Development of a Synthetic Plant Volatile-Based Attracticide for Female Noctuid Moths

Australian Journal of Entomology (2010) 49, 10–20

Development of a synthetic plant volatile-based attracticide for female noctuid moths. I. Potential sources of volatiles attractive to Helicoverpa

armigera (Hübner) (Lepidoptera: Noctuidae)aen_733 10..20

Alice P Del Socorro,1,2* Peter C Gregg,1,2 Daniel Alter2 and Chris J Moore3

1Cotton Catchment Communities Cooperative Research Centre, Narrabri, NSW 2390, Australia. 2School of Environmental and Rural Science, University of New England, Armidale, NSW 2351, Australia. 3Animal Research Institute, Queensland Department of Primary Industries and Fisheries, Yeerongpilly, Qld 4105, Australia.

Abstract This paper is the first of a series which will describe the development of a synthetic plant volatile-based attracticide for noctuid moths. It discusses potential sources of volatiles attractive to the cotton bollworm, Helicoverpa armigera (Hübner), and an approach to the combination of these volatiles in synthetic blends. We screened a number of known host and non-host (for larval development) plants for attractiveness to unmated male and female moths of this species, using a two-choice olfactometer system. Out of 38 plants tested, 33 were significantly attractive to both sexes. There was a strong correlation between attractiveness of plants to males and females. The Australian natives, Angophora floribunda and several Eucalyptus species were the most attractive plants. These plants have not been recorded either as larval or oviposition hosts of Helicoverpa spp., suggesting that attraction in the olfactometer might have been as nectar foraging rather than as oviposition sources. To identify potential compounds that might be useful in developing moth attractants, especially for females, collections of volatiles were made from plants that were attractive to moths in the olfactometer. Green leaf volatiles, floral volatiles, aromatic compounds, monoterpenes and sesquiterpenes were found. We propose an approach to developing synthetic attractants, here termed ‘super-blending’, in which compounds from all these classes, which are in common between attractive plants, might be combined in blends which do not mimic any particular attractive plant. Key words kairomone, moth attractant, olfactometer, plant volatile.

INTRODUCTION 2002; Meagher 2002; Meagher & Landolt 2008). In this series of papers, we discuss the theoretical underpinning of this Plants emit volatile compounds that mediate insect–plant development as well as the demonstrations of field efficacy, interactions. These compounds can have either attractant or non-target impacts and other data required for registration of repellent effects which can potentially be exploited to aid pest products such as Magnet®. This paper describes the identifi- management. We have recently registered an attract-and-kill cation of potential sources of attractive volatiles, and a novel product (Magnet®) for use in integrated pest management approach to their combination in attractive blends. of the important noctuid pest species Helicoverpa armigera The cotton bollworm, H. armigera, is an important eco- (Hübner) and H. punctigera (Wallengren) in Australian cotton nomic pest of cotton and many other summer crops in Austra- and other crops (Australian Pesticides & Veterinary Medicines lia. The larvae are highly polyphagous, usually feeding on Authority 2009). To our knowledge, this is the first synthetic the fruiting bodies, and the moths are attracted to many plants plant volatile-based attracticide which has been commercial- either as food sources or oviposition sites (Zalucki et al. 1986; ised for noctuid pests of agriculture, despite the identification Firempong & Zalucki 1990). In non-transgenic cotton, control in the laboratory of several attractive volatile compounds in of this pest has usually been through insecticides targeted at various host plants (Landolt 1989; Haynes et al. 1991; Heath the larvae, which has led to H. armigera developing resistance et al. 1992; Hartlieb & Rembold 1996; Bruce & Cork 2001; to a number of insecticides (Forrester et al. 1993). To help curb Rajapakse et al. 2006), and experimental demonstrations of resistance as well as to reduce environmental problems with the attractiveness of both crude plant extracts and synthetic insecticides, the cotton industry has adopted an integrated mixtures in the field (Zhu et al. 1993; Shaver et al. 1998; pest management approach including a resistance manage- Landolt & Alfaro 2001; Landolt et al. 2001; Landolt & Higbee ment scheme for H. armigera (Farrell 2008). More recently, the extensive use of transgenic cotton expressing Bt toxins in Australia (Fitt & Cotter 2005) has required the widespread *[email protected] adoption of resistance management plans (Andow et al. 2008). © 2010 The Authors Journal compilation © 2010 Australian Entomological Society doi:10.1111/j.1440-6055.2009.00733.x Plant volatiles as moth attractants 11

