Tiny predators avoid herbivorous caterpillar traces to prevent revolutionary predation

Tiny predators avoid herbivorous caterpillar traces to prevent revolutionary predation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Tiny predators avoid herbivorous caterpillar traces to prevent revolutionary predation Shiori Kinto, Shuichi Yano This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6643361/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract We report the first example of predators having a strategy to avoid ‘revolutionary predation’ by herbivores, i.e. predation on the higher trophic levels by the lower ones. The predatory mites Neoseiulus womersleyi and Euseius sojaensis are smaller than 0.5 mm and lay eggs on plant leaf surfaces; thus, their immobile eggs would be incidentally consumed along with leaves by voracious lepidopteran caterpillars. We experimentally demonstrated that eggs of both mite species were preyed upon by tested hawkmoth caterpillars (Theretra oldenlandiae, Theretra japonica) along with leaves. Therefore, the ability to avoid such revolutionary predation should confer a selective advantage to mites. We further demonstrated that adult females of both mite species avoided laying eggs on leaves with traces of all tested caterpillars (T. oldenlandiae, T. japonica, Papilio xuthus and Bombyx mori), indicating that eggs may avoid revolutionary predation by voracious caterpillars that may be nearby. This is the first demonstration of a repellent effect of herbivore traces on carnivores. Considering previous studies showing that spider mites as small as predatory mites also avoid caterpillar traces, the same need to avoid predation by huge caterpillars may have led to the development of the same solutions for both spider mites and predatory mites. Biological sciences/Ecology Earth and environmental sciences/Ecology revolutionary predation caterpillar predatory mite trace predation avoidance Figures Figure 1 Figure 2 Figure 3 Introduction Since some tiny herbivores can be subject to intraguild predation (IGP) when they are incidentally consumed by large herbivores along with shared host plants, they have developed strategies to avoid it 1 , 2 . Tiny herbivores experience IGP because they cannot immediately evacuate while large herbivores feed on leaves, and likewise, tiny predators that prey upon them are also likely to become involved in such predation. Therefore, the ability to avoid such incidental predation should also confer a selective advantage to tiny predators. It has been reported that some predators can be killed by counterattacks by herbivores 3 – 5 and hence some of these predators have strategies to avoid it 6 – 8 . However, there are no reports on predators avoiding one-sided ‘attacks’ by large herbivores. The predatory mites Neoseiulus womersleyi Schicha and Euseius sojaensis Ehara are typically < 0.4 mm in size. The former is a specialist predator which prey only upon spider mites and the latter is a generalist predator which feed on spider mites as well as non-prey resources such as pollen and pearl bodies 9 , 10 . These mites lay eggs on leaf hairs and in domatia of plant leaves infested by spider mites 11 , 12 . Lepidopteran larvae (hereafter ‘caterpillars’) such as Theretra oldenlandiae Fabricius and Papilio xuthus L. share food plants with spider mite that are preyed upon by predatory mites 13 – 15 . These caterpillars grow to 30–100 mm, and consume food leaves along with the spider mites 16 . For example, a final instar T. oldenlandiae consumes ca. 20 leaflets of Cayratia japonica (Thunb.) Gagnep. per day 1 . Adult predatory mites can run fast and can escape quickly when a leaf-eating caterpillar approaches, while their eggs laid on the leaves cannot escape. Therefore, any trait that prevents predatory mite eggs from encountering caterpillars should confer a selective advantage to the mites. Since some predatory mite species exhibit the ability to detect their prey spider mite traces 17 – 19 , we hypothesized that egg-laying female predatory mites may avoid caterpillar traces, which signal ongoing caterpillar activity. Here, we provide the first report of caterpillars predating the eggs of predatory mites and predator avoiding traces of herbivorous caterpillars. Materials and Methods All the materials followed relevant institutional and national guidelines and legislation. Predatory mites We collected E. sojaensis individuals from kudzu vines, Pueraria lobata (Willd) Ohwi, in 2011 in Kyoto and