In contrast to the dominance of methionine derived glucosinolates in Arabidopsis thaliana (Gigolashvili et al., 2008), the major glucosinolates in B. vulgaris are derived from homo-phenylalanine, and their biosynthesis is largely unknown. Collectively, these glucosinolates are best named phenethyl glucosinolates. A number of genetic variants contain various phenethyl glucosinolates and yield different hydrolysis products upon damage (Agerbirk et al., 2014; Agerbirk and Olsen, 2015), conferring differential effects on insect herbivores (van Leur et al., 2008). The G- and P- type B. vulgaris differ in their content of stereochemical isomers (diastereomers with respect to hydroxyl groups and hence termed epimers) of 2-hydroxy-2 phenylethylglucosinolate (**Figure 1**; Kuzina et al., 2011). The Gtype mainly contains the (2S)-epimer (glucobarbarin, **2S**) and the P-type mainly contains the (2R)-epimer (epiglucobarbarin, **2R**), which are both assumed to be biosynthesized by hydroxylation of a common precursor, 2-phenylethylglucosinolate (**1**) (Kuzina et al., 2011). Two previous reports have investigated the genetics of this hydroxylation. In the first report, the gene coding for biosynthesis of **2S** was found to be a single dominant gene, while a rare phenotype dominated by **1**, devoid of a hydroxyl group, was controlled by a recessive allele of the same locus (van Leur et al., 2006). In the second report, quantitative trait locus mapping was applied to identify the genes involved in G- and P-type B. vulgaris glucosinolate polymorphism using a P × G-type derived F2 population (Kuzina et al., 2011). The genes determining the **2S**/**2R** difference between G-type and P-type were mapped on two chromosome regions spanning 20–60 cM (Kuzina et al., 2011). Also in this study, recombinant plants dominated by **1** rather than **2R** or **2S** were reported, in accordance with separate loci for biosynthesis of **2R** and **2S**, respectively, from **1**.

Despite these pioneering genetic investigations, the glucosinolate biosynthesis pathway has not been compared in molecular detail between the two plant types, neither has their DBM-feeding-responses been studied. RNA-seq allows simultaneous acquisition of sequences for gene discovery, as well as transcript identification involved in specific biological processes (Wang et al., 2013). In recent papers, using Illumina paired-end sequencing, we reported the transcriptome profile of G-type B. vulgaris at 0, 1, 4, 8, 12, 24, and 48 h (Wei et al., 2013) and P-type at 0 and 4 h after infestation by DBM (Zhang et al., 2015). These data offered sufficient information to study glucosinolate synthesis related genes and their regulation in different types of B. vulgaris in response to DBM.

In the present work, we evaluated the impact of constitutive and DBM-induced changes in glucosinolates of B. vulgaris, using G-type and P-type plants, which contain resistant and non-resistant saponins, respectively. Consequently, we identified genes that were likely to be involved in phenethyl glucosinolate biosynthesis in G- and P-type B. vulgaris, based on RNA-seq data by comparison with the known pathway of methionine derived glucosinolates in A. thaliana (Wittstock and Halkier, 2002). We then characterized the gene expression patterns in response to DBM. The sequences and expression of some genes in the pathway were additionally validated by molecular cloning and quantitative PCR (qPCR). Two genes controlling the S and R epimers of glucosinolates in the two genotypes were identified. These data revealed a correlation between glucosinolate production and gene expression, and provide a better understanding of the defensive strategy of DBM resistant and susceptible B. vulgaris in terms of glucosinolates. At the same time, these results will deepen our understanding of the biosynthesis of phenethyl glucosinolates at the molecular level.

## MATERIALS AND METHODS

## Plant Material and Cultivation

Seeds of B. vulgaris accessions B4 (P-type) and B44 (G-type) were obtained from the University of Copenhagen, Denmark (Agerbirk and Olsen, 2015). Accession B4 is identical to accession NGB23547 publicly available at www.nordgen.org. The F1 hybrid was generated by a G-type (male) × P-type (female) cross in the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, China. Plant growth conditions were the same as in previous pyrosequencing studies (Wei et al., 2013; Zhang et al., 2015). Barbarea vulgaris seeds were surfacedsterilized in 1% NaClO and sown into 10 × 10 cm pots filled with a mixture of peat soil. Plants were kept in a growth chamber at 25◦C/20◦C (light/dark), and 60% relative humidity, at 225µmol·m−2 ·s −1 light intensity, and on a 16 h:8 h (light:dark) photoperiod. Plants were watered as needed and fertilized with half-strength Hoagland's nutrient solution. Plants at 10 weeks old were used in this study.

## Insect Feeding Treatment

Diamondback moth larvae were originally obtained from a cabbage field in Taigu, Shanxi, China in the autumn of 2010 and reared on cabbage at 25◦C, 12 h:12 h photoperiod and 60% relative humidity. Three DBM third-instar larvae were inoculated on each fully extended leaf of the 10 weeks old B. vulgaris plants from time zero until the time of sampling (8 or 48 h); seven leaves per plant were inoculated. The transcriptome results showed that most genes were significantly induced after 4 h of infestation by DBM; therefore, this time point was set for qPCR. To obtain optimal glucosinolate induction, 8 and 48 h of infestation by DBM were used for glucosinolate analysis. Plants with DBM and DBM-free control plants were kept in a growth chamber under the same condition. After 48 h, the leaves of control plants (not including the petiole) were cut and immediately flash frozen in liquid nitrogen. For DBM-treated plants, the DBM larvae were removed with a brush and the leaves were harvested using the same method used to harvest the control. Five plants were used as biological replicates. All material was stored at −80◦C.

#### Glucosinolate Extraction and Analysis

Glucosinolates were extracted according to the method of La et al. (2009). Two hundred milligrams of freezedried leaf powder was weighed in a 15 mL tube and 5 mL of boiling 100% methanol was added. Glucotropaeolin (benzylglucosinolate) was added as an internal standard. Samples were then incubated at 80◦C for 15 min in a water bath. The mixture was centrifuged at 7000 × g for 10 min at 4 ◦C and the supernatant was decanted into another tube. The extraction was repeated twice from residues using the same procedure with 70% methanol (v/v). The three supernatants were combined and 2 mL of each glucosinolate extract was added to a mini-column filled with diethylaminoethanol (DEAE) Sephadex A-25 (Amersham Bio-sciences, Uppsala, Sweden) activated with 0.02 M NaAc (Sinopharm Chemical Reagent, Beijing, China), and desulfated by sulfatase (Dikma Technologies, CA, USA). After reaction at room temperature overnight (16 h), the desulfated glucosinolates were eluted with 2 mL de-ionized water and stored at −20◦C before to high performance liquid chromatography (HPLC) analysis.

Samples were analyzed by HPLC on an Agilent HP 1100 Series instrument equipped with a C-18 reversed-phase column (Nova-PakR, 3.9 × 150 mm, 5µm particle size) using 0.5 g·L −1 ammonium acetate (solvent A)–a mixture of 1 L 0.1 mol·L −1 ammonium acetate and 300 mL methanol (solvent B) gradient at a flow rate of 1 mL·min−1 (injection volume 20µL). The gradient was as follows: constant 100% (A) at 0–6 min, a linear gradient from 100 to 30% (A) at 6–21 min, a linear gradient from 30% to 0 (A) at 21–24 min, constant 100% (B) at 24–28 min, a linear gradient from 0 to 100% (A) at 28– 30 min, and constant 100% (A) at 30–35 min. The eluent was monitored by diode array detection between 200 and 400 nm. Desulfoglucosinolates were identified by comparing retention times and UV absorption spectra with those of known standards. Results are given asµmol·g −1 dry weight, calculated using peak areas and generally agreed relative response factors for UV detection at 229 nm: 0.95 in case of **1**, **2R**, **2S** and the internal standard benzylglucosinolate, 0.50 in case of the phenolic **3R**, 0.29 in case of **4**, 0.25 in case of **5**, and 0.20 in case of **6** (Agerbirk et al., 2015).

## Database for Glucosinolate Biosynthetic Genes Identification in *B. vulgaris*

Sequences representing the complete set of glucosinolate biosynthetic genes in A. thaliana were acquired from the TAIR database (www.arabidopsis.org). Data for G- and P-type B. vulgaris transcriptome sequence were obtained from previous pyrosequencing studies<EMAIL_ADDRESS>and EMBL/NCBI/SRA (accession numbers SRR1582492 and SRR1583630); Wei et al., 2013; Zhang et al., 2015]. We identified candidate genes related to glucosinolate biosynthesis and transcription factors of B. vulgaris using BLASTN with a cutoff E-value ≤ 1E-10.

## RNA Extraction and First-Strand cDNA Synthesis

Total RNA of the samples was isolated using an EasyPure R Plant RNA Kit (TransGen Biotech, China), according to the manufacturer's instructions. 800 ng of total RNA was reverse transcribed to synthesize first-strand cDNA using oligo dT primers and EasyScript R One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, China) and diluted 20 fold as templates for molecular cloning and qPCR.

#### Quantitative Real-Time PCR (qPCR)

Quantitative real-time PCR was performed on a StepOne™ Real-Time PCR System (Applied Biosystems), using the TransStart R Green qPCR SuperMix (TransGen Biotech), following the manufacturer's instructions. Primers were designed using Primer3web (version 4.0.0, http://primer3.ut. ee/; Untergasser et al., 2012). A list of genes and primers is shown in Table S1. The reaction volume was 20µL, including 0.4µL of 10 mM Forward and Reverse primer respectively, 10µL of 2 × TransStart Green qPCR SuperMix, 2.0µL of the cDNA sample, 0.4µL of Passive Reference Dye I, and 6.8µL of ddH2O. The thermal cycling profile was: 95◦C for 10 min; 40 cycles of 95◦C for 15 s, 59◦C for 15 s, 72◦C for 10 s; then 95◦C for 15 s, 60◦C for 1 min, ramping to 95◦C for 15 s. Three independent biological and technical replicates were performed. Data were analyzed using StepOne™ Software v.2.0 (Applied Biosystems). Tubulin was used as an internal control. The relative expression level were estimated by the 2−11CT method (Livak and Schmittgen, 2001).

## Gene Cloning, Sequencing, and Sequence Analysis

Reverse transcription (RT)-PCR cloning was performed to confirm the assembly quality of genes involved in glucosinolate biosynthesis. Specific PCR primers for the six selected genes were designed corresponding to the ends of longer unigenes of G-type and P-type B. vulgaris, using Primer3web (version 4.0.0, http://primer3.ut.ee/; Untergasser et al., 2012). The list of genes and primers is shown in Table S1. PCR was performed in a total volume of 50µL, including 5µL of 10 × PCR buffer, 5µL of 25 mM MgSO4, 3µL of 2 mM dNTPs, 1.5µL of each 10 mM primer, 1µL of 1.0 U/µL KOD-Plus-Neo polymerase (Toyobo, Osaka, Japan), 2µL of cDNA and 31µL of ddH2O, with the following reactions: an initial denaturation step at 94◦C for 2 min; followed by 35 cycles of 98◦C for 10 s and 68◦C for 60 s. The PCR products were separated on 1% (w/v) agarose gel and isolated using a MaxiGel Extraction Kit (CoWin Biotech, Beijing, China), ligated into the pEASY **R** -Blunt Cloning vector (TransGen Biotech, Beijing, China), and then transformed into Escherichia coli DH 5α. Positive clones were confirmed by PCR and sequenced using an ABI 3730 instrument (Applied Biosystems, CA, USA). Sequence data alignment and amino acid deduce and alignment were performed using DNAMAN version 8 (Lynnon, Quebec, Canada) with the default parameters.

#### Statistical Analysis

The SPSS 17.0 software package for Windows was used for all statistical analyses. The data were analyzed for significant differences using Tukey's HSD test at a significance threshold of p = 0.05.

## RESULTS

## A Pair of Phenethyl Glucosinolate Epimers is Dominant in G- and P-Type *B. vulgaris* and Inducible by DBM

We examined leaf damage, glucosinolate types and their quantities of G- and P-type B. vulgaris over 0, 8, and 48 h after DBM infestation. The DBM-resistance ability differed significantly between the G- and P-type B. vulgaris, as evaluated by the fraction of leaf area damaged. G-type plants were more resistant and only suffered minor injuries, while about a quarter of P-type leaves were damaged at 48 h after DBM infestation (**Figure 2**). Glucosinolate profiling indicated that seven types of major glucosinolates were present in the leaves of B. vulgaris (**Table 1** and **Figure 3**). The dominant class was aromatic glucosinolates with a phenethyl backbone, of which the most abundant compound was glucobarbarin (**2S**) in Gtype and epiglucobarbarin (**2R**) in the P-type (**Figures 1**, **3**); both of them were induced by DBM infestation (**Table 1**). As previously reported for the P-type, the phenolic glucosinolate **3R**, apparently a ring-oxidized derivative of **2R**, was also detected but at moderate levels. Neither **3R** nor its S-epimer has ever been detected in the G-type by specific HPLC-MS. However, for purely statistical reasons a trace signal with similar retention time was quantitated for the G-type (**Table 1**, **Figure 3**). Glucobrassicin (**4**) was the most abundant indole glucosinolate in both types, accompanied by the 4-methoxy derivative (**5**) and traces of the N-methoxy derivative (**6**). Indole glucosinolates accumulated more abundant in the G-type. Only levels of three individual glucosinolates could be induced by DBM infestation: **2S** (only in G-type), **2R** (only in P-type) and **4** (only in Gtype). Because the dominating glucosinolate in each plant type was induced, total glucosinolate levels were also "induced" in both types, but only due to changes in the three mentioned individual glucosinolates. The contents of both **2S** and **4** in the G-type were gradually induced at 8 and 48 h, while the Ptype's response to DBM infestation was more rapid: significant induction of epiglucobarbarin was observed at 8 h, with no significant difference with 48 h (**Table 1**). In relative terms, the induction of the glucobarbarins (**2R** and **2S**) was similar, around 1.4-fold in 48 h. This extend of induction was comparable to the mean induction of the indole glucosinolate glucobrassicin in the G-type (around 1.5-fold). In absolute terms the induction of **2R** in the P-type was moderately higher than the induction of **2S** in the G-type, reflecting a higher glucosinolate level in this type at our growth conditions (**Table 1**).

