Environmental Entomology, 48(1), 2019, 189–192 doi: 10.1093/ee/nvy164 Advance Access Publication Date: 4 December 2018 Chemical Ecology Research

Evidence of Aggregation–Sex Pheromone Use by Longhorned (Coleoptera: Cerambycidae) Species

Native to Africa Downloaded from https://academic.oup.com/ee/article-abstract/48/1/189/5229558 by Technical Services Serials user on 13 February 2019

Bridget Bobadoye,1 Baldwyn Torto,1 Ayuka Fombong,1 Yunfan Zou,2 Karl Adlbauer,3 Lawrence M. Hanks,4 and Jocelyn G. Millar2,5,6

1Behavioural and Chemical Ecology Department, International Centre of Physiology and Ecology, Nairobi, Kenya, 2Department of Entomology, University of California, Riverside, CA 92521, USA, 3Kasernstraße 84, Graz, Austria, 4Department of Entomology, University of Illinois at Urbana–Champaign, Urbana, IL 61801, USA, 5Department of Chemistry, University of California, Riverside, CA 92521, USA, and 6Corresponding author, e-mail: [email protected]

Subject Editor: Dong H. Cha

Received 30 July 2018; Editorial decision 15 October 2018

Abstract During field bioassays in Kenya of 10 chemicals that are common pheromone components of cerambycid beetles, six species in the subfamily were significantly attracted to 3-hydroxyhexan-2-one, and one species in the subfamily was significantly attracted to 2-(undecyloxy)ethanol (known as monochamol). These results further demonstrate that the former compound is highly conserved as a cerambycid pheromone because it has now been identified or implicated in the pheromones of numerous cerambycine species native to all six habitable continents. Similarly, monochamol has been identified or implicated as a pheromone component for species in the subfamily Lamiinae native to Asia, Europe, North America, and now Africa. The eight other compounds tested, including (E)-6,10-dimethyl-5,9-undecadien-2-ol (fuscumol), the corresponding (E)-6,10-dimethyl-5,9-undecadien- 2-yl acetate (fuscumol acetate), syn- and anti-(2,3)-hexanediols and (2,3)-octanediols, 3-hydroxyoctan-2-one, and 3-hydroxydecan-2-one, did not attract significant numbers of any species.

Key words: Cerambycidae, semiochemical, 3-hydroxyhexan-2-one, 2-(undecyloxy)ethanol, management

Cerambycid beetles comprise a large insect family, with ~35,000 have also shown that many cerambycid pheromone components are described species worldwide (Švácha and Lawrence 2014). The highly conserved within related taxonomic groups, to the extent that larvae usually develop in the tissues of woody plants, with host species of the same subfamily but from different continents, which preferences ranging from living and apparently healthy trees, to have been separated for many million years, may still share the same stressed and dying trees, through to decomposing logs and branches pheromone components. For example, (R)-3-hydroxyhexan-2-one (Hanks 1999). The beetles are crucial members of forest commu- has been shown to be a pheromone component or likely pheromone nities, performing valuable ecosystem services by culling diseased component for cerambycid species in Asia, Europe, Australia, and and weakened trees, and by initiating the process of degradation North and South America (summarized in Hanks and Millar 2016). and recycling of dead woody tissues. However, the larvae also can However, to our knowledge, pheromones have not yet been reported cause significant damage to living trees and cut logs, and some spe- from any African cerambycid species. cies, such as the Asian longhorned borer, Anoplophora glabripennis Africa has a rich cerambycid fauna (Duffy 1957, Gardner 1957), (Motschulsky), or Phoracantha species that attack eucalyptus, con- but to date, most research on African cerambycids has focused on stitute important invasive species that can cause massive damage in their , occurrence, and distribution, especially in North novel ecosystems into which they have been introduced (Eyre and Africa (Algeria, Libya, Morocco, Tunisia: Maican and Serafim 2015; Haack 2017). Consequently, in recent years, there has been increased Egypt: Ismail et al. 2009), West Africa (Ghana, Sierra Leone, Senegal: interest in identifying semiochemical lures that could be exploited Wagner et al. 1991, Schabel 2006), Southern Africa (Namibia, for detection and monitoring of invasive cerambycid species, result- Swaziland, Zimbabwe, and South Africa: Rice 2008), and Kenya ing in the identification of pheromones and related semiochemical (Sakalian and Georgiev 2011). A few African species have been doc- attractants for nearly 300 species to date, including several known umented to be serious pests of timber, coffee, and other economically invasive species (review, Hanks and Millar 2016). Overall, these data important woody plants (e.g., Schabel 2006, Ismail et al. 2009).

