1. Ecology

A gustatory receptor tuned to the steroid plant hormone brassinolide in Plutella xylostella (Lepidoptera: Plutellidae)

  1. Ke Yang
  2. Xin-Lin Gong
  3. Guo-Cheng Li
  4. Ling-Qiao Huang
  5. Chao Ning
  6. Chen-Zhu Wang  Is a corresponding author
  1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, China
  2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, China
https://doi.org/10.7554/eLife.64114
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Cite this article
  1. Ke Yang
  2. Xin-Lin Gong
  3. Guo-Cheng Li
  4. Ling-Qiao Huang
  5. Chao Ning
  6. Chen-Zhu Wang
(2020)
A gustatory receptor tuned to the steroid plant hormone brassinolide in Plutella xylostella (Lepidoptera: Plutellidae)
eLife 9:e64114.
https://doi.org/10.7554/eLife.64114
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Abstract

Feeding and oviposition deterrents help phytophagous insects to identify host plants. The taste organs of phytophagous insects contain bitter gustatory receptors (GRs). To explore their function, the GRs in Plutella xylostella were analyzed. Through RNA sequencing and qPCR, we detected abundant PxylGr34 transcripts in the larval head and adult antennae. Functional analyses using the Xenopus oocyte expression system and 24 diverse phytochemicals showed that PxylGr34 is tuned to the canonical plant hormones brassinolide (BL) and 24-epibrassinolide (EBL). Electrophysiological analyses revealed that the medial sensilla styloconica of 4th instar larvae are responsive to BL and EBL. Dual-choice bioassays demonstrated that BL inhibits larval feeding and female oviposition. Knock-down of PxylGr34 by RNAi attenuates the taste responses to BL, and abolishes BL-induced feeding inhibition. These results increase our understanding of how herbivorous insects detect compounds that deter feeding and oviposition, and may be useful for designing plant hormone-based pest management strategies.

eLife digest

Plant-eating insects use their sense of taste to decide where to feed and where to lay their eggs. They do this using taste sensors called gustatory receptors which reside in the antennae and legs of adults, and in the mouthparts of larvae. Some of these sensors detect sugars which signal to the insect that the plant is a nutritious source of food. While others detect bitter compounds, such as poisons released by plants in self-defense.

One of the most widespread plant-eating insects is the diamondback moth, which feeds and lays its eggs on cruciferous vegetable crops, like cabbage, oilseed rape and broccoli. Before laying its eggs, female diamondback moths pat the vegetable’s leaves with their antennae, tasting for the presence of chemicals. But little was known about the identity of these chemicals.

Cabbages produce large amounts of a hormone called brassinolide, which is known to play a role in plant growth. To find out whether diamondback moths can taste this hormone, Yang et al. examined all their known gustatory receptors. This revealed that the adult antennae and larval mouthparts of these moths make high levels of a receptor called PxylGr34.

To investigate the role of PxylGr34, Yang et al. genetically modified frog eggs to produce this receptor. Various tests on these receptors, as well as receptors in the mouthparts of diamondback larvae, showed that PxylGr34 is able to sense the hormone brassinolide. To find out how this affects the behavior of the moths, Yang et al. investigated how adults and larvae responded to different levels of the hormone. This revealed that the presence of brassinolide significantly decreased both larval feeding and the amount of eggs laid by adult moths.

Farmers already use brassinolide to enhance plant growth and protect crops from stress. These results suggest that the hormone might also help to shield plants from insect damage. However, more research is needed to understand how this hormone acts as a deterrent. Further studies could improve understanding of insect behavior and potentially identify more chemicals that can be used for pest control.

Introduction

Many phytophagous insects have evolved to select a limited range of host plants. Understanding the ultimate and proximate mechanisms underlying this selection strategy is a crucial issue in the field of insect–plant interactions. Insects’ decisions to feed and oviposit are mainly based on information carried via chemosensory systems (Schoonhoven et al., 2005). Insects discriminate among potential hosts after perceiving a combination of lineage-specific and more ubiquitous chemicals synthesized by plants. Some nutrients are ubiquitous across plant taxa. For example, various sugars and amino acids are feeding stimulants for the majority of herbivorous insects. Secondary plant metabolites occur in certain plant taxa at much higher concentrations than in others, and therefore are of greater significance in host-plant selection by insects (Jermy, 1966). They are usually deterrent or ‘bitter’ compounds for many phytophagous insects, except for some specialized species that use them as token stimuli (Schoonhoven et al., 2005).

Phytophagous insects have sophisticated taste systems to recognize deterrent or stimulant compounds, which direct their feeding and oviposition behavior (Yarmolinsky et al., 2009). The taste sensilla of insects are mainly situated on the mouthparts, tarsi, ovipositor, and antennae (Bernays and Chapman, 1994). These sensilla take the form of hairs or cones with a terminal pore in the cuticular structure, and often contain the dendrites of three or four gustatory sensory neurons (GSNs). The axons of GSNs synapse directly onto the central nervous system. Analyses using the tip-recording technique for taste sensilla led to the discovery of a ‘deterrent neuron’ in the larvae of Bombyx mori (Ishikawa, 1966) and Pieris brassicae (Ma, 1969). Since then, GSNs coding for secondary plant metabolites have been identified in maxillary sensilla in larvae and tarsal sensilla of adults of many Lepidopteran species (Glendinning et al., 2002; Zhou et al., 2009). However, the molecular basis of these deterrent neurons remains unclear.

Gustatory receptors (GRs) expressed in the dendrites of GSNs determine the selectivity of the response of GSNs (Thorne et al., 2004; Wang et al., 2004). Since the first insect GRs were identified in the model organism Drosophila melanogaster (Clyne et al., 2000), the function of some of its bitter GRs have been revealed (Dweck and Carlson, 2020; Freeman and Dahanukar, 2015). Five D. melanogaster GRs (Gr47a, Gr32a, Gr33a, Gr66a, and Gr22e) are involved in sensing strychnine (Lee et al., 2010; Lee et al., 2015; Moon et al., 2009; Poudel et al., 2017). Nicotine-induced action potentials are dependent on Gr10a, Gr32a, and Gr33a (Rimal and Lee, 2019). Gr8a, Gr66a, and Gr98b function together in the detection of L-canavanine (Shim et al., 2015). Gr33a, Gr66a, and Gr93a participate in the responses to caffeine and umbelliferone (Lee et al., 2009; Moon et al., 2006; Poudel et al., 2015). Gr28b is necessary for avoiding saponin (Sang et al., 2019). However, only a few studies have focused on the function of bitter GRs in phytophagous insects. PxutGr1, a bitter GR in Papilio xuthus, was found to respond specifically to the oviposition stimulant, synephrine (Ozaki et al., 2011). In recent studies, the insect bitter GRs BmorGr16, BmorGr18, and BmorGr53 showed response to coumarin and caffeine in vitro, and coumarin was found to have a feeding deterrent effect on B. mori larvae (Kasubuchi et al., 2018); another bitter GR in B. mori, Gr66, was reported to be responsible for the mulberry-specific feeding preference of silkworms (Zhang et al., 2019). Although some progress has been made in determining the functions of such receptors in Drosophila and a few herbivorous insects, we still lack basic mechanistic information about the functions of bitter GRs from most herbivorous insect lineages.

Plutella xylostella (L.) is the most widespread Lepidopteran pest species, causing losses of US$ 4–5 billion per year (You et al., 2020; Zalucki et al., 2012). It has developed resistance to the usual insecticides because of its short life cycle (14 days) (Furlong et al., 2013). P. xylostella mainly selects Brassica species as its host plants, and its females pat the leaf surfaces with their antennae before egg laying (Qiu et al., 1998). This behavior may be related to certain chemical components in leaves of Brassica species, including sugars, sugar alcohols, amino acids, amines, glucosinolates, and plant hormones. Among these compounds, sinigrin and brassinolide (BL) have relatively higher concentrations in Brassica than in many other plant species (Fahey et al., 2001; Lv et al., 2014). Sinigrin is known to be a feeding/oviposition stimulant for P. xylostella (Gupta and Thorsteinson, 1960). The medial sensilla styloconica in the maxillary galea of P. xylostella larvae contain a GSN sensitive to sinigrin and other glucosinolates (van Loon et al., 2002). BL is a ubiquitous plant hormone that has been widely studied in relation to its role in plant growth and development (Clouse and Sasse, 1998), but little is known about its effects on the behavior of phytophagous insects.

To uncover the molecular basis of the perception of feeding/oviposition stimulants and deterrents by P. xylostella, we re-examined all the GRs reported in previous studies on this insect. Through transcriptome analysis and qPCR, we identified one bitter GR (PxylGr34) highly expressed in the larval head and the adult antennae. We functionally analyzed this GR with the Xenopus oocyte expression system and RNAi, and found that PxylGr34 is tuned to BL as a feeding and oviposition deterrent in P. xylostella.

Results

Identification of PxylGr34, a highly expressed GR gene in P. xylostella

To search for candidate GRs that may be involved in host selection by P. xylostella, we searched for candidates among those that had been annotated in the P. xylostella genome (Engsontia et al., 2014; You et al., 2013), the transcriptome (Yang et al., 2017), and the P. xylostella GRs deposited in GenBank (Supplementary file 1). We obtained 79 annotated GRs (69 GRs from the genome, 7 GRs from the transcriptome, and 3 GRs from GenBank). After removing repetitive sequences and deleting paired sequences with amino acid identity greater than 99%, we validated 67 of the GR sequences (61 GRs from the genome, 3 GRs from the transcriptome, and 3 GRs from GenBank) (Figure 1 and Supplementary file 1). On this basis, we first analyzed the transcriptomes of the antennae, forelegs (only tibia and tarsi), and head (without antennae) of adults, and the mouthparts of 4th instar larvae although the genomic data and antennae transcriptome of P. xylostella had been reported previously (Yang et al., 2017; You et al., 2013). Next, we looked specifically for candidates that were highly expressed in these chemosensory organs by calculating the transcripts per million (TPM) values of these 67 GRs (Figure 2A). Those GRs with high expression levels in both adult and larval taste organs were considered to be candidates for those with key roles in the host-plant selection of this species. Intriguingly, the TPM value of PxylGr34, which clustered with bitter GRs, was much higher than those of other GR genes in the antennae, head and forelegs of adults, as well as the mouthparts of larvae (Figure 2A). PxylGr34 was originally annotated from genomic data (Engsontia et al., 2014), and then detected in the antennal transcriptomic data of P. xylostella (named as ‘PxylGr2’ in Yang et al., 2017). However, both studies provided only its partial coding sequences (Figure 1—figure supplement 1). Based on our transcriptomic data, we obtained the full-length coding sequence of PxylGr34 through gene cloning and Sanger sequencing (Figure 1—figure supplement 1). The protein encoded by PxylGr34 is typical of most GRs with seven transmembrane domains, and a full open reading frame (ORF) of 418 amino acids (Figure 1—figure supplement 2).

Figure 1 with 2 supplements see all
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Phylogenetic tree of gustatory receptors (GRs).

