Male confusion of the nun moth with disparlure at high and low population densities

The feasibility of using the synthetic sex pheromone, disparlure, as a mating confusant of the nun moth (Lymantria monacha L.) was investigated on wild populations occurring in outbreak proportions in Poland and in low density in Czechoslovakia. The size of experimental plots ranged from 1 to 12 hectares. The amounts of pheromone applied were 10 or 20 g/hectare. Three types of slow-release formulations of a racemic mixture of disparlure were tested: (1) a spray formulation with latex as a carrier, (2) a tubing of natural rubber, and (3) sawdust of a porous material used for making tips of fiber-tip pens. All three pheromone formulations prevented significant numbers (98.6–100% in the low density situation, 90–96.2% during the outbreak) of the males from locating a discrete source of pheromone (a trap containing synthetic pheromone or a female) during the whole flight period, indicating long-lasting efficiency of the formulations.     

(7R,8S)-cis-7,8-epoxy-2-methyloctadec-17-ene: a novel trace component from the sex pheromone gland of gypsy moth, Lymantria dispar

Considering the vast Eurasian distribution of gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae), the many subspecies, and their presence in different lymantriid communities, we tested the hypothesis that L. dispar populations in eastern Asia employ one or more pheromone components in addition to the previously known single component pheromone (7R,8S)-cis-7,8-epoxy-2-methyloctadecane [= (+)-disparlure]. Coupled gas chromatographic–electroantennographic detection (GC–EAD) analyses of pheromone gland extracts of female L. dispar sensu lato (including both AGM and NAGM) on four GC columns (DB-5, DB-23, DB-210, and SP-1000) revealed a new trace component that eluted just before (DB-5; DB-210) or after (DB-23, SP-1000) disparlure, and elicited strong antennal responses. Isolation of this compound by high-performance liquid chromatography and hydrogenation produced disparlure, suggesting that the new component had the molecular skeleton of disparlure, with one or more double bonds. Of all possible monounsaturated cis-7,8-epoxy-2-methyloctadecenes, only cis-7,8-epoxy-2-methyloctadec-17-ene co-chro- matographed with the insect-produced compound on all GC columns and elicited comparable antennal responses. In field experiments in Honshu (Japan) with enantioselectively synthesized compounds, (7R,8S)-cis-7,8-epoxy-2- methyloctadec-17-ene (7R8S-epo-2me-17-ene-18Hy) was weakly attractive to male L. dispar, but was less effective as a trap bait than (+)-disparlure, and failed to enhance attractiveness of (+)-disparlure when tested in blends. The antipode, (7S,8R)-cis-7,8-epoxy-2-methyloctadec-17-ene, was not attractive, and when added to (+)-disparlure and/or 7R8S-epo-2me-17-ene-18Hy reduced attractiveness. Thus, the biological role of 7R8S-epo-2me-17-ene-18Hy remains unclear. It may enhance pheromone attractiveness or specificity in other L. dispar populations within their vast Eurasian distribution.     

Pheromone-mediated behavior of the gypsy moth

The pheromone-mediated behavior of gypsy moth males was studied in both natural and simulated populations in central Pennsylvania. Feral males released into 50-m-diam plots, each with 2 feral females around the perimeter, oriented initially to trees and not to females. Neither exposure to virgin females nor exposure to wicks baited with approx 6 mg disparlure affected the subsequent sexual activity of males released into the 0.2-hectare plots. Males released into untreated plots, following 24 hr exposure in an area treated with approx 37 g/hectare of microencapsulated disparlure, located and mated with feral females within 4 min after release. None of the released males was caught in disparlure-baited Delta traps. In the disparlure-treated plot none of the females was mated. Males within this treated plot continued to search actively but did not settle down on the bark surface and initiate short-range (< 15 cm) search behavior. In plots testing the effect of various ratios of baited wicks to virgin females on disruption, there was no evidence of mating disruption due to point-source confusion. There were no significant differences in the responses of feral males to either virgin females or the various portions of Hercon wicks placed out in 0.2-hectare plots. In a series of tests using feral virgin females given various treatments to alter their physical and chemical characteristics (i.e., removed wings, denuded abdomen, washed in xylene, etc.), all females elicited the full range of sexual behavior responses of the male moths in natural populations. Apparently, males stimulated by pheromone are capable of using a number of different additional stimuli to initiate and terminate short-range sexual behavior patterns. Lymantria dispar (L.); Lymantriidae: Lepidoptera.Authorized for publication on April 19, 1976 as paper no. 5066 in the journal series of the Pennsylvania Agricultural Experiment Station, Experiment Station Project No. 2044.     

Design and Synthesis of Fluorophore-Tagged Disparlure Enantiomers to Study Pheromone Enantiomer Discrimination in the Pheromone-Binding Proteins from the Gypsy Moth,Lymantria dispar

Fluorescent analogues of the gypsy moth sex pheromone (+)-disparlure (1) and its enantiomer (?)-disparlure (ent-1) were designed, synthesized, and characterized. The fluorescently labelled analogues 6-FAM (+)-disparlure and 1a 6-FAM (?)-disparlure ent-1a were prepared by copper-catalyzed azide-alkyne cycloaddition of disparlure alkyne and 6-FAM azide. These fluorescent disparlure analogues 1a and ent-1a were used to measure disparlure binding to two pheromone-binding proteins from the gypsy moth, LdisPBP1 and LdisPBP2. The fluorescence binding assay showed that LdisPBP1 has a stronger affinity for 6-FAM (?)-disparlure ent-1a, whereas LdisPBP2 has a stronger affinity for 6-FAM (+)-disparlure 1a, consistent with findings from previous studies with disparlure enantiomers. The 6-FAM disparlure enantiomers appeared to be much stronger ligands for LdisPBPs, with binding constants (K_d) in the nanomolar range, compared to the fluorescent reporter 1-NPN (which had K_d values in the micromolar range). Fluorescence competitive binding assays were used to determine the displacement constant (K_i) for the disparlure enantiomers in competition with fluorescent disparlure analogues binding to LdisPBP1 and LdisPBP2. The K_i data show that disparlure enantiomers can effectively displace the fluorescent disparlure from the binding pocket of LdisPBPs and, therefore, occupy the same binding site.

