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Chemical Senses, 2016, Vol 41, 325–338 doi:10.1093/chemse/bjw005 Original Article Advance Access publication February 8, 2016

Original Article Formic and Acetic in Degradation Products of Plant Volatiles Elicit Olfactory and Behavioral Responses from an Insect Vector Justin George1, Paul S. Robbins1, Rocco T. Alessandro1,

Lukasz L. Stelinski2 and Stephen L. Lapointe1 Downloaded from

1Subtropical Insects and Horticultural Research Unit, United States Horticultural Research Laboratory, USDA-ARS, 2001 South Rock Road, Fort Pierce, FL 34945, USA and 2Entomology and Nematology Department, Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA

Correspondence to be sent to: Stephen L. Lapointe, Subtropical Insects and Horticultural Research Unit, United States Horticul- http://chemse.oxfordjournals.org/ tural Research Laboratory, USDA-ARS, 2001 South Rock Road, Fort Pierce, FL 34945, USA. e-mail: [email protected]

Accepted 11 January 2016.

Abstract Volatile phytochemicals play a role in orientation by phytophagous insects. We studied antennal and behavioral responses of the Asian citrus psyllid, Diaphorina citri, vector of the citrus greening disease pathogen. Little or no response to citrus leaf volatiles was detected by electroantennography. at University of Florida on April 22, 2016 Glass cartridges prepared with β-ocimene or citral produced no response initially but became stimulatory after several days. Both compounds degraded completely in air to a number of smaller . Two peaks elicited large antennal responses and were identified as acetic and formic acids. Probing by D. citri of a wax substrate containing odorants was significantly increased by a blend of formic and acetic acids compared with either compound separately or blends containing β-ocimene and/or citral. Response surface modeling based on a 4-component mixture design and a 2-component mixture-amount design predicted an optimal probing response on wax substrate containing a blend of formic and acetic acids. Our study suggests that formic and acetic acids play a role in host selection by D. citri and perhaps by phytophagous insects in general even when parent compounds from which they are derived are not active. These results have implications for the investigation of arthropod olfaction and may to elaboration of attract-and-kill formulations to reduce nontarget effects of chemical control in agriculture.

Key words: Asian citrus psyllid, electroantennography, insect feeding behavior, insect–plant interactions

Introduction attack, plant–atmosphere interactions, etc., and can have important implications for plant-plant signaling and plant–insect interactions. Plants produce volatile organic compounds (VOCs) such as monoter- For example, plants produce as a byproduct of cell wall penes, sesquiterpenes, terpenoid alcohols, acetates, and by that is emitted at high levels during plant growth (Fall multiple metabolic pathways. They contribute to plant plumes and Benson 1996; Hüve et al. 2007). Enzyme-mediated oxidation of involved in attraction of insects (Beyaert and Hilker 2014). Plant methanol in plants produces that can be further oxi- catabolic processes can break down and release these compounds dized to formic and CO (Cossins 1964). , as lower chain aldehydes, ketones and carboxylic acids by oxidative 2 and are also produced during primary metabolism. Under pathways (Oikawa and Lerdau 2013). Catabolism and degradation stress conditions, ethanol can be transported from roots to leaves of VOCs can result from plant stress, environmental stress, herbivore

Published by Oxford University Press on behalf of US Government 2016. 325 326 Chemical Senses, 2016, Vol. 41, No. 4 and undergo enzyme-mediated oxidation to acetaldehyde and acetic Very little is known about the orientation of D. citri to host plant acid (Kreuzwieser et al. 1999, 2001). The emission levels of these volatiles or of insects in general to the degradation products of host catabolic byproducts can be regulated by (O3) levels, stress plant volatiles. In the literature of insect–plant interactions, degrada- conditions, pollution and freezing events (Kimmerer and Kozlowski tion products of headspace volatiles have not been considered as 1982) as well as pests or diseases. Measurements of net ecosystem insect kairomones. Instead, the primary focus has been on phyto- exchange () of ethanol, acetaldehyde and acetic acid above an chemicals as they are released by the plant (Metcalf and Metcalf orange grove (Citrus sinensis) found higher net fluxes of acetalde- 1992). The current study reports a convenient method for recording hyde and acetic acid compared with ethanol. The ratio of catabo- electroantennograms (EAGs) from whole insect preparations of D. lite (acetaldehyde, acetic acid) was found to be 676% and 311% citri. We screened common host plant volatiles previously identified greater, respectively, than the precursor (ethanol) (Park et al. 2013). from headspace collected over young leaves of Rutaceae species of Increased flux rates of catabolic or degradation byproducts such as varying susceptibility to D. citri (Patt et al. 2011; Poerwanto et al. acetic acid and formic acid may act as significant olfactory cues for 2012). We report for the first time antennal response of D. citri to insect herbivores including pathogen vectors. formic and acetic acids formed as degradation products of constitu- Host plant finding is of paramount importance for phytophagous tive terpenoids. Oxidative byproducts such as formic and acetic acids insects to fulfill their nutritional requirements and to find suitable produced from citrus VOCs may act as olfactory cues for D. citri oviposition sites (Bruce et al. 2005). Orientation of Diaphorina citri to locate stressed plants that are more susceptible to insect attack Kuwayama to host plants appears to be multimodal and includes and infection by the HLB pathogen. Antennal responses of D. citri Downloaded from visual, auditory and as yet unidentified chemical cues (Wenninger to plant volatiles and their degradation products are discussed. Our et al. 2009a, 2009b; Rohde et al. 2013). Olfactory cues based on results suggest that formic and acetic acids, possibly arising as deg- VOCs and their catabolic byproducts or abiotic degradation prod- radation products from host plant volatiles or directly emitted by ucts may play an important role in host finding and feeding behavior plants, may serve as important orientation cues for phytophagous of insects (Visser and Yan 1995; Raguso and Light 1998; Hansson insects and disease vectors. et al. 1999; Tol and Visser 2002). Peripheral olfactory systems of http://chemse.oxfordjournals.org/ insects consist of antennae and maxillary or labial palps that house Materials and methods olfactory receptor neurons (ORNs) important in the detection of host volatiles. Electrophysiological responses from insect antennae Asian citrus psyllids are processed and integrated within neural glomeruli in the insect A healthy (HLB-free) colony of D. citri was maintained at the US brain resulting in behavior (Bruce et al. 2005). Onagbola et al. Horticultural Research Laboratory, Fort Pierce, FL. Insects were (2008) characterized eleven morphologically unique sensillae on the reared on seedlings of Citrus macrophylla Wester and maintained antennae of male and female D. citri. Some of these were identified at 28 °C, 14:10 L: D. All insects used for recordings were adults as porous sensilla trichoidea and rhinarial plates with a likely olfac- between 8 and 10 days old. tory function (Onagbola et al. 2008). The olfactory funtion of these was subsequently proven electrophysiologically with single sensil- Electroantennography at University of Florida on April 22, 2016 lum recordings (Coutinho-Abreu et al. 2014a). Individual D. citri adults were placed in the large end of 10 µL plastic Diaphorina citri is a vector of the causal pathogen of the dev- pipette tips (USA Scientific) from which the tip had been cut so the astating citrus greening disease (aka huanglongbing in Chinese or resultant opening provided room for the head and antennae of the HLB). The disease is caused by fastidious, phloem-restricted bacte- psyllid to project (Figure 1). Psyllids were placed in the large opening rium (Candidatus Liberibacter spp.) whose symptoms include chlo- of the pipette tip and allowed to move upward toward the smaller rotic leaf mottling, shoot dieback, small, misshapen and bitter fruit opening. Using a small piece of cotton wool, psyllids were gently and eventual tree death (Gottwald et al. 2007). The vector was first pushed partially through the small end of the plastic pipette tip until reported in Florida in 1998 (Halbert 1998) followed by the confir- the head and antennae were exposed. The pipette tip was threaded mation of HLB in Florida in 2005 (Halbert 2005). Adult D. citri to a custom stage (Figure 1) that held the psyllid in place prior to feed on various leafy parts of rutaceous plants but oviposition and placement of electrodes. A filtered and humidified airstream passed nymphal development require tender young leaves and shoots. Host over the antenna at 30 cm/s. Glass electrodes were created by pull- selection and feeding behavior are influenced by host plant vola- ing glass capillary cartridges (World Precision Instruments, TW150- tiles (Halbert and Manjunath 2004; Bove 2006). Constitutive host 4, 1.5 mm OD, 1.12 mm ID) in a capillary puller (World Precision plant volatiles and their role in host plant finding have been studied Instruments) and then filling them with 0.5 M KCl. Lengths of sil- extensively for members of the Liviidae (Moran and Brown 1973; ver wire (0.5 mm diameter, Alfa Aesar) were inserted through the Nehlin et al. 1994; Soroker et al. 2004; Cen et al. 2005; Nissinen glass electrode holder and into the KCl solution. Using microman- et al. 2008; Rouseff et al. 2008; Patt and Sétamou 2010; Onagbola ipulators, the indifferent electrode was inserted into the back of the et al. 2011; Patt et al. 2011; Poerwanto et al. 2012; Robbins et al. insect’s head; the recording electrode encircled the distal end of the 2012). antenna as described by Wenninger et al. (2009b). A Kombi-Probe Volatile stimuli emitted by flushing shoots may play an impor- amplifier (Syntech) was used to acquire the signal from the antenna. tant role in the detection, location, and evaluation of potential host The signal from the amplifier was conditioned using a Syntech plants by D. citri (Patt and Sétamou 2010; Sule et al. 2012). Mann IDAC-4 interface (Syntech). Signals acquired from the IDAC-4 were et al. (2012) reported that citrus plants infected with HLB were more displayed and stored on a computer running Syntech software. attractive to D. citri than noninfected plants, possibly due to higher amounts of methyl salicylate released from infected compared with Odorant preparation and EAG recordings healthy plants. Induced release of methyl salicylate by psyllid feeding An initial survey of volatile plant produced by young emergent may act as a conspecific cue that could lead to increased transmis- leaves of C. macrophylla was conducted. A sample of leaves from pot- sion of the HLB pathogen (Mann et al. 2012). ted plants was ground to a powder using liquid . A portion Chemical Senses, 2016, Vol. 41, No. 4 327

