Identification of Pheromone Synergists for Rhynchophorus ferrugineus

Article pubs.acs.org/JAFC

Identification of Pheromone Synergists for Rhynchophorus ferrugineus Trapping Systems from Phoenix canariensis Palm Volatiles Sandra Vacas,* María Abad-Payá, Jaime Primo, and Vicente Navarro-Llopis Centro de Ecología Quı ́mica Agrícola, Instituto Agroforestal del Mediterráneo, Universitat Politècnica de València, edificio 6C, 5a planta, Cmno. de Vera s/n, 46022 Valencia, Spain ABSTRACT: Trapping systems for the red palm weevil, Rhynchophorus ferrugineus Olivier, rely on the use of natural plant odor sources to boost the attractiveness of the aggregation pheromone. The identification of the key odorants involved in attraction is essential in the development of a synthetic pheromone synergist to replace the nonstandardized use of plant material in traps. Canary Islands date palms (Phoenix canariensis) have become preferred hosts for R. ferrugineus in Europe; thus, the volatile profile of different P. canariensis plant materials, including healthy and infested tissues, is investigated in the present work by means of solid phase microextraction (SPME-GC-MS), aimed to identify pheromone synergists. The electroantennography (EAG) response of the compounds identified was recorded, as well as the preliminary field response of several EAG-active compounds. The so-called “palm esters” (ethyl acetate, ethyl propionate, ethyl butyrate, and propyl butyrate) elicit the strongest EAG responses but performed poorly in the field. Mixtures of esters and alcohols give evidence of better performance, but release rates need further optimization. KEYWORDS: red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae), ferrugineol, kairomone, host attractants, Phoenix canariensis (Arecales: Arecaceae), Canary Islands date palm, SPME

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INTRODUCTION Integrated pest management strategies play an important role in controlling the red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), given this pest’s concealed nature and its exceptional colonization capability. Larvae are the most destructive stage of this weevil as they penetrate deep into the stem, damage its internal tissues, disrupt nutrient transport, and even lead to tree collapse and death.1 Red palm weevil was first described to attack coconut palms, but it has been reported to affect 30 palm species belonging to 17 different genera.2−4 Since R. ferrugineus invaded the Mediterranean Basin and reached southern Europe in 1993,5 it has become the most damaging pest for Canary Islands date palms [Phoenix canariensis (hort. ex Chabaud) (Arecales: Arecaceae)].6−8 In the past 20 years in Europe, R. ferrugineus has demonstrated a strong preference for P. canariensis compared with other palm species such as Phoenix dactylifera L. or Washingtonia spp. In our region (the east coast of Spain), local government institutions have reported this preference on the basis of inspections made of thousands of palm trees. By way of example, 16% of a total of 2699 P. canariensis in the city of Valencia (eastern Spain) were infested during 2011−2012, whereas only 0.7% of a total 5925 P. dactylifera specimens were infested.9 Moreover, the red palm weevil seems to prefer male to female Canary Islands palms. According to the Detection and Eradication Campaign developed by the regional Valencian government, 76.2% of the infested P. canariensis were male specimens, taking into account that the sex ratio in this area is ca. 60:40 (males/ females) (data provided by Conselleriá de Agricultura, Peixca i Alimentació, Generalitat Valenciana). The same was observed in the city of Valencia, where 74.7% of the attacked P. © 2014 American Chemical Society

canariensis were males and 18.5% were females, with 6.8% undetermined.9 Generally, attack is detected only after the palm has been seriously damaged; thus, most efforts are made to develop methods that detect early stages of infestations, such as visual inspection, sniffing dogs,10,11 and acoustic12−14 and thermal detection.15 Preventive and curative procedures have been investigated and proposed as control methods, which include chemical control16−20 and biological control with entomopathogenic organisms, nematodes,21−23 and fungi.24,25 However, trapping systems through mass trapping play an important role for both detection and control purposes. This system does not create resistances, but allows population monitoring and helps enhance the efficacy of other control methods to reduce weevil populations and the number of insecticide applications. The introduction of pheromone-based trapping systems was possible thanks to the discovery of the red palm weevil aggregation pheromone component 4-methyl-5-nonanol (ferrugineol).26 In general, male-produced aggregation pheromones for palm weevil consist of 8−10 carbon, methylbranched secondary alcohols.27 However, the attractant power of these pheromones tends to be weak in the traps deployed in the field, and the presence of synergistic plant tissues or other food attractants is required.28−30 Unfortunately, this is a nonstandardized, costly, and cumbersome practice because the plant material needs to be replaced frequently to maintain the attractant level. Received: Revised: Accepted: Published: 6053

February 26, 2014 June 11, 2014 June 14, 2014 June 14, 2014 dx.doi.org/10.1021/jf502663y | J. Agric. Food Chem. 2014, 62, 6053−6064

Journal of Agricultural and Food Chemistry

Article

Table 1. Preliminary Field Tests no.

date

location

no. of blocks

attractanta

referenceb

1

May−Aug 2013

Godelleta (Valencia)