A range of approaches, including chemical, cultural and feeding on nectar from these plants. For the purpose of devel- behavioural manipulation, is incorporated in these plans. oping attract-and-kill formulations, the ecological basis of the However, there remains a need for new tools, including attrac- attraction is of less consequence than the level of attraction, ticides targeted at female moths (Gregg et al. 1998). In non- and whether it varies according to the sex or mated status transgenic cotton, removing female moths from the population of the moths. could reduce oviposition, thus reducing the need for insec- In this paper, we report the attractiveness of various host ticides and allowing other components of integrated pest and non-host plants to unmated H. armigera moths in the management to work more efficiently. For transgenic cotton, laboratory using an olfactometer. We document the volatile selective removal of potentially resistant moths using attracti- compounds emitted by these plants which might be used in cides could reduce the frequency of resistance alleles. Alter- attract-and-kill formulations, and describe an approach to natively attraction of female moths to refuge crops where they developing these formulations which does not involve mim- might subsequently oviposit could enhance the production of icking the volatile emissions of particular attractive plants. moths which have not been exposed to Bt toxin, an important objective of the resistance management strategy (Farrell 2008). MATERIALS AND METHODS The attractiveness of plants and plant odours to Helicoverpa spp. and other noctuid moths is well documented. Extracts Experimental insects from pigeon pea, Cajanus cajan, were shown to be attractive Laboratory-reared H. armigera moths from an insect culture to H. armigera moths (Rembold & Tober 1987; Hartlieb & maintained in an insectary were used in the bioassays. The Rembold 1996). Rembold et al. (1991) demonstrated the culture originated from larvae collected from chickpeas in the attractiveness of a synthetic chickpea (Cicer arietinum) Darling Downs region of southern Queensland, and had been kairomone to H. armigera moths in laboratory and field maintained in the laboratory for at least 10 generations. Larvae experiments. Floral compounds identified in the African mari- were reared individually in 35 mL plastic containers and gold, Tagetes erecta, and their synthetic equivalents were provided with a small block of soybean-based artificial diet found to be attractive to H. armigera females (Bruce & Cork (Teakle 1991). Rearing conditions were 25–26°C, approxi- 2001). In the USA, volatiles emitted by the night-blooming mately 50% humidity and 16L : 8D photoperiod, with the dark Gaura spp. have been shown to be highly attractive to Heli- period between 09:30 and 17:30 h Australian Eastern Standard coverpa zea (Boddie) and other noctuid moths (Beerwinkle Time (AEST). Day lighting was provided by six 40 W white et al. 1996; Shaver et al. 1998). The attraction of the cabbage fluorescent tubes, and four 150 W incandescent bulbs. The looper, Trichoplusia ni (Hübner), to various host plants and latter were connected to a time-controlled dimming system host plant odours and to the floral compounds from night- providing 30 min transitions between the light regimes, to blooming jessamine, Cestrum nocturnum, has also been simulate dusk and dawn (H. armigera moths fly throughout reported (Landolt 1989; Heath et al. 1992). Zhu et al. (1993) the night, but simulating the natural transitions between day demonstrated the attractiveness of various flowering plants to and night could be important in initiating flight). Pupae were the cutworm, Agrotis ipsilon (Hufnagel), the armyworm, Pseu- sexed and upon emergence, moths were individually held in daletia unipuncta (Haworth) and the corn earworm, H. zea. 150 mL plastic containers and fed distilled water only until Additional work has documented the existence of volatile they were used in the olfactometer between 1 and 4 days of and non-volatile compounds which enhance oviposition. age. Unmated moths were used because targeting females Breeden et al. (1996) recorded oviposition stimulants for H. which had not laid eggs would be expected to produce the zea from the isolated acids and alkane wax fractions of various greatest reduction in oviposition. Moths were used once only Lycopersicon (Solanaceae) species. Jallow et al. (1999) dem- in the bioassays. onstrated that methanol, ethanol, acetone and pentane extracts from leaves, squares and flowers of different cotton genotypes Bioassay system influenced oviposition behaviour in H. armigera females in the laboratory. Female moths laid more eggs on pentane extracts A two-choice olfactometer based on the design of Beerwinkle of cotton flowers than extracts of leaves from pre-flowering, et al. (1996) was used in laboratory bioassays (Fig. 1). It con- early flowering and peak-flowering plants. sisted of a perspex box measuring 60 ¥ 25 ¥ 25 cm which had Plants which are not oviposition or larval hosts might also two choice chambers on the floor in the upwind end. Entrance provide a useful source of plant volatiles for use in attract- by moths to each chamber was through a metal gauze funnel and-kill formulations. Wilted (or fresh) leaves of the Chinese (6 cm diameter) leading to a holding cylinder (10 cm diameter wingnut tree Pterocarya stenoptera do not support larval and 17 cm high). Beneath this, and separated from it by a growth, but wilted leaves are attractive to H. armigera in China metal gauze floor, was a sub-chamber which held either the (Xiao et al. 2002), as is also the case for wilted leaves of black test plant or nothing (as control). Air was supplied from a poplar (Wang et al. 2003). Gregg (1993) demonstrated the compressor located outside the building, and passed sequen- widespread presence of pollen from non-larval host plants in tially through two large PVC cylinders, one containing acti- the genus Eucalyptus and family Brassicaceae on the pro- vated charcoal to remove extraneous volatiles, and the other boscis of H. armigera moths, which indicates that moths were containing distilled water to humidify the air (approximately © 2010 The Authors Journal compilation © 2010 Australian Entomological Society 12 A P Del Socorro et al.