#### Prediction of Glucosinolate Metabolic Pathway in *B. vulgaris* and its Candidate Genes Response to DBM Infestation

To investigate the molecular basis of the glucosinolate biosynthesis in B. vulgaris, a glucosinolates metabolic pathway was deduced according to the KEGG pathway (PATHWAY: map00966) and previous reports (Sønderby et al., 2010; **Figure 4A**). The pathway is generally characterized into four stages: (i) chain elongation of selected precursor amino

acids (e.g., Met and Phe), (ii) core structure formation, (iii) secondary modification of the side chain, and (iv) degradation of glucosinolate. Forty-two G-type and 33 P-type unigenes were identified as candidates of the 30 enzymes in the pathway, by homology searching from our former reported B. vulgaris transcriptome dataset with the glucosinolate metabolic genes from A. thaliana as baits. The gene list and their corresponding Arabidopsis homolog AGI codes, as well as the sequence similarities with G- or P-type, are shown in Tables S2 and S4.

for 8 h; (C,F), leaves treated with three DBM third-instar larvae for 48 h.

The biosynthesis pathway of methionine derived glucosinolates starts from the side-chain elongation process catalyzing the parent amino acid deamination to form 2 oxo acid by a branched-chain amino acid aminotransferase (BCAT4; **Figure 4A** and Table S3). In our annotated B. vulgaris transcriptome unigene dataset, a unigene corresponding to BCAT4 was identified in both G- and P-type plants. It was significantly induced by DBM in the P-type, but not significantly in the G-type plant (**Figure 4B** and Table S3). The 2-oxo acid then enters a cycle of three successive transformations (Sønderby et al., 2010): (1) condensation with acetyl-CoA by MAM (methylthioalkylmalate synthase); (2) isomerization by IPMI LSU (isopropylmalate isomerase large subunit) and IPMI SSU (isopropylmalate isomerase small subunit); and (3) oxidative decarboxylation by IPMDH (isopropylmalate dehydrogenase). A similar chain elongation is also needed for biosynthesis of phenethyl glucosinolates (**Figure 1**, **1**–**3**), but would not be relevant for the tryptophan derived (**Figure 1**, **4**–**6**). One MAM, two IPMI LSU, two IPMI SSU, four IPMDH transcripts were identified in G-type while one MAM, one IPMI LSU, two IPMI SSU, and two IPMDH transcripts were identified in P-type plants. All of these genes were upregulated by DBM infestation in P-type plants but not in the G-type (**Figure 4B** and Table S3). Thereafter, the molecules are transaminated by BCAT3. Two unigenes encoding BCAT3 were identified in both G- and P-type plants, amongst, the genes in the P-type were induced by

FIGURE 3 | Quantification of glucosinolates from G-type, P-type, an d hybrid F 1 plants by HPLC after desulfation of the native metabolites and an added standard. The desulfated glucosinolates, in order of elution, are: 3R, (2 R)-2-hydroxy-2-(4-hydroxyphenylethyl) GSL (4-hydroxyepiglucobarbarin); 2R, (2 R)-2-hydroxy-2-phenylethyl GSL (epiglucobarbarin); 2S, (2 S)-2-hydroxy-2-phenylethyl GSL (glucobarbarin); I.S, Glucotropaeolin (internal standard); 4, 3-indolylmethyl GSL (glucobrassicin); 1. 2-phenylethyl GSL (gluconasturtiin); 5, 4-methoxy-3-indolylmethyl GSL (4-methoxyglucobrassicin); 6, N-methoxy-3-indolylmethyl. Trace levels of 3R in G-type represents maximum estimates based on integration of a trace signal at the same retention time, and is not proof of presence of this glucosinolate in the G-type.

DBM. The products of the above reactions then enter the core glucosinolate structure pathway.

The formation of the glucosinolate core structure is accomplished by 13 enzymes catalyzing five biochemical steps (glutathione S-transferase (GSTF), glutathione S-transferase tau (GSTU) are predicted and gamma-glutamyl peptidase (GGP) is a partially characterized enzyme; they are considered as one step along with CYP83 in previous and current reports; Grubb and Abel, 2006; Sønderby et al., 2010). It begins with the oxidation of the side-chain elongated amino acids to convert them to aldoximes by cytochromes P450 of the CYP79 family. There are seven CYP79 genes in the Arabidopsis genome and one of them (CYP79A2) uses Phe as substrate. A high similarity homolog of CYP79A2 was not found in B. vulgaris . However, three and two CYP79s were identified in G- and Ptype B. vulgarisrespectively. Among them, CL10668.Contig1−All in G-type and T\_Unigene\_BMK.15233 in P-type had higher expression levels and were induced by DBM. They are most

represents maximum estimates based on integration of a trace signal at the same retention time, and is not proof of presence of this glucosinolate

 in the G-type. Glucosinolate

 numbers according to *Figure 1*.

TABLE 1 |

Glucosinolate

 contents in rosette leaves of G-type and P-type

*Barbarea*

diamondback

 moth larvae infestation.

 *vulgaris* under likely the candidate genes for the enzyme involved in phenethyl glucosinolate biosynthesis. Next step is catalyzed by cytochrome P450 of the CYP83 family. Two unigenes in each G- and P-type B. vulgaris were identified as homologs to the two Arabidopsis CYP83 genes (CYP83A1 and CYP83B1). The CYP83B1 were classified and named CYP83B1v1 (G-type) and CYP83B1v2 (P-type) based on sequence comparisons. This was also done for additional B. vulgaris CYPs mentioned below. The next step involves conjugation with a sulfur donor to form a GSH conjugate. Two predicted enzymes, encoded by GSTF and GSTU, may be involved in this reaction, but this reaction can also happen non-enzymatically (Sønderby et al., 2010). Three homolog unigenes of GSTF, two homolog unigenes of GSTU were identified in G- and P-type B. vulgaris. The above steps were not significantly affected by DBM infestation. In Arabidopsis, there is good evidence that the sulfur donor is glutathione, γ-Glu-Cys-Gly, and its biosynthesis involves five committed enzymes, including ATPs, APR, OASTL, GSH1, and GSH2 (Geu-Flores et al., 2009, 2011). One unigene was discovered encoding GSH in G- and P-type, respectively. The GSH conjugate is then hydrolyzed by GGP (Geu-Flores et al., 2009) to form an Salkyl-thiohydroximate, which is subsequently degraded by C-S lyase SUPERROOT1 (SUR1) to form a thiohydroximate, which is in turn S-glucosylated by glucosyltransferase UGT74 to form a desulfoglucosinolate. One GGP, one SUR1 and two UGT74 homolog sequences were discovered in G- and P-type B. vulgaris, respectively. All of them were significantly induced by DBM infestation in both types of plants, except UGT74B1, in Gtype plants. The final step is catalyzed by desulfoglucosinolate sulfotransferase (SOT) to generate the glucosinolate itself, with 3-phosphoadenosine-5′ -phosphosulfate (PAPS) as the sulfate donor. Four and three unigenes were identified as SOTs in Gand P-type B. vulgaris. One of them, T\_Unigene\_BMK.12426 in the P-type had the highest expression level and was upregulated by DBM (**Figure 4B** and Table S3). PAPS is produced from adenosine-5′ -phosphosulfate (APS) through a two-step catalysis by ATP sulfurylase (ATPS) and APS kinase (APK; Sønderby et al., 2010). In the present study, two DBM inducible unigenes were discovered encoding APKs in G- and P-type B. vulgaris, respectively. By the end of these steps, the parent phenethyl glucosinolate, 2-phenylethylglucosinolate (**1**) would be produced.

Both methionine derived glucosinolates and phenethyl glucosinolates are further subjected to hydroxylation modification, resulting in increased structural diversity. However, existing genetic and enzymological knowledge concerns methionine-derived glucosinolates. The flavinmonooxygenase glucosinolate S-oxygenase (FMO-GSOX), alkenyl hydroxalkyl producing (AOP), Fe (II)-dependent oxygenase superfamily protein (GS-OH) and CYP81F2 are reported to take part in these processes and other side chain modification (Hansen et al., 2007; Wentzell et al., 2007; Li et al., 2008; Bednarek et al., 2009; Clay et al., 2009; Pfalz et al., 2009). Assuming that related genes would be responsible for hydroxylation of phenethyl glucosinolates, we searched for homologs in our transcriptome. Two FMO-GSOX and one CYP81F2 homolog sequences were discovered in G- and P-type B. vulgaris, respectively. These CYPs were classified and named CYP81F1v1 (G-type) and CYP81F1v2 (P-type). However, unigenes responsible for AOP were not detected. In this study, CL12207.Contig1−All in G- and T−Unigene−BMK.14596 in P-type B. vulgaris were homologous to the Arabidopsis GS-OH, which is responsible for modification of 3-butenyl glucosinolate to produce 2-hydroxy-3-butenyl glucosinolate. These two genes were expressed at high levels and were upregulated by 2.5- and 4.4-fold in G- and P-types, respectively, under DBM infestation (**Figure 4B**, Tables S2, S3).

Five unigenes in G-type plants were identified as homologs of genes encoding myrosinase, while only one unigene was found in P-type. All of these genes were slightly upregulated by DBM but did not fulfill the two-fold cutoff in both types (**Figure 4B** and Table S3).

#### Candidate Regulatory Transcription Factor Genes and their Response to DBM Infestation

It was reported that three transcription factors (IQD1-1, Dof1.1 and MYB) could regulate the expression of genes involved in glucosinolate metabolism in A. thaliana (Wang et al., 2011). Two and one homolog sequences were discovered encoding Dof1.1 in G- and P-type B. vulgaris, respectively. One unigene was predicted to encode IQD1-1 in both plant types. These two types of transcription factors were not significantly affected by DBM in both plant types (**Figure 4C** and Table S3). Six members of the MYB family (MYB28, 29, 34, 51, 76, and 122) are reported to regulate the biosynthesis of glucosinolates (Gigolashvili et al., 2008; Sonderby et al., 2010). From our B. vulgaris transcriptome, one member of each of MYB28, 29, 34, and 51 homolog was found in the G-type and one homolog of each of MYB28, 34, 51, and 76 and two homologs of MYB29 were identified in Ptype plants. MYB28 was downregulated in the P-type but not in the G-type B. vulgaris. MYB29 was significantly upregulated in both types, but accumulated more than 27-fold higher in the Ptype compared with the G-type plants after 4 h of infestation by DBM. While MYB34 accumulated more than nine-fold more in the G-type than in the P-type. MYB51 was downregulated in both plant types. MYB76 was only identified in P-type pants and was induced by DBM (**Figure 4C** and Table S3). Thus, the induction of the glucosinolate pathway is possibly regulated by MYB34 in the G-type and by MYB29 and MYB76 in the P-type.

## qPCR Confirmation of Glucosinolate Genes Expression Patterns

We confirmed the expression patterns of six genes responsible for glucosinolates biosynthesis in leaves of B. vulgaris before and after (4 h) DBM infestation using qPCR. The expression patterns of these genes are shown in **Figure 5**. Most genes showed good correlation with the profiles from transcriptome sequencing. The unigenes encoding MAM1, GGP1, and UGT74B1 showed the same expression patterns between the two genotypes, both were upregulated at 4 h after DBM infestation. While the transcription of myrosinases was suppressed in both plant types. The BCAT4 genes were significantly downregulated in G-type plants, but

FIGURE 4 | Potential glucosinolates biosynthesis and degradation pathway in *Barbarea vulgaris*. (A) Glucosinolate biosynthesis and degeneration pathway adopted from Arabidopsis thaliana. Because the enzymology in B. vulgaris is yet unknown, enzyme names (e.g., BCAT) in A. thaliana are indicated even if corresponding enzymes involved in aromatic glucosinolate biosynthesis in B. vulgaris would have other names and specificities (B) Differentially expressed genes in B. vulgaris types potentially involved in glucosinolate biosynthesis and degeneration. (C) Differentially expressed transcription factors potentially involved in regulating glucosinolate biosynthesis in B. vulgaris types. The color code indicates differential expression values and the scale on the right represents gene expression ration values, log2 of reads per kilo bases per million reads (RPKM) of plants treated 0 and 4 h of G-type and P-type after DBM feeding.  In contrast to BCAT4, the APK2 gene was stable in the G-type, while they were significantly upregulated in the P-type by DBM infestation.

## Molecular Cloning and Comparison of Genes Involved in Glucosinolates Metabolism

Several RT-PCR cloning experiments were performed to check the quality of the unigene assembly. cDNA sequences of six selected genes [BCAT4, CYP83A1, GGP1, SUR1, GS-OH (SHO in G-type and RHO in P-type)] from the glucosinolate biosynthetic pathway were isolated from both G- and P-type B. vulgaris by blunt end cloning and were subsequently sequenced using the Sanger method. The sequence similarities between the clones and corresponding unigenes were more than 99.2% pairwise identity, which validated the reliability of RNA-seq assembly (**Table 2**). Additionally, sequences similarities between G- and P-type were also compared, showing that the genes in both types were highly conserved (>99.0% pairwise identity) except for GS-OH, which showed significantly sequence variation (77.50% pairwise identity in coding DNA sequences and 65.48% identity in deduced amino acid sequences) between the two types (**Table 2**, **Figure 6**). The sequence diversity of GS-OH could be responsible for the different glucobarbarin epimers of the G-type and Ptype glucosinolates (glucobarbarin, **2S**, vs. epiglucobarbarin, **2R**). Furthermore, the known biochemical function of GS-OH is equivalent to the functions envisioned for SHO and RHO:

β-hydroxylation (**Figure 1**) of a glucosinolate side chain (Hansen et al., 2008). Thus, we named the GS-OH in G- and P-type separately as SHO and RHO, respectively, in accordance with a previous hypothesis put forward by Agerbirk et al. (2015).