(2 g) was extracted with 100 mL of methanol: (3:1). The aqueous by SPME for 1 h. Controls consisted of a chamber containing an methanolic extract was back-extracted 3 times with 50 mL aliquots of identical pot with soil but no plant and a chamber into which 10 µL 1:1 ether:hexane. The combined extracts were dried over anhydrous each of acetic and formic acid were introduced and allowed to vola- sodium sulfate, concentrated and analyzed by GC-MS (Figure 2). tilize. Samples were analyzed by coupled gas chromatograph-mass To test for direct release of acetic and/or formic acids by citrus (GC-MS) using a ThermoElectron Trace GC coupled plants, potted plants of three citrus genotypes were placed in sepa- to a DSQII quadrupole MS. The GC-MS was equipped with a PTV rate glass collection chambers (0.2 m × 0.8 m, Analytical Research injector operated in CT-splitless mode at 250 °C. After 2 min, the Systems, Inc.): C. macrophylla and two varieties of sweet orange, split and purge vents were pened with the split vent flow at 20 mL/ C. sinensis (Valencia and Madam Vinous). Headspace volatiles were min and purge vent flow at 3.5 mL/min. The oven program started at collected using SPME (50 mm Carboxen/PDMS/DVB fiber) exposed 10 °C for 4 min then increased at 4 °C/min to 280 °C with a 10 min for 1 h through a sampling port 1 h after introduction of the plants hold. The MS was scanned for the first 5.5 min from m/z 10 to 200, into the chambers and daily thereafter up to day 7. Condensate then from 5.5 to 40 min from m/z40 to 400, and 40 min from m/ that appeared on the glass walls of the collection chamber was col- z50 to 550. The MS source was maintained at 220 °C and the GC lected on filter paper, placed in a 20 mL sampling vial and sampled transfer line at 300 °C. Downloaded from http://chemse.oxfordjournals.org/ at University of Florida on April 22, 2016

Figure 1. Custom stage for positioning whole insect preparations of Diaphorina citri for electroantennogram recording. Inset shows an adult psyllid constrained in a plastic pipette tip cut to allow protrusion of the insect’s head.

Figure 2. Chromatogram showing major odorant peaks from of a concentrated 1:1 ether hexane back extraction of aqueous methanolic extracts of young leaves of Citrus macrophylla, a preferred host of Diaphorina citri. Neral and geranial (double bond isomers together known as citral) and (Z) and (E)- β-ocimene were used in odorant degradation studies as they consistently showed degradation in glass cartridges under laboratory conditions. 1, α-pinene; 2, β-myrcene; 3, limonene; 4, (Z)-β-ocimene; 5, γ-terpinene; 6, linalool; 7, β-citronellal; 8, neral; 9, geranial; 10, citral; 11, δ-elemene; 12, isocaryophyllene; 13, α-humulene; 14, murolene; 15, dibutylhydroxytoluene; 16, germacrene B. 328 Chemical Senses, 2016, Vol. 41, No. 4

To determine antennal responses to individual odorants, log EAG experiments were also performed using log doses (100 ng, doses of purchased odorants were prepared in chloride. 1 µg, 10 µg, 100 µg, 1 mg, 10 mg) of citral, β-ocimene, geranyl ace- Stimulus cartridges were prepared for electroantennography by tate, linalyl acetate, neryl acetate, β-caryophyllene, acetic acid, and pipetting 10 µL of each of the various odorant doses onto 0.75 cm formic acid (Sigma–Aldrich). Citral is any blend of its geometric × 3 cm strips of filter paper (Whatman No. 1) before insertion into isomers (Z, E) referred as neral and geranial, respectively. For odor- glass Pasteur pipettes (14.5 cm). Pipettes were capped at both ends preparations and detection by EAG of citral and β-ocimene, the with 1000 µL disposable plastic pipette tips briefly exposed to flame commercially available isomeric blends were used. Detection by gas to close the distal end. Stimulus cartridges were prepared fresh for chromatogram-coupled electroantennography (GC-EAD) allowed each recording or allowed to age as described under EAG response for antennal discrimination of the individual isomers. The individual to degradation products of citral and β-ocimene. The volatile con- odorants for EAG were prepared by serial dilutions in methylene tents of stimulus cartridges were wafted over the insect preparation chloride. Ten microliters of each dilution were placed on filter paper by removing the pipette tip caps and connecting the large end of the strips and allowed to dry in a fume hood before insertion into glass Pasteur pipette to tubing connected to a Syntech Stimulus Controller Pasteur pipettes. One antenna from each of 4 male and 4 female (Syntech) that, upon actuation, delivered a 1 mL puff of filtered and D. citri were tested against log doses of each odorant. A new set of humidified air through the stimulus cartridge and into the airstream log dose pipettes was used for each insect. Log doses were tested 2 cm upwind from the insect. beginning with a methylene chloride control, followed by 100 ng, For each recording, a blank stimulus cartridge containing filter 1 µg, 10 µg, 100 µg, 1 mg, and 10 mg of odorants, and finally fol- Downloaded from paper was puffed over the antennae to control for nonspecific signals lowed by 1 mg of formic acid as a positive control. from mechanoreceptors reacting to pressure changes associated with the stimulus puff. The nonspecific response from the corresponding run EAG response to degradation products of citral and was subtracted from antennal response values. One mg of formic acid β-ocimene (10 µL of 100 µg/µL) pipetted on to filter paper was puffed at the end Stimulus cartridges were prepared with citral or β-ocimene [(Z-) of each recording as a positive control, given its known activity. In the and (E-) isomers] tested at 0, 3, 6, and 9 days after preparation. http://chemse.oxfordjournals.org/ initial experiment, 24 chemical odorants known to be present in citrus Both compounds were tested at 100 µg, 10 mg, and neat by applying leaf extracts or volatiles, or that had elicited behavioral response from 10 µL of each dilution to filter paper strips. For the neat dose, 10 µL psyllids were tested (Table 1). All compounds were tested against one of pure odorant (99.7% or 95%) (Sigma–Aldrich) was applied antenna of each of 3 male and 3 female D. citri. directly to the filter paper strips. The aged cartridges were puffed over one antenna of each of 4 male and 4 female D. citri. A new set Table 1. Mean ± SEM (n = 3) electroantennogram responses from of pipettes was used for each individual. A negative control (meth- male and female Diaphorina citri to odorants present in headspace ylene chloride) was puffed at the beginning of each run. One mil- volatile collection from Citrus macrophylla leaf flushes ligram of formic acid was puffed at the end of each run as a positive Citrus odorants (100 µg) Male response (mV) Female response (mV) control. at University of Florida on April 22, 2016