09:30 h. After 8 h the moths which had entered each chamber, and those which remained in the box, were counted. Moths which died during the 8 h run (usually <2%) were excluded from the counts. We used two measures to determine attractiveness in the olfactometer: % positive response (100*T/N), and % total response (100*(T + C)/N), where T = number of moths entering the test chamber C = number of moths entering control (blank) chamber N = total number of moths in the olfactometer We considered % positive response to be our primary criterion of attractiveness. The extent to which moth activity, especially upwind movement, is stimulated by the presence of volatiles in the body of the olfactometer might influence % total response. However, % positive response is the best indi- cator of the choice of moths to enter the test chamber. We preferred the use of two separate measures of attraction over the common approach of using ratios such as the coeffi- cient of discrimination, CD = (T - C)/(T + C), because it is less susceptible to the effects of low numbers entering either chamber, as occurred in the blank olfactometer and with some plants. Also the use of these two % response parameters rec- ognises that moths which remain downwind, in the body of the olfactometer, may not be neutral – they may in fact be responding negatively to volatiles from the test plants. The analysis of the parameters, implemented by GLM models, is discussed by Hern and Dorn (2001). Each trial for a given test plant was replicated three times for each sex. The sub-chamber which held the plant material (left or right) was rotated between replicate trials to remove the effects of any positional bias which may have occurred in the olfactometer. A set of blank experiments (three replicates) was done in which both sub-chambers had no test plant material. Fig. 1. The two-choice olfactometer system used in laboratory One chamber was designated as ‘test’ and the other as bioassays. ‘control’, and the two were alternated between replicates. After each run, the olfactometer was dismantled and the component parts thoroughly cleaned using Pyroneg alkaline deter- 50%), and bring it to room temperature (25–26°C). It was then gent in hot water followed by rinsing in cool water to remove metered into each sub-chamber at the rate of 12 L/min and any volatiles originating from either plant or insect sources in extracted from the downwind end of the box to the outside the previous run. by a fan set to maintain a slight negative pressure (about -10 kPa), to avoid any volatile contamination in the room. Plant materials The bioassay room had the same temperature and photo- periodic conditions as the insect rearing room. During the A total of 38 host and non-host plants (for larvae) of Helicov- scotophase (09:30–17:30 h AEST), the bioassay room was lit erpa spp. were tested in the olfactometer. These plants were by two 40 W red fluorescent photographic safelights. Day grouped into crops, weeds, ornamentals, Australian natives lighting with simulated dusk and dawn similar to that in the and a Chinese tree (Table 1). Plants were collected from the insectary was provided for the olfactometers in the bioassay university glasshouses and farms, natural environments or room. household gardens around the Armidale region. The Chinese In most cases, 50 moths were tested as a group in the wingnut tree, P. stenoptera, originated from the Royal Botanic olfactometer for each run of each sex. They were placed in the Gardens at Mount Tomah, NSW. In most cases, plant materials olfactometer at 08:30 h AEST (i.e. beginning of the simulated were collected within 30–60 min before testing. Fresh bou- dusk period) and a barrier separating them from the choice quets (about 50 g, containing both flowers and leaves) of chambers was removed at the start of full scotophase at plants in small containers of water were held in the sub- © 2010 The Authors Journal compilation © 2010 Australian Entomological Society Plant volatiles as moth attractants 13

Table 1 List of plants tested for attractiveness to H. armigera moths in the olfactometer

Group Plant name Common name Family name Australian native plants Angophora floribunda‡ Rough barked apple Myrtaceae Eucalyptus viminalis† Manna gum Myrtaceae Eucalyptus caliginosa† Broad-leaved stringybark Myrtaceae Eucalyptus nova-anglica‡ New England peppermint Myrtaceae Eucalyptus melliodora† Yellow box Myrtaceae Acacia subulata Awl-leaf wattle Mimosaceae Acacia cambadgeii Gidgee Mimosaceae Acacia sp. (not identified) Mimosaceae Eremophila gilesii Charleville turkey bush Myoporaceae Eremophila sturtii Turpentine Myoporaceae Helipterum floribundum Large white sunray Asteraceae Ixiolaena brevicompta Plains plover daisy Asteraceae Nicotiana velutina Wild tobacco Solanaceae Crops Helianthus annuus† Sunflower Asteraceae Sorghum bicolor Sorghum Poaceae Zea mays† Corn Poaceae Lablab purpureus‡ Dolichos lablab cv. Koala Fabaceae Cajanus cajan† Pigeon pea Fabaceae Cicer arietinum† Chickpea Fabaceae Medicago sativa Lucerne Fabaceae Linum usitatissimum† Linseed Linaceae Gossypium hirsutum† Cotton Malvaceae Brassica napus‡ Canola Brassicaceae Malus domestica† Apple Rosaceae Weeds Sonchus oleraceus‡ Sow thistle Asteraceae Galinsoga parviflora† Yellow weed Asteraceae Bidens pilosa Cobbler’s peg Asteraceae Chicorium intybus† Chicory Asteraceae Echium plantagineum† Paterson’s curse Boraginaceae Hirschfeldia incana‡ Buchan weed Brassicaceae Araujia hortorum† Moth vine Asclepidiaceae Ornamentals Calendula officinalis† Marigold Asteraceae Gaura lindheimerii† Butterfly bush Onagraceae Westringia fruticosa Coast rosemary Lamiaceae Jasminum officinale† Jasmine Oleaceae Lonicera japonica Honeysuckle Caprifoliaceae Oenothera stricta Evening primrose Onagraceae Chinese tree (wilted leaves) Pterocarya stenoptera Chinese wingnut tree Juglandaceae