## A Genetic Model in Which SHO and RHO Produce the S and R Epimers of Glucobarbarins in *B. vulgaris*

To test the hypothesis that CL12207.Contig1−All and T−Unigene−BMK.14596 are the GS-OH genes responsible for the glucosinolate epimers in the G-type (SHO) and P-type (RHO) B. vulgaris, we generated F1 plants by hybridization of G- and P-type plants and analyzed the correlation between gene expression and glucosinolate production. The SHO and **2S**, RHO and **2R** co-occurred in G-, P-type, and F1 plants (**Figure 7**). For testing co-segregation, cooccurrence of gene and suggested glucosinolate product in F2 plants was needed. Unfortunately, F2 plants were not available in this project. Indeed, an F2 generation from a G-type × P-type cross appears to have been obtained only once in the literature (Kuzina et al., 2011), while further F2 progenies have not been published, possibly due to a highly frequent sterility barrier between the accessions of the types that have so far been tested. Based on the available results, we established a double-codominance gene model to explain the **2S** and **2R** inheritance in the two types of B. vulgaris. In this model, the double heterozygote SsRr plants (F1) generated by hybridization of SSrr (G-type, mainly containing **2S**) and ssRR (P-type, mainly containing **2R**) will contain both epimers (**2S** and **2R**; **Figures 3**, **7**). Therefore, F2 populations should contain recessive homozygous plants (ssrr), which only accumulated precursor glucosinolate, **1** (or traces of the glucobarbarin epimers if additional minor genes were present). A previous study showed that the profile of five plants in 129 F2 B. vulgaris population were dominated by **1** (Kuzina et al., 2011), which supports our codominance-gene model. The model is further supported by observation of low levels of epiglucobarbarin in so-called "NAS-forms" of the G-type devoid of glucobarbarin (van Leur et al., 2006; Agerbirk et al., 2015).

## DISCUSSION

Glucosinolates can be divided into groups according to their amino acid precursor, of which we consider three: methionine (Met), phenylalanine (Phe), and tryptophan (Trp) including chain-elongated homologs of the former two. Glucosinolates derived from Met (including chain elongated homologs) and Trp are well-studied and their biosynthetic pathways have been identified in the model plant A. thaliana (Sønderby et al., 2010). These served as a model for our search for genes involved in the biosynthetic pathway of aromatic Phe-derived glucosinolates. This is currently unknown, although a recent review lists a couple of genes that may be involved in their biosynthesis in natural or engineered systems (CYP79A2, CYP83A1, CYP83B1, SUR1, UGT74B1, and SOT16; Baskar et al., 2012). In an evolutionary perspective, Phe-derived glucosinolates should be divided in non-chain elongated glucosinolates directly derived from Phe and named "benzyl glucosinolates," and chain elongated derived from homoPhe and named "phenethyl glucosinolates." The former, without chain elongation, are believed to be an ancient character, while the latter seem more recent and occur as the core structure gluconasturtiin (**1**) in roots of most cruciferous crops, and as oxidized derivatives in Barbarea and occasionally elsewhere. B. vulgaris is dominated by oxidized phenethyl glucosinolates (Agerbirk et al., 2015), making it an ideal plant for the study of the phenethyl glucosinolate biosynthetic pathway including side chain decoration. In this study, a potential glucosinolate biosynthesis pathway was manually predicted by homology with biosynthesis of Met-derived glucosinolates in Arabidopsis and a number of candidate genes in this pathway were discovered in G- and P-type B. vulgaris. As Met-derived glucosinolates are not known from Barbarea, the genes would seem to be involved in biosynthesis of phenethyl glucosinolates. Our model will serve as a starting point for exploring the biosynthesis of phenethyl glucosinolates at the molecular level. Furthermore, we found the GS-OH candidate genes SHO and RHO in G- and P-type plants, which are most likely responsible for the stereospecific biosynthesis of glucobarbarin and its epimer, epiglucobarbarin. The functions of the genes identified in this study require confirmation by further studies, such as expression in A. thaliana. The transcription factors involved in regulating the glucosinolate biosynthetic pathway, including Dof1.1, IQD1-1, and MYB, were also identified successfully in G- and P-type B. vulgaris. Based on their expression patterns, the MYB34 in the G-type and MYB29 and MYB76 in the P-type were deduced as regulators of the glucosinolate induction in the DBM response.

Recently, it has been speculated that resistant and susceptible plant types could have diverged during the ice age because of geographical isolation and adaption to their new environment, leading to the formation of different evolutionary lineages and taxa, which differ in their resistance, hairiness, saponins, flavonoids, and glucosinolates (Hauser et al., 2012; Christensen et al., 2014). The results of the present study showed that

upstream genes of GS-OH were highly conserved during evolution: the sequence similarity between the two genotypes exceeded 98.9%. Unexpectedly, the SHO and RHO have significantly sequence variation between G- and P-type B. vulgaris, as low as 77.5% in coding DNA sequences and 65.48% identity in deduced amino acid sequences (**Figure 6**). Thus, we consider them as two independent genes that may have diverged during the separation of the two types.

There is ample evidence that glucosinolates not only function in defense against generalist herbivores, but also play a key role in host recognition for crucifer specialist insects (Mewis et al., 2005; Badenes-Pérez et al., 2011). Previous reports on A. thaliana indicated that both generalist and specialist insects can induce glucosinolate synthesis pathways, while the transcription of myrosinases was suppressed (Ku´snierczyk et al., 2007). In our previous study using the same data set, the P-type

glucosinolate biosynthesis pathway was not over-represented among the upregulated pathways by a hypergeometric test, mainly because many differentially expressed genes have not been annotated to the glucosinolate biosynthesis pathway by the automatic KEGG annotation pipeline. In our present study, the glucosinolate biosynthesis pathways were constructed manually and refined, and the expression level of the most glucosinolate synthesis genes were increased in G- and P-type B. vulgaris after DBM infestation, as revealed by transcriptome and qPCR experiments, which is consistent with previous reports in Arabidopsis and Brassica plants (Ku´snierczyk et al., 2007).

Herbivory is probably a multifaceted challenge of plants given that the wounds from the herbivore provide a direct access for pathogenic microbes as well as increased evaporation, stress from released phytochemicals, etc. Hence, it is likely that plant responses to insect herbivory should include not only defenses against the herbivore, but also defenses against a variety of microbes and other stresses. Furthermore, induction responses may not be specific for each herbivore, but produce

a response that on average has defensive properties against a range of frequent herbivores. For these reasons, it is not surprising that some glucosinolates were induced by DBM larvae despite the resistance of the larvae to this defense. Indeed, a similar induction of **2S** and **2R** by a flea beetle was recently reported (van Mölken et al., 2014). Quantitatively, the reported induction was similar to the induction reported here. However, we find it striking that the massive tissue damage in the P-type (**Figure 1**) did not result in induction of the indole glucosinolate **4**, which is in many plant species a highly inducible glucosinolate (Bodnaryk, 1992; Hopkins et al., 1998; Bartlet et al., 1999). In contrast, the modest tissue damage in the G-type (**Figure 2**) never-the-less induced **4**. Furthermore, it is interesting that the phenolic **3R**, believed to be biosynthesized from **2R**, was not induced in the P-type, although the apparent precursor was induced. In contrast, the phenolic **3R** was reported to be many fold induced in the transition from summer to fall (Agerbirk and Olsen, 2015) suggesting that environmental regulation of glucosinolate hydroxylation in B. vulgaris is complex. A gene sequence (PHO) for this hydroxylation of **2R** to **3R** is not suggested here, but the availability of the P-type transcriptome (Zhang et al., 2015), and the present refinement, now provide candidates for future investigations of glucosinolate regulation in the species. From this investigation, the relevant gene would be expected to be unique for the P-type and not be induced by DBM herbivory. This hydroxylation is known to have functional significance, as the hydrolysis product of **3R** is a thiazolidine-2 one, in contrast to the oxazolidine-2-thione produced from **2R** and **2S**.

The DBM is one of the most destructive pests of crucifer crops causing about \$ 4-5 billion loss annually in the world (Furlong et al., 2013). It is reported to have developed resistance to all major classes of insecticides including Bacillus thuringiensis (Bt) insecticidal proteins (Shelton, 2004; Furlong et al., 2013). In China, high dose of insecticide with short interval time are commonly used to control DBM, which causes severe environmental damage and food contamination. Therefore, an integrated pest management is urgently needed. Previous studies on agricultural uses of B. vulgaris mostly focus on the use as a "dead-end" trap crop (Lu et al., 2004; Badenes-Pérez et al., 2005) and mining resistance genes for breeding insect resistant cultivars (Wei et al., 2013; Khakimov et al., 2015; Zhang et al., 2015). In recent reports, we have confirmed the resistance-properties of G-type B. vulgaris to a contemporary Chinese field-isolate

#### REFERENCES

of DBM (Wei et al., 2013; Liu et al., 2015b; Zhang et al., 2015), further implying the application potential of this wild crucifer in the DBM controlling. Our present research shows that DBM infection induced the content of glucosinolate, an oviposition attracting signal for DBM, in agreement with usage of B. vulgaris as a "dead-end" trap for DBM control. On the other hand, DBM chewing can be envisioned to expose the plant to pathogenic microbes which may lead to additional loss for Brassicaceae vegetable production. The DBM induction of glucosinolates possibly could reduce pathogenic microbes' access via the wounds rather than waste of resources, thus improving insect resistance or tolerance, respectively, of G-type and P-type B. vulgaris.

In conclusion, the present study identified genes involved in glucosinolate biosynthesis of G- and P-type B. vulgaris, and characterized the relationship between gene expression patterns and glucosinolate contents in response DBM. These findings will deepen our understanding of the biosynthesis of the phenethyl group of aromatic glucosinolates at the molecular level and provide the basis for further investigation of the molecular ecology of insect resistance in B. vulgaris plants.

#### AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: XL, TL, XZ. Performed the experiments: TL, XZ, HY, JS. Analyzed the data: TL, XZ, XL, NA, YQ, HW, DS. Wrote the paper: TL, XZ, XL, NA. All authors read and approved the final manuscript.

#### ACKNOWLEDGMENTS

This work was supported by grant from the National Key Technology R & D Program of the Ministry of Science and Technology of China (2013BAD01B04-2), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS). We thank Dr. David Nelson for naming the discovered B. vulgaris CYPs.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 00083

in watercress types with contrasting chromosome number. J. Agric. Food Chem. 62, 9586–9596. doi: 10.1021/jf5032795

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Liu, Zhang, Yang, Agerbirk, Qiu, Wang, Shen, Song and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Development of a Susceptibility Index of Apple Cultivars for Codling Moth, *Cydia pomonella* (L.) (Lepidoptera: Tortricidae) Oviposition

*Neelendra K. Joshi1,2\*†, Edwin G. Rajotte1, Clayton T. Myers3, Greg Krawczyk2 and Larry A. Hull2*

*1 Department of Entomology, Pennsylvania State University, University Park, PA, USA, 2 Entomology, Fruit Research and Extension Center, Pennsylvania State University, Biglerville, PA, USA, 3 United States Department of Agriculture – Agricultural Research Service, Appalachian Fruit Research Station, Kearneysville, WV, USA*

#### *Edited by:*

*Alison Jane Karley, The James Hutton Institute, UK*

#### *Reviewed by:*

*Shimat Villanassery Joseph, University of California, USA Jhalendra Rijal, University of California, USA*

> *\*Correspondence: Neelendra K. Joshi<EMAIL_ADDRESS>
#### *†Present address:*

*Neelendra K. Joshi, Department of Entomology, University of Arkansas, 319 Agriculture Building, Fayetteville, AR 72701, USA*

#### *Specialty section:*

*This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science*

*Received: 30 June 2015 Accepted: 29 October 2015 Published: 13 November 2015*

#### *Citation:*

*Joshi NK, Rajotte EG, Myers CT, Krawczyk G and Hull LA (2015) Development of a Susceptibility Index of Apple Cultivars for Codling Moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) Oviposition. Front. Plant Sci. 6:992. doi: 10.3389/fpls.2015.00992* Codling moth (CM), *Cydia pomonella* (L.) (Lepidoptera: Tortricidae) is a major fruit feeding pest of apples. Understanding susceptibility differences of various apple cultivars to CM oviposition is an important step in developing resistant varieties as well as monitoring and management strategies for this pest in apple orchards planted with mixed-cultivars. In this context, oviposition preferences of CM for the fruits of different apple cultivars were studied in laboratory bioassays using a series of no-choice and multiple-choice tests in 2006, 2007, and 2008. In 2006 and 2007, 10 apple cultivars, *viz*., Arlet, Fuji, Gala, Golden Delicious, Honeycrisp, Pristine, Delicious, Stayman, Sunrise, and York Imperial were evaluated, while in the 2008 tests, Golden Delicious, Honeycrisp, and York Imperial were evaluated. During the 2006 tests, preferred apple cultivars for CM oviposition were Golden Delicious and Fuji, while the least preferred were Arlet, Pristine, Sunrise, and Honeycrisp. Similarly, during the 2007 tests, Golden Delicious, Fuji and Stayman remained the preferred cultivars, while Arlet, Honeycrisp, Pristine, and Sunrise remained the least preferred cultivars. In the 2008 tests, Golden Delicious and Honeycrisp were the most and least preferred cultivars, respectively. Based on the oviposition preferences from these bioassays, a susceptibility index for each cultivar was developed. This index may be used as a standard measure in cultivar evaluations in breeding programs, and may assist fruit growers and crop consultants to select the most appropriate cultivar(s) for monitoring and detecting the initial signs of fruit injury from CM in an apple orchard planted with mixed-cultivars.

Keywords: apple cultivars, codling moth, oviposition, susceptibility, host preference, Honeycrisp, Gala, Golden Delicious

## INTRODUCTION

The codling moth (CM), *Cydia pomonella* (L.) (Lepidoptera: Tortricidae), probably originating in Europe (Pashely and Bush, 1979), is a serious pest of apples worldwide (Dean, 1989; Barnes, 1991; Witzgall et al., 2008), and causes significant economic damage to pome fruits. CM is closely associated with apple, *Malus pumila* Miller (Rosaceae), however, other species belonging to various plant families, such as pears (*Pyrus* sp.), quinces (*Cydonia oblonga* Mill.), peaches (*Prunus persica*)

**138**

(L.), wild haws (*Crataegus* sp.), English walnuts (*Juglans regia* L.) (Shelford, 1927), plum (*Prunus* sp.), nectarines (*Prunus* sp.), and sweet cherry (*Prunus avium* L.) are also reported as host plants (Madsen and Borden, 1954; Barnes, 1991).

