Neotrop Entomol DOI 10.1007/s13744-016-0372-3
ECOLOGY, BEHAVIOR AND BIONOMICS
Identification of the Female Sex Pheromone of the Leafroller Proeulia triquetra Obraztsov (Lepidoptera: Tortricidae) J BERGMANN1, L REYES-GARCIA1,2, C BALLESTEROS3, Y CUEVAS3, MF FLORES1, T CURKOVIC3 1
Instituto de Química, Pontificia Univ Católica de Valparaíso, Valparaíso, Chile Depto de Ciencias Básicas, Univ Santo Tomás, Viña del Mar, Chile 3 Depto de Sanidad Vegetal, Fac de Ciencias Agronómicas, Univ de Chile, Santiago, Chile 2
Keywords Euliini, electroantennographic detection, gas chromatography, male attractant, mass spectrometry, reproductive isolation Correspondence J Bergmann, Instituto de Química, Pontificia Univ Católica de Valparaíso, Valparaíso, Chile; [email protected] Edited by Stefano Colazza – Univ of Palermo Received 2 July 2015 and accepted 19 January 2016 * Sociedade Entomológica do Brasil 2016
Abstract Proeulia triquetra Obraztsov (Lepidoptera: Tortricidae) is an occasional pest in fruit orchards in central-southern Chile. In order to develop species-specific lures for detection and monitoring of this species, we identified the female-produced sex pheromone. (Z)-11-Tetradecenyl acetate (Z11-14:OAc), (E)-9-dodecenyl acetate (E9-12:OAc), and (E)-11Tetradecenyl acetate (E11-14:OAc) were identified as biologically active compounds present in female pheromone glands by solvent extraction of the gland and analysis of the extracts by gas chromatographyelectroantennographic detection and gas chromatography-mass spectrometry. In field tests, lures baited with synthetic Z11-14:OAc and E912:OAc in a 10:1 ratio were highly attractive to males of the species.
Introduction Agriculture is an important economic activity in Chile, contributing ca. 2.6% to the gross domestic product of the country, and the production and exportation of fresh and processed fruit makes up about 40–45% of this contribution. The majority of fruit plantations are located in central-southern Chile, and the most important products are grapes, stone and pip fruits (peach, plum, nectarine, apple, etc.), avocados, and berries, among others (ODEPA 2014). There are several exotic and endemic species threatening the production, and among them are native leafrollers of the genus Proeulia (Lepidoptera: Tortricidae). Proeulia triquetra Obraztsov, together with Proeulia auraria (Clarke) and Proeulia chrysopteris (Butler) have been recognized as the species of greatest potential economic impact within this genus, as these species are causing occasional but increasing damage in fruit orchards (González 2003). Proeulia triquetra is a polyphagous species found on grapes (Vitis vinifera), apples (Malus domestica),
mandarins (Citrus reticulata), and berry (Rubus spp.) orchards, some weeds (Convolvulus arvensis, Galega officinalis), several ornamentals (Buddleja davidii, Hebe sp., Lonicera japonica), and some native plants (Fuchsia magellanica, Maytenus boaria, Myoschilos oblonga) (Cepeda & Cubillos 2011). Despite the fact that its distribution has been reported from San Felipe (32°45′S, 70°43′W) to Los Lagos (40°13′S, 74°49′W), economic damage has been reported mainly in orchards (vineyards, berries, apples) south of Maule (35°06′S, 71°16′W) (González 2003, Cepeda & Cubillos 2011). Several natural enemies of Proeulia are known (González 2003, Ripa & Larral 2008), but biological control has not been developed against this species. Chemical control of P. triquetra is usually a side effect of pesticide applications aimed at the control of other tortricid species, mainly Cydia pomonella L. and Cydia molesta Busck. The increasing use of pheromone based tactics (i.e., mating disruption) for the control of the latter two species and the consequential withdrawal of insecticides targeting Lepidoptera pests, however, has led to local and occasional outbreaks of Proeulia species
Bergmann et al.
(Curkovic, unpublished data). Another major problem is the quarantine status of Proeulia spp., and the presence of these insects has been a permanent cause of rejection for the exportation of fresh fruits (including blueberries, grapes, and apples) during the last years, mainly from the Maule and the Biobio regions. In particular, P. triquetra has been listed within quarantine species by the respective plant protection agencies in Australia and the USA. An associated problem is the correct identification of these morphologically similar species at the larval or pupal stages, although some progress has been recently made (Cepeda & Cubillos 2011). Besides, adult characters are available from descriptions by Razowski (1999), González (2003), and Razowski & Pelz (2010). Traps baited with synthetic sex pheromones are useful tools for detection and monitoring of insect pests, and the specificity of pheromone-baited traps can help in the identification of the species present in fruit orchards. The sex pheromone of P. auraria has been identified recently by our group as a four-component blend consisting of (E)-11tetradecenyl acetate (E11-14:OAc), (E)-11-tetradecenol (E1114:OH), tetradecyl acetate (14:OAc), and (Z)-11-tetradecenyl acetate (Z11-14:OAc) (Reyes-Garcia et al 2014). Knowing the sex pheromone of P. triquetra would set the basis for the development of monitoring lures for this species, thus enabling a more precise determination of the actual species present in fruit orchards and provide information for optimal timing of insecticide application. Furthermore, P. triquetra is sympatric and synchronic with P. auraria, and we hypothesized that a factor for the reproductive isolation of these two species is the use of different sex pheromones (Cardé et al 1977). Therefore, in the present work, we identified the female-produced sex pheromone of P. triquetra by means of gas-chromatography-electroantennographic detection (GC-EAD), gas chromatography-mass spectrometry (GCMS), and by carrying out field tests with blends of synthetic compounds.
