Identification and field attraction of the female sex pheromone of a kiwifruit pest, Nokona feralis (Lepidoptera: Sesiidae)

ABSTRACT

Female sex pheromone of a clearwing moth Nokona feralis (Leech) (Lepidoptera: Sesiidae), a pest of kiwifruit, was identified to be a 7:3 mixture of (3E,13Z)-3,13-octadecadienyl acetate (E3,Z13-18:OAc) and (3E,13Z)-3,13-octadecadien-1-ol (E3,Z13-18:OH) by GC-EAD and GC/MS analyses. Males were attracted to wide-range mixtures of E3,Z13-18:OAc and E3,Z13-18:OH, and a 7:3 mixture of those two compounds strongly attracted the males in the field.

KEYWORDS:

• 3, 13-octadecadienyl acetate
• 3, 13-octadecadien-1-ol
• clearwing moth
• borer
• Actinidiaceae

Once a clearwing moth Nokona feralis (Leech) (Lepidoptera: Sesiidae) was a rare species in Japan; until 2004, a few adults were collected, and the life history of this species was fragmentally reported that a female laid eggs on Actinidia arguta (Sieb. et Zucc.) and a last-instar larvae from the stem of Actinidia kolomikta (Maxim. et Rupr.) were found [1]. However, recently the damage to the branches of cultivated kiwifruit, Actinidia deliciosa (A. Chev.) C. F. Liang & A. R. Ferguson, by the larvae of N. feralis is a serious problem in the southwest Japan [2,3].

The eggs overwinter, and the hatched 1st-instar-larvae bore into the current shoots from the base of the petiole in the next April. N. feralis is only the species, which overwinter in egg stage, in Sesiidae [2,3]. Almost leaves on the current shoots are withered when larvae bore into, and larval feeding cause significant losses of fruit production. The mining behavior of the larvae protects them from sprayed insecticide; therefore the sex pheromone facilitates monitoring and mating disruption for integrated pest management (IPM) of the moth in kiwifruit orchards.

So far, the sex pheromones of 25 other sesiid species were revealed. Most of them are composed of (3E,13Z)-, (3Z,13Z)- and/or (2E,13Z)-octadecadienyl compounds. Furthermore, male attractants for more than 100 sesiid species have been found by many random screening tests of the synthetic dienyl compounds [4]. In the genus Nokona, female sex pheromones of N. regalis and N. pernix were identified [5–7]. Both these two species use only alcohols to their sex pheromone; N. regalis uses single (3E,13Z)-3,13-octadecadien-1-ol (E3,Z13-18:OH) and N. pernix uses a 9:1 mixture of E3,Z13-18:OH and its (3Z,13Z)-isomer (Z3,Z13-18:OH). However, the sex pheromone or sex attractant of N. feralis have not reported yet.

Overwintering eggs of N. feralis were collected at a kiwifruit orchard in Kouhoku Town, Saga Prefecture, Japan (33.23°N, 130.13°E) in January 2008. The eggs were put to potted kiwifruit trees in Saga Fruit Research Laboratory and kept until July 2008. About 20 larvae were transported to Tottori University with stems which the larvae bored into, and placed individually in a plastic cage (CleanCase®L, 375 mm × 220 mm × 280 mm height, Sanko Co., Ltd., Osaka, Japan) under laboratory conditions (25 ± 2ºC, 15L:9D) until emergence of the adults. Their sexes were discriminated at the adult stage based on their antennae and hair tufts [1]. The females showed calling-position on 9–11 h after the start of the photophase under the laboratory conditions. The abdominal tip of one or two-day-old virgin females keeping the calling posture were excised and separately immersed in hexane (250 μL/female) for 5 min to extract the sex pheromone. The crude extract which was kept at −20°C was concentrated at 1 μL/female by a gentle nitrogen stream for the instrumental analysis.

The electroantennogram (EAG) activity of natural pheromone components was measured by a gas chromatography equipped with an electroantennographic detector (GC-EAD). The GC involved an HP-5890 Series II gas chromatograph (Hewlett-Packard, Wilmington, DE) and a DB-23 capillary column (0.25 mm ID × 30 m length, 0.25 μm film thickness, J&W Scientific, Folsom, CA). The carrier gas was He. The oven temperature was maintained at 100ºC for 2 min, programmed at a rate of 20ºC/min up to 175ºC, 6ºC/min up to 220ºC, and maintained 220ºC for 15 min, with the GC-inlet temperature at 220ºC. A split/splitless injector was used in the splitless mode for 1 min and kept at 220ºC. The effluent from the column was split into two lines, which were led to a flame ionization detector (FID) and EAD in a ratio of 1:1. While the crude extract of the pheromone glands (0.5 females equivalent; FE) included many compounds detected by FID, the male antennae responded reproducibly to only two components: I, retention time (t_R) 11.32 min, Kovats retention index (KI) 2626; and II, t_R 11.51 min, KI 2648. The t_R values for I and II were respectively the same as those of synthetic E3,Z13-18:OAc and E3,Z13-18:OH (Figure 1(A)).