CM completes its life cycle in four different stages, viz., egg, larva, pupa, and adult. The eggs of CM are disk-shaped, flattened, or ovate, and measure about 0.98 by 1.25 mm in diameter (Putman, 1963; Dean, 1989). Development time for eggs largely depends on temperature, and upon hatching on or near fruits, the CM larva penetrates the epidermis of the fruit, feeds on the fruit pulp and eventually making its way to the core, where the larva feeds on the seeds. After feeding on the seeds, mature larvae (fifth instar) make their way to the periphery of the fruit and make a hole to exit from the fruit. Larvae then search for a suitable place for spinning a cocoon to pupate or enter into diapause in order to avoid unfavorable environmental conditions (for instance, winter). Upon emergence, adult moths feed on the exudates from fruits and other parts of their host plants (Geier, 1963), and copulate during the dusk period (Van Leeuwen, 1929). Multiple mating occurs in both sexes (Gehring and Madsen, 1963; Howell et al., 1978), and fecundity of the female varies from host to host (Phillips and Barnes, 1975).

The fruits and leaves of apple tree release different volatiles that attract female moths to the host tree and regulate hostfinding mechanisms (Wearing et al., 1973; Sutherland et al., 1974; Hern and Dorn, 1999). The main source of attraction of CM to apple trees and other host plants are kairomones (i.e., *E, E* α-farnesene and *Z, E* α-farnesene), which are naturally occurring sesquiterpene compounds (Wearing and Hutchins, 1973). Kairomones likely induce female moths to lay their eggs directly on fruit or in close vicinity of fruits and fruit clusters (Wildbolz, 1958). Oviposition in CM is mainly stimulated by a sesquiterpene compound known as α-farnesene (Wearing and Hutchins, 1973). Most eggs (up to 90%) are laid within 10 cm of a fruit (Blomfield et al., 1997). The size of fruit clusters also has significant impacts on the distribution of eggs. The number of eggs laid on fruit and nearby leaves increases with an increase in the size of the fruit cluster (Jackson, 1979; Blomfield et al., 1997). In a field environment with different apple cultivars, CM females deposit eggs on fruits, as well as both sides of spur and shoot leaves (Joshi et al., 2009; Joshi, 2011).

Female CM may discriminate among apple cultivars for oviposition as they do for other hosts such as walnut (Shelton and Anderson, 1990). The fruit size of walnut and its chemical composition varies across different commercial cultivars (Tulecke and McGranahan, 1994) and are known to affect oviposition preferences (Bezemer and Mills, 2001). In addition, the maturity level of fruits of different walnut cultivars is also known to affect the oviposition preferences of CM, as the female moths prefer to oviposit on mature rather than immature fruits (Olson, 1977; Shelton and Anderson, 1990). However, in the case of apple, such studies related to oviposition/host preference are restricted to several cultivars with very few published reports (Phillips and Barnes, 1975; Blomfield et al., 1997). Considering the importance of oviposition preferences in understanding host plant resistance, in this study we investigated susceptibility of 10 apple cultivars for CM oviposition in the laboratory. In particular, we determined if oviposition and oviposition-site preference of CM varies among apple cultivars, and if there are any differences in the susceptibility of apple cultivars for CM oviposition during the early and late crop season. Based on the results from these studies, a susceptibility index of apple cultivars for CM oviposition was developed. This index may be used as a standard measure in cultivar evaluations and breeding programs to develop future resistant varieties as well as assisting fruit growers and pest management consultants select the most appropriate cultivar(s) for monitoring and detecting the initial signs of a CM infestation.

## MATERIALS AND METHODS

Over three years, a series of laboratory experiments were conducted to study the susceptibility of 10 commercial apple cultivars, *viz*., 'Arlet,' 'Gala,' 'Golden Delicious,' 'Fuji,' 'Honeycrisp,' 'Pristine,' 'Delicious,' 'Stayman,' 'Sunrise,' and 'York Imperial' for CM oviposition. Two sets of experiments, based on fruit maturity, were conducted each year with the fruits collected from trees during the second week of July and either the first or second week of August in 2006, 2007, and 2008. General descriptions of bloom time, harvest time and an estimated range of fruit maturity in days after full bloom of the apple cultivars used in this study are given in **Table 1**.

#### Experimental Fruits

Fruits of all cultivars were collected from unsprayed (without insecticide application) trees (10–33 years old) in apple orchards established in south facing slopes with typical well-drained soils of the Appalachian region. Fruits were stored in small cardboard boxes in a cold room (0◦C). Fruits were removed from the cold room approximately 4–5 h before the start of each experiment. All fruits were washed three times with clean cold water and were carefully inspected via a 10X Opti-VisorR lens (Donegan

TABLE 1 | Description of bloom time, harvest time and an estimated range of fruit maturity in terms of days after full bloom of apple cultivars used in multiple-choice and no-choice experiments.

∗*DAFB, Days after full bloom.*

*Source: Pennsylvania Tree Fruit Production Guide (2006–2007).*

Optical Co., Lenexa, KS, USA) for field oviposition/infestation by CM and other insects. Fruits of approximately similar size were vertically suspended in oviposition chambers by tying the stem to the top of the oviposition chamber using aluminum wire. Fruits damaged while being placed in oviposition chambers were discarded and replaced by new fruits from the same lot.

#### Experimental Insects

Codling moth adults used in this study were obtained from a laboratory colony established from adults or larvae collected from a block of apples located at The Pennsylvania State University, Fruit Research and Extension Center (FREC), Biglerville, PA, USA. Green thinning apples of various cultivars were used to maintain the laboratory colony/insect culture throughout the year during this study year. Pupae were collected from rearing containers in cardboard strips, and kept in environmentally controlled chambers (18–20◦C) till their use. CM pupae of similar age were selected and sexed, and placed into the oviposition chambers. The adult moths were allowed to emerge, mate, and freely oviposit on fruits. Pupae were regularly monitored for adult emergence. If there was no adult emergence from a pupa within 3 days of release, then it was replaced by an adult (2–3 days old) of the same sex from the same pupal lot.

## Experimental Design (Multiple-choice and No-choice Tests)

Multiple-choice oviposition preference tests and no-choice preference tests were conducted for both fruit maturity sets. In the no-choice tests, the oviposition chamber consisted of transparent plastic cups (1.0 L) internally lined with charcoal-colored fiberglass screen. In the multiple-choice tests, a cylindrical chamber (length = 0.81 m, diameter = 0.17 m) made of transparent fiberglass internally lined with fine aluminum mesh screening served as the oviposition chamber. In the multiple-choice tests, fruits of each cultivar were allocated to one of several locations at random in the oviposition chamber. During the study period, insects were maintained under laboratory conditions (temperature ∼21–23◦C, relative humidity ∼70%, and photoperiod 11:10 h light:dark with an ∼3 h period of dim light for oviposition induction). The year-wise description of these bioassays is as follows:

#### 2006 Bioassays

Nine cultivars ('Arlet,' 'Golden Delicious,' 'Fuji,' 'Honeycrisp,' 'Pristine,' 'Delicious,' 'Stayman,' 'Sunrise,' and 'York Imperial') were evaluated in multiple-choice and no-choice tests during the first set (July) of experiments. In the second set of experiments (August), all cultivars (except 'Pristine' which was replaced by 'Gala') were again evaluated. Each treatment (cultivar) was replicated at least eight times in the multiple-choice tests and 10 times in the no-choice tests. All fruits were collected during 14–17 July and 12–15 August for the first set (19 July) and second set (25 August) of experiments, respectively. Fruits of all cultivars (except 'Arlet,' 'Pristine,' and 'Sunrise') were collected from non-insecticide sprayed trees at FREC, Biglerville. Fruits of 'Arlet,' 'Pristine,' and 'Sunrise' cultivars (collected from an orchard partially sprayed with common orchard pesticides for the

purpose of general maintenance) were received from the USDA Appalachian Fruit Research Station, Kearneysville, WV, USA. In both no-choice experiments (early and late), one pair of unmated male and female adults was placed per cup, and the number of deposited eggs was counted after 8 days. In multiple-choice tests, seven and six pairs of unmated adults were utilized in the early and late experiments, respectively. Total numbers of eggs were counted after 15 days (early), and 10 days (late). The position of each egg on fruit (stem, calyx, or lateral) was recorded.

#### 2007 Bioassays

All 10 cultivars were evaluated in multiple-choice and no-choice tests conducted during the months of July and August. Each treatment (cultivar) had 8 and 10 replicates in the no-choice and multiple-choice tests, respectively. Fruits were collected during 12–15 July (early) 11–14 August (late). Fruits of all cultivars (except 'Arlet,' 'Pristine,' and 'Sunrise') were collected from non-insecticide sprayed trees at FREC, Biglerville. Fruits of 'Arlet,' 'Pristine,' and 'Sunrise' cultivars (collected from partially sprayed orchards) were received from the USDA Appalachian Fruit Research Station, Kearneysville, WV, USA for the early set of experiments, and for the late set of experiments from The Russell E. Larson Agricultural Research farm, Rock Springs, PA, USA. The early set of multiple-choice and no-choice tests were conducted on 17 July, while the late set of experiments were conducted on 16 August. In the no-choice tests, two pairs of unmated male and female adults were used in both sets of nochoice tests. In multiple-choice tests, three pairs of unmated male and female adults were used in both sets of multiple-choice tests. In all tests, the total numbers of deposited eggs on fruits were counted after 10 days. The position of eggs on the fruits was recorded as per the procedure used in the 2006 bioassays.

#### 2008 Bioassays

Based on the results of bioassays conducted during the first two years, only three cultivars ('Golden Delicious,' 'Honeycrisp,' and 'York Imperial') were further evaluated in the third year. Fruits of similar size were collected from non-insecticide sprayed trees at FREC, Biglerville, and utilized the same day for both the no-choice and multiple-choice experiments. The study was replicated 15 and 8 times in the no-choice and multiple-choice tests, respectively. In the multiple-choice tests, two fruits of each treatment/cultivar were used in each replication. In the early experiment, fruits were collected on 22 July, and used in both types of tests on the same day, and observations on eggs were taken after 10 days. In the late set, fruits were collected on 28 August, and observations were recorded after 11 days in both nochoice and multiple-choice tests. Similar to previous years, the position of eggs on the fruits was recorded in 2008.

## Statistical Analysis and Development of Oviposition-based Susceptibility Index

A general linear mixed-model analysis of variance (ANOVA) was used to analyze the data. In the analysis, two similar statistical models were used to address the study objectives. The first model (**Table 2**) was used to determine: (a) the oviposition preference of CM among apple cultivars; (b) differences in oviposition

TABLE 2 | Mix model ANOVA results of the sum of number of eggs per pair of codling moth and covariates (year, apple cultivar, and season [early and late]).

*Response variable in this analysis is eggs per pair of codling moth per fruit. All data and all years pooled together.*

preferences during the early and late season (i.e., based on time of fruit collection: early [July] versus late season [August]); and (c) differences in oviposition preferences in the multiple-choice and no-choice tests. The second model, which includes the egg counts by position on the fruit, was used to determine CM ovipositionsite preferences across different cultivars (**Table 3**). The mixedmodel ANOVA analysis was performed using R software (ISBN 3-900051-07-0; R Development Core Team, 2005).

Oviposition preference based on the mean number of eggs (per pair of CM per fruit) was determined for each cultivar. The data sets were transformed (to achieve the assumptions of parametric analysis) by taking the natural log of the "eggs per pair" variable. Pairwise comparisons were done among all cultivars, and means were separated using Tukey's honest significant differences *post hoc* test (*P <* 0.05) when ANOVA was significant (Zar, 1999).

TABLE 3 | Mix model ANOVA results of the sum of mean number of eggs per pair of codling moth and covariates (year, apple cultivar, position of eggs on apple [calyx, stem, and lateral sites], and season [early and late]).

*Response variable in this analysis is eggs per pair of codling moth per fruit. All data and all years pooled together.*

The CM oviposition susceptibility index (based on oviposition preferences of CM) for each cultivar was characterized as:

$$SI = \frac{1}{n} \sum\_{i,j,k,t} SEPP(i,j,k,t) \tag{1}$$

Where, *SI* = Susceptibility index; *SEPP* = Standardized mean eggs per pair of moths for an apple cultivar [*i*]; *j* = Time of fruit collection (early or late); *k* = year of observation; and *t* = type of tests (i.e., no-choice and multiple-choice tests).

Standardized mean eggs per pair of moths for an apple cultivar were determined as following:

$$SEPP[i] = \frac{EPP[i]}{EPP\_{\text{max}}[i]} \tag{2}$$

Where, *SEPP* = Standardized mean eggs per pair of moths for an apple cultivar [*i*]; *EPP* [*i*] = Mean number of eggs per pair of moths on an apple cultivar [*i*]; and *EPP*max [*i*] = Maximum number of eggs per pair of moths on an apple cultivar [*i*].

Mean total number of eggs per pair of moths (EPP) on a cultivar was calculated by the following equation:

$$EPP\left[i\right] \ = \ EPP\_C\left[i\right] \ + \ EPP\_S\left[i\right] \ + \ EPP\_L\left[i\right] \tag{3}$$

Where *EPP* [*i*] = Mean number of eggs per pair of moths on an apple cultivar [*i*]; *EPPC*[*i*] = Mean number of eggs per pair of moths on calyx side of an apple cultivar [*i*]; *EPPS*[*i*] = Mean number of eggs per pair of moths on stem side of an apple cultivar [*i*]; and *EPPL*[*i*] = Mean number of eggs per pair of moths on lateral side of an apple cultivar [*i*].

Oviposition susceptibility index of all cultivars was compared and means were separated using Fisher's protected least significant differences *post hoc* test (*P <* 0.05) when ANOVA was significant (Zar, 1999). The analysis was performed using SPSS-13 statistical software (SPSS Inc., Chicago, IL, USA).