3

ph + acetoin ph + esters mix ph + esters mix + acetoin

ph

2

Sept−Oct 2013

Manises (Valencia)

3

ph + EtAc + EtOH ph + EtAc + alcohol mix

ph

3

Oct−Nov 2013

Valencia

3

ph + EtAc + phenyl-EtOH ph + EtAc + EtOH

ph

4

Sept−Nov 2013

Elche (Alicante)

5

ph + EtAc + alcohol mix ph + EtAc + phenyl-EtOH only ph

complete bait

5

Nov 2013−March 2014

Elche (Alicante)

5

ph + EtAc:EtOH (1:3) ph + EtAc:EtOH (3:1) ph + EtAc only ph

complete bait

a

Attractant contained in Picusan traps: ph, aggregation pheromone dispenser (Ferosan RF); esters mix, (ethyl acetate + ethyl propionate + ethyl butyrate + propyl butyrate); EtAc, ethyl acetate; EtOH, ethanol; alcohol mix, (ethanol + 3-methyl-1-butanol); phenyl-EtOH, 2-phenylethanol. b Reference attractant employed for the main comparison: ph, aggregation pheromone; complete bait, molasses (10%) + water (2 L) + plant material (P. canariensis stem pieces). Inc.) in the middle. Chambers were connected to the outlet of an air compressor (Jun-air Intl. A/S, Norresundby, Denmark) coupled with an AZ 2020 air purifier system (Claind Srl, Lenno, Italy) to provide ultrapure air (amount of total hydrocarbons 90%, sum of enantiomers); ocimene and farnesene (90%, mixture of isomers); β-caryophyllene (≥80%); and 3-hydroxy-2-butanone (>96%). Unfortunately, it was not possible to obtain the commercial standards of some compounds, so their identification was tentative and based on high-probability matches (>80%) according to the NIST MS Search routine (NIST Mass Spectral Search Program for the NIST \EPA\NIH Mass Spectral Library, version 2.0, build 4/2005). Electrophysiological Antennal Activity. Once identified, the electroantennographic response triggered by the compounds was

Adult female weevils are attracted to and oviposit in healthy, stressed, or damaged palms31 as they are attracted by a range of stimuli. However, attractiveness for weevils is generally boosted by the fermentation volatiles emanating from wounds or those resulting from infestation.27,32 Ethyl esters are common volatile constituents in fermenting palm oils, sap, and plant tissues.27,29,30 Many of these esters elicit significant electrophysiological responses in palm weevil species. Currently, however, they still prove less effective than the plant material used in traps when employed alone or combined. This work investigates the volatile profile of different P. canariensis plant materials, including healthy and infested tissues, by means of solid phase microextraction (SPME), with a view to identifying pheromone synergists for red palm weevil. For this purpose, both the electroantennography (EAG) response of the identified volatiles and the preliminary field response of the most EAG-active compounds were recorded.

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MATERIALS AND METHODS

Plant Material. Different P. canariensis palm materials were sampled for volatile collection and compound identification: (1) freshly cut petiole and rachis pieces of nonsprayed female and male P. canariensis palm leaves; (2) fresh healthy meristem stem tissues; (3) male inflorescence, including rachis and flowers; (4) infested stem tissues, upon which weevil larvae were feeding; (5) leaf pieces fermented in a 10% sugar water solution under field conditions; and (6) fermented stem pieces from the traps of the monitoring system deployed in Elche (Alicante, eastern Spain), which were also baited with a 10% molasses water solution. Fermented materials were collected when they still displayed attractant activity (1 or 2 weeks old). Volatile Collection. The three replicates of each experimental case were simultaneously collected by SPME using Supelco SPME holders equipped with a polydimethylsiloxane/divinylbenzene fiber (PDMS/ DVB), film thickness = 100 μm (Supelco Inc., Bellefonte, PA, USA). SPME fibers were conditioned before volatile sampling in a GC injector at 250 °C for 10 min under a 20 mL/min helium flow rate. Each sample was placed inside a 1.3 L glass chamber with a screwtop polytetrafluoroethylene (PTFE)−silicone septum cap (Supelco 6054

dx.doi.org/10.1021/jf502663y | J. Agric. Food Chem. 2014, 62, 6053−6064

acetic acid propanoic acid butanoic acid isobutanoic acid isopentanoic acid pentanoic acid

C2H4O2 C3H6O2 C4H8O2 C4H8O2 C5H10O2 C5H10O2

C2H6O C4H10O C5H12O C5H12O C5H12O C8H10O

C6H12O C7H14O C8H16O C8H8O C9H18O C10H20O

C12H26 C13H28 C14H30 C15H32 C16H34 C17H36 C18H38 C19H40

C15H30 C17H34 C19H38

C8H8O2 C9H10O2 C8H8O3 C10H12O2 C9H10O3

0.71 2.67 5.57 6.11 7.85 9.10

0.33 0.51 1.02 2.53 2.61 16.30

4.47 7.94 12.73 14.28 16.50 19.65

19.58 21.96 24.33 26.56 28.40 30.25 31.53 32.64

6055

26.33 29.92 32.37

16.39 18.84 19.35 19.85 21.34

methyl benzoate ethyl benzoate methyl salicylate isopropyl benzoate ethyl salicylate