Material and Methods Insects Larvae and pupae of P. triquetra were collected from G. officinalis and Rubus spp. in an infested raspberry orchard, near Linares (35°48′S, 71°40′W) in central Chile and transported to the laboratory, where they were kept in rearing chambers (25 ± 1°C, 50 ± 5% RH, 16L:8D photoperiod). Larvae were supplied with host plant foliage (V. vinifera and G. officinalis leaves). The pupae were sexed, and both males and females were kept separately under rearing chamber conditions. After adult emergence, virgin moths were kept individually and provided with a 5% sugar solution through a cotton wick.
Preparation of gland extracts The activity of female P. triquetra was observed by placing the insects individually in 150-mL sterilized glass containers that were covered with screen and maintained under ambient conditions. Calling behavior (wing fanning, curving of the abdomen, and extrusion of the abdominal gland) was observed mainly during the last 2 h of the scotophase. Virgin females (1–3 days old) were killed at that time of the day by freezing them at −20°C for 10 min. The pheromone glands were extruded by gently pressing the abdominal tips, excised, and immersed in 10 μL of hexane (SupraSolv, Merck, Darmstadt, Germany) that was previously cooled to −20°C. After 10 min, the hexane was carefully removed and stored at −20°C until use. Extracts of two to three glands were pooled for analysis. Chemicals Z11-14:OAc, E11-14:OAc, and (E)-9-dodecenyl acetate (E912:OAc) were purchased from Bedoukian Inc. (Danbury, CT, USA). For the field tests, each compound was purified by column chromatography on silica gel impregnated with silver nitrate, and showed purities >99% (GC) after this procedure. Chemical analyses GC-EAD analyses were carried out using a Shimadzu GC-2014 AFSC gas chromatograph (Shimadzu, Kyoto, Japan) coupled to an electroantennographic detector (Syntech, Kirchzarten, Germany). The column effluent was split in two equal parts, with one part going to a flame ionization detector (FID) and the other through a heated transfer line into a humidified airstream (400 mL min−1) that was directed to the male antennal preparation. The antennae were prepared by decapitating the insect (1- to 3-day-old males) and connecting the base of the head and the tips of both of the antennae to the two electrodes of the probe covered with conducting gel (Syntech probe). The signal was acquired with the signal acquisition interface IDAC-2 (Syntech) and recorded and processed using the software GC-EAD 2010 v1.2.2 (Syntech). The GC was equipped with either a fused silica RTX-5 capillary column (30 m × 0.25 mm id, 0.25 µm film, Restek, Bellefonte, PA, USA) or a fused silica RTX-Wax capillary column (30 m × 0.32 mm id, 0.25 µm film, Restek). For the RTX5 column, the oven was programmed from 50°C for 5 min to 270°C at 8°C min−1; and for the RTX-Wax column, the conditions were 50°C, 2 min hold, 10°C min−1 to 220°C. The GC was operated in split/splitless mode (30 s sampling time) with an injector temperature of 250°C, and helium was used as the carrier gas. GC-MS analyses were carried out using a Shimadzu GCMS-QP2010 Ultra combination using the same
Sex pheromone of Proeulia triquetra
GC columns and conditions as above. Electron impact mass spectra were acquired at 70 eV.
Pairwise comparisons were realized with Tukey-Kramer’s test (p ≤ 0.05). All statistical analyses were carried out using JMP ver. 11 (SAS).
Dimethyl disulfide derivatization of pheromone gland extracts
Results Dimethyl disulfide (DMDS) (50 μL) and 50 μL of a 5% solution of iodine in diethyl ether were added to a hexane gland extract containing one female equivalent in ca. 50 μL (Buser et al 1983). The reaction mixture was kept at 50°C overnight. After addition of a drop of a solution of 10% sodium thiosulfate in water, the mixture was extracted with 200 μL hexane, concentrated to ca. 20 μL, and submitted to GC-MS analysis using the RTX-5 column as described above. Field tests Field tests with synthetic compounds were carried out in an organically managed orchard (including raspberry hybrids, vineyards, and raspberries) near Linares (central Chile). Blends of synthetic compounds were prepared in hexane and white rubber septa (Sigma-Aldrich, St. Louis, MO, USA, catalog #Z553905) were loaded with 100 μL of the respective solutions. Septa were placed inside Delta traps (Pherocon II B, Trécé Inc., USA). Septa treated with 100 μL of hexane were used as controls. The traps were placed at 1.0- to 1.5-m height on the orchard rows, with a distance of 30 to 35 m between the traps. A completely randomized block design was used in all experiments. Preliminary tests (not shown) indicated that the simultaneous presence of Z11-14:OAc and E9-12:OAc was needed for attraction, and hence, the first field test was designed to evaluate different blends of these two compounds. It was conducted from November 14 to 29, 2014, using 1:0, 10:0.3, 10:1, 10:3, 1:1, and 0:1 blends of Z1114:OAc and E9-12:OAc at a dose of 500 μg of the main compound. Four replicates per treatment were used. The second field experiment was conducted from February 17 to 27, 2015, testing 10:1 blends of Z11-14:OAc and E9-12:OAc, to which different amounts of E11-14:OAc were added (0, 1, 4, 10, and 100%, respectively, in relation to Z11-14:OAc). Again, the dose of the main compound Z11-14:OAc was 500 μg, and four replicates per treatment were used. Data analysis The control treatments (no compounds) and the treatment with only E9-12:OAc in the first trial failed to capture males and were excluded from the statistical analysis. Data of captures per trap per day were transformed to log (x + 1) before analysis, and the homogeneity of variances was confirmed by Levene’s test. Data were analyzed by ANOVA with treatment as an independent variable and block as a random factor.