β-Pinene 0.00 ± 0.00 0.03 ± 0.01 GC-EAD analysis Sabinene 0.00 ± 0.00 0.16 ± 0.27 The GC-EAD system consisted of an Agilent 7890A GC equipped D-Limonene 0.18 ± 0.28 0.10 ± 0.21 (Z)&(E)-β-Ocimene 0.10 ± 0.23 0.04 ± 0.12 with a SOLGEL-WAX capillary column 30 m × 0.25 mm (ID) × γ-Terpinene 0.00 ± 0.00 0.07 ± 0.11 0.25 μm film (SGE Incorporated) and a post-column glass Y-tube α-Humulene 0.00 ± 0.00 0.04 ± 0.08 splitter (Supelco) dividing the column effluent in a 1:1 ratio between β-Caryophyllene 0.00 ± 0.00 0.00 ± 0.00 the flame ionization detector (FID) and a heated (200 °C) EAD trans- (Z)-2-Hexenol 0.04 ± 0.18 0.06 ± 0.02 fer line (Syntech). The heated transfer line emptied into a charcoal- (Z)-3-Hexenol 0.00 ± 0.00 0.23 ± 0.33 filtered, humidified air stream (30 cm/s) that carried the effluent over (R)-(−)-Linalool 0.05 ± 0.19 0.00 ± 0.00 the antennal preparation. Lengths of deactivated column (0.32 mm β-Citronellol 0.03 ± 0.19 0.04 ± 0.08 ID) were used to carry the column effluent from the Y-tube splitter Geraniol 0.00 ± 0.00 0.04 ± 0.01 to the FID and into the humidified air stream. EAG and FID sig- (E)-2-Hexenal 0.00 ± 0.00 0.01 ± 0.02 nals acquired from the IDAC-4 interface were displayed and stored Nonanal 0.00 ± 0.00 0.08 ± 0.02 Citral 0.30 ± 0.31 0.02 ± 0.13 on a computer running Syntech GC-EAD software. At the start of Methyl salicylate 0.05 ± 0.23 0.09 ± 0.20 each GC-EAD run, the GC oven temperature was held at 35 °C for Linalyl acetate 0.11 ± 0.03 0.24 ± 0.11 3 min, increased to 260 °C at 15 °C/min with a 10 min final hold. Geranyl acetate 0.21 ± 0.15 0.16 ± 0.24 Injector and FID temperatures were 220 °C and 300 °C, respectively. Neryl acetate 0.04 ± 0.09 0.12 ± 0.14 Splitless injection was used with helium as the carrier gas at 2 mL/ Lauric acid 0.14 ± 0.23 0.02 ± 0.01 min. Additional makeup helium gas was added before the effluent Myristic acid 0.00 ± 0.00 0.00 ± 0.00 splitter at 10 mL/min. Pentadecanoic acid 0.00 ± 0.00 0.11 ± 0.12 Antennally active peaks from the aged citral or β-ocimene were Palmitic acid 0.58 ± 0.57 0.02 ± 0.00 identified via GC-EAD by extracting filter paper strips from stimulus Stearic acid 0.00 ± 0.00 0.09 ± 0.13 cartridges that had been aged for 10 days. Filter paper strips from Formic acid (1 mg) 3.10 ± 0.57 3.00 ± 0.36 10 aged cartridges of each compound were soaked in 4 mL of meth- ylene chloride for 24 h. Filter paper strips were removed and Responses were adjusted by subtracting responses to clean air during the corresponding run from odorant responses. 100 µg of synthetic odorant was the remaining extract was concentrated to 1 mL under nitrogen. For applied to filter paper in glass stimulus cartridges and puffed into a filtered and each GC-EAD analysis, 1 μL of extract was injected. One antenna humidified airflow upstream from the antenna. Formic acid (1mg) was puffed from each of 2 male and 2 female D. citri were tested against each at the end of each odorant run as a positive control. extract. Chemical Senses, 2016, Vol. 41, No. 4 329

GC-MS analysis 8-day-old D. citri adults were starved for 6 h and then released into Chemical analysis of degradation products was accomplished by each cage and allowed to probe for 21 h. Cages were held in a tem- GC-MS using a ThermoElectron Trace GC coupled to a DSQII perature and humidity controlled incubator at 26 °C, 75% RH, 24 h quadrupole MS controlled by Xcalibur software. The GC-MS was light conditions L:D. To visualize stylet sheaths produced by feeding equipped with a PTV injector, operated in the CTSPLIT mode and attempts on the SPLAT beads, the cover slips were removed from the a 30 m × 0.25 mm × 0.25 µm film DB5MS fused silica open tubular cages and beads were stained with Coomassie blue dye, washed in column (Agilent) using helium carrier gas at 1 mL/min. The injector water, and allowed to air-dry. The number of salivary sheaths in each temperature was maintained at 220 °C with a split flow of 10 mL/ bead was counted under a stereomicroscope at 4× magnification. min and a constant septum purge. The oven temperature program Two experiments were performed using response surface meth- started at 30 °C with a 1 min hold and then increased at 4 °C/min ods to identify primary drivers in a 4-component mixture and then to 240 °C. The transfer line from the GC to the MS was maintained in a 2-component mixture-amount design to identify an optimal at 250 °C. The MS source was maintained at 200 °C and the MS blend of the primary drivers for maximum probing by D. citri. was scanned from m/z 10 to 200 for the first 5min of the run then Experiment designs were generated and analyzed with Design- scanned from m/z 40 to 400 for the remainder of the run. Compounds Expert software (v9, Stat-Ease, Inc.). For the 4-component mixture, were identified by retention index (RI) of the eluted peaks and the the total amount of odorant was constant at 40 µL/10 mL SPLAT. background-subtracted mass spectrum averaged across the total Odorants were formic acid, acetic acid, β-ocimene, and citral. A modified D-optimal mixture design sufficient to satisfy a Scheffé

