Female Attraction to Click Beetle Sex Pheromone: Development of Bisexual Lures - A Summary

1. Female attraction to click beetle (Coleoptera: Elateridae) sex pheromone: development of bisexual lures – a summary Miklós Tóth1 and János Molnár2 1Plant Protection Institute, Ctr. Agric. Res. Hung. Acad. Sci., Budapest, Hungary 2freelance, Budapest, Hungary This presentation is dedicated to the memory of the late V.G. Yatsynin, who led a determining role in click beetle pheromone research Krasnodar, 2018

2. Females responding to the pheromone 2 Click beetle pheromone traps capture large numbers of males. In some species, however, users reported on the presence of females in the catch! YATLOR funnel trap The phenomenon most frequently was reported in: A. sordidus and A. brevis – Lorenzo Furlan, Italy A. ustulatus – István Szarukán, Hungary

3. Females responding to the pheromone 3 On closer scrutiny, beetle catches in pheromone baited traps indeed contained a sizeable proportion of female beetles in these species! A. brevis A. sordidus A. ustulatus pheromone components: (E,E)-farnesyl butyrate + geranyl butyrate pheromone component: geranyl hexanoate pheromone component: (E,E)-farnesyl acetate Berton-Greggio, Italy, 2001 Eraclea, Italy, 2000 www.elateridae.com Veneto, Italy, 2001 Total caught: 114 beetles Total caught: 367 beetles Total caught: 1173 beetles

4. Females responding to the pheromone 4 3 2 1 0 Female catches in pheromone baited traps were significantly higher than in unbaited traps. A. brevis A. sordidus A. ustulatus pheromone components: (E,E)-farnesyl butyrate + geranyl butyrate pheromone component: geranyl hexanoate pheromone component: (E,E)-farnesyl acetate Berton-Greggio, Italy, 2001 Debrecen, Hungary, 2003 www.elateridae.com Eraclea, Italy, 2000 Total caught: 42 beetles Total caught: 116 beetles Total caught: 194 beetles P< 0.0001 P= 0.0001 P= 0.0011 Mean catches (+SE) pheromone bait

5. Females responding to the pheromone 5 In EAG screenings of synthetic pheromone compounds of click beetles, female antennae showed similar response spectra as male antennae, responding best to the respective pheromone component(s). % pheromone 800 700 600 500 standard control 400 300 200 Agriotes sordidus 100 0 mean normalized responses (+SE) geranyl isovalerate geranyl butyrate (E,E)-farnesyl butyrate geranyl hexanoate (E,E)-farnesyl acetate geranyl octanoate (E,E)-farnesol geraniol air 0 50 100 150 200 250 300 350

6. Females responding to the pheromone 6 This suggested that females were capable of detecting their respective pheromone component(s). % pheromone 400 pheromone 350 300 250 standard control 200 150 Agriotes brevis 100 50 mean normalized responses (+SE) 0 geranyl isovalerate geranyl butyrate (E,E)-farnesyl butyrate geranyl hexanoate (E,E)-farnesyl acetate geranyl octanoate (E,E)-farnesol geraniol air 0 50 100 150 200 250 300 350

7. Females responding to the pheromone 7 1.5 1 0.5 25 20 15 10 5 0 Female catches in pheromone baited traps showed dose dependence, similar to catches of male beetles. A. sordidus A. ustulatus Berton-Greggio, Italy, 2001 Cessalto, Italy, 2003 2.5 (total caught: 109beetles) (total caught: 91beetles) 2 1.5 mean / trap (+SE) 1 0.5 a a b c c ab bc c 0 0 10 30 100 mg 0 10 30 100 mg a b bc c a b bc c 2 4 mean / trap (+SE) 6 8 10 (total caught: 709beetles) (total caught: 1708beetles) 12 14

8. Females responding to the pheromone 8 1.5 1 0.5 25 20 15 10 5 0 The demonstrated female attraction to the resp. pheromone brings up the possibility of developing a trap catching females also. A. sordidus A. ustulatus Berton-Greggio, Italy, 2001 Cessalto, Italy, 2003 2.5 (total caught: 109beetles) (total caught: 91beetles) 2 mean / trap (+SE) 1.5 1 0.5 a a b c c ab bc c 0 0 10 30 100 mg 0 10 30 100 mg a b bc c a b bc c 2 4 mean / trap (+SE) 6 8 10 (total caught: 709beetles) (total caught: 1708beetles) 12 14

9. Efficient click beetle traps for both sexes? 9 First development efforts started on: A. ustulatus Since A. ustulatus adults can frequently be seen visiting flowers for feeding, we supposed that the localization of flowers is assisted by chemical stimuli of floral scents. Floral compounds constituting such scents, could be exploited to develop a female-targeted lure.