reared them on tea pollen on expanded primary leaves of the kidney bean, Phaseolus vulgaris L. The leaf was pressed onto water-saturated cotton wool in Petri dishes (90 mm diameter, 14 mm deep; hereafter referred to as ‘leaf disc’). Water-saturated cotton wool served as a barrier to prevent mites from escaping. The leaf discs were placed in transparent plastic containers. The dishes were placed in transparent plastic containers and reared at a constant temperature of 25°C, 50% r.h., and L16:D8 photoperiod (hereafter described as “laboratory conditions”). The N. womersleyi individuals were collected from bushkiller plants (C. japonica) in 2015 in Kyoto and maintained on leaf discs, which were heavily infested with Tetracnychus urticae as prey (30–50 female adults and individuals of other stages per leaf). Herbivorous caterpillars We used caterpillars of four lepidopteran species T. japonica, T. oldenlandiae, P. xuthus and Bombyx mori L. We collected eggs and larvae of T. japonica and T. oldenlandiae from C. japonica in 2022 in Kyoto, Japan, and reared them on C. japonica leaves until pupation. We collected eggs and larvae of P. xuthus from Poncirus trifoliata L. in 2022 in Kyoto, Japan, and reared them on Citrus unshiu Markovich leaves until pupation. We obtained commercial populations of the B. mori w1-pnd strain. We reared B. mori larvae on an artificial diet produced at the Kyoto Institute of Technology. We reared caterpillars of T. japonica, T. oldenlandiae and P. xuthus in 900 mL transparent plastic cups and caterpillars of B. mori in transparent plastic containers (140 × 220 × 35 mm). All caterpillars were maintained under laboratory conditions. Plants In the following tests, we used expanded leaflets of C. japonica for both hornworm species, and expanded primary leaves of P. vulgaris for P. xuthus and B. mori. Although the latter two caterpillars do not feed on P. vulgaris, we used the plant because the following procedures were difficult by using their original food plants, i.e. rutaceous and mulberry leaves. Cayratia japonica is a common perennial vine that reproduces via both seed production and vegetatively. We collected C. japonica leaves on the campus of Kyoto University. Do caterpillars consume predatory mite eggs together with food leaves? To examine whether caterpillars consume predatory mite eggs together with food leaves, we cut 20 × 20 mm leaf squares from C. japonica leaflets and placed them on water-saturated cotton. We then introduced two mated adult females of E. sojaensis or N. womersleyi onto each leaf square, and maintained the setup under laboratory conditions. After 24 h, when the mites had laid an egg on the leaf square, we introduced the leaf square and a fifth instar hornworm (T. japonica or T. oldenlandiae) in a 200 mL transparent plastic cup. The caterpillars had previously been starved by isolation for more than 3 h. We repeated the procedure 50 times using 13 T. japonica individuals and 65 times using 11 T. oldenlandiae individuals, respectively. Each leaf square and predatory mites were used only once. Oviposition avoidance of predatory mites against caterpillar traces (dup: abstract ?) To examine whether predatory mites (E. sojaensis and N. womersleyi) avoid laying eggs on plant surfaces bearing caterpillar traces, we conducted dual-choice tests using paired adjacent leaf squares with and without caterpillar traces. We did not use whole plants because it was practically difficult to induce caterpillar traces thoroughly on whole plants. For two hornworm species (T. japonica and T. oldenlandiae), we cut a 10 × 20 mm leaf piece from a C. japonica leaf and then cut the piece into two equal squares (10 × 10 mm). To introduce caterpillar traces to one square, we arranged them on a separate piece of paper towel on water-saturated cotton. This procedure was necessary because the caterpillars used were larger than individual leaf squares. Then we placed a final instar caterpillar on the squares and induced the caterpillar to walk across every leaf square three times (Fig. 1 ). We carefully removed all caterpillar-produced silk threads from the squares. Within 30 min, we arranged the square (trace +) to touch against the other square (trace −) on water-saturated cotton in a Petri dish. Subsequently, a 2- to 4-day-old mated adult female of E. sojaensis or N. womersleyi was introduced onto a pointed piece of Parafilm in contact with both leaf edges using a fine brush (Fig. 1 ). We recorded the number of eggs laid on each leaf