Chemical analyses GC-EAD analyses of a pheromone gland extract using male antennae showed three EAD-active compounds 1, 2, and 3 (Fig 1). The retention indices on both columns (Table 1) and the mass spectra obtained upon GC-MS analysis suggested compound 1 to be dodecenyl acetate (m/z 43, 61, 166) and compounds 2 and 3 to be tetradecenyl acetates (m/z 43, 61, 194). DMDS derivatization of the gland extract resulted in the formation of adducts, which indicated a double bond at the 9-position in case of the dodecenyl acetate (diagnostic fragments: m/z 89, 231, 320) and in 11-position for the tetradecenyl acetates (diagnostic fragments for both derivatives: m/z 89, 259, 348). Comparison of the mass spectra and the retention times of the natural products with those of authentic reference samples on both columns confirmed compound 1 to be E9-12:OAc, and compounds 2 and 3 were identified as E11-14:OAc and Z11-14:OAc, respectively. Other structurally related, minor compounds were identified from the gland extracts in addition to compounds 1, 2, and 3 (Table 1). Due to the co-elution with other compounds on both columns (in particular, Z11-14:OAc with 14:OAc on the RTX-5 column, and E11-14:OAc with 16:Ald on the RTX-Wax column), the ratio of the EAD-active compounds could only be estimated. The ratio of E9-12:OAc in relation to the main compound Z11-14:OAc was approximately 0.1:1, while the ratio of E11-14:OAc (relative to Z11-14:OAc) varied in different extracts between approximately 0.04–0.3:1 (n = 4). Field tests The first field test was designed to evaluate binary mixtures of Z11-14:OAc and E9-12:OAc in different ratios. The results showed that there were significant differences between treatments (F4,12 = 42.89, p < 0.0001). Traps baited with 10:0.3, 10:1, and 10:3 mixtures were equally attractive to male P. triquetra, while traps baited with a 1:1 mixture attracted significantly fewer males than traps baited with the 10:1 mixture (Table 2). In the second experiment, different amounts of E11-14:OAc were added to a 10:1 mixture of Z11-14:OAc and E9-12:OAc. Again, significant differences between treatments were found (F4,12 = 10.96, p < 0.001). Traps containing 0, 1, 4, and 10% of E11-14:OAc (relative to the main compound) were equally attractive to males, and traps containing 100% E11-14:OAc captured significantly fewer males (Table 3).
Bergmann et al.
Fig 1 Gas chromatogram (RTX-Wax column) of an extract of the abdominal glands of female Proeulia triquetra with simultaneous detection by FID (upper trace) and EAD (lower trace).
Discussion We have identified the female sex pheromone of P. triquetra by analyzing abdominal gland extracts of 1- to 3-day-old adult virgin females by GC-EAD and GC-MS, followed by field tests with synthetic compounds. DMDS derivatization and GC-MS analysis allowed the identification of Z11-14:OAc, E9-12:OAc, and E11-14:OAc. Field test with lures baited with synthetic compounds confirmed the biological activity of these compounds by attracting large numbers of males. A preliminary experiment had shown that none of the three compounds was attractive to males on its own, and the simultaneous Table 1 Compounds identified from gland extracts of Proeulia triquetra and their retention data on two different stationary phases. RTX-5
presence of Z11-14:OAc and E9-12:OAc was essential for attractiveness. While these two compounds were found in the gland in a 10:1 ratio, males are also attracted to other ratios of these two compounds, as traps with lower (10:0.3 ratio) and higher (10:3 ratio) amounts of E9-12:OAc were as attractive as the 10:1 mixture (Table 2). A 1:1 mixture still captured about half of the number of males, but this result differed significantly from the 10:1 mixture. The second field test, in which different amounts of E11-14:OAc were added to a 10:1 mixture of the other two compounds, was designed in consideration of the strongly E-biased pheromone of the closely related species P. auraria, where addition of small amounts of the geometric isomer Z11-14:OAc to the main component E1114:OAc had either a synergistic (at the 1% level) or an Table 2 Captures of Proeulia triquetra males in traps baited with different blends of synthetic pheromone compounds.
RTX-Wax
Compound
Rt (min)
RI
Rt (min)
RI
Lure composition (μg)
E9-12:OAc (1) 12:OAc Z9-12:OAc 14:Ald E11-14:OAc (2) 14:OAc Z11-14:OAc (3) 16:Ald
21.34 21.40 n.d. 21.48 24.10 24.16 24.17 24.29
1606 1611 n.d. 1616 1806 1809 1810 1818
13.21 12.75 13.32 13.13 15.24 14.82 15.35 15.21
1928 1884 1938 1920 2135 2091 2147 2132
Z11-14:OAc
E9-12:OAc
500 500 500 500 500 0 0
0 15 50 150 500 500 0
Rt retention time, RI retention index, n.d. not detected, E9-12:OAc (E)-9dodecenyl acetate, 12:OAc dodecyl acetate, Z9-12:OAc (Z)-9-dodecenyl acetate, 14:Ald tetradecanal, E11-14:OAc (E)-11-tetradecenyl acetate, 14:OAc tetradecyl acetate, Z11-14:OAc (Z)-11-tetradecenyl acetate, 16:Ald hexadecanal.
No. of males per trap per day (mean ± SD)a
0.2 ± 0.4 c 10.4 ± 2.3 ab 11.8 ± 0.2 a 10.6 ± 1.7 ab 5.9 ± 3.5 b 0 0
The experiment was carried out from November 14–29, 2014 using four replicates per treatment. a Values followed by different letters are different at p < 0.05 according to ANOVA followed by Tukey-Kramer’s test.
Sex pheromone of Proeulia triquetra Table 3 Captures of Proeulia triquetra males in traps baited with different blends of synthetic pheromone compounds.