Amino acid sequences are based on previously reported GRs. Bootstrap values are based on 1000 replicates. Abbreviations: Hmel, Heliconius melpomene; Bmor, Bombyx mori; Pxyl, Plutella xylostella. ○, GRs of P. xylostella; ●, PxylGR34.

Figure 1—source data 1

Amino acid sequences of GRs in Plutella xylostella.

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Figure 2
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Tissue expression pattern of gustatory receptors (GRs) in Plutella xylostella as determined by Illumina read-mapping and qPCR analysis.

(A) Transcripts per million (TPM) value of each GR is indicated in box. Color scales were generated using Microsoft Excel. Antenna, head, and foreleg were from the moth; larval mouthpart was from 4th instar larvae. The GRs that undetectable in the TPM analysis were not listed. Relative PxylGr34 transcript levels in (B) 4th instar larval tissues and (C) mated moth tissues of Plutella xylostella as determined by qPCR. Data are mean ± SEM. n = 3 replicates of 40–200 tissues each. For 4th instar larvae, p=0.0004; for the moth, p<0.0001 (one-way ANOVA, Tukey’s HSD test).

Figure 2—source data 1

Source data for Figure 2B and C.

https://cdn.elifesciences.org/articles/64114/elife-64114-fig2-data1-v2.xlsx

High PxylGr34 transcript levels in the larval head and adult antennae

To further confirm the expression patterns of PxylGr34 in the larvae and adults, we detected its relative transcript levels in different tissues of adults and the 4th instar larvae of P. xylostella using quantitative real-time PCR (qPCR). The larvae eat the most in the 4th instar and can forage around the plant more easily (Harcourt, 1957). The high levels of PxylGr34 transcripts were detected in the larval head. They were also detected in the larval thoracic legs and gut (Figure 2B). In the adults, PxylGr34 transcripts were restricted to the antennae (Figure 2C).

BL and EBL induced a strong response in the oocytes expressing PxylGr34

We used the Xenopus laevis oocyte expression system and two-electrode voltage-clamp recording to study the function of PxylGr34. Among 24 tested phytochemicals belonging to sugars, sugar alcohols, amino acids, amines, glucosinolates, and plant and insect hormones (Key Resources Table), BL induced a strong response in the oocytes expressing PxylGr34, as did its racemate EBL at a concentration of 10–4 M (Figure 3A and Figure 3B). The currents induced by BL increased from the lowest threshold concentration of 10–4 M in a dose-dependent manner (Figure 3C and Figure 3D). Oocytes expressing PxylGr34 showed weak responses to methyl jasmonate and allyl isothiocyanate, but no response to 20-hydroxyecdysone and other tested compounds (Figure 3A and Figure 3B). As negative controls, the water-injected oocytes failed to respond to any of the tested chemical stimuli (Figure 3—figure supplement 1).

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Brassinolide and 24-epibrassinolide induced a strong response in the oocytes expressing PxylGr34.

(A) Representative inward current responses of Xenopus oocytes expressing PxylGr34 in response to ligands at 10−4 M. (B) Response profiles of Xenopus oocytes expressing PxylGr34 in response to ligands at 10−4 M. Data are mean ± SEM. n = 7 replicates of cells. p<0.0001 (one-way ANOVA, Tukey's HSD test). (C) Representative inward current responses of Xenopus oocytes expressing PxylGr34 in response to BL at a range of concentrations. (D) Response profiles of Xenopus oocytes expressing PxylGr34 in response to BL at a range of concentrations. Data are mean ± SEM; n = 6–8 replicates of cells. p<0.0001 (one-way ANOVA, Tukey’s HSD test).

Figure 3—source data 1

Source data for Figure 3B and D.

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The larval medial sensilla styloconica exhibited vigorous responses in P. xylostella to BL and EBL

Next, using the tip-recording technique, we examined whether any gustatory sensilla in the mouthparts of larvae of P. xylostella could respond to BL and EBL. Of the two pairs of sensilla styloconica in the maxillary galea of 4th instar larvae, the lateral sensilla styloconica had no response to BL and EBL (Figure 4A,B,C and D); the medial sensilla styloconica exhibited vigorous responses to BL and EBL at 3.3 × 10−4 M, and the spike amplitudes induced by BL and EBL were about the same (Figure 4A,B,C and D). As previously reported, the medial sensilla styloconica also exhibited vigorous responses to sinigrin. However, the spike amplitudes induced by sinigrin were larger than those induced by BL and EBL (Figure 4E and F). These results suggest that BL and EBL activate the same neuron, while sinigrin activates a different neuron in the sensillum. The medial sensilla styloconica showed a dose-dependent response to BL, although the testing concentrations were limited because of the low solubility of BL in water (highest concentration approximately 3.3 × 10−4 M) (Figure 5).

Figure 4
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The larval medial sensilla styloconica exhibited vigorous responses in P.

xylostella to brassinolide (BL) and 24-epibrassinolide (EBL). Typical electrophysiological recordings in response to water and BL (A), EBL (C), and sinigrin (E) at 3.3 × 10−4 M for 1 s obtained by tip-recording from a neuron innervating the lateral and medial sensillum styloconica on maxillary galea of P. xylostella 4th instar larvae. Response profiles of the two lateral and medial sensilla styloconica on the maxilla of P. xylostella 4th instar larvae to water and BL (B), EBL (D), and sinigrin (F) at 3.3 × 10−4 M. Data are mean ± SEM. For lateral sensilla styloconica, n = 14, p=0.0905 to BL; n = 10, p=0.3869 to EBL; n = 10, p=0.0672 to sinigrin; for medial sensilla styloconica, n = 16, p<0.0001 to BL; n = 10, p<0.0001 to EBL; n = 10, p<0.0001 to sinigrin. Data were analyzed by paired-samples t-test.

Figure 4—source data 1

Source data for Figure 4B,D and F.

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Figure 5
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The medial sensilla styloconica showed a dose-dependent response to brassinolide (BL).

(A) Typical electrophysiological recordings in response to water and BL at a series of concentrations for 1 s obtained by tip-recording from a neuron innervating the medial sensillum styloconicum on the maxillary galea of P. xylostella 4th instar larvae. (B) Response profiles of medial sensilla styloconica on maxilla of P. xylostella 4th instar larvae to water and BL at a series of concentrations. Data are mean ± SEM. n = 8–14 replicates of larvae, p<0.0001 (one-way ANOVA, Tukey’s HSD test).

Figure 5—source data 1

Source data for Figure 5B.

https://cdn.elifesciences.org/articles/64114/elife-64114-fig5-data1-v2.xlsx

BL and EBL-induced feeding deterrence effect on P. xylostella larvae

We further tested the effects of BL and EBL on the larval feeding behavior of P. xylostella on pea leaves in the dual-choice leaf disc assay. In a dual-choice feeding test with 4th instar larvae, the feeding areas of larvae were significantly smaller on the leaf discs treated with BL and EBL at concentrations of 10−4 M and above than on the control leaf discs. In addition, the feeding preference of larvae to BL and EBL tended to decrease with increasing BL and EBL concentrations (Figure 6 and Figure 6—figure supplement 1), indicating that both BL and EBL function as feeding deterrents to P. xylostella larvae.

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Brassinolide (BL)-induced feeding deterrence effect on P.xylostella larvae.

Dual-choice feeding tests were conducted using pea leaf discs. Total feeding area of control (white bars) and BL-treated discs (green bars). BL was diluted in 50% ethanol to 10−6, 10−5, 10−4, 10−3, and 10−2 M. Data are mean ± SEM. For 10−6 M, n = 18, p=0.3947; for 10−5 M, n = 21, p=0.5967; for 10−4 M, n = 30, p<0.0001; for 10−3 M, n = 20, p<0.0001; for 10−2 M, n = 19, p<0.0001. Data were analyzed by paired-samples t-test.

Figure 6—source data 1

Source data for Figure 6.

https://cdn.elifesciences.org/articles/64114/elife-64114-fig6-data1-v2.xlsx

BL-induced oviposition deterrence to P. xylostella females

We also tested the effects of BL on the female ovipositing behavior of P. xylostella. In a dual-choice oviposition test with mated females, significantly fewer eggs were laid on the sites treated with BL at 10−4 M, 10−3 M, and 10−2 M than on the control sites. In addition, the oviposition preference tended to decrease as the BL concentrations increased (Figure 7).

Figure 7
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Brassinolide (BL)-induced oviposition deterrence to P. xylostella females.

Dual-choice oviposition tests were conducted using plastic film coated with cabbage leaf juice. Both control (50% ethanol) and BL (diluted in 50% ethanol) were painted evenly onto plastic film. After 24 hr, total number of eggs laid by a single mated female on control films (white bars) and BL-treated films (green bars) were counted. Data are mean ± SEM. For 10−5 M, n = 9, p=0.4364; for 10−4 M, n = 12, p=0.0275; for 10−3 M, n = 13, p=0.0015; 10−2 M, n = 8, p=0.0038. Data were analyzed using paired-samples t-test.

Figure 7—source data 1

Source data for Figure 7.

https://cdn.elifesciences.org/articles/64114/elife-64114-fig7-data1-v2.xlsx

PxylGr34 siRNA-treated P. xylostella larvae show attenuated taste responses to BL and alleviated feeding deterrent effect of BL

To clarify whether PxylGr34 mediates the electrophysiological and behavioral responses of P. xylostella larvae to BL in vivo, we tested the effect of siRNA targeting PxylGr34 on the responses of medial sensilla styloconica and the feeding behavior of the 4th instar larvae. The relative transcript level of PxylGr34 in the head of larvae treated with PxylGr34 siRNA was half that in the head of larvae treated with green fluorescent protein (GFP) siRNA or ddH2O (Figure 8A). This confirmed that feeding with siRNA is an effective method for RNAi-inhibition of PxylGr34 in the larval head.

Figure 8
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PxylGr34 siRNA-treated P. xylostella larvae inhibit the responses of sensilla to brassinolide (BL).

(A) Effect of PxylGr34 siRNA on transcript levels of PxylGr34 in 4th instar larval head. Heads were collected after feeding larvae with cabbage leaf discs coated with PxylGr34 siRNA, GFP siRNA, or ddH2O. Relative transcript levels of PxylGr34 in each treatment were determined by qPCR. Data are mean ± SEM. n = 3 replicates of 21–24 heads each, p=0.0237 (one-way ANOVA, Tukey’s HSD test). (B) Typical electrophysiological recordings in response to water and BL at 3.3 × 10−4 M for 1 s obtained by tip-recording from a neuron innervating the medial sensillum styloconica, on maxillary galea of P. xylostella 4th instar larvae with cabbage leaf discs coated with PxylGr34 siRNA, GFP siRNA, or ddH2O. (C) Response profiles of the medial sensilla styloconica on the maxilla of P. xylostella 4th instar larvae with cabbage leaf discs coated with PxylGr34 siRNA, GFP siRNA, or ddH2O, to water and BL at 3.3 × 10−4 M. Data are mean ± SEM. The data of each treatment to water and BL at 3.3 × 10−4 M were analyzed by paired-samples t-test. For ddH2O treatment, n = 9, p<0.0001; for GFP siRNA treatment, n = 10, p<0.0001; for PxylGr34 siRNA treatment, n = 12, p=0.0019. The data of different treatments to BL at 3.3 × 10−4 M were analyzed by one-way ANOVA, Tukey’s HSD test, p=0.0002.