1-pentadecene 3-heptadecene 1-nonadecene

dodecane tridecane tetradecane pentadecane hexadecane heptadecane octadecane nonadecane

hexanal heptanal octanal 2-phenylethanal nonanal decanal

ethanol 1-butanol 2-methyl-1-butanol 3-methyl-1-butanol 1-pentanol 2-phenylethanol

name

formula

RT

b

C C C T C

T T C

C C C C C C C C

C C C C C C

C C C C C C

C C C C C C

ID

c

0.76 ± 0.08

0.43 ± 0.06

1.52 ± 0.37

female

nd

Aldehydes

Alcohols

Acids

male

Benzoates

Alkenes

1.70 ± 0.40

Alkanes 2.80 ± 0.60

palm leaves

Table 2. Compounds Detected in the Different P. canariensis Samples

8.21 85.42 1.91 0.11 1.24

± ± ± ± ±

0.50 1.43 0.98 0.08 0.45

0.06 ± 0.01

0.09 ± 0.03

healthy tissue

± ± ± ± ± ±

0.02 0.05 0.02 0.02 0.23 0.03

± ± ± ± ±

0.28 0.00 0.20 0.00 0.03

0.51 ± 0.10 0.06 ± 0.02

1.60 0.03 0.48 0.04 0.24

0.03 ± 0.01

0.04 0.16 0.02 0.02 0.24 0.04

inflorescence

0.16 ± 0.04

2.90 ± 0.45 5.47 ± 0.56

0.66 ± 0.16

0.38 ± 0.14 0.39 ± 0.04

2.62 ± 0.94 2.07 ± 0.18 24.82 ± 1.34

affected stem

percentage of the total chromatogram areaa (mean ± SE)

0.13 ± 0.07

0.30 ± 0.05 0.18 ± 0.02 2.74 ± 1.12

0.07 ± 0.01 0.61 ± 0.13 0.65 ± 0.12

3.76 ± 0.55

0.31 ± 0.11

fermented leaves

0.58 ± 0.13

3.31 ± 0.92 6.09 ± 2.48 0.24 ± 0.15

fermented stem

Journal of Agricultural and Food Chemistry Article


C10H12O2 C11H14O2 C11H14O2 C12H16O2

C4H8O2 C5H10O2 C6H12O2 C6H12O2 C6H12O2 C6H12O2 C7H14O2 C7H14O2 C7H14O2 C7H14O2 C7H14O2 C7H14O2 C7H14O2 C7H14O2 C7H14O2 C8H16O2 C8H16O2 C8H16O2 C8H16O2 C8H16O2 C9H18O2 C9H18O2 C9H18O2 C9H18O2 C9H16O2 C10H20O2 C9H10O2 C10H20O2 C10H20O2 C10H12O2 C11H22O2 C10H12O2 C11H22O2 C11H14O2

0.91 2.03 3.18 3.49 3.95 4.90 6.12 6.39 7.28 7.51 8.10 8.33 8.75 8.92 8.95 10.36 10.80 11.51 12.73 12.96 14.24 14.89 15.14 16.58 17.64 18.30 18.30 18.35 19.26 20.92 21.00 21.10 21.83 22.45

formula

21.35 22.79 23.89 25.37

RT

b

Table 2. continued

ethyl acetate ethyl propionate sec-butyl acetate isobutyl acetate ethyl butyrate butyl acetate ethyl 2-methylbutyrate ethyl 3-methylbutyrate sec-butyl propionate isopentyl acetate propyl butyrate ethyl pentanoate pentyl acetate butyl propionate unknown (1) sec-butyl butyrate isobutyl butyrate isopentyl propionate butyl butyrate ethyl hexanoate isobutyl isopentanoate unknown (2) isopentyl butyrate ethyl heptanoate ethyl cyclohexanecarboxylate pentyl pentanoate benzyl acetate butyl hexanoate ethyl octanoate ethyl phenylacetate isopentyl hexanoate β-phenylethyl acetate ethyl nonanoate ethyl 3-phenylpropionate