chromatogram (TIC) peak or across a characteristic extracted ion Downloaded from current peak when co-elution of multiple compounds was detected. cubic polynomial response surface model (Cornell 2002) consisting The RI database used for identification was generated at USHRL of 24 odorant combinations was used (Table 2). The entire experi- using the same column and GC-MS system. Mass spectra were com- ment was replicated 6 times for a total of 144 individual cover slips pared with those recorded in the NIST Library. For identification in 6 blocks (cages). The same design was used in each cage but with purposes, RIs had to match within 5 RI units of the average database unique randomizations of treatments within each cage. All cages value and the reverse search of the sample spectrum had to achieve were kept in the same environmental chamber at 26 °C and popu- http://chemse.oxfordjournals.org/ a match of >500 of 1000. lated simultaneously with cohorts of D. citri. In addition to those Degradation of citral and β-ocimene to oxidative byproducts needed to satisfy model terms, points (odorant combinations) were was studied over a period of 9 days by collecting headspace vola- added to estimate lack of fit and several points were duplicated to tiles from the Pasteur pipettes stored under laboratory conditions. attain sufficient degrees of freedoms to estimate pure error across was used as a solvent to wash the inside of glass pipettes the design space, provide estimates of block effects, and to attain a when needed. Headspace volatiles in Pasteur pipettes loaded with near uniform leverage for all points (Weisberg 1985). The resulting citral or β-ocimene were analyzed by GC-MS at 0, 3, 6, and 9 days quadratic design had 5 block, 9 model, 87 lack of fit, and 42 pure after loading. A 1 μL injection in the splitless mode was made using error degrees of freedom (Myers and Montgomery 2002). Based a gas-tight syringe to sample cartridges through a at University of Florida on April 22, 2016 septum capping the large end of the Pasteur pipette. Identification Table 2. Proportions of 4 odorant components varied in a 3-dimen- of antennally active peaks detected by GC-EAD was done using sional mixture design to study probing response of Diaphorina citri GC-MS by injecting 1 μL of each of the previously described filter paper extracts of aged citral or β-ocimene. Blend Formic acid Acetic acid (Z)-β-Ocimene Citral Statistical analyses of antennal responses to headspace over aged 1 0.00 0.00 0.00 1.00 stimulus tubes containing citral and ß-ocimene, and to log doses of 2 0.00 0.00 0.00 1.00 8 citrus volatiles were performed using Statistix software (Statistix 3 0.00 0.00 0.50 0.50 9). Tukey’s HSD test was used for post hoc means comparison 4 0.00 0.00 1.00 0.00 (α = 0.05) after a significant ANOVA using GLM procedures. 5 0.00 0.00 1.00 0.00 6 0.00 0.33 0.33 0.33 Psyllid probing behavior 7 0.00 0.50 0.00 0.50 8 0.00 0.50 0.50 0.00 A choice was designed to study the probing behavior of D. citri 9 0.00 1.00 0.00 0.00 in response to odorants. This assay measured both insect orientation 10 0.00 1.00 0.00 0.00 from several cm to the source of volatiles (olfaction) and subsequent 11 0.13 0.13 0.13 0.63 probing behavior that may result from a combination of olfaction 12 0.13 0.13 0.63 0.13 and gustation upon contact with a wax substrate containing odor- 13 0.13 0.63 0.13 0.13 . Test compounds were incorporated in a slow-release wax 14 0.25 0.25 0.25 0.25 matrix for volatiles (SPLAT, ISCA Technologies, Inc.) and offered to 15 0.25 0.25 0.25 0.25 caged D. citri adults (Patt et al. 2013). Release matrix blends were 16 0.38 0.13 0.13 0.38 prepared by adding 6 µL of green food coloring to 10 mL of white 17 0.50 0.00 0.00 0.50 18 0.50 0.00 0.50 0.00 SPLAT (resulting in a yellow-green mixture), which was mixed in 19 0.50 0.00 0.50 0.00 a vortex rotor for 5 min and combined with odorant compounds. 20 0.50 0.50 0.00 0.00 Treatments (SPLAT plus odorants) were applied as narrow strips 21 0.50 0.50 0.00 0.00 or beads (2.0 cm × 0.5 cm × 0.1 cm) to glass cover slips (22 mm × 22 0.63 0.13 0.13 0.13 22 mm, Fisherbrand Microscope Cover Glass 12-542-B). Each cover 23 1.00 0.00 0.00 0.00 slip received approximately 0.17 mL of SPLAT and 0.7 µL of the 24 1.00 0.00 0.00 0.00 odorant(s). The beads were air-dried for 2 h and placed in a com- pletely randomized pattern with replications on the floor of a cubi- Total amount of odorant was constant at 0.7 µL incorporated into each cal cage (60 cm × 60 cm × 60 cm, BioQuip). Cohorts of 200 5- to bead of wax substrate. 330 Chemical Senses, 2016, Vol. 41, No. 4 on the results from the 4-component blend experiment, we built a in the 2-component mixture-amount experiment and the predicted 2-component mixture-amount design to identify the optimal blend optimum was compared with the control treatments to determine and of formic and acetic acids to encourage probing if increased probing resulted from inclusion of odorants in the wax of the wax substrate by D. citri. An I-optimal design sufficient to matrix. satisfy a Scheffé cubic × cubic polynomial consisting of 40 runs was used (Table 3) with 4 replications of the compete design in separate cages. The total amount of odorant varied between 10 and 40 µL. Results The design had 8 model, 12 lack of fit, and 19 pure error degrees of Methanolic extracts of citrus leaf flushes contained a variety of freedom. Blend proportions selected by the design were produced volatile compounds including α-pinene, β-myrcene, limonene, and assayed for probing response. (Z)-β-ocimene, linalool, citral (neral + geranial), β-caryophyllene and others (Figure 2). These and other odorants were also identi- Statistical analyses of response surfaces fied from the headspace volatiles collected by SPME over 3 citrus Lapointe et al. (2008) provided a detailed description of the statisti- varieties (Supplementary Figure S1). β-Ocimene, neral and geranial cal methods used to evaluate similar blend designs. For the probing were detected in most plant headspace samples. Formic and acetic assays, models were calculated with Design-Expert. Model selec- acids were not detected in the headspace samples, nor were they tion was then based on a lack of aliased terms, low residuals, a detected in the samples of the moisture condensate from the sides 2 of the sampling jars. Acetic and formic acids were readily detected low P value, nonsignificant lack of fit, a low SD, high R and a low Downloaded from PRESS (prediction error sum of squares) value. The selected models after spiking the collection chamber with the acids (Supplementary were then evaluated by adequacy tests described by Anderson and Figure S1). Whitcomb (2005). A Box–Cox plot (Box and Cox 1964) was used Odorants associated with citrus leaf volatile components to select the square-root transformation of count data. The optimal did not elicit significant EAG responses from D. citri antennae blend of formic and acetic acids was calculated with Design-Expert (Table 1). Antennae from male or female D. citri did not respond using a simplex hill-climbing algorithm (Anderson and Whitcomb to stimulus cartridges loaded with citral or β-ocimene (<3 h-old). http://chemse.oxfordjournals.org/ 2005) in a multi-dimensional pattern search that converges at a sta- However, the same sealed stimulus cartridges elicited large tionary point. The parameter for optimization was maximum num- responses from antennae of both male and female D. citri after 3 ber of probes within the range of odorant proportions tested. The to 6 days at ambient laboratory conditions (23 °C) suggesting that control (no odorant) was, by definition, not included in the 4-com- degradation products were responsible for the electrophysiologi- ponent mixture design space because there is no possible intercept cal response. in a mixture. To test the hypothesis that the best blend resulted in a GC-MS analysis revealed a large and rapid decline in both (Z)- greater number of probes than the unscented control, the number of and (E)-β-ocimene (Figure 3A) and citral (Figure 3C) in stimulus probes on the control was compared by ANOVA with the number cartridges after 3 days and concomitant appearance of degradation of probes observed on the actual blend design point that fell closest products. Significantly lower amounts of (Z)- and (E)-β-ocimene at University of Florida on April 22, 2016 to the predicted optimum blend. This prediction was then validated (P = 0.0007 and P = 0.0005, respectively) were present in the stimu- lus cartridges on day 3 and successive days compared with fresh stimulus cartridges prepared on day 0. The stimulus cartridges ini- Table 3. Proportion and amounts of formic and acetic acids var- tially loaded with β-ocimene contained higher amounts of acetic ied in a 2-component mixture-amount design to study probing re- acid than formic acid and these degradation products were great- sponse of Diaphorina citri est on day 6 (Figure 3B). The peak areas were similar for formic Blend Proportion Amount (µL) Replicates and acetic acids in the stimulus cartridges initially loaded with citral (Figure 3D). Peak areas of acetaldehyde, , α,α-dimethyl allyl Formic acid Acetic acid , acetic acid and formic acid increased over the duration of the experiment (Figure 4). 1 1.00 0.00 10 3 2 1.00 0.00 25 2 3 1.00 0.00 40 3 EAG response to degradation products of citral and 4 0.75 0.25 18 1 β-ocimene 5 0.75 0.25 30 1 EAG recordings of male and female D. citri showed responses 6 0.75 0.25 36 1 to citral and β-ocimene degradation products on days 3, 6, and 7 0.50 0.50 10 2 9. Antennae of both sexes were not responsive to fresh citral or 8 0.50 0.50 25 4 β-ocimene (day 0) at 100 µg, 10 mg, or neat doses (Figure 5). The 9 0.50 0.50 32 1 degradation of citral elicited significantly higher antennal responses 10 0.50 0.50 40 2 on day 9 (F = 7.64; P = 0.01) from males compared with females, 11 0.38 0.63 10 2 2,11 12 0.38 0.63 20 2 but no significant antennal responses were observed on day 0, 3, and

13 0.38 0.63 30 2 6 [(F2,11 = 1.08; P = 0.38) (F2,11 = 3.50; P = 0.07) and (F2,11 = 4.16; 14 0.38 063 40 2 P = 0.05), respectively] to the odorant doses tested (Figure 5A). The 15 0.25 0.75 14 1 overall male antennal responses showed a dose effect (F2,47 = 15.08; 16 0.25 0.75 36 1 P = 0.000) and no effects of day (F3,47 = 1.43; P = 0.25) or dose × day 17 0.13 0.88 20 1 interaction (F6,47 = 0.94; P = 0.48) were observed. However, the dif- 18 0.00 1.00 10 3 ferent citral odorant doses did not elicit significant female antennal 19 0.00 1.00 25 2 responses on days 0, 3, 6, and 9 [(F = 0.38; P = 0.69) (F = 0.50; 20 0.00 1.00 32 1 2,11 2,11 P = 0.62) (F = 0.47; P = 0.63) and (F = 4.11; P = 0.054), respec- 21 0.00 1.00 40 3 2,11 2,11 tively] (Figure 5B). Chemical Senses, 2016, Vol. 41, No. 4 331 Downloaded from

Figure 3. Mean ± SEM (n = 6 for β-ocimene, n = 2 for citral) relative amount by TIC of neat β-ocimene (A, n = 6) and citral (C, n = 2) in glass stimulus cartridges http://chemse.oxfordjournals.org/ over 9 days and concurrent formation of formic and acetic acids in the same stimulus cartridges initially containing β-ocimene (B, n = 6) and citral (D, n = 2). at University of Florida on April 22, 2016

Figure 4. Chromatogram showing degradation products in the headspace of glass cartridges initially containing neat (Z) and (E)-ß-ocimene after 6 days. Inset shows the TIC (top) and, below are extracted ion current profiles used for identification of formic acid (mol.wt = 46) and acetic acid (mol.wt = 60) of toluene washes of glass cartridges.