square at 24 h after its introduction. Each female mite and pair of leaf squares were used only once. There were 31 replicates using traces of T. japonica and 18 of T. oldenlandiae for E. sojaensis, as well as 27 and 14, respectively, for N. womersleyi. The numbers of eggs on each leaf square were compared in pairs using the Wilcoxon signed-rank test (SAS 9.22 software, SAS Institute Inc., Cary, NC, USA). For other two caterpillar species (P. xuthus and B. mori), we used fully expanded primary kidney bean leaves for substrates. We prepared bean leaf squares with caterpillar traces in the same manner described above. Then we arranged the square (trace +) to lie in close proximity to the control square (trace −), and compared the avoidance response of predatory mite females in the same manner described above. Results Do caterpillars consume predatory mite eggs together with food leaves? All predatory mite eggs observed were consumed together with C. japonica leaves by both hornworms, indicating that hornworm caterpillars do not care at all for consuming predatory mite eggs. On the other hand, all predatory mite adults escaped safely from leaves either by stepping down from the leaves or by traveling over the body surface of the hornworms (Fig. 2 ). Oviposition avoidance of predatory mites against caterpillar traces Significantly fewer E. sojaensis and N. womersleyi females laid eggs onto C. japonica leaf squares with traces of both hornworm species (T. oldenlandiae and T. japonica), indicating that both predatory mites avoided laying eggs on leaves with hornworm traces (Fig. 3 ). Similarly, significantly fewer E. sojaensis and N. womersleyi females laid eggs onto bean leaf squares with traces of B. mori or P. xuthus, indicating that both predatory mites also avoided laying eggs on leaves with traces of these caterpillars (Fig. 3 ). Discussion Herbivorous hawkmoth larvae incidentally fed on the eggs of predatory mites, but the mites never prey on huge hawkmoth larvae, so a trophic level reversal has occurred here. For convenience, we shall refer to predation of the higher trophic levels by the lower trophic levels as ‘revolutionary predation.’ All eggs of both predatory mite species were eaten by both tested hawkmoth species along with food leaves, suggesting that predatory mites have no means of protecting their eggs from revolutionary predation by the approaching caterpillars. Encountering a final instar of hawkmoth, which consumes ca. 20 leaflets of C. japonica per day, 1 would pose a major threat to the predatory mites. Both predatory mite species avoided laying eggs on plant leaves with traces of all tested caterpillar species. This avoidance will reduce the likelihood of revolutionary predation by voracious caterpillars that may be nearby. This study is the first report to find that predators have strategies to avoid revolutionary predation by herbivores. Less mobile larvae of predatory mites are vulnerable to intraguild predation and cannibalism 20 – 22 ; therefore, they can prevent such predation by taking refuge in leaf structures (e.g., domatia, pubescence), and spider mite webs 23 – 27 . Moreover, female adults protect their eggs from such predation by avoiding oviposition in patches shared with such intraguild predators, and by watching over unhatched eggs 28 , 29 . However, these predation avoidance strategies are likely meaningless against voracious caterpillars, which can consume an entire leaf within 10 minutes. We previously reported that spider mites as small as the predatory mites avoid the chemical components of these caterpillars’ traces 1 . In this study, all silk threads produced by the caterpillars were removed prior to testing, so it is likely that the trace effects of the caterpillars are not mediated by silk threads but by chemicals common to many caterpillar traces, which will need to be verified in the future. The importance of incidental predation due to predator-prey size differences has been largely ignored in ecological studies 2 , but may be one of the major forces influencing habitat use by tiny animals regardless of their trophic levels. The avoidance of caterpillar traces by both spider mites 1 and predatory mites may reflect the same need that led to the same solution of trace avoidance. In an applied context, it is not necessarily a disadvantage that biological control agents for spider mites (predatory mite) avoid caterpillar traces as spider mites do. A previous study has shown that when UV-B irradiation and