Introduction

Some predators exposed to intraguild predation (IGP) have developed strategies to avoid it1,2,3,4. By contrast, tiny herbivores are sometimes consumed along with plants by much larger herbivores5,6,7, which can be considered incidental IGP. Therefore, the ability to avoid such incidental IGP should confer a selective advantage to tiny herbivores. However, little is known about IGP avoidance by tiny herbivores except for examples of aphids that immediately drop off a plant in response to the breath of a mammalian herbivore5.

The spider mites Tetranychus kanzawai Kishida and Tetranychus urticae Koch are typically < 0.5 mm in size, and feed on a variety of wild and cultivated plant species8,9,10. These mites construct protective webs on host plant leaves and usually live beneath them11,12. Lepidopteran caterpillars such as Papilio xuthus L. and Theretra oldenlandiae Fabricius that share host plants with these mites10,13,14,15 grow to 30–100 mm, and indiscriminately consume spider mite-infested and uninfested leaves6. For example, a final instar T. oldenlandiae consumes approximately 20 Cayratia japonica (Thunb.) Gagnep. leaflets per day (Kinto, personal observation). Even if some mites can successfully escape caterpillar attack, all eggs and quiescent mites along with webs will be lost6. Therefore, any trait that prevents such loss should confer a selective advantage to the mites. Host plant use by T. kanzawai and T. urticae is ultimately determined by adult females that disperse from their webs and found new webs on uninfested leaves14,15,16,17 Moreover, adult females of these mites exhibit the ability to detect predator traces on leaves18,19,20,21. Therefore, we hypothesized that adult female spider mites should avoid caterpillar traces on host plants, which would indicate ongoing caterpillar activity. Here, we provide the first report of spider mite avoidance of caterpillar traces on host plants as well as chemical extracts of these traces.

Materials and methods

All the materials followed relevant institutional and national guidelines and legislation.

Mites

We used a T. kanzawai population collected from trifoliate orange trees (Poncirus trifoliata [L.] Raf.) in 2018 in Kyoto, Japan, and a T. urticae population collected from chrysanthemum plants (Chrysanthemum morifolium Ramat.) in 1998 in Nara, Japan. These populations were reared on adaxial surfaces of kidney bean (Phaseolus vulgaris L.) primary leaves, which were pressed onto water-saturated cotton in Petri dishes (90 mm diameter, 14 mm depth). The water-saturated cotton served as a barrier to prevent mites from escaping. The dishes were maintained at 25 °C, 50% relative humidity, and a 16L:8D photoperiod. All experiments were conducted under these conditions. We only used mated adult females (i.e., the dispersal stage) of T. kanzawai or T. urticae mites.

Caterpillars

We used caterpillars of four lepidopteran species: Bombyx mori L., P. Xuthus, Spodoptera litura Fabricius and T. oldenlandiae. We collected eggs and larvae of T. oldenlandiae from C. japonica in 2021 in Kyoto, Japan, and reared them on C. japonica leaves until pupation. Theretra oldenlandiae shares Vitaceae host plants with T. kanzawai and T. urticae8,15. We collected eggs and larvae of P. xuthus from Ptelea trifoliata in 2021 in Kyoto, Japan, and reared them on Citrus unshiu Markov. leaves until pupation. Papilio. xuthus and T. kanzawai share P. trifoliata as a host plant in Kyoto (Kinto, personal observation).

We obtained commercial populations of the B. mori Kinshu × Showa strain (Ueda-sanshu Co., Ltd, Nagano, Japan) or the w1-pnd strain. We reared B. mori larvae on an artificial diet produced at the Kyoto Institute of Technology. Although T. kanzawai use Morus alba, a food plant for the B. mori strain, the mite and the strain never encounter one another in the wild, because the B. mori strain has been domesticated for hundreds of years.

We obtained a sub-cultured population of S. litura from the Kyoto Institute of Technology. We reared first to fourth instars of S. litura on an artificial diet (Insecta LFM, Nosan Insect Materials, Kanagawa, Japan), while final instars were fed P. vulgaris leaves. Because S. litura feeds on various wild and cultivated plants22,23, it may share some host plants with T. kanzawai and T. urticae, both of which also feed on many host plant species8,9,10.