10. Regular A. ustulatus catches in traps baited with synthetic floral compounds – in tests aimed at other insects – appeared to support this hypothesis 10

11. After having screened several floral combinations we concluded as preliminary result that the addition of cinnamic compounds to (E)-anethole increased female catches 11

12. When cinnamic compounds were added singly, this also incresed catches. Numerically highest catches were observed with the blend of (E)-anethole plus (E)-cinnamaldehyde. 12 (E)-anethole (E)-cinnamic acetate (E)-cinnamaldehyde (E)-cinnamyl alcohol

13. Search for most efficient blend 10 10 5 5 0 0 In further tests the blend of (E)-cinnamaldehyde plus (E)-anethole was used as the basic female-targeted floral bait for A. ustulatus 13 Debrecen, Hungary, 2006 c (total caught: 935beetles) (total caught: 839beetles) c Sex ratio in catch of the 2 component floral lure (total caught1232beetles) mean / trap (+SE) b b a a (E)-anethole (E)-cinnam- aldehyde Composition of baits: = present = absent

14. When 1) testing the floral bait in lower and higher dose and 2) studying the effect of the addition of other compounds with high EAG activity, in MALES, the activity of the pheromone was dominant. 14 Veneto, 2007 Debrecen, 2007

15. FEMALES: the addition of compounds with high EAG activity did not influence catch of the basic floral bait 15

16. FEMALES: triplicating the dose had no significant effect 16

17. FEMALES: the effect of white colour (a known attractive visual cue) was very weak as compared to the effect of chemical stimuli 17

18. FEMALES: in presence of the pheromone the floral lure catches more females???? 18

19. FEMALES: YES, confirmed the next year, in presence of the pheromone the floral lure catches more females!!! 19

20. FEMALES: the effect of white colour was still very moderate 20

21. FEMALES: the effect of white colour was still very moderate 21

22. FEMALES: the effect of white colour was still very moderate 22

23. In MALES, again the effect of the pheromone was dominant 23

24. Most beetle specimens were caught in traps with both floral AND pheromonal baits 24

25. That the presence of the pheromone increases the effect of floral bait on females is unusual with sex pheromones 25

26. However, such a phenomenon is frequently reported with aggregation pheromones 26

27. This suggests that the pheromone of A. ustulatus is not a “classical” sex pheromone 27

28. All in all, the combination of the floral and pheromonal lure can be used by farmers as a powerful bisexual lure for A. ustulatus 28

29. Acknowledgements This presentation is based on published results from the following papers 29: Tóth, M., Furlan, L., Szarukán, I., Vuts, J. 2011. Development of a female-targeted attractant for the click beetle Agriotes ustulatus Schwarz. Acta Phytopath. Entomol Hung. 46:235-245. Vuts, J., Furlan, L., Bálintné-Csonka, É., Woodkock, C.M., Caulfield, J.C., Mayon, P., Pickett, J.A., Birkett, M.A., Tóth, M. 2014. Development of a female attractant for the click beetle pest Agriotes brevis. Pest Manag. Sci. 70:610-614. Tóth, M., Furlan, L., Vuts, J., Szarukán, I., Ujváry, I., Yatsynin, VG., Tolasch, T., Francke, W. 2015. Geranyl hexanoate, the female produced pheromone of Agriotes sordidus Illiger (Coleoptera: Elateridae) and its activity on both sexes. Chemoecology 25:1-10. Vuts, J., Furlan, L., Tóth, M. 2018. Female responses to synthetic pheromone and plant compounds in Agriotes brevis Candeze (Coleoptera: Elateridae). J. Insect Behav. 31:106-117.

30. Acknowledgements Scientists cooperating in unpublished results shown 30: Bálintné Csonka, É. (Hungary) Francke, W. (Germany) Furlan, L. (Italy) Subchev, M. (Bulgaria) Szarukán, I. (Hungary) Tolasch, T. (Germany) Toshova, T. (Bulgaria) Vuts, J. (Hungary) Yatsynin, V.G. (Russia)