Figure 8—source data 1

Source data for Figure 8A and C.

https://cdn.elifesciences.org/articles/64114/elife-64114-fig8-data1-v2.xlsx

To test the effects of RNAi-inhibition of PxylGr34 on the taste responses, the medial sensilla styloconica from siRNA-treated larvae were subjected to a tip-recording analysis as described above, with 3.3 × 10−4 M BL or water. As shown in Figure 8, although the medial sensilla styloconica of PxylGr34 siRNA-treated larvae still showed some response to BL, the frequency of spikes to BL elicited in the medial sensilla styloconica of the PxylGr34 siRNA-treated larvae was decreased (Figure 8B and Figure 8C).

To test the effects of RNAi-knockdown of PxylGr34 on the feeding behavior of 4th instar larvae, the siRNA-treated larvae were subjected to a dual-choice leaf disc feeding assay as described above, with leaf discs of pea treated with 10−4 M BL or untreated (control). As shown in Figure 9, both the water-treated larvae and the GFP siRNA-treated larvae preferred control leaf discs over those treated with BL, whereas the PxylGr34 siRNA-treated larvae showed no significant preference (Figure 9). Thus, the knock-down of PxylGr34 by RNAi attenuated the electrophysiological responses of the medial sensilla styloconica to BL, and alleviated the deterrent effect of BL on the feeding of P. xylostella larvae.

Figure 9
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PxylGr34 siRNA-treated P. xylostella larvae alleviated the feeding deterrent effect of brassinolide (BL).

Choice assay using 4th instar larvae fed on cabbage leaf discs treated with PxylGr34 siRNA, GFP siRNA, or ddH2O. In dual-choice assay, larvae chose between control pea leaf discs (treated with 50% ethanol) and those treated with BL at 10−4 M (diluted in 50% ethanol). Figure shows total feeding area of control (white bars) and treated discs (green bars). Data are mean ± SEM. For ddH2O treatment, n = 19, p=0.0093; for GFP siRNA treatment, n = 18, p=0.0044; for PxylGr34 siRNA treatment, n = 19, p=0.1864. Data were analyzed by paired-samples t-test.

Figure 9—source data 1

Source data for Figure 9.

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Discussion

In this study, we identified the full-length coding sequence of PxylGr34 from our transcriptome data, and found that this gene is highly expressed in the head of the 4th instar larvae and in the antennae of females. Our results show that PxylGr34 is specifically tuned to BL and its racemate EBL, and that the medial sensilla styloconica of 4th instar larvae have electrophysiological responses to BL and EBL. Our results also show that BL inhibits larval feeding and female oviposition of P. xylostella, and that knock-down of PxylGr34 by RNAi can attenuate the responses of sensilla to BL, and abolish the feeding inhibition effect of BL. This is the first study to show that an insect can detect and react to this steroid plant hormone. The results of the systematic functional analyses of the GR, the electrophysiological responses of the sensilla, behavioral assays, and behavioral regulation in vivo show that PxylGr34 is a bitter GR specifically tuned to BL. This receptor mediates the deterrent effects of BL on feeding and ovipositing behaviors of P. xylostella.

The larvae of Lepidopteran species have two pairs of gustatory sensilla (medial and lateral sensilla styloconica) located in the maxillae galea; these sensilla play a decisive role in larval food selection (Dethier, 1937; Schoonhoven and van Loon, 2002). Each sensillum usually contains four GSNs, of which one is often responsive to deterrents. Ishikawa, 1966 described a ‘deterrent neuron’ in the medial sensillum styloconicum of silkworm, Bombyx mori, and showed that it responds to several plant alkaloids and phenolics (Ishikawa, 1966). Similar neurons have been found in other Lepidopteran species, but their profiles vary. For example, the tobacco hornworm, Manduca sexta, has a deterrent neuron in the medial sensillum styloconicum that responds to aristolochic acid, and another deterrent neuron in the lateral sensillum styloconicum that responds to salicin, caffeine, and aristolochic acid (Glendinning et al., 2002). The diversity of GRs facilitates the detection of, and discrimination among, a wide range of diverse taste stimuli, implying that different sets of bitter GRs are expressed in these neurons. In this study, we proved that one GSN in the medial maxillary sensillum styloconicum of the 4th instar larvae of the diamondback moth responds to sinigrin (van Loon et al., 2002), and we identified one neuron responding to BL and EBL in the same sensillum. For P. xylostella larvae, sinigrin is a feeding stimulant whereas BL and EBL are feeding deterrents, together with sinigrin and BL/EBL have different spike amplitudes, we speculate that the GRs tuned to sinigrin and BL/EBL are located in different GSNs in the medial sensilla styloconica.

The GR family is massively expanded in moth species, and most of the GRs are bitter GRs (Cheng et al., 2017). However, few studies have functionally characterized bitter GRs. In D. melanogaster, the loss of bitter GRs was found to eliminate repellent behavior in response to specific noxious compounds. For example, Gr33a mutant flies could not avoid non-volatile repellents like quinine and caffeine (Moon et al., 2009), and mutation of Gr98b impaired the detection of L-canavanine (Shim et al., 2015). When the bitter GR PxutGr1 of P. xuthus was knocked-down by RNAi, the oviposition behavior in response to synephrine was strongly reduced (Ozaki et al., 2011). Knock-out of the bitter GR BmorGr66 in B. mori larvae resulted in a loss of feeding specificity for mulberry (Zhang et al., 2019). In this study, BL and EBL induced a strong response in the oocytes expressing PxylGr34, knock-down of PxylGr34 in the larvae of P. xylostella eliminated the feeding deterrence of BL, indicating that this bitter GR is specifically tuned to BL and EBL, and mediates the aversive response of larvae to BL and related compounds. Although the 24 tested compounds represent a wide range of compound profiles, the ligands of PxylGr34 could be more than BL and EBL. Given the high expression level of PxylGr34 in the taste organs of P. xylostella, we could not rule out the possibility that this gene also functions together with other GRs to perceive other compounds.

BL was the first brassinosteroid (BR) hormone to be discovered in plants. It was first isolated and identified from a crude extract of pollen from oilseed rape (Brassica napus L.), and was found to induce rapid elongation of pinto bean Phaseolus vulgaris internodes distinct from gibberellin-mediated stem elongation (Mitchell et al., 1970; Grove et al., 1979). Almost all plant tissues contain BRs, and they function to promote elongation and stimulate cell division, participate in vascular differentiation and fertilization, and affect senescence (Clouse and Sasse, 1998). As a C28 BR, BL exhibits the highest activity among all BRs and is distributed widely in the plant kingdom, along with other biosynthetically related compounds (Clouse and Sasse, 1998). Exogenous application of BL and its analog 24-epibrassinolide (EBL) to plants has been shown to increase their stress resistance (Clouse and Sasse, 1998).

Plant hormones, although generally found in small amounts and rarely toxic, play a key role in regulating plant growth, development, and resistance to biotic and abiotic stresses (Bari and Jones, 2009; Krouk et al., 2011; Wu and Baldwin, 2010). Jasmonic acid, salicylic acid, ethylene, and abscisic acid have been shown to be involved in priming plant defense responses against herbivorous insects and plant pathogens by activating related signal transduction pathways and changing the expression of defense-related genes (Bari and Jones, 2009). Jasmonic acid plays an important role in plant resistance to insects (Wang et al., 2019). Plants accumulate jasmonic acid and its derivatives upon wounding. Exogenous treatment with jasmonic acid activates the expression of hundreds of defense-related genes. Application of exogenous jasmonic acid to cabbage plants was shown to indirectly retard the development of P. xylostella larvae and reduce the pupal weight and female fecundity (Lv and Liu, 2005). Therefore, signaling molecules associated with induced plant defenses may be used as reliable cues by herbivorous insects. Up to now, only one study showed that Helicoverpa zea reacts to jasmonate and salicylate in plants, resulting in the activation of four of its cytochrome P450 genes that are associated with detoxification (Li et al., 2002). However, how the caterpillars eavesdrop the hormone signals remains a mystery. The present study provides the first evidence that P. xylostella can detect the plant hormone BL with a bitter GR. This reflects a new adaptation of insects to plant defenses.

Herbivorous insects have evolved counter adaptations against the chemical defenses of plants. The perception of bitter substances is an adaptation to avoid potentially toxic secondary plant metabolites. We can speculate that BL and EBL may influence insect development because BL and EBL have strikingly similar structures to that of ecdysteroid hormones in arthropods, such as 20-hydroxyecdysone (Fujioka and Sakurai, 1997). Their structures are so similar that BL and EBL show agonistic activity with 20-hydroxyecdysone in many insect species (Zullo and Adam, 2002). It has been shown that injection with 20 µg EBL was fatal to mid last-instar larvae of Spodoptera littoralis (Smagghe et al., 2002). The BL content differs widely among plant species; for example, it is 1.37 × 10−4 g/kg in Brassica campestris L. leaves and 1.25 × 10−6 g/kg in Arabidopsis thaliana leaves (Lv et al., 2014). Our results show that BL has the inhibitory effects on feeding and oviposition of P. xylostella, and the threshold concentration of BL for such behavioral inhibitions of P. xylostella is in the range of 10−4–10−3 g/kg, suggesting that BL plays a dual role of plant hormones and insect feeding/oviposition deterrents in plants.

There is a rich variety of bitter GRs in phytophagous insects, but only a few have been functionally characterized (Kasubuchi et al., 2018; Zhang et al., 2019). In this study, we showed that PxylGr34, a bitter GR highly expressed in larval head and adult antennae of P. xylostella, is tuned to the plant hormones BL and EBL, which mediates the aversive feeding/oviposition responses of P. xylostella to these compounds. These findings not only increase our understanding of the gustatory coding mechanisms of feeding/oviposition deterrents in phytophagous insects, but also offer new perspectives for using plant hormones as potential agents to suppress pest insects.

