Loss of olfaction reduces caterpillar performance and increases susceptibility to a natural enemy

Many caterpillars are major pests in agriculture, feeding on a variety of crops. They constantly face threats from both predators and the toxic defenses of the plants they eat. While scientists have long studied how predators find their prey, much less is known about how prey – like caterpillars – manage to survive and defend themselves.

Plants have evolved various strategies to fend off insects. When attacked by herbivores, some plants release specific chemical signals known as herbivore-induced plant volatiles. These scents act as a distress call, attracting natural enemies of the herbivores, such as parasitoid wasps, which use the caterpillars for reproduction, killing the herbivores in the process.

In turn, caterpillars have evolved different defense strategies. They have a sophisticated sense of smell, which may help them detect not only the scent of host plants but also the presence of predators or other caterpillars nearby.

Despite how common and important caterpillars are in farming systems, it is still not fully understood how they choose where to feed or how they avoid being eaten. To explore this, Wang et al. studied how much caterpillars rely on their sense of smell to survive and find food.

In insects, the ability to smell relies on a group of sensory proteins, most of which need a key gene called Orco to function. Without this gene, an insect’s ability to smell is severely impaired. Wang et al. used a gene editing tool known as CRISPR/Cas9 to ‘turn off’ the Orco gene in caterpillars of the large cabbage white butterfly (Pieris brassicae), effectively disabling their sense of smell. The researchers then compared the development, survival performance, and behavioral preferences of these mutant caterpillars with normal ones.

This revealed that caterpillars without a working sense of smell gained less weight and were less successful at finding suitable food sources. They were also more likely to be killed by parasitoid wasps. Behavioral experiments showed that caterpillars with an intact Orco gene avoided plants where other caterpillars were under attack by detecting the warning signals in the air. In contrast, mutant caterpillars could not recognize these danger cues and were unable to find suitable host plants or select enemy-free spaces.

The study of Wang et al. shows that caterpillars use smell not only to find food, but also to avoid danger. These insights could help farmers develop new, environmentally friendly ways to manage pests. For example, by planting companion plants that produce specific scents, or by breeding crops that naturally repel pests, we might be able to steer caterpillars away from valuable crops without relying on chemical pesticides.

Chemoreception plays a critical role in the insect life cycle. For instance, insects exploit chemical cues to locate food plants, to find and select mates, to evaluate oviposition sites, and to avoid natural enemies (Dicke and Grostal, 2001). Olfaction, a pivotal component of chemoreception, involves multiple processes in insects and has been extensively investigated in many species (Haverkamp et al., 2018), including its role in the interactions with conspecifics and natural enemies (Kannan et al., 2022). Thus far, the study of olfaction in insects has largely centered upon imagos, the final life stage of insects. However, chemical information likely also plays an essential role in host-plant choice and natural-enemy avoidance during the immature life stage, which is especially pronounced in holometabolous insects. Nonetheless, the olfactory system in larval herbivores remains largely unexplored.

Tri-trophic interactions among plants, herbivores, and natural enemies have been widely investigated in the last few decades, and chemoreception is considered to play an important role in these systems (Vet and Dicke, 1992; Turlings and Erb, 2018). In particular, abundant research has demonstrated that olfactory information, such as plant volatiles, is crucial in the coevolution of herbivores and natural enemies (Turlings and Erb, 2018; Dicke and Baldwin, 2010; Haverkamp and Smid, 2020). In addition to plant volatiles, chemicals emitted by the insects themselves also play an important role (Venugopal et al., 2020; Ebrahim et al., 2015). Under strong selection pressure from natural enemies, herbivores have developed several integrated avoidance strategies based on the detection of chemical cues. Drosophila melanogaster fruit flies, for example, prefer to lay eggs on citrus fruits because the volatiles emitted by citrus are repellent to the endoparasitoid Leptopilina boulardi, a natural enemy of fruit fly larvae (Dweck et al., 2013). Similarly, the brown planthopper Nilaparvata lugens deposits eggs on rice plants infested by the striped stem borer Chilo suppressalis, which is repellent to the egg parasitoid Anagrus nilaparvatae (Hu et al., 2020).