†Volatile collections were done from these plants using solid-phase micro extraction. ‡Volatile collections were done from these plants using Tenax collection. chambers below the choice chambers. Leaves of P. stenoptera used in the presence of these plants in the olfactometer. were wilted at room temperature for 3 days prior to use Volatiles were collected for 30–60 min. In earlier testing of the (including transit time from Mount Tomah), because this is the other seven plants, volatile collection was done by headspace method used in China, where fresh leaves proved unattractive methods using adsorption onto the commercial substrate to H. armigera moths (Xiao et al. 2002). Tenax, followed by thermal desorption in the GCMS (Tholl et al. 2006). To identify and quantify the volatiles, analysis was done by Identification of plant volatiles conventional GCMS techniques on a Hewlett Packard 6890 Collections of plant volatiles were done for 23 of the plant series GC and HP 5973 mass selective detector (Hewlett- species tested. For 16 plants, collection was done by means of Packard, Palo Alto, USA). The column used was a HP-5MS solid-phase micro extraction (SPME) in the test air stream (5% phenyl methyl siloxane, 30 m ¥ 0.25 mm i.d., 0.25 mm of the olfactometer, followed by thermal desorption from the film thickness; J & W Scientific, Folsom, USA) fused capillary SPME fibre in the gas chromatograph-mass spectrometer column. The carrier gas was ultrapure helium set at a flow rate (GCMS) (Tholl et al. 2006). Freshly cut flowering bouquets of 0.8 m/s. The column temperature was programmed to (also about 50 g of flowers and leaves) of the test plants were increase from 40°C (0.50 min hold) to 250°C at 20°C/min. held in the test sub-chamber for volatile collection. This was Temperatures of the splitless injector and the GCMS interface done to ensure that the conditions of the plant materials during were set at 280°C and 300°C respectively. Total run time was the collection process were similar to those when moths were 30 min. A mass spectrum was scanned from m/z 30–300 and © 2010 The Authors Journal compilation © 2010 Australian Entomological Society 14 A P Del Socorro et al. acquired data were collected and analysed on a Hewlett- (Male = 7.63 + 0.98 ¥ Female, P < 0.001, R2 = 0.87). A Packard workstation using HP Chem/Station software, with similar correlation (Male = 10.7 + 0.96 ¥ Female, P < 0.001, mass spectra from the NIST database and additional spectra R2 = 0.84) was found using the % total response criterion. In derived from authentic compounds. both cases, both the intercept and the slope were significant. Outliers from these regressions are indicated in Table 2 by Statistical analyses the presence of separate means for each sex. In most cases, outliers represent cases where even more males entered the For each test plant, the numbers of moths that entered the test test and control chambers than would be expected from the and control chambers in the three replicate runs for each sex regressions. were compared with those in the ‘blank’ olfactometer using R-analyses (Dalgaard 2002). Two sets of three blank runs Identification of plant volatiles (nothing in either chamber of the olfactometer, with one set for females and the other for males) were done for comparison Plant volatiles were grouped into floral volatiles (fatty acid with the runs using test plants. Two statistical analyses were derivatives, mostly short-chain alcohols and acetates, which done, using the GLM procedure in R with a quasibinomial are products of nectar fermentation), green leaf volatiles (C6 distribution appropriate to proportional data. The first one fatty acid derivatives, straight chain alcohols, aldehydes and dealt with the number of moths caught in the test chamber as esters mostly present in leaves), aromatic compounds (cyclic a percentage of the total moths placed in the olfactometer (% C6 compounds and their derivatives, found in flowers and positive response), while the second one, with the number of leaves) and isoprenoids (mono- and sesquiterpenes which can moths caught in both the test and control chambers as a per- be found in both leaves and flowers) (Table 3). These group- centage of the total (% total response). The advantages of the ings were based on the classifications of plant volatiles by GLM approach using multiple measures in behavioural assays Knudsen et al. (1993) and Metcalf (1987). are discussed by Hern and Dorn (2001). Among the floral volatiles, ethanol and ethyl acetate were the most commonly found, and were abundant in the highly attractive Eucalyptus and Angophora spp. Green leaf volatiles RESULTS were found in most plants, whether highly attractive or not, frequently in large quantities. Aromatic compounds were Attractiveness of plants in the olfactometer usually found in small quantities, and rarely in the most attractive plants. The plants which produced the greatest quantities Helicoverpa armigera moths showed significantly greater % of aromatic compounds, pigeon peas and jasmine, were among positive response to the test compared with the blank chamber the least attractive in the olfactometer. Monoterpenes were in 33 of the 38 plants tested (Table 2). Using the % total found, often in large quantities, in the most attractive plants, response criterion, 27 of 38 plants were significantly more including (but not limited to) Eucalyptus and Angophora attractive than the blank olfactometer. No plants were signifi- spp. Prominent monoterpenes included cineole, a-pinene, cantly less attractive than the blank olfactometer (i.e. repellent) limonene, E-b-ocimene and g-terpinene. Sesquiterpenes were using the % positive response criterion, and only one (Acacia also abundant, but less clearly associated with attractive plants. cambadgeii) was repellent using the % total response criterion. Prominent ones included aromadendrene and caryophyllene. The three most attractive plants were the Australian native plants, Eucalyptus nova-anglica, Angophora floribunda and Eucalyptus melliodora. The other two Eucalyptus spp., viminalis and caliginosa, were also highly attractive to moths. DISCUSSION These plants are not known larval or oviposition hosts of H. armigera. All the crop plants which are known hosts of H. Most plants tested in the olfactometer were significantly armigera as well as the seven weed plants showed significant attractive to one or both sexes of H. armigera moths (Table 2). attraction to moths. Four of the six ornamentals tested were Of the 38 plants tested, only five (Acacia subulata, Lonicerus also significantly attractive to moths. The non-attractive plants japonicum, P. stenoptera wilted leaves, J. officinale and included the native plants, Acacia subulata and A. cambadgeii, A. cambadgeii) were not significantly different from the blank the ornamentals L. japonica and Jasminum officinale and olfactometer, using the % positive response criterion. This wilted leaves of the Chinese wingnut tree, P. stenoptera. result suggests that volatile compounds generating at least some level of attraction to Helicoverpa moths are widespread in plants. Responses of males compared with females In general, plants attractive to female moths were also Our two criteria to determine attractiveness in the olfactometer attractive to males. The significance of the intercepts in the (% positive response and % total response) were strongly regression lines described above, the fact that the slopes of the correlated with each other, and on both criteria the responses regressions were close to 1, and the results in the blank olfac- of the two sexes were strongly correlated across all plants. tometer (where the % total response was significantly different On the % positive response criterion, attractiveness to between the sexes) all suggest that the main reason for signifi- males was strongly correlated with attractiveness to females cant sex differences in % total response was that males were © 2010 The Authors Journal compilation © 2010 Australian Entomological Society Plant volatiles as moth attractants 15