© The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: [email protected]. 189 190 Environmental Entomology, 2019, Vol. 48, No. 1

The aim of the work described here was to conduct field Table 1. Cerambycid species trapped during field bioassays trials in Kenya with several chemicals that are known phero- in Kenya that tested for attraction to known pheromones mone components of cerambycid beetles native to other regions Taxonomy Karura Taita Sum of the world, as a possible first step toward providing phero- mone-based methods of detecting and managing African species. 2014 2015 2014 2015 Our specific objectives were 1) to determine whether the demon- strated high degree of conservation of pheromone structures Cerambycinae Callichromatini among related cerambycid species on other continents was also Compsomera elegantissima White 1 1 true of Africa and 2) to develop leads as to likely pheromones of Clytini particular species, so that they could be subsequently targeted Calanthemis conradti Kolbe 5 1 6 Downloaded from https://academic.oup.com/ee/article-abstract/48/1/189/5229558 by Technical Services Serials user on 13 February 2019 for full identification of their pheromones. To our knowledge, Calanthemis hauseri Jordan 21 21 the results from this study constitute the first report of phero- Calanthemis myops Thompson 1 3 4 mone-based attractants for any cerambycid species native to the Calanthemis saltator Kolbe 4 14 12 30 continent of Africa. Calanthemis sp. 1 5 5 Xylotrechus ansorgei Latreille 2 7 9 Xylotrechus sp. 10 1 4 5 Materials and Methods Xylotrechus sp. 12 3 3 Obriini Sources of Chemicals Oemida gahani Distant 7 5 12 Racemic 3-hydroxyhexan-2-one, racemic (E)-6,10-dimethyl- Psathyrus sp. 1 8 8 5,9-undecadien-2-ol (henceforth “fuscumol”), the correspond- Tragocephalini ing (E)-6,10-dimethyl-5,9-undecadien-2-yl acetate (“fuscumol Chariesthes sp. 1 1 1 acetate”), and 2-(undecyloxy)ethanol (monochamol) were pur- Unknown chased from Bedoukian Research, Inc. (Danbury, CT). Racemic Cerambycid sp. 1 1 1 syn- and anti-(2,3)-hexanediols were synthesized by osmium Cerambycine sp. 1 3 3 Cerambycine sp. 2 1 12 13 tetroxide catalyzed oxidation of (E)- and (Z)-hexenes, respec- Cerambycine sp. 3 1 1 tively, as described in Lacey et al. (2004, 2007), and the Cerambycine sp. 4 1 1 same method was used to synthesize the analogous syn- and Cerambycine sp. 5 1 1 1 3 anti-(2,3)-octanediols from (E)- and (Z)-octenes, respectively. Lamiinae Racemic 3-hydroxyoctan-2-one and 3-hydroxydecan-2-one were Acanthocinini synthesized from 1-octyn-3-ol and 1-decyn-3-ol as described in Monoxenus kenyensis Breuning 3 1 4 Allison et al. (2012). Ceroplesini Crossotus albicollis Guérin-Méneville 2 1 3 Crossotus plumicornis Audinet-Serville 1 1 General Methods of Trapping Frea marmorata Gerstaecker 5 2 7 Field experiments were conducted at two forest study sites in Kenya, Frea sp. 1 3 3 namely Karura and Taita Hills. Karura is an urban forest located Desmiphorini within the city of Nairobi (1.23442°S, 36.8347°E; 1,041 ha, 1,600 Diadelia sp. 1 5 5 m elevation) and is predominantly a second-growth and old-growth Sophronisca grisea Aurivillius 1 2 3 mix of primarily indigenous and a few exotic tree species as listed in Sophronisca sp. 1 4 4 Nyambane et al. (2016). Taita Hills forest is a remnant of the Eastern Eunidiini Eunidia sp. 1 1 1 2 Arc Mountains and is considered a biodiversity hotspot (Adriaensen Eunidia sp. 2 2 1 3 et al. 2007). Our study site in Taita Hills, Ngangao (3.36528°S, Eunidia sp. 3 1 1 38.3425°E, 120 ha, 1,700 m elevation) is one of its largest surviving Eunidia sp. 4 1 1 hotspots and contains a large diversity of plant species as described Monochamini in Wilder et al. (1998) and Omoro et al. (2010). Monochamus spectabilis (Perroud) 7 16 4 27 Black cross-vane panel traps (AlphaScents, Portland, OR) (Noserocera) sp. 1 6 6 coated with the fluoropolymer dispersion Fluon PTFE (AGC Parmenini Chemicals Americas, Inc., Exton, PA) were used to trap beetles. Kenyavelleda jirouxi Téocchi 7 1 8 Traps were hung ~1.2 m above the ground, close to standing Tetraulaxini trees, from inverted L-shaped frames constructed of polyvinyl- Tetraulax minor Breuning 1 1 Tragocephalini chloride irrigation pipe. Trap basins were partially filled with Pseudochariesthes sp. 1 1 1 water containing a detergent (10%) to preserve captured beetles. 68 69 58 12 207 Pheromone lures consisted of clear polyethylene sachets (press- seal bags, Bagette model 14770, 5.1 × 7.6 cm, 0.05 mm thick, Cousin Corp., Largo, FL) loaded with 50 mg of a test compound deployed from 7 November 2014 to 3 January 2015, and 10 in 1 ml of isopropanol. Control lures contained 1 ml of isopro- April to 26 June 2015, representing the major dry and wet sea- panol. Lures were replaced every 2–3 wk. Traps were deployed sons, respectively. in random order across slopes within each forest with a 10-m Beetles were tentatively identified to family level by the entomo- spacing, and their positions were rotated weekly to minimize logical unit of the National Museums of Kenya (NMK) and later positional bias. The traps were monitored twice weekly (i.e., on confirmed to species level by K.A., followingAdlbauer (2018). days 4 and 7). Captured were transferred into containers Voucher specimens have been deposited in the NMK and the ICIPE with 50% ethanol for short-term preservation. Bioassays were Biosystematics Reference Collection. Environmental Entomology, 2019, Vol. 48, No. 1 191