Materials and methods

Key resources table
Reagent type
(species) or
resource		DesignationSource or
reference		IdentifiersAdditional
information
Gene (Plutella xylostella)Muscle actin geneNCBIGenBank: AB282645.1
Commercial assay or kitRNeasy Plus Universal Mini KitQiagenCat# 73404
Commercial assay or kitQ5 High-Fidelity DNA PolymeraseNEBCat# M0491
Commercial assay or kitM-MLV reverse transcriptasePromegaCat# M1701
Commercial assay or kitSYBR Premix Ex TaqIITakaraCat# RR820
Commercial assay or kitmMESSAGE mMACHINE SP6AmbionCat# AM1340
Chemical compound, drug(+/−)-Abscisic acidSigma-AldrichCAS: 21293-29-8
Chemical compound, drugL-AlanineSigma-AldrichCAS: 56-41-7
Chemical compound, drugAllyl isothiocyanateSigma-AldrichCAS: 57-06-7
Chemical compound, drugAristolochic acidShanghai Macklin Biochemical Co.,Ltd, ChinaCAS: 313-67-7
Chemical compound, drugBrassinolideYuanyeshengwu Co., Ltd, ChinaCAS: 72962-43-7
Chemical compound, drug(+/−)-CamphorBeijing Mreda Technology Co., Ltd, ChinaCAS: 76-22-2
Chemical compound, drug(−)-CitronellalTokyo Chemical Industry Co., Ltd, JapanCAS: 5949-05-3
Chemical compound,
drug		24-EpibrassinolideShanghai Macklin Biochemical Co., Ltd, ChinaCAS: 78821-43-9
Chemical compound, drugD-FructoseSigma-AldrichCAS: 7660-25-5
Chemical compound, drugGibberellic acidYuanyeshengwu Co., Ltd, ChinaCAS: 77-06-5
Chemical compound, drugcis-3-Hexenyl salicylateShanghai Macklin Biochemical Co., Ltd, ChinaCAS: 65405-77-8
Chemical compound, drug20-HydroxyecdysoneYuanyeshengwu Co., Ltd, ChinaCAS: 5289-74-7
Chemical compound, drugIndole-3-acetic acidYuanyeshengwu Co., Ltd, ChinaCAS: 87-51-4
Chemical compound, drug(+/−)-Jasmonic acidTokyo Chemical Industry Co., Ltd, JapanCAS: 6894-38-8
Chemical compound, drugKinetinSigma-AldrichCAS: 525-79-1
Chemical compound, drugMethyl jasmonateSigma-AldrichCAS: 1211-29-6
Chemical compound, drugMethyl salicylateSigma-AldrichCAS: 119-36-8
Chemical compound, drugMyo-inositolSigma-AldrichCAS: 87-89-8
Chemical compound, drug(+/−)-NicotineSigma-AldrichCAS: 22083-74-5
Chemical compound, drugPutrescineAlfa AesarCAS: 110-60-1
Chemical compound, drugSinigrinSigma-AldrichCAS: 3952-98-5
Chemical compound, drugSpermidineSigma-AldrichCAS: 124-20-9
Chemical compound, drugSpermineSigma-AldrichCAS: 71-44-3
Chemical compound, drugD-SucroseSigma-AldrichCAS: 57-50-1
Software, algorithmGraphPad PrismGraphPad PrismRRID:SCR_0027988.3
Software, algorithmAdobe IllustratorAdobe systemsRRID:SCR_014198CC2018
Software, algorithmpCLAMP softwarepCLAMP softwareRRID:SCR_011323

Insects and plants

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P. xylostella was originally collected from the cabbage field of Institute of Plant Protection, Shanxi Academy of Agricultural Sciences, China. It is not specialized on peas. The insects were reared at the Institute of Zoology, Chinese Academy of Sciences, Beijing. The larvae were fed only with cabbage (Brassica oleracea L.) and kept at 26 ± 1°C with a 16L:8D photoperiod and 55–65% relative humidity. The diet for adults was a 10% (v/v) honey solution. Pea (Pisum sativum L.) plants were grown in an artificial climate chamber at 26 ± 1°C with a 16L:8D photoperiod and 55–65% relative humidity. The plants were grown in nutrient soil in pots (8 × 8 × 10 cm) and were 4–5 weeks old when they were used in experiments.

Care and use of X. laevis

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All procedures were approved by the Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences, and followed The Guidelines for the Care and Use of Laboratory Animals (protocol number IOZ17090-A). Female X. laevis were provided by Prof. Qing-Hua Tao (MOE Key Laboratory of Protein Sciences, Tsinghua University, China) and reared in our laboratory with pig liver as food. Six healthy naive X. laevis 18–24 months of age were used in these experiments. They were group-housed in a box with purified water at 20 ± 1°C. Before experiments, each X. laevis individual was anesthetized by bathing in an ice–water mixture for 30 min before surgically collecting the oocytes.

Sequencing and expression analysis of GR genes in P. xylostella

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We conducted transcriptome analyses of the P. xylostella moth antennae, foreleg (only tibia and tarsi), head (without antenna), and the 4th instar larval mouthparts. Total RNA was extracted using QIAzol Lysis Reagent (Qiagen, Hilden, Germany) and treated with DNase I following the manufacturer’s protocol. Poly(A) mRNA was isolated using oligo dT beads. First-strand complementary DNA was generated using random hexamer-primed reverse transcription, followed by synthesis of second-strand cDNA using RNaseH and DNA polymerase I. Paired-end RNA-seq libraries were prepared following Illumina’s protocols and sequenced on the Illumina HiSeq 2500 platform (Illumina, San Diego, CA). High-quality clean reads were obtained by removing adaptors and low-quality reads, then de novo assembled using the software package Trinity v2.8.5 (Haas et al., 2013). The GR genes were annotated by NCBI BLASTX searches against a pooled insect GR database, including GRs from P. xylostella (Engsontia et al., 2014; You et al., 2013; Yang et al., 2017) and other insect species (Guo et al., 2017; Xu et al., 2016; Robertson et al., 2003). The translated amino acid sequences of the identified GRs were aligned manually by NCBI BLASTP and tools at the T-Coffee web server (Notredame et al., 2000). The TPM values were calculated using the software package RSEM v1.2.28 (Li and Dewey, 2011) to analyze GR gene transcript levels.

Phylogenetic analysis

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Phylogenetic analysis of P. xylostella GRs was performed based on amino acid sequences, together with those of previously reported GRs of Heliconius melpomene (Briscoe et al., 2013) and B. mori (Guo et al., 2017). Amino acid sequences were aligned with MAFFT v7.455 (Rozewicki et al., 2019), and gap sites were removed with trimAl v1.4 (Capella-Gutiérrez et al., 2009). The maximum likelihood phylogenetic tree was constructed using RAxML v8.2.12 (Stamatakis, 2014) with the Jones-Taylor-Thornton amino acid substitution model. Node support was assessed using a bootstrap method based on 1000 replicates. The tree was visualized in FigTree Version 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

RNA isolation and cDNA synthesis

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The adult antennae, head (without antennae), forelegs (only tarsi and tibia), and ovipositor, and the larval head, thorax (without wing disc, thoracic legs, gut, or other internal tissues), thoracic leg, abdomen (without gut or other internal tissues) and gut were dissected immediately placed in a 1.5 mL Eppendorf tube containing liquid nitrogen, and stored at −80°C until use. Total RNA was extracted using QIAzol Lysis Reagent following the manufacturer’s protocol (including DNase I treatment), and RNA quality was checked with a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA). The single-stranded cDNA templates were synthesized using 2 μg total RNAs from various samples with 1 μg oligo (dT) 15 primer (Promega, Madison, WI, USA). The mixture was heated to 70°C for 5 min to melt the secondary structure of the template, then M-MLV reverse transcriptase (Promega) was added and the mixture was incubated at 42°C for 1 hr. The products were stored at −20°C until use.

PCR amplification of PxylGr34 from P. xylostella

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Based on the candidate full-length nucleotide sequences of PxylGr34 identified from our transcriptome data, we designed specific primers (Supplementary file 1). All amplification reactions were performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Beverly, MA, USA). The PCR conditions for amplification of PxylGr34 were as follows: 98°C for 30 s, followed by 30 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 1 min, and final extension at 72°C for 2 min. Templates were obtained from antennae of female P. xylostella. The sequences were further verified by Sanger sequencing.

Quantitative real-time PCR

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The qPCR analyses were conducted using the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) with SYBR Premix Ex Taq (TaKaRa, Shiga, Japan). The gene-specific primers to amplify an 80–150 bp product were designed by Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Supplementary file 1). The thermal cycling conditions were as follows: 10 s at 95°C, followed by 40 cycles of 95°C for 5 s and 60°C for 31 s, followed by a melting curve analysis (55–95°C) to detect a single gene-specific peak and confirm the absence of primer dimers. The product was verified by nucleotide sequencing. PxylActin (GenBank number: AB282645.1) was used as the control gene (Teng et al., 2012). Each reaction was run in triplicate (technical replicates) and the means and standard errors were obtained from three biological replicates. The relative copy numbers of PxylGr34 were calculated using the 2–ΔΔCt method (Livak and Schmittgen, 2001).

Receptor functional analysis

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The full-length coding sequence of PxylGr34 was first cloned into the pGEM-T vector (Promega) and then subcloned into the pCS2+ vector. cRNA was synthesized from the linearized modified pCS2+ vector with mMESSAGE mMACHINE SP6 (Ambion, Austin, TX, USA). Mature healthy oocytes were treated with 2 mg mL−1 collagenase type I (Sigma-Aldrich, St Louis, MO, USA) in Ca2+-free saline solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH = 7.5) for 20 min at room temperature. Oocytes were later microinjected with 55.2 ng cRNA. Distilled water was microinjected into oocytes as the negative control. Injected oocytes were incubated for 3–5 days at 16°C in a bath solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH = 7.5) supplemented with 100 mg mL−1 gentamycin and 550 mg mL−1 sodium pyruvate. Whole-cell currents were recorded with a two-electrode voltage clamp. The intracellular glass electrodes were filled with 3 M KCl and had resistances of 0.2–2.0 MΩ. Signals were amplified with an OC-725C amplifier (Warner Instruments, Hamden, CT, USA) at a holding potential of −80 mV, low-pass filtered at 50 Hz, and digitized at 1 kHz. Each of 24 compounds listed in Key Resources Table was diluted and the pH was adjusted to 7.5 in Ringer’s solution before being introduced to the oocyte recording chamber using a perfusion system. Data were acquired and analyzed using Digidata 1322A and pCLAMP software (RRID:SCR_011323) (Axon Instruments Inc, Foster City, CA, USA). Dose-response data were analyzed using GraphPad Prism software (RRID:SCR_002798 6) (GraphPad Software Inc, San Diego, CA, USA).

Electrophysiological responses of contact chemosensilla on the maxilla of larvae to BL, EBL, and sinigrin

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The tip-recording technique was used to record action potentials from the lateral and medial sensilla styloconica on the maxillary galea of larvae, following the protocols described elsewhere (Hodgson et al., 1955; van Loon, 1990; van Loon et al., 2002). Distilled water served as the control stimulus, as KCl solutions elicited considerable responses from the galeal styloconic taste sensilla (van Loon et al., 2002), and sinigrin served as the positive control stimulus (van Loon et al., 2002). Experiments were carried out with larvae that were 1–2 days into their final stadium (4th instar). The larvae were starved for 15 min before analysis. The larvae were cut at the mesothorax, and then silver wire was placed in contact with the insect tissue. The wire was connected to a preamplifier with a copper miniconnector. A glass capillary filled with the test compound, into which a silver wire was inserted, was placed in contact with the sensilla. Electrophysiological responses were quantified by counting the number of spikes in the first second after the start of stimulation. The interval between two successive stimulations was at least 3 min to avoid adaptation of the tested sensilla. Before each stimulation, a piece of filter paper was used to absorb the solution from the tip of the glass capillary containing the stimulus solution to avoid an increase in concentration due to evaporation of water from the capillary tip. The temperature during recording ranged from 22° to 25°C. Neural activity was sampled with a computer equipped with a Metrabyte DAS16 A/D conversion board. An interface was used (GO-box) for signal conditioning. This involved a second order band pass filter (−3 dB frequencies: 180 and 1700 Hz) (van Loon et al., 2002). The electrophysiological signals were recorded by SAPID Tools software version 16.0 (Smith et al., 1990), and analyzed using Autospike software version 3.7 (Syntech).