Table 2 Summary of results for the different plants used in the olfactometer

Plant material % positive response (test/total) % total response ((test + control)/total) % response† Sex Attractant Sex ¥ % total Sex Attractant Sex ¥ attractant response† attractant E. nova-anglica 67.4 ns <0.001 ns 72.6 ns <0.001 ns A. floribunda 64.3 ns <0.001 ns 73.2 ns <0.001 ns E. melliodora 54.6 ns <0.001 ns 68.3, 59.1 0.042 <0.001 ns A. hortorum 52.3 ns <0.001 ns 60.3 ns <0.001 ns E. caliginosa 51.0 ns <0.001 ns 66.4 ns <0.001 ns N. velutina‡ 48.8, 32.3 0.004 <0.001 ns 59.8, 48.7 <0.001 <0.001 ns C. officinalis‡46.8ns<0.001 ns 70.0, 58.5 <0.001 <0.001 ns H. annuus‡46.0ns<0.001 ns 62.7, 51.5 0.008 <0.001 ns E. viminalis 50.7, 39.5 0.030 <0.001 ns 68.7, 55.1 <0.001 <0.001 ns B. pilosa 43.8 ns <0.001 ns 52.6 ns <0.001 ns Z. mays‡42.6ns<0.001 ns 61.6 ns <0.001 ns H. floribundum‡ 53.5, 31.3 0.018 <0.001 ns 62.5, 48.2 0.017 <0.001 ns G. lindhemerii 42.0 ns <0.001 ns 52.3 ns 0.001 ns H. incana 49.3, 33.3 <0.001 <0.001 ns 60.0, 46.0 <0.001 <0.001 ns M. domestica‡ 50.3, 32.0 0.002 <0.001 ns 64.5, 41.9 <0.001 <0.001 0.001 L. usitatissimum‡ 45.5, 35.2 0.009 <0.001 ns 57.7, 45.9 <0.001 <0.001 ns M. sativa‡39.2ns<0.001 ns 49.8 ns 0.001 ns S. oleraceus‡ 48.6, 29.2 <0.001 <0.001 ns 58.6, 43.0 <0.001 <0.001 ns E. plantagineum‡ 38.8 ns 0.009 ns 65.9, 46.6 0.013 0.003 ns C. arietinum‡ 38.2 ns 0.004 ns 63.6 ns 0.010 ns L. purpureus‡ 48.5, 26.6 0.006 0.002 ns 58.9, 43.1 0.020 0.017 ns W. fruticosa 37.3 ns 0.001 ns 55.4, 48.8 0.040 <0.001 ns C. intybus‡35.4ns<0.001 ns 39.3, 44.5 ns ns 0.011 E. gilesii 34.9 ns <0.001 ns 48.9, 41.6 0.040 0.001 ns Acacia spp. 2 39.8, 27.9 0.033 0.0001 ns 49.3, 37.6 0.020 0.040 ns I. brevicompta‡33.3ns<0.001 ns 45.3 ns 0.050 ns E. sturtii 39.6, 25.9 0.025 0.001 ns 63.6, 41.3 <0.001 <0.001 0.003 G. hirsutum‡ 30.8 ns 0.001 ns 48.0, 38.6 0.010 0.010 ns G. parviflora 29.7 ns 0.002 ns 45.7 ns ns ns S. bicolor‡ †28.6 ns 0.010 ns 43.4 ns ns ns C. cajan‡ 27.7 ns 0.020 ns 40.6 ns ns ns O. stricta‡ 25.7 ns 0.010 ns 33.8 ns ns ns B. napus‡ 30.7, 19.4 0.010 0.030 ns 47.9, 30.3 <0.001 ns ns A. subulata 19.9 ns ns ns 30 ns ns ns L. japonica 24.7, 14.6 0.003 ns ns 42.0, 30.6 <0.001 ns ns P. stenoptera 15.8 ns ns ns 34.6, 36.4 ns ns 0.010 J. officinale 15.3 ns ns ns 48.4, 38.0 0.020 ns ns A. cambadgeii 17.1, 11.1 0.05 ns ns 31.2, 20.3 <0.001 <0.001 ns Blank 18.9, 14.0 ns – – 32.9, 29.3 0.036 – –

