The avoidance of conspecific cues during egg-laying decision-making in the Asian long-horned beetle Anoplophora glabripennis

Abstract

For insects whose larvae are incapable of moving between food resources, the selection of oviposition sites by females is critical to the survival and development of their offspring. In such insects, it is known that females utilise and benefit from conspecific cues for oviposition choice. Studying how information from the behaviour of conspecifics affects egg-laying decision-making is crucial for understanding the biology of insects, which can lead to novel strategies for pest management. We focused on the reproductive behaviour of the Asian long-horned beetle Anoplophora glabripennis, which has become an invasive pest species throughout the world. Here, we show that A. glabripennis avoids sites already containing conspecific cues during egg-laying decision-making. The field survey measuring the distance between neighbouring oviposition scars (in this species, females make scars through the bark of host branches for laying eggs) suggested that the selection of oviposition sites by females is not random. In laboratory oviposition-choice bioassays, females made less oviposition scars on branches containing scars made by other females than those without scars. In addition, female oviposition was also deterred by the presence of their own oviposition scars. Our results indicate that avoiding sites containing conspecific (and their own) traces realises fitness benefit such as reducing resource competition among larvae. This study provides insights into the reproductive behaviour of this invasive longhorn beetle, which is useful for developing environmentally friendly control methods such as oviposition deterrents.

Introduction

Egg-laying decision-making by females is critical to the survival of offspring for insects whose larvae have little or no possibility to move from their initial food resources, e.g. seeds, fruits, and trees (Anbutsu and Togashi, 1997a; Deas and Hunter, 2012; Mitchell, 1975). In many insect species, females utilise and benefit from conspecific cues for oviposition choice (Prokopy and Roitberg, 2001). By joining conspecifics and engaging in group oviposition, some insects enjoy fitness benefits such as protection from natural enemies and overcoming a plant’s defensive response (Desurmont and Weston, 2011; Fletcher and Miller, 2008; Raitanen et al., 2014). Conversely, for some insects, avoiding conspecifics and engaging in spaced oviposition also realises fitness benefits such as reducing resource competition (Anbutsu and Togashi, 1996; Kiflawi et al., 2003; Zhang et al., 2019). Studying how information from the behaviour of conspecifics affects egg-laying decision-making is crucial for understanding the biology of insects, which can lead to novel strategies for pest management. However, at present, such interindividual interactions are poorly studied aspects of the research on pest science.

Invasive longhorn beetles are significant threats, causing economic and ecological damage through tree mortality in forests, agricultural lands, and urban areas. The Asian long-horned beetle Anoplophora glabripennis (Coleoptera: Cerambycidae) (fig. 1a), which is native to China and the Korean Peninsula, has become a high profile invasive pest species in North America, Europe, and Japan (Haack et al., 2010; Hu et al., 2009; Wang et al., 2023). This species uses various broadleaf trees such as genera Acer, Populus, Salix, and Ulmus as its hosts and kills them through mass infestation (Sjöman et al., 2014; Straw et al., 2015; van der Gaag and Loomans, 2014; Yasui et al., 2024). Eggs are laid within ‘oviposition scars’ made by females through the bark of host branches (fig. 1b,c), and larvae tunnel into the woody tissues, causing physical damage through their feeding activities (Faccoli et al., 2015; Keena and Sánchez, 2018; Li et al., 2024). In the United States, for example, potential damage to urban sugar maples was valued at up to approximately $669 billion (Nowak et al., 2001). To develop management strategies for A. glabripennis, it is necessary to understand their biological characteristics, including reproductive behaviour and egg-laying patterns.

Figure 1. The Asian long-horned beetle Anoplophora glabripennis and its reproductive behaviours. (a) A pair in copula, where the male mounts on the female back. (b) A female chewing an oviposition scar through the bark. (c) An oviposition scar (enclosed by a white dashed line) on the Chinese elm Ulmus parvifolia. A single egg is laid just under the bark in the cambium. (Online version in colour.)

In this study, we investigated the effects of information from the behaviour of conspecifics (specifically, whether conspecifics have already oviposited in the site) on egg-laying decision-making in A. glabripennis. Considering the observations of intraspecific predation in longhorn beetle larvae (Dodds et al., 2001; Ware and Stephen, 2006), we hypothesised that females of this species avoid sites already containing conspecific cues. First, we observed the oviposition pattern in the field, by measuring the distance between neighbouring oviposition scars. Because the field survey suggested that selection of oviposition sites by females is not random, we then performed laboratory bioassays to examine the response of females to conspecific (and their own) oviposition scars.