Dual-choice feeding bioassays

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Dual-choice feeding assays were used to quantify the behavioral responses of P. xylostella larvae to BL and EBL on pea leaves. Pea (Pisum sativum L.) is a neutral host plant of P. xylostella and widely used in the feeding preference analysis in this species (Thorsteinson, 1953; van Loon et al., 2002). These assays were based on the protocol reported by van Loon et al., 2002, with modifications. The axisymmetric pinnate leaf was freshly picked from 4-week-old pea plants grown in a climate-controlled room. One leaf was folded in half, and two leaf discs (diameter, 7 mm) were punched from the two halves as the control (C) and treated (T) discs, respectively. For the treated discs, 5 µL (13 µL/cm2) of the test compound diluted in 50% ethanol was spread on the upper surface using a paint brush. For the control discs, 5 µL 50% ethanol was applied in the same way. Control (C) and treated (T) discs were placed in a C-T-C-T sequence around the circumference of the culture dish (60 mm diameter ×15 mm depth; Corning, NY, USA). After the ethanol had evaporated (15 min later), a single 4th instar caterpillar (day 1), which had been starved for 6 hr, was placed in each dish. The dishes were kept for 24 hr at 23°–25°C in the dark, to avoid visual stimuli. Each dish was covered with a circular filter paper disc (diameter 7 cm) moistened with 200 µL ddH2O to maintain humidity. At the end of the test, the leaf discs were scanned using a DR-F120 scanner (Canon, Tokyo, Japan) and the remaining leaf area was quantified with ImageJ software (NIH). Paired-sample t-test was used to detect differences in the consumed leaf area between control and treated leaf discs.

Dual-choice oviposition bioassays

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A dual-choice oviposition bioassay was used to quantify the behavioral responses of P. xylostella mated female moths to BL. This assay was modified from the protocol reported by Gupta and Thorsteinson, 1960 and Justus and Mitchell, 1996. A paper cup (10 cm diameter ×8 cm height) with a transparent plastic lid (with 36 pinholes for ventilation) was used for ovipositing of the mated females. Fresh cabbage leaf juice was centrifuged at 3000 rpm for 5 min, and 60 μL of the supernatant was spread with a paintbrush onto polyethylene (PE) film from clinical gloves for 10 min to evaporate. Four culture dishes (35 mm diameter) (Corning, New York, NY, USA) covered with these PE films were placed on the bottom of each cup. This oviposition system was developed based on the biology of P. xylostella (Harcourt, 1957). On each of two diagonally positioned treatment films, 125 µL BL (13 µL/ cm2) diluted in 50% ethanol was evenly spread on the upper surface using a paint brush. On the other two diagonally positioned films, 125 µL 50% ethanol (control) was spread in the same way. After the ethanol had evaporated (30 min later), a small piece of absorbent cotton soaked with 10% honey-water mixture was placed in the center of the cup.

The pupae of P. xylostella were selected and newly emerged adults were checked and placed in a mesh cage (25 × 25 × 25 cm), with a 10% honey-water mixture supplied during the light phase. The female:male ratio was 1:3 to ensure that all the females would be mated. After at least 24 hr of mating time, the mated females were removed from the cage and placed individually into the oviposition cup during the light phase. After 24 hr at 26 ± 1°C with a 16L: 8D photoperiod and 55–65% relative humidity, the number of eggs on each plastic film was counted. Paired-samples t-test was used to detect significant differences in the number of eggs laid between the control and treatment films. siRNA preparation.

A unique siRNA region specific to PxylGr34 was selected guided by the siRNA Design Methods and Protocols (Takasaki, 2013). The siRNA was prepared using the T7 RiboMAX Express RNAi System kit (Promega, Madison) following manufacturer’s protocol. The GFP (GenBank: M62653.1) siRNA was designed and synthesized using the same methods. We tested three different siRNAs of PxylGr34 based on different sequence regions, and selected the most effective and stable one for further analyses. The oligonucleotides used to prepare siRNAs are listed in Supplementary file 2.

Oral delivery of siRNA

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The siRNAs were supplied to the larvae by oral delivery as reported elsewhere (Chaitanya et al., 2017; Gong et al., 2011), with some modifications. Each siRNA was spread onto cabbage leaf discs (Brassica oleracea) and fed to 4th-instar larvae. Freshly punched cabbage leaf discs (diameter 0.7 cm) were placed into 24-well clear multiple well plates (Corning, NY, USA). For each disc, 3 µg siRNA diluted in 6 µL 50% ethanol was evenly distributed on the upper surface using a paint brush. Both 50% ethanol and GFP siRNA were used as negative controls. After the ethanol had evaporated (20 min later), one freshly molted 4th-instar larva, which had been starved for 6 hr, was carefully transferred onto each disc and then allowed to feed for 12 hr. Each well was covered with dry tissue paper to maintain humidity. The larvae that had consumed the entire disc were selected and starved for another 6 hr, and then these larvae were used in the dual-choice behavioral assay, electrophysiological responses of contact chemosensilla on the maxilla, or for qPCR analyses as described above. The larvae that did not consume the treated discs were discarded.

Data analysis

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Data were analyzed using GraphPad Prism 8.3. Figures were created using GraphPad Prism 8.3 and Adobe illustrator CC 2018 (Adobe systems, San Jose, CA). Two-electrode voltage-clamp recordings, electrophysiological dose-response curves, and the square-root transformed qPCR data were analyzed by one-way ANOVA and Tukey’s HSD tests with two distribution tails. These analyses were performed using GraphPad prism 8.3. Electrophysiological response data and all dual-choice test data were analyzed using the two-tailed paired-samples t-test. Statistical tests and the numbers of replicates are provided in the figure legends. In all statistical analyses, differences were considered significant at p<0.05. Asterisks represent significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant. Response values are indicated as mean ± SEM; and n represents the number of samples in all cases.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all the figures.

References

  2. Book
    1. Bernays EA
    2. Chapman RF
    (1994)
    Host-Plant Selection by Phytophagous Insects
    New York: Chapman and Hall.
    1. Lv Y
    2. Liu S
    (2005)
    Effects of exogenous jasmonic acid-induced plant responses on development and growth of Plutella xylostella
    Chinese Journal of Applied Ecology 16:193–195.
  50. Book
    1. Schoonhoven LM
    2. van Loon JJA
    3. Dicke M
    (2005)
    Insect-Plant Biology
    New York: Oxford University Press.
    1. Schoonhoven LM
    2. van Loon JJA
    (2002)
    An inventory of taste in caterpillars: each species its own key
    Acta Zoologica Academiae Scientiarum Hungaricae 48:215–263.

Decision letter

  1. Kristin Scott
    Reviewing Editor; University of California, Berkeley, United States
  2. Meredith C Schuman
    Senior Editor; University of Zurich, Switzerland
  3. Sonja Bisch-Knaden
    Reviewer; Max Planck Institute for Chemical Ecology, Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The vast majority of herbivorous insects are specialized on a set of host plants and use a combination of lineage-specific and more ubiquitous chemicals synthesized by plants to discriminate among potential hosts. This study identifies a gustatory receptor in a mustard-specialized moth that responds to the plant steroid brassinide. This is an important advance as very few Grs have been implicated in the role of detecting plant compounds among truly herbivorous insects.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "A gustatory receptor tuned to the steroid plant hormone brassinolide in Plutella xylostella (Lepidoptera: Plutellidae)" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Noah Whiteman (Reviewer #1).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife in its current form. Please note that eLife aims for an efficient peer review process and rejects submissions in cases where revisions are likely to take longer than two months. However, should you address the concerns summarized in the following paragraph, we would be happy to consider a revised study as a new submission.

As you will see from the reviews below, the reviewers thought that the topic is interesting but raised conceptual and technical concerns which would require additional experiments to fully address. Reviewers 1 and 2 raised concerns about the framing of the study that require addressing. Reviewer 3 raised concerns about the tip recordings and behavioral experiments that also require addressing. The reviewers agree that it is essential for the authors to perform additional SSR experiments including knockdown animals and e.g. sinigrin as a positive control. The reviewers also recommend that the authors revise the text and figures, including some reorganization (e.g. move EBL experiments to supplementals). Reviewer three noted that a more detailed molecular characterization of PxylGr34 would be interesting, but the reviewers and editors agree that this would be beyond the scope of the study and do not consider it to be an essential revision.

Reviewer #1:

The vast majority of herbivorous insects are specialized on a set of host plants and use a combination of lineage-specific and more ubiquitous chemicals synthesized by plants to discriminate among potential hosts. Mechanistic information on this question is lacking, specifically regarding the identify and evolution of chemoreceptors that are responsible for mediating these behaviors, which are central to understanding the evolution of herbivorous insects. Although progress has been made on non-herbivorous drosophilids and two herbivorous drosophilids, we still lack basic mechanistic information from most herbivorous lineages. Here, Yang et al. focus on this question using the mustard-specialized diamondback moth Plutella xylostella. Through RNA sequencing and qPCR, they identified PxylGr34 as being highly expressed in adult female antennae and in larval tissues. By expressing PxylGr34 in Xenopus oocytes and subjecting them to 24 diverse phytochemicals, they found that the canonical plant hormones brassinolide and 24-epibrassinolide induce a strong response at increasing concentrations. This electrophysiological response is similarly seen from recordings from larval medial (but not lateral) sensilla styloconica. Behavioral assays with larvae and mated adult females in which individuals are given a choice between pea discs/plastic film, respectively, treated and untreated with BL, show aversion to BL in both life stages. In larvae, this aversion is reduced by feeding them PxylGr34 siRNA, which significantly, but not completely, reduced mRNA expression of PxylGr34 in their heads. Collectively, these results show compelling evidence that PxylGr34 very likely mediates the response to BL. While there is clearly still much to learn in terms of understanding the coding mechanisms of Grs, i.e. whether other Grs are necessary for this BL-induced response, this is significant because very few Grs have been implicated in the role of detecting plant compounds among truly herbivorous insects. This is also the first time a chemoreceptor has been identified that seems to be tuned to this particular plant hormone, a ubiquitous plant steroid. Where this manuscript can improve is the general framework and discussion and the importance of discriminating diverse phytochemicals and in particular how insects can use host cues associated with stress, including plant hormones, to avoid particular plants.