†For plants with significant effect of sex or a significant interaction between sex and attractant, the data in the format x, y are for males (x) and females (y). For plants where no such effects existed, the single figures are means for combined male and female runs. ‡Known host plant for larvae (Zalucki et al. 1986, 1994 or PC Gregg unpubl. data 1993). Plants are listed in order of attractiveness (average of males and females). ‘Sex’ indicates the significance (P-value) of a GLM R analysis comparing males and females. ‘Attractant’ indicates the significance (P-value) of a GLM R analysis comparing the plant with the blank olfactometer. ns, not significant (P > 0.05). generally more active in the olfactometer, and more likely to tion. Helicoverpa spp. has a wide host range for oviposition move upwind into either chamber. Thus, plants which were and larval development. Zalucki et al. (1986, 1994) recorded differentially attractive to one or other sex could only be iden- H. armigera from 101 plants in 30 families and the endemic tified by a significant sex–attractant interaction. No such plants species H. punctigera from 172 plants in 40 families. Less is were identified using the primary criterion of % positive known about hosts for adult feeding, but studies on moth- response, and only four were indentified using the secondary borne pollen have shown that Helicoverpa moths feed on criterion of % total response. These four plants were Malus flowers from many non-host plants such as Eucalyptus spp. domestica, Chicorium intybus, Eremophila sturtii and P. and weeds in the Brassicaceae, as well as many larval host stenoptera. plants (Gregg 1993; A Del Socorro & P Gregg unpubl. data Potential sources of moth attractants which might be used in 2001). managing H. armigera include any plant which is attractive for The attraction of H. armigera moths to some plants in the adult nectar foraging and, in the case of females, for oviposi- olfactometer in our study was probably for adult feeding rather © 2010 The Authors Journal compilation © 2010 Australian Entomological Society ora oplto 00Asrla noooia Society Entomological Australian 2010 © compilation Journal Authors The 2010 © Table 3 List of compounds identified from various plants 16

Compound† Class En Af Em Ah Ec Co Ev Ha Zm Gl Hi Md Lu So Ep Ca Lp Ci Gh Gp Cc Bn Jo Socorro Del P A Butanoic acid F ++ Butyrolactone F ++ Ethanol F ++ ++ + ++ Ethyl acetate F +++ ++ + +++ +++ Octanoic acid F tr 1-dodecanol F + 1-octen-3-ol F +++ al. et Decanal F ++ Nonanal F tr ++

(E)-2 hexenal C6F +++ + +++ (E)-2-hexen-1-ol C6F +++ (E)-2 hexenyl acetate C6F +++

(E)-2 hexenyl hexanoate C6F + (E)-3 hexenol C6F ++

(Z)-3 hexenol C6F + +++ ++ +++ +++ +++ ++ ++ (Z)-3 hexenyl acetate C6F +++ +++ ++ ++ +++ (Z)-3 hexenyl butyrate C6F ++ (Z)-3 hexenyl formate C6F ++ +++

Ethyl hexanoate C6F ++ Hexenal C6F ++ + +++

Hexyl hexanoate C6F ++ + ++ Benzoic acid A tr Benzyl acetate A ++++ Benzaldehyde A +++ + ++ tr + Benzyl alcohol A ++ + Benzyl propanal A + Cinnamaldehyde A +++ Durene A +++ Indole A ++ Methyl salicylate A +++ Phenol A ++ Phenylacetaldehyde A +++ ++ 2-phenoxyethanol A tr Phenylethanol A + p-acetylethylbenzene A tr p-cresol A + ++++ 2,4-tert-butylphenol A ++ Azulene M + +++ (E,E) alloocimene M + Camphene M + Cineole M + +++ +++ +++ + tr +++ Geraniol M +++ Geraniol acetate M ++ Geranylacetone M + Isoeugenol M ++ Isopulegol M + a-guajene M + Limonene M tr +++ tr + tr ++ ++ + +++ Linalool M ++ tr +++ ++ E–b–ocimene M tr tr tr + tr Sabinene M ++ Terpinolene M ++ a-terpinolene M ++ a-phellandrene M + b-phellandrene M ++ + a-pinene M ++ +++ +++ +++ tr +++ tr ++ +++ + b-pinene M tr ++ ++ b-myrcene M ++ ++ o-cymene M +++ ++ ++ p-cymene M tr tr +++ ++ ++ a-terpinene M ++ g-terpinene M + tr ++ ++ a-terpineol M ++ a-thujene M +++ ++ Aromadendrene S ++ ++ + +++ Copaene S ++ +++ Viridiﬂorene S + Ylangene S ++

ora oplto 00Asrla noooia Society Entomological Australian 2010 © compilation Journal g-cadinene S ++ d-cadinene S ++ + a-Caryophyllene S ++ tr ++ +++ ++ b-Caryophyllene S tr + ++

a-himachalene S ++ attractants moth as volatiles Plant b-elemene S +++ Cyclohexene O ++ Cyclopentene O + Dimethyl disulphide O ++ Allyl isothiocyanate O ++