Materials and methods

Sample collection

Trees infested by A. glabripennis were obtained at three locations in the Kanto region, Japan, during 2023. Logs of the goat willow Salix caprea (Malpighiales: Salicaceae) were collected in Sakuragawa-shi, Ibaraki (29 logs from one tree). Logs of the katsura tree Cercidiphyllum japonicum (Saxifragales: Cercidiphyllaceae) were collected in Kasama-shi, Ibaraki (9 logs from one tree) and Hama-cho, Chuo-ku, Tokyo (5 logs from one tree). All logs were placed in outdoor cages at the Forestry and Forest Products Research Institute in Tsukuba-shi, Ibaraki. To collect emerged adults, we monitored the cages between late May and early August in 2024. The beetles were individually maintained in plastic cups (∼130 mm in diameter and 100 mm in height) under 14L:10D at 25°C in the laboratory and fed fresh twigs with leaves of the Chinese elm Ulmus parvifolia (Urticales: Ulmaceae). The sex of the adult was distinguished by the morphology (males have longer antennae relative to their body and are generally smaller than females) following a previous study (Meng et al., 2015). To avoid the decline in fecundity with age, all individuals were used for the experiments within 30 days of collection, referring to past research that performed similar oviposition assays (Keena and Sánchez, 2018; Smith et al., 2002). All animal care procedures were authorised by the Ministry of the Environment of Japan (registered under number 24000004), and the experiments described here complied with relevant institutional, national, and international guidelines and legislation.

Oviposition pattern in the field

To investigate whether the presence of conspecific cues affects the egg-laying decision-making by females, we observed oviposition patterns in the field by focusing on the distance between neighbouring oviposition scars (in this species, females make scars through the bark of host branches for laying eggs). We measured the distance from a given oviposition scar to the nearest scar using a tape measure. This was performed on all oviposition scars found on the surface of the obtained logs after the emergence of adults: 29 logs of S. caprea measuring 49.186 ± 1.360 cm (mean ± SE) in length and 14 logs of C. japonicum measuring 63.193 ± 6.909 cm (mean ± SE) in length. Oviposition scars were easily distinguished from mere chew marks not containing room for eggs or bark cracks due to the deposition of larval frass. If the distance between neighbouring oviposition scars does not follow a normal distribution, even with a sufficient sample size, we can expect that the selection of oviposition sites by females is not random. Here, we did not use typical analysis for determining spatial patterns (e.g. uniform distribution, clumped distribution or Poisson distribution) in ecological studies because the obtained logs were only a part of each infested tree.

Oviposition-choice bioassays

Two behavioural assays were conducted to examine how the presence of exisiting oviposition scars affects the egg-laying decision-making by females. In the first experiment, a female was given a choice between a control branch and a branch containing oviposition scars from a different female. As egg-laying substrates, fresh branches of U. parvifolia (150 mm in length) were used. Because this species shows a preference for branch diameter when laying eggs (Huang et al., 2024), we confirmed there were no significant differences in the branch diameters between the treatments (see the Results section). The branches used in each replication were contiguous from the same tree to provide similar bark thickness, phloem quality, and moisture. To obtain the treated branches (i.e. those with oviposition scars), a pair of adults were introduced into a plastic container made by connecting two plastic cups (∼130 mm in diameter and 100 mm in height) with a U. parvifolia branch (for oviposition) and excised twigs (as food). After 24 h, we removed the males and allowed the females to lay eggs for 1 week. The sequence of oviposition events by A. glabripennis was carefully described in previous studies (Faccoli et al., 2015; Keena and Sánchez, 2018; Li et al., 2024); females make scars through the bark of host branches and lay single eggs at the cambial interface in some, but not all of the scars. Before the choice tests, we removed the female and examined the treatment branch to determine the number of oviposition scars. We then added a control branch into each plastic container. Next, we introduced a different female from the same population of origin into the plastic container and allowed them to lay eggs for 1 week, after which we counted the oviposition scars on both branches. The second experiment consisted of a choice between a control branch and one that contained her own oviposition scars under the same conditions as the above method. We performed 20 replicates for each experiment. During the oviposition-choice bioassays, 16 of 20 females in the choice between a control branch and a treated branch with conspecific scars and 11 of 20 females in the choice between a control branch and a treated branch with own scars made oviposition scars. Individuals who did not make any oviposition scars during the experiments were removed from the dataset.