#### RESULTS

#### 2006 Early Season (July)

In the multiple-choice test, on the calyx site of fruits (**Table 4**), CM females laid significantly higher numbers of eggs on 'York Imperial,' 'Golden Delicious,' and 'Delicious' than other cultivars (*P <* 0.05; **Figure 1A**). In contrast, the lowest numbers of eggs were laid on the 'Honeycrisp' cultivar (*P <* 0.05; **Figure 1A**). On the stem site of fruits, CM females laid significantly more eggs on 'Stayman,' 'York Imperial,' 'Golden Delicious,' and 'Delicious' than all other cultivars (*P <* 0.05; **Figure 1B**). On the lateral site of fruits, CM females preferred 'York Imperial,' 'Golden Delicious,' and 'Delicious' than the cultivars 'Pristine,' 'Honeycrisp,' 'Arlet,' and 'Sunrise' (*P <* 0.05; **Figure 1C**).

In the no-choice test, on the calyx site (**Table 4**), the female moths significantly preferred to oviposit on 'Golden Delicious' (*P* = 0.009), 'Fuji' (*P* = 0.005), and 'Delicious' (*P* = 0.008), compared to 'Pristine' (**Figure 1D**). On the stem site, 'Golden Delicious' was significantly more preferred than

#### TABLE 4 | Statistical details of oviposition site preferences of codling moth across different cultivars.

∗*df values are same across different oviposition sites.*

#### FIGURE 1 | Relative susceptibility of different apple cultivars for oviposition of codling moth during July 2006 (early season/Set 1). Mean number of eggs per pair of moths per fruit on calyx, stem, and lateral sides of fruits of different cultivars are shown in multiple-choice tests (A–C) and no-choice tests (D–F). St, Stayman; YI, York Imperial; GD, Golden Delicious; Fu, Fuji; RD, Delicious; Pr, Pristine; Ho, Honeycrisp; Ar, Arlet; Su, Sunrise. *N* = 8 for all the multiple-choice tests, and *N* = 10 for all the no-choice tests. Each bar represents standard error of mean. Different letters over bars indicate significant difference (*P <* 0.05).

'Delicious' (*P* = 0.014), 'Pristine' (*P* = 0.013), 'Honeycrisp' (*P <* 0.001), 'Arlet' (*P* = 0.047), and 'Sunrise' (*P* = 0.023; **Figure 1E**). On the lateral site, 'Golden Delicious' was significantly more preferred than 'Honeycrisp' (*P* = 0.049), 'Arlet' (*P* = 0.004), and 'Sunrise' (*P* = 0.008; **Figure 1F**); however, the total number of eggs on 'Golden Delicious' was not significantly different from that of all other cultivars (*P >* 0.05; **Figure 1F**).

#### 2006 Late Season (August)

In the multiple-choice test, on the calyx site (**Table 4**), the oviposition preference of CM was not significantly different for all cultivars (*P >* 0.05), except for 'Fuji', when compared to 'Delicious' (*P* = 0.042), and 'Sunrise' (*P* = 0.015; **Figure 2A**). On the stem (**Figure 2B**) and lateral (**Figure 2C**) sites of fruits, 'Stayman,' 'Golden Delicious,' 'Fuji,' 'Honeycrisp,' and 'Arlet' rather than 'Delicious,' 'Gala,' and 'Sunrise' were the significantly preferred cultivars (*P <* 0.05).

In the no-choice test, on the calyx site (**Table 4**), CM deposited more eggs on 'Golden Delicious' than on 'Gala' (*P* = 0.011), otherwise, there was no significant differences between 'Golden Delicious' and all other cultivars (*P >* 0.05; **Figure 2D**). On the stem (**Figure 2E**) and lateral (**Figure 2F**) sites of fruits, CM deposited more eggs on 'Golden Delicious' and 'Fuji' than on 'Gala,' 'Honeycrisp,' and 'Sunrise' (*P <* 0.05).

#### 2007 Early Season (July)

In the multiple-choice test of early season 2007, on the calyx site (**Table 4**), CM significantly preferred 'Golden Delicious' for oviposition over 'Pristine' (*P* = 0.006), 'Arlet' (*P* = 0.032), 'Sunrise' (*P* = 0.006), and 'Gala' (*P* = 0.032; **Figure 3A**). On the stem site of fruits, 'Golden Delicious' was again the significantly preferred cultivar for oviposition over other cultivars, *viz*., 'Pristine' (*P <* 0.001), 'Honeycrisp' (*P <* 0.001), 'Arlet' (*P <* 0.001), 'Sunrise' (*P <* 0.001), and 'Gala' (*P <* 0.001; **Figure 3B**). However, the preference for 'Golden Delicious' was similar to 'Stayman' (*P* = 0.092), 'York Imperial' (*P* = 0.457),

'Fuji' (*P* = 0.777), and 'Delicious' (*P* = 0.064; **Figure 3B**). On the lateral site, 'Golden Delicious' was significantly more preferred than all other cultivars (*P <* 0.05), except 'Stayman' (*P* = 0.996), 'York Imperial' (*P* = 0.777), and 'Delicious' (*P* = 0.109; **Figure 3C**). In contrast, 'Arlet' was the least preferred cultivar for oviposition on the lateral site of fruits (*P <* 0.05; **Figure 3C**).

In the no-choice test (July 2007), on the calyx site of fruits (**Table 4**), CM deposited higher numbers of eggs on 'Golden Delicious' and 'Gala' than on 'Pristine,' 'Honeycrisp,' 'Arlet,' and 'Sunrise' (*P <* 0.05; **Figure 3D**). On the stem (**Figure 3E**) and lateral (**Figure 3F**) sites of fruits, 'Golden Delicious' and 'Fuji' received the highest number of eggs over 'Pristine,' 'Honeycrisp,' 'Arlet,' and 'Sunrise' (*P <* 0.05).

#### 2007 Late Season (August)

In the multiple-choice test conducted during the late season study of 2007, on the calyx site (**Table 4**), 'Golden Delicious' was more preferred for oviposition than 'York Imperial' (*P* = 0.036) and 'Sunrise' (*P* = 0.022; **Figure 4A**). On the stem site of fruits, the moths again preferred 'Golden Delicious' for oviposition over all other cultivars (*P <* 0.05), except 'Stayman' (*P* = 0.978) and 'Fuji' (*P* = 0.563; **Figure 4B**). In contrast, 'Pristine' was the least preferred cultivar (*P <* 0.05; **Figure 4B**). On the lateral site, 'Stayman,' 'Golden Delicious,' and 'Fuji' were the most preferred cultivars for oviposition (*P <* 0.05), except for 'York Imperial' and 'Delicious' (*P >* 0.05; **Figure 4C**).

In the no-choice test, on the calyx site (**Table 4**), CM deposited less eggs on 'Pristine' than 'Gala' (*P* = 0.047), however, such lower preference for 'Pristine' was not significantly different from all other cultivars (*P >* 0.05; **Figure 4D**). On the stem (**Figure 4E**) and lateral (**Figure 4F**) sites of fruits, CM showed less preference for 'Pristine' (*P <* 0.05) than all other cultivars, except for 'Honeycrisp,' 'Arlet,' and 'Sunrise' (*P >* 0.05).

#### 2008 Early Season (July)

In the multiple-choice test (**Table 4**), on the calyx (**Figure 5A**) and stem (**Figure 5B**) sites of fruits, 'Golden Delicious' was the most preferred cultivar for oviposition over the other two cultivars

(*P <* 0.05). On the lateral site, 'Honeycrisp' was less preferred for oviposition than 'Golden Delicious' (*P* = 0.001; **Figure 5C**).

In the no-choice test (July 2008), on the calyx site (**Table 4**), the oviposition preference of CM did not differ significantly across all the cultivars (*P >* 0.05; **Figure 5D**). On stem (**Figure 5E**) and lateral sites of fruits (**Figure 5F**), CM deposited more eggs on 'Golden Delicious' over 'Honeycrisp' and 'York Imperial' (*P <* 0.05).

#### 2008 Late Season (August)

In the multiple-choice test (**Table 4**), on the calyx site, 'Golden Delicious' was the most preferred cultivar over 'Honeycrisp' (*P* = 0.004), but it was not more preferred over 'York Imperial' (*P* = 0.475; **Figure 5G**). On stem (**Figure 5H**) and lateral (**Figure 5I**) sites of fruits, 'Honeycrisp' was the least preferred cultivar when compared to 'Golden Delicious' and 'York Imperial' (*P <* 0.05).

In the no-choice test (August 2008), on calyx (**Figure 5J**), stem (**Figure 5K**), and lateral (**Figure 5L**) sites of fruits, 'Golden Delicious' was the most preferred cultivar for oviposition over that of 'Honeycrisp' and 'York Imperial' (*P <* 0.05).

## Interaction Effects of Apple Cultivar, Choice (Type of Test), Season (Early or Late), Study Year and Oviposition Sites on CM Oviposition

All covariates (cultivar, season [early or late], and study year) had a significant influence on the oviposition of CM on different apple cultivars (*P <* 0.001; **Table 2**). All types of interactions presented in **Table 2** had a significant impact on the oviposition preference of CM (*P <* 0.05). Oviposition sites (i.e., calyx, stem, and lateral) on fruits had a highly significant influence on CM oviposition (*P <* 0.001; **Table 3**). All the

interactions of oviposition sites with other covariates, except Site:Season (*P* = 0.458), Site:Cultivar:Season (*P* = 0.115) and Site:Cultivar:Year:Season (*P* = 0.615), displayed a significant interactive impact on the oviposition of CM (*P <* 0.05; **Table 3**).

## Susceptibility Index for Different Apple Cultivars for CM Oviposition

In terms of the CM oviposition susceptibility index (on a scale of 0 – 1, where, '0' = the least susceptible and '1' = the most susceptible), 'Golden Delicious' had a significantly higher susceptibility index than 'Stayman' (*P* = 0.002), 'York Imperial' (*P* = 0.002), 'Fuji' (*P* = 0.011), 'Delicious' (*P <* 0.001), 'Pristine' (*P <* 0.001), 'Honeycrisp' (*P <* 0.001), 'Arlet'(*P <* 0.001), 'Sunrise' (*P <* 0.001), and 'Gala' (*P <* 0.001; **Figure 6**). In contrast, 'Pristine,' 'Honeycrisp,' 'Arlet,' and 'Sunrise' were noticeably less susceptible to oviposition by CM (*P <* 0.05; **Figure 6**).

## DISCUSSION

In the majority of bioassays conducted across different years, CM females preferred to oviposit on 'Golden Delicious,' 'Fuji,' 'Delicious,' 'Stayman,' and 'York Imperial' over other cultivars, *viz*., 'Pristine,' 'Honeycrisp,' 'Arlet,' 'Sunrise,' and 'Gala.' Different volatile fruit-coat constituents likely affect the ovipositional preferences by CM for apple fruits. For instance, the production of the sesquiterpene α-farnesene, an ovipositional stimulant for female CM, and an important constituent in the outer skin of apple fruits, varies greatly across different cultivars and changes as fruit mature (Wearing and Hutchins, 1973; Sutherland et al., 1977). Such variation could be an important factor in helping explain the differential cultivar ovipositional preferences of CM found in this study. However, such hypothesis needs further evaluation.

Results from the no-choice and multiple-choice tests across different years in this study showed that CM females deposited significantly more eggs on 'Golden Delicious' over other cultivars, *viz*., 'Pristine,' 'Honeycrisp,' 'Arlet,' and 'Sunrise.' Similar trends in the ovipositional preferences of a closely related tortricid pest, oriental fruit moth [*Grapholita molesta* (Busck)] for these different apple cultivars are also reported (Joshi et al., 2007; Myers et al., 2007). In the one study by Joshi et al. (2007), the oriental fruit moth preferred 'Golden Delicious' for oviposition compared to the cultivars 'Pristine,' 'Arlet,' and 'Sunrise.' Based on the ovipositional preferences exhibited by CM, these preferred cultivars are highly likely more susceptible to CM infestations, especially if these laboratory results reflect field behaviors. The choice of a preferred suitable substrate or host for oviposition plays a key role in the survival and completion of different life stages of lepidopteran insects (Chew and Robbins, 1984; Renwick, 1989). Similarly, in the case of CM, judicious selection of an appropriate host for depositing eggs might play a key role in determining the initial fate of a neonate larva that feeds internally in fruits of the selected host(s). Upon hatching, the larva enters the fruit, and remains inside the fruit till the pre-pupal stage. The larva developing inside the fruit is usually incapable of moving from one fruit to other, so the oviposition preferences of female CM most likely determine larval survival by selecting the most suitable host/cultivar. In oviposition preference studies of a closely related fruit pest species (i.e., oriental fruit moth), Myers et al. (2006a) found higher percentages of larval entry in fruits of preferred (in terms of oviposition) cultivars like 'Golden Delicious' and 'Delicious' during their early and late season experiments. Therefore, it is likely that the oviposition preference of CM for these different apple cultivars might be related to larval survival. The percent larval survival on the most preferred cultivar (i.e., 'Golden Delicious') was higher than one of the less preferred cultivars (i.e., 'Arlet') when neonate larvae were individually exposed to these different cultivars (NKJ et al., unpublished data). Such preferences for 'Golden Delicious' were also revealed in the present oviposition bioassays, as 'Golden Delicious' was the preferred cultivar over 'Arlet,' 'Sunrise,' and 'Pristine' cultivars. In a related study on relative susceptibility of different apple cultivars to various arthropod pests that was conducted in an orchard, Hogmire and Miller (2005) reported 'Golden Delicious' as a highly susceptible cultivar to CM infestations versus other cultivars such as 'Pristine,' 'Honeycrisp,' 'Arlet,' and 'Sunrise.'