We reared caterpillars of T. oldenlandiae, P. xuthus, and S. litura in 900 mL transparent plastic cups and caterpillars of B. mori in transparent plastic containers (140 × 220 × 35 mm). All caterpillars were maintained under the same laboratory conditions described above.

Plants

We used several parts of P. vulgaris plants in the following experiments. This species is a preferred food for both mite species16,17 and S. litura24, but the other three caterpillar species do not feed on it (Kinto, personal observation). We thus used P. vulgaris rather than shared host plants, because some caterpillars and mites (T. urticae and P. xuthus, for example) do not share any host plant.

Avoidance of caterpillar traces on leaf surfaces by spider mites

To examine whether spider mites avoid settling on host 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, in practice, it was difficult to induce caterpillar traces on whole plants. We used two spider mite species (T. kanzawai and T. urticae) and four caterpillar species (T. oldenlandiae, P. xuthus, B. mori, and S. litura). We cut a 10 × 20 mm leaf piece from a fully expanded primary kidney bean 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 fourth or final instar caterpillar on the squares and induced the caterpillar to walk across every leaf square three times (Fig. 1a). 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 T. kanzawai or T. urticae was introduced onto a pointed piece of Parafilm in contact with both leaf edges using a fine brush (Fig. 1a). We recorded the leaf square onto which the mite had settled at 2 h after its introduction, as preliminary observations confirmed that all females would settle on a particular leaf within that period. Each female mite and pair of leaf squares were used only once. All tests described below were conducted between 13:00 and 17:00 h, when adult female spider mites actively disperse by walking. There were 14 replicates using traces of T. oldenlandiae, 48 of P. xuthus, 20 of B. mori, and 26 of S. litura for T. kanzawai, as well as 18, 32, 16, and 47, respectively, for T. urticae. Data were subjected to two-tailed binomial tests with the common null hypothesis that a spider mite would settle on the two squares with equal probability (i.e., 0.5).

Figure 1
figure 1

(a) Procedure used to observe avoidance of caterpillar traces by spider mites. (b) Experimental setup used to observe avoidance of B. mori traces on plant stems by T. kanzawai. (c) Experimental setup used to observe avoidance of B. mori trace extracts by T. kanzawai.

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Duration of B. mori trace avoidance by T. kanzawai

To examine whether the effects of caterpillar traces on spider mite avoidance decline over time, we used T. kanzawai mites and B. mori caterpillars. We used B. mori because populations can be easily maintained over many generations. We prepared bean leaf squares with B. mori traces in the same manner descried above and preserved the traced square on water-saturated cotton for 0 h (n = 30), 24 h (n = 29), 48 h (n = 28), or 72 h (n = 28). Then we arranged the square (trace +) to lie in close proximity to the control square (trace −) that had been preserved for the same periods of time. Then we compared the avoidance response of T. kanzawai females in the same manner described above.

Avoidance of B. mori traces on plant stems by T. kanzawai

To examine whether T. kanzawai females avoid walking along plant stems bearing caterpillar traces, we used Y-shaped kidney bean stems (Fig. 1b). We cut symmetric bean plants ca. 15 days after sowing from their base and inserted them perpendicularly into a 5 mL glass bottle filled with water and wet cotton. To induce caterpillar traces on one branch of the stem, we allowed a silkworm to crawl from the branching point to the far end of one branch three times for each stem (n = 20). Then we introduced a T. kanzawai adult female at a release point 35 mm below the branch point (Fig. 1b). We recorded the branch along which the female walked to the far end. Each female mite and each Y-shaped stem were used only once. The numbers of females were compared using binomial tests in the same manner described above.

Avoidance of B. mori trace extracts by T. kanzawai

To extract chemical traces of caterpillar, we introduced 10 third instar B. mori to a glass Petri dish (120 mm diameter, 60 mm depth). After 1 h, we removed all caterpillars and washed the inside bottom of the dish with 1.0 mL acetone. We replicated the procedure twice using different individuals to combine all extracts and to acquire enough extract for the following experiment.