predatory mites are used together for spider mite control in a greenhouse, a synergistic effect is created because both spider mites and predatory mites avoid UV-B irradiation, making it easier for them to encounter in places on plants where UV-B irradiation cannot reach 30 . Similarly, if there are caterpillar traces or their chemical components on the plant, spider mites and their biological control agents that avoid them are more likely to be encountered in places free of traces and their chemicals. Declarations Competing Interests: The authors declare no competing interests. Funding Japan Society for the Promotion of Science KAKENHI, JP24KJ1495 and JP25K09122. Author Contribution S.K. and S.Y. conceived and designed experiments. S.K. conducted experiments and analyzed data. S.K. and S.Y. wrote the manuscript. Acknowledgement We would like to express my deepest appreciation to Drs. T. Akino and Dr. S. Nagaoka of Kyoto Institute of Technology for generously providing me with the caterpillars and the artificial diet. We also thank Dr. N. Hinomoto of Kyoto university who gave me valuable advice and encouragement. This work was supported by JSPS KAKENHI Grant Numbers JP24KJ1495 and JP25K09122. Data Availability Data is provided within the manuscript or supplementary information file. References Kinto, S., Akino, T. & Yano, S. Spider mites avoid caterpillar traces to prevent intraguild predation. Sci. Rep. 13 , 1841 (2023). Gish, M., Dafni, A. & Inbar, M. Mammalian herbivore breath alerts aphids to flee host plant. Curr. Biol. 20 , R628–R629 (2010). Saitō, Y. Prey kills predator: Counter-attack success of a spider mite against its specific phytoseiid predator. Exp. Appl. Acarol . 2 , 47–62 (1986). Ono, M., Igarashi, T., Ohno, E. & Sasaki, M. Unusual thermal defence by a honeybee against mass attack by hornets. Nature 377 , 334–336 (1995). Choh, Y., Ignacio, M., Sabelis, M. 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Entomol. 36 , 435–441 (2011). Ferreira, J. M., Eshuis, B., Janssen, A. & Sabelis, M. W. Domatia reduce larval cannibalism in predatory mites. Ecol. Entomol. 33 , 374–379 (2008). Prado, J., Witte, A. R., Frank, S. & Sadof, C. S. Do leaf domatia mediate intraguild predation and host plant resistance to Oligonychus aceris (Shimer) on Red Sunset Maple (Acer rubrum)? Biol. Control 90 , 187–192 (2015). Roda, A., Nyrop, J., Dicke, M. & English-Loeb, G. Trichomes and spider-mite webbing protect predatory mite eggs from intraguild predation. Oecologia 125 , 428–435 (2000). Seelmann, L., Auer, A., Hoffmann, D. & Schausberger, P. Leaf pubescence mediates intraguild predation between predatory mites. Oikos 116 , 807–817 (2007). Walzer, A. & Schausberger, P. Integration of multiple intraguild predator cues for oviposition decisions by a predatory mite. Anim. Behav. 84 , 1411–1417 (2012). Saitoh, F., Janssen, A. & Choh, Y. Predatory mites protect own eggs against predators. Entomol. Exp. Appl. 169 , 501–507 (2021). Tanaka, M., Yase, J., Kanto, T. & Osakabe, M. Combined nighttime ultraviolet B irradiation and phytoseiid mite application provide optimal control of the spider mite on greenhouse strawberry plants. Pest Manag Sci. 80 , 698–707 (2024). Additional Declarations No competing interests reported. Supplementary Files KintoSupplementaryinformationfile.xlsx 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-6643361","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":463450625,"identity":"320b3bdd-aaeb-48ee-9270-12b49fd603d3","order_by":0,"name":"Shiori 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13:32:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18623,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of \u003cem\u003eC. japonica\u003c/em\u003e leaves eaten up by \u003cem\u003eT. japonica\u003c/em\u003e or \u003cem\u003eT. oldenlandiae. \u003c/em\u003eThe numbers of leaf squares eaten up by hawkmoth caterpillars are provided in each bar.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6643361/v1/70032dfa6f184e29b96f1243.png"},{"id":83615406,"identity":"f7b94817-ecbe-45fd-8512-2344f0d43e05","added_by":"auto","created_at":"2025-05-29 13:32:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31964,"visible":true,"origin":"","legend":"\u003cp\u003ePredatory mite avoidance of traces left by caterpillars: (a) T. japonica, (b) T. oldenlandiae, (c) B. moriand (d) P. xuthus.