Early maturing varieties have been considered less susceptible to CM infestations (Isely, 1943). In the present study, for the majority of oviposition bioassays, CM least preferred to oviposit on early maturing cultivars, *viz*., 'Pristine,' 'Honeycrisp,' 'Arlet,' 'Sunrise,' and 'Gala' as compared to later maturing cultivars such as 'Stayman,' 'York Imperial,' 'Golden Delicious,' 'Fuji,' and 'Delicious.' Such preference could be related to the presence or emission of fruit volatiles from these cultivars, since fruit volatiles are known to play a crucial role in guiding female moths to oviposit on or near fruits (Wildbolz, 1958; Lombarkia and Derridj, 2002; Reed and Landolt, 2002). The oviposition preferences of CM across different cultivars may also vary in relation to the time during the season that an apple matures and to its fruit maturity at any specified time during the season, because the release of volatiles from fruits increases from early to late season (Sutherland et al., 1977; Mattheis et al., 1991). In general, more eggs per pair of CM adults per fruit were observed in bioassays conducted with fruits collected later in the season (August) than those collected earlier in the season (July). During the early stages of fruit development, fruits are reported to release only a few ester type compounds as compared to ripening and mature fruits (late season), which are reported to release many ester type compounds plus a few terpenoids (Bengtsson et al., 2001). Such changes in volatile emissions may be the reason for the variations in the ovipositional preferences of CM for fruits collected in July (early season) and August (late season). During the early stages of fruit development, CM females are reported to deposit more eggs on neighboring leaves (i.e., shoot and spur) around fruits and fruit clusters (Wildbolz, 1958; Blomfield et al., 1997) than directly on fruits (NKJ et al. unpublished data), while during the fruit maturation and ripening period, more eggs are deposited directly on fruits compared to the early stages of fruit development (Summerland and Steiner, 1943). This type of oviposition pattern/preference may be helpful in increasing the likelihood of larval survival upon hatching. Sutherland et al. (1977) found that the production of α-farnesene (which is known to influence the oviposition behavior of CM) increases as fruit maturity increases. Consequently, CM females deposit more eggs on fruits as fruit maturity increases during the season. The variability among different apple cultivars in the production of the oviposition stimulant α-farnesene could be a major factor affecting the ovipositional preference of CM for different apple cultivars during the early and latter part of the growing season. Other strong possibilities causing such early and late season variation in the oviposition preferences of CM could be the differential developmental stages (maturity level) and other characteristics (such as chemical composition of fruit-coat, fruit color, etc.) of fruits of these different cultivars during the two different time periods of a season.

Apart from the chemical constituents of the fruit coat, physical characteristics of the apple fruit surface may vary from one cultivar to other, as well as within the calyx, stem and lateral sides of fruit of different cultivars (Belding et al., 1998; Verardo et al., 2003). Such microtopographic properties can be categorized on the basis of roughness and smoothness of host surface, and play a crucial role in the attachment ability of CM (Al Bitar et al., 2010), and may influence its oviposition behavior, particularly the oviposition site selection (Al Bitar et al., 2014). Friction forces, which affect the attachment ability of CM eggs to these different types of surfaces, had been reported to be higher on oviposition substrates with smooth surfaces (Al Bitar et al., 2009, 2010), and could be main factors behind the CM oviposition preferences for the smooth substrates (e.g., fruits) over rough surfaces (e.g., leaves with trichomes). Variation in the CM oviposition on calyx, stem and lateral sides of fruits of apple cultivars in this study could be due to differences in fruit surface properties such as amorphous wax layer (comprised of microcracks and epicuticular wax crystals) favoring CM egg adhesion to oviposition substrates. Composition and abundance of microcracks (Al Bitar et al., 2014) and epicticular wax (Belding et al., 1998) on fruit surfaces vary across different apple cultivars. CM egg adhesion to different oviposition substrates of the fruit of different cultivars (for instance, 'Golden Delicious,' 'Elstar,' 'Jonica,' 'Boskoop,' 'Topaz') had been reported to vary within upper (stem), middle (lateral), and lower (calyx) sections of fruits (Al Bitar et al., 2014). Regardless of test type, year and season, in general, we recorded higher number of eggs on stem and lateral sites compared to calyx end of fruit. It could be due to higher abundance of microcracks as well as stronger bonding between CM eggs and fruit surfaces on stem and lateral fruit surfaces than calyx end (Al Bitar et al., 2014).

Cultivar, season (early or late), study year and oviposition sites (i.e., calyx, stem, and lateral) on fruits had a significant influence on the oviposition of CM on different apple cultivars. Covariate interactions (except, Site:Season, Site:Cultivar:Season, and Site:Cultivar:Year:Season) were also significant. Ovipositionsites on fruits may vary from one cultivar to another. In the multiple-choice and no-choice tests, CM deposited more eggs on lateral and stem sites than on the calyx site of fruits. Such patterns of egg deposition could be due to the physical characteristics of the apple fruit surface as discussed earlier or due to the 'vertical' placement of fruits, as in all these tests, fruits were vertically placed in oviposition chambers. In contrast, oriental fruit moth adult females preferred to oviposit on the calyx and stem sites of apple fruit, and their oviposition site preferences are also reported to vary between different apple cultivars (Myers et al., 2006b).

Susceptibility to various pest infestations may vary among cultivated varieties as well as wild varieties (e.g., crab apples). In the past, susceptibility of apple cultivars/germplasms to different arthropod pests has been studied using several methods, such as their impact on pest developmental rate and pest survival rate (Mackenzie and Cummins, 1982; Myers et al., 2006b), damage (in terms of fruit injury) caused by pests (Dean and Chapman, 1973; Goonewardene et al., 1979; Straub, 2003; Hogmire and Miller, 2005) and the occurrence of pests (Goonewardene et al., 1976; Straub, 2003; Hogmire and Miller, 2005; Myers et al., 2007). However, using a standardized oviposition-based susceptibility index of apple cultivars as developed in this study reveals important information about the relative susceptibility of cultivars when evaluated under different seasons and times during the season. The newly developed CM oviposition susceptibility index for apple cultivars showed that susceptibility is linked to the oviposition preferences of CM, as female moths least preferred 'Pristine,' 'Sunrise,' 'Arlet,' and 'Honeycrisp' (less susceptible cultivars) for oviposition than 'Golden Delicious' (highly susceptible cultivar). Similarly, Hogmire and Miller (2005) reported that 'Golden Delicious' was significantly more susceptible than 'Pristine,' 'Honeycrisp,' 'Arlet,' and 'Sunrise' to injury by CM in the field environment. Straub (2003) studied the relative susceptibility of some new apple cultivars in New York to different orchard pests, and found that cultivars such as 'Sunrise,' 'Pristine,' 'McIntosh' (Pioneer), and 'Honeycrisp' were comparatively resistant to CM larval damage compared to 'Golden Delicious'. The CM oviposition susceptibility index could be useful to researchers/research extension workers and fruit growers in IPM decision-making in apple orchards.

To summarize, CM preferred to oviposit on later maturing cultivars 'Golden Delicious,' 'Stayman,' 'York Imperial,' 'Fuji,' and 'Delicious' (preferred cultivars) than early maturing cultivars, *viz*., 'Pristine,' 'Honeycrisp,' 'Arlet,' 'Sunrise,' and 'Gala' (less preferred cultivars) in the majority of the multiple-choice tests. In the no-choice tests, CM deposited more eggs on these preferred cultivars than the less preferred cultivars. Regardless of choice test type and season, CM deposited significantly more eggs on 'Golden Delicious' over other cultivars, *viz*., 'Pristine,' 'Honeycrisp,' 'Arlet,' and 'Sunrise.' In both types of tests, more eggs were laid on lateral and stem sites than the calyx site of fruits across different cultivars. In terms of a CM oviposition susceptibility index, 'Golden Delicious' was the most susceptible cultivar to oviposition, while 'Pristine,' 'Honeycrisp,' 'Arlet,' and 'Sunrise' were least susceptible. From an integrated pest management perspective, the newly developed susceptibility index can assist fruit growers and consultants select the most appropriate cultivar(s) for monitoring and detecting the initial signs of fruit injury from this pest. For instance, 'Golden Delicious' is the most preferred cultivar for oviposition, therefore it should be the cultivar of choice for monitoring CM injury in mixed-cultivar planted orchards. If it is not present in a block/orchard, then the next preferred cultivar for oviposition should be selected for examining CM injury or oviposition. In addition, results of these studies would be helpful in breeding programs, particularly in developing CM resistant apple varieties. As previously discussed, oviposition by CM is likely stimulated by fruit volatiles, and variations in the production and release of these volatiles from different apple cultivars may result in different oviposition preferences. Further investigations are needed to understand the biochemical as well as physical aspects of fruits and other factors involved in determining apple cultivar susceptibility for CM oviposition.

#### ACKNOWLEDGMENTS

The authors would like to thank the State Horticultural Association of Pennsylvania and the Garretson Endowment for providing funding, and Teresa Krawczyk, Fruit Research and Extension Center- Penn State, for providing the insect colony. The authors are also thankful to the Statistical Consulting Center, Department of Statistics at Penn State University for

#### REFERENCES

their assistance with the statistical analyses and consultation, and Dr. Robert M. Crassweller (Penn State University) and Dr. James Schupp (Penn State Fruit Research and Extension Center) for providing fruits of certain cultivars. The authors are also thankful to Dr. James Schupp and Dr. Dennis Calvin for their insightful comments on the previous version of this manuscript. Publication of this article was funded in part by The Pennsylvania State University Libraries Open Access Publishing Fund.

Howell, J. F., Hutt, R. B., and Hill, W. B. (1978). Codling moth: mating behavior in the laboratory. *Ann. Entomol. Soc. Am.* 71, 891–895. doi: 10.1093/aesa/71.6.891

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Joshi, Rajotte, Myers, Krawczyk and Hull. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Plant–Aphid Interactions Under Elevated CO2: Some Cues from Aphid Feeding Behavior

Yucheng Sun† , Huijuan Guo† and Feng Ge\*

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

Although the increasing concentration of atmospheric carbon dioxide (CO2) accelerates the accumulation of carbohydrates and increases the biomass and yield of C3 crop plants, it also reduces their nitrogen concentration. The consequent changes in primary and secondary metabolites affect the palatability of host plants and the feeding of herbivorous insects. Aphids are phloem feeders and are considered the only feeding guild that positively responds to elevated CO2. In this review, we consider how elevated CO2 modifies host defenses, nutrients, and water-use efficiency by altering concentrations of the phytohormones jasmonic acid, salicylic acid, ethylene, and abscisic acid. We will describe how these elevated CO2-induced changes in defenses, nutrients, and water statusfacilitate specific stages of aphid feeding, including penetration, phloem-feeding, and xylem absorption. We conclude that a better understanding of the effects of elevated CO2 on aphids and on aphid damage to crop plants will require research on the molecular aspects of the interaction between plant and aphid but also research on aphid interactions with their intra- and inter-specific competitors and with their natural enemies.

#### Edited by:

Scott Nicholas Johnson, University of Western Sydney, Australia

#### Reviewed by:

James Michael William Ryalls, Hawkesbury Institute for the Environment, Australia Jonathan A. Newman, University of Guelph, Canada

#### \*Correspondence:

Feng Ge<EMAIL_ADDRESS>†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science

Received: 07 December 2015 Accepted: 29 March 2016 Published: 13 April 2016

#### Citation:

Sun Y, Guo H and Ge F (2016) Plant–Aphid Interactions Under Elevated CO2: Some Cues from Aphid Feeding Behavior. Front. Plant Sci. 7:502. doi: 10.3389/fpls.2016.00502 Keywords: elevated CO2, aphid, nitrogen metabolism, plant defenses, water potential, legumes

## INTRODUCTION

Since the industrial revolution, atmospheric CO2 concentrations have increased from 280 ppm to approximately 400 ppm due to anthropogenic effects, i.e., deforestation and fossil fuel combustion. These increases in atmospheric CO2 concentrations have serious implications for global warming and climate change (Stocker et al., 2013). Although changes in climate have been anticipated to greatly affect agricultural ecosystems (Fuhrer, 2003), increases in atmospheric CO2 concentration alone can also be very important because they can directly affect plant physiology and indirectly alter interactions between plants and herbivores and plant pathogens (Robinson et al., 2012). These altered interactions may then lead to more severe and frequent outbreaks of pest insects and plant diseases in agricultural ecosystems (Percy et al., 2002).

To understand how elevated concentrations of atmospheric CO2 could increase pest problems, we must first recognize that increases in CO2 tends to increase the growth of plants by enhancing their photosynthetic rate, resulting in higher yields for most C3 crops (Ainsworth and Rogers, 2007). Under elevated CO2, however, C3 crop plants exhibit decreases in nitrogen (N) and other trace elements, i.e., zinc and iron (Bloom et al., 2010). These decreases reduce the nutritional value for herbivorous insects and may therefore change their feeding behaviors (Myers et al., 2014). For

those insects that chew leaves, a reduction in the N concentration in crop tissue and the resulting increase in the carbon/nitrogen ratio (C:N ratio) under elevated CO2 could cause these insect pests to consume more leaves to meet their N needs (Bezemer and Jones, 1998; Sun and Ge, 2011). In addition, leaves grown under elevated CO2 decrease their ability to produce jasmonic acid (JA), a hormone that contributes to plant defenses against chewing insects (Zavala et al., 2008).

Elevated CO2 may also increase the damage to crops caused by phloem-sucking insects including aphids. Aphids feed exclusively on the phloem sap and are very sensitive to changes in plant quality caused by climate change (Pritchard et al., 2007). Recent meta-analysis result shows that aphids tend to perform better under elevated CO2 on average (Robinson et al., 2012). The conclusions from many statsitically signficant researches, however, exhibit idiosyncratic responses of aphids in terms of population abundance, fecundity as well as survival (summarized in **Table 1**). Although predictions are difficult, it is nevertheless useful to determine why some aphids are more fit while others are less fit under elevated CO2. A mechanistic understanding can help make sense of these contradictory results. Previous study demonstrates that the effect of elevated CO2 on plant, which includes C and N assimilation, secondary metabolism, plant stomatal conductance as well as leaf temperature, could in turn affect aphid population numbers and growth (Ainsworth et al., 2006; May et al., 2013). Futhermore, the feeding behavior of aphids and their interaction with host plant under elevated CO2 are largely ignored but should be crucial to the understanding of idiosyncratic responses. The aim of this review is to highlight overlooked processes and new discoveries that how elevated CO2 affects the components of plant leaves and how these effects alter the different feeding phases of aphids. We also suggest some possible molecular mechanisms underlying the interactions between aphids and their host plants under elevated CO2.