propyl benzoate isobutyl benzoate butyl benzoate isopentyl benzoate

name

6056

C C T C T T C C T C C T

T C C C C T

C C C C C C C T T C C C C T

C C C T

ID

c

female

palm leaves

Esters

Benzoates

male ± ± ± ± 0.27 0.08 0.22 0.01

0.34 ± 0.16

1.44 0.63 0.41 0.04

healthy tissue

inflorescence

± ± ± ± ± ± ± ± 0.53 0.30 0.15 0.02 2.97 0.17 0.06 0.12

0.37 0.03 0.06 0.09 0.26 0.14 0.24 0.21 0.10 0.02

1.24 0.13 0.01 0.08 0.23 0.02

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ±

1.29 0.37 0.65 1.64 3.48 2.40 5.45 1.06 0.74 0.12

3.95 0.36 0.05 0.38 0.38 0.09

0.17 ± 0.06

1.65 ± 0.36 0.46 ± 0.17 1.50 ± 0.45

4.41 1.95 0.54 0.12 21.95 0.56 0.76 0.77

affected stem


0.22 ± 0.03

1.16 ± 0.37 32.33 ± 1.17

0.03 ± 0.01

0.08 ± 0.07

0.14 ± 0.02

20.46 ± 1.76

0.09 ± 0.01

0.27 ± 0.08

33.47 ± 0.74 0.05 ± 0.01

fermented leaves

0.16 ± 0.10

0.58 ± 0.40

0.67 ± 0.16 9.65 ± 4.82

5.25 ± 1.79

0.44 ± 0.17

fermented stem



C6H6O C7H8O C8H10O2

12.31 15.68 19.26

C10H16 C10H14 C10H16 C10H16 C10H16 C10H16 C10H18O C15H22 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H22 C15H24 C15H22 C15H24 C15H24 C15H22 C15H24 C15H24 C15H24

C4H8O C4H8O2 C5H10O2 C7H14O C9H18O C11H22O

0.93 2.11 4.68 8.10 16.14 21.77

12.48 13.81 13.97 14.33 14.72 15.90 16.44 22.72 23.29 23.45 23.57 23.81 24.11 24.33 24.45 24.52 24.62 24.85 25.01 25.24 25.65 25.67 25.72

C12H24O2 C12H16O2 C14H28O2 C16H32O2

formula

24.20 25.36 28.20 31.36

RT

b

Table 2. continued

β-myrcene p-cymene limonene Z-β-ocimene E-β-ocimene α-terpinolene linalool 9,10-dehydroisolongifolene α-cubebene unknown (3) (mw = 204) cyclosativene α-copaene β-cubebene unknown (4) (mw = 204) unknown (5) (mw = 202) α-gurjunene unknown (6) (mw = 202) β-caryophyllene unknown (7) (mw = 204) unknown (8) (mw = 202) α-caryophyllene aromadendrene alloaromadendrene

phenol p-cresol p-methylguaiacol

2-butanone 3-hydroxy-2-butanone 2-hydroxy-3-pentanone 2-heptanone 2-nonanone 2-undecanone

ethyl decanoate 2-phenylethyl butyrate ethyl laurate ethyl myristate

name

6057

2.03 ± 0.17

2.10 ± 0.30

3.30 ± 0.50 0.70 ± 0.05

2.72 ± 0.27 0.40 ± 0.27

C

C C T

1.00 ± 0.20

1.05 ± 0.04

1.60 1.90 0.20 0.10

T

8.50 33.20 0.50 0.40

12.30 38.00 0.52 0.44

1.19 2.24 0.28 0.14

± ± ± ±

1.30 0.80 0.60 0.60 5.10 nd

± ± ± ±

0.30 0.15 0.32 0.07 0.59 0.07

1.20 ± 0.30

± ± ± ± ± ±

Terpenes ± 1.20 ± 0.50 ± 0.30 ± 0.30 ± 0.90

Phenols

Ketones

Esters

male

1.11 ± 0.07

1.06 0.15 0.86 0.10 1.39 0.26

female

palm leaves

T C T

C C C C C C C T C

C C C

C C T T C T

C T C C

ID

c

0.09 ± 0.07

healthy tissue

3.32 0.01 0.05 0.19 0.02 0.21 0.02

± ± ± ± ± ± ± 45.10 0.45 0.59 0.68 0.07 1.81 0.11

5.72 ± 0.40

0.03 0.43 0.02 0.07 2.54 0.49

± ± ± ± ± ± 0.10 2.22 0.11 0.70 32.73 1.78

0.01 ± 0.00

inflorescence

0.05 ± 0.02

0.07 ± 0.05

0.20 ± 0.13

2.14 ± 0.28

1.13 ± 0.48 0.11 ± 0.03 0.17 ± 0.10

affected stem


0.02 ± 0.01

0.17 ± 0.18

0.19 ± 0.10

0.01 ± 0.01

0.10 ± 0.05

0.22 ± 0.06

0.72 ± 0.12

fermented leaves

± ± ± ± ± ±

12.11 2.23 0.19 0.10 0.62 0.06

0.03 ± 0.01

4.69 ± 1.22

0.16 ± 0.03 11.47 ± 3.97

0.46 ± 0.09 0.64 ± 0.13

40.76 10.38 0.94 0.25 1.70 0.11

fermented stem



C8H8 C7H10N2 C8H10O C12H12 C15H20

7.96 13.01 13.67 24.92 27.40 C T C C T

T T T T C T T T

τ-muurolene germacrene D τ-elemene α-muurolene α-farnesene τ-cadinene β-cadinene δ-cadinene unknown (9) (mw = 204)

styrene 2,3,5-trimethylpyrazine 4-methylanisole 2,6-dimethylnaphthalene calacorene

