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Orientation of rusty grain beetles, Cryptolestes ferrugineus (Coleoptera: Laemophloeidae), to semiochemicals in field and laboratory experiments
Journal of Stored Products Research 84 (2019) 101513
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Orientation of rusty grain beetles, Cryptolestes ferrugineus (Coleoptera: Laemophloeidae), to semiochemicals in field and laboratory experiments Stephen M. Losey a, Gregory J. Daglish b, c, Thomas W. Phillips a, c, * a b c
Department of Entomology, Kansas State University, Manhattan, KS, 66506, USA Department of Agriculture and Fisheries, Queensland, Ecosciences Precinct, GPO Box 267, Brisbane, QLD, 4001, Australia Plant Biosecurity Cooperative Research Centre, GPO Box 5012, Bruce, ACT, 2617, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2019 Received in revised form 16 August 2019 Accepted 10 September 2019 Available online xxx
Cryptolestes ferrugineus is a common beetle pest of stored grain for which techniques to monitor dispersing beetles are limited. Early research found several male-produced aggregation pheromones, but there has been little related work since that time. This paper reports experiments on orientation of C. ferrugineus in response to synthetic pheromones and other semiochemicals via flight and walking. Field trapping studies showed that flying beetles were caught on the western sides of grain bins in Kansas compared to the other three cardinal directions. Work with synthetic formulations of the two male-produced aggregation pheromones found that flying beetles were attracted to traps with the pheromone and wheat compared to traps with wheat only. Walking bioassays in the laboratory determined that either of the two aggregation pheromones, known as Cucujolide I and Cucujolide II, were attractive whether deployed singly or in combination. Laboratory bioassays showed that volatiles from grains, other grain-based materials and two commercial food attractants used in traps were attractive to C. ferrugineus. Further laboratory assays demonstrated that C. ferrugineus would orient to synthetic pheromones of three other beetle species and one moth species, all common pests of stored products. These new data on semiochemicals for C. ferrugineus suggest future work that could be done toward developing new tools for detecting and monitoring this serious pest. Crown Copyright © 2019 Published by Elsevier Ltd. All rights reserved.
Keywords: Pheromones Food attractants Traps Stored grain Cucujolide
1. Introduction The rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae), is among the most common grain pests wherever cereal grains are stored and marketed (Canadian Grain Commission, 2013; Hagstrum et al., 2012; Cuperus et al., 1986). Cryptolestes ferrugineus can infest all cereal grains in storage, and there are records of this pest infesting up to 69 different commodities worldwide such as black pepper, chili, cocoa, coffee bean, cotton seed, dried fruits, hemp, licorice, oilseeds, peanuts, rice, dried tomatoes, and yams (Hagstrum and Subramanyam, 2009). Cryptolestes ferrugineus can cause heating when infesting stored grain (Sinha, 1961), and it can damage wheat by consuming
* Corresponding author. Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS, 66506, USA. E-mail address: [email protected] (T.W. Phillips). https://doi.org/10.1016/j.jspr.2019.101513 0022-474X/Crown Copyright © 2019 Published by Elsevier Ltd. All rights reserved.
the germ. Both adults and larvae feed mostly on damaged kernels, grain dust, and fungi in stored grain. Cryptolestes ferrugineus populations can increase rapidly requiring effective mitigation before grain value is lost. Unfortunately, C. ferrugineus populations from different parts of the world have evolved resistance to the commonly used grain fumigant phosphine (e.g. Nayak et al., 2013; Konemann et al., 2017), necessitating good methods for early detection of this pest before populations surpass fumigation action thresholds. Detection and monitoring of C. ferrugineus in stored grain can be done by sifting samples of grain either at point of sale or over the course of storage, though collection of samples from stored grain can be very challenging and dangerous. Perforated probe traps (Subramanyam et al., 1993) inserted below the grain surface are effective in capturing C. ferrugineus adults (Reed et al., 1991; Phillips et al., 2000). Adults of C. ferrugineus walking through the grain mass encounter a probe trap, fall through a hole to a receptacle at the bottom where they can be counted several days or weeks after trap
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placement. There are several factors that affect the capture of C. ferrugineus adults in probe traps. These factors include the temperature of the grain mass that can affect insect activity, density of insects, and trap location within the grain mass (White and Loschiavo, 1986; Fargo et al. 1989, 1994; Subramanyam et al., 1993; Hagstrum, 2000). Toews et al. (2005) showed that the number of C. ferrugineus captured in probe traps at the grain surface and 1 m below the surface were similar. Unbaited sticky traps have been used to monitor the flight activity of adult C. ferrugineus near grain bins (Nansen et al., 2004). Although attractant pheromones have been described for over 50 pest species of stored-product insects, and commercial pheromone lures are available for about half of those, pheromone-baited traps for monitoring species of Cryptolestes have received limited commercial development if any at this time, and are not widely marketed or used, to our knowledge (Swords and Van Ryckeghem, 2010). Borden et al. (1979) were the first to report on the aggregation pheromones produced by male C. ferrugineus while feeding that are attractive to both sexes. These pheromones act as intraspecific signals used in mate-finding and host colonization and were first isolated and identified by Wong et al. (1983). The aggregation pheromones consist of two macrolide lactones, and these were identified as ferrulactone I and II, (E, E)-4, 8-decadien-10-olide and (S)-(Z)-3-dodecen-11-olide. These chemicals are commonly referred to as cucujolide I and II, respectively (hereafter referred to as CeI and C-II). A 9:1 mixture of CeI and C-II was determined to generate the highest response in laboratory experiments compared with other mixture ratios (Wong et al., 1983). Loschiavo et al. (1986) conducted the first field study using synthetic versions of the maleproduced aggregation pheromone against adults in a grain bin in Canada and found that the trap containing synthetic pheromone did not catch more adults than the control trap. Those experiments used baited and non-baited probe traps inserted into stored grain and did not directly assess flight or walking responses of beetles outside of grain bins. Research and applications with the aggregation pheromones of C. ferrugineus have been limited due to the lack of synthetic pheromone for use by researchers. Oehlschlager et al. (1983) reported on the synthesis of the two macrolides, and a useful method for synthesis of one of the macrolides was published soon after (Czeskis et al., 1994), but synthetic pheromones for commercial uses were not available until recently (Norwood, 2015). Holloway et al. (2018) recently published results from field experiments using synthetic pheromone lures containing CeI and C-II of C. ferrugineus and provided new information on seasonal and spatial patterns of flight for this species in Australia. Nansen et al. (2004a,b) provided information about flight of C. ferrugineus outside grain bins in Oklahoma, but information on flight of this
pest outside grain bins in North America is otherwise limited. The objectives of the research reported here were to: 1) evaluate orientation of C. ferrugineus in flight near bins of stored wheat; 2) determine if C. ferrugineus would respond in flight to traps baited with a synthetic pheromone, and 3) evaluate responses of C. ferrugineus to synthetic pheromones and to other potential semiochemicals in laboratory bioassays. 2. Materials and methods 2.1. Flight near grain bins assessed with wheat-filled bucket traps Bucket traps with wheat were used to capture flying C. ferrugineus adults near infested grain bins and to assess the feasibility of such field sites for subsequent testing of synthetic pheromones. Bucket traps were 18.9 L opened plastic paint buckets protected from rain with a modified bird-feeder squirrel baffle cover that was hung from a trap stand (Fig. 1). A piece of hardware cloth with 1.9 cm openings was clipped down over the top of the bucket to prevent small vertebrates from infesting the grain while allowing entry of beetles into the bucket. The bucket trap assembly was suspended from its trap stand at approximately 12.7 cm above the ground (Fig. 1). This design allowed the bucket trap to move with the wind but not be blown away or overturned, to keep the majority of rainwater and debris out with the squirrel baffle so as to also allow responding beetles go inside the bucket. Bucket traps contained 670 g of soft white wheat to provide food for and retain responding C. ferrugineus, and possibly serve as a natural attractant for the flying beetles. Bucket trap experiments were conducted near the cities of Junction City and Abilene in north-central Kansas from 24 July to 12 September 2014. Four bucket traps were placed around a single grain bin at the Abilene site that was approximately 18 m in diameter and 15 m tall and contained wheat that was known to be infested with many species of stored-product insects including C. ferrugineus. A single bucket trap was placed at each of the four cardinal directions around the bin, approximately 6 m from the bin sidewall. Spacing of grain bins at the Junction City location allowed for only two bucket traps to be placed around a bin with infested rice Oryza sativa L., that was approximately 15 m diameter x 22 m tall. One trap was placed on the west side of the bin and one on the north side of the bin. Bucket traps at each location were checked weekly for 7 weeks, with each week serving as an experimental replicate by time at each location in lieu of the lack of multiple bins at a site to serve as true replicates. Insects were sifted, removed and counted from each bucket. The old wheat was discarded and replaced with new wheat for each subsequent week of trapping. Numbers of C. ferrugineus trapped in a week at a given bin were
Fig. 1. Materials used to assess orientation of C. ferrugineus: bucket trap containing wheat (left), 4-unit funnel trap (center) and the two-choice pitfall bioassay (right). See text for details.