No. of males per trap per day (mean ± SD)a
Lure composition (μg) Z11-14:OAc
E9-12:OAc
E11-14:OAc
500
50
0
500
50
5
9.3 ± 1.8 a
500 500
50 50
20 50
8.6 ± 2.3 a 4.9 ± 2.6 a
8.5 ± 3.0 a
500
50
500
1.0 ± 1.5 b
0
0
0
0
The experiment was carried out from February 17–27, 2015 using four replicates per treatment. a
Values followed by different letters are different at p < 0.05 according to ANOVA followed by Tukey-Kramer’s test.
antagonistic effect (at the 4% level). In the case of P. triquetra, our results showed that neither the absence of E11-14:OAc, nor its presence in amounts up to 10% in 10:1 mixtures of the two other compounds, affected the trap captures (Table 3). In this experiment, the mean number of males captured with the 10:1:1 ratio was lower than the treatments with less amount of E11-14:OAc; however, this data was not statistically different due to the variance of the result caused by a single trap with lower captures. When E11-14:OAc was present in higher amounts (10:1:10 ratio), however, the number of males in traps was significantly reduced. Variations of the relative amount of this compound in the range from at least 0–10% of the main component thus seem to have no effect on the attractiveness of the pheromone mixture, which coincides with the variability of the relative amounts detected in different gland extracts. These results suggest that E11-14:OAc is not essential for attraction in the tested three-component blends, but males are able to detect this compound and respond to unusual high concentrations of it in the pheromone mixture by avoiding the respective traps. The compounds identified here are commonly found in tortricid species (Ando et al 2004, El-Sayed 2014). It is known that closely related lepidopteran species may share the same compounds as part of their respective sex pheromones, and that species specificity is achieved by specific mixtures of two or more compounds, rather than by a single compound (Ando et al 2004). In this context, it is noteworthy that the synthetic lures attractive to P. triquetra attracted exclusively males of this species, in spite of the presence of P. auraria at the test site. The sex pheromone of P. auraria has recently been identified as a four-component mixture composed of E11-14:OAc, E11-14:OH, Z11-14:OAc, and 14:OAc in a 100:37:1:19 ratio (Reyes-Garcia et al 2014). In this species, E11-14:OAc has been shown to be attractive on its own, but the other three compounds act synergistically. In particular, the amount of Z11-14:OAc was critically important for the attractiveness of traps, as the addition of 1% had a synergistic effect on trap catches, while the addition of 4% resulted in almost
complete loss of attractiveness. It seems likely that the different composition of the respective sex pheromones of P. triquetra and P. auraria is largely responsible for the reproductive isolation of these two closely related, sympatric, and synchronic species. Proeulia auraria males will not be attracted to P. triquetra females due to the presence of large amounts of Z11-14:OAc in the pheromone of the latter species, while P. auraria females do not produce E9-12:OAc, which is necessary for the attraction of P. triquetra males. Furthermore, the avoidance of high amounts of E11-14:OAc in pheromone blends by male P. triquetra might be an additional mechanism ensuring reproductive isolation, as this is the main component of the pheromone of P. auraria. Razowski & Pelz (2010) listed a total of 38 species of the genus Proeulia present in Chile, many of them of recent description. Except for P. auraria and now P. triquetra, no pheromones or sex attractants are known for these species. Larvae of different Proeulia species are difficult to distinguish and are often simply referred to as “Eulia.” Species-specific lures using sex pheromones would be useful tools for the identification of populations, and enable a more precise study of the geographical distribution of Proeulia species in central-southern Chile. It would also be interesting to conduct a survey of sex pheromones and/or sex attractants for species of the tribe Euliini, in order to learn more about the evolution of chemical communication and other ecological aspects in this group. The only other sex pheromone of a member of the Euliini is known from the neotropical species Bonagota cranaodes, a species not present in Chile, which consists of an unusual four-component blend containing (3E, 5Z)-dodecadienyl acetate, (Z)-5-dodecenyl acetate, (3E,5Z)tetradecadienyl acetate, and (Z)-9-hexadecenyl acetate (Unelius et al 1996, Eiras et al 1999, Coracini et al 2001). In summary, our results indicate that a 10:1 mixture of Z1114:OAc and E9-12:OAc is optimal for attracting P. triquetra males, and the high attractiveness of traps suggest that this pheromone can be a useful tool to monitor the presence of this species in Chilean fruit orchards.
Bergmann et al. Acknowledgments Financial support from Fondo Nacional de Desarrollo Científico y Tecnológico (grants 1110365 and 1140779 to JB) and from Deutscher Akademischer Austauschdienst (via material resources program to JB) is acknowledged. LRG is grateful for a doctoral fellowship from Programa de Mejoramiento de la Calidad y la Equidad de la Educación Superior (grant UCH0601).