Male and female psyllids showed higher antennal responses (F2,11 = 10.33; P = 0.004), respectively] and females (Figure 5D) on day 3 onwards with log doses of citral and β-ocimene degrad- [(F2,11 = 12.42; P = 0.002) (F2,11 = 9.19; P = 0.006) (F2,11 = 52.00; ing to new byproducts. Degradation of β-ocimene over 3, 6, and P = 0.0001), respectively] on day 3, 6, and 9. Male antennal

9 days produced significantly higher antennal responses com- responses to log doses of β-ocimene were significant (F2,35 = 29.32; pared with day 0 (α = 0.05). There were significant differences P < 0.0001; n = 4), and no effects of day (F2,35 = 0.75; P = 0.48) in antennal responses between the different odorant doses from or day × dose interaction (F2,35 = 1.01; P = 0.42) were observed. males (Figure 5C) [(F2,11 = 7.29; P = 0.01) (F2,11 = 15.35; P = 0.001) Female responses to log doses of β-ocimene treatment were also 332 Chemical Senses, 2016, Vol. 41, No. 4 Downloaded from http://chemse.oxfordjournals.org/

Figure 5. Electroantennogram responses of antennae of male (A and C) and female (B and D) Diaphorina citri to the content of glass stimulus cartridges initially containing neat citral (A and B) or (Z) and (E)-β-ocimene (C and D) over 9 days expressed as percent of antennal response to a positive control (1 mg formic acid) at the end of each recording. N.S.: no significant difference between mean antennal responses on a given day by ANOVA; means within day followed by the

same letter do not differ by Tukey’s HSD following a significant ANOVA (α = 0.0125 corrected for multiple comparisons). at University of Florida on April 22, 2016

significant (F2,35 = 35.01; P < 0.001; n = 4), but no effects of day mixture design produced a highly significant (P < 0.0001) special

(F2,35 = 0.62; P = 0.5432) or day × dose interaction (F4,35 = 0.96; cubic model that was improved by backward reduction (Table 4). P = 0.45) were observed. There was a significant decline in the The square root transformation was applied to the data (number amount of citral and β-ocimene over time (days) and an increase in of probes) as per Box–Cox analysis. The model diagnostics were all oxidation products that elicited antennal responses from both male within acceptable limits. The data appeared normal and displayed and female D. citri (Figure 3). constant variance with no outliers, no points that exceeded a Cook’s GC-EAD recordings were performed using male and female distance of one, and the predicted versus actual value plots showed 2 2 2 2 D. citri to study their response to different degradation products close agreement. The R statistics (R , Radj, and Rpred ) were clus- formed from the oxidative breakdown of citral and β-ocimene. tered with a difference less than 0.12. The lack-of-fit test was not sig- D. citri antennae responded to the volatile degradation products nificant (Prob > F = 0.1274). Based on these diagnostics, the special of citral and β-ocimene, but not to the parent volatiles. Antennae cubic model was selected and indicated significant factor effects on from male and female D. citri responded to acetic and formic acid the probing response; the model was of sufficient quality to navigate presumably formed as oxidation products from citral (Figure 6A) the experimental design space and for predicting new observations. or β-ocimene (Figure 6B). Antennae of both males and females pro- The ANOVA indicated significant terms for component effects and duced significantly higher EAG responses to acetic acid (Figures 7A interactions for formic × acetic acids (P < 0.0001) and for formic × and 8) and formic acid (Figures 7B and 8) at 1 and 10 mg compared citral, and for the 3 factor interaction acetic acid × β-ocimene × citral with the lower doses (ANOVA, orthogonal contrast, α = 0.05). None (P < 0.01). The optimization protocol predicted 3 points on the for- of the other compounds, including citral or β-ocimene, produced mic-acetic axis with desirability coefficients >0.5. All 3 were blends significant antennal responses from D. citri at any dose within the of formic and acetic acids with the highest score at an approximately range tested (Figure 8). 2:1 blend followed by a 1:1 blend of the 2 acids (Figure 9). To test the hypothesis that the best blend tested in the 4-component design Psyllid probing behavior actually received a larger number of probes than the control, we A preliminary probing assay showed no significant differences compared (ANOVA) the number of probes observed on the control

(ANOVA, F7,59 = 0.89) in the number of probes observed in lines of (no odorant in yellow SPLAT) (n = 24) with the number on the 1:1 SPLAT blended with acetic acid, β-ocimene, citral, or formic acid as blend (n = 12). There was no effect of block (cage) (P > F = 0.27) or single compounds. More probes were observed on SPLAT containing interaction between block and treatment (P > F = 0.86). The mean ± 20 µL of a 1:1 blend of acetic and formic acid compared with 20 µL SEM number of probes observed on the 1:1 blend of formic and of either acid separately or other compounds. The 4-component acetic acids (36 ± 3, n = 12) was significantly greater (P > F = 0.0002) Chemical Senses, 2016, Vol. 41, No. 4 333 Downloaded from

Figure 6. GC-EAD recording showing typical antennal responses of male and female Diaphorina citri to extracts of filter paper containing (A) citral or (B) β-ocimene aged for 10 days in glass. http://chemse.oxfordjournals.org/ at University of Florida on April 22, 2016

Figure 7. EAG responses of male and female Diaphorina citri to log doses of (A) acetic acid or (B) formic acid applied to filter paper in glass stimulus cartridges and puffed over the antennae. Filtered and humidified air (blank) was puffed at the beginning of each recording and 1 mg of formic acid was puffed at the end of each run as a positive control.

Figure 8. Mean (±SEM, n = 4) EAG responses of (A) male and (B) female Diaphorina citri to log doses of 8 citrus volatiles from 100 ng to 10 mg. Y axis represents EAG responses as percentage of the positive control (1 mg formic acid) puffed at the end of each recording (n = 4). Responses were indistinguishably low to citral, ß-ocimene, neryl acetate, geranyl acetate, linalyl acetate, and ß-caryophyllene. Means for acetic and formic acids with the same letter were not significantly different (α = 0.05, Tukey’s HSD). 334 Chemical Senses, 2016, Vol. 41, No. 4 than the number of probes observed on the control (19 ± 2, n = 24). <0.2. The lack-of-fit test was not significant (P > F = 0.128). Based The 2-component mixture-amount experiment resulted in a signifi- on these diagnostics, a cubic × linear model (Table 5) was selected cant cubic × linear model (P > F = 0.0002). The square root transfor- and indicated significant factor effects; the model was of sufficient mation was applied to the count data. The model diagnostics were quality to navigate the experimental design space and for predicting all within acceptable limits. The data appeared normal with constant new observations. The results from this experiment closely validated variance and no outliers, no points exceeded a Cook’s distance of one, the prediction resulting from the 4-component mixture experiment. and predicted versus actual value plots showed close agreement. The The linear effect of amount was not large (P = 0.02) compared with 2 2 2 2 R statistics (R , Radj, and Rpred ) were clustered with a difference the other significant effects (Table 5) and more evident for the pure

Table 4. P values, regression coefficients, and response surface model fitting diagnostic statistics for a reduced special cubic model of probing by Diaphorina citri in response to varying proportions 4 odorants

Sum of squares df Mean square F P value Coefficient estimate

Model 136.69 9 15.19 14.57 <0.0001 Linear mixture 89.14 3 29.71 28.50 <0.0001 A 4.95

B 2.78 Downloaded from C 2.90 D 2.60 AB 29.45 1 29.45 28.25 <0.0001 7.35 AD 8.07 1 8.07 7.74 0.0062 −4.44 BC 0.61 1 0.61 0.58 0.4463 −1.39

BD 0.26 1 0.26 0.25 0.6198 −0.91 http://chemse.oxfordjournals.org/ CD 0.16 1 0.16 0.16 0.6943 −0.72 BCD 6.93 1 6.93 6.65 0.0110 35.05 Lack of fit 99.90 87 1.15 1.39 0.1174

2 2 2 A, formic acid; B, acetic acid; C, β-ocimene; D, citral. R = 0.5041; Radj = 0.4694; Rpred = 0.3933. at University of Florida on April 22, 2016

Figure 9. A planar projection of the 3-dimensional tetrahedral design space created by varying the proportions (%) of 4 components. The response surface is plotted by color and contour lines indicating the number of individual stylet sheaths (probes) by Diaphorina citri on a wax substrate containing 4 odorant compounds: acetic acid, citral, formic acid, and β-ocimene. Dotted arrows indicate decreasing proportion of the corresponding compound. The projection is flattened for printing but can be folded along the 3 black axes to produce the 3-dimensional design space as shown in the cartoon inset on the left. The star indicates the predicted optimal blend for maximum number of probes (1.9:1.0 formic acid:acetic acid).