ID

name

c

25.69 ± 4.00

0.99 ± 0.41

27.90 ± 4.70

1.42 ± 0.31 5.07 ± 0.84

2.10 ± 0.30

Other

1.20 ± 0.30 4.40 ± 0.70

1.72 ± 0.18

male Terpenes 1.00 ± 0.10

female

palm leaves healthy tissue 0.16 0.51 0.19 0.05 0.03 0.50 0.02 0.07

± ± ± ± ± ± ± ±

0.35 1.12 0.40 0.30 0.09 1.82 0.05 0.23

inflorescence

0.08 ± 0.04

0.03 ± 0.01

affected stem


0.43 ± 0.06 0.13 ± 0.04 0.92 ± 0.32

0.02 ± 0.01

0.43 ± 0.14

fermented leaves

0.29 ± 0.05

0.56 ± 0.30

fermented stem

a

Means of three replicates. bRT, retention time (min) on ZB-5 column (30 m × 0.25 mm i.d. × 0.25 μm; Phenomenex Inc., Torrance, CA, USA). cIdentification of the compound: C, confirmed with commercial standard; T, tentative with spectra and high probability matches (>80%) according to NIST mass spectral database.

C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24

formula

26.02 26.16 26.47 26.54 26.67 26.84 26.95 27.06 27.26

RT

b

Table 2. continued


6058



Article

evaluated with a Syntech IDAC 2 acquisition controller and a Syntech CS-55 stimulus controller (Ockenfels Syntech GmbH, Kirchzarten, Germany), whereas GC-EAD 32 (v 4.3) software was employed for data acquisition. The EAG tests for each compound had seven replicates per sex, with each antenna belonging to seven different weevils. Individuals were collected from the field, maintained at 26 ± 2 °C under photoperiod conditions (10 h light and 14 h dark), and fed apples. Antennae were removed from living insects, cut off as close as possible to their base with fine-point stainless steel entomology scissors, whereas the club remained unaltered. After excision, the antenna was placed immediately on a fork holder, the flat tips of which had been previously covered with a small bead of Spectra 360 electrode gel (Parker Laboratories Inc., Fairfield, NJ, USA). The holder was then mounted in an EAG probe type PRG-2 (Syntech) to be connected directly to the IDAC-2 system. Test and control samples were prepared in hexane solution, and 1 μL aliquots were applied to filter paper strips. The solvent was allowed to evaporate for 30 s before the strip was placed into a disposable Pasteur pipet. The olfactory stimulus (1 s duration) was delivered into a continuous humidified charcoal-filtered air stream (400 mL/min) toward the antenna, after placing the pipet tip into the hole of the tube carrying the air stream. Each antenna was stimulated successively with the solvent, the positive reference (1 μg of ferrugineol), and no more than four different test compounds (1 μg). Stimuli were applied at 3 min intervals. To eliminate the variability of the response due to the different antennae conditions, the EAG test results are provided as the ratio: mVC/mVF, where C is the response for each compound and F is the response of the reference stimulus, which was 1 μg of ferrugineol in all cases. Preliminary Field Tests. The compounds that triggered the most significant electrophysiological responses were included in the preliminary field tests to study the weevils’ actual response under field conditions, using Picusan traps (Sansan Prodesing SL, Náquera, Valencia, Spain), the bases of which were filled with water.33 All of the trials were designed as randomized block assays and were carried out in different locations in the Valencian community (on the Spanish eastern coast): Godelleta, Manises, Valencia, and Elche. The revision of catches and rotation of traps were performed on a weekly basis. In all of the trials, the traps within each block were separated by at least 30 m and the distance between blocks was at least 100 m. The different attractants compared are detailed in Table 1, which also includes the number of blocks in each trial and the dates when the tests were conducted. The aggregation pheromone (ph) dispenser employed in all cases was Ferosan RF (Sansan Prodesing SL), loaded with 1 g of ferrugineol. Mixtures of compounds were formulated in low-density polyethylene vials to obtain controlled release rates. The gravimetric method was employed to assess the amount of active ingredients released in relation to the aging time. In parallel to the field trials, three corresponding dispensers of each type were aged under the same field conditions inside the same type of trap. These dispensers were weighed weekly in the laboratory on a precision balance. The weight differences over a period were referred to as the active mixture released from the dispenser. To obtain the mean emission level for each dispenser, recorded weights were fitted to linear regression models, y = a + bx, where y is the weight of the dispenser and x is the corresponding aging days. The resulting mean release rates refer to the mixtures released from the polyethylene vials. In addition, several samples of aged dispensers were also analyzed by gas chromatography (GC-FID) to check the proportions at which ethyl acetate and ethanol were emitted in the mixtures tested in trial 5. The number of weevils captured with each experimental attractant was compared with that obtained in the traps baited only with ferrugineol (ph) or with the complete bait employed in the Valencian community red palm weevil monitoring network, which is composed of water, 10% molasses solution, and P. canariensis palm stem pieces. The number of males, females, or total weevils recorded during each trapping period was divided by the number of days between dates to calculate the weevils per trap and day index (WTD). Log (WTD+1) transformation was applied to normalize the errors distribution prior to the analysis of variance (ANOVA), followed by LSD test (at P < 0.05). The Statgraphics Centurion XVI package was employed to

perform the statistical analysis (Statpoint Technologies Inc., Warranton, VA, USA).