## APHID FEEDING BEHAVIOR

Recent advances indicate that complex molecular interactions occur when aphids feed on plants. Unlike chewing insects that remove large pieces of plant tissues, aphids use their flexible and long stylets to obtain nutrients from the phloem sap and only inflict slight physical damage (Jaouannet et al., 2014). The specialized feeding behavior of aphids can be detected with electrical penetration graph (EPG) methods, i.e., EPG methods can be used to determine the locations and activities of aphid stylets, including pooled pathway phase activities, probing, salivation into sieve elements, passive uptake of the phloem sap, and xylem absorption (Tjallingii and Esch, 1993). Data on the initiation and duration of these feeding phases provide valuable cues regarding aphid activities and plant responses (Alvarez et al., 2006). Rather than simply withdrawing food from hosts, aphids can change their feeding location to avoid plant defenses or can secrete 'effector' proteins to suppress plant defenses (Hogenhout and Bos, 2011). To enhance their feeding, aphids can also alter host physiological traits, e.g., they can induce changes in host primary metabolism and in stomatal movement, and suppress the plant defenses (Giordanengo et al., 2010). Thus, a better understanding of aphid feeding behavior, its effects on hosts, and host responses is critical for understanding how elevated CO2 is likely to affect plant–aphid interactions.

## APHID PROBING AND PENETRATION STAGE AND ITS RELATION TO PLANT RESISTANCE

#### Influence of Plant Physical Barriers

Once they have arrived on a plant leaf, aphids must conquer host physical defenses including trichomes and waxes before they can insert their stylets into the host (Wang et al., 2004). Surface resistance is the first barrier of plant defense against aphid attack. The time that aphids spend between arriving on a leaf and making their first probe mainly reflects the physical barriers of the leaf surface including trichomes, repellent volatiles, and a thick or tough leaf surface (van Helden and Tjallingii, 1993). Plants can deter aphid attack by releasing secondary metabolites such as glucose esters and sesquiterpenes from glandular trichomes (Avé et al., 1987; Goffreda et al., 1989; Neal et al., 1990). Furthermore, a specifically expressed gene, NtLTP1, in the glandular trichomes of Nicotiana tabacum could enhance the plant's defense against aphids (Choi et al., 2012). The changes in trichome density in response to CO2 are idiosyncratic. For example, trichome density increased in Brassica rape and Medicago truncatula (Karowe and Grubb, 2011; Guo et al., 2014a) but decreased in Arabidopsis and wheat under elevated CO2 (Masle, 2000; Bidart-Bouzat et al., 2005; Lake and Wade, 2009). In the legume M. truncatula under elevated CO2, the increased density of non-glandular and glandular trichomes caused aphids to spend more time before they made their first probe and to experience a prolonged pathway phase (Guo et al., 2014a). CO2 concentrations may affect trichome development by affecting the levels of gibberellic acid (GA), JA, and the microRNA molecule miR156. Elevated CO2 tends to increase plant GA content and decrease plant JA content (Teng et al., 2006; Zavala et al., 2008) and to decrease expression of miR156 (May et al., 2013). Additional research is needed, however, to clarify whether the effects of elevated CO2 on glandular trichome development and surface resistance to aphids is due to changes in GA, JA, and miR156.

#### Phytohormone-Mediated Defenses

When the aphid stylet penetrates the plant epidermis and mesophyll, it forms a channel that permits the delivery of saliva into the phloem (Jaouannet et al., 2014). On the one hand, elicitors in aphid saliva could trigger the formation of reactive oxygen species (ROS), which in turn could induce plant defenses (Giordanengo et al., 2010). On the other hand, "effectors" in aphid saliva could suppress plant resistance and manipulate host cell processes to favor aphid feeding and colonization (Bos

et al., 2010a,b; McLellan et al., 2013; Gimenez-Ibanez et al., 2014; King et al., 2014). Parameters of aphid feeding behavior revealed by EPG could reflect the intensity of plant resistance; these parameters include the minimum duration of pathway phase activity, the number of test probes, and the total time before phloem ingestion begins (Alvarez et al., 2006). In the M. truncatula–pea aphid system, elevated CO2 increased the number of test probes but decreased the total time before phloem ingestion began (Guo et al., 2014a). The inconsistent effects of elevated CO2 on aphid feeding parameters may result from the contrasting effects of elevated CO2 on the defense signaling pathways involving the phytohormones JA, salicylic acid (SA), and ethylene (ET) (Guo et al., 2014a). Elevated CO2 tends to enhance SA-dependent defense but reduce JA- and ET-dependent defenses in plants (Zavala et al., 2009; Guo et al., 2012; Sun et al., 2013). Furthermore, the enhanced SA signaling pathway under elevated CO2 caused aphids to spend more time before the first probe and reduced aphid fitness (Casteel et al., 2012; Guo et al., 2014a). The suppression of the JA signaling pathway under elevated CO2, however, reduces the time required by aphids to reach the phloem. In addition, elevated CO2 downregulates the expression of the ET signaling pathway genes ACC, SKL, and ERF in M. truncatula under attack by the pea aphid system; this downregulation, decreases the accumulation of H2O2 and the activities of key enzymes related to ROS (Guo et al., 2014b).

Moreover, elevated CO2 potentially disrupts the homeostatic cross-talk between SA and JA/ET pathways by directly activating the NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1) gene (DeLucia et al., 2012; Zavala et al., 2013). NPR1-mediated suppression of JA signaling is regulated by glutathione biosynthesis (Spoel and Loake, 2011). Elevated CO2 changes the expression of genes that encode thioredoxins and glutathione S-transferase, which may activate the expression

of NPR1 (DeLucia et al., 2012). However, Sun et al. (2013) found that when the NPR1 gene was knocked down, the JA-dependent defenses of Arabidopsis were not enhanced by elevated CO2, suggesting that the activation of NPR1 may not explain the response of SA, JA, and ET signaling pathways to elevated CO2. Clearly, the upstream network regulating plant immunity against aphids is complex. The elicitors secreted from aphid salivary glands were recognized by the host coreceptor BRI-ASSOCIATED RECEPTOR KINASE 1 (BAK1) which subsequently phosphorylates BOTRYTIS INDUCED KINASE1 (BIK1). The BAK1 and the BIK1 complexes could jointly modulate the downstream phytohormone-mediated defense signaling pathway (Chaudhary et al., 2014; Lei et al., 2014; Prince et al., 2014). In addition to BAK/BIK, other kinases such as mitogen protein kinases (MAPKs) are also important for regulating plant defense responses against insect herbivores (Hettenhausen et al., 2015). A number of studies reported that MAPKs could regulate the JA, SA, and ET signaling pathways by activating WRKY genes (Zavala et al., 2013). It is still unknown whether elevated CO2 affects the JA- and SA-dependent signaling pathways by regulating upstream BAK/BIK or MAPK signaling. Thus, additional research is needed to determine how elevated CO2 affects these regulatory molecules in phytohormone signaling networks.

## Secondary Metabolite-Mediated Resistance

Many plant secondary metabolites may help plants resist aphid attack by negatively affecting the penetration pathway stage of aphid feeding. These secondary metabolites include alkaloids, steroids, foliar phenolic esters (rutin, cholorogenic acid, etc.), terpenoids, cyanogenic glycosides, glucosinolates, saponins, flavonoids, and pyrethrins (Sharma et al., 2000; Urbanska et al., 2002). For example, aphids that fed on highsaponin lines of alfafa required a prolonged time to penetrate the epidermis and mesophyll (pattern C wave) and showed a significant reduction in phloem sap ingestion (Goławska, 2007). Furthermore, different phenolic compounds seem to have different effects on the feeding parameters of aphids. Caffeic and gallic acids in cereals, for example, drastically shortened the probing phase of the grain aphid, whereas catechin prolonged the pathway phase and also decreased the number of probes by the grain aphid (Urbanska et al., 2002). On average, elevated CO2 increases the total phenolicsin plants by an average of 19%, condensed tannins by 22%, and flavonoids by 27% (Robinson et al., 2012). The excess of secondary metabolites in plants may help explain the increased epidermis and mesophyll resistance of plants during pathway and probing feeding stages of aphids under elevated CO2 (Guo et al., 2014a). Despite of increasing tannin content and phenolic compounds in whole host plant leaves, the bird cherry-oat aphid Rhopalosiphum padi performed better under elevated CO2 (Bezemer and Jones, 1998; Zhang et al., 2003). This result suggested that the tricky feeding strategy of the aphid allows it to avoid some potential defensive components. Thus, it is hard to predict the impact on aphid fitness only through surface or pathway effects.

Phenylalanine ammonialyase (PAL) and polyphenol oxidase (PPO) are two key enzymes involved in the synthesis of phenolic compounds that may be absorbed by the salivary sheath of the aphid stylet. The further polymerization of phenolic compounds causes browning of cells in contact with the saliva; such browning was associated with aphid probing activity during penetration of the epidermal and mesophyll tissues (Jiang and Miles, 1993; Urbanska et al., 1998; Han et al., 2009). PAL and PPO activities are changed by elevated CO2. For example, elevated CO2 tends to increase PAL activity but decrease PPO activity in M. truncatula. However, it is still unknown how changes in PPO and PAL activities under elevated CO2 affect the penetration phase of aphid feeding (Guo et al., 2014b).

#### Resistance Expressed in the Phloem

After overcoming defenses associated with the plant epidermis and mesophyll, the aphid stylet may finally reach the phloem, but plants have ways to prevent or reduce the ingestion of phloem sap. Phloem sap contains carbohydrates, proteins, and amino acids that are essential for plant development (Gündüz and Douglas, 2009). If the phloem is impaired, plants could suffer loss of nutrients, disturbance of translocation, and increased vulnerability to infection by microbial pathogens (Dinant et al., 2010). Therefore, plants have evolved a range of defenses to inhibit phloem feeding by aphids (Will et al., 2013). The most common defense involves the occlusion of sieve tubes by the plugging of sieve pores (Knoblauch and van Bel, 1998). Two groups of sieve-tube occlusion mechanisms can be found in plants: callose deposition and protein plugging (e.g., Will and van Bel, 2006; Furch et al., 2007). The Ca2+ signaling pathway in plants plays a key role in sieve-tube occlusion during aphid penetration. When the stylet penetrates the sieve membrane, the high concentration gradient of Ca2+ between the apoplast and the sieve element lumen leads to an influx of Ca2+into the sieve element lumen, which induces occlusion (Knoblauch and van Bel, 1998). When this occurs, aphids must secret watery saliva into the phloem; the saliva contains proteins that bind Ca2+ and counteract sieve element occlusion. Thus, the time spent during salivary secretion into sieve elements reflects the defenses located in the phloem (Will et al., 2013).

Phloem resistance against aphids may be affected by elevated CO2. The key gene involved in callose biosynthesis is upregulated in Arabidopsis under elevated CO2 (Li et al., 2008). Furthermore, cytosolic free Ca2+ is increased by elevated CO2 in Commelina communis (Webb et al., 1996). The increased production of callose and free Ca2+ in cells may cause aphids to spend more time in overcoming phloem resistance. EPG data consistently showed that elevated CO2 increased the time of salivary secretion into sieve elements when pea aphids fed on M. truncatula (Guo et al., 2013). Still, there is no direct evidence confirming that elevated CO2 increases the phloem resistance against pea aphids because of increases in callose deposition and in the Ca2+ signaling pathway.

## Aphid Phloem Ingestion and Its Relation to Plant Nutrition

The efficiency with which aphids feed on phloem sap is determined by the nutritional composition of the sap (Douglas, 2003). Sucrose is the dominant organic compound in the phloem sap and is a crucial C source for aphids (Fisher and Cash-Clark, 2000; Douglas, 2003). Sucrose is the principal energy source for aphids and also provides the C skeleton for lipid, amino acid, and protein synthesis (Rhodes et al., 1996; Febvay et al., 1999). In potato, a mutation of the sucrose transporter StSUT1 gene reduced the phloem sucrose content and simultaneously reduced the performance of the potato aphid (Pescod et al., 2007). Nevertheless, high concentrations of soluble carbohydrates in plant tissues often reduce aphid performance because they dilute other nutrients such as amino acids and proteins; as a consequence, the aphids must increase their consumption of phloem sap and excrete the excess sucrose as honeydew (Wilkinson et al., 1997). In contrast to carbohydrate-based nutrients, N nutrition is a limiting factor for aphid growth. The phloem sap ingested by aphids has a protein/carbohydrate ratio (w/w) as low as 0.1 while the leaf tissue ingested by chewing insects has a protein/carbohydrate ratio ranging from 0.8 to 1.5 (Behmer, 2008).

Increases in atmospheric CO2 accelerate photosynthesis and synthesize and transport of sucrose into the phloem, which dilutes the N concentration and increases the C:N ratio in the phloem of non-legumes (Barbehenn et al., 2004). The decreased nitrogen concentration of plants could prolonged the pre-reproductive period and decrease the fecundity of some aphids under elevated CO2 (Dáder et al., 2016). However, Sun et al. (2009) found that although amino acid relative concentration in the phloem of cotton plants was lower under elevated CO2 than under ambient CO2, higher amounts of free amino acids were found in cotton aphids fed on cotton grown in elevated CO2. These results suggested that cotton aphids under elevated CO2 will ingest increased quantities of phloem sap to satisfy their nutritional requirements. Moreover, the relative concentrations of predominantly essential amino acids in the phloem of barley are increased under elevated CO2 (Ryan et al., 2015). The latter result is consistent with the large increases in the levels of minor amino acids (most of which are considered essential) in tobacco seedlings under elevated CO2 (Geiger et al., 1998). These results suggest that although the total N concentration of plants is decreased, amino acids biosynthesis and translation in some non-legumes may increase under elevated CO2. Moreover, the mathematic model constructed by Newman et al. (2003) predicted that aphid populations tend to be larger under elevated CO2 if host plants have higher N supplementation, that the nitrogen requirement of aphids is low and that the density-dependent response is weak. Thus, a general explanation for the speciesspecific responses of aphids to elevated CO2 remains to be elucidated.