Figure 1. A GC-EAD analysis (DB-23 capillary column) of a crude pheromone extract from N. feralis females (0.5 FE) with a flame ionization detector (FID) and an electroantennographic detector (EAD). Male antennae responded to two components (component I: t_R 11.32 min, KI 2626; II: t_R 11.51 min, KI 2648), and did not responded to five synthetic compounds which were recorded as sex pheromone components of other Sesiidae species [4]. B Mass spectrum of component I (t_R 10.90 min, KI 2627) by GC/MS analysis (DB-23 capillary column) of a crude pheromone extract from N. feralis females (2 FE). C Mass spectrum of component II (t_R 11.08 min, KI 2648).

The crude extract was next analyzed by GC combined with mass spectrometry (GC/MS) in an electron impact ionization (EI) mode (70 eV) under the same GC conditions as those for the GC-EAD measurement. By GC/MS analysis of a crude extract (2 FE), the mass spectra of components I (t_R 10.90 min, KI 2627) and II (t_R 11.08 min, KI 2648) were successfully recorded. The mass chromatograms of the ions at m/z 248 ([M-60]⁺), 219, 205, 191, 81, 55, and 61 ([AcOH+1]⁺) and mass spectrum of component I indicated that those characteristic ions and t_R of component I were quite similar to those of synthetic E3,Z13-18:OAc (Figure 1(B)) [7]. In the same way, while M⁺ at m/z 266 and [M-18]⁺ at m/z 248 were not detected, the mass chromatograms of some characteristic fragment ions, mass spectrum, and t_R of component II were quite similar to those of synthetic E3,Z13-18:OH (Figure 1(C)). In addition to 2,13-dienyl compounds, four geometrical isomers of 3,13-dienyl compounds, which show almost the same mass spectra, can be distinguishable by their KI values [7]. On the other hand, mass chromatograms at m/z 264 (M⁺), 246 ([M-18]⁺), 235, 221 and 207 for 3,13- or 2,13-octadecadienals [8], any ion peaks were not detected at their expected t_Rs. On three replications of GC/MS analyses using other extracts (2 FE each), the titers of E3,Z13-18:OAc was about 11.8 ± 1.6 ng/female and of E3,Z13-18:OH was about 4.8 ± 0.7 ng/female, and the ratio of those two compounds were about 7:3.

The pheromone candidates, which were supplied by Shin-Etsu Chemical Co., and their geometrical isomers, which we had previously synthesized [7], were evaluated in the field to confirm the identification. All tests were conducted at a kiwifruit orchard in Ainan Town, Ehime Prefecture (32.98°N, 132.04°E) from August 2 to November 9 2009. Synthetic compounds were dissolved in hexane (20 mg/ml) and applied to rubber septa (white rubber, for o.d. 8mm, Sigma-Aldrich Co., St. Louis, MO, USA) as dispensers, and placed at the center of sticky board traps (SE Trap®, 30 × 27 cm bottom plate with a roof, Sankei Chemical Co., Kagoshima, Japan), and were hung separately 1.5 m above ground level at intervals of 10 m. Different blends of E3,Z13-18:OAc and E3,Z13-18:OH were tested for attractiveness to N. feralis males (Table 1). Males attracted to wide-range mixtures of E3,Z13-18:OAc and E3,Z13-18:OH, and the 7:3 blend of E3,Z13-18:OAc and E3,Z13-18:OH attracted most males (9.3 ± 8.3 males/trap); the optimal ratio of E3,Z13-18:OAc and E3,Z13-18:OH for attraction is estimated as 7:3. Only a male (0.3 ± 0.3 males/trap) was captured to single E3,Z13-18:OAc, whereas single E3,Z13-18:OH was not attracted any males.

Table 1. Attraction of N. feralis males by traps baited with a mixture of E3,Z13-18:OAc and E3,Z13-18:OH in different ratios.