To examine avoidance of B. mori trace extracts by T. kanzawai females, we conducted dual-choice experiments using T-shaped pathways of filter paper (35 × 35 mm; width, 2 mm; Fig. 1c). Using disposable micropipettes (Drummond Scientific Co., PA, USA), 1.75 caterpillar equivalents (i.e., 60 µL) of acetone extract were applied to an alternately selected branch (17.5 mm long) of each pathway (i.e., 0.10 caterpillar equivalent/mm), with control acetone applied to the other branch. We applied each solution dropwise at the junction point to minimize mixing. After evaporating the solvent from those pathways, we perpendicularly suspended them (Fig. 1c) and introduced an adult female mite at 2 days post-maturation onto the bottom of each pathway using a fine brush and recorded the branch along which the female first walked to the far end. Each female mite and each T-shaped filter paper were used only once, with 19 replicates. Each female mite made a choice within 10 min. The avoidance response of T. kanzawai was analysed in the same manner described above.

Indirect effects of B. mori traces on T. kanzawai via plants

To determine whether B. mori traces on plants indirectly affect the performance of T. kanzawai on plants, we introduced 70–80 randomly selected quiescent female deutonymphs of T. kanzawai onto kidney bean leaf disks. Immediately after synchronized adult emergence, we introduced the same number of adult males to allow mating; the detailed procedure is described elsewhere25. After 24 h, we transferred the females singly onto 10 × 10 mm bean leaf squares with or without B. mori traces prepared as described above. Because the number of eggs laid within a certain period is considered the most sensitive performance index of spider mite females26,27, any plant-mediated indirect interaction, such as defence induction in response to caterpillar traces, should result in lower egg numbers laid by the test females. We counted the eggs laid on the leaf squares 24 h after their introduction. One female that laid no eggs during the 24 h period (n = 1, trace +) was excluded from the analysis. We obtained 33 and 36 replicates for the trail+ and trail– conditions, respectively. We compared the numbers of eggs laid on leaves with and without B. mori traces using a generalized linear model with a Poisson error distribution using the SAS 9.22 software (SAS Institute Inc., Cary, NC, USA).

Ethics

This article does not contain any studies with human participants or animals.

Results

Avoidance of caterpillar traces on leaf surfaces by spider mites

Significantly fewer T. kanzawai and T. urticae females settled onto bean leaf squares with traces of all tested caterpillars (T. oldenlandiae, P. xuthus, B. mori, and S. litura), indicating that both spider mites avoided traces of these caterpillars (Fig. 2).

Figure 2
figure 2

Avoidance of caterpillar traces by (a) T. kanzawai and (b) T. urticae. The numbers of females settled onto each leaf square are provided in each bar.

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Duration of B. mori trace avoidance by T. kanzawai

Tetranychus kanzawai females significantly avoided B. mori traces that were aged 0 h (26:4, binomial test P = 0.0001), 24 h (26:3, binomial test P < 0.0001), and 48 h (20:8, binomial test P = 0.0357), but not 72 h (19:9, binomial test P = 0.0872, Fig. 3a). These results suggest that avoidance lasted for more than 48 h but less than 72 h.

Figure 3
figure 3

(a) Duration of caterpillar trace avoidance by T. kanzawai. (b) Avoidance of B. mori traces on plant stems by T. kanzawai. (c) Avoidance of B. mori trace extracts by T. kanzawai.

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Avoidance of B. mori traces on plant stems by T. kanzawai

Significantly fewer T. kanzawai females walked along branches bearing B. mori traces (16:4, binomial test P = 0.0118, Fig. 3b), indicating that mites avoided walking along caterpillar traces on plant stems.

Avoidance of B. mori trace extracts by T. kanzawai

Significantly fewer T. kanzawai females walked along the branch to which B. mori trace extracts had been applied (18:1, binomial test P = 0.0001, Fig. 3c), indicating that mites avoided caterpillar trace extracts.

Indirect effects of B. mori traces on T. kanzawai via plants

We found no significant difference in the number of eggs (mean ± standard error [SE]) laid by T. kanzawai on leaf pieces with (9.273 ± 0.449, n = 33) and without (9.778 ± 0.378, n = 36) B. mori traces (P = 0.4972, GLM), suggesting that any indirect effects of B. mori traces on T. kanzawai performance on plants were negligible (see Supplementary Data 2).