References Ando T, Inomata S, Yamamoto M (2004) Lepidopteran sex pheromones. Top Curr Chem 239:51–96 Buser H-R, Arn H, Guerin P, Rauscher S (1983) Determination of double bond position in mono-unsaturated acetates by mass spectrometry of dimethyl disulfide adducts. Anal Chem 55:818–822 Cardé RT, Cardé AM, Hill AS, Roelofs WL (1977) Sex attractant specificity as a reproductive isolating mechanism among the sibling species Archips argyrospilus and mortuans and other sympatric tortricine moths (Lepidoptera: Tortricidae). J Chem Ecol 3:71–84 Cepeda D, Cubillos G (2011) Description of the last larval stage and a compilation of host records, of seven species of tortricids of economic importance in Chile (Lepidoptera: Tortricidae). Gayana 75:39–70 Coracini MDA, Bengtsson M, Reckziegel A, Löfqvist J, Francke W, Vilela EF, Eiras AE, Kovaleski A, Witzgall P (2001) Identification of a four-
component sex pheromone blend in Bonagota cranaodes (Lepidoptera: Tortricidae). J Econ Entomol 94:911–914 Eiras AE, Kovaleski A, Vilela EF, Chambon J-P, Unelius CR, Borg-Karlson AK, Liblikas I, Mozuraitis R, Bengtsson M, Witzgall P (1999) Sex pheromone of the Brazilian Apple leafroller, Bonagota cranaodes Meyrick (Lepidoptera: Tortricidae). Z Naturforsch 54c:595–601 El-Sayed AM (2014) The Pherobase: database of pheromones and semiochemicals. http://www.pherobase.com Accessed 14 Dec 2014 González RH (2003) Las polillas de la fruta en Chile. Universidad de Chile, Serie Ciencias Agronómicas N° 9, Santiago, Chile, p 188 ODEPA (2014). Oficina de Estudios y Políticas Agrarias, http://www. odepa.cl Accessed 15 Nov 2014 Razowski J (1999) Euliini (Lepidoptera: Tortricidae) of Chile. Pol J Entomol 68:69–90 Razowski J, Pelz V (2010) Tortricidae from Chile (Lepidoptera: Tortricidae). SHILAP Revta Lepidop 38:5–55 Reyes-Garcia L, Cuevas Y, Ballesteros C, Curkovic T, Löfstedt C, Bergmann J (2014) A 4-component sex pheromone of the Chilean fruit leafroller Proeulia auraria (Lepidoptera: Tortricidae). Cien Inv Agr 41:187–196 Ripa R, Larral P (eds) (2008) Manejo de plagas en paltos y cítricos. Instituto de Investigaciones Agropecuarias (INIA), Colección Libros INIA N° 23. La Cruz, Chile Unelius CR, Eiras AE, Witzgall P, Bengtsson M, Kovaleski A, Vilela EF, Borg-Karlson A-K (1996) Identification and synthesis of the sex pheromone of Phtheocroa cranaodes (Lepidoptera: Tortricidae). Tetrahedron Lett 37:1505–1508
ECOLOGY, BEHAVIOR AND BIONOMICS
Identification of the Female Sex Pheromone of the Leafroller Proeulia triquetra Obraztsov (Lepidoptera: Tortricidae) J BERGMANN1, L REYES-GARCIA1,2, C BALLESTEROS3, Y CUEVAS3, MF FLORES1, T CURKOVIC3 1
Instituto de Química, Pontificia Univ Católica de Valparaíso, Valparaíso, Chile Depto de Ciencias Básicas, Univ Santo Tomás, Viña del Mar, Chile 3 Depto de Sanidad Vegetal, Fac de Ciencias Agronómicas, Univ de Chile, Santiago, Chile 2
Keywords Euliini, electroantennographic detection, gas chromatography, male attractant, mass spectrometry, reproductive isolation Correspondence J Bergmann, Instituto de Química, Pontificia Univ Católica de Valparaíso, Valparaíso, Chile; [email protected] Edited by Stefano Colazza – Univ of Palermo Received 2 July 2015 and accepted 19 January 2016 * Sociedade Entomológica do Brasil 2016
Abstract Proeulia triquetra Obraztsov (Lepidoptera: Tortricidae) is an occasional pest in fruit orchards in central-southern Chile. In order to develop species-specific lures for detection and monitoring of this species, we identified the female-produced sex pheromone. (Z)-11-Tetradecenyl acetate (Z11-14:OAc), (E)-9-dodecenyl acetate (E9-12:OAc), and (E)-11Tetradecenyl acetate (E11-14:OAc) were identified as biologically active compounds present in female pheromone glands by solvent extraction of the gland and analysis of the extracts by gas chromatographyelectroantennographic detection and gas chromatography-mass spectrometry. In field tests, lures baited with synthetic Z11-14:OAc and E912:OAc in a 10:1 ratio were highly attractive to males of the species.
Introduction Agriculture is an important economic activity in Chile, contributing ca. 2.6% to the gross domestic product of the country, and the production and exportation of fresh and processed fruit makes up about 40–45% of this contribution. The majority of fruit plantations are located in central-southern Chile, and the most important products are grapes, stone and pip fruits (peach, plum, nectarine, apple, etc.), avocados, and berries, among others (ODEPA 2014). There are several exotic and endemic species threatening the production, and among them are native leafrollers of the genus Proeulia (Lepidoptera: Tortricidae). Proeulia triquetra Obraztsov, together with Proeulia auraria (Clarke) and Proeulia chrysopteris (Butler) have been recognized as the species of greatest potential economic impact within this genus, as these species are causing occasional but increasing damage in fruit orchards (González 2003). Proeulia triquetra is a polyphagous species found on grapes (Vitis vinifera), apples (Malus domestica),
mandarins (Citrus reticulata), and berry (Rubus spp.) orchards, some weeds (Convolvulus arvensis, Galega officinalis), several ornamentals (Buddleja davidii, Hebe sp., Lonicera japonica), and some native plants (Fuchsia magellanica, Maytenus boaria, Myoschilos oblonga) (Cepeda & Cubillos 2011). Despite the fact that its distribution has been reported from San Felipe (32°45′S, 70°43′W) to Los Lagos (40°13′S, 74°49′W), economic damage has been reported mainly in orchards (vineyards, berries, apples) south of Maule (35°06′S, 71°16′W) (González 2003, Cepeda & Cubillos 2011). Several natural enemies of Proeulia are known (González 2003, Ripa & Larral 2008), but biological control has not been developed against this species. Chemical control of P. triquetra is usually a side effect of pesticide applications aimed at the control of other tortricid species, mainly Cydia pomonella L. and Cydia molesta Busck. The increasing use of pheromone based tactics (i.e., mating disruption) for the control of the latter two species and the consequential withdrawal of insecticides targeting Lepidoptera pests, however, has led to local and occasional outbreaks of Proeulia species
Bergmann et al.