In legumes, elevated CO2 leads to a 38% increase in the quantity of N fixed from the atmosphere, which can compensate for decreases in plant N under elevated CO2 and cause the legumes to maintain a C:N ratio similar to that under ambient CO2 (Lam et al., 2012). When M. truncatula was infested by pea aphids, elevated CO2 significantly increased the concentration of total amino acids in leaves and of most individual amino acids in the phloem by enhancing the enzyme activities of N transamination (Guo et al., 2013). The increased amino acids, however, are mostly nonessential, and require the aphid endosymbiont Buchnera to convert them into essential amino acids (Nikoh et al., 2010). When the N-fixation ability was reduced by artificially induced mutation, the individual amino acid relative concentration in the phloem of M. truncatula was decreased such that Buchnera could no longer convert the nonessential amino acids into essential amino acids (Guo et al., 2013). These results with legumes suggest that elevated CO2 may increase the phloem feeding time of the pea aphid by altering amino acid metabolism, and that this response depends on a functional N fixation system. Responses of different cultivars, varieties, or genotypes of the same species to elevated CO2, however, can also vary. For example, Johnson et al. (2014) found elevated CO2 increased 86% and 56% essential amino acid concentrations and pea aphid colonization success on the high resistant cultivar 'Sequel' of M. sativa. However, elevated CO2 decreased 53% and 33% essential amino acid concentrations and aphid colonization on the moderate resistant cultivar 'Genesis'. This result suggested some cultivars may become more or less susceptible to aphid attack under climate change conditions, an important consideration for determining future outcomes (McKenzie et al., 2013).

The ability to fix N is regulated by several hormone signaling pathways including the ET signaling pathway (Ma et al., 2002; Penmetsa et al., 2008). When the key gene Mtskl in the ET-perception pathway was mutated in M. truncatula, the nitrogenase activity was increased about two times (Penmetsa and Cook, 1997). Previous study has shown that elevated CO2 decreases the ET signaling pathway in Arabidopsis (Sun et al., 2013). The suppression of the ET signaling pathway in M. truncatula increased nodulation and N fixation ability, which thereby satisfied the increased demand for N by plants growing under elevated CO2. The down-regulation of the ET signaling pathway, however, is accompanied by decreased ET-mediated host resistance against the pea aphid (Guo et al., 2014b). This result suggested that in the M. truncatula–pea aphid system under elevated CO2, both nutritional and resistance effects would increase the fitness of the pea aphid by suppressing the ET signaling pathway (**Figure 1**).

#### APHID XYLEM ABSORPTION AND ITS RELATION TO PLANT WATER STATUS

Aphids occasionally ingest xylem to increase their phloem feeding efficiency (Tjallingii and Esch, 1993; Douglas, 2006). Because the sugar-enriched phloem sap has an osmotic pressure that is as much as 4 to 5 times greater than that of the aphid's haemolymph, continuously passive uptake of the phloem sap could result in aphid dehydration. To avoid self-dehydration and

osmotic stress in the haemolymph during the phloem-feeding phase (i.e., to balance haemolymph osmolarity), aphids must consume a certain amount of xylem sap, which has a lower osmolarity than phloem sap (Pompon et al., 2011). This xylemfeeding behavior requires that the host plant has a relatively high plant water potential because the feeding is passive, i.e., fluid moves from plant to aphid because of a water potential gradient (Huberty and Denno, 2004; Daniels et al., 2009; Nalam et al., 2012). Aphids, like pathogens, can trigger stomatal closure, decrease leaf transpiration, and maintain the water content of the host plant by up-regulating the ABA signaling pathway. This manipulation of host stomata helps aphids absorb water from the xylem to neutralize phloem osmotic pressure (Sun et al., 2015).

Under elevated CO2, plants also exhibit reduced stomatal apertures and stomatal conductance. In FACE experiments, elevated CO2 has decreased stomatal conductance by an average of 22% for five functional plant groups that include 285 plant species (Ainsworth and Rogers, 2007). As noted, the decreased stomatal conductance reduces water loss from plants and increases plant water potential and water content (Wullschleger et al., 2002; Pritchard et al., 2007). Sun et al. (2015) found that the decreases in stomatal aperture and increases in plant water potential induced by elevated CO2 facilitated xylem feeding by aphids and thereby decreased aphid haemolymph osmolarity, which indicated a decreased cost of osmoregulation in aphids under elevated CO2.

## CONCLUSION AND PERSPECTIVES

Recent studies have provided evidence that elevated CO2 alters plant resistance, nutritional value, and water status and that these changes affect certain feeding stages of aphids (**Figure 1**). The evidence also indicates that such changes and effects could be mediated by the phytohormones JA, SA, ET, and ABA (Guo et al., 2013, 2014a,b; Sun et al., 2015). In these and related studies, elevated CO2 stimulated the SA signaling pathway and thereby increased the epidermis and mesophyll resistance of plants. However, elevated CO2 decreased JA and ET signaling pathways, which reduced the total time required by aphids to reach the phloem. The decreased ET signaling pathway also increased the N fixation ability of legumes and thereby increased their synthesis of amino acids, which in turn increased amino acid acquisition by aphids (Guo et al., 2013). Moreover, elevated CO2 decreased stomatal aperture and increased plant water potential, which thereby increased aphid xylem absorption and enhanced aphid osmoregulation (Sun et al., 2015). Nevertheless, transcriptomic evidence shows that elevated CO2 has a wide range of effects on plant metabolism (including C and N assimilation, secondary metabolism, and transportation), all of which may affect aphid performance (Ainsworth et al., 2006; May et al., 2013). Thus, the effects of elevated CO2 on the interaction between plants and aphids cannot be understood by simply relating one aspect of plant quality to a specific feeding phase of the aphid. Because the responses to elevated CO2 differ among plant species, it is currently difficult to generalize about

how further increases in concentrations in atmospheric CO2 affects aphid feeding and damage. An increased understanding of the molecular mechanisms underlying the recognition and interactions between plants and aphids should increase our ability to predict aphid damage under elevated CO2.

In addition to changes in aphid feeding behavior, changes in aphid physiology must be considered to understand how aphid performance is affected by elevated CO2. Some studies have reported increases in aphid growth rate and fecundity under elevated CO2, which suggests that elevated CO2 increases aphid fitness and increases the probability of aphid outbreaks. At present, we have some understanding of what happens but we do not know how it happens. Like chewing insects, aphids could sense and respond to nutritional changes in host plants by regulating a complex regulatory network involving the insulinrelated peptides, the target of rapamycin (TOR), ecdysteroids, and juvenile hormone (Badisco et al., 2013). For example, TOR acts as a central regulator of protein synthesis by sensing and integrating signals from amino acid nutrition, while the insulin signaling pathway is responsible for sensing carbohydratederived nutrients (Grewal, 2009). Thus, research is needed on how these two nutrient-sensing and regulatory pathways in aphids affect vitellogenins and juvenile hormone/ecdysone when aphids feed on plants with increased C:N ratios under elevated CO2.

Herbivorous insects can be affected by environmental change via changes in host physiology and chemical composition or via changes in competitors or natural enemies (Awmack and Leather, 2002). Elevated CO2 affects aphid performance from the level of individual physiology or even molecular function to the level of the ecosystem (Sun and Ge, 2011). The effects of elevated CO2 on individual plants and aphids may differ from the effects involving the entire ecosystem and multiple trophic levels because responses to elevated CO2 may differ among trophic levels. It is well known that elevated CO2 has bottom-up effects on the feeding behavior and population size of aphids, but the situation becomes more complicated when aphid–aphid interactions or top–down effects involving natural enemies are considered. For example, aphid species, or even different genotypes within the same species, differ in their responses to elevated CO2 (Mondor et al., 2005), and these differences might affect the outcome of intra- or inter-specific competition between aphid species or genotypes (Stacey and Fellowes, 2002; Sun et al., 2009). Furthermore, some reports indicate that parasitoids and predators are more abundant or

#### REFERENCES

effective under elevated CO2 (Percy et al., 2002; Chen et al., 2005) and that aphids are less sensitive to alarm pheromones under elevated CO2 (Awmack et al., 1997; Mondor et al., 2004). It seems that enhanced top-down effects on aphids under elevated CO2 may strongly alter the effects of aphids on host plants (Hentley et al., 2014).

The different feeding strategies evident in aphid responses to environmental changes are possibly driven by synchronous adaptation to host and environment. Because it directly affects herbivorous only weakly, elevated CO2 mainly influences herbivorous insect by altering the host plant (Yin et al., 2010). Thus, understanding plant–aphid interactions is likely to be central to understanding how aphids respond to elevated CO2. We suggest that molecular tools be used to better understand how the host plant 'recognizes' the aphid and vice versa; this research might focus on salivary secretions (the most obvious 'signal' available), which could trigger various molecular responses in the host that then affect the aphid in various ways. Although the knowledge from literatures shows that aphids may have species-specific molecular interaction with the hosts, it is believed that the genetics and physiology governing plant– aphid interactions have many commonalities rooted in their phylogenies so that understanding the complexity of interaction will provide meaningful insights into aphids acting on different kinds of plants and aid us in using them to our best advantage. Given increasing concentrations of atmospheric CO2 and climate change, new crop varieties will be needed that can produce sustainable yields in spite of the changing environment and the potential for increased pressure from aphids and other pests. The development of such crop varieties will be facilitated by a better understanding of the interactions between plants and aphids at molecular, community, and ecosystem levels.

#### AUTHOR CONTRIBUTIONS

YS and HG wrote this article, FG revised it.

#### ACKNOWLEDGMENTS

This project was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (no. XDB11050400) and the National Nature Science Fund of China (nos. 31500332 and 31221091).

wild tuber-bearing Solanum species to the aphid Myzus persicae. Entomol. Exp. Appl. 121, 145–157. doi: 10.1111/j.1570-8703.2006.00464.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Sun, Guo and Ge. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fpls-07-00502 April 11, 2016 Time: 18:21 # 10

# Root Herbivores Drive Changes to Plant Primary Chemistry, but Root Loss Is Mitigated under Elevated Atmospheric CO2

Scott W. McKenzie1,2,3,4, Scott N. Johnson5 , T. Hefin Jones4 , Nick J. Ostle6 , Rosemary S. Hails3 and Adam J. Vanbergen1 \*

1 Centre for Ecology and Hydrology, Edinburgh, UK, 2 The James Hutton Institute, Dundee, UK, 3 Centre for Ecology and Hydrology, Wallingford, UK, 4 School of Biosciences, Cardiff University, Cardiff, UK, 5 Hawkesbury Institute for the Environment, University of Western Sydney, Sydney, NSW, Australia, 6 Lancaster Environment Centre, Lancaster University, Lancaster, UK

Above- and belowground herbivory represents a major challenge to crop productivity and sustainable agriculture worldwide. How this threat from multiple herbivore pests will change under anthropogenic climate change, via altered trophic interactions and plant response traits, is key to understanding future crop resistance to herbivory. In this study, we hypothesized that atmospheric carbon enrichment would increase the amount (biomass) and quality (C:N ratio) of crop plant resources for above- and belowground herbivore species. In a controlled environment facility, we conducted a microcosm experiment using the large raspberry aphid (Amphorophora idaei), the root feeding larvae of the vine weevil (Otiorhynchus sulcatus), and the raspberry (Rubus idaeus) host-plant. There were four herbivore treatments (control, aphid only, weevil only and a combination of both herbivores) and an ambient (aCO2) or elevated (eCO2) CO2 treatment (390 versus 650 ± 50 µmol/mol) assigned to two raspberry cultivars (cv Glen Ample or Glen Clova) varying in resistance to aphid herbivory. Contrary to our predictions, eCO2 did not increase crop biomass or the C:N ratio of the plant tissues, nor affect herbivore abundance either directly or via the host-plant. Root herbivory reduced belowground crop biomass under aCO2 but not eCO2, suggesting that crops could tolerate attack in a CO2 enriched environment. Root herbivory also increased the C:N ratio in leaf tissue at eCO2, potentially due to decreased N uptake indicated by lower N concentrations found in the roots. Root herbivory greatly increased root C concentrations under both CO2 treatments. Our findings confirm that responses of crop biomass and biochemistry to climate change need examining within the context of herbivory, as biotic interactions appear as important as direct effects of eCO2 on crop productivity.

Keywords: aphid, vine weevil, carbon, nitrogen, plant productivity, aboveground, belowground

## INTRODUCTION

Root herbivory is very damaging to plants, especially when combined with multiple biotic and abiotic stresses (Zvereva and Kozlov, 2012) that can lead to substantial losses of crop yields (Villani and Wright, 1990; Blossey and Hunt-Joshi, 2003; Blackshaw and Kerry, 2008). Crop traits such as compensatory growth are key to crop survival and primary productivity in the face of herbivore

#### Edited by:

Susana M. P. Carvalho, University of Porto, Portugal

#### Reviewed by:

Zhilong Bao, University of Florida, USA Piotr Trebicki, Agriculture Victoria, Australia

> \*Correspondence: Adam J. Vanbergen<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science

Received: 12 February 2016 Accepted: 27 May 2016 Published: 14 June 2016

#### Citation:

McKenzie SW, Johnson SN, Jones TH, Ostle NJ, Hails RS and Vanbergen AJ (2016) Root Herbivores Drive Changes to Plant Primary Chemistry, but Root Loss Is Mitigated under Elevated Atmospheric CO2. Front. Plant Sci. 7:837. doi: 10.3389/fpls.2016.00837

pest pressure (Strauss and Agrawal, 1999; Watts et al., 2011; Huang et al., 2012; Robert et al., 2014). Plants, however, generally are less able to compensate for root herbivory compared to shoot herbivory (Johnson et al., 2016a). Moreover, even in simple agroecosystems insect herbivores occur as part of an above–belowground community (Megías and Müller, 2010; Soler et al., 2012). Consequently, the direct and indirect (mediated by host-plant plasticity) interactions among plants and herbivores occupying different guilds or niches, are key to understanding crop resistance and resilience to herbivory (Johnson et al., 2009; Huang et al., 2013; McKenzie et al., 2013; Hagenbucher et al., 2014).