Compounds (μg)
E3,Z13-18:OAc	E3,Z13-18:OH	N	Captures males (mean ± SE)^a				Total
1,000	0	3	0.3	±	0.3	a b	1
900	100	3	1	±	0.6	a b	3
700	300	3	9.3	±	8.3	a	28
500	500	3	3.3	±	1.2	a b	10
300	700	3	6	±	2.6	a b	18
100	900	3	0.7	±	0.3	a b	2
0	1,000	3	0				0
0	0	3	0				0

Tested at a kiwifruit orchard in Ainan Town, Ehime Prefecture from August 2 to November 9, 2009.

^aMales/trap. Values followed by a different letter are significantly different by the Tukey’s test (P < 0.05) after GLMM (family = Poisson, link = log) performed with R version 3.4.1.9) [9].

While 7:3 mixtures of the acetate and alcohol with a different configuration were tested, only the mixture of (3E,13Z)-isomers strongly attracted N. feralis males (11.3 ± 5.2 males/trap), and other isomers did not attract any males (Table 2). This suggests that male moths strictly discriminated the configurations at the 3- and 13-positions.

Table 2. Attraction of N. feralis males by traps baited with a synthetic lure, including a geometrical isomer of 3,13-octadecadienyl acetates and 3,13-octadecadien-1-ols (7: 3 mixture, totally 1,000 μg).

Compounds (μg)	N	Captures males (mean ± SE)^a			Total
E3,Z13-18:OAc (700) + E3,Z13-18:OH (300)	3	11.3	±	5.2	34
E3,E13-18:OAc (700) + E3,E13-18:OH (300)	3	0			0
Z3,E13-18:OAc (700) + Z3,E13-18:OH (300)	3	0			0
Z3,Z13-18:OAc (700) + Z3,Z13-18:OH (300)	3	0			0
Control	3	0			0

Tested at a kiwifruit orchard in Ainan Town, Ehime Prefecture from August 2 to November 9, 2009. E3,Z13-18:OAc and:OH are natural-type isomers, and other mixtures are unnatural-type isomers.

^aMales/trap.

Genus Nokona is included into a tribe Paranthrenini. In the tribe Paranthrenini, female sex pheromones have been identified from 5 species. Four of the five Paranthrenini species use only alcohols for their sex pheromone, but only Vitacea polistiformis use acetates [4,10]. Adding them, while sex attractants of 7 Paranthrenini species were reported, 3 species attracted to alcohols and 4 species to acetates. However, all of these species require either alcohols or acetates for male attraction. N. feralis is the first species which use both alcohols and acetates for female sex pheromone in Paranthrenini.

For control of two clearwing moth pests, Synanthedon hector (Butler) and S. tenuis (Butler), a mating disruptant (Synanthelure, Sukashiba-con®-L) was developed and registered in Japan, and this mating disruptant has been used to control N. feralis at kiwifruits orchards from 2014 [3]. This disruptant contains a 42.9:40.9:3.5 mixture of E3,Z13-18:OAc, Z3,Z13-18:OAc and E2,Z13-18:OAc to reproduce the sex pheromone of S. hector [11]. E3,Z13-18:OAc, the major component of this disruptant, also is the major pheromone component of N. feralis. Because generally partial blend of sex pheromone effectively plays a roll of mating disruptant [12], this disruptant may be useful in the control of N. feralis in kiwifruit orchards. However, partial blend of sex pheromone, not a full blend, occasionally causes of control failure by the resistant population for mating disruptant. In the smaller tea tortrix Adoxophyes honmai, continuous using of a mating disruptant made by single-component formulation made a resistant population for the mating disruptant, and finally an alternative mating disruptant contain the full blend of the sex pheromone components of this species [13]. When the mating disruption of N. feralis by Synanthelure was failed, a mating disruptant contain both E3,Z13-18:OAc and E3,Z13-18:OH would be required.

Author contributions

Conceived this work: H. N., and S. K. Designed the work: H. N. Rearing insects: K. F., and H. N. Chemical analysis: H. S., M. Y., H. N., and T. A. Synthesize compounds: H. S., M. Y., and T. A. Identification of insect specimen: Y. A., and H. N. Field tests. S. K., Y. S., F. K., and H. N. Statistics: H. N. Wrote the paper: H. N., and T. A.

Acknowledgments

We are grateful to Dr. F. Mochizuki of Shin-Etsu Chemical Co., for providing the synthetic 3,13-octadecadienyl compounds. We also thank Drs. Yohei Izumi at Shimane University, Seiya Kudo at Hirosaki University, and Messrs. Yasuhiro Ushida at Kagawa Prefectural Agricultural Experiment Station, Takahiro Mitarai at JA Fukuoka Yame, Tadashi Kudo in Hirosaki City, who kindly helped field experiments and supplied relevant information.

Disclosure statement

No potential conflict of interest was reported by the authors.