Insect olfaction is dominated by two classes of olfactory receptor genes, odorant receptors (ORs) and ionotropic receptors (IRs). ORs detect the majority of volatiles in the insect habitat. In all ORs, the odorant receptor co-receptor (Orco) is required to form a functional ion channel in the membrane of the odorant receptor neurons (ORNs) (Sato et al., 2008; Wicher et al., 2008; Jones et al., 2005). Previous research indicates that silencing the Orco gene can lead to a severe olfaction deficiency, causing a loss of social behavior, impaired mating, and significantly reduced foraging behavior (Trible et al., 2017; Yan et al., 2017; Fandino et al., 2019). Previous reports have also clearly shown that adult insect behavior and neuronal development were greatly affected when Orco is knocked out (Trible et al., 2017; Yan et al., 2017). Yet, potential changes in olfaction-deficient larvae remain largely unexplored, and little is known about how olfaction contributes to the co-adaptation of larvae and their natural enemies, especially in the larvae of non-model insects such as lepidopteran caterpillars.

Pieris brassicae is a very common butterfly species that causes substantial agricultural production loss across Europe and the Indian subcontinent (Feltwell, 1982; Hasan and Ansari, 2011; Hasan and Shafiq Ansari, 2010). The butterflies utilize chemoreception to evaluate oviposition sites and host plants (van Loon et al., 1992a; van Loon et al., 1992b; Wang et al., 2023). The caterpillars also have a well-developed olfactory system (Wang et al., 2024) and chemical communication has been demonstrated to be involved in the interaction between herbivores and their natural enemies (Stam et al., 2014; Dicke and Baldwin, 2010). Natural enemies such as parasitoid wasps are highly attracted to volatiles from cabbage plants infested by P. brassicae caterpillars (Mattiacci et al., 1994). P. brassicae caterpillars are thus under strong selection pressure from natural enemies, though caterpillars can defend themselves by spitting at natural enemies while being attacked (Müller et al., 2003).

To gain comprehensive insight into the role of olfaction in P. brassicae, and to understand the role of olfaction in their coevolution with natural enemies, we employed a well-established research system utilizing Brassica oleracea as host plants, P. brassicae caterpillars as herbivores and Cotesia glomerata parasitoid wasps as natural enemies. In this study, we knocked out Orco by CRISPR/Cas9 in P. brassicae to investigate how olfaction guides the host-plant choice and natural-enemy avoidance of this insect herbivore. The knockout (KO) was verified in caterpillars by staining Orco and glomeruli in larval antennae and the larval antennal center (LAC), respectively, and was further confirmed in butterflies by electrophysiological investigations. We also examined the egg-laying behavior of KO butterflies, which is the basis of establishing a homozygous KO insect colony for our subsequent studies. The development of caterpillars on cabbage plants was evaluated, and the performance of caterpillars under the threat of natural enemies was studied to investigate the ecological significance of larval olfaction in interacting with their natural enemies. The larval host-plant seeking behaviors were further evaluated to determine the importance of olfaction in locating food sources. Volatiles emitted by caterpillars and parasitoids were analyzed to identify candidate chemicals that might play a role in this interaction. Our study provides further knowledge on the olfactory system in insect herbivores and might help to clarify the ecological significance of olfaction in the interaction with their natural enemies.

Chemical communication plays a significant role in the interaction between plants, herbivores, and their natural enemies. Infested host plants emit herbivore-induced plant volatiles (HIPVs) and thereby attract natural enemies such as parasitoid wasps (Mattiacci et al., 1994). While the role of adult herbivores’ olfaction in mediating these tri-trophic interactions has been investigated, the significance of caterpillar olfaction in tri-trophic interactions of plants, herbivores, and their natural enemies has remained largely underexplored. In this study, we severely impaired the olfaction of P. brassicae by knocking out Orco using CRISPR/Cas9 to investigate the ecological importance of olfaction in host-plant choice and enemy avoidance by P. brassicae larvae. The KO of olfaction was successful, as we did not find Orco proteins in the antennae of Orco KO caterpillars and showed an altered brain structure in the Orco KO caterpillars in comparison to WT caterpillars. Furthermore, the near-complete lack of response to odors by Orco KO butterfly antennae compared to WT confirms that olfaction was largely lost. Intriguingly, although mating and oviposition were significantly reduced, KO butterflies still produce some homozygous offspring. After this characterization of our mutant line, we aimed to test the role of larval olfaction in a multitrophic framework. We found that the loss of olfaction had a significant effect on the foraging behavior of the caterpillars, which exhibited a reduced growth rate and a greater vulnerability to the parasitoid wasp C. glomerata.