†Classifications were based from those of Knudsen et al. (1993) and Metcalf (1987).F–floral volatiles, C6F–C6 fatty acid derivatives found in leaves (green leaf volatiles), A – aromatic compounds, M – monoterpenes, S – sesquiterpenes,O–other. Relative abundance of the volatiles is denoted by tr = < 0.1%, +=0.1 - 1%, ++ = 1 - 10%, +++ = >10% of total volatile compounds identified for that plant. Plants are arranged left to right in order of attractiveness in the olfactometer (Table 2). En – Eucalyptus nova-anglica, Af – Angophora floribunda, Em – Eucalyptus melliodora, Ah – Araujia hortorum, Ec – Eucalyptus

00TeAuthors The 2010 © caliginosa, Co – Calendula officinalis, Ev – Eucalyptus viminalis, Ha – Helianthus annuus, Zm – Zea mays (silk), Gl – Gaura lindhemerii, Hi – Hirschfeldia incana, Md – Malus domestica, Lu – Linum usitatissimum, So – Sonchus oleraceus, Ep – Echium plantagineum, Ca – Cicer arietinum, Lp – Lablab purpureus, Ci – Chicorium intybus, Gh – Gossypium hirsutum, Gp – Galinsoga parviflora, Cc – Cajanus cajan, Bn – Brassica napus, Jo – Jasminum officinale. 17 18 A P Del Socorro et al. than oviposition particularly as unmated females were used would seem to be serendipitous, as is the case for wilted poplar in the experiments. This is suggested by the attractiveness of leaves (Wang et al. 2003). We tested wilted P. stenoptera plants like the four Eucalyptus spp. and Angophora floribunda. leaves sourced from the Royal Botanic Gardens at Mount These plants are not known hosts for either larval feeding or Tomah, NSW but they were not attractive to Australian H. oviposition of Helicoverpa spp. The attractiveness of these armigera moths. Australian H. armigera moths were also not non-host plants as well as most of the other plants tested in the attracted to steam distilled extracts of P. stenoptera sourced olfactometer could have been largely due to the presence of from China (A Del Socorro & P Gregg unpubl. data 2002). flowers in the test plant materials, as floral odours release Whether there are physiological differences between the food-seeking or feeding behaviour in moths (Brantjes 1973, Chinese and Australian H. armigera in response to plant 1978 as cited by Dobson 1994). A similar lack of correlation volatiles from P. stenoptera is not known. between the larval host status and attractiveness in the olfac- The chemical composition of volatile emissions from tometer is indicated by the two native Asteraceae we tested, flowers and other plant parts varies according to whether the Helipterum floribundum and Ixiolaena brevicompta. Both are parts are picked or cut, or left attached to the plant. Mookher- good larval hosts for H. armigera and H. punctigera (Zalucki jee et al. (1990) showed that the composition and quantity of et al. 1994), and I. brevicompta has been proposed as a odours emitted by picked flowers differed significantly from ‘primary host’ for the latter species (Walter & Benfield 1994). those left intact or attached to the plant. Matile and Alten- However, in our assays I. brevicompta was much less attractive burger (1988) also reported that periodicity of flower fragrance than H. floribundum. changes in detached or intact flowers. In our olfactometer It is not surprising that all the crop plants tested were studies, freshly cut bouquets of flowers and leaves were used significantly attractive to both sexes. They are mostly known as it was not physically feasible to use whole or intact plants hosts of larvae. Crops such as corn and sunflower are preferred with the olfactometer design. Hence, it is possible that the oviposition hosts (Firempong & Zalucki 1990; Fitt 1991). volatile profiles of our test plant materials would have been Pigeon pea extracts and synthetic chickpea kairomones have different from those of intact plant materials. The degree of been reported to be attractive to mated H. armigera female damage and the time between cutting and testing in the olfac- moths (Rembold & Tober 1987; Rembold et al. 1991; Hartlieb tometer may have influenced the amount of some volatiles. For & Rembold 1996), and these plants are highly attractive for this reason, caution must be applied when extrapolating from oviposition in the field, so much so that pigeon pea is a pre- our results to the relative attractiveness of crops and other ferred species for refuge crops, which are used to breed sus- plants in the field. However, the primary purpose of our study ceptible moths for the management of resistance to transgenic was to identify plant volatiles which might be useful in syn- cotton (Baker et al. 2008; Farrell 2008). However, they were thetic attractants for management of H. armigera. The plant not especially attractive in our study, perhaps indicating that materials used for volatile collections were in the same con- they are not good adult feeding hosts. Similarly, cotton, the ditions as the test plants used in the olfactometer (i.e. freshly crop which suffers the greatest economic losses to Helicov- cut bouquets), and the collection source was identical (the erpa spp. in Australia (Adamson et al. 1997), was not espe- airstream of the olfactometer) so we would have expected cially attractive by comparison with other hosts. Cotton has similar volatile profiles from the same plants, whether used for also been shown to be a less preferred plant for oviposition by volatile collection or for moth response testing. H. armigera females under laboratory conditions (Firempong In developing moth attractants for a highly polyphagous & Zalucki 1990; Jallow & Zalucki 1996). species like H. armigera, the question of which plant to mimic The majority of