1) The expression patterns of PxylGr34 is really striking, which supports their interest in focusing on it. I think the framing of the paper isn't doing it justice. In the Results section under "Identification of the GR gene PxylGr34 in P. xylostella" they would benefit from framing it in a question driven way, i.e. they were interested in searching for candidate Grs that may be involved in host selection within a model herbivore, and they looked for candidates among those that had been annotated from the P. xylostella genome. They then looked specifically for candidates that were highly expressed, comparing several chemosensory organs. Intriguingly, PxylGr34 was highly expressed in the antennae, as well as in other gustatory tissues, relative to other Grs. Because the expression data across Grs provides the biggest piece of evidence for focusing on this Gr, I would pull Figure 1—figure supplement 2 out from the supplement and stick it in Figure 1 as an additional panel. That being said, just because it is highly expressed, doesn't mean that it is the most important Gr for enabling feeding discrimination on their host plants – it would certainly be interesting to do the same experiments with all the other Grs but that's out of the scope of this paper for now.

2) "Brassinolide (BL) is a C28 brassinosteroid (BR)…" this whole paragraph needs to be restructured so that it's clearer why they focused on brassinolides, a plant hormone, rather than the typical anti-herbivore plant defenses, as mentioned in the previous sentence, or rather than other plant hormones. They outline some interesting reasons, i.e. their ubiquity in the plant kingdom, their structural similarity to molting hormones, but more explanation would benefit their study-there is still a way to frame this that makes the choice to focus on BLs clearer. After reading the rest of the paper, it's clear now that they tested 24 different phytochemicals of various groups, and brassinolide was the one that activated PxylGr34. But it makes sense for the Introduction to discuss more generally what their initial thoughts or hypotheses were about the kinds of compounds that should be important for herbivorous insects. If they had an a priori reason to be interested in brassinolides, then discuss that here; otherwise, leave it for the Discussion section.

3) Subsection “BL and its analog EBL induced a strong response in the oocytes expressing PxylGr34” – it would be great to mention earlier in this paper that 24 phytochemicals were tested, in the Abstract and Introduction. It would have answered my earlier questions about why there was a focus on brassinolides. Also, it might be worth mentioning that brassinolides might not be the only stimulant of Gr34; although 24 is a rather large number of compounds to test, there still may be others. Also, I only see the chemical listed in Figure 2. It would be great to list them at least in the Materials and methods, with source information.

4) Discussion paragraph six – it would make sense if this last paragraph or some part of the discussion was framed around why insects would want to detect brassinolides. That's the main question I've had throughout this paper. Is there evidence that insects routinely detect plant hormones to regulate their behavior (I know this to be true, but a paragraph on this topic csould be important)? Do these hormones correlate well with the nutritional content, growth, stress, defense concentrations, etc. within the plants to provide a reliable cue for where to feed? From there, you can then speculate that perhaps the BL may influence insect development because of the structural similarity with ecdysteroids, and then that discussion flows well from there.

5) Subsection “Phylogenetic analysis” – neighbor joining (NJ) method – NJ is not a true phylogenetic method per se (it ignores character information completely), and so I suggest you repeated this analysis by using maximum likelihood inference or Bayesian inference (BI). NJ is based on genetic distances, whereas ML or BI uses the character information and a model of sequence evolution to explore the tree space before selecting the most likely tree. The general consensus is that more robust phylogenetic methods should be used for datasets like these.

6) Figure 1 and Figure 3: For Figure 1, I'd like to see all datapoints overlaid on each histogram. Same with Figure 3B and 3D and Figure 4, 5, 6 where practicable. All of the raw data should be deposited in an excel sheet that is easily accessible, so we can link each figure to a dataset readily (data for each figure on a different sheet in same file).

Reviewer #2:

The manuscript describes the expression pattern of gustatory receptors in taste organs of larvae and females of the cabbage moth. One of these receptors (GR34) belonging to the bitter receptors was found to have an especially high expression level. Heterologous expression of GR34 in Xenopus oocytes revealed a ubiquitous plant hormone, brassinolide, as the best ligand. Brassinolide turned out to be deterrent for foraging larvae and egg laying females. After silencing the expression of GR34 in larvae, the deterrent effect of the plant hormone was abolished. Although the manuscript adds valuable information about the function of gustatory receptors in insects, several points are not clearly enough explained to reach a broader audience.

1) Novelty of the research: The authors stress in the Introduction that the molecular basis of deterrent gustatory neurons in phytophagous insects has not been investigated yet. However, one publication they cite at the end of their discussion (Kasubuchi et al., 2018) could deorphanize several bitter GRs in the silk moth, and show the deterrent effect of a ligand of these GRs in larvae. It would be reasonable to mention this study already in the Introduction, and state in which way the present manuscript wants to add further insights, e.g. by silencing the receptor and testing oviposition behavior.

2) Role of the plant hormone brassinolide: It should be explained why it is interesting to test if insects can detect this ubiquitous plant hormone. Is brassinolide regularly tested in insect gustatory research? What kind of information might high doses of brassinolide that are necessary to elicit physiological and behavioral responses convey for the hungry larvae or egg-laying female? As the hormone is present across the plant kingdom and in almost every plant tissue it is difficult to imagine how it could be used to identify host plants.

3) Molecular work: A) How were the 21 GRs analyzed in Figure 1—figure supplement 2 selected from the 42 bitter GRs identified the phylogenetic tree? B) Why is there a difference in the expression pattern of GR34 in female tissue in Figure 1-S3 (antenna, head and foreleg) and in Figure 1 (only antenna)?

4) Effect of GR34 in egg laying behavior: The focus of the study was on larvae; however, it would be worth exploring the role of GR34 in female moths. Would it be possible to record from antennal gustatory sensilla of the female, and to silence GR34 also in females and investigate the effect on oviposition? It should be mentioned in the Introduction or Discussion that Plutella females touch the leaf surfaces with their antenna before egg laying to explain why gustatory receptors on the antenna might be useful for host plant choice.

Reviewer #3:

In this manuscript, the authors characterized PxylGr34 of P. xylostella. They identified the full-length coding sequencing of PxylGr34 based on public data and their unpublished RNA-seq data. They found that PxylGr34 is highly expressed in larva head as well as female antennae in a real-time PCR. Through functional studies, they show that PxylGr34 is specifically tuned to BL and EBL in Xenopus oocyte in vitro system. They further show that BL evoked electrophysiological response in the medial sensilla styloconica on maxillary galea of larvae. Finally, they show BL inhibited larvae feeding and female oviposition of P. xylostella.

The authors have done a good job of characterizing PxylGr34 in vitro analysis. However, this manuscript has to remedy several limitations to be published in eLife.

1) It will be great if they can show the molecular properties of PxylGr34- for example, whether they are ligand-gated channels or G-protein coupled receptors.

2) There are several issues with tip-recording.

i) It is not clear whether PxylGr34 is required for BL-evoked in vivo electrophysiological responses. A knockdown experiment is recommended.

i) Representative traces are hard to read, making it difficult to determine whether these are actual spikes upon BL stimulation. Also, please add a y-axis scale bar.

iii) Since it has been reported that Sinigrin and glucosinolates stimulate the same sensilla, it is beneficial to show the spikes evoked by these chemicals as a control.

iv) Please show whether EBL can also stimulate the GSNs in the medial sensilla.

v) It will be beneficial to show the schematic of larval taste organs.

3) The effect of EBL is not convincing. It will be helpful to provide a dose-response profile with a wide range of EBL.

4) It is hard to tell if the knockdown of PxylGr34 has an effect on feeding behavior. It seems that only control food feeding increased, in contrast to BL-containing food.

5) It is interesting whether PxylGr34 is only expressed in adult female antenna and involved in ovipositing. Is it expressed in male antenna? If it is, what is the role of PxylGr34 in male?

6) There is no description for Figure 2D in the main text.

7) The authors have to provide the experimental condition and data of the survival test with BL.

8) Typo Figure 1 legend “4th instar larvae” to “adult female.”

In general, this manuscript is premature and does not have a significant scientific impact to general readers of eLife.

https://doi.org/10.7554/eLife.64114.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

The vast majority of herbivorous insects are specialized on a set of host plants and use a combination of lineage-specific and more ubiquitous chemicals synthesized by plants to discriminate among potential hosts. Mechanistic information on this question is lacking, specifically regarding the identify and evolution of chemoreceptors that are responsible for mediating these behaviors, which are central to understanding the evolution of herbivorous insects. Although progress has been made on non-herbivorous drosophilids and two herbivorous drosophilids, we still lack basic mechanistic information from most herbivorous lineages. Here, Yang et al. focus on this question using the mustard-specialized diamondback moth Plutella xylostella. Through RNA sequencing and qPCR, they identified PxylGr34 as being highly expressed in adult female antennae and in larval tissues. By expressing PxylGr34 in Xenopus oocytes and subjecting them to 24 diverse phytochemicals, they found that the canonical plant hormones brassinolide and 24-epibrassinolide induce a strong response at increasing concentrations. This electrophysiological response is similarly seen from recordings from larval medial (but not lateral) sensilla styloconica. Behavioral assays with larvae and mated adult females in which individuals are given a choice between pea discs/plastic film, respectively, treated and untreated with BL, show aversion to BL in both life stages. In larvae, this aversion is reduced by feeding them PxylGr34 siRNA, which significantly, but not completely, reduced mRNA expression of PxylGr34 in their heads. Collectively, these results show compelling evidence that PxylGr34 very likely mediates the response to BL. While there is clearly still much to learn in terms of understanding the coding mechanisms of Grs, i.e. whether other Grs are necessary for this BL-induced response, this is significant because very few Grs have been implicated in the role of detecting plant compounds among truly herbivorous insects. This is also the first time a chemoreceptor has been identified that seems to be tuned to this particular plant hormone, a ubiquitous plant steroid. Where this manuscript can improve is the general framework and discussion and the importance of discriminating diverse phytochemicals and in particular how insects can use host cues associated with stress, including plant hormones, to avoid particular plants.

Thank you very much for your positive comments on our study. The summarization is very thoughtful and constructive. We agree that there is clearly still much to learn in terms of understanding the coding mechanisms of GRs. Based on our results, single GR (PxylGr34) expression is enough to cause Xenopus oocytes to respond to BL, implying other Grs are not necessary for this BL-induced response, but there is a possibility that this GR coupled with other GRs may have some new functions, which is what we would focus on in the next. In the new version, we revise the general framework, and rewrite two paragraphs in the Discussion about the insects using phytohormones associated with induced plant defenses as reliable cues, and their counter adaptations against the chemical defenses of plants.

1) The expression patterns of PxylGr34 is really striking, which supports their interest in focusing on it. I think the framing of the paper isn't doing it justice. In the Results section under "Identification of the GR gene PxylGr34 in P. xylostella" they would benefit from framing it in a question driven way, i.e. they were interested in searching for candidate Grs that may be involved in host selection within a model herbivore, and they looked for candidates among those that had been annotated from the P. xylostella genome. They then looked specifically for candidates that were highly expressed, comparing several chemosensory organs. Intriguingly, PxylGr34 was highly expressed in the antennae, as well as in other gustatory tissues, relative to other Grs. Because the expression data across Grs provides the biggest piece of evidence for focusing on this Gr, I would pull Figure 1—figure supplement 2 out from the supplement and stick it in Figure 1 as an additional panel. That being said, just because it is highly expressed, doesn't mean that it is the most important Gr for enabling feeding discrimination on their host plants – it would certainly be interesting to do the same experiments with all the other Grs but that's out of the scope of this paper for now.