The perception of chemical cues is mediated by the chemosensory signal transduction in insect antennae and further processing of the information in the brain. In P. brassicae KO caterpillar antennae, no Orco protein was detected, and chemical signals can therefore not be converted into electrical signals (Sato et al., 2008; Wicher et al., 2008). Interestingly, the non-functional Orco decreased the number of glomeruli in the LAC, indicating that Orco is likely to play a role in the glomerular organization of the LAC (Figure 1C–I). Similar changes have been reported for adult insects of a few hymenopteran species: the ants Harpegnathos saltator and Ooceraea biroi and the honeybee Apis mellifera, where the number and total size of glomeruli decreased while the volume of individual glomeruli increased in Orco KO mutants (Trible et al., 2017; Yan et al., 2017; Chen et al., 2021). In contrast, the antennal lobe morphology of D. melanogaster was not significantly changed when Orco was knocked out (Larsson et al., 2004). In Lepidoptera, the size and number of glomeruli in Helicoverpa armigera were found to be comparable between WT and KO adult moths without any significant difference (Fan et al., 2022). However, silencing the pheromone receptor, SlitOR5, led to a decreased size of a glomerulus in the adult moth Spodoptera littoralis, and knocking out Orco also led to a reduced size of OR-related glomeruli in adult Manduca sexta (Koutroumpa et al., 2022; Fandino et al., 2019). The loss of olfactory glomeruli in the caterpillar might, therefore, either indicate that the formation of glomeruli in the embryo is mediated by a different mechanism than during pupation (Oland and Tolbert, 2011), or that the loss of neuronal activity reduces axonal innervations and thereby the size of the glomeruli to an extent that they could no longer be visualized by our method. However, the mechanism of glomerular formation and the roles that olfactory genes play in this process need to be further investigated in both immature and adult insects (Williams et al., 2022).

The role of olfaction in the behavioral ecology of adult insects has already been studied in quite some detail, and silencing Orco has been an important tool for unraveling the importance of chemical communication in adults of various insect species. For instance, in Orco KO adults of several insect species, there was a significant reduction in the electrophysiological responses to volatile chemicals such as alcohols and esters (Fandino et al., 2019; Sun et al., 2020). Consequently, Orco mutants exhibited notable changes in host-plant location and severely reduced fecundity due to impaired pheromone perception and mating, which also resulted in significantly reduced egg-laying behavior (Fan et al., 2022; Fandino et al., 2019; Liu et al., 2023). Similar to other species, Orco KO P. brassicae butterflies exhibited a loss of electrophysiological responses to most plant volatiles we tested (Fan et al., 2022; Sun et al., 2020; Figure 1J, K), and the mating behavior of P. brassicae Orco KO butterflies was also disrupted (Figure 1—figure supplement 3). However, we observed some differences: P. brassicae Orco KO butterflies were still able to mate to a limited degree (Figure 1—figure supplement 2). Whereas Orco KO adults in some other insect species did not exhibit any mating behavior (Fandino et al., 2019; Fan et al., 2022). This finding suggests that additional sensory systems beyond olfaction, such as vision, may be involved in the mating processes of this day-active butterfly (Carpenter and Sparks, 1982; Obara et al., 2008). Interestingly, while many P. brassicae butterflies with impaired olfaction failed to mate, several KO butterflies still mated and deposited fertilized eggs when the oviposition substrate was supplied in our laboratory setting. The mated Orco KO butterflies laid a comparable number of fertilized eggs on cabbage plants to WT butterflies, which is in line with the results from different moth species (Fandino et al., 2019; Fan et al., 2022; Sun et al., 2023), suggesting oviposition decisions are mostly guided by non-volatile compounds when no host-plant location is required (van Loon et al., 1992a). Furthermore, the hatching rate of eggs laid by mated Orco KO butterflies was similar to that of WT butterflies (Figure 1—figure supplement 2), indicating that the reduced fecundity in these insects is primarily due to impaired mating behavior and reduced mating frequency, which may result from a lack of pheromonal communication.