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converted to proportion of beetles trapped at given direction relative to the bin, and proportion data were then subjected to the angular transformation (Zar, 1984) before subjecting to analysis of variance (ANOVA; SAS, 2002). 2.2. In-flight field responses to cucujolide-II Four-unit Lindgren funnel traps (Phero Tech Inc., Delta, BC, Canada) were used in experiments at the Junction City and Abilene sites from 7 August to 17 September 2015 to assess the activity of synthetic C-II provided by Alpha Scents Inc. (West Linn, OR, USA). Funnel traps capture insects that fly into a vertically oriented series of funnels and then fall through the funnels into a collection cup at the bottom (Fig. 1; Lindgren, 1983). Sets of two traps spaced 20 m apart and placed at least 30 m away from any other pair of traps, were deployed at two different locations. The Abilene site had two sets of traps arranged in a line from east to west 50 m south of a grouping of five steel grain bins. The Junction City location had four sets of funnel traps arranged in an east-west line, with two sets at the north edge of the property and two sets 100 m away at the south edge of the property. Each trap had a small mesh bag that contained approximately 10 g of partially crushed wheat wired to the vertical connector between the 3rd and 4th funnels allowing the bag to hang freely inside the free space of the funnel above the collection cup. One trap within each two-trap set was randomly selected as the experimental control and had wheat only, while the second trap in a set was the experimental treatment with a cucujolide-II lure and a bag of wheat. Pheromone lures with synthetic pheromone absorbed onto a rubber stopper septum (Fisher Scientific, Pittsburg, PA, USA) containing either 25 mg of cucujolideII applied in hexane, or hexane only as a control, were added inside each trap such that the septum did not touch the trap. Each trap contained a piece of crumpled paper towel in the collection cup to provide a harborage for trapped beetles and a 1 cm2 piece, 0.7 g, of a Hot Shot® No-Pest® Strip2 (Spectrum Brands, Inc., Middleton, Wisconsin USA) with the active ingredient of dichlorvos at 18.6%, in the collection cup as a killing agent. Traps at each location were checked every 1e2 days. After collecting and counting the trapped beetles in each trap in a set, the trap position in each set was randomly re-assigned before the next trapping period. Numbers trapped in the traps with C-II and in the number in control traps were summed across all sets of traps for a given trapping time, with each of seven trapping times serving as a replicate (n ¼ 7). Total beetles trapped for treatment and control traps were converted into proportions of trapped C. ferrugineus from the total nubmer trapped for that time replicate, and were then subjected to the angular transformation (Zar, 1984) before analysis with Student's ttest (SAS, 2002). 2.3. Laboratory assays of semiochemicals Laboratory experiments were conducted to examine orientation to synthetic pheromones and to additional semiochemicals by C. ferrugineus. Laboratory experiments were conducted using beetles from an established colony of C. ferrugineus obtained from the USDA Center for Grain and Animal Health Research in Manhattan, KS. These insects were reared on a diet consisting of rolled oats and brewer's yeast (95:5) in 508 ml glass jars covered using filter paper in the screw-on ring lid to ensure air and moisture diffusion. Colonies were maintained in a growth chamber at 28 C and 50e70% RH. Adult insects used in bioassays were 3e4 weeks old and were separated from their food substrate using a US number 12 sieve (Fisher Scientific). Beetles were then held without food in ventilated class vials in groups of 20 for 2e3 h before being used in bioassays to ensure uniform starvation and
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to possibly enhance their olfactory response after being deprived of food. We used a two-choice pitfall bioassay for these experiments (Fig. 1), like that used by Pierce et al. (1981). The bioassay used two 11 ml shell vials, 19 mm in diameter x 50 mm tall, and a glass Petri dish of 100 mm in diameter x 20 mm in height (Fisher Scientific). Each dish had two 6 mm diameter holes in the floor, placed 40 mm apart and 15 mm from the side walls positioned along a mid-line of the dish floor. Each dish had its vials stabilized in 21-mm holes that were drilled into a wooden board and spaced so that the glass dishes could be placed directly over the vials. Up to 10 bioassay dishes were set up at a given time in a small room free of insect or grain materials and maintained at 22e24 C, 30% RH in complete darkness. Glassware was washed using soap and water, rinsed with deionized water, and then rinsed with acetone and dried in an oven at 65 C for a minimum of 2 h prior to use in all assays. Each twochoice dish assay evaluated the orientation of 20 mixed sex adult C. ferrugineus by the number falling into a given vial or remaining on the arena floor. Treatment and control were assigned at random to a given vial in each two-choice bioassay. A water suspension of polytetrafluoroethylene (Sigma-Aldrich; St. Louis, MA, USA) was applied to the top inside-wall of each dish and vial to prevent escape of beetles by walking up the vertical glass surfaces. The cover was removed from each Petri dish, the vial holding the 20 beetles was carefully overturned on the dish-bottom at the midpoint between the two holes, and the vial kept in place for 10 min to allow beetles to acclimate. The overturned vial was then removed and the dish cover replaced to allow beetles to move freely in the dish under complete darkness for 18 h, after which the lights in the bioassay room were turned on and the number of beetles in the main dish arena and the treatment and control vials was recorded for each bioassay unit. Laboratory bioassays studied the responses of C. ferrugineus to different food or insect-produced volatiles, some of which were grains or grain-related materials and others that were pheromones. Food baits included the commercially marketed “Storgard Oil” used ce Inc. (Adair, OK, USA); a commercial in traps and provided by Tre food lure for traps from Russell IPM Ltd (Deeside, Flintshire, UK) and laboratory grade ethanol (Fisher Scientific, Waltham, MA, USA), which is known as a fermentation product from grains and other foods that can attract insects (e.g. Moeck, 1970). The whole or processed grains of wheat, corn as corn meal or oats, and a grainbased dog food (Purina, St. Louis) were also tested. We evaluated the synthetic C-II pheromone of C. ferrugineus (Alpha Scents, Inc., West Bend, OR, USA), diluted in hexane and applied at 25 ng to 5,000 ng to 1.0 cm2 pieces of filter paper vs hexane only on filter paper as a control. We also evaluated rubber septum lures already formulated with CeI, C-II or a combination of CeI and C-II purchased from Research Directions Pty Ltd (Queensland, Australia) vs. rubber septa with hexane only as controls. Synthetic pheromones of the following species formulated in rubber septa and provided by ce Inc. were as follows: Lasioderma serricorne (F.) (Coleoptera: Tre Anobiidae), Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae), Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). All two-choice assays with pheromone lures had a rubber septum not formulated with pheromone as a control. Test materials for the two-choice assays were placed in the bottom of one shell vial and the second vial was either empty or contained a neutral material (e.g., blank septum or hexane on filter paper) to serve as the untreated control. Each bioassay experiment had 20 replicate dishes. Numbers of beetles responding to treatment and control vials for each experiment were analyzed using a paired t-test (R Core Team, 2014).
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3. Results 3.1. Orientation to bucket traps in the field A total of 410 C. ferrugineus were captured in the two wheatfilled bucket traps at the Junction City location. On average, 63.1% (±7.4% SE) of beetles were captured on the west side and 37.0% (±7.4% SE) on the north side over seven 1-week trapping periods, with the proportions captured on the two sides being significantly different (Fig. 2; F ¼ 6.225, df ¼ 1, 6, P < 0.05). A total of 66 C. ferrugineus were captured in the four bucket traps at the Abilene location over the seven replicate weeks. A significantly higher proportion of beetles was captured on the west side (Fig. 3; F value ¼ 48.77, df ¼ 3, 6; P < 0.01). On average, 4.2% (±4.1% SE) of beetles were captured on the north, compared with 9.6% (±6.0% SE) on the east, 7.9% (±3.4% SE) on the south and 78.3% (±7.1% SE) on the west. On the basis of the capture of C. ferrugineus at Junction City and Abilene, we concluded that the sites were feasible for subsequent testing of synthetic pheromones.
3.2. Orientation to funnel traps with synthetic C-II Although the studies with bucket traps revealed active populations at both the Abilene and Junction City in 2014, the funnel trap experiment with synthetic pheromone in 2015 trapped very low numbers of beetles at these same locations. The low numbers prevented application of statistical analyses directly on all the raw data because many traps caught no C. ferrugineus. Therefore, any experimental set of two traps that captured one or no beetles was not included in the analysis. For the remaining two-trap sets in a given trapping period the numbers of beetles in the treatment traps were summed, as were the total numbers in the control traps, and then proportions of C. ferrugineus in treatment traps and control traps were calculated and the treatment effect was evaluated with a t-test following an arcsine transformation of the proportion data. Of the total 201 C. ferrugineus trapped at the Junction City location, funnel traps with wheat only caught on average 30.9% (±5.0%) of all beetles compared to an average of 69.1% (±5.0%) of all beetles from traps containing a synthetic C-II rubber septum and wheat, which was significantly higher (P ¼ 0.0015). The same treatments were
compared at the Abilene location with a total of 54 beetles trapped. Traps with wheat alone caught a mean percent (±SE) of 7.4% (±6.5%) beetles while traps containing synthetic C-II rubber septa and wheat caught 92.3% (±6.5%) beetles (P ¼ 0.0013; Fig. 4).
3.3. Two-choice laboratory bioassays Two-choice orientation bioassays with grains and grain-based products showed positive responses of beetles to both wheat (P ¼ 0.02) and corn meal (P ¼ 0.07), but there was no significant response to rolled oats or crushed dog food (P > 0.10 for both; ce Storgard Oil, a material used as a host volatile Fig. 5). Tre attractant in traps for grain beetles, elicited a significant positive response (P < 0.001), as did the Russell food lure (P < 0.01; Fig. 6). Ethanol was included in these laboratory assays because preliminary field trials (Losey, 2015) suggested its attractive activity for C. ferrugineus. Ethanol at 15 ml on filter paper elicited a positive response (P < 0.05) from responding beetles compared to filter paper only, but at the higher dose of 25 ml there was no significant effect (P > 0.10; Fig. 6). Three experiments were conducted to determine if semiochemicals released by C. ferrugineus adults feeding on grain would elicit a positive orientation response from other conspecific beetles (Fig. 7). In the two experiments using oats as a food odor source, beetles responded positively only when the control vial was empty (P < 0.05), thus the positive response to oats with beetles relative to an empty vial could have simply been from oats alone. As with the oats experiment, that with males on wheat vs wheat alone found no significant response to the vials containing beetles. Assays with synthetic CeI, C-II and a mixture of both compounds loaded on to rubber septa (Research Directions, Brisbane, Australia) versus blank septa elicited strong positive responses (P < 0.001 in three cases) from adult C. ferrugineus (Fig. 8). Bioassays with synthetic cucujolide II (Alpha Scents, Bend OR) dissolved in hexane in three different doses revealed significant positive orientation at 2500 ng (P < 0.05) and 5000 ng (P < 0.01) applied to filter paper, but not at 25 ng (P > 0.10; Fig. 8). When C. ferrugineus were assayed against synthetic pheromones from five different stored-product insect species we found positive orientation to pheromones of four of these species, including three beetle species
Fig. 2. Average percentage of C. ferrugineus caught in bucket traps on the north and west sides of a bin filled with rice at the Junction City location. A higher proportion were caught on the west side of the bin (F ¼ 6.225, df ¼ 1, 6, P < 0.05).
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Fig. 3. Average percentage of C. ferrugineus caught in bucket traps at four cardinal directions around a grain bin at the Abilene location. ANOVA found a significant treatment effect: F value ¼ 48.77, df ¼ 3, 6; P < 0.01. Compass directions with same letter above error bars are considered not statistically different from each other (Tukey HSD post hoc analysis).
Fig. 4. Average percent C. ferrugineus caught in Lindgren multiple funnel traps baited with synthetic C-II at the Junction City and Abilene locations. Paired t-tests found difference to be significant (p < 0.01) at both locations.
and one species of moth. Rubber septa containing the synthetic pheromone of the cigarette beetle, L. serricorne, elicited the strongest positive responses (P < 0.001) and lures for the lesser grain borer, R. dominica (P < 0.05) the larger grain borer, P. truncates (P < 0.01)and the Indian meal moth, P. interpunctella (P < 0.05) elicited lower but significantly positive responses from adult C. ferrugineus. Lures for the red flour beetle, T. castaneum, did not elicit a significant response compared to unbaited septa (P > 0.10) (see Fig. 9). 4. Discussion Conclusions
from
our
experiments
are
as
follows. 1)
C. ferrugineus adults flying near grain bins in north-central Kansas were more likely to be trapped on the west side of the bin compared to other directions. 2) Beetles in flight responded more to traps baited with the synthetic pheromone C-II with wheat compared to lower numbers responding to wheat only. 3) Walking beetles in laboratory assays oriented preferentially to the synthetic pheromones CeI and C-II. 4) Walking beetles oriented preferentially to volatiles from certain grains and grain-based materials. 5) Walking C. ferrugineus responded positively to pheromones from other species of stored product insects. These experiments provide new information about the activity of semiochemicals in host and mate-location behavior by C. ferrugineus as discussed below. We conducted preliminary studies on C. ferrugineus flight in
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Fig. 5. Responses of Cryptolestes ferrugineus to cracked wheat kernels, corn meal, rolled oats and commercial dog food using a two-choice pitfall bioassay. Data were analyzed using paired t-tests: p ¼ 0.02 for Wheat vs Blank, p ¼ 0.07 for Corn Meal vs Blank, and p > 0.10 (NS) for the other two comparisons (n ¼ 20).