Data analysis

All statistical analyses were performed using R software (v4.4.2) (R Core Team, 2024). In the field survey, the distribution normality of the distance between neighbouring oviposition scars was tested using the Shapiro–Wilk test. We also examined the effect of the perimeter length of logs on the distance between neighbouring oviposition scars using Spearman’s rank correlation coefficient. In the laboratory oviposition-choice bioassays, we analysed the numbers of oviposition scars using generalised linear mixed models (GLMMs) with a Poisson distribution, and the branch diameters using GLMMs with a Gaussian distribution. In the GLMM, treatment (i.e. scar-present or scar-absent) was a fixed factor and female ID was a random factor. We also examined the effect of the numbers of initial oviposition scars on the numbers of oviposition scars added using generalised linear models (GLMs) with a Poisson distribution. Likelihood ratio tests were conducted to determine the significance of each fixed effect. A significance value of p < 0.05 was considered to indicate statistical significance. Experimental data analysed during this study are included in the Supplementary Information file (Dataset S1).

Results

Oviposition pattern in the field

The distance between neighbouring oviposition scars did not follow a normal distribution in both tree species. In S. caprea, the distance from a given oviposition scar to the nearest scar was 4.383 ± 0.091 cm (mean ± SE, range 1.0–14.7 cm, n = 594 oviposition scars from one tree, fig. 2a). The data were significantly deviated from a normal distribution (Shapiro–Wilk test, p < 0.001), showing a skewness of 1.730 (positive skew). No significant differences were observed in the distance between neighbouring oviposition scars depending on the perimeter length of the logs (Spearman’s rank correlation, ρ = 0.069, p = 0.094). In C. japonicum, the distance from a given oviposition scar to the nearest scar was 7.516 ± 0.416 cm (mean ± SE, range 2.0–26.2 cm, n = 116 oviposition scars from two trees, fig. 2b). The data was significantly deviated from a normal distribution (Shapiro–Wilk test, p < 0.001), showing a skewness of 1.380 (positive skew). No significant differences were observed in the distance between neighbouring oviposition scars depending on the perimeter length of the logs (Spearman’s rank correlation, ρ = −0.073, p = 0.435).

Figure 2. Oviposition pattern of Anoplophora glabripennis in the field. Each histogram shows the frequency distribution of the distance from a given oviposition scar to the nearest scar by tree species. (a) Distance between neighbouring oviposition scars found on the branches of the goat willow Salix caprea. (b) Distance between neighbouring oviposition scars found on the branches of the katsura tree Cercidiphyllum japonicum. (Online version in colour.).

Oviposition-choice bioassays

Females avoided both conspecific and their own oviposition scars during the choice tests. In the first experiment, females made less oviposition scars on branches containing scars made by other females than those without scars (likelihood ratio test, df = 1, χ ² = 63.80, p < 0.001, fig. 3a). Females showed stronger avoidance of the treated branches when they had more conspecific oviposition scars (likelihood ratio test, df = 1, χ ² = 83.14, p < 0.001, fig. 3b). We also confirmed that there were no significant differences in the branch diameters (likelihood ratio test, df = 1, χ ² = 1.07, p = 0.294, fig. S1a). In the second experiment, females also avoided making oviposition scars on branches containing already-made own scars (likelihood ratio test, df = 1, χ ² = 55.24, p < 0.001, fig. 3c). Females showed stronger avoidance of the treated branches containing more of their own oviposition scars (likelihood ratio test, df = 1, χ ² = 52.65, p < 0.001, fig. 3d). We also confirmed that there were no significant differences in the branch diameters (likelihood ratio test, df = 1, χ ² = 1.15, p = 0.273, fig. S1b).