■

RESULTS AND DISCUSSION Overview of Detected Compounds. Table 2 reports the 122 compounds detected in the different P. canariensis palm samples. Of these, 81 were confirmed with commercial standards and 32 were tentatively identified (most of them being sesquiterpenes with no available standards). The main constituents of the palm leaf volatile samples were identified as cyclosativene, α-copaene, and δ-cadinene which, all together, assumed ∼70% of the total chromatogram area. Terpene hydrocarbons (mostly monoterpenes C10H16 and sesquiterpenes C15H24) are the largest group of airborne natural products emitted by vegetation and are responsible for both floral and fruity aromas and a mild, wood-like odor.34 The remaining compounds detected in palm leaves were other terpenes, except for ∼5%, which comprised alkanes, aldehydes, and 2,6-dimethylnaphthalene (probably a contaminant from the aeration system). The volatile profiles of the female and male P. canariensis palm leaves were not significantly different if compared to the reported qualitative and relative composition (Table 2); only decanal and α-terpinolene were not detected in the male profile. Consequently, the volatile profile does not seem to explain the greater susceptibility of male palms observed in the field. Thus, other physical factors could affect the oviposition preferences of females or make larvae’s feeding habits difficult. Benzoates, such as ethyl and methyl benzoate, were the main constituents of the healthy tissue volatile profile (∼99% of the total chromatogram area), which are reported to confer floral and fruity odors.35,36 As expected, male inflorescence presented traces of several aldehydes (0.5%), which are commonly found in essential oils and contribute to their favorable odors (citrusy, floral, and green aromas).37 Several alkanes were also detected (2.4%), but sesquiterpenes were the main constituents. Unfortunately, it was not possible to identify the most abundant compound (45.1%), but the mass spectrum suggests a sesquiterpene structure [m/z 91 (50), 105 (45), 119 (100), 131 (40), 132 (50), 145 (38), 159 (25), 187 (21), 202 (12)] with a molecular weight of 202. Nonetheless, some GC-EAD coupled tests performed with SPME replicates of these samples did not evidence any electrophysiological activity for this unidentified compound (data not shown). The volatile profile of the palm material dramatically changes when tissues are either affected by the pest or subjected to fermentation processes in traps; then esters, acids, alcohols, and ketones are the main constituents of the volatile profile of this plant material (Table 2). Short-chain fatty acids (acetic, propanoic, butanoic, etc.), associated with off-flavors, are the main products of the fermentative microorganisms responsible for anaerobic plant fiber degradation. A substantial part of many arthropods’ diet consists of plant-derived polymers, such as lignin, hemicellulose, and cellulose,38 which is the case for R. ferrugineus larvae. After egg hatching, larvae bore galleries into the palm and leave a frass, composed of excrements and plant debris with a high water content. The production of CO2, 3hydroxy-2-butanone (acetoin), ethanol, and ethyl lactate, among others, is associated with the bacterial community present in the frass.39 In general, the relative distribution of the volatiles in degraded samples can be determined by several factors during fermentation, including microorganism species and/or strain, fermentation temperature, nutrient availability, 6059



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Table 3. Relativea Electrophysiological Antennal Responses (Mean ± SE)b Triggered by Compounds Detected in the Different Palm Materials compound propyl butyrate ethyl propionate ethyl butyrate ethyl 2-methylbutyrate ethyl valerate ethyl acetate ethyl hexanoate ethyl heptanoate ethyl lactate isoamyl propionate propyl propionate sec-butyl acetate butyl butyrate ethyl isobutyrate methyl butyrate ethyl decanoate isoamyl butyrate methyl isovalerate octyl acetate isoamyl acetate pentyl acetate ethyl octanoate heptyl acetate ethyl laurate benzyl acetate ethyl myristate decyl acetate butyl acetate

2-phenylethanol 2-methyl-1-butanol 1-butanol 3-methyl-1-butanol 1-pentanol ethanol 2,3-butanediol 1,3-butanediol

acetic acid butyric acid isovaleric acid a

ratio males Esters 1.24 1.20 1.17 1.11 1.01 0.77 0.98 0.92 0.90 0.89 0.89 0.88 0.85 0.84 0.84 0.81 0.78 0.74 0.72 0.70 0.70 0.63 0.58 0.57 0.52 0.49 0.49 0.47

ratio females

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.11 0.16 0.07 0.08 0.05 0.07 0.11 0.09 0.05 0.08 0.04 0.08 0.08 0.06 0.04 0.08 0.06 0.07 0.12 0.04 0.06 0.02 0.08 0.07 0.04 0.04 0.05 0.03

0.75 1.06 0.93 0.83 0.74 1.01 0.78 0.68 0.77 0.79 0.88 0.71 0.77 0.89 0.91 0.59 0.72 0.69 0.66 0.66 0.57 0.85 0.54 0.53 0.56 0.47 0.72 0.61