Fig. 6. Responses of C. ferrugineus to commercial food-based lures and ethanol using two-choice pitfall bioassays. All data were analyzed using paired t-test, n ¼ 20 for each: *, P < 0.05; **, P < 0.01; ***, P < 0.001 NS, P > 0.1.
agricultural areas and found very few beetles were trapped at distances greater than 500 m away from stored grain (Losey, 2015). An Australian study (Holloway et al., 2018) also found that C. ferrugineus could be trapped in higher numbers with unbaited (passive) flight traps closest to grain silos compared to trapping very low numbers just 280 m (on average) away from the silos. We therefore decided to do pheromone experiments in open areas within 100 m from grain bins. Our bucket trap data showed that C. ferrugineus could be monitored very close to grain bins, which led us to do our subsequent field experiments with synthetic pheromone at those bin locations of Abilene and Junction City. The bucket trap results suggest a directional effect on captures for relative numbers near the bin, as the largest proportion of beetles were trapped on the westward side of the grain bin at each location. This
directional effect could be due to the flight behavior of C. ferrugineus relative to wind direction. The most common direction of wind flow at the Abilene and Junction City trapping locations during the weeks of this study was from the west or southwest, according to the US National Weather Service (www. weather.gov). Beetles flying with the wind from the west or southwest may have landed in the western bucket trap as they encountered the gran bin and were not flying around the bin to the east and north. Alternatively, beetles flying from the east, or those dispersing from the grain at the top if the bin may have encountered the westward bucket trap while flying upwind. Nansen et al. (2004a,b) working in Oklahoma hung unbaited sticky traps at different heights on the outside of stored wheat bins at the four cardinal directions and found significantly more C. ferrugineus
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Fig. 7. Responses of Cryptolestes ferrugineus to volatiles from conspecific adults feeding on grains using a two-choice pitfall bioassay. All data were analyzed using paired t-test, n ¼ 20 in each case: NS, P > 0.1; *, P < 0.05; n ¼ 20.
Fig. 8. Responses of C. ferrugineus to rubber septa loaded with the synthetic pheromones CeI and C-II, or to C-II applied in hexane to filter paper in different concentration in twochoice pitfall bioassays. Data were subjected to paired t-tests, n ¼ 20 for each experiment; NS, P > 0.1; *, P < 0.1; **, P < 0.01; ***, P < 0.001.
trapped on the north sides at two locations and these places had much of their wind coming from the south. However, that same study reported beetles were caught predominantly on the south side at other bins, and concluded the there was no consistent trend in beetle flight relative to wind or geographic aspect. We found that C. ferrugineus in flight will respond more to funnel traps with the synthetic pheromone C-II when compared side-by-side to traps without the pheromone. Field research with the male-produced aggregation pheromones of C. ferrugineus is very limited. The pheromones were first studied, identified and synthesized in the late 1970s and early 1980s (Borden et al., 1979; Wong et al., 1983; Oehlschlager et al., 1983) and at most there was just one field trial published at that time with inconclusive results (Loschiavo et al., 1986). The Canadian study by Loschiavo et al.
(1986) tested response of walking C. ferrugineus to pitfall-probe traps inserted into the top of a grain mass, so flight response was not assessed. Holloway et al. (2018), did field experiments in Australia with significant attraction of flying C. ferrugineus to 4-unit multiple funnel traps baited with both CeI and C-II compared to traps without pheromone. The Australian work reported low numbers of beetles trapped in flight, as in our experiments here, and with variation among several field sites. Nevertheless, the response to pheromone-baited traps had 3-folde20-fold more beetles responding to pheromones compared to traps without lures. Our experiments found pheromone-baited traps (C-II) had 2fold or 9-fold more beetles in pheromone vs non-pheromone traps across the two field sites. The Holloway et al. (2018) study and our experiments reported here are the most recent research on
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Fig. 9. Responses of Cryptolestes ferrugineus to rubber septum lures loaded with synthetic pheromones of five species of stored product pests using a two-choice pitfall bioassay. Data were analyzed using paired t-test; NS, P > 0.1; *, P ¼ 0.05; **, P < 0.01; ***, P < 0.001; n ¼ 20 for each experiment. Names of synthetic pheromones and the species they are intended to monitor are: DL-1þ2 for Rhyzopertha dominica, CB¼ Cigarette beetle (Lasioderma serricorne), RFB ¼ Red Flour Beetle (Tribolium castaneum), PT¼ Larger grain borer (Prostephanus truncatus), IMM¼ Indian meal moth (Plodia interpunctella).
pheromones for C. ferrugineus, and apparently the first and only reports, of pheromone responses by feral beetles flying in agricultural landscapes. Our experiments suggest that the relative importance of CeI and C-II, whether alone or separately, in orientation behavior and reproductive success of C. ferrugineus deserves further study. Our fieldwork found that C-II alone attracted beetles to traps, and the laboratory assays reported in Fig. 8 suggest the CeI and C-II illicit similar responses whether released separately or together. Although multiple pheromone systems are well known in other species to assure species-specificity in responses or to elicit a maximum response in combination, pheromones released as two or more isomers, or two or more structurally similar molecules, are known to be redundant in their activity (e.g., McBrien et al., 2002). Several species of phytophagous beetles, including pests of forests (Landolt, 1997; Borden, 1989; Byers, 1992; Raffa et al., 1993) and stored products (Burkholder and Ma, 1985; Phillips, 1997; Phillips et al., 2000), utilize aggregation pheromones that are produced by males feeding on host material that attract both males and females for mating and oviposition. The first study to demonstrate an aggregation pheromone for C. ferrigineus used a laboratory assay to show that males feeding on grain produced an attractant for both males and females (Borden et al., 1979), but we were unable to demonstrate the same effect with our laboratory assays (Fig. 6). Bioassays with mixed-sex adults feeding on oats, or males only feeding on wheat, did not attract beetles at a higher rate than either host material alone. The one case of more beetles attracted to adults with oats vs a blank vial did not control for the presence of oats, which could have been the source of attraction. It is possible that our two-choice assay did not reveal a significant effect for some comparisons, and that the bioassay of Borden et al. (1979), which differed from ours as being a no-choice response of beetles walking to test odors in an airstream, was more sensitive to beetle behavior when odors were tested singly than in a paired comparison. Although we could not show a pheromone effect with odors from feeding C. ferrugineus, we did show significant attraction of walking beetles to the synthetic CeI and C-II in the twochoice pitfall bioassays. Results of the first three experiments in Fig. 8 show that over twice as many beetles responded to rubber
septum lures containing either CeI, C-II, or a combination of both septa in a response vial compared to septa without these chemicals. We also observed positive responses of beetles responding to C-II applied to filter paper in the amounts of either 2,500 ng or 5,000 ng compared to untreated filter paper. These lab results corroborate the field trapping studies reported here and those of Holloway et al. (2018) that used lures with synthetic aggregation pheromone. The concentrations of synthetic pheromone released in these laboratory assays were most likely extremely high compared to amounts that may be released by living males. Rubber septa pheromone lures release synthetic pheromone at a stable rate, perhaps for several weeks to attract insects from long distances. Lures may release synthetic pheromone at rates of 1000s of ng per day, which could easily be two orders of magnitude higher than an insects natural release rate (McNeil, 1991). Information on the amounts of pheromone released by single insects is not available for C. ferrugineus, but we can assume it is much lower than the amounts of CeI or C-II released in our laboratory assays. Nevertheless, we demonstrated orientation of C. ferrugineus to the synthetic pheromones in the laboratory; the biology of production and release of pheromone in this species, and details on behavioral responses to pheromones, will require more research in the future. The results here support the practice of using food-based materials as attractants for C. ferrugineus. Some grain-based foods, such as cracked wheat and corn meal, were attractive to C. ferrugineus, while odors from rolled oats or dog food were not (Fig. 4). All four of these foods can serve as suitable feeding and reproductive media for this species (Arbogast, 1991), but the twochoice bioassays suggest they may not all have similar stimuli for orientation. Odors from grain-derived food materials such as Storgard Oil, the Russell Food Lure and ethanol elicited positive orientation in our experiments (Fig. 5). The commercial Russell food lure reportedly contains wheat germ oil and extracts of other foods used by storage pests and the Storgard Oil also contains wheat germ oil and additional materials, and both products are used with traps for stored product insects like C. ferrugineus (Wakefield et al., 2006; Collins et al., 2007). Ethanol, a common fermentation product of grain-based foods, elicited a significant positive response at 15 ml
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applied to filter paper (Fig. 6). Ethanol is associated with moist and decomposing grain, typically a result of microbial activity, and is likely to be associated with preferred food sources for this species (Nout and Bartelt, 1998). This result suggests ethanol may be a useful semiochemical for detecting or monitoring C. ferrugineus. Interspecific attraction by one species of insect to a pheromone of another species is known to occur in many cases, including the chemical ecology of stored product insects (e.g., Edde and Phillips, 2006; Phelan and Baker, 1986). We report here C. ferrugineus displayed positive responses to the synthetic pheromones of three beetle species and a common pheromone of several moths in laboratory tests (Fig. 9). It is possible that these results reflect some adaptive response of C. ferrugineus for locating suitable host material that is already infested by other stored product pests releasing their pheromones in a given ecosystem. Interspecific attraction to a common and presumably rare host resource can be considered maladaptive for the impact of competition. However, such responses are proposed to be mutually adaptive when considering increased reproductive fitness for the responders (e.g. Baker, 1989 for moths) or for intraspecific successful invasion of a new food source by multiple species (e.g., Kausrud, et al., 2011 for tree-killing bark beetles). Nevertheless, the results here are very limited, but suggest that future work on interspecific attraction for C. ferrugineus may elaborate these interactions and provide additional semiochemical tools for use in IPM applications. Pheromones for many stored-product pests were identified and synthesized over the past 50 years, and several are commercially available or used commercially for pest detection, monitoring and mating disruption (Phillips, 1997; Phillips et al., 2000; Phillips and Throne, 2010). Although pheromones for C. ferrugineus and other beetle pests in the Laemophloeidae and Silvanidae were identified starting in the 1980s (e.g. Oehlschlager et al., 1983), the lack of commercial pheromone lures has limited pheromone research and applications for this and related species up to this time. The availability of synthetic pheromones like those studied here should now allow for more research on flight behavior, pheromone-directed orientation and perhaps for developing new tools for detecting, monitoring and managing this pest. Acknowledgements The authors gratefully acknowledge the support of Cooperative Research Centre for National Plant Biosecurity (Project CRC3039), established and supported under the Australian Government’s Cooperative Research Centres program (http://www. crcplantbiosecurity.com.au). This research was supported by the Professor Donald Wilbur Endowed Professorship for StoredProduct Protection at Kansas State University. The authors are grateful to Darek Czokajlo of Alpha-Scents for providing the synthetic C-II. Dr. Ching Kang provided valuable advice and consultation on statistical analyses. This article represents publication number 20-039-J from the Kansas Agricultural Experiment Station. References Arbogast, R.T., 1991. Beetles: Coleoptera, pp. 131-176. In: Gorham, J.R. (Ed.), Ecology and Management of Food-Industry Pests. FDA Technical Bulletin 4. Association of Analytical Chemists, VA. Baker, T.C., 1989. Sex pheromone communication in Lepidoptera: new research progress. Experientia 45, 248e262. Borden, J.H., 1989. Semiochemicals and bark beetle populations: exploitation of natural phenomena by pest management strategists. Holarctic Ecology 12, 501e510. Borden, J.H., Dolinski, M.G., Chong, L., Verigin, V., Pierce Jr., H.D., Oehlschlager, A.C., 1979. Aggregation pheromone in the rusty grain beetle, Cryptolestes ferrugineus (Coleoptera: Cucujidae). Can. Entomol. 111, 681e688. Burkholder, W.E., Ma, M., 1985. Pheromones for monitoring and control of storedproduct insects. Annu. Rev. Entomol. 30, 257e272.