Figure 3. Effects of already-made oviposition scars on the egg-laying decision-making in Anoplophora glabripennis. (a) Comparison of the number of oviposition scars added by a female between a branch without scars and a branch containing scars from a different female. (b) Correlation between the number of initial oviposition scars made by a female and the number of scars added by a different female. (c) Comparison of the number of oviposition scars added by a female between a branch without scars and a branch containing scars from the same female. (d) Correlation between the number of initial oviposition scars by a female and the number of scars added by the same female. In bar plots (a, c), each straight line represents that two connected data points belong to the same replication. Error bars represent the standard error of the mean (SE). Asterisks indicate significant differences (likelihood ratio test, *p < 0.05, **p < 0.01, ***p < 0.001). In scatter plots (b, d), GLM-fitted curves are shown together. (Online version in colour.).

Discussion

We demonstrated that females avoid conspecific cues during egg-laying decision-making in A. glabripennis. This is the first study to report interindividual interactions in this invasive longhorn beetle when females chose an oviposition site. Oviposition deterrence caused by conspecific cues can be interpreted as a part of social information use (Prokopy and Roitberg, 2001). Examples from various taxa illustrate that joining (Desurmont and Weston, 2011; Fletcher and Miller, 2008; Raitanen et al., 2014) or avoidance (Anbutsu and Togashi, 1996; Kiflawi et al., 2003; Zhang et al., 2019) behaviour differs among species. Moreover, within a given species, the decision to join or avoid conspecifics can be affected by the physiological and informational state of the individual and by contextual response thresholds to resource availability (Otake and Dobata, 2018). The fitness benefit of oviposition deterrence associated with conspecific cues has often been discussed in terms of avoiding food shortage and cannibalism. In longhorn beetles, which are internally feeding insects, newly hatched larvae have little or no possibility to move from their initial food resources. In such cases, if an adult lays eggs on a site already occupied by conspecific larvae, their progeny could experience increased mortality (Anbutsu and Togashi, 2000). Previous studies reported that females avoid hosts already occupied by conspecific cues (presence of oviposition scars, eggs, larvae, and their frass) in some longhorn beetles such as Monochamus alternatus (Anbutsu and Togashi, 1996, 2000, 2002), M. saltuarius (Anbutsu and Togashi, 1997b), M. scutellatus (Peddle et al., 2002), and Paraglenea fortunei (Wang et al., 1990). In this study, females avoided both conspecific and their own oviposition scars, in contrast to Peddle et al. (2002), who concluded that females did not avoid their own eggs in M. scutellatus. The adaptive advantage for females to avoid their own oviposition scars may be different depending on the species.

How do females recognise and avoid conspecific oviposition scars in A. glabripennis? It is possible that females use semiochemical signals to mark their own oviposition sites and detect hosts occupied by conspecifics. Oviposition deterrence is induced by a marking pheromone released by females in some insects (Nufio and Papaj, 2001; Roitberg and Prokopy, 1987). In A. glabripennis, the female trail secretions (two major components: 2-methyldocosane and (Z)-9-tricosene, two minor components: (Z)-9-pentacosene and (Z)-7-pentacosene) were identified (Graves et al., 2016; Hoover et al., 2014). These authors proposed that trail secretions may act as a spacing pheromone for females. Intriguingly, in this study, females showed stronger avoidance of the treated branches when they had more conspecific oviposition scars. This suggests that females can recognise oviposition scars themselves or oviposition-related substances. In other longhorn beetles, M. alternatus females were deterred from oviposition by the jelly-like secretion that the conspecific females deposited in the oviposition scars immediately after oviposition, which shows that females’ recognition of the egg-containing scars and departure from such scars were elicited by semiochemicals contained in the jelly-like secretion they deposited (Anbutsu and Togashi, 2000, 2001). It is also possible that A. glabripennis females perceive the shape of oviposition scars tactually or visually. Future studies should investigate the detailed mechanisms of oviposition deterrence caused by conspecific cues, e.g. chemical analysis of oviposition scars and further bioassays using artificial scars. Identifying the key to the avoidance can lead to ‘oviposition deterrents’, which discourage the pests from laying eggs on treated trees.

This study provides insights into the reproductive behaviour of this invasive longhorn beetle, which is useful for developing environmentally friendly control methods such as oviposition deterrents. Oviposition deterrents could be a viable option for reducing established and incipient populations in urban and natural forests, where intensive management methods such as spraying pesticides and destructing infested trees may be environmentally undesirable (Prokopy, 1972, 1981). Here, we point out that taking account of interindividual interactions is important for future technical innovations in applied entomology.