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.09 0.04 0.04 0.05 0.16 0.05 0.05 0.04 0.03 0.02 0.05 0.10 0.06 0.06 0.11 0.10 0.05 0.07 0.05 0.11 0.04 0.05 0.06 0.08 0.02 0.12 0.06

Alcohols 0.85 ± 0.87 ± 0.79 ± 0.79 ± 0.78 ± 0.63 ± 0.63 ± 0.53 ±

0.06 0.06 0.11 0.03 0.07 0.05 0.03 0.06

1.10 0.76 0.89 0.82 0.86 0.73 0.64 0.43

± ± ± ± ± ± ± ±

0.12 0.05 0.08 0.04 0.05 0.04 0.06 0.06

Acids 0.73 ± 0.12 0.56 ± 0.08 0.45 ± 0.06

compound

ratio females

± ± ± ± ± ± ± ± ±

0.11 0.06 0.05 0.04 0.04 0.05 0.06 0.04 0.06

1.01 0.68 0.70 0.73 0.68 0.67 0.58 0.51 0.47

± ± ± ± ± ± ± ± ±

0.03 0.06 0.03 0.08 0.04 0.04 0.06 0.02 0.05

nonanal hexanal decanal heptanal octanal

Aldehydes 0.82 ± 0.64 ± 0.59 ± 0.46 ± 0.40 ±

0.12 0.05 0.08 0.03 0.05

0.81 0.69 0.60 0.44 0.47

± ± ± ± ±

0.06 0.11 0.06 0.04 0.06

methyl benzoate methyl salicylate ethyl salicylate isobutyl benzoate propyl benzoate butyl benzoate ethyl benzoate

Benzoates 0.79 ± 0.70 ± 0.68 ± 0.60 ± 0.59 ± 0.57 ± 0.52 ±

0.07 0.05 0.02 0.05 0.04 0.02 0.04

0.58 0.61 0.67 0.61 0.55 0.51 0.49

± ± ± ± ± ± ±

0.06 0.06 0.05 0.05 0.04 0.04 0.04

p-ethylguaiacol p-cresol p-methylguaiacol phenol

Phenols 0.60 0.51 0.52 0.50

0.06 0.05 0.06 0.06

0.61 0.44 0.51 0.58

± ± ± ±

0.07 0.07 0.04 0.05

0.06 0.05 0.05 0.04 0.04 0.06

0.61 0.46 0.59 0.53 0.51 0.53

± ± ± ± ± ±

0.05 0.07 0.03 0.05 0.08 0.10

0.03 0.06 0.05 0.06

0.80 0.69 0.58 0.50

± ± ± ±

0.05 0.06 0.04 0.08

3-hydroxy-2-butanone 6-methyl-5-hepten-2-one acetophenone 2-pentanone 2-hexanone 2-nonanone 2-butanone p-methylacetophenone p-ethylacetophenone

limonene α-phellandrene α-pinene aromadendrene β-caryophyllene α-copaene

0.74 ± 0.10 0.78 ± 0.12 0.46 ± 0.08

ratio males Ketones 1.10 0.83 0.71 0.70 0.67 0.58 0.57 0.61 0.59

anisole 2-phenethyl acetate dimethyl sulfide dimethyl disulfide

± ± ± ±

Terpenes 0.69 ± 0.68 ± 0.67 ± 0.65 ± 0.53 ± 0.43 ± Other 0.75 ± 0.76 ± 0.61 ± 0.65 ±

Relative response: ratio = (mV compound)/(mV ferrugineol). bMeans of seven replicates for each sex.

pH, unsaturated fatty acid levels, etc.40−42 The formation of fatty acid ethyl esters and acetate esters confers floral and fruity sensory properties. Fatty acid esters (such as ethyl butyrate, ethyl hexanoate, and ethyl octanoate) result from the ethanolysis of acylCoA, which is produced during fatty acid synthesis or degradation. Acetate esters (such as butyl acetate, isoamyl acetate, hexyl acetate, and phenethyl acetate) are formed from the reaction of acetylCoA with the alcohols that result from the degradation of amino acids, carbohydrates, and lipids.40,43 Biodegradation of lignin from plant tissues by rot fungi and bacteria can also generate the phenolic compounds

detected (guaiacol, p-methylguaiacol, etc.),44 which are associated with smoky or spicy trait.45 Electrophysiological Antennal Activity. Seventy-four compounds were tested for EAG responses (Table 3). The largest number of significant responses was elicited by the esters identified in the degraded samples for both male and female weevils, followed by alcohols and ketones. Propyl butyrate, ethyl propionate, ethyl butyrate, ethyl 2-methylbutyrate, ethyl valerate, and 3-hydroxy-2-butanone (acetoin) elicited the most significant responses, which were comparable to the reference ferrugineol (ratio ≥1) for males, whereas 2phenylethanol, ethyl propionate, ethyl acetate, and acetoin 6060