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Byers, J.A., 1992. Attraction of bark beetles Tomicus piniperda, Hylurgus palliates, and Trypodendron domesticus and other insects to short-chain alcohols monoterpenes. J. Chem. Ecol. 18, 2385e2402. Canadian Grain Commission, 2013. Rusty Grain Beetle Cryptolestes ferrugineus. Steph.. https://www.grainscanada.gc.ca/storage-entrepose/pip-irp/rgb-cr-eng. htm Collins, L.E., Bryning, G.P., Wakefield, M.E., Chambers, J., Cox, P.D., 2007. Progress towards a multi-species lure: identification of components of food volatiles as attractants for three storage beetles. J. Stored Prod. Res. 43, 53e63. Cuperus, G.W., Prickett, C.K., Bloome, P.D., Pitts, J.T., 1986. Insect populations in aerated and unaerated stored wheat in Oklahoma. J. Kans. Entomol. Soc. 59, 620e627. Czeskis, B.A., Shpiro, N.A., Moiseenkov, A.M., 1994. Efficient Synthesis of (S,Z)Dodec-3-en-11-olide (Ferrulactone II) Using 2-Carboxyethyltriphenylphosphonium Bromide. ChemInform 25 (Abstract). Edde, P.A., Phillips, T.W., 2006. Field responses of non-target species to semiochemicals of stored-product Bostrichidae. Ann. Entomol. Soc. Am. 99, 175e183. Fargo, W.S., Epperly, D., Cuperus, G.W., Clary, B., Noyes, R., 1989. Effects of temperature and duration of trapping on four stored grain insect species. J. Econ. Entomol. 82, 970e973. Fargo, W.S., Cuperus, G.W., Bonjour, E.L., Burkholder, W.E., Clary, B.L., Payton, M.E., 1994. Influence of probe trap type and attractants on the capture of four storedgrain Coleoptera. J. Stored Prod. Res. 30, 237e241. Hagstrum, D.W., 2000. Using five sampling methods to measure insect distribution and abundance in bins storing wheat. J. Stored Prod. Res. 36, 253e262. Hagstrum, D.W., Subramanyam, B., 2009. Table 1.1 Insects and their natural enemies associated with stored products, p 183. In: Hagstrum, D.W., Subramanyam, B. (Eds.), Stored-product Insect Resource. AACC International Inc., St. Paul, MN. Hagstrum, D.W., Phillips, T.W., Cuperus, G. (Eds.), 2012. Stored Product Protection. Kansas State University, Manhattan, KS, p. 352. http://ksre.ksu.edu/library/p. aspx?tabid¼70. Holloway, J.C., Mayer, D.G., Daglish, G.J., 2018. Flight activity of Cryptolestes ferrugineus in southern New South Wales, Australia. J. Pest Sci. 91, 1353e1362. https://doi.org/10.1007/s10340-018-0981-1. goire, J.C., Skarpaas, O., Erbilgin, N., Gilbert, M., Økland, B., Kausrud, K.L., Gre Stenseth, N.C., 2011. Trees wantedddead or alive! Host selection and population dynamics in tree-killing bark beetles. PLoS One 6 (5), e18274. https:// doi.org/10.1371/journal.pone.0018274. Konemann, C.E., Hubhachen, Z., Opit, G.P., Gautam, S., Bajracharya, N.S., 2017. Phosphine resistance in Cryptolestes ferrugineus (Coleoptera: Laemophloeidae) collected from grain storage facilities in Oklahoma, USA. J. Econ. Entomol. 110 (3), 1377e1383. https://doi.org/10.1093/jee/tox101. Landolt, J.P., 1997. Sex attractant and aggregation pheromones of male phytophagous insects. Am. Entomol. 43e 1. Lindgren, B.S., 1983. A multiple-funnel trap for Scolytid beetles (Coleoptera). Can. Entomol. 115, 299e302. Loschiavo, S.R., Wong, J., White, N.D.G., Pierce Jr., H.D., Borden, J.H., Oehlschlager, A.C., 1986. Field-Evaluation of a pheromone to detect adult rusty grain beetles, Cryptolestes ferrugineus (Coleoptera: Cucujidae), in stored grain. Can. Entomol. 118, 1e8. Losey, S., 2015. Orientation and dispersal of Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae) is response to various semiochemicals. http:// krex.k-state.edu/dspace/bitstream/handle/2097/20566/StephenLosey2015.pdf? sequence¼1&isAllowed¼y, 51 pp. McBrien, H.L., Millar, J.G., Rice, J.G., McElfresh, J.S., Cullen, E., Zalom, F.G., 2002. Sex attractant pheromone of the red-shouldered stink bug Thyanta pallidovirens: a pheromone blend with multiple redundant components. J. Chem. Ecol. 28, 1797e1818. McNeil, J.N., 1991. Behavioral ecology of pheromone-mediated communication in moths and its importance in the use of pheromone traps. Annu. Rev. Entomol. 36, 407e430. Moeck, H., 1970. Ethanol as the primary attractant for the ambrosia beetle Trypodentron lineatum (Coleoptera: Scolytidae). Can. Entomol. 102, 985e995. Nansen, C., Bonjour, E.L., Phillips, T.W., Vela-Coiffier, E.L., Gates, M.W., 2004a. Timedelayed relationship between flight activity outside grain bins and probe trap catches inside grain bins of Cryptolestes ferrugineus. Environ. Entomol. 33, 1465e1470. Nansen, C., Bonjour, E.L., Gates, M.W., Phillips, T.W., Cuperus, G.W., Payton, M.E., 2004b. Model of Cryptolestes ferrugineus flight activity outside commercial steel grain bins in central Oklahoma. Environ. Entomol. 33, 426e434. Norwood, C., 2015. New lure to trap rusty grain beetle. https://grdc.com.au/ resources-and-publications/groundcover/ground-cover-supplements/groundcover-issue-119-grain-storage/new-lure-to-trap-rusty-grain-beetle. (Accessed 5 July 2018). Nayak, M.K., Holloway, J.C., Emery, R.N., Pavic, H., Bartlet, J., Collins, P.J., 2013. Strong resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae): its characterization, a rapid assay for diagnosis and its distribution in Australia. Pest Manag. Sci. 69, 48e53. https:// doi.org/10.1002/ps.3360, 2013. Nout, M.J.R., Bartelt, R.J., 1998. Attraction of a flying nitidulid (Carpophilus humeralis) to volatiles produced by yeasts grown on sweet corn and a corn-based medium. J. Chem. Ecol. 24, 1217e1239. Oehlschlager, A.C., Wong, J.W., Verigin, V.G., Pierce, H.D.J., 1983. Synthesis of two macrolide pheromones of the rusty grain beetle, Cryptolestes ferrugineus (Stephens). J. Org. Chem. 4, 5009e5017.
10
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Phelan, P.L., Baker, T.C., 1986. Cross-attraction of five species of stored-product Phycitinae (Lepidoptera: Pyralidae) in a wind tunnel. Environ. Entomol. 15, 369e372. Phillips, T.W., 1997. Semiochemicals of stored-product insects: research and applications. J. Stored Prod. Res. 33, 17e30. Phillips, T.W., Cogan, P.M., Fadamiro, H.Y., 2000. Pheromones. Pages 273-302. In: Subramanyam, Bh, Hagstrum, D.W. (Eds.), Alternatives to Pesticides in StoredProduct IPM. Kluwer Academic Publishers, Boston. Phillips, T.W., Throne, J.E., 2010. Biorational approaches to managing stored-product insects. Annu. Rev. Entomol. 55, 375e397. Pierce, A.M., Borden, J.H., Oehlschlager, A.C., 1981. Olfactory response to beetleproduced volatiles and host-food attractants by Oryzaephilus surinamensis and O. mercator. Can. J. Zool. 59, 1980e1990. R Core Team, 2014. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project. org/. Raffa, K.F., Phillips, T.W., Salom, S.M., 1993. Mechanisms and strategies of host colonization by bark beetles. In: Schowalter, T., Filip, G. (Eds.), Interactions Among Bark Beetles, Pathogens and Conifers in North American Forests. Academic Press, London, pp. 103e128, 252. Reed, C.R., Wright, V.F., Mize, T.W., Pedersen, J.R., Brockschmidt-Evans, J., 1991. Pitfall traps and grain samples as indicators of insects in farm-stored wheat. J. Econ. Entomol 84, 1381e1387. SAS, 2002. User’s Guide, v. 8. SAS Institute, Cary, NC. Sinha, R.N., 1961. Insects and mites associated with hot spots in farm stored grain. Can. Entomol. 93, 609e621.
Subramanyam, Bh, Hagstrum, D.W., Schenk, T.C., 1993. Sampling adult beetles (Coleoptera) associated with stored grain: comparing detection and mean trap catch efficiency of two types of probe traps. Environ. Entomol 22, 33e42. Swords, P., Van Ryckeghem, A., 2010. Summary of commercially available pheromones of common stored-product beetles. In: Carvalho, M.O., Fields, P.G., Adler, C.S., Arthur, F.H., Athanassiou, C.G., Campbell, J.F., et al. (Eds.), Proceedings of the 10th International Working Conference on Stored-Product Protection, 27 June- 2 July, 2010 2010 Estoril, Portugal. Julius Kuhn-Institut, Berlin, Germany, pp. 1004e1007. Toews, M.D., Phillips, T.W., Payton, M.E., 2005. Estimating populations of grain beetles using probe traps in wheat-filled concrete silos. Environ. Entomol. 34, 712e718. Wakefield, M.E., Collins, L.E., Bryning, G.P., Chambers, J., Cox, P.D., Fennah, K., Zaidi, S., Cross, D., Braithwaite, M., 2006. Improved Detection and Monitoring of Beetle Pests in Stored Grain through Use of a Multi-Species Lure (LK0929). Project Report No. 396. Home Grown Cereals Authority. White, N.D.G., Loschiavo, S.R., 1986. Effects of insect density, trap depth, and attractants on the capture of Tribolium castaneum (Coleoptera: Tenebrionidae) and Cryptolestes ferrugineus (Coleoptera: Cucujidae) in stored wheat. J. Econ. Entomol. 79, 1111e1117. Wong, J.W., Verigin, V., Oehlschlager, A.C., Borden, J.H., Pierce, H.D., Pierce, A.M., Chong, L., 1983. Isolation and identification of two macrolide pheromones from the frass of Cryptolestes ferrugineus (Coleoptera: Cucujidae). J. Chem. Ecol. 9, 451e474. Zar, J.H., 1984. Biostatistical Analysis, second ed. Prentice-Hall Inc., Englewood Cliffs, NJ. 718 p.