Table 2. Mean (± SE) number of cerambycid beetles captured per treatment and replicate during field bioassays of known cerambycid pheromones in Kenya

Treatment Calanthemis conradti Calanthemis Calanthemis Calenthemis Psathyrus Xylotrechus Monochamus hauseri saltator sp. 1 sp. 1 sp. 10 spectabilis

3-Hydroxyhexan-2-one 1.2 ± 0.2 2.7 ± 0.3 2.0 ± 0.5 2.5 ± 1.5 1.2 ± 0.5 1.7 ± 0.7 0.08 ± 0.08 3-Hydroxyoctan-2-one 0 0.17 ± 0.1 0.08 ± 0.08 0 0 0 0 3-Hydroxydecan-2-one 0 0 0.17 ± 0.1 0 0 0 0.25 ± 0.2 syn-(2,3)-Hexanediol 0 0.33 ± 0.3 0 0 0 0 0 syn-(2,3)-Octanediol 0 0 0 0 0 0 0.17 ± 0.1 anti-(2,3)-Hexanediol 0 0 0 0 0.4 ± 0.2 0 0.08 ± 0.08 Downloaded from https://academic.oup.com/ee/article-abstract/48/1/189/5229558 by Technical Services Serials user on 13 February 2019 anti-(2,3)-Octanediol 0 0.33 ± 0.3 0 0 0 0 0.08 ± 0.08 Fuscumol 0 0 0 0 0 0 0 Fuscumol acetate 0 0 0 0 0 0 0 Monochamol 0 0 0.08 ± 0.08 0 0 0 1.6 ± 0.5 Solvent control 0 0 0.08 ± 0.08 0 0 0 0 Blank 0 0 0 0 0 0 0

Included are the seven species that had statistically significant overall treatment effects (see text). Bold text indicates treatment means that were significantly greater than means for all remaining treatments for that species (REGWQ test, P < 0.05). Results of statistical tests are as follows (subscripted letters with Q represent degrees of freedom for the treatment effect and total number of replicates included): the cerambycines Calanthemis conradti (Friedman’s Q11,60 = 58.9,

P < 0.0001), Calanthemis hauseri (Q11,72 = 49.6, P < 0.0001), Calanthemis saltator (Q11,144 = 66.1, P < 0.0001), Calenthemis sp. 1 (Q11,24 = 23.0, P = 0.018),

Psathyrus sp. 1 (Q11,60 = 37.4, P < 0.0001), Xylotrechus sp. 10 (Q11,36 = 35.0, P = 0.0003), and the lamiine Monochamus spectabilis (Q11,144 = 70.7, P < 0.0001).