Table 5. P values, regression coefficients, and response surface model fitting diagnostic statistics for a cubic × linear model of probing by Diaphorina citri in response to varying proportions and amounts of formic and acetic acids

Sum of squares df Mean square F P value Coefficient Estimate

Model 15.04 4 3.76 7.56 0.0002 Linear mixture 3.02 1 3.02 6.09 0.0187 A 1 — 3.40 B 1 — 3.82 AB 1.41 1 1.41 2.83 0.1012 1.69 AC 4.00 1 4.00 8.05 0.0075 −0.70 AB(A-B) 4.60 1 4.60 9.25 0.0044 −8.62 Lack of fit 10.30 16 0.64 1.73 0.1280

2 2 2 A, acetic acid; B, formic acid; C, amount. R = 0.4636; Radj = 0.4023; Rpred = 0.3192. Chemical Senses, 2016, Vol. 41, No. 4 335 acetic acid blend (Figure 10). The optimization algorithm predicted a when the cartridges were stored on a laboratory bench. We also narrow range of points clustered around a 2:1 blend of formic:acetic observed an identical effect when filter paper was not used and the acids (Figure 10). compounds were placed into sealed glass cartridges. GC-MS analysis by SPME and extraction of aged filter paper revealed thatβ -ocimene and citral began to disappear from the cartridges within 3 days Discussion (Figure 3). GC-EAD of extracts of aged filter paper allowed us to Insect vectors and the plant pathogens they transmit limit food identify formic and acetic acids as the stimulatory compounds that production worldwide (Purcell and Almeida 2005). Citrus greening accumulated in the cartridges, presumably the result of oxidative disease, transmitted by D. citri, is a major threat to the economic degradation of β-ocimene and citral. This is the first observation that sustainability of the citrus industry worldwide. Currently, citrus pro- degradation products of constitutive plant volatiles are stimulatory duction in the United States and elsewhere is dependent on intensive to insect antennae and the first instance of GC-EAD used to identify insecticide applications to minimize vector and movement chemical compounds stimulatory to the antenna of a psyllid species. (Tiwari et al. 2011) and nutritional supplementation to ameliorate The existence of plant airborne signals has been known for cen- disease symptoms and extend productive tree (Schumann et al. turies and has been well documented for (Lin et al. 2009), 2011). Identifying semiochemicals from host plants and conspecifics methyl jasmonate, linalool, and β-ocimene (Holopainen and Blande for use in traps or other control applications would help to control 2012). Monoterpenes such as limonene, α-pinene, β-myrcene, lin- the vector and the disease, potentially reducing the use of pesticides. alool, neral, geranial, and β-ocimene (Figure 2) are common citrus Downloaded from Most of the volatile compounds previously identified from head- volatiles found in the atmosphere surrounding citrus orchards (Park space of young leaves of susceptible citrus genotypes (Robbins et al. et al. 2013). Some monoterpenes such as isomers of β-ocimene are 2012) did not produce a measureable antennal response from male known to be unstable in air (Fahlbusch et al. 2003). Recent stud- or female D. citri under the conditions of our study by GC-EAD ies (Oikawa and Lerdau 2013; Park et al. 2013) have shown that analysis or EAG response to individual neat compounds. This may degradation of VOCs is a common phenomenon and that oxidation be related to the relative paucity of chemoreceptors on D. citri of acetaldehyde and formaldehyde can produce acetic and formic http://chemse.oxfordjournals.org/ antennae (Onagbola et al. 2008) and the possibility that olfaction acids that are released into the atmosphere. The emission rates of plays a subordinate or secondary role in mating and host plant ori- these catabolites (formic or acetic acid) are 200–300% higher than entation in this species. Attraction to visual cues and orientation to the precursor compounds (ethanol, methanol, acetaldehyde, formal- sex-specific stridulation (Wenninger et al. 2009a; Rohde et al. 2013) dehyde, etc.). Park et al. (2013) showed that monoterpenes com- may condition or supersede volatile stimuli. prised approximately 24% of VOC compounds above an orange Several citrus volatiles have been suggested as possible attract- grove (C. sinensis). They also measured significant amounts of ace- ants or repellents for D. citri (Wenninger et al. 2009b; Patt and tic acid (8.4%) either from photochemical degradation of parent Sétamou 2010; Mann et al. 2012; Robbins et al. 2012). We tested compounds or direct plant emission. Blande et al. (2014) suggested many citrus volatile odorants by EAG and only a few elicited anten- that emitted plant signals are modified by the environment and that nal responses (Table 1). During our attempts to study these by EAG biotic responses are collectively determined by emitted signals and at University of Florida on April 22, 2016 and GC-EAD, we discovered that antennal responses to the contents their degradation products. of glass cartridges containing filter paper loaded with small amounts Understanding the mechanisms involved in insect sensory percep- of 2 common citrus compounds, β-ocimene [(Z) and (E)-β-ocimene] tion, integration, and behavior is complex and has advanced along a and citral (neral and geranial), increased dramatically over time number of paths in recent years. Integration of the knowledge