(Curkovic, unpublished data). Another major problem is the quarantine status of Proeulia spp., and the presence of these insects has been a permanent cause of rejection for the exportation of fresh fruits (including blueberries, grapes, and apples) during the last years, mainly from the Maule and the Biobio regions. In particular, P. triquetra has been listed within quarantine species by the respective plant protection agencies in Australia and the USA. An associated problem is the correct identification of these morphologically similar species at the larval or pupal stages, although some progress has been recently made (Cepeda & Cubillos 2011). Besides, adult characters are available from descriptions by Razowski (1999), González (2003), and Razowski & Pelz (2010). Traps baited with synthetic sex pheromones are useful tools for detection and monitoring of insect pests, and the specificity of pheromone-baited traps can help in the identification of the species present in fruit orchards. The sex pheromone of P. auraria has been identified recently by our group as a four-component blend consisting of (E)-11tetradecenyl acetate (E11-14:OAc), (E)-11-tetradecenol (E1114:OH), tetradecyl acetate (14:OAc), and (Z)-11-tetradecenyl acetate (Z11-14:OAc) (Reyes-Garcia et al 2014). Knowing the sex pheromone of P. triquetra would set the basis for the development of monitoring lures for this species, thus enabling a more precise determination of the actual species present in fruit orchards and provide information for optimal timing of insecticide application. Furthermore, P. triquetra is sympatric and synchronic with P. auraria, and we hypothesized that a factor for the reproductive isolation of these two species is the use of different sex pheromones (Cardé et al 1977). Therefore, in the present work, we identified the female-produced sex pheromone of P. triquetra by means of gas-chromatography-electroantennographic detection (GC-EAD), gas chromatography-mass spectrometry (GCMS), and by carrying out field tests with blends of synthetic compounds.
Material and Methods Insects Larvae and pupae of P. triquetra were collected from G. officinalis and Rubus spp. in an infested raspberry orchard, near Linares (35°48′S, 71°40′W) in central Chile and transported to the laboratory, where they were kept in rearing chambers (25 ± 1°C, 50 ± 5% RH, 16L:8D photoperiod). Larvae were supplied with host plant foliage (V. vinifera and G. officinalis leaves). The pupae were sexed, and both males and females were kept separately under rearing chamber conditions. After adult emergence, virgin moths were kept individually and provided with a 5% sugar solution through a cotton wick.
Preparation of gland extracts The activity of female P. triquetra was observed by placing the insects individually in 150-mL sterilized glass containers that were covered with screen and maintained under ambient conditions. Calling behavior (wing fanning, curving of the abdomen, and extrusion of the abdominal gland) was observed mainly during the last 2 h of the scotophase. Virgin females (1–3 days old) were killed at that time of the day by freezing them at −20°C for 10 min. The pheromone glands were extruded by gently pressing the abdominal tips, excised, and immersed in 10 μL of hexane (SupraSolv, Merck, Darmstadt, Germany) that was previously cooled to −20°C. After 10 min, the hexane was carefully removed and stored at −20°C until use. Extracts of two to three glands were pooled for analysis. Chemicals Z11-14:OAc, E11-14:OAc, and (E)-9-dodecenyl acetate (E912:OAc) were purchased from Bedoukian Inc. (Danbury, CT, USA). For the field tests, each compound was purified by column chromatography on silica gel impregnated with silver nitrate, and showed purities >99% (GC) after this procedure. Chemical analyses GC-EAD analyses were carried out using a Shimadzu GC-2014 AFSC gas chromatograph (Shimadzu, Kyoto, Japan) coupled to an electroantennographic detector (Syntech, Kirchzarten, Germany). The column effluent was split in two equal parts, with one part going to a flame ionization detector (FID) and the other through a heated transfer line into a humidified airstream (400 mL min−1) that was directed to the male antennal preparation. The antennae were prepared by decapitating the insect (1- to 3-day-old males) and connecting the base of the head and the tips of both of the antennae to the two electrodes of the probe covered with conducting gel (Syntech probe). The signal was acquired with the signal acquisition interface IDAC-2 (Syntech) and recorded and processed using the software GC-EAD 2010 v1.2.2 (Syntech). The GC was equipped with either a fused silica RTX-5 capillary column (30 m × 0.25 mm id, 0.25 µm film, Restek, Bellefonte, PA, USA) or a fused silica RTX-Wax capillary column (30 m × 0.32 mm id, 0.25 µm film, Restek). For the RTX5 column, the oven was programmed from 50°C for 5 min to 270°C at 8°C min−1; and for the RTX-Wax column, the conditions were 50°C, 2 min hold, 10°C min−1 to 220°C. The GC was operated in split/splitless mode (30 s sampling time) with an injector temperature of 250°C, and helium was used as the carrier gas. GC-MS analyses were carried out using a Shimadzu GCMS-QP2010 Ultra combination using the same
Sex pheromone of Proeulia triquetra
GC columns and conditions as above. Electron impact mass spectra were acquired at 70 eV.
Pairwise comparisons were realized with Tukey-Kramer’s test (p ≤ 0.05). All statistical analyses were carried out using JMP ver. 11 (SAS).