Thank you so much for your suggestions. Accordingly, we reframe the Results section under “Identification of PxylGr34, a highly expressed GR gene in P. xylostella” in a question driven way. We also pull Figure 1—figure supplement 2 out from the supplement and make it as Figure 2A in the revised version.

2) "Brassinolide (BL) is a C28 brassinosteroid (BR)…" this whole paragraph needs to be restructured so that it's clearer why they focused on brassinolides, a plant hormone, rather than the typical anti-herbivore plant defenses, as mentioned in the previous sentence, or rather than other plant hormones. They outline some interesting reasons, i.e. their ubiquity in the plant kingdom, their structural similarity to molting hormones, but more explanation would benefit their study-there is still a way to frame this that makes the choice to focus on BLs clearer. After reading the rest of the paper, it's clear now that they tested 24 different phytochemicals of various groups, and brassinolide was the one that activated PxylGr34. But it makes sense for the Introduction to discuss more generally what their initial thoughts or hypotheses were about the kinds of compounds that should be important for herbivorous insects. If they had an a priori reason to be interested in brassinolides, then discuss that here; otherwise, leave it for the Discussion section.

We accept these good suggestions and add the related information in the revised Introduction. We move the paragraph about BL information to the Discussion.

3) Subsection “BL and its analog EBL induced a strong response in the oocytes expressing PxylGr34” – it would be great to mention earlier in this paper that 24 phytochemicals were tested, in the Abstract and Introduction. It would have answered my earlier questions about why there was a focus on brassinolides. Also, it might be worth mentioning that brassinolides might not be the only stimulant of Gr34; although 24 is a rather large number of compounds to test, there still may be others. Also, I only see the chemical listed in Figure 2. It would be great to list them at least in the Materials and methods, with source information.

We revise as “Functional analyses using the Xenopus oocyte expression system and 24 diverse phytochemicals showed that PxylGr34 is tuned to the canonical plant hormones brassinolide (BL) and 24-epibrassinolide (EBL).” in the Abstract. We also add several sentences in the Introduction as follows: “The chemical components in leaves of Brassica species, including sugars, sugar alcohols, amino acids, amines, glucosinolates and plant hormones, may be involved in such a process. Among these compounds, sinigrin and brassinolide (BL) have relatively higher concentrations in Brassica than in many other plant species (Fahey et al., 2001; Lv et al., 2014). Sinigrin has been proved as a feeding/ oviposition stimulant for P. xylostella (Gupta and Thorsteinson, 1960). The medial sensilla styloconica in the maxillary galea of P. xylostella larvae contain a GSN sensitive to sinigrin and other glucosinolates (van Loon et al., 2002). BL as a ubiquitous plant hormone has been widely studied in plant growth and development (Clouse and Sasse, 1998), but little is known about its behavioral effects on phytophagous insects.”

We add “It is worth pointing out that although the tested 24 compounds are a rather large compound profiles, the ligands of PxylGr34 could be more than BL and EBL. Given the high expression of PxylGr34 in the taste organs of P. xylostella, we could not rule out the possibility that this gene also functions together with other GRs.” in the Discussion.

We add “Each of 24 compounds listed in Table 1 was diluted and the pH was adjusted to 7.5 in Ringer’s solution before being introduced to the oocyte recording chamber using a perfusion system.” in the Materials and methods, and list the tested 24 phytochemicals with source information in the revised Table 1.

4) Discussion paragraph six – it would make sense if this last paragraph or some part of the discussion was framed around why insects would want to detect brassinolides. That's the main question I've had throughout this paper. Is there evidence that insects routinely detect plant hormones to regulate their behavior (I know this to be true, but a paragraph on this topic csould be important)? Do these hormones correlate well with the nutritional content, growth, stress, defense concentrations, etc. within the plants to provide a reliable cue for where to feed? From there, you can then speculate that perhaps the BL may influence insect development because of the structural similarity with ecdysteroids, and then that discussion flows well from there.

We accept this suggestion. We add a new paragraph in Discussion as follows.

“Plant hormones, although generally found in small amounts and rarely toxic, play a key role in regulating plant growth, development, and resistance to biotic and abiotic stresses (Bari and Jones, 2009; Krouk et al., 2011; Wu and Baldwin, 2010). […] However, how the caterpillars eavesdrop the hormone signals remains a mystery. The present study provides the first evidence that P. xylostella could detect the plant hormone BL with a bitter gustatory receptor, which reflects a new adaptation of insects to plant defenses.”

5) Subsection “Phylogenetic analysis” – neighbor joining (NJ) method – NJ is not a true phylogenetic method per se (it ignores character information completely), and so I suggest you repeated this analysis by using maximum likelihood inference or Bayesian inference (BI). NJ is based on genetic distances, whereas ML or BI uses the character information and a model of sequence evolution to explore the tree space before selecting the most likely tree. The general consensus is that more robust phylogenetic methods should be used for datasets like these.

We accept your suggestion and repeat the phylogenetic analysis using the maximum likelihood inference. The original Figure 1—figure supplement 1 is updated as a new figure (Figure 1) in the revised version based on the new phylogenetic analysis. The related sentences about the phylogenetic method was also revised in Materials and methods.

6) Figure 1 and Figure 3: For Figure 1, I'd like to see all datapoints overlaid on each histogram. Same with Figure 3B and 3D and Figure 4, 5, 6 where practicable. All of the raw data should be deposited in an excel sheet that is easily accessible, so we can link each figure to a dataset readily (data for each figure on a different sheet in same file).

We accept the suggestion and show all datapoints overlaid on each histogram in the related Figures (Figure 2, Figure 4B, 4D, 4F, and Figure 5, Figure 6, Figure 6—figure supplement 1, Figure 7, 8, and 9 in the revised version). All of the raw data are provided as Source data files in the revised version.

Reviewer #2:

The manuscript describes the expression pattern of gustatory receptors in taste organs of larvae and females of the cabbage moth. One of these receptors (GR34) belonging to the bitter receptors was found to have an especially high expression level. Heterologous expression of GR34 in Xenopus oocytes revealed a ubiquitous plant hormone, brassinolide, as the best ligand. Brassinolide turned out to be deterrent for foraging larvae and egg laying females. After silencing the expression of GR34 in larvae, the deterrent effect of the plant hormone was abolished. Although the manuscript adds valuable information about the function of gustatory receptors in insects, several points are not clearly enough explained to reach a broader audience.

1) Novelty of the research: The authors stress in the Introduction that the molecular basis of deterrent gustatory neurons in phytophagous insects has not been investigated yet. However, one publication they cite at the end of their Discussion (Kasubuchi et al., 2018) could deorphanize several bitter GRs in the silk moth, and show the deterrent effect of a ligand of these GRs in larvae. It would be reasonable to mention this study already in the Introduction, and state in which way the present manuscript wants to add further insights, e.g. by silencing the receptor and testing oviposition behavior.

Thank you for your suggestion. We accept it and cite the mentioned study as “Most recently, BmorGr16, BmorGr18, and BmorGr53 showed response to coumarin and caffeine in vitro, and the coumarin had feeding deterrent effect on B. mori larvae (Kasubuchi et al., 2018); …” in Introduction.

We rephrase the related statement in the revised version as follows:

“In order to uncover the molecular basis of P. xylostella perceiving feeding/ oviposition stimulants and deterrents, we re-examined all the GRs reported from the previous studies of P. xylostella. Through transcriptome analysis and qPCR, we identified one bitter GR (PxylGr34) highly expressed in the larval head and the adult antennae.

Subsequently, we functionally analyzed this GR with Xenopus oocyte expression system and RNAi, and found that PxylGr34 is tuned to BL as a feeding and oviposition deterrent of P. xylostella.”

2) Role of the plant hormone brassinolide: It should be explained why it is interesting to test if insects can detect this ubiquitous plant hormone. Is brassinolide regularly tested in insect gustatory research? What kind of information might high doses of brassinolide that are necessary to elicit physiological and behavioral responses convey for the hungry larvae or egg-laying female? As the hormone is present across the plant kingdom and in almost every plant tissue it is difficult to imagine how it could be used to identify host plants.

Thank you for your suggestions. We add the information in the Introduction as follows:

“The chemical components in leaves of Brassica species, including sugars, sugar alcohols, amino acids, amines, glucosinolates and plant hormones, may be involved in such a process. Among these compounds, sinigrin and brassinolide (BL) have relatively higher concentrations in Brassica than in many other plant species (Fahey et al., 2001; Lv et al., 2014). Sinigrin has been proved as a feeding/ oviposition stimulant for P.

xylostella (Gupta and Thorsteinson, 1960). The medial sensilla styloconica in the maxillary galea of P. xylostella larvae contain a GSN sensitive to sinigrin and other glucosinolates (van Loon et al., 2002). BL as a ubiquitous plant hormone has been widely studied in plant growth and development (Clouse and Sasse, 1998), but little is known about its behavioral effects on phytophagous insects.”

As far as we known, brassinolide (BL) was not tested in previous insect taste studies although BL shows agonistic activity with 20hydroxyecdysone in many insect species (Zullo and Adam, 2002).

This study shows that the hungry larvae avoid feeding and the egg-laying females avoid ovipositing on the plant with the high concentration of BL. We suggest that this is an adaptation of this insect species to potential toxic substances in plants because BL has a similar structure with insect molting hormones such as 20-hydroecdysone and may have detrimental effect on survival and development of P. xylostella.

The plant hormone BRs are present across the plant kingdom and in almost every plant tissue, but their content differs widely among plant 4 species. For example, the concentration of BL is 1.37×10-4 g/kg in Brassica campestris L. leaves, while it is 1.25×10-6 g/kg in Arabidopsis thaliana leaves (Lv et al., 2014). Our results show that the threshold concentration of BL for behavioral inhibition of P. xylostella is in the range of 10-4 –10-3 g/kg, so we suggest that P. xylostella could detect the plants or plant tissues with higher concentration of BL with this receptor.

3) Molecular work: A) How were the 21 GRs analyzed in Figure 1—figure supplement 2 selected from the 42 bitter GRs identified the phylogenetic tree? B) Why is there a difference in the expression pattern of GR34 in female tissue in Figure 1-S3 (antenna, head and foreleg) and in Figure 1 (only antenna)?

We analyzed the expression of all the 67 validated GRs of P. xylostella, by calculating the transcripts per million (TPM) values based on our transcriptome data. However, 46 GRs showed very low expression and TPM was zero, so we only list the 21 GRs. We add “The GRs that undetectable in the TPM analysis were not listed.” in the figure legend.

Generally, most genes showed consistent results between RNAsequencing and qPCR data. However, the inconsistent expression results would be observed between them especially when the genes were lower expressed in some tissues (Everaert et al., 2017). This fits the case of Gr34 here. In this study, the expression of Gr34 in the head and foreleg are much lower than in the antennae although Gr34 also has some expression in female head and foreleg based on the qPCR analysis.