the weed plants we tested in the olfactome- arises. Previous attempts to develop synthetic plant volatile ter are known larval or oviposition hosts of H. armigera, attractants have mimicked the volatile profiles of particular and were observed to be attractive to the moths. Sow thistle, larval host plants, such as Gaura suffulta (Shaver et al. 1998), Sonchus oleraceus, which is well known as a weed host for pigeon pea or chickpea (Hartlieb & Rembold 1996) and larval H. armigera (Gu & Walter 1999) was highly attractive to the African marigold, Tagetes erecta (Bruce & Cork 2001). the adults. However, Buchan weed (Hirschfeldia incana) However, we have shown here that non-host plants can be which is not a larval host was also highly attractive. Of further highly attractive, at least to unmated unfed moths such as those interest among the weeds is the attractiveness of moth vine, used in our experiments. For attract-and-kill of females, tar- Araujia hortorum. This species is closely related to A. sericof- geting unmated females is advantageous because they have not era (bladder flower), which has been reported to produce the already laid eggs. Each female killed represents a loss to the volatile compound phenylacetaldehyde and was found to be population of her entire potential fecundity (1000–2000 eggs attractive to noctuid moths (Cantelo & Jacobson 1979). in the case of Helicoverpa spp.; Zalucki et al. 1986). Clearly, In China, farmers trap H. armigera moths in cotton fields volatiles from non-host plants should be considered as poten- using branches of wilted leaves of the wingnut tree, P. tial candidates for inclusion in synthetic attractant blends. stenoptera (J-W Du pers. comm. 1997). Extracts of this plant Another possible complication in using plant mimics as were also found to be attractive to the Chinese H. armigera attractants for polyphagous insects is learning. Learning in moths in an olfactometer (Xiao et al. 2002). As this tree does feeding and oviposition behaviour has been demonstrated in not appear to provide nectar for adults, and is not a suitable H. armigera (Cunningham et al. 2004, 2006). In natural habi- larval host, the presence of volatiles attractive to H. armigera tats, preferences of moths for different hosts (with different © 2010 The Authors Journal compilation © 2010 Australian Entomological Society Plant volatiles as moth attractants 19 volatile profiles) are likely to be influenced by the abundance transgenic cotton plantings in eastern Australia. Australian Journal of these hosts (Cunningham et al. 1999). Developing a syn- of Agricultural Research 59, 723–732. Beerwinkle KR, Shaver TN, Lingren PD & Raulston JR. 1996. Free- thetic blend which mimics a particular host could be ineffec- choice olfactometer bioassay system for evaluating the attractiveness tive if that host is rare in a region where the formulation is of plant volatiles to adult Helicoverpa zea. Southwestern Entomolo- to be used. Conversely, even a less attractive host in a mono- gist 21, 395–405. culture may be preferred to a synthetic attractant mimic, if Brantjes NBM. 1973. Sphingophilous flowers, function of their scent. In: Pollination and Dispersal (eds NBM Brantjes & HF Linskens), the insects are conditioned to respond to that plant. 27. Publ. Botany, Univ. Nijmegen. Our approach to developing synthetic attractant blends, Brantjes NBM. 1978. Sensory responses to flowers in night-flying moths. which we term ‘super-blending’, does not involve mimicking In: The Pollination of Flowers by Insects (ed. AJ Richards). Linn. the composition of volatiles in any one host plant. Instead, we Soc. Symp. Ser. No. 6, 13. Academic Press, London, UK. Breeden DC, Young TE, Coates RM & Juvik JA. 1996. Identification and propose identifying compounds that are in common between bioassay of kairomones for Helicoverpa zea. Journal of Chemical the highly attractive plants (regardless of the relative quantities Ecology 22, 513–539. in these plants), and then blending those compounds in com- Bruce TJ & Cork A. 2001. Electrophysiological and behavioral responses binations which may not occur in nature. This approach could of female Helicoverpa armigera to compounds identified in flowers of African marigold, Tagetes erecta. Journal of Chemical Ecology potentially avoid any difficulties of learned responses, and 27, 1119–1131. allow the inclusion of volatiles from non-host plants which Cantelo WW & Jacobson M. 1979. Phenylacetaldehyde attracts moths to might enhance the attractiveness of the blend to unmated bladder flower and to blacklight traps. Environmental Entomology 8, 444–447. females. In such an approach, volatiles worth considering Cribb BW, Hull CD, Moore CJ, Cunningham JP & Zalucki MP. 2007. could include representatives from all the classes we consid- Variability in odour reception in the peripheral sensory system of ered, but especially monoterpenes such as cineole, limonene Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). Australian and a-pinene, most of which have not previously been used in Journal of Entomology 46, 1–6. Cunningham JP, Zalucki MP & West SA. 1999. Learning in Helicoverpa noctuid moth attractants. Additional candidate volatiles could armigera (Lepidoptera: Noctuidae): a new look at the behaviour and be derived from electroantennogram studies, either using control of a polyphagous pest. Bulletin of Entomological Research 89, single cell responses (Røstelien et al. 2005) or whole antennae 201–207. (Cribb et al. 2007). However, caution is required in extrapo- Cunningham JP, Moore CJ, Zalucki MP & West SA. 2004. 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