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were the most active for females. The palm esters (ethyl propionate, ethyl acetate, ethyl butyrate, etc.) that elicited significant antennal responses have already been reported by Gries et al.29 for R. phoenicis L., R. palmarum L., R. cruentatus (Fabricius), R. ferrugineus, R. vulneratus (Panz.), and R. bilineatus (Montrouzier). Later, while testing the antennal responses of several palm esters (ethyl acetate, ethyl butyrate, ethyl isobutyrate, and ethyl lactate), Guarino et al.46 reported that ethyl propionate was the ester that elicited the strongest response for R. ferrugineus and for both females and males. Aldehydes and terpene hydrocarbons, these being characteristic compounds of inflorescence and palm leaves, elicited only intermediate responses in our tests. Preliminary Field Activity. Nevertheless, the EAG tests do not provide conclusive ideas about the attractiveness and potential of each compound as semiochemical under field conditions. Indeed, the mechanisms responsible for EAG and field activity are different, and possible correlations between them should be considered with caution, as should the possible synergistic effects of blends for single compounds. For example, Giblin-Davis et al.47 reported the attraction of R. cruentatus to some fermentation products. Yet, whereas isopropyl acetate elicited a major EAG response but not significant attractiveness in traps, ethanol elicited a minor relative EAG response, but proved attractive in the field when combined with the pheromone cruentol. This scenario coincides with the main findings of our preliminary field tests. In trial 1 (Figure 1A), when traps were baited with a mixture of the most EAG-active esters (ethyl acetate, ethyl propionate, ethyl butyrate, and propyl butyrate), the attractant power of the traps did not significantly differ from that of the traps baited only with pheromone for female, male, and total weevil catches (the significance of the factors is provided in Table 4 for the ANOVA performed using the data collected per sex and for total catches). Thus, the addition of these esters, in the proportion and at the emission level employed (Table 5), did not significantly improve weevil catches. The same was observed when ferrugineol was combined with acetoin or with esters and acetoin (Figure 1A and Table 4), these being the compounds that elicited the strongest EAG responses. Whereas the combination of esters and ketones did not offer any advantage if compared to using the aggregation pheromone alone, the total weevil catches significantly increased when the ethyl acetate ester was combined with ethanol (Figure 1B), which was due mainly to the significant female captures obtained (trial 2) (Table 4). The total weevil catches almost doubled with this kairomonal mixture. When a second alcohol (3-methyl-1-butanol) was added to the mixture (alcohol mix in Figure 1B), catches did not significantly improve with regard to ethyl acetate + ethanol, suggesting that addition of this alcohol is irrelevant, at least under our particular test conditions (trial 2) (Table 5). Although 2-phenylethanol elicited a strong antennal response for female R. ferrugineus, no synergistic effect was observed when it was added to traps in combination with ethyl acetate in trial 3 (Figure 1C and Table 4), whereas the combination (ethyl acetate + ethanol) still achieved significantly larger female, male, and total catches. The same was observed in Elche for trial 4 (Figure 1D and Table 4). Whereas the female catches obtained in traps baited with 2-phenylethanol combined with ethyl acetate did not significantly differ from those baited with only pheromone, the results obtained with ethyl acetate + ethanol + 3-methyl-1-butanol were significantly

Figure 1. Number of weevils (females, males, and total) captured per trap and day in the different traps deployed in (A) trial 1, (B) trial 2, (C) trial 3, (D) trial 4, and (E) trial 5. All compounds combined with the aggregation pheromone (ph). Bars labeled with the same letter for each weevil case are not significantly different (ANOVA, LSD test at P < 0.05).

better than when using the pheromone alone. Moreover, the mean number of females trapped using this ester + alcohols 6061



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Table 4. Statistical Parameters for the ANOVA Performed for Each Preliminary Field Trial sexa

factorb

trial 1

trial 2

trial 3

trial 4

trial 5

females

attractant week block

F3,39 = 0.50; P = 0.683 F3,39 = 1.65; P = 0.193 F2,39 = 7.51; P = 0.002

F2,39 = 7.00; P = 0.003 F5,39 = 1.91; P = 0.115 F2,39 = 7.59; P = 0.002

F2,28 = 9.60; P = 0.001 F3,28 = 2.52; P = 0.078 F2,28 = 3.00; P = 0.066

F3,52 = 6.98; P < 0.001 F3,52 = 3.71; P = 0.017 F3,52 = 3.08; P = 0.035

F4,143 = 6.41; P < 0.001 F7,143 = 28.54; P < 0.001 F3,143 = 5.63; P < 0.001

males

attractant week block attractant week block

F3,39 F3,39 F2,39 F3,39 F3,39 F2,39

F2,39 F5,39 F2,39 F3,39 F5,39 F2,39

F2,28 F3,28 F2,28 F2,28 F3,28 F2,28

F3,52 F3,52 F3,52 F3,52 F3,52 F3,52

F4,143 F7,143 F3,143 F4,143 F7,143 F3,143

total

a

= = = = = =

0.64; 0.26; 2.63; 1.14; 1.46; 8.40;

P P P P P P

= = = = = =

0.591 0.851 0.085 0.347 0.240 0.001

= = = = = =

3.89; 1.98; 3.83; 8.37; 0.99; 9.03;

P P P P P P

= = = = =

Identification of Pheromone Synergists for Rhynchophorus ferrugineus

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