Contents lists available at ScienceDirect
Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
Orientation of rusty grain beetles, Cryptolestes ferrugineus (Coleoptera: Laemophloeidae), to semiochemicals in field and laboratory experiments Stephen M. Losey a, Gregory J. Daglish b, c, Thomas W. Phillips a, c, * a b c
Department of Entomology, Kansas State University, Manhattan, KS, 66506, USA Department of Agriculture and Fisheries, Queensland, Ecosciences Precinct, GPO Box 267, Brisbane, QLD, 4001, Australia Plant Biosecurity Cooperative Research Centre, GPO Box 5012, Bruce, ACT, 2617, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2019 Received in revised form 16 August 2019 Accepted 10 September 2019 Available online xxx
Cryptolestes ferrugineus is a common beetle pest of stored grain for which techniques to monitor dispersing beetles are limited. Early research found several male-produced aggregation pheromones, but there has been little related work since that time. This paper reports experiments on orientation of C. ferrugineus in response to synthetic pheromones and other semiochemicals via flight and walking. Field trapping studies showed that flying beetles were caught on the western sides of grain bins in Kansas compared to the other three cardinal directions. Work with synthetic formulations of the two male-produced aggregation pheromones found that flying beetles were attracted to traps with the pheromone and wheat compared to traps with wheat only. Walking bioassays in the laboratory determined that either of the two aggregation pheromones, known as Cucujolide I and Cucujolide II, were attractive whether deployed singly or in combination. Laboratory bioassays showed that volatiles from grains, other grain-based materials and two commercial food attractants used in traps were attractive to C. ferrugineus. Further laboratory assays demonstrated that C. ferrugineus would orient to synthetic pheromones of three other beetle species and one moth species, all common pests of stored products. These new data on semiochemicals for C. ferrugineus suggest future work that could be done toward developing new tools for detecting and monitoring this serious pest. Crown Copyright © 2019 Published by Elsevier Ltd. All rights reserved.
Keywords: Pheromones Food attractants Traps Stored grain Cucujolide
1. Introduction The rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae), is among the most common grain pests wherever cereal grains are stored and marketed (Canadian Grain Commission, 2013; Hagstrum et al., 2012; Cuperus et al., 1986). Cryptolestes ferrugineus can infest all cereal grains in storage, and there are records of this pest infesting up to 69 different commodities worldwide such as black pepper, chili, cocoa, coffee bean, cotton seed, dried fruits, hemp, licorice, oilseeds, peanuts, rice, dried tomatoes, and yams (Hagstrum and Subramanyam, 2009). Cryptolestes ferrugineus can cause heating when infesting stored grain (Sinha, 1961), and it can damage wheat by consuming
* Corresponding author. Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS, 66506, USA. E-mail address: [email protected] (T.W. Phillips). https://doi.org/10.1016/j.jspr.2019.101513 0022-474X/Crown Copyright © 2019 Published by Elsevier Ltd. All rights reserved.
the germ. Both adults and larvae feed mostly on damaged kernels, grain dust, and fungi in stored grain. Cryptolestes ferrugineus populations can increase rapidly requiring effective mitigation before grain value is lost. Unfortunately, C. ferrugineus populations from different parts of the world have evolved resistance to the commonly used grain fumigant phosphine (e.g. Nayak et al., 2013; Konemann et al., 2017), necessitating good methods for early detection of this pest before populations surpass fumigation action thresholds. Detection and monitoring of C. ferrugineus in stored grain can be done by sifting samples of grain either at point of sale or over the course of storage, though collection of samples from stored grain can be very challenging and dangerous. Perforated probe traps (Subramanyam et al., 1993) inserted below the grain surface are effective in capturing C. ferrugineus adults (Reed et al., 1991; Phillips et al., 2000). Adults of C. ferrugineus walking through the grain mass encounter a probe trap, fall through a hole to a receptacle at the bottom where they can be counted several days or weeks after trap
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placement. There are several factors that affect the capture of C. ferrugineus adults in probe traps. These factors include the temperature of the grain mass that can affect insect activity, density of insects, and trap location within the grain mass (White and Loschiavo, 1986; Fargo et al. 1989, 1994; Subramanyam et al., 1993; Hagstrum, 2000). Toews et al. (2005) showed that the number of C. ferrugineus captured in probe traps at the grain surface and 1 m below the surface were similar. Unbaited sticky traps have been used to monitor the flight activity of adult C. ferrugineus near grain bins (Nansen et al., 2004). Although attractant pheromones have been described for over 50 pest species of stored-product insects, and commercial pheromone lures are available for about half of those, pheromone-baited traps for monitoring species of Cryptolestes have received limited commercial development if any at this time, and are not widely marketed or used, to our knowledge (Swords and Van Ryckeghem, 2010). Borden et al. (1979) were the first to report on the aggregation pheromones produced by male C. ferrugineus while feeding that are attractive to both sexes. These pheromones act as intraspecific signals used in mate-finding and host colonization and were first isolated and identified by Wong et al. (1983). The aggregation pheromones consist of two macrolide lactones, and these were identified as ferrulactone I and II, (E, E)-4, 8-decadien-10-olide and (S)-(Z)-3-dodecen-11-olide. These chemicals are commonly referred to as cucujolide I and II, respectively (hereafter referred to as CeI and C-II). A 9:1 mixture of CeI and C-II was determined to generate the highest response in laboratory experiments compared with other mixture ratios (Wong et al., 1983). Loschiavo et al. (1986) conducted the first field study using synthetic versions of the maleproduced aggregation pheromone against adults in a grain bin in Canada and found that the trap containing synthetic pheromone did not catch more adults than the control trap. Those experiments used baited and non-baited probe traps inserted into stored grain and did not directly assess flight or walking responses of beetles outside of grain bins. Research and applications with the aggregation pheromones of C. ferrugineus have been limited due to the lack of synthetic pheromone for use by researchers. Oehlschlager et al. (1983) reported on the synthesis of the two macrolides, and a useful method for synthesis of one of the macrolides was published soon after (Czeskis et al., 1994), but synthetic pheromones for commercial uses were not available until recently (Norwood, 2015). Holloway et al. (2018) recently published results from field experiments using synthetic pheromone lures containing CeI and C-II of C. ferrugineus and provided new information on seasonal and spatial patterns of flight for this species in Australia. Nansen et al. (2004a,b) provided information about flight of C. ferrugineus outside grain bins in Oklahoma, but information on flight of this
pest outside grain bins in North America is otherwise limited. The objectives of the research reported here were to: 1) evaluate orientation of C. ferrugineus in flight near bins of stored wheat; 2) determine if C. ferrugineus would respond in flight to traps baited with a synthetic pheromone, and 3) evaluate responses of C. ferrugineus to synthetic pheromones and to other potential semiochemicals in laboratory bioassays. 2. Materials and methods 2.1. Flight near grain bins assessed with wheat-filled bucket traps Bucket traps with wheat were used to capture flying C. ferrugineus adults near infested grain bins and to assess the feasibility of such field sites for subsequent testing of synthetic pheromones. Bucket traps were 18.9 L opened plastic paint buckets protected from rain with a modified bird-feeder squirrel baffle cover that was hung from a trap stand (Fig. 1). A piece of hardware cloth with 1.9 cm openings was clipped down over the top of the bucket to prevent small vertebrates from infesting the grain while allowing entry of beetles into the bucket. The bucket trap assembly was suspended from its trap stand at approximately 12.7 cm above the ground (Fig. 1). This design allowed the bucket trap to move with the wind but not be blown away or overturned, to keep the majority of rainwater and debris out with the squirrel baffle so as to also allow responding beetles go inside the bucket. Bucket traps contained 670 g of soft white wheat to provide food for and retain responding C. ferrugineus, and possibly serve as a natural attractant for the flying beetles. Bucket trap experiments were conducted near the cities of Junction City and Abilene in north-central Kansas from 24 July to 12 September 2014. Four bucket traps were placed around a single grain bin at the Abilene site that was approximately 18 m in diameter and 15 m tall and contained wheat that was known to be infested with many species of stored-product insects including C. ferrugineus. A single bucket trap was placed at each of the four cardinal directions around the bin, approximately 6 m from the bin sidewall. Spacing of grain bins at the Junction City location allowed for only two bucket traps to be placed around a bin with infested rice Oryza sativa L., that was approximately 15 m diameter x 22 m tall. One trap was placed on the west side of the bin and one on the north side of the bin. Bucket traps at each location were checked weekly for 7 weeks, with each week serving as an experimental replicate by time at each location in lieu of the lack of multiple bins at a site to serve as true replicates. Insects were sifted, removed and counted from each bucket. The old wheat was discarded and replaced with new wheat for each subsequent week of trapping. Numbers of C. ferrugineus trapped in a week at a given bin were
Fig. 1. Materials used to assess orientation of C. ferrugineus: bucket trap containing wheat (left), 4-unit funnel trap (center) and the two-choice pitfall bioassay (right). See text for details.
