Influence of visual cues on oviposition site searching and learning behavior in the parasitic beetle Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae)

Host foraging by parasitic insects, including parasitoid wasps and parasitic beetles, can be considered a form of habitat selection¹. This process is often viewed as a step-by-step searching process, in which female parasitoids must first locate plants inhabited by their hosts, select those that present a high probability of finding a host, and finally search within this microhabitat for the host^2–5. Parasitic insects use olfactory, visual, vibrational, tactile, and acoustic cues from plants, hosts, or host frass to recognize host habitats^2,6–8. Chemical cues play major roles in the host-searching behavior of parasitic insects, and a great deal of research has been devoted to this subject in recent years^9,10. However, although Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is an important parasitoid of many longhorn beetles, there is relatively little information related to the use of visual cues by this species during host and host habitat searching.

The longhorn beetles comprise more than 35,000 species in approximately 4,000 genera. Many species are important pests of forest, plantation, orchard and urban trees and may act as vectors of tree diseases¹¹. These beetles cause heavy economic losses in forests around the world, as their larvae typically burrow in the tissues of woody plants¹¹. This burrowing behavior limits the effectiveness of chemical pesticides in controlling the larvae. Therefore, biological control using existing natural enemies is an important research direction. The parasitic beetle D. helophoroides is an important parasitoid of several longhorn beetle species in its native range, including Anoplophora glabripennis Motschulsky, Monochamus alternatus (Hope), Massicus raddei Blessig and Batocera horsfieldi (Hope)^12–14. D. helophoroides is distributed widely in Kyushu and Osaka, Japan¹⁵; Korea¹⁶; and China¹⁷. Females deposit egg clusters on the outer surface of the bark near a host entrance or frass extrusion hole or on larval tunnel walls, and the hatched larvae search for hosts and paralyze them¹⁸. This behavior raises interesting questions related to the host habitat and host detection by D. helophoroides, including the following fundamental question: Do adult D. helophoroides use olfactory and visual cues from the host and host habitat to locate suitable oviposition sites? Olfactory cues from frass and tunnels produced in wood by longhorn beetle larvae have been identified; these cues are used by D. helophoroides to identify host habitats and find hosts within them^6,19,20. However, nothing is known about the visual cues involved in the identification of hosts and host habitats by D. helophoroides adults.

Dastarcus helophoroides is classified as a generalist or polyphagous parasitoid because it can parasitize the larvae of many longhorn beetle species^6,12, which typically burrow in the living or dead tissues of woody plants in various habitats¹¹. Generalist species must typically contend with a larger variety of cues than must specialists during host and host habitat selection². Stephens²¹ and Aquino et al.⁷ suggested that if a host or its habitat is variable, the evolution of learning behaviors can allow parasitoids to explore different hosts and reduce environmental uncertainty. Generalist parasitoids can retain information to allow them to distinguish novel hosts and host microhabitats by associative learning. For example, visual cues (color and shape) are acquired by female Diachasmimorpha longicaudata (Ashmead) through associative learning and play an important role in their search for host habitats². However, the visual cues were used and learned by female D. helophoroides to search for hosts and host microhabitats and identify appropriate oviposition sites have rarely been addressed in previous research.

As female D. helophoroides deposit egg clusters near the host entrance or frass extrusion hole in natural environments¹⁸, they may use the color of the bark near the host entrance or frass extrusion hole and the visual and olfactory cues from larval frass to locate hosts and host habitats. The larval host entrance and frass extrusion holes are created in bark of different colors, and the bark also varies in color according to the species of plant inhabited by the host larvae. Many researchers have reported that female parasitoids can learn to differentiate the visual or olfactory cues associated with oviposition sites³. Therefore, the aims of this study were to investigate whether D. helophoroides females use visual cues (color substrate) to locate oviposition sites and able to locate and identify special color substrates during the search for oviposition sites, as well as to demonstrate their ability to associate the presence of simulated oviposition sites with substrate color through associative learning.

The results show that parasitic D. helophoroides females have an innate preference for ovipositing on black substrates. The preference for this color was changed by training on other colors with artificial oviposition sites, as D. helophoroides females laid more eggs on substrates of the color used in training than on black substrates. For example, the parasitic beetles laid eggs predominantly on the black substrate in the innate preference test, but when the insects were trained using a black substrate without wood and another color with wood, the parasitic beetles chose the conditioned substrate for oviposition, and the number of eggs laid on that substrate was significantly greater than that laid on the black substrate in the test session.

