Sex Attractant of a Clearwing Moth Synanthedon Nashivora (Lepidoptera: Sesiidae)

A sex attractant of Synanthedon nashivora Naka and Yano, a pest of Asian pear discovered in Kyoto, Japan in 2014 and described as a novel species in 2019 was revealed. Pheromone lures baited mixtures with one or two of the seven compounds used as sex pheromones by Sesiidae species were prepared, and screening tests were conducted using these lures in pear orchards in Kyotango City, Kyoto Prefecture, Japan, from 2017 to 2018. Males were attracted to various mixture ratios, especially 7:3 mixture ratio, of (3Z,13Z)-3,13-octadecadienyl acetate (Z3,Z13-18:OAc) and (2E,13Z)-2,13-octadecadienyl acetate (E2,Z13-18:OAc) mixtures. This nding will enable monitoring of this species in Asian pear orchards. By attempting to search for males in the eld using this pheromone lure, it will be possible to understand the distribution of this species in Japan and neighboring countries, its microhabitat, and the seasonal occurrence of this species.


Introduction
A clearwing moth Synanthedon nashivora Naka and Yano (Lepidoptera: Sesiidae) was found in 2014 as a pest of Asian pear [Pyrus pyrifolia (Burm.f.) Nakai] and then described as a novel species in 2019 (Naka et al. 2019). Currently, this species is found only in the Tango Peninsula, Japan. The larvae attack pear trees in pear orchards in the Tango Peninsula. The larvae burrow alone under the bark, and larval feces are observed at the site of larval attack. If the base of a thin branch is damaged, the branch is more likely to break off and new buds are less likely to develop from the damaged area. When the large part of the main branch is damaged, the bark becomes rough. When the dead part of the tree expands, the tree's vigor declines (Kyoto Prefecture 2017;Kukizaki unpubl.).
This species is a recently discovered pest. Therefore, the seasonal trend and domestic distribution are not known. The status of damage to Asian pears by this species in the Tango Peninsula can be determined by careful investigation of traces of damage caused by the larvae of this species. However, it is very di cult to know the seasonal trend and to reveal the distribution of this species except for the Tango Peninsula. Because clearwing moths are diurnal, they are not attracted to light traps. Moreover, populations might be very low. If we want to collect adult clearwing moths by only visually searching for and catching them, we will suffer from encounters with numerous wasps and bees (Naka 2018).
The sex pheromone of clearwing moths has been studied for a long time and is used for monitoring and mating disruption all over the world. In Japan, pheromone lures for monitoring Synanthedon hector (Butler), S. tenuis (Butler), and Glossosphecia romanovi (Leech) are commercially available (Japan Plant Protection Association 2020). Also, mating disruptant has been used to control S. hector, S. tenuis and Nokona feralis (Leech) (Japan BioControl Association 2020).
This study aimed to develop a sex attractant that can effectively attract males of this species to reveal the seasonal trend and the distribution of this species. To this end, eld screening tests using pheromone traps with seven compounds commonly used as sex pheromones in clearwing moths as an attraction source were conducted at Asian pear orchards in the Tango Peninsula.

Materials And Methods
Chemicals.
with purity levels greater than 98%, were utilized as analytical standards and lures for eld tests after puri cation to purify level > 99.5% by column chromatography on silver nitrate-impregnated silica gels.
Field trapping test.
Based on the results of test I, test II was conducted at the same orchards from May 1 to June 13, 2018. In this test, 11 types of pheromone lures were used; 10:0, 9:1, 5:5, 1:9, and 0:10 mixtures of E2,Z13-18:OAc and Z3,Z13-18:OAc, 9:1, 5:5, and 1:9 mixtures of E2,Z13-18:OAc and E3,Z13-18:OAc, and 9:1, 5:5, and 1:9 mixtures of E2,Z13-18:OAc and E2,Z13-18:OH (N = 4) ( In all eld screening tests, the numbers of males captured by each trap were checked every about a week. 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. The traps were rotated after counting the captured moths to eliminate any position effect. Identi cation of the attracted males was following Naka et al. (2019). The sternites 4, 5, and 6 in this species were covered with white scales, and these white scales are important to identify this species.
The numbers of captured males in tests II and III, excluding zeroes, were analyzed using a generalized linear model (GLM) with Poisson distribution and log link function, applying treatment as a xed factor. Signi cant explanatory variables were compared by Tukey's test. These analyses were performed with R version 4.0.3 (R Development Core Team 2020).

Discussion
In the three eld attraction tests conducted from 2017 to 2018 with 7 compounds used as sex pheromones by clearwing moths, males of Synanthedon nashivora were attracted to various mixture ratios, especially the 7:3 mixture ratio, of Z3,Z13-18:OAc and E2,Z13-18:OAc mixtures (Tables 1-3). These results suggest that the sex pheromone components of this species contain at least two compounds, Z3,Z13-18:OAc and E2,Z13-18:OAc, which are essential for attraction.
Both the sex attractant of this species and the sex pheromone of S. haitangvora, a very closely related species, are composed of Z3,Z13-18:OAc and E2,Z13-18:OAc. The fact that both males of S. nashivora and S. haitangvora are attracted to a mixture of these two components reminds us of two hypotheses for the relationship between the two species. The rst is that S. nashivora may be a vicarious species of S. haitangvora. As mentioned above, S. haitangvora is widely distributed in the Korean Peninsula to China and Japan (Hokkaido), but so far there is no co-distribute with S. nashivora. This reinforces the possibility that S. nashivora is a vicarious species in Honshu (Tango Peninsula) derived from S. haitangvora by geographical isolation. The second possibility is that S. nashivora and S. haitangvora are not sibling species (despite overlapping host plants and similar external morphology), but have different distributions as a result of reproductive interference (Ribeiro and Spielman 1986;Kuno 1992). Although S. nashivora is closely related to S. haitangvora, a closely related species, S. unocingulata Bartel, whose host plants are Elaeagnaceae, also occurs in Japan, China, and the Korean Peninsula. Although S. unocingulata occurs sympatrically in the habitats of S. nashivora and S. haitangvora, no S. unocingulata males were attracted in this study, Yang et al (2009), or monitoring conducted in various areas of Japan using pheromone lures containing these two components (Ohno et al. in prep.). This means that the sex pheromone of S. unocingulata may be different from the sex attractant of S. nashivora and the sex pheromone of S. haitangvora. If S. nashivora or S. haitangvora were sympatrically speciated from S. unocingulata by using different components of the host plant or sex pheromone, reproductive interference could explain why S. nashivora and S. haitangvora are not sympatrically distributed. In any case, a nationwide monitoring study using pheromone traps with Z3,Z13-18:OAc and E2,Z13-18:OAc as attractants would allow us to know the detailed distribution of S. nashivora and S. haitangvora simultaneously. This will reveal which of the two hypotheses for the relationship between the two species is more plausible.
A pheromone lure containing a mixture of Z3,Z13-18:OAc and E2,Z13-18:OAc would be an effective tool for monitoring S. nashivora. By attempting to search for males in the eld using this pheromone lure, it will be possible to understand the distribution of this species in Japan and neighboring countries, its microhabitat, and the seasonal occurrence of this species. b Males/trap. Values not zero followed by a different letter are signi cantly different by the Tukey's test (P < 0.05) after GLM (family = Poisson, link = log) performed with R version 4.0.3.