Figure 10. Predicted 3-dimensional surface response plot generated by a 2-component mixture-amount design to validate the predicted optimal blend of formic acid:acetic acid to maximize the number of probes on a wax substrate by Diaphorina citri. Circles are data for design points above (red) or below (pink) the predicted value. 336 Chemical Senses, 2016, Vol. 41, No. 4 is a challenge, but recent efforts to do so (Renou 2014) emphasize and how acetic and formic acids interact with other odorants at the the importance of understanding interactions inherent in mixtures level of perception and behavior. between odorant signals and between odorant receptors (Baker No pheromone has been identified for D. citri. Formic acid, 2009; Su et al. 2012). Our finding suggests that an additional level of known as an allomone in ants, is also a sex pheromone for 6 ant spe- complexity may exist in the volatile environment surrounding insect cies (El-Sayed 2014). The list of semiochemical uses for acetic acid is habitats. Although it has been generally accepted that many phy- extensive (El-Sayed 2014). It is designated as an allomone for one spe- tophagous insects orient to their host plants based on plant-emitted cies of Phasmida, one species of Uropygi, 5 species of Coleoptera, one odors (Bruce et al. 2005; Renou 2014), the peripheral sensory system species of Hymenoptera and 10species of Heteroptera. Additionally, of D. citri appears to be sensitive to compounds that arise through it is an attractant for 136 species including Diptera, Heteroptera, degradation of common plant volatiles that, by themselves, do not Hymenoptera, Lepidoptera, and Neuroptera. It is listed as a sex elicit antennal or behavioral responses. As often occurs, serendip- pheromone in 9 species of Heteroptera, 1 species of Hymenoptera, 4 ity contributed to our discovery that common plant volatiles appear species of Lepidoptera, and 1 species of Orthoptera (El-Sayed 2014). to degrade into other distinct small molecules in the environment Male and female psyllids responded consistently to formic and external to the plant and act as primary physiological and behav- acetic acids by EAG and GC-EAD. Incorporation of those com- ioral cues for an arthropod vector. This phenomenon may occur pounds into the feeding assay slow-release matrix allowed us to more widely than we currently appreciate. The host range of D. citri examine behavioral response to odorant blends. Probing by D. citri is restricted to genera of the family Rutaceae. Despite this rather of the feeding substrate increased with the addition of a blend of Downloaded from restricted host range, there has been no report of taxonomically spe- formic and acetic acids compared with the unscented control. We are cific compound(s) that play a role in the orientation of this species now pursuing the hypothesis that additional odorant compounds, to its host. Antennae of D. citri bear a small number of olfactory not stimulatory by themselves, may augment the probing response sensilla that could play a role in mate or host location (Onagbola when combined with formic and acetic acids. The 4-component et al. 2008). However, evidence has not been found to document blend experiment suggested that β-ocimene and citral did not con- orientation to host or conspecific odors from a distance. Coutinho- tribute to increased probing. The model predicted that an optimal http://chemse.oxfordjournals.org/ Abreu et al. (2014b) performed extensive single-cell recording from response (greatest number of probes) would occur in response to a antennal sensilla of D. citri. They identified a 3-component blend 2-component blend along the formic-acetic acid axis even though that modestly increased trap catch on sticky yellow cards in the field, that particular combination was not included as a design point but such traps remain highly inefficient. It would appear that sen- (Figure 9). Validation of the predicted optimum was obtained by sitive olfactory discrimination of suitable host plant tissue from a the 2-component mixture-amount experiment (Figure 10) that con- distance requires a higher degree of olfactory sense sophistication firmed a 2:1 formic:acetic blend as optimal for stimulating the prob- than the insect’s morphology suggests. It is more likely that color is ing response. Current work concentrates on introducing additional the stronger attractant for flighted D. citri to young, emergent and/or odorant compounds into the design space to identify mixtures capa- diseased foliage (Mann et al. 2012). Host location by adult D. citri ble of eliciting greater substrate probing than we have obtained with may resemble the “degenerate” mechanism proposed by Kennedy the 2:1 formic:acetic acid blend. The most evident application of at University of Florida on April 22, 2016 and Stroyan (1959) to explain host plant discovery by species of that knowledge could be an attract-and-kill formulation that would aphids: random dispersal, visual orientation to yellow wavelengths, greatly increase the specificity of pesticide applications and thereby and gustatory perception of an arrestant stimulus after landing. This reduce nontarget effects of insecticides currently used to control may explain our results wherein formic and acetic acids are highly D. citri in commercial citrus. These methods could be applied to stimulatory to antennal receptors but do not elicit orientation from other phytopathogen vectors for practical management. a distance. Rather, they may condition a behavioral response (prob- We were unable to detect acetic and formic acids in the headspace ing) only after alightment on a potential host. This report is the first over healthy potted citrus under the conditions of our study. However, to identify compounds that elicit antennal responses when presented we have not demonstrated that D. citri in nature responds to acetic to the antenna individually by GC-EAD or EAG. The strength and and formic acids arising as degradation products or simply as natu- concomitant weakness of these methods is that the odorants are rally produced secondary metabolites directly emitted by citrus trees as presented to the antennae individually. The perception of a mixture may occur under field conditions. Nonetheless, the role of degradation results from the interaction between its individual components giv- products of parent volatile compounds in the orientation of organisms ing rise to a neural representation that acquires new characteristics to their hosts may be an area worthy of increased investigation. relevant to the odorant mixture (Renou 2014). Diaphorina citri may respond to odorant blends in the context of a particular olfactory background as demonstrated for Manduca sexta (Patt et al. 2013; Supplementary material Riffel et al. 2014). Acetic and formic acids have been shown to be Supplementary material can be found at http://www.chemse.oxford- present in the air above citrus orchards (Park et al. 2013). These journals.org/ acids are the only elicitors of antennal response we have been able to consistently demonstrate for D. citri and we hypothesize a role for these compounds in host location or selection. They may play a pri- Funding mary role as attractants or a secondary role as additive or synergistic Partial funding was provided by the Citrus Research and agents of other, as yet unidentified, odorants. Our inability to dem- Development Foundation [853]. onstrate antennal responses to an array of citrus volatiles suggests that acetic and formic acids may act as primary olfactory cues for D. citri and may have practical use for management of this pathogen Acknowledgments vector. A great deal more effort will be required to determine how We thank Larry Markle, Jiemin Liu, and Evan Koester (USDA-ARS, Ft. Pierce, this finding is relevant to movement of the adult psyllids in the field FL) for technical assistance and Anna Sara Hill (USDA-ARS, Ft. Pierce, FL) for Chemical Senses, 2016, Vol. 41, No. 4 337 insect rearing. USDA is an equal opportunity provider and employer. Mention Kreuzwieser J, Scheerer U, Rennenbergg H. 1999. Metabolic origin of acet- of a trademark or proprietary product does not constitute a guarantee or war- emitted by poplar (Populus tremula × P. alba) trees. J Exp Bot. ranty of the product by the US Department of Agriculture and does not imply 50:757–765. its approval to the exclusion of other products that may also be suitable. Lapointe SL, Evens TJ, Niedz RP. 2008. Insect diets as mixtures: optimization for a polyphagous weevil. J Insect Physiol. 54(7):1157–1167. References Lin Z, Zhong S, Grierson D. 2009. Recent advances in ethylene research. J Exp Bot. 60(12):3311–3336. Anderson MJ, Whitcomb PJ. 2005. RSM simplified: optimizing processes Mann RS, Ali JG, Hermann SL, Tiwari S, Pelz-Stelinski KS, Alborn HT, Ste- using response surface methods for design of experiments. New York: Pro- linski LL. 2012. Induced release of a plant-defense volatile ‘deceptively’ ductivity Press. p. 292. attracts insect vectors to plants infected with a bacterial pathogen. PLoS Baker TC. 2009. Nearest neural neighbors: moth sex pheromone receptors Pathog. 8:e1002610. doi:10.1371/journal.ppat.1002610. HR11 and HR13. Chem Senses. 34(6):465–468. Metcalf RL, Metcalf ER. 1992. Plant Kairomones in insect ecology and con- Beyaert I, Hilker M. 2014. Plant odour plumes as mediators of plant-insect trol. Contemporary topics in entomology 1. New York: Chapman and interactions. Biol Rev. 89:68–81. Hall. p. 168. Blande JD, Holopainen JK, Niinemets U. 2014. Plant volatiles in polluted Moran VC, Brown RP. 1973. The antennae, host plant chemoreception and atmospheres: stress responses and signal degradation. Plant Cell Environ. probing activity of the citrus psylla, Trioza erytreae (Del Guerico) (Hom- 37(8):1892–1904. optera: Psyllidae). J Entomol Soc Southern Africa. 36:191–202. Bove JM. 2006. Huanglongbing: a destructive, newly-emerging, century-old Myers RH, Montgomery DC. 2002. Response surface methodology: process

disease of citrus. J Plant Pathol. 88:7–37. and product optimization using designed experiments. 2nd ed. New York: Downloaded from Box GEP, Cox DR. 1964. An analysis of transformations (with discussion). J Wiley & Sons. Roy Stat Soc Series B. 26:211–246. Nehlin G, Valterová I, Borg-Karlson AK. 1994. Use of conifer volatiles to Bruce TJ, Wadhams LJ, Woodcock CM. 2005. Insect host location: a volatile reduce injury caused by carrot psyllid, Trioza apicalis, Förster (Homop- situation. Trends Plant Sci. 10(6):269–274. tera, Psylloidea). J Chem Ecol. 20(3):771–783. Cen YJ, Ye JM, Xu CB, Feng AW. 2005. The taxis of Diaphorina citri to the vol- Nissinen A, Kristoffersen L, Anderbrant O. 2008. Physiological state of female

atile oils extracted from non-host plants. J S China Agric Univ. 26:41–44. and light intensity affect the host-plant selection of carrrot psyllid, Trioza http://chemse.oxfordjournals.org/ Cornell JA. 2002. Experiments with Mixtures. 3rd ed. New York: Wiley & apicalis (Hemiptera: Triozidae). Euro J Entomol. 105:227–232. Sons. NIST/NIH/EPA (2005) NIST/NIH/EPA. Mass Spectral Library, Standard Ref- Cossins EA. 1964. The utilization of carbon-1 compounds from plants. Can J erence Database 1, NIST 05. Gaithersburg (MD): Standard ReferenceData Biochem. 42:1793–1802. Program, National Institute of Standards and Technology. Coutinho-Abreu IV, Forster L, Guda T, Ray A. 2014a. Odorants for surveil- Oikawa PY, Lerdau MT. 2013. Catabolism of volatile organic compounds lance and control of the Asian Citrus Psyllid (Diaphorina citri). PLoS One. influences plant survival. Trends Plant Sci. 18:12. 9(10):e109236. Onagbola EO, Meyer WL, Boina DR, Stelinski LL. 2008. Morphological char- Coutinho-Abreu IV, McInally S, Forster L, Luck R, Ray A. 2014b. Odor cod- acterization of the antennal sensilla of the Asian citrus psyllid, Diaphorina ing in a disease-transmitting herbivorous insect, the Asian citrus psyllid. citri Kuwayama (Hemiptera: Psyllidae), with reference to their probable Chem Senses. 39(6):539–549. functions. Micron. 39(8):1184–1191.