Two hymenopteran parasitoids were previously shown to prefer yellow over other colors⁷. Hymenopterans exhibit maximum spectral sensitivity to radiation in the range of UV, blue, green/yellow, and yellow, as surfaces emit radiation in the range of the visible spectrum similar to that of the young foliage of plants; accordingly, these parasitoids and some phytophagous insects use color cues (e.g., yellow) to locate appropriate hosts and host microhabitats^7,22. However, in the multi-choice experiments, we found that parasitic D. helophoroides females have an innate preference for ovipositing on black substrates. The very low reflectance of the substrates preferred by these parasitic beetles could also represent the use of a cue related to the habitat of the host (i.e., the coloration of bark beneath the larval frass). In the current study, the reflectance spectrum range of the black substrates might be more similar than those of the other substrates to the spectrum reflected by tree bark. The parasitic beetle D. helophoroides is the predominant species among the natural enemies of the wood-boring tree insects, and it shows outstanding effectiveness in controlling large- and medium-bodied longhorn beetles, such as A. glabripennis, Apriona germari, and M. alternatus^13,14. In previous studies on many forest pests, trunk pests particularly clearly prefer black to other colors in the field. For example, Campbell et al.²³ suggested that the wood-boring beetle and the ambrosia beetle showed greater preferences for black traps than for white traps, and the female European woodwasp Sirex noctilio was trapped more frequently by a black flight intercept trap than by a clear jar trap²⁴. In addition, many longhorn beetle species preferentially feed on plants that are suitable host plants for their larvae¹¹. Thus, D. helophoroides, as a parasitic beetle, may depend on visual cues from damaged plants when searching the host site for a suitable egg-laying location. Therefore, our findings suggest that D. helophoroides may use visual cues to find appropriate oviposition sites during the initial stages of their search for and selection of host habitats.

In the dual-choice experiments, we aimed to determine the ability of these beetles to distinguish the color substrates at appropriate oviposition sites locations and expected that the beetles would be able to identify special colors. However, these beetles still showed an oviposition preference for black substrates. The parasitic wasps prefer yellow to the other colors because they have maximum spectral sensitivity to radiation in the range of UV, blue, green/yellow, and yellow (see above). However, some species of insect show a preference for black. For example, in the color trap experiments with the old house borer (Hylotrupes bajulus), the results showed that black colored traps were more effective than brown, yellow, red, grey, blue, white, and green traps²⁵. The mosquitoes Toxorhynchites moctezuma and Tx. amboinensis also oviposited preferentially into black containers rather than white, red, yellow, green or blue containers²⁶. For these species that show a black preference for oviposition or orientation, future surveys of the maximum spectral sensitivity range will be necessary. However, our results form dual-choice tests are consistent with the results of multi-choice tests, which further demonstrate that the beetles prefer black to the tested substrates of colors for oviposition.

Orange and yellow both belong to the approximate yellow region of the color spectrum. Although the female beetles in this experiment laid significantly more eggs on the black substrate than on the orange substrate, there was no significant difference between the orange and black substrates. However, females laid significantly fewer eggs on the yellow substrate than on the black one, indicating that the female beetles recognize slight differences in wavelength and intensity. However, yellow is reported to be a typical foliage stimulus for aphids, psyllid species and others, and numerous insect species show a preference towards yellow^27–29. The response to color depends on the consumption of certain parts of host by a given insect species³⁰. For insects that specialize in feeding on leaves and flowers, yellow may be more important than black or green. By contrast, for D. helophoroides, which parasitize the larvae of longicorn beetles, black and orange colors similar to those of bark and larval frass may be preferred over green and yellow. Further research should focus on enhancing our knowledge of host-finding behavior in the ability of D. helophoroides to recognize different intensities of yellow and black, especially because this beetle is potentially an excellent natural enemy of longhorn beetles. Furthermore, in Aquino et al.⁷ used sample sizes of 19–25 female adults, and found that two parasitoids continued to show a preference for yellow substrates after being trained on substrates of different colors, whereas Lucchetta et al.,³¹ used sample sizes of 20–168 Venturia canescens females and trained them to associate an orange-colored stimulus with a food reward. In our research, a sample size of 20 replicates was used, and the small number of sample replicates might have been the reason that the female beetles did not show a significant difference in color preference between black and orange substrates; however, Rosenberg (2010)³² provided an equation for calculating effect sizes for Chi-square tests and found that the effect size was 0.36 in the comparison between black with orange substrates, which indicates that if the sample size were to be increased, the mean number of eggs laid on the black substrate might be significantly greater than that on the orange substrate³³. Moreover, we speculate that similarity of the orange color to the larval frass color was the reason for this finding.

