formulated the idea.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgrawal AA, Laforsch C, Tollrian R (1999) Transgenerational induction of defences in animals and plants. Nature 401:60\u0026ndash;63. doi: 10.1038/43425\u003c/li\u003e\n\u003cli\u003eBarton KE, Hanley ME (2013) Seedling-herbivore interactions: insights into plant defence and regeneration patterns. Ann. Bot. 112:643\u0026ndash;650. doi: 10.1093/aob/mct139\u003c/li\u003e\n\u003cli\u003eBrown PD, Tokuhisa, J.G., Reichelt, M., and Gershenzon, J. (2003) Variation of glucosinolate accumulation among different organs and developmental stages of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. 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ONE 9:e86500. doi: 10.1371/journal.pone.0086500\u003c/li\u003e\n\u003cli\u003eMeldau S, Kastner J, von Knorre D, Baldwin IT (2014) Salicylic acid-dependent gene expression is activated by locomotion mucus of different molluscan herbivores. Commun. Integr. Biol. 7:e28728. doi: 10.4161/cib.28728\u003c/li\u003e\n\u003cli\u003eOrians CM, Fritz RS, Hochwender CG, Albrectsen BR, Czesak ME (2013) How slug herbivory of juvenile hybrid willows alters chemistry, growth and subsequent susceptibility to diverse plant enemies. Ann. Bot. 112:757\u0026ndash;765. doi: 10.1093/aob/mct002\u003c/li\u003e\n\u003cli\u003eOrrock JL (2013) Exposure of unwounded plants to chemical cues associated with herbivores leads to exposure-dependent changes in subsequent herbivore attack. PLoS. 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Behaviour 152:167\u0026ndash;179\u003c/li\u003e\n\u003cli\u003eTanaka K, Heil M (2021) Damage-associated molecular patterns (DAMPs) in plant innate immunity: applying the danger model and evolutionary perspectives. Annu. Rev. Phytopathol. 59:53\u0026ndash;75. doi: 10.1146/annurev-phyto-082718-100146\u003c/li\u003e\n\u003cli\u003eWieski K, Slusarczyk M (2022) On the different role of alarm substances and fish kairomones in diapause induction in a freshwater planktonic crustacean. J. Plankton Res. 44:278\u0026ndash;287. doi: 10.1093/plankt/fbac004\u003cstrong\u003e\u003cstrong\u003e\u003c/strong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Kairomone, signal, mollusk, risk, induced defense","lastPublishedDoi":"10.21203/rs.3.rs-7041192/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7041192/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile plant defense against herbivory is primarily thought to occur following attack, there is also evidence that plants can detect and respond to pre-attack cues. These cues include chemicals released from damaged conspecifics and kairomones, non-attack-related substances emitted by an herbivore that plants can detect and use to their benefit. It is unknown, however, whether or how plants react to the interaction of these pre-attack cues. We measured germination, growth, and herbivore susceptibility of \u003cem\u003eB. nigra\u003c/em\u003e seedlings in an experiment that crossed the presence/absence of crushed \u003cem\u003eB. nigra\u003c/em\u003e leaves with the presence/absence of mucus of a generalist herbivore, \u003cem\u003eA. subfuscus\u003c/em\u003e. Seeds exposed to both crushed leaves and slug mucus germinated 8% more quickly than control seeds; neither risk cue increased germination speed when tested individually. The same pattern was found in herbivore bioassays: \u003cem\u003eSpodoptera exigua\u003c/em\u003e ate almost 10x more foliage from control seedlings than from from seedlings exposed to both crushed leaves and slug mucus. There was no difference in the final biomass of mature plants, suggesting that plants exposed to herbivore cues early in their development can increase defense without a measurable cost in size at maturity.\u003c/p\u003e","manuscriptTitle":"Herbivore cues and plant damage-associated-compounds jointly alter seed germination and seedling herbivory","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 14:01:24","doi":"10.21203/rs.3.rs-7041192/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57320f3d-c428-4b6c-9335-2602601bce15","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-26T22:09:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-22 14:01:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7041192","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7041192","identity":"rs-7041192","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}