Statistical Analysis Antarctica. Similarly, monochamol now has been shown to be an For each species that was represented by at least five specimens, aggregation-sex pheromone (sensu Cardé 2014) or attractant (and differences between treatment means, blocked by site and col- hence likely pheromone) for at least 15 Monochamus species native lection date, were tested with the nonparametric Friedman’s test to Asia, North America, Europe, and now Africa, as well as spe- (PROC FREQ, option CMH; SAS Institute 2011) because data vio- cies from four different genera and two other tribes (Wickham et al. lated assumptions of ANOVA (Sokal and Rohlf 1995). Replicates 2014). Conversely, the lack of significant attraction to any of the that contained no beetles of the species of interest in any trap, for other compounds tested, all of which have been shown to be pher- example because of inclement weather, were dropped from analyses. omone components for at least several species on other continents, For all analyses with significant overall treatment effects, pairs of suggests that African species may share only a limited number of treatment means were compared using the nonparametric Ryan– components with their congeners from different continents. Einot–Gabriel–Welsch Q (REGWQ) multiple comparison test (SAS The attraction of both endemic African Monochamus species and Institute 2011). potentially invasive Monochamus species from other continents to monochamol could be very important. Specifically, the pinewood nematode Bursaphelenchus xylophilus (Nematoda: Aphelenchida; Results and Discussion Steiner and Buhrer 1934) is vectored by Monochamus spp. (Sousa In total, 207 cerambycid beetles were trapped during the experi- et al. 2015), and if it is introduced into Africa, it could cause major ments (Table 1), including 18 species in at least four tribes of the damage in forestry plantations planted with introduced conifer spe- Cerambycinae and 17 species in four tribes of the Lamiinae. It was cies (Evans and Turnbull 2004). not possible to identify all specimens to the species level, and some The mutual attraction of the six cerambycine species to may represent undescribed species. 3-hydroxyhexan-2-one suggests that this compound is the dom- Significant numbers of several species were attracted to 2 of the 10 inant if not sole pheromone component for these species. Among compounds tested (Table 2). Thus, six species, including Calanthemis other sympatric cerambycines, species with similar or even iden- conradti Kolbe, Calanthemis hauseri Jordan, Calanthemis saltator tical pheromones may maintain reproductive isolation by differ- Kolbe, Calanthemis sp. 1, Xylotrechus sp. 10 (all Cerambycinae, ences in seasonal and/or diel flight phenology (e.g., Mitchell et al. tribe Clytini), and Psathyrus sp. 1 (Cerambycinae, tribe Oemini) 2013, 2015). The six species in the present report appeared to were significantly attracted to traps baited with the very com- overlap broadly in seasonal activity period (data not presented), mon cerambycine pheromone component 3-hydroxyhexan-2-one. but it is possible and even likely that they differed in the time of In addition, Monochamus spectabilis (Perroud) (Lamiinae, tribe day that the adults were active. Monochamini) was significantly attracted to monochamol. Small In summary, these trials have suggested that, as in other con- numbers of these and other species were caught in traps baited with tinents, use of attractant pheromones may be common among other components, but none of these means were significantly differ- the African cerambycid fauna. The demonstrated attraction of ent from the means for solvent control or unbaited traps, which with seven species to known pheromone components has also pro- one exception, caught no beetles at all. vided strong leads for follow-up work to formally identify the Overall, these results suggest that at least two common cer- pheromones of those species. However, the fact that 8 out of ambycid pheromone components, shared by a variety of species the 10 compounds tested did not attract significant numbers of worldwide, are also conserved within African species. Specifically, any species also suggests that many African species may have 3-hydroxyhexan-2-one has now been found or implicated as a evolved as yet unknown pheromones. Thus, the African ceramby- pheromone component for more than 70 species in 12 tribes in cid fauna may represent a rich and completely untapped source the subfamily Cerambycinae, occurring on all continents except of novel pheromone structures. 192 Environmental Entomology, 2019, Vol. 48, No. 1

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