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6643361/v1/8f2850ba0649a5b0f5e59cd3.png"},{"id":88105688,"identity":"c0efa90a-6bc9-4fb0-9bdb-94c176c7196a","added_by":"auto","created_at":"2025-08-01 12:23:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":597267,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6643361/v1/d0b6bacf-0ddb-416a-85f4-626dce4fca44.pdf"},{"id":83615409,"identity":"3c79f2a3-d86d-472a-ba07-9e166a40424d","added_by":"auto","created_at":"2025-05-29 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Tiny herbivores experience IGP because they cannot immediately evacuate while large herbivores feed on leaves, and likewise, tiny predators that prey upon them are also likely to become involved in such predation. Therefore, the ability to avoid such incidental predation should also confer a selective advantage to tiny predators. It has been reported that some predators can be killed by counterattacks by herbivores\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and hence some of these predators have strategies to avoid it\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, there are no reports on predators avoiding one-sided \u0026lsquo;attacks\u0026rsquo; by large herbivores.\u003c/p\u003e \u003cp\u003eThe predatory mites Neoseiulus womersleyi Schicha and Euseius sojaensis Ehara are typically\u0026thinsp;\u0026lt;\u0026thinsp;0.4 mm in size. The former is a specialist predator which prey only upon spider mites and the latter is a generalist predator which feed on spider mites as well as non-prey resources such as pollen and pearl bodies\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These mites lay eggs on leaf hairs and in domatia of plant leaves infested by spider mites\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLepidopteran larvae (hereafter \u0026lsquo;caterpillars\u0026rsquo;) such as Theretra oldenlandiae Fabricius and Papilio xuthus L. share food plants with spider mite that are preyed upon by predatory mites\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These caterpillars grow to 30\u0026ndash;100 mm, and consume food leaves along with the spider mites\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. For example, a final instar T. oldenlandiae consumes ca. 20 leaflets of Cayratia japonica (Thunb.) Gagnep. per day\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdult predatory mites can run fast and can escape quickly when a leaf-eating caterpillar approaches, while their eggs laid on the leaves cannot escape. Therefore, any trait that prevents predatory mite eggs from encountering caterpillars should confer a selective advantage to the mites. Since some predatory mite species exhibit the ability to detect their prey spider mite traces\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, we hypothesized that egg-laying female predatory mites may avoid caterpillar traces, which signal ongoing caterpillar activity. Here, we provide the first report of caterpillars predating the eggs of predatory mites and predator avoiding traces of herbivorous caterpillars.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAll the materials followed relevant institutional and national guidelines and legislation.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePredatory mites\u003c/h2\u003e \u003cp\u003eWe collected E. sojaensis individuals from kudzu vines, Pueraria lobata (Willd) Ohwi, in 2011 in Kyoto and reared them on tea pollen on expanded primary leaves of the kidney bean, Phaseolus vulgaris L. The leaf was pressed onto water-saturated cotton wool in Petri dishes (90 mm diameter, 14 mm deep; hereafter referred to as \u0026lsquo;leaf disc\u0026rsquo;). Water-saturated cotton wool served as a barrier to prevent mites from escaping. The leaf discs were placed in transparent plastic containers. The dishes were placed in transparent plastic containers and reared at a constant temperature of 25\u0026deg;C, 50% r.h., and L16:D8 photoperiod (hereafter described as \u0026ldquo;laboratory conditions\u0026rdquo;).\u003c/p\u003e \u003cp\u003eThe N. womersleyi individuals were collected from bushkiller plants (C. japonica) in 2015 in Kyoto and maintained on leaf discs, which were heavily infested with Tetracnychus urticae as prey (30\u0026ndash;50 female adults and individuals of other stages per leaf).