Dimethyl disulfide derivatization of pheromone gland extracts
Results Dimethyl disulfide (DMDS) (50 μL) and 50 μL of a 5% solution of iodine in diethyl ether were added to a hexane gland extract containing one female equivalent in ca. 50 μL (Buser et al 1983). The reaction mixture was kept at 50°C overnight. After addition of a drop of a solution of 10% sodium thiosulfate in water, the mixture was extracted with 200 μL hexane, concentrated to ca. 20 μL, and submitted to GC-MS analysis using the RTX-5 column as described above. Field tests Field tests with synthetic compounds were carried out in an organically managed orchard (including raspberry hybrids, vineyards, and raspberries) near Linares (central Chile). Blends of synthetic compounds were prepared in hexane and white rubber septa (Sigma-Aldrich, St. Louis, MO, USA, catalog #Z553905) were loaded with 100 μL of the respective solutions. Septa were placed inside Delta traps (Pherocon II B, Trécé Inc., USA). Septa treated with 100 μL of hexane were used as controls. The traps were placed at 1.0- to 1.5-m height on the orchard rows, with a distance of 30 to 35 m between the traps. A completely randomized block design was used in all experiments. Preliminary tests (not shown) indicated that the simultaneous presence of Z11-14:OAc and E9-12:OAc was needed for attraction, and hence, the first field test was designed to evaluate different blends of these two compounds. It was conducted from November 14 to 29, 2014, using 1:0, 10:0.3, 10:1, 10:3, 1:1, and 0:1 blends of Z1114:OAc and E9-12:OAc at a dose of 500 μg of the main compound. Four replicates per treatment were used. The second field experiment was conducted from February 17 to 27, 2015, testing 10:1 blends of Z11-14:OAc and E9-12:OAc, to which different amounts of E11-14:OAc were added (0, 1, 4, 10, and 100%, respectively, in relation to Z11-14:OAc). Again, the dose of the main compound Z11-14:OAc was 500 μg, and four replicates per treatment were used. Data analysis The control treatments (no compounds) and the treatment with only E9-12:OAc in the first trial failed to capture males and were excluded from the statistical analysis. Data of captures per trap per day were transformed to log (x + 1) before analysis, and the homogeneity of variances was confirmed by Levene’s test. Data were analyzed by ANOVA with treatment as an independent variable and block as a random factor.
Chemical analyses GC-EAD analyses of a pheromone gland extract using male antennae showed three EAD-active compounds 1, 2, and 3 (Fig 1). The retention indices on both columns (Table 1) and the mass spectra obtained upon GC-MS analysis suggested compound 1 to be dodecenyl acetate (m/z 43, 61, 166) and compounds 2 and 3 to be tetradecenyl acetates (m/z 43, 61, 194). DMDS derivatization of the gland extract resulted in the formation of adducts, which indicated a double bond at the 9-position in case of the dodecenyl acetate (diagnostic fragments: m/z 89, 231, 320) and in 11-position for the tetradecenyl acetates (diagnostic fragments for both derivatives: m/z 89, 259, 348). Comparison of the mass spectra and the retention times of the natural products with those of authentic reference samples on both columns confirmed compound 1 to be E9-12:OAc, and compounds 2 and 3 were identified as E11-14:OAc and Z11-14:OAc, respectively. Other structurally related, minor compounds were identified from the gland extracts in addition to compounds 1, 2, and 3 (Table 1). Due to the co-elution with other compounds on both columns (in particular, Z11-14:OAc with 14:OAc on the RTX-5 column, and E11-14:OAc with 16:Ald on the RTX-Wax column), the ratio of the EAD-active compounds could only be estimated. The ratio of E9-12:OAc in relation to the main compound Z11-14:OAc was approximately 0.1:1, while the ratio of E11-14:OAc (relative to Z11-14:OAc) varied in different extracts between approximately 0.04–0.3:1 (n = 4). Field tests The first field test was designed to evaluate binary mixtures of Z11-14:OAc and E9-12:OAc in different ratios. The results showed that there were significant differences between treatments (F4,12 = 42.89, p < 0.0001). Traps baited with 10:0.3, 10:1, and 10:3 mixtures were equally attractive to male P. triquetra, while traps baited with a 1:1 mixture attracted significantly fewer males than traps baited with the 10:1 mixture (Table 2). In the second experiment, different amounts of E11-14:OAc were added to a 10:1 mixture of Z11-14:OAc and E9-12:OAc. Again, significant differences between treatments were found (F4,12 = 10.96, p < 0.001). Traps containing 0, 1, 4, and 10% of E11-14:OAc (relative to the main compound) were equally attractive to males, and traps containing 100% E11-14:OAc captured significantly fewer males (Table 3).
Bergmann et al.
Fig 1 Gas chromatogram (RTX-Wax column) of an extract of the abdominal glands of female Proeulia triquetra with simultaneous detection by FID (upper trace) and EAD (lower trace).
Discussion We have identified the female sex pheromone of P. triquetra by analyzing abdominal gland extracts of 1- to 3-day-old adult virgin females by GC-EAD and GC-MS, followed by field tests with synthetic compounds. DMDS derivatization and GC-MS analysis allowed the identification of Z11-14:OAc, E9-12:OAc, and E11-14:OAc. Field test with lures baited with synthetic compounds confirmed the biological activity of these compounds by attracting large numbers of males. A preliminary experiment had shown that none of the three compounds was attractive to males on its own, and the simultaneous Table 1 Compounds identified from gland extracts of Proeulia triquetra and their retention data on two different stationary phases. RTX-5
presence of Z11-14:OAc and E9-12:OAc was essential for attractiveness. While these two compounds were found in the gland in a 10:1 ratio, males are also attracted to other ratios of these two compounds, as traps with lower (10:0.3 ratio) and higher (10:3 ratio) amounts of E9-12:OAc were as attractive as the 10:1 mixture (Table 2). A 1:1 mixture still captured about half of the number of males, but this result differed significantly from the 10:1 mixture. The second field test, in which different amounts of E11-14:OAc were added to a 10:1 mixture of the other two compounds, was designed in consideration of the strongly E-biased pheromone of the closely related species P. auraria, where addition of small amounts of the geometric isomer Z11-14:OAc to the main component E1114:OAc had either a synergistic (at the 1% level) or an Table 2 Captures of Proeulia triquetra males in traps baited with different blends of synthetic pheromone compounds.
RTX-Wax
Compound
Rt (min)
RI
Rt (min)
RI
Lure composition (μg)
E9-12:OAc (1) 12:OAc Z9-12:OAc 14:Ald E11-14:OAc (2) 14:OAc Z11-14:OAc (3) 16:Ald
21.34 21.40 n.d. 21.48 24.10 24.16 24.17 24.29
1606 1611 n.d. 1616 1806 1809 1810 1818
13.21 12.75 13.32 13.13 15.24 14.82 15.35 15.21
1928 1884 1938 1920 2135 2091 2147 2132
Z11-14:OAc
E9-12:OAc
500 500 500 500 500 0 0
0 15 50 150 500 500 0
Rt retention time, RI retention index, n.d. not detected, E9-12:OAc (E)-9dodecenyl acetate, 12:OAc dodecyl acetate, Z9-12:OAc (Z)-9-dodecenyl acetate, 14:Ald tetradecanal, E11-14:OAc (E)-11-tetradecenyl acetate, 14:OAc tetradecyl acetate, Z11-14:OAc (Z)-11-tetradecenyl acetate, 16:Ald hexadecanal.