Discussion

Both spider mite species avoided traces of all tested caterpillar species. As all silk threads produced by caterpillars were removed before the tests, the caterpillar trace effect seems to be mediated by chemical(s) rather than silk. Although these spider mites avoid predatory mite traces18,19,20, this study is the first to report the avoidance of herbivore traces by spider mites. Spider mites prevent attacks by generalist predators using their protective webs28,29,30,31. They also prevent attacks by specialist predators that penetrate the webs by dispersing from invaded patches18,19,32,33 or by seeking refuge and laying eggs on webs that cannot be easily accessed by the predators34,35,36,37,38. However, spider mites would be defenceless against large caterpillars that incidentally, rather than intentionally, consume spider mites along with host plant leaves6. For example, Phytoseiulus persimilis Athias-Henriot, the most voracious predatory mite species, consumes ca. 20 spider mite eggs per day39, whereas final instar larvae of T. oldenlandiae consume tens to hundreds of spider mite eggs on C. japonica leaves within as little as 10 min (Kinto, personal observation). Therefore, caterpillars would be more harmful as intraguild predators than as real predators of spider mites under some conditions. As the performance index of T. kanzawai females did not differ according to the presence or absence of B. mori traces on leaves, the avoidance of B. mori traces by T. kanzawai females may be promoted by factors other than indirect effects of B. mori traces via plants. Thus, we suggest that the avoidance of herbivore traces is driven by the prevention of incidental IGP by voracious caterpillars. Caterpillar trace avoidance would prevent spider mites from encountering caterpillars at the cost of abandoning available plant resources. Such repellence appeared to decline over time, but did last for a few days, suggesting that spider mites can avoid encountering caterpillars that likely remain in the vicinity. We did not use whole plants in the experiments described above. However, the data on avoidance of stem caterpillar traces by mites (Figs. 1b and 3b) are robust. The aboveground parts of many plants consist of leaves and stems, and all leaves are connected by stems, along which mites ambulate to access food leaves. It is thus logical to extrapolate spider mite avoidance of caterpillar traces on such stems to avoidance on entire plants.

Interestingly, spider mites avoided not only traces of caterpillars that they could potentially encounter in the wild, but also those of caterpillars that they never encounter in the wild. For example, T. urticae and P. xuthus do not share any host plants, and the domesticated B. mori strain is not distributed in the wild. Because spider mites are extremely polyphagous and may therefore encounter many caterpillar species on host plants, it is likely that spider mites avoid substances that are commonly contained in the traces of many caterpillar species.

Tetranychus kanzawai avoided caterpillar traces on both food plant leaves and plant stems. Because most plant leaves are hierarchically connected by stems, spider mite females that avoid walking along caterpillar traces on a plant stem must abandon all food leaves on that stem, which in turn reduces available food resources for their offspring. Although both T. kanzawai and T. urticae exploit a wide range of host plants9,10, available food resources for spider mites may be largely limited by the ongoing traces of a great number of caterpillars as well as by many predators. The need to avoid IGP and caterpillar traces may be shared by leaf-mining or sessile herbivores. By examining these possibilities, we may be able to partially answer the long-standing question of why only a portion of available resources are used by herbivores40,41.

Tetranychus kanzawai also avoided acetone extracts of B. mori traces, indicating that spider mites avoid chemical components of caterpillar traces. This study provides the first demonstration of a repellent effect of herbivore trace chemicals on heterospecific herbivores. Spider mites use sensory receptors on the forelegs and mouthparts to locate feeding sites42. They also detect chemical cues left by predators such as predatory mites and ants18,19,20,21, suggesting that they may detect such cues left by caterpillars. Although spider mites have developed resistance against many synthetic pesticides43,44, it may be possible to manufacture spider mite repellents that simulate caterpillar traces, i.e., natural compounds that are seemingly harmless to humans. Such chemicals, which appear to be effective for a few days, may be valuable for repelling spider mites from agricultural crops. As all experiments were conducted in the laboratory, it is essential to conduct field experiments that confirm the effects of caterpillar traces and to identify the chemicals that mediate avoidance.