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHerbivorous caterpillars\u003c/h3\u003e\n\u003cp\u003e \u003cstrong\u003eWe used caterpillars of four lepidopteran species\u003c/strong\u003e \u003cp\u003eT. japonica, T. oldenlandiae, P. xuthus and Bombyx mori L. We collected eggs and larvae of T. japonica and T. oldenlandiae from C. japonica in 2022 in Kyoto, Japan, and reared them on C. japonica leaves until pupation. We collected eggs and larvae of P. xuthus from Poncirus trifoliata L. in 2022 in Kyoto, Japan, and reared them on Citrus unshiu Markovich leaves until pupation. We obtained commercial populations of the B. mori w1-pnd strain. We reared B. mori larvae on an artificial diet produced at the Kyoto Institute of Technology.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eWe reared caterpillars of T. japonica, T. oldenlandiae and P. xuthus in 900 mL transparent plastic cups and caterpillars of B. mori in transparent plastic containers (140 \u0026times; 220 \u0026times; 35 mm). All caterpillars were maintained under laboratory conditions.\u003c/p\u003e\n\u003ch3\u003ePlants\u003c/h3\u003e\n\u003cp\u003eIn the following tests, we used expanded leaflets of C. japonica for both hornworm species, and expanded primary leaves of P. vulgaris for P. xuthus and B. mori. Although the latter two caterpillars do not feed on P. vulgaris, we used the plant because the following procedures were difficult by using their original food plants, i.e. rutaceous and mulberry leaves. Cayratia japonica is a common perennial vine that reproduces via both seed production and vegetatively. We collected C. japonica leaves on the campus of Kyoto University.\u003c/p\u003e\n\u003ch3\u003eDo caterpillars consume predatory mite eggs together with food leaves?\u003c/h3\u003e\n\u003cp\u003eTo examine whether caterpillars consume predatory mite eggs together with food leaves, we cut 20 \u0026times; 20 mm leaf squares from C. japonica leaflets and placed them on water-saturated cotton. We then introduced two mated adult females of E. sojaensis or N. womersleyi onto each leaf square, and maintained the setup under laboratory conditions. After 24 h, when the mites had laid an egg on the leaf square, we introduced the leaf square and a fifth instar hornworm (T. japonica or T. oldenlandiae) in a 200 mL transparent plastic cup. The caterpillars had previously been starved by isolation for more than 3 h. We repeated the procedure 50 times using 13 T. japonica individuals and 65 times using 11 T. oldenlandiae individuals, respectively. Each leaf square and predatory mites were used only once.\u003c/p\u003e\n\u003ch3\u003eOviposition avoidance of predatory mites against caterpillar traces (dup: abstract ?)\u003c/h3\u003e\n\u003cp\u003eTo examine whether predatory mites (E. sojaensis and N. womersleyi) avoid laying eggs on plant surfaces bearing caterpillar traces, we conducted dual-choice tests using paired adjacent leaf squares with and without caterpillar traces. We did not use whole plants because it was practically difficult to induce caterpillar traces thoroughly on whole plants. For two hornworm species (T. japonica and T. oldenlandiae), we cut a 10 \u0026times; 20 mm leaf piece from a C. japonica leaf and then cut the piece into two equal squares (10 \u0026times; 10 mm). To introduce caterpillar traces to one square, we arranged them on a separate piece of paper towel on water-saturated cotton. This procedure was necessary because the caterpillars used were larger than individual leaf squares. Then we placed a final instar caterpillar on the squares and induced the caterpillar to walk across every leaf square three times (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We carefully removed all caterpillar-produced silk threads from the squares. Within 30 min, we arranged the square (trace +) to touch against the other square (trace \u0026minus;) on water-saturated cotton in a Petri dish. Subsequently, a 2- to 4-day-old mated adult female of E. sojaensis or N. womersleyi was introduced onto a pointed piece of Parafilm in contact with both leaf edges using a fine brush (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We recorded the number of eggs laid on each leaf square at 24 h after its introduction. Each female mite and pair of leaf squares were used only once. There were 31 replicates using traces of T. japonica and 18 of T. oldenlandiae for E. sojaensis, as well as 27 and 14, respectively, for N. womersleyi. The numbers of eggs on each leaf square were compared in pairs using the Wilcoxon signed-rank test (SAS 9.22 software, SAS Institute Inc., Cary, NC, USA). For other two caterpillar species (P. xuthus and B. mori), we used fully expanded primary kidney bean leaves for substrates. We prepared bean leaf squares with caterpillar traces in the same manner described above. Then we arranged the square (trace +) to lie in close proximity to the control square (trace \u0026minus;), and compared the avoidance response of predatory mite females in the same manner described above.