4) Effect of GR34 in egg laying behavior: The focus of the study was on larvae; however, it would be worth exploring the role of GR34 in female moths. Would it be possible to record from antennal gustatory sensilla of the female, and to silence GR34 also in females and investigate the effect on oviposition? It should be mentioned in the Introduction or Discussion that Plutella females touch the leaf surfaces with their antenna before egg laying to explain why gustatory receptors on the antenna might be useful for host plant choice.

Thank you for your suggestions. We agree that it would be worth exploring the role of Gr34 in female moths. However, the gustatory sensilla in the female moth antenna are very small and dense, we had tried but it is very hard to get recordings from the single gustatory sensillum from female antennae.

We totally agree that it would be very meaningful to silence Gr34 also in females and investigate the effect on oviposition. We had tried to inject the siRNA into the female pupae, but all failed because the pupae of P. xylostella is so small that the injection harmed the pupae severely. Therefore, we only successfully carried out the larval RNAi experiment and measured physiological and feeding effects in this work.

Sure, P. xylostella uses antennae to pat leaf surfaces before egg laying. We add “P. xylostella mainly selects Brassica species as its host plants, and its females pat the leaf surfaces with their antennae before egg laying (Qiu et al., 1998).” in Introduction.

Reviewer #3:

In this manuscript, the authors characterized PxylGr34 of P. xylostella. They identified the full-length coding sequencing of PxylGr34 based on public data and their unpublished RNA-seq data. They found that PxylGr34 is highly expressed in larva head as well as female antennae in a real-time PCR. Through functional studies, they show that PxylGr34 is specifically tuned to BL and EBL in Xenopus oocyte in vitro system. They further show that BL evoked electrophysiological response in the medial sensilla styloconica on maxillary galea of larvae. Finally, they show BL inhibited larvae feeding and female oviposition of P. xylostella.

The authors have done a good job of characterizing PxylGr34 in vitro analysis. However, this manuscript has to remedy several limitations to be published in eLife.

1) It will be great if they can show the molecular properties of PxylGr34- for example, whether they are ligand-gated channels or G-protein coupled receptors.

Thank you for your suggestion. The molecular properties of GRs are a very important aspect in GR studies. Insect GRs are evolutionarily related to the insect olfactory receptors (ORs). The insect ORs have an inverted topology relative to GPCRs, and functions both in ligand-gated channels and G-protein coupled (Sato et al., 2008; Wicher et al., 2008). Similarly, insect GRs also have an inverted topology relative to GPCRs (Xu et al., 2012; Zhang et al., 2011), and may function in ligandgated ion channel (Sato et al., 2011). Given the close relationship between insect GRs and ORs, it could be that PxylGr34 functions both in ligandgated channel and G-protein coupled. We would further analyze the molecular properties of PxylGr34, and study its structure and function relationship in the next.

2) There are several issues with tip-recording.

i) It is not clear whether PxylGr34 is required for BL-evoked in vivo electrophysiological responses. A knockdown experiment is recommended.

We accept your suggestion. We carried out a new knockdown experiment and tested the electrophysiological responses, which result in the new figures, Figure 8B and 8C. We found that the frequency of spikes to BL elicited in the medial sensilla styloconica of the PxylGr34 siRNA treated larvae was decreased. We add this result in revised version.

i) Representative traces are hard to read, making it difficult to determine whether these are actual spikes upon BL stimulation. Also, please add a y-axis scale bar.

Good suggestions and we accept both of them. We rearrange the representative traces of BL stimulation in the revised Figure 4A; add the y-axis scale bar in the revised Figure 4A, 4C, 4E, and Figure 5A, Figure 8B.

iii) Since it has been reported that Sinigrin and glucosinolates stimulate the same sensilla, it is beneficial to show the spikes evoked by these chemicals as a control.

It is a very good suggestion and we accept it. We carried out a new electrophysiological experiment using sinigrin as a positive control. New data are present in two new figures, Figure 4E and 4F. As previously reported, the medial sensilla styloconica also exhibited vigorous responses to sinigrin. However, the spike amplitudes induced by sinigrin were larger than those induced by BL and EBL (Figure 4E and 4F). These results suggest that BL and EBL activate the same neuron, while sinigrin activates a different neuron in the sensillum. We add this result in the revised version.

iv) Please show whether EBL can also stimulate the GSNs in the medial sensilla.

We accept your suggestion and run a new experiment to test the electrophysiological responses of the GSNs in the medial and lateral sensilla to EBL. The results show that of the two pairs of sensilla styloconica in the maxillary galea of 4th instar larvae, the lateral sensilla styloconica had no response to EBL (Figure 4C and 4D); the medial sensilla styloconica exhibited vigorous responses to EBL at 3.3×10-4 M, and the spike amplitudes induced by BL and EBL were about the same (Figure 4A, B, C, and D). The spike amplitudes induced by sinigrin were larger than those induced by BL and EBL (Figure 4E and 4F). These results suggest that BL and EBL activate the same neuron, while sinigrin activates a different neuron in the sensillum. We add this result in the revised Figure 4C and 4D, and in the text.

v) It will be beneficial to show the schematic of larval taste organs.

It is a very nice suggestion, we accept it and add the schematic of larval taste organs in the revised Figure 4.

3) The effect of EBL is not convincing. It will be helpful to provide a dose-response profile with a wide range of EBL.

We accept your suggestion and run a new experiment. In the revised version, we provide a dose-response profile with a wide range of EBL in the Figure 6—figure supplement 1. We found that the feeding areas of larvae were significantly smaller on the leaf discs treated with EBL at concentrations of 10-4 M and above than on the control leaf discs, which is similar with BL. We add this result in the revised version.

4) It is hard to tell if the knockdown of PxylGr34 has an effect on feeding behavior. It seems that only control food feeding increased, in contrast to BL-containing food.

It is an important question, and then we repeated the treatment of the knockdown of PxylGr34 on the feeding behavior of 4th instar larvae. The results show that the PxylGr34 siRNA-treated larvae had no significant preference for control leaf discs over those treated with BL, which is consistent with the result in the original manuscript. It proves that the knock-down of PxylGr34 by RNAi abolishes BL-induced feeding inhibition, which is not caused by control food feeding increased.

5) It is interesting whether PxylGr34 is only expressed in adult female antenna and involved in ovipositing. Is it expressed in male antenna? If it is, what is the role of PxylGr34 in male?

It is a nice question. We newly tested the expression of PxylGr34 in the male antennae, and found that PxylGr34 also has high expression in male antennae. For P. xylostella is a typical specialist and mate detection is closely related with the chemical cues of the host plants, we speculate that PxylGr34 may take part in the judgement of host plants in both male and females. We add this result in the revised Figure 2C, and in the text.

6) There is no description for Figure 2D in the main text.

Sorry. Now we add the description for it (now Figure 3D) in revised version.

7) The authors have to provide the experimental condition and data of the survival test with BL.

Thank you for your suggestion. The experimental condition is provided in Materials and methods as “The axisymmetric pinnate leaf was freshly picked from 4-week-old pea plants grown in a climate-controlled room. One leaf was folded in half, and two leaf discs (diameter, 7 mm) were punched from the two halves as the control (C) and treated (T) discs, respectively. For the treated discs, 5 µL (13 µL/cm2 ) of the test compound diluted in 50% ethanol was spread on the upper surface using a paint brush. For the control discs, 5 µL 50% ethanol was applied in the same way. Control (C) and treated (T) discs were placed in a C-T-C-T sequence around the circumference of the culture dish (60 mm diameter × 15 mm depth; Corning, NY, USA). After the ethanol had evaporated (15 min later), a single 4th instar caterpillar (day 1), which had been starved for 6 h, was placed in each dish. The dishes were kept for 24 h at 23°–25°C in the dark, to avoid visual stimuli. Each dish was covered with a circular filter paper disc (diameter 7 cm) moistened with 200 µL ddH2O to maintain humidity.”

Considering BL did not affect larval survival but may reduce development time or have other detrmental effects because of its simularity with ecdysone, we delete the survival test with BL in the revised version, as suggested by Reviewer 1.

8) Typo Figure 1 legend “4th instar larvae” to “adult female.”

Sorry for this mistake. We correct it in the revised version.

https://doi.org/10.7554/eLife.64114.sa2

Article and author information

Author details

  1. Ke Yang

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing, Experiment design
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4138-3373

  2. Xin-Lin Gong

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Validation, Investigation
    Competing interests
    No competing interests declared

  3. Guo-Cheng Li

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Formal analysis, Validation
    Competing interests
    No competing interests declared

  4. Ling-Qiao Huang

    State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    Contribution
    Resources, Validation, Investigation
    Competing interests
    No competing interests declared

  5. Chao Ning

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Formal analysis, Validation
    Competing interests
    No competing interests declared

  6. Chen-Zhu Wang

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing, Experiment design
    For correspondence
    czwang@ioz.ac.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0418-8621

Funding

National Natural Science Foundation of China (31830088)

  • Chen-Zhu Wang

China Postdoctoral Science Foundation (2019M660792)

  • Ke Yang

National Key R and D Program of China (2017YFD0200400)

  • Chen-Zhu Wang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank the lab members Hao Guo, Jun Yang, Nan-Ji Jiang, and Rui Tang for their helps in the data analysis and comments, Yan Chen, Zi-Lin Li, Shuai-Shuai Zhang, and Ruo-Xi Shi for their assistance in tip-recording analysis. We thank Xi-Zhong Yan from Shanxi Agricultural University for the assistance in insect rearing, Prof. Qing-Hua Tao from MOE Key Laboratory of Protein Sciences, Tsinghua University for providing Xenopus laevis frogs. We also thank Prof. Bill Hansson from Max Planck Institute for Chemical Ecology, Germany, for his comments on this work. This work is funded by National Key R and D Program of China (Grant No. 2017YFD0200400), the National Natural Science Foundation of China (Grant No. 31830088), and China Postdoctoral Science Foundation (Grant No. 2019M660792).

Ethics

Animal experimentation: All procedures were approved by the Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences, and followed The Guidelines for the Care and Use of Laboratory Animals (protocol number: IOZ17090-A).

Senior Editor

  1. Meredith C Schuman, University of Zurich, Switzerland

Reviewing Editor

  1. Kristin Scott, University of California, Berkeley, United States

Reviewer

  1. Sonja Bisch-Knaden, Max Planck Institute for Chemical Ecology, Germany

Publication history

  1. Received: October 17, 2020
  2. Accepted: December 10, 2020
  3. Accepted Manuscript published: December 11, 2020 (version 1)
  4. Version of Record published: January 13, 2021 (version 2)

Copyright

© 2020, Yang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Ke Yang
  2. Xin-Lin Gong
  3. Guo-Cheng Li
  4. Ling-Qiao Huang
  5. Chao Ning
  6. Chen-Zhu Wang
(2020)
A gustatory receptor tuned to the steroid plant hormone brassinolide in Plutella xylostella (Lepidoptera: Plutellidae)
eLife 9:e64114.
https://doi.org/10.7554/eLife.64114

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