When directly placed on their host plant, we found that WT caterpillars gained more weight than Orco KO caterpillars (Figure 2A). We speculate that olfaction is needed to evaluate plant tissues and to select different leaves or leaf parts in order to feed efficiently, even when the caterpillar is already on the plant. Secondary metabolites are often unevenly distributed even within a single leaf, and these concentration differences have been shown to influence the performance of different caterpillars (Kester et al., 2002; Yuan et al., 2022). It is conceivable that these localized differences in plant secondary metabolites are also detectable by the herbivore through its olfactory system (Hanson and Dethier, 1973). The difference between WT and KO caterpillars was amplified in the presence of disarmed parasitoids, which were able to attack but not injure the caterpillars, indicating that KO caterpillars were more susceptible to harassment by their natural enemies (Figure 2E). We hypothesize that this further reduction in caterpillar weight was due to the less efficient foraging behavior of the KO caterpillars, which resulted in an extended time period in which the caterpillars were vulnerable to the harassment by the disarmed parasitoids. This harassment would then further reduce the foraging efficiency of the KO caterpillars and thereby exacerbate the effect seen in Figure 2A.

P. brassicae caterpillars are most vulnerable to C. glomerata during the first two larval instars. Due to their reduced growth rate, KO caterpillars spent a longer time in these vulnerable stages and, therefore, in a natural setting would likely have suffered more strongly from parasitoid attack than WT caterpillars (Brodeur et al., 1996; Haverkamp and Smid, 2020). In addition, caterpillars might have been avoiding plant parts with natural enemies similar to choosing infested plants without parasitoids over infested plants with parasitoids present (Figure 2D). This suggests that caterpillars might be able to use their olfactory system to find enemy-free spaces based on volatiles emitted by the plant or the frass and spit of conspecifics. Similarly, when the caterpillars were exposed to unmanipulated parasitoids, the KO caterpillars were more susceptible and exhibited a higher mortality rate, demonstrating again that olfaction is crucial in larvae to survive under the pressure of natural enemies (Figure 2F). Subsequently, we inferred that the differences in weight gain and survival rate between WT and KO caterpillars are due to different sensitivity to plant volatiles. WT caterpillars can sooner reach a body size in which they are no longer vulnerable to attack by C. glomerata by locating an available food source efficiently (Brodeur et al., 1996). The higher mortality rate of KO caterpillars might, therefore, be partly due to a reduced foraging efficiency caused by the lack of olfactory information, which in turn led to an extended period in which the caterpillars were vulnerable to attack by their natural enemies. In addition, the WT caterpillars might have been better able to use olfactory cues to avoid plant parts where conspecifics are under attack by parasitoids, again reducing their mortality in comparison to KO caterpillars (Figure 2D).

Searching for host plants suitable for feeding and free of natural enemies is largely accomplished via the olfactory system of herbivorous insects (Carrasco et al., 2015; Hu et al., 2020). Without functional ORs, P. brassicae caterpillars could still discriminate plant tissues from paper discs; however, Orco KO caterpillars preferred the non-host tomato plant over their natural food plant (Figure 2B). We surmise that OR-mediated olfaction is required in caterpillars to locate and identify their host plants, and losing this part of their olfactory receptor repertoire would make it challenging to distinguish host from non-host plants (Liu et al., 2023). Nonetheless, Orco KO caterpillars still retain ORNs expressing ionotropic receptors, which commonly detect acids and amines that have been found to elicit aversion in insect herbivores (Zhang et al., 2019). Fatty acids are present at the edge of both artificially damaged and caterpillar-infested cabbage leaves (Horikoshi et al., 1997), which, in the absence of ORs detecting attractive plant compounds, may have caused avoidance behavior in the KO caterpillar to their natural host plant. Interestingly, the parasitoid C. glomerata also uses the fatty acids produced by the plant to locate Pieris caterpillars, and it has been argued that herbivores might avoid these compounds to escape from their natural enemies (Horikoshi et al., 1997; Zhang et al., 2019).