El-Sayed AM. 2014. Pherobase: database of pheromones and semiochemicals. Onagbola EO, Rouseff RL, Smoot JM, Stelinski LL. 2011. Guava leaf volatiles at University of Florida on April 22, 2016 Available from: http://www.pherobase.com and dimethyl disulphide inhibit response of Diaphorina citri Kuwayama Fahlbusch KG, Hammerschmidt FJ, Panten J, Pickenhagen W, Schatkowski D, to host plant volatiles. J Appl Entomol. 135:404–414. Bauer K, Garbe D, Surburg H. 2003. Flavors and fragrances. Ullmann’s Park JH, Goldstein AH, Timkovsky J, Fares S, Weber R, Karlik J, Holzinger Encyclopedia of industrial chemistry. Weinheim: John Wiley & Sons. R. 2013. Active atmosphere-ecosystem exchange of the vast majority of Fall R, Benson AA. 1996. Leaf methanol—the simplest natural product from detected volatile organic compounds. Science. 341(6146):643–647. plants. Trends Plant Sci. 9:296–301. Patt JM, Meikle WG, Mafra-Neto A, Sétamou M, Mangan R, Yang C, Malik N, Gottwald TR, Graca JV, Bassanezi RB. 2007. Citrus huanglongbing: the path- Adamczyk JJ. 2011. Multimodal cues drive host-plant assessment in Asian ogen, its epidemiology and impact. Plant Health Prog. 9: 1–36. citrus psyllid (Diaphorina citri). Environ Entomol. 40(6):1494–1502. Halbert SE. 1998. Entomology section. Tri-ology. 37:6–7. Patt JM, Sétamou M. 2010. Responses of the Asian citrus psyllid to volatiles Halbert SE. 2005. The discovery of huanglongbing in Florida. Florida Citrus emitted by the flushing shoots of its rutaceous host plants. Environ Ento- Mutual, Orlando, FL, 7–11 Nov. p. 50. mol. 39(2):618–624. Halbert SE, Manjunath KL. 2004. Asian citrus psyllids (Sternorrhyncha: Psyl- Patt JM, Woods D, Dimitratos S, Meikle W, Stockton D, Lapointe SL, Mafra- lidae) and greening disease of citrus: a literature review and assessment of Neto A. 2013. Novel synthetic ligands enhance the behavioral responses of risk in Florida. Fla Entomol. 87:330–353. Asian citrus psyllid to naturally occurring host-plant volatiles. In: Beck J, Hansson BS, Larsson MC, Leal WS. 1999. Green leaf volatile-detecting olfac- et al., editors. Pest management with natural products. Washington (DC): tory receptor neurons display very high sensitivity and specificity in a ACS Symposium Series. p.111–124, Chap. 8. scarab . Physiol Entomol. 24:121–126. Poerwanto ME, Trisyono YA, Beattie GAC, Subandiyah S, Martono E, Holford Holopainen JK, Blande JD. 2012. Molecular plant volatile communication. P. 2012. Olfactory responses of the Asiatic citrus psyllid (Diaphorina citri) Adv Exp Med Biol. 739:17–31. to mineral oil-treated mandarin leaves. Amer J Agric Biol Sci. 7(1):50–55. Hüve K, Christ MM, Kleist E, Uerlings R, Niinemets U, Walter A, Wildt J. Purcell HA, Almeida RPP. 2005. Insects as vectors of disease agents. In: Good- 2007. Simultaneous growth and emission measurements demonstrate an man RM, editors. Encyclopedia of plant and crop science. London (UK): interactive control of methanol release by leaf expansion and stomata. J Taylor & Francis Group. p. 1–5. Exp Bot. 58(7):1783–1793. Raguso RA, Light DM. 1998. Electroantennogram responses of male Sphinx Kennedy JS, Stroyan, HLG. 1959. Biology of aphids. Nature. 235:108–109. perelegans hawkmoths to floral and green-leaf volatiles. Entomol Exp Kimmerer TW, Kozlowski TT. 1982. Ethylene, ethane, acetaldehyde and etha- Appl. 86:287–293. nol production under stress. Plant Physiol. 69:840–847. Renou M. 2014. Pheromones and general odor perception in insects. In: Kreuzwieser J, Harren FJM, Laarhoven LJJ, Boamfa I, Lintel-Hekkert ST, Mucignat-Caretta C, editors. Neurobiology of chemical communication. Scheerer U, Huglin C, Rennenberg H. 2001. Acetaldehyde emission by the Boca Raton: CRC Press. leaves of trees—correlation with physiological and environmental param- Riffel JA, Shilzerman E, Sanders E, Abrell L, Medina B, Hinterwirth AJ, Kutz eters. Physiol Plantarum. 113:41–49. JN. 2014. Flower discrimination by pollinators in a dynamic chemical environment. Science. 344:1515–1518. 338 Chemical Senses, 2016, Vol. 41, No. 4

Robbins PS, Alessandro RT, Stelinski LL, Lapointe SL. 2012. Volatile profiles Sule H, Rita M, Omar D, Kah-Wei AH. 2012. Response of Diaphorina citri of young leaves of Rutaceae spp. varying in susceptibility to the Asian Kuwayama (Hemiptera: Psyllidae) to volatiles emitted from leaves of two citrus psyllid (Hemiptera: Psyllidae). Fla Entomol. 95:774–776. rutaceous plants. J Agric Sci. 4:152–159. Rohde B, Paris TM, Heatherington EM, Hall DG, Mankin RW. 2013. Tiwari S, Mann RS, Rogers ME, Stelinski LL. 2011. Insecticide resistance in field Responses of Diaphorina citri (Hemiptera: Psyllidae) to conspecific populations of Asian citrus psyllid in Florida. Pest Management Sci. 67:1258. vibrational signals and synthetic mimics. Ann Entomol Soc Am. Tol VR, Visser JH. 2002. Olfactory antennal responses of the vine weevil Otio- 106:392–393. rhynchus sulcatus to plant volatiles. Entomol Exp Appl. 102:49–64. Rouseff RL, Onagbola EO, Smoot JM, Stelinski LL. 2008. Sulfur volatiles in Visser JH, Yan FS. 1995. Electroantennogram responses of the grain aphids guava (Psidium guajava L.) leaves: possible defense mechanism. J Agric Sitobion avenae (F) and Metopolophium dirhodum (Walk) (Hom, Aphidi- Food Chem. 56(19):8905–8910. dae) to plant odor components. J Appl Entomol. 119:539–542. Schumann A, Spann T, Rouse R. 2011. Maintaining productivity in the pres- Weisberg S. 1985. Applied linear regression. 2nd ed. New York: Wiley & Sons. ence of HLB. Citrus Industry, Nov: 6–9. Ocala (FL): AgNet Media, Inc. Wenninger EJ, Hall DG, Mankin RW. 2009a. Vibrational communication Soroker V, Talebaev S, Hararai AR, Wesley SD. 2004. The role of chemical cues between the sexes in Diaphorina citri (Hemiptera: Psyllidae). Ann Ento- in host and mate location in the pear psylla Cacopsylla bidens (Homop- mol Soc Am. 102:547–555. tera: Psyllidae). J Insect Behav. 17:613–626. Wenninger EJ, Stelinski LL, Hall DG. 2009b. Roles of olfactory cues, visual cues, Su CY, Menuz K, Reisert J, Carlson JR. 2012. Non-synaptic inhibition between and mating status in orientation of Diaphorina citri Kuwayama (Hemiptera: grouped neurons in an olfactory circuit. Nature. 492(7427):66–71. Psyllidae) to four different host plants. Environ Entomol. 38(1):225–234. Downloaded from http://chemse.oxfordjournals.org/ at University of Florida on April 22, 2016