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDo caterpillars consume predatory mite eggs together with food leaves?\u003c/h2\u003e \u003cp\u003eAll predatory mite eggs observed were consumed together with C. japonica leaves by both hornworms, indicating that hornworm caterpillars do not care at all for consuming predatory mite eggs. On the other hand, all predatory mite adults escaped safely from leaves either by stepping down from the leaves or by traveling over the body surface of the hornworms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOviposition avoidance of predatory mites against caterpillar traces\u003c/h3\u003e\n\u003cp\u003eSignificantly fewer E. sojaensis and N. womersleyi females laid eggs onto C. japonica leaf squares with traces of both hornworm species (T. oldenlandiae and T. japonica), indicating that both predatory mites avoided laying eggs on leaves with hornworm traces (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilarly, significantly fewer E. sojaensis and N. womersleyi females laid eggs onto bean leaf squares with traces of B. mori or P. xuthus, indicating that both predatory mites also avoided laying eggs on leaves with traces of these caterpillars (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHerbivorous hawkmoth larvae incidentally fed on the eggs of predatory mites, but the mites never prey on huge hawkmoth larvae, so a trophic level reversal has occurred here. For convenience, we shall refer to predation of the higher trophic levels by the lower trophic levels as \u0026lsquo;revolutionary predation.\u0026rsquo; All eggs of both predatory mite species were eaten by both tested hawkmoth species along with food leaves, suggesting that predatory mites have no means of protecting their eggs from revolutionary predation by the approaching caterpillars. Encountering a final instar of hawkmoth, which consumes ca. 20 leaflets of C. japonica per day,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e would pose a major threat to the predatory mites. Both predatory mite species avoided laying eggs on plant leaves with traces of all tested caterpillar species. This avoidance will reduce the likelihood of revolutionary predation by voracious caterpillars that may be nearby. This study is the first report to find that predators have strategies to avoid revolutionary predation by herbivores.\u003c/p\u003e \u003cp\u003eLess mobile larvae of predatory mites are vulnerable to intraguild predation and cannibalism\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e; therefore, they can prevent such predation by taking refuge in leaf structures (e.g., domatia, pubescence), and spider mite webs\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Moreover, female adults protect their eggs from such predation by avoiding oviposition in patches shared with such intraguild predators, and by watching over unhatched eggs\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, these predation avoidance strategies are likely meaningless against voracious caterpillars, which can consume an entire leaf within 10 minutes.\u003c/p\u003e \u003cp\u003eWe previously reported that spider mites as small as the predatory mites avoid the chemical components of these caterpillars\u0026rsquo; traces\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In this study, all silk threads produced by the caterpillars were removed prior to testing, so it is likely that the trace effects of the caterpillars are not mediated by silk threads but by chemicals common to many caterpillar traces, which will need to be verified in the future.\u003c/p\u003e \u003cp\u003eThe importance of incidental predation due to predator-prey size differences has been largely ignored in ecological studies\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, but may be one of the major forces influencing habitat use by tiny animals regardless of their trophic levels. The avoidance of caterpillar traces by both spider mites\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and predatory mites may reflect the same need that led to the same solution of trace avoidance.