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converted to proportion of beetles trapped at given direction relative to the bin, and proportion data were then subjected to the angular transformation (Zar, 1984) before subjecting to analysis of variance (ANOVA; SAS, 2002). 2.2. In-flight field responses to cucujolide-II Four-unit Lindgren funnel traps (Phero Tech Inc., Delta, BC, Canada) were used in experiments at the Junction City and Abilene sites from 7 August to 17 September 2015 to assess the activity of synthetic C-II provided by Alpha Scents Inc. (West Linn, OR, USA). Funnel traps capture insects that fly into a vertically oriented series of funnels and then fall through the funnels into a collection cup at the bottom (Fig. 1; Lindgren, 1983). Sets of two traps spaced 20 m apart and placed at least 30 m away from any other pair of traps, were deployed at two different locations. The Abilene site had two sets of traps arranged in a line from east to west 50 m south of a grouping of five steel grain bins. The Junction City location had four sets of funnel traps arranged in an east-west line, with two sets at the north edge of the property and two sets 100 m away at the south edge of the property. Each trap had a small mesh bag that contained approximately 10 g of partially crushed wheat wired to the vertical connector between the 3rd and 4th funnels allowing the bag to hang freely inside the free space of the funnel above the collection cup. One trap within each two-trap set was randomly selected as the experimental control and had wheat only, while the second trap in a set was the experimental treatment with a cucujolide-II lure and a bag of wheat. Pheromone lures with synthetic pheromone absorbed onto a rubber stopper septum (Fisher Scientific, Pittsburg, PA, USA) containing either 25 mg of cucujolideII applied in hexane, or hexane only as a control, were added inside each trap such that the septum did not touch the trap. Each trap contained a piece of crumpled paper towel in the collection cup to provide a harborage for trapped beetles and a 1 cm2 piece, 0.7 g, of a Hot Shot® No-Pest® Strip2 (Spectrum Brands, Inc., Middleton, Wisconsin USA) with the active ingredient of dichlorvos at 18.6%, in the collection cup as a killing agent. Traps at each location were checked every 1e2 days. After collecting and counting the trapped beetles in each trap in a set, the trap position in each set was randomly re-assigned before the next trapping period. Numbers trapped in the traps with C-II and in the number in control traps were summed across all sets of traps for a given trapping time, with each of seven trapping times serving as a replicate (n ¼ 7). Total beetles trapped for treatment and control traps were converted into proportions of trapped C. ferrugineus from the total nubmer trapped for that time replicate, and were then subjected to the angular transformation (Zar, 1984) before analysis with Student's ttest (SAS, 2002). 2.3. Laboratory assays of semiochemicals Laboratory experiments were conducted to examine orientation to synthetic pheromones and to additional semiochemicals by C. ferrugineus. Laboratory experiments were conducted using beetles from an established colony of C. ferrugineus obtained from the USDA Center for Grain and Animal Health Research in Manhattan, KS. These insects were reared on a diet consisting of rolled oats and brewer's yeast (95:5) in 508 ml glass jars covered using filter paper in the screw-on ring lid to ensure air and moisture diffusion. Colonies were maintained in a growth chamber at 28 C and 50e70% RH. Adult insects used in bioassays were 3e4 weeks old and were separated from their food substrate using a US number 12 sieve (Fisher Scientific). Beetles were then held without food in ventilated class vials in groups of 20 for 2e3 h before being used in bioassays to ensure uniform starvation and
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to possibly enhance their olfactory response after being deprived of food. We used a two-choice pitfall bioassay for these experiments (Fig. 1), like that used by Pierce et al. (1981). The bioassay used two 11 ml shell vials, 19 mm in diameter x 50 mm tall, and a glass Petri dish of 100 mm in diameter x 20 mm in height (Fisher Scientific). Each dish had two 6 mm diameter holes in the floor, placed 40 mm apart and 15 mm from the side walls positioned along a mid-line of the dish floor. Each dish had its vials stabilized in 21-mm holes that were drilled into a wooden board and spaced so that the glass dishes could be placed directly over the vials. Up to 10 bioassay dishes were set up at a given time in a small room free of insect or grain materials and maintained at 22e24 C, 30% RH in complete darkness. Glassware was washed using soap and water, rinsed with deionized water, and then rinsed with acetone and dried in an oven at 65 C for a minimum of 2 h prior to use in all assays. Each twochoice dish assay evaluated the orientation of 20 mixed sex adult C. ferrugineus by the number falling into a given vial or remaining on the arena floor. Treatment and control were assigned at random to a given vial in each two-choice bioassay. A water suspension of polytetrafluoroethylene (Sigma-Aldrich; St. Louis, MA, USA) was applied to the top inside-wall of each dish and vial to prevent escape of beetles by walking up the vertical glass surfaces. The cover was removed from each Petri dish, the vial holding the 20 beetles was carefully overturned on the dish-bottom at the midpoint between the two holes, and the vial kept in place for 10 min to allow beetles to acclimate. The overturned vial was then removed and the dish cover replaced to allow beetles to move freely in the dish under complete darkness for 18 h, after which the lights in the bioassay room were turned on and the number of beetles in the main dish arena and the treatment and control vials was recorded for each bioassay unit. Laboratory bioassays studied the responses of C. ferrugineus to different food or insect-produced volatiles, some of which were grains or grain-related materials and others that were pheromones. Food baits included the commercially marketed “Storgard Oil” used ce Inc. (Adair, OK, USA); a commercial in traps and provided by Tre food lure for traps from Russell IPM Ltd (Deeside, Flintshire, UK) and laboratory grade ethanol (Fisher Scientific, Waltham, MA, USA), which is known as a fermentation product from grains and other foods that can attract insects (e.g. Moeck, 1970). The whole or processed grains of wheat, corn as corn meal or oats, and a grainbased dog food (Purina, St. Louis) were also tested. We evaluated the synthetic C-II pheromone of C. ferrugineus (Alpha Scents, Inc., West Bend, OR, USA), diluted in hexane and applied at 25 ng to 5,000 ng to 1.0 cm2 pieces of filter paper vs hexane only on filter paper as a control. We also evaluated rubber septum lures already formulated with CeI, C-II or a combination of CeI and C-II purchased from Research Directions Pty Ltd (Queensland, Australia) vs. rubber septa with hexane only as controls. Synthetic pheromones of the following species formulated in rubber septa and provided by ce Inc. were as follows: Lasioderma serricorne (F.) (Coleoptera: Tre Anobiidae), Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae), Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). All two-choice assays with pheromone lures had a rubber septum not formulated with pheromone as a control. Test materials for the two-choice assays were placed in the bottom of one shell vial and the second vial was either empty or contained a neutral material (e.g., blank septum or hexane on filter paper) to serve as the untreated control. Each bioassay experiment had 20 replicate dishes. Numbers of beetles responding to treatment and control vials for each experiment were analyzed using a paired t-test (R Core Team, 2014).
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3. Results 3.1. Orientation to bucket traps in the field A total of 410 C. ferrugineus were captured in the two wheatfilled bucket traps at the Junction City location. On average, 63.1% (±7.4% SE) of beetles were captured on the west side and 37.0% (±7.4% SE) on the north side over seven 1-week trapping periods, with the proportions captured on the two sides being significantly different (Fig. 2; F ¼ 6.225, df ¼ 1, 6, P < 0.05). A total of 66 C. ferrugineus were captured in the four bucket traps at the Abilene location over the seven replicate weeks. A significantly higher proportion of beetles was captured on the west side (Fig. 3; F value ¼ 48.77, df ¼ 3, 6; P < 0.01). On average, 4.2% (±4.1% SE) of beetles were captured on the north, compared with 9.6% (±6.0% SE) on the east, 7.9% (±3.4% SE) on the south and 78.3% (±7.1% SE) on the west. On the basis of the capture of C. ferrugineus at Junction City and Abilene, we concluded that the sites were feasible for subsequent testing of synthetic pheromones.
3.2. Orientation to funnel traps with synthetic C-II Although the studies with bucket traps revealed active populations at both the Abilene and Junction City in 2014, the funnel trap experiment with synthetic pheromone in 2015 trapped very low numbers of beetles at these same locations. The low numbers prevented application of statistical analyses directly on all the raw data because many traps caught no C. ferrugineus. Therefore, any experimental set of two traps that captured one or no beetles was not included in the analysis. For the remaining two-trap sets in a given trapping period the numbers of beetles in the treatment traps were summed, as were the total numbers in the control traps, and then proportions of C. ferrugineus in treatment traps and control traps were calculated and the treatment effect was evaluated with a t-test following an arcsine transformation of the proportion data. Of the total 201 C. ferrugineus trapped at the Junction City location, funnel traps with wheat only caught on average 30.9% (±5.0%) of all beetles compared to an average of 69.1% (±5.0%) of all beetles from traps containing a synthetic C-II rubber septum and wheat, which was significantly higher (P ¼ 0.0015). The same treatments were
compared at the Abilene location with a total of 54 beetles trapped. Traps with wheat alone caught a mean percent (±SE) of 7.4% (±6.5%) beetles while traps containing synthetic C-II rubber septa and wheat caught 92.3% (±6.5%) beetles (P ¼ 0.0013; Fig. 4).
3.3. Two-choice laboratory bioassays Two-choice orientation bioassays with grains and grain-based products showed positive responses of beetles to both wheat (P ¼ 0.02) and corn meal (P ¼ 0.07), but there was no significant response to rolled oats or crushed dog food (P > 0.10 for both; ce Storgard Oil, a material used as a host volatile Fig. 5). Tre attractant in traps for grain beetles, elicited a significant positive response (P < 0.001), as did the Russell food lure (P < 0.01; Fig. 6). Ethanol was included in these laboratory assays because preliminary field trials (Losey, 2015) suggested its attractive activity for C. ferrugineus. Ethanol at 15 ml on filter paper elicited a positive response (P < 0.05) from responding beetles compared to filter paper only, but at the higher dose of 25 ml there was no significant effect (P > 0.10; Fig. 6). Three experiments were conducted to determine if semiochemicals released by C. ferrugineus adults feeding on grain would elicit a positive orientation response from other conspecific beetles (Fig. 7). In the two experiments using oats as a food odor source, beetles responded positively only when the control vial was empty (P < 0.05), thus the positive response to oats with beetles relative to an empty vial could have simply been from oats alone. As with the oats experiment, that with males on wheat vs wheat alone found no significant response to the vials containing beetles. Assays with synthetic CeI, C-II and a mixture of both compounds loaded on to rubber septa (Research Directions, Brisbane, Australia) versus blank septa elicited strong positive responses (P < 0.001 in three cases) from adult C. ferrugineus (Fig. 8). Bioassays with synthetic cucujolide II (Alpha Scents, Bend OR) dissolved in hexane in three different doses revealed significant positive orientation at 2500 ng (P < 0.05) and 5000 ng (P < 0.01) applied to filter paper, but not at 25 ng (P > 0.10; Fig. 8). When C. ferrugineus were assayed against synthetic pheromones from five different stored-product insect species we found positive orientation to pheromones of four of these species, including three beetle species
Fig. 2. Average percentage of C. ferrugineus caught in bucket traps on the north and west sides of a bin filled with rice at the Junction City location. A higher proportion were caught on the west side of the bin (F ¼ 6.225, df ¼ 1, 6, P < 0.05).
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Fig. 3. Average percentage of C. ferrugineus caught in bucket traps at four cardinal directions around a grain bin at the Abilene location. ANOVA found a significant treatment effect: F value ¼ 48.77, df ¼ 3, 6; P < 0.01. Compass directions with same letter above error bars are considered not statistically different from each other (Tukey HSD post hoc analysis).
Fig. 4. Average percent C. ferrugineus caught in Lindgren multiple funnel traps baited with synthetic C-II at the Junction City and Abilene locations. Paired t-tests found difference to be significant (p < 0.01) at both locations.
and one species of moth. Rubber septa containing the synthetic pheromone of the cigarette beetle, L. serricorne, elicited the strongest positive responses (P < 0.001) and lures for the lesser grain borer, R. dominica (P < 0.05) the larger grain borer, P. truncates (P < 0.01)and the Indian meal moth, P. interpunctella (P < 0.05) elicited lower but significantly positive responses from adult C. ferrugineus. Lures for the red flour beetle, T. castaneum, did not elicit a significant response compared to unbaited septa (P > 0.10) (see Fig. 9). 4. Discussion Conclusions
from
our
experiments
are
as
follows. 1)
C. ferrugineus adults flying near grain bins in north-central Kansas were more likely to be trapped on the west side of the bin compared to other directions. 2) Beetles in flight responded more to traps baited with the synthetic pheromone C-II with wheat compared to lower numbers responding to wheat only. 3) Walking beetles in laboratory assays oriented preferentially to the synthetic pheromones CeI and C-II. 4) Walking beetles oriented preferentially to volatiles from certain grains and grain-based materials. 5) Walking C. ferrugineus responded positively to pheromones from other species of stored product insects. These experiments provide new information about the activity of semiochemicals in host and mate-location behavior by C. ferrugineus as discussed below. We conducted preliminary studies on C. ferrugineus flight in
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Fig. 5. Responses of Cryptolestes ferrugineus to cracked wheat kernels, corn meal, rolled oats and commercial dog food using a two-choice pitfall bioassay. Data were analyzed using paired t-tests: p ¼ 0.02 for Wheat vs Blank, p ¼ 0.07 for Corn Meal vs Blank, and p > 0.10 (NS) for the other two comparisons (n ¼ 20).