We found that WT caterpillars prefer caterpillar-infested plants free of wasps over caterpillar-infested plants with wasps, which suggests that caterpillars are able to detect certain volatiles that are derived from the interaction with caterpillars and wasps. By analyzing headspace samples of caterpillars, parasitoids, as well as the interaction of caterpillars and parasitoids, we successfully identified some chemical compounds which were already known plant volatiles, such as alcohols and aldehydes. However, we did not find any compounds that were specifically abundant in Pb-Cg or Cg. This suggests that a volatile signal serving as a ‘danger signal’ directly emitted by the wasps or caterpillars might not exist, or it might not be detectable with our methods (Figure 3B). Alternatively, indirect cues such as the volatiles emitted from the caterpillar spit during their defensive behavior might have triggered the avoidance behavior of the WT caterpillars to infested plants with natural enemies. When we tested the most abundant compounds in a two-choice behavioral assay against a solvent control, we found that P. brassicae WT larvae showed a preference to the plant volatiles (Z)-3-hexen-1-ol and (E)-2-hexenal (Figure 3D), which are known HIPVs. Previous studies on other species have demonstrated that caterpillars are capable of detecting different HIPVs (Di et al., 2017; de Fouchier et al., 2018). For caterpillars, HIPVs can serve as indicators for the location of potential food plants and the presence of conspecifics (Di et al., 2017; Zhang et al., 2019), but they also attract natural enemies and enhance the risk of being attacked (Bernays, 1997; Ngumbi and Fadamiro, 2012; Yang et al., 2016). Most HIPVs identified in this study were found abundantly in caterpillar saliva that caterpillars spit onto parasitoids to defend against attack. In our study, caterpillars were attracted by HIPVs emitted by plants and by individual HIPVs (Figure 3D). We speculate that caterpillars use low to intermediate concentrations of HIPVs to find suitable host plants and high concentrations to avoid sites where other caterpillars are under attack by their natural enemies. Therefore, perceiving plant leaf volatiles and approaching the food plants accordingly will increase the herbivores’ feeding efficiency and enhance survival chances. Interestingly, ITCs, which are hydrolyzed from glucosinolates after leaf damage in crucifer plants, did not elicit strong responses from the butterfly antenna (Figure 1J, K). Because all receptors expressed in the caterpillar are also present in the butterfly and because of the limited number of ORs and ORNs in larvae and low expression levels of ORs (Wang et al., 2024), we found it less likely that ITCs are used by P. brassicae caterpillars as foraging cues, even though these compounds are recognized by Plutella xylostella ORs (Liu et al., 2020). In our study, volatile cues of wasps were only found in very low quantities, and these compounds hardly elicited any behavioral response in the caterpillars; however, these chemicals might still play a role in the interaction between the caterpillars and wasps at a close range (Ebrahim et al., 2015).

The way that larval insects react to the potential threat from their natural enemies is still largely unknown for most insects. Our study that exploited CRISPR/Cas9 shows that olfaction is of high significance to caterpillars for locating food sources and likely affects their survival when under the selection pressure from natural enemies. We provided novel insights into the tri-trophic interactions of plants, herbivores, and their natural enemies from the perspective of a caterpillar and highlighted the role of olfaction for both foraging and escaping natural enemies.

All data generated during this study and the corresponding scripts for the statistical analysis are available in the supplementary files and under DOI: https://doi.org/10.4121/610e707d-b10a-467e-8afe-ead477827914.v1.

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Author details

1. Qi Wang

   Laboratory of Entomology, Wageningen University and Research, Wageningen, Netherlands

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2718-5856

2. Yufei Jia

   Laboratory of Entomology, Wageningen University and Research, Wageningen, Netherlands

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

3. Hans M Smid

   Laboratory of Entomology, Wageningen University and Research, Wageningen, Netherlands

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5867-2799

4. Berhane T Weldegergis

   Laboratory of Entomology, Wageningen University and Research, Wageningen, Netherlands

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8838-7876

5. Liana O Greenberg

   Biosystematics Group, Wageningen University & Research, Wageningen, Netherlands

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7404-2674

6. Maarten Jongsma

   Business Unit Bioscience, Wageningen University & Research, Wageningen, Netherlands

   Contribution

   Resources, Software, Validation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

7. Marcel Dicke

   Laboratory of Entomology, Wageningen University and Research, Wageningen, Netherlands

   Contribution

   Conceptualization, Resources, Supervision, Validation, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8565-8896

8. Alexander Haverkamp

   Laboratory of Entomology, Wageningen University and Research, Wageningen, Netherlands

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration, Writing – review and editing

   For correspondence

   alexander.haverkamp@wur.nl

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3512-9659

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