\u003c/p\u003e \u003cp\u003eIn an applied context, it is not necessarily a disadvantage that biological control agents for spider mites (predatory mite) avoid caterpillar traces as spider mites do. A previous study has shown that when UV-B irradiation and predatory mites are used together for spider mite control in a greenhouse, a synergistic effect is created because both spider mites and predatory mites avoid UV-B irradiation, making it easier for them to encounter in places on plants where UV-B irradiation cannot reach\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Similarly, if there are caterpillar traces or their chemical components on the plant, spider mites and their biological control agents that avoid them are more likely to be encountered in places free of traces and their chemicals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eJapan Society for the Promotion of Science KAKENHI, JP24KJ1495 and JP25K09122.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.K. and S.Y. conceived and designed experiments. S.K. conducted experiments and analyzed data. S.K. and S.Y. wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to express my deepest appreciation to Drs. T. Akino and Dr. S. Nagaoka of Kyoto Institute of Technology for generously providing me with the caterpillars and the artificial diet. We also thank Dr. N. Hinomoto of Kyoto university who gave me valuable advice and encouragement. This work was supported by JSPS KAKENHI Grant Numbers JP24KJ1495 and JP25K09122.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKinto, S., Akino, T. \u0026amp; Yano, S. Spider mites avoid caterpillar traces to prevent intraguild predation. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 1841 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGish, M., Dafni, A. \u0026amp; Inbar, M. Mammalian herbivore breath alerts aphids to flee host plant. \u003cem\u003eCurr. 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Combined nighttime ultraviolet B irradiation and phytoseiid mite application provide optimal control of the spider mite on greenhouse strawberry plants. \u003cem\u003ePest Manag Sci.\u003c/em\u003e \u003cb\u003e80\u003c/b\u003e, 698\u0026ndash;707 (2024).\u003c/span\u003e\u003c/li\u003e\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":"revolutionary predation, caterpillar, predatory mite, trace, predation avoidance","lastPublishedDoi":"10.21203/rs.3.rs-6643361/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6643361/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe report the first example of predators having a strategy to avoid \u0026lsquo;revolutionary predation\u0026rsquo; by herbivores, i.e. predation on the higher trophic levels by the lower ones. The predatory mites Neoseiulus womersleyi and Euseius sojaensis are smaller than 0.5 mm and lay eggs on plant leaf surfaces; thus, their immobile eggs would be incidentally consumed along with leaves by voracious lepidopteran caterpillars. We experimentally demonstrated that eggs of both mite species were preyed upon by tested hawkmoth caterpillars (Theretra oldenlandiae, Theretra japonica) along with leaves. Therefore, the ability to avoid such revolutionary predation should confer a selective advantage to mites. We further demonstrated that adult females of both mite species avoided laying eggs on leaves with traces of all tested caterpillars (T. oldenlandiae, T. japonica, Papilio xuthus and Bombyx mori), indicating that eggs may avoid revolutionary predation by voracious caterpillars that may be nearby. This is the first demonstration of a repellent effect of herbivore traces on carnivores. Considering previous studies showing that spider mites as small as predatory mites also avoid caterpillar traces, the same need to avoid predation by huge caterpillars may have led to the development of the same solutions for both spider mites and predatory mites.\u003c/p\u003e","manuscriptTitle":"Tiny predators avoid herbivorous caterpillar traces to prevent revolutionary predation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-29 13:32:53","doi":"10.21203/rs.3.rs-6643361/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":"e1dc60bf-e125-4ba1-9c7c-aaceff109aed","owner":[],"postedDate":"May 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49210970,"name":"Biological sciences/Ecology"},{"id":49210971,"name":"Earth and environmental sciences/Ecology"}],"tags":[],"updatedAt":"2025-08-01T12:23:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-29 13:32:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6643361","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6643361","identity":"rs-6643361","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}