Fig. 6. Responses of C. ferrugineus to commercial food-based lures and ethanol using two-choice pitfall bioassays. All data were analyzed using paired t-test, n ¼ 20 for each: *, P < 0.05; **, P < 0.01; ***, P < 0.001 NS, P > 0.1.
agricultural areas and found very few beetles were trapped at distances greater than 500 m away from stored grain (Losey, 2015). An Australian study (Holloway et al., 2018) also found that C. ferrugineus could be trapped in higher numbers with unbaited (passive) flight traps closest to grain silos compared to trapping very low numbers just 280 m (on average) away from the silos. We therefore decided to do pheromone experiments in open areas within 100 m from grain bins. Our bucket trap data showed that C. ferrugineus could be monitored very close to grain bins, which led us to do our subsequent field experiments with synthetic pheromone at those bin locations of Abilene and Junction City. The bucket trap results suggest a directional effect on captures for relative numbers near the bin, as the largest proportion of beetles were trapped on the westward side of the grain bin at each location. This
directional effect could be due to the flight behavior of C. ferrugineus relative to wind direction. The most common direction of wind flow at the Abilene and Junction City trapping locations during the weeks of this study was from the west or southwest, according to the US National Weather Service (www. weather.gov). Beetles flying with the wind from the west or southwest may have landed in the western bucket trap as they encountered the gran bin and were not flying around the bin to the east and north. Alternatively, beetles flying from the east, or those dispersing from the grain at the top if the bin may have encountered the westward bucket trap while flying upwind. Nansen et al. (2004a,b) working in Oklahoma hung unbaited sticky traps at different heights on the outside of stored wheat bins at the four cardinal directions and found significantly more C. ferrugineus
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Fig. 7. Responses of Cryptolestes ferrugineus to volatiles from conspecific adults feeding on grains using a two-choice pitfall bioassay. All data were analyzed using paired t-test, n ¼ 20 in each case: NS, P > 0.1; *, P < 0.05; n ¼ 20.
Fig. 8. Responses of C. ferrugineus to rubber septa loaded with the synthetic pheromones CeI and C-II, or to C-II applied in hexane to filter paper in different concentration in twochoice pitfall bioassays. Data were subjected to paired t-tests, n ¼ 20 for each experiment; NS, P > 0.1; *, P < 0.1; **, P < 0.01; ***, P < 0.001.
trapped on the north sides at two locations and these places had much of their wind coming from the south. However, that same study reported beetles were caught predominantly on the south side at other bins, and concluded the there was no consistent trend in beetle flight relative to wind or geographic aspect. We found that C. ferrugineus in flight will respond more to funnel traps with the synthetic pheromone C-II when compared side-by-side to traps without the pheromone. Field research with the male-produced aggregation pheromones of C. ferrugineus is very limited. The pheromones were first studied, identified and synthesized in the late 1970s and early 1980s (Borden et al., 1979; Wong et al., 1983; Oehlschlager et al., 1983) and at most there was just one field trial published at that time with inconclusive results (Loschiavo et al., 1986). The Canadian study by Loschiavo et al.
(1986) tested response of walking C. ferrugineus to pitfall-probe traps inserted into the top of a grain mass, so flight response was not assessed. Holloway et al. (2018), did field experiments in Australia with significant attraction of flying C. ferrugineus to 4-unit multiple funnel traps baited with both CeI and C-II compared to traps without pheromone. The Australian work reported low numbers of beetles trapped in flight, as in our experiments here, and with variation among several field sites. Nevertheless, the response to pheromone-baited traps had 3-folde20-fold more beetles responding to pheromones compared to traps without lures. Our experiments found pheromone-baited traps (C-II) had 2fold or 9-fold more beetles in pheromone vs non-pheromone traps across the two field sites. The Holloway et al. (2018) study and our experiments reported here are the most recent research on
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Fig. 9. Responses of Cryptolestes ferrugineus to rubber septum lures loaded with synthetic pheromones of five species of stored product pests using a two-choice pitfall bioassay. Data were analyzed using paired t-test; NS, P > 0.1; *, P ¼ 0.05; **, P < 0.01; ***, P < 0.001; n ¼ 20 for each experiment. Names of synthetic pheromones and the species they are intended to monitor are: DL-1þ2 for Rhyzopertha dominica, CB¼ Cigarette beetle (Lasioderma serricorne), RFB ¼ Red Flour Beetle (Tribolium castaneum), PT¼ Larger grain borer (Prostephanus truncatus), IMM¼ Indian meal moth (Plodia interpunctella).
pheromones for C. ferrugineus, and apparently the first and only reports, of pheromone responses by feral beetles flying in agricultural landscapes. Our experiments suggest that the relative importance of CeI and C-II, whether alone or separately, in orientation behavior and reproductive success of C. ferrugineus deserves further study. Our fieldwork found that C-II alone attracted beetles to traps, and the laboratory assays reported in Fig. 8 suggest the CeI and C-II illicit similar responses whether released separately or together. Although multiple pheromone systems are well known in other species to assure species-specificity in responses or to elicit a maximum response in combination, pheromones released as two or more isomers, or two or more structurally similar molecules, are known to be redundant in their activity (e.g., McBrien et al., 2002). Several species of phytophagous beetles, including pests of forests (Landolt, 1997; Borden, 1989; Byers, 1992; Raffa et al., 1993) and stored products (Burkholder and Ma, 1985; Phillips, 1997; Phillips et al., 2000), utilize aggregation pheromones that are produced by males feeding on host material that attract both males and females for mating and oviposition. The first study to demonstrate an aggregation pheromone for C. ferrigineus used a laboratory assay to show that males feeding on grain produced an attractant for both males and females (Borden et al., 1979), but we were unable to demonstrate the same effect with our laboratory assays (Fig. 6). Bioassays with mixed-sex adults feeding on oats, or males only feeding on wheat, did not attract beetles at a higher rate than either host material alone. The one case of more beetles attracted to adults with oats vs a blank vial did not control for the presence of oats, which could have been the source of attraction. It is possible that our two-choice assay did not reveal a significant effect for some comparisons, and that the bioassay of Borden et al. (1979), which differed from ours as being a no-choice response of beetles walking to test odors in an airstream, was more sensitive to beetle behavior when odors were tested singly than in a paired comparison. Although we could not show a pheromone effect with odors from feeding C. ferrugineus, we did show significant attraction of walking beetles to the synthetic CeI and C-II in the twochoice pitfall bioassays. Results of the first three experiments in Fig. 8 show that over twice as many beetles responded to rubber
septum lures containing either CeI, C-II, or a combination of both septa in a response vial compared to septa without these chemicals. We also observed positive responses of beetles responding to C-II applied to filter paper in the amounts of either 2,500 ng or 5,000 ng compared to untreated filter paper. These lab results corroborate the field trapping studies reported here and those of Holloway et al. (2018) that used lures with synthetic aggregation pheromone. The concentrations of synthetic pheromone released in these laboratory assays were most likely extremely high compared to amounts that may be released by living males. Rubber septa pheromone lures release synthetic pheromone at a stable rate, perhaps for several weeks to attract insects from long distances. Lures may release synthetic pheromone at rates of 1000s of ng per day, which could easily be two orders of magnitude higher than an insects natural release rate (McNeil, 1991). Information on the amounts of pheromone released by single insects is not available for C. ferrugineus, but we can assume it is much lower than the amounts of CeI or C-II released in our laboratory assays. Nevertheless, we demonstrated orientation of C. ferrugineus to the synthetic pheromones in the laboratory; the biology of production and release of pheromone in this species, and details on behavioral responses to pheromones, will require more research in the future. The results here support the practice of using food-based materials as attractants for C. ferrugineus. Some grain-based foods, such as cracked wheat and corn meal, were attractive to C. ferrugineus, while odors from rolled oats or dog food were not (Fig. 4). All four of these foods can serve as suitable feeding and reproductive media for this species (Arbogast, 1991), but the twochoice bioassays suggest they may not all have similar stimuli for orientation. Odors from grain-derived food materials such as Storgard Oil, the Russell Food Lure and ethanol elicited positive orientation in our experiments (Fig. 5). The commercial Russell food lure reportedly contains wheat germ oil and extracts of other foods used by storage pests and the Storgard Oil also contains wheat germ oil and additional materials, and both products are used with traps for stored product insects like C. ferrugineus (Wakefield et al., 2006; Collins et al., 2007). Ethanol, a common fermentation product of grain-based foods, elicited a significant positive response at 15 ml
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applied to filter paper (Fig. 6). Ethanol is associated with moist and decomposing grain, typically a result of microbial activity, and is likely to be associated with preferred food sources for this species (Nout and Bartelt, 1998). This result suggests ethanol may be a useful semiochemical for detecting or monitoring C. ferrugineus. Interspecific attraction by one species of insect to a pheromone of another species is known to occur in many cases, including the chemical ecology of stored product insects (e.g., Edde and Phillips, 2006; Phelan and Baker, 1986). We report here C. ferrugineus displayed positive responses to the synthetic pheromones of three beetle species and a common pheromone of several moths in laboratory tests (Fig. 9). It is possible that these results reflect some adaptive response of C. ferrugineus for locating suitable host material that is already infested by other stored product pests releasing their pheromones in a given ecosystem. Interspecific attraction to a common and presumably rare host resource can be considered maladaptive for the impact of competition. However, such responses are proposed to be mutually adaptive when considering increased reproductive fitness for the responders (e.g. Baker, 1989 for moths) or for intraspecific successful invasion of a new food source by multiple species (e.g., Kausrud, et al., 2011 for tree-killing bark beetles). Nevertheless, the results here are very limited, but suggest that future work on interspecific attraction for C. ferrugineus may elaborate these interactions and provide additional semiochemical tools for use in IPM applications. Pheromones for many stored-product pests were identified and synthesized over the past 50 years, and several are commercially available or used commercially for pest detection, monitoring and mating disruption (Phillips, 1997; Phillips et al., 2000; Phillips and Throne, 2010). Although pheromones for C. ferrugineus and other beetle pests in the Laemophloeidae and Silvanidae were identified starting in the 1980s (e.g. Oehlschlager et al., 1983), the lack of commercial pheromone lures has limited pheromone research and applications for this and related species up to this time. The availability of synthetic pheromones like those studied here should now allow for more research on flight behavior, pheromone-directed orientation and perhaps for developing new tools for detecting, monitoring and managing this pest. Acknowledgements The authors gratefully acknowledge the support of Cooperative Research Centre for National Plant Biosecurity (Project CRC3039), established and supported under the Australian Government’s Cooperative Research Centres program (http://www. crcplantbiosecurity.com.au). This research was supported by the Professor Donald Wilbur Endowed Professorship for StoredProduct Protection at Kansas State University. The authors are grateful to Darek Czokajlo of Alpha-Scents for providing the synthetic C-II. Dr. Ching Kang provided valuable advice and consultation on statistical analyses. This article represents publication number 20-039-J from the Kansas Agricultural Experiment Station. References Arbogast, R.T., 1991. Beetles: Coleoptera, pp. 131-176. In: Gorham, J.R. (Ed.), Ecology and Management of Food-Industry Pests. FDA Technical Bulletin 4. Association of Analytical Chemists, VA. Baker, T.C., 1989. Sex pheromone communication in Lepidoptera: new research progress. Experientia 45, 248e262. Borden, J.H., 1989. Semiochemicals and bark beetle populations: exploitation of natural phenomena by pest management strategists. Holarctic Ecology 12, 501e510. Borden, J.H., Dolinski, M.G., Chong, L., Verigin, V., Pierce Jr., H.D., Oehlschlager, A.C., 1979. Aggregation pheromone in the rusty grain beetle, Cryptolestes ferrugineus (Coleoptera: Cucujidae). Can. Entomol. 111, 681e688. Burkholder, W.E., Ma, M., 1985. Pheromones for monitoring and control of storedproduct insects. Annu. Rev. Entomol. 30, 257e272.