No. of males per trap per day (mean ± SD)a
0.2 ± 0.4 c 10.4 ± 2.3 ab 11.8 ± 0.2 a 10.6 ± 1.7 ab 5.9 ± 3.5 b 0 0
The experiment was carried out from November 14–29, 2014 using four replicates per treatment. a Values followed by different letters are different at p < 0.05 according to ANOVA followed by Tukey-Kramer’s test.
Sex pheromone of Proeulia triquetra Table 3 Captures of Proeulia triquetra males in traps baited with different blends of synthetic pheromone compounds.
No. of males per trap per day (mean ± SD)a
Lure composition (μg) Z11-14:OAc
E9-12:OAc
E11-14:OAc
500
50
0
500
50
5
9.3 ± 1.8 a
500 500
50 50
20 50
8.6 ± 2.3 a 4.9 ± 2.6 a
8.5 ± 3.0 a
500
50
500
1.0 ± 1.5 b
0
0
0
0
The experiment was carried out from February 17–27, 2015 using four replicates per treatment. a
Values followed by different letters are different at p < 0.05 according to ANOVA followed by Tukey-Kramer’s test.
antagonistic effect (at the 4% level). In the case of P. triquetra, our results showed that neither the absence of E11-14:OAc, nor its presence in amounts up to 10% in 10:1 mixtures of the two other compounds, affected the trap captures (Table 3). In this experiment, the mean number of males captured with the 10:1:1 ratio was lower than the treatments with less amount of E11-14:OAc; however, this data was not statistically different due to the variance of the result caused by a single trap with lower captures. When E11-14:OAc was present in higher amounts (10:1:10 ratio), however, the number of males in traps was significantly reduced. Variations of the relative amount of this compound in the range from at least 0–10% of the main component thus seem to have no effect on the attractiveness of the pheromone mixture, which coincides with the variability of the relative amounts detected in different gland extracts. These results suggest that E11-14:OAc is not essential for attraction in the tested three-component blends, but males are able to detect this compound and respond to unusual high concentrations of it in the pheromone mixture by avoiding the respective traps. The compounds identified here are commonly found in tortricid species (Ando et al 2004, El-Sayed 2014). It is known that closely related lepidopteran species may share the same compounds as part of their respective sex pheromones, and that species specificity is achieved by specific mixtures of two or more compounds, rather than by a single compound (Ando et al 2004). In this context, it is noteworthy that the synthetic lures attractive to P. triquetra attracted exclusively males of this species, in spite of the presence of P. auraria at the test site. The sex pheromone of P. auraria has recently been identified as a four-component mixture composed of E11-14:OAc, E11-14:OH, Z11-14:OAc, and 14:OAc in a 100:37:1:19 ratio (Reyes-Garcia et al 2014). In this species, E11-14:OAc has been shown to be attractive on its own, but the other three compounds act synergistically. In particular, the amount of Z11-14:OAc was critically important for the attractiveness of traps, as the addition of 1% had a synergistic effect on trap catches, while the addition of 4% resulted in almost
complete loss of attractiveness. It seems likely that the different composition of the respective sex pheromones of P. triquetra and P. auraria is largely responsible for the reproductive isolation of these two closely related, sympatric, and synchronic species. Proeulia auraria males will not be attracted to P. triquetra females due to the presence of large amounts of Z11-14:OAc in the pheromone of the latter species, while P. auraria females do not produce E9-12:OAc, which is necessary for the attraction of P. triquetra males. Furthermore, the avoidance of high amounts of E11-14:OAc in pheromone blends by male P. triquetra might be an additional mechanism ensuring reproductive isolation, as this is the main component of the pheromone of P. auraria. Razowski & Pelz (2010) listed a total of 38 species of the genus Proeulia present in Chile, many of them of recent description. Except for P. auraria and now P. triquetra, no pheromones or sex attractants are known for these species. Larvae of different Proeulia species are difficult to distinguish and are often simply referred to as “Eulia.” Species-specific lures using sex pheromones would be useful tools for the identification of populations, and enable a more precise study of the geographical distribution of Proeulia species in central-southern Chile. It would also be interesting to conduct a survey of sex pheromones and/or sex attractants for species of the tribe Euliini, in order to learn more about the evolution of chemical communication and other ecological aspects in this group. The only other sex pheromone of a member of the Euliini is known from the neotropical species Bonagota cranaodes, a species not present in Chile, which consists of an unusual four-component blend containing (3E, 5Z)-dodecadienyl acetate, (Z)-5-dodecenyl acetate, (3E,5Z)tetradecadienyl acetate, and (Z)-9-hexadecenyl acetate (Unelius et al 1996, Eiras et al 1999, Coracini et al 2001). In summary, our results indicate that a 10:1 mixture of Z1114:OAc and E9-12:OAc is optimal for attracting P. triquetra males, and the high attractiveness of traps suggest that this pheromone can be a useful tool to monitor the presence of this species in Chilean fruit orchards.
Bergmann et al. Acknowledgments Financial support from Fondo Nacional de Desarrollo Científico y Tecnológico (grants 1110365 and 1140779 to JB) and from Deutscher Akademischer Austauschdienst (via material resources program to JB) is acknowledged. LRG is grateful for a doctoral fellowship from Programa de Mejoramiento de la Calidad y la Equidad de la Educación Superior (grant UCH0601).
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