To date, there have been no relevant studies regarding whether D. helophoroides has clear preferences for particular visual cues (color, shape, etc.) from its host plants or the frass of its host larvae^13,14. Many studies have focused on determining whether the combination of visual and olfactory stimuli, compared to visual or olfactory stimuli alone, may enhance the accuracy with which insects discriminate hosts and host habitats. Indeed, many insects have been shown to respond to host plants and mates via synergistic visual, olfactory and tactile cues^34–37. Moreover, in a previous study, Wei et al.⁶ showed that (S)-(−)-limonene from cerambycid larval frass was an important component of the kairomone attracting D. helophoroides adults in their search for larval A. glabripennis. A great deal of work has suggested that chemicals play a major role in the host-searching behavior of D. helophoroides. Therefore, the responses of D. helophoroides adults to the interaction between visual (particularly color) and olfactory cues from their hosts and host habitats will be the focus of our future research. The use of visual cues combined with chemical cues or cues of other modalities may provide better and more reliable information than any single modality for parasitic beetles in search of hosts and host habitats.

Because D. helophoroides is an important parasitoid of several species of longhorn beetles in China, these beetles are considered generalist or polyphagous parasitoids^13,14. These parasitic beetles have a wide distribution in China¹⁷ and have different preferences for visual and chemical cues in different environmental contexts. For example, (S)-(−)-limonene was found to be an important component of the kairomone attracting D. helophoroides adults to parasitize larvae and pupae of A. glabripennis in Xi’an, China, while (R)-(−)-limonene and limonene were not important. However, D. helophoroides adults were attracted by all three of the abovementioned monoterpenes from parasitized Apriona swainsoni (Hope) in Qufu, Shandong, China. Wei et al. suggested that this difference in preference may have arisen because populations have different geographic origins, necessitating their adaptation to different ecological environments⁶. In agreement with this assertion, our experiments showed that 10 consecutive days of associative learning changed the choice patterns of the parasitic beetles in terms of the substrate colors on which they oviposited. Although no information has previously been reported about learning behavior in D. helophoroides adults, we found that their innate preference for black substrate was changed by associative learning with oviposition sites and successful oviposition on substrates of other colors as rewards. These parasitic beetles also have a long lifespan³⁸, and Giunti et al.³ suggested that the habitat of a host may vary from one generation to the next, whereas it is typically constant within a generation. Therefore, many female parasitoids can learn to associate visual and olfactory information with oviposition sites. In our experiment, we found that D. helophoroides females are also able to learn visual associations; this capability enables them to rapidly adapt to different environments.

Non-host-foraging parasitoids encounter a trade-off between two mutually exclusive activities: searching for hosts and searching for food. Lucchetta et al.³¹ speculated that these parasitic insects can integrate information on three key parameters, i.e., the probability of finding food (food availability), metabolic reserves and host availability, to decide how they should allocate their time in searching for food and hosts. In the present study, a substitute host was used to artificially rear D. helophoroides, following established methods; however, over the long term, such rearing techniques could lead to recognition of the substitute host instead of the natural host. This study suggests that D. helophoroides females can use and learn visual cues from their experience in searching for oviposition locations. Therefore, if relevant cues of the target host were provided to adults to learn, the biological control abilities of these parasitic beetles for the target pests (food availability) could be enhanced.

Understanding the biology, ecology, and behavior of these parasitic beetles and their target pest species is essential for identifying and rearing high-quality natural enemies to ensure the success of biological control programs targeting specific pests¹. Our results allow an improved understanding of the visual cues used by adult D. helophoroides during the search for oviposition sites. Giunti et al.³ indicated that visual cues, especially colors, often serve as valuable information for parasitic enemies at both close and long range when they are reliably associated with food or oviposition opportunities. Our experiment showed that adult D. helophoroides have the ability to learn using visual cues. D. helophoroides is the most effective natural enemy of large- and medium-bodied longhorn beetles^13,14. Associative training with chemical and visual cues might be used to enhance the efficiency of biological control agents against a given target in mass-rearing protocols³.