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Byers, J.A., 1992. Attraction of bark beetles Tomicus piniperda, Hylurgus palliates, and Trypodendron domesticus and other insects to short-chain alcohols monoterpenes. J. Chem. Ecol. 18, 2385e2402. Canadian Grain Commission, 2013. Rusty Grain Beetle Cryptolestes ferrugineus. Steph.. https://www.grainscanada.gc.ca/storage-entrepose/pip-irp/rgb-cr-eng. htm Collins, L.E., Bryning, G.P., Wakefield, M.E., Chambers, J., Cox, P.D., 2007. Progress towards a multi-species lure: identification of components of food volatiles as attractants for three storage beetles. J. Stored Prod. Res. 43, 53e63. Cuperus, G.W., Prickett, C.K., Bloome, P.D., Pitts, J.T., 1986. Insect populations in aerated and unaerated stored wheat in Oklahoma. J. Kans. Entomol. Soc. 59, 620e627. Czeskis, B.A., Shpiro, N.A., Moiseenkov, A.M., 1994. Efficient Synthesis of (S,Z)Dodec-3-en-11-olide (Ferrulactone II) Using 2-Carboxyethyltriphenylphosphonium Bromide. ChemInform 25 (Abstract). Edde, P.A., Phillips, T.W., 2006. Field responses of non-target species to semiochemicals of stored-product Bostrichidae. Ann. Entomol. Soc. Am. 99, 175e183. Fargo, W.S., Epperly, D., Cuperus, G.W., Clary, B., Noyes, R., 1989. Effects of temperature and duration of trapping on four stored grain insect species. J. Econ. Entomol. 82, 970e973. Fargo, W.S., Cuperus, G.W., Bonjour, E.L., Burkholder, W.E., Clary, B.L., Payton, M.E., 1994. Influence of probe trap type and attractants on the capture of four storedgrain Coleoptera. J. Stored Prod. Res. 30, 237e241. Hagstrum, D.W., 2000. Using five sampling methods to measure insect distribution and abundance in bins storing wheat. J. Stored Prod. Res. 36, 253e262. Hagstrum, D.W., Subramanyam, B., 2009. Table 1.1 Insects and their natural enemies associated with stored products, p 183. In: Hagstrum, D.W., Subramanyam, B. (Eds.), Stored-product Insect Resource. AACC International Inc., St. Paul, MN. Hagstrum, D.W., Phillips, T.W., Cuperus, G. (Eds.), 2012. Stored Product Protection. Kansas State University, Manhattan, KS, p. 352. http://ksre.ksu.edu/library/p. aspx?tabid¼70. Holloway, J.C., Mayer, D.G., Daglish, G.J., 2018. Flight activity of Cryptolestes ferrugineus in southern New South Wales, Australia. J. Pest Sci. 91, 1353e1362. https://doi.org/10.1007/s10340-018-0981-1. goire, J.C., Skarpaas, O., Erbilgin, N., Gilbert, M., Økland, B., Kausrud, K.L., Gre Stenseth, N.C., 2011. Trees wantedddead or alive! Host selection and population dynamics in tree-killing bark beetles. PLoS One 6 (5), e18274. https:// doi.org/10.1371/journal.pone.0018274. Konemann, C.E., Hubhachen, Z., Opit, G.P., Gautam, S., Bajracharya, N.S., 2017. Phosphine resistance in Cryptolestes ferrugineus (Coleoptera: Laemophloeidae) collected from grain storage facilities in Oklahoma, USA. J. Econ. Entomol. 110 (3), 1377e1383. https://doi.org/10.1093/jee/tox101. Landolt, J.P., 1997. Sex attractant and aggregation pheromones of male phytophagous insects. Am. Entomol. 43e 1. Lindgren, B.S., 1983. A multiple-funnel trap for Scolytid beetles (Coleoptera). Can. Entomol. 115, 299e302. Loschiavo, S.R., Wong, J., White, N.D.G., Pierce Jr., H.D., Borden, J.H., Oehlschlager, A.C., 1986. Field-Evaluation of a pheromone to detect adult rusty grain beetles, Cryptolestes ferrugineus (Coleoptera: Cucujidae), in stored grain. Can. Entomol. 118, 1e8. Losey, S., 2015. Orientation and dispersal of Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae) is response to various semiochemicals. http:// krex.k-state.edu/dspace/bitstream/handle/2097/20566/StephenLosey2015.pdf? sequence¼1&isAllowed¼y, 51 pp. McBrien, H.L., Millar, J.G., Rice, J.G., McElfresh, J.S., Cullen, E., Zalom, F.G., 2002. Sex attractant pheromone of the red-shouldered stink bug Thyanta pallidovirens: a pheromone blend with multiple redundant components. J. Chem. Ecol. 28, 1797e1818. McNeil, J.N., 1991. Behavioral ecology of pheromone-mediated communication in moths and its importance in the use of pheromone traps. Annu. Rev. Entomol. 36, 407e430. Moeck, H., 1970. Ethanol as the primary attractant for the ambrosia beetle Trypodentron lineatum (Coleoptera: Scolytidae). Can. Entomol. 102, 985e995. Nansen, C., Bonjour, E.L., Phillips, T.W., Vela-Coiffier, E.L., Gates, M.W., 2004a. Timedelayed relationship between flight activity outside grain bins and probe trap catches inside grain bins of Cryptolestes ferrugineus. Environ. Entomol. 33, 1465e1470. Nansen, C., Bonjour, E.L., Gates, M.W., Phillips, T.W., Cuperus, G.W., Payton, M.E., 2004b. Model of Cryptolestes ferrugineus flight activity outside commercial steel grain bins in central Oklahoma. Environ. Entomol. 33, 426e434. Norwood, C., 2015. New lure to trap rusty grain beetle. https://grdc.com.au/ resources-and-publications/groundcover/ground-cover-supplements/groundcover-issue-119-grain-storage/new-lure-to-trap-rusty-grain-beetle. (Accessed 5 July 2018). Nayak, M.K., Holloway, J.C., Emery, R.N., Pavic, H., Bartlet, J., Collins, P.J., 2013. Strong resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae): its characterization, a rapid assay for diagnosis and its distribution in Australia. Pest Manag. Sci. 69, 48e53. https:// doi.org/10.1002/ps.3360, 2013. Nout, M.J.R., Bartelt, R.J., 1998. Attraction of a flying nitidulid (Carpophilus humeralis) to volatiles produced by yeasts grown on sweet corn and a corn-based medium. J. Chem. Ecol. 24, 1217e1239. Oehlschlager, A.C., Wong, J.W., Verigin, V.G., Pierce, H.D.J., 1983. Synthesis of two macrolide pheromones of the rusty grain beetle, Cryptolestes ferrugineus (Stephens). J. Org. Chem. 4, 5009e5017.
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S.M. Losey et al. / Journal of Stored Products Research 84 (2019) 101513
Phelan, P.L., Baker, T.C., 1986. Cross-attraction of five species of stored-product Phycitinae (Lepidoptera: Pyralidae) in a wind tunnel. Environ. Entomol. 15, 369e372. Phillips, T.W., 1997. Semiochemicals of stored-product insects: research and applications. J. Stored Prod. Res. 33, 17e30. Phillips, T.W., Cogan, P.M., Fadamiro, H.Y., 2000. Pheromones. Pages 273-302. In: Subramanyam, Bh, Hagstrum, D.W. (Eds.), Alternatives to Pesticides in StoredProduct IPM. Kluwer Academic Publishers, Boston. Phillips, T.W., Throne, J.E., 2010. Biorational approaches to managing stored-product insects. Annu. Rev. Entomol. 55, 375e397. Pierce, A.M., Borden, J.H., Oehlschlager, A.C., 1981. Olfactory response to beetleproduced volatiles and host-food attractants by Oryzaephilus surinamensis and O. mercator. Can. J. Zool. 59, 1980e1990. R Core Team, 2014. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project. org/. Raffa, K.F., Phillips, T.W., Salom, S.M., 1993. Mechanisms and strategies of host colonization by bark beetles. In: Schowalter, T., Filip, G. (Eds.), Interactions Among Bark Beetles, Pathogens and Conifers in North American Forests. Academic Press, London, pp. 103e128, 252. Reed, C.R., Wright, V.F., Mize, T.W., Pedersen, J.R., Brockschmidt-Evans, J., 1991. Pitfall traps and grain samples as indicators of insects in farm-stored wheat. J. Econ. Entomol 84, 1381e1387. SAS, 2002. User’s Guide, v. 8. SAS Institute, Cary, NC. Sinha, R.N., 1961. Insects and mites associated with hot spots in farm stored grain. Can. Entomol. 93, 609e621.
Subramanyam, Bh, Hagstrum, D.W., Schenk, T.C., 1993. Sampling adult beetles (Coleoptera) associated with stored grain: comparing detection and mean trap catch efficiency of two types of probe traps. Environ. Entomol 22, 33e42. Swords, P., Van Ryckeghem, A., 2010. Summary of commercially available pheromones of common stored-product beetles. In: Carvalho, M.O., Fields, P.G., Adler, C.S., Arthur, F.H., Athanassiou, C.G., Campbell, J.F., et al. (Eds.), Proceedings of the 10th International Working Conference on Stored-Product Protection, 27 June- 2 July, 2010 2010 Estoril, Portugal. Julius Kuhn-Institut, Berlin, Germany, pp. 1004e1007. Toews, M.D., Phillips, T.W., Payton, M.E., 2005. Estimating populations of grain beetles using probe traps in wheat-filled concrete silos. Environ. Entomol. 34, 712e718. Wakefield, M.E., Collins, L.E., Bryning, G.P., Chambers, J., Cox, P.D., Fennah, K., Zaidi, S., Cross, D., Braithwaite, M., 2006. Improved Detection and Monitoring of Beetle Pests in Stored Grain through Use of a Multi-Species Lure (LK0929). Project Report No. 396. Home Grown Cereals Authority. White, N.D.G., Loschiavo, S.R., 1986. Effects of insect density, trap depth, and attractants on the capture of Tribolium castaneum (Coleoptera: Tenebrionidae) and Cryptolestes ferrugineus (Coleoptera: Cucujidae) in stored wheat. J. Econ. Entomol. 79, 1111e1117. Wong, J.W., Verigin, V., Oehlschlager, A.C., Borden, J.H., Pierce, H.D., Pierce, A.M., Chong, L., 1983. Isolation and identification of two macrolide pheromones from the frass of Cryptolestes ferrugineus (Coleoptera: Cucujidae). J. Chem. Ecol. 9, 451e474. Zar, J.H., 1984. Biostatistical Analysis, second ed. Prentice-Hall Inc., Englewood Cliffs, NJ. 718 p.