     

Gypsy moth mating disruption: Dosage effects

Small (1-hectare) plots in a dense gypsy moth (Lymantria dispar) infestation were treated with 5, 50, or 500 g racemic disparlure, and effects on male trap catch and mating behavior were studied. Capture of males in traps baited with 1, 10, 100, or 1000 g (+)-disparlure declined as disruptant dosages increased. Traps with high levels of attractant caught moths when capture in those with lower baitings was dramatically reduced. While all disruptant dosages reduced trap catch, it was reducd at least 95% at all attractant levels at the 500-g disruptant application rate. Visual estimates indicated that male moth density was similar in treated and control plots; female mating success was reduced 6.5, 34.5, and 84% in plots with 5, 50, and 500 g/ hectare disruptant, respectively. The duration of precopulatory and copulatory periods was similar for all females that were observed mating, regardless of disruptant treatment. It is proposed that reduced trap catch and female mating success are due to effects of atmospheric synthetic disparlure (disruptant) camouflaging natural attractant point sources.Lepidoptera: Lymantriidae.     

Determining enantiomeric composition of disparlure

A method has been developed to convert disparlure (2-methyl-7,8-epoxyoctadecane) to the correspondingN-(-methylbenzyl)aziridine with inversion of configuration at both the 7 and 8 positions. The diastereomeric aziridines can be separated on an efficient gas chromatography column, permitting determination of the enantiomeric constitution of the starting disparlure. As little as ca. 0.1% of the minor enantiomer can be detected.     

Pheromone Components and Diel Periodicity of Pheromonal Communication in Lymantria fumida

Extracts of pheromone glands from female Lymantria fumida were analyzed by coupled gas chromatographic–electroantennographic detection (GC-EAD) and by coupled GC–mass spectrometry (MS). The two compounds that elicited responses from male L. fumida antennae were identified as cis-7,8-epoxy-2-methyloctadecane (disparlure) and 2-methyl-Z7-octadecene (2me-Z7–18Hy). Field experiments in northern Japan demonstrated that synthetic (7R,8S)-cis-7,8-epoxy-2-methyloctadecane [(+)-disparlure] and 2me-Z7–18Hy are synergistic sex pheromone components of L. fumida. (7S,8R)-cis-7,8-Epoxy-2-methyloctadecane [(–)-disparlure] had no behavioral effect on male L. fumida. Traps baited with (+)-disparlure and 2me-Z7–18Hy captured male L. fumida between 21:00 and 24:00 hr, whereas traps baited with (+)-monachalure [(7R,8S)-cis-7,8-epoxy-octadecane], (+)-disparlure and 2me-Z7–18Hy attracted males of the nun moth, L. monacha L., between 02:00 and 04:00 hr. Both temporal separation of pheromonal communication and specificity of pheromone blends seem to contribute to the reproductive isolation of sympatric and coseasonal L. fumida and L. monacha.     

Control of low-density gypsy moth (Lepidoptera: Lymantriidae) populations by mating disruption with pheromone

This four-year study demonstrated that low-density populations of the gypsy moth,Lymantria dispar (L.), were effectively suppressed by annual aerial application of 75 g of racemic disparlure per hectare formulated in plastic laminate flakes. These tests also showed that, when plots were treated with 150 g of pheromone per hectare in 1990 only and left untreated for the following three years, populations continued to be suppressed in 1991–1993 as compared with the controls. Although none of the plots were treated in 1994, population assessment continued and showed that the gypsy moth population density remained low in the plots that had been treated annually for the preceding four years. The laminate flakes released an average of 0.48 g disparlure per day per hectare from each of the two applications in 1990, and 0.72 g per day per hectare from the single application in each of the following three years (1991–1993). Only 27–40% of the applied pheromone dose was emitted during male moth flight.     

Synthesis of deuterated fatty acids to investigate the biosynthetic pathway of disparlure,the sex pheromone of the Gypsy Moth, <Emphasis Type="Italic">Lymantria dispar</Emphasis>

The preparation and characterization of a series of deuterium-labeled intermediates used in the study of the biosynthetic pathway for disparlure, the sex pheromone of Lymantria dispar, is reported. The synthetic route starts with propargyl alcohol, and the deuterium atoms are introduced by deuteration of an alkyne precursor in the presence of Wilkinson's catalyst. The olefinic bond was created by the Wittig reaction of a suitable aldehyde with a common tetradeuterated phosphonium ylide intermediate. The presence of the expected label and its correct location were confirmed by both MS and ¹³C NMR. These compounds were successfully used to elucidate the disparlure biosynthetic pathway.     

Scented Males and Choosy Females: Does Male Odor Influence Female Mate Choice in the Mediterranean Fruit Fly?

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann), displays a lek mating system characterized by a high level of female discrimination among potential mates. The basis of female choice is not understood, but recent studies indicate that male exposure to the aroma of certain plant structures or essential oils may increase mating success. In particular, exposure to the aroma of ginger root oil (GRO) enhances male mating frequency, and several sterile-male release programs against C. capitata have incorporated ‘aromatherapy’ (large-scale exposure of pre-release insects to GRO) to increase the effectiveness of control efforts. We investigated the mechanism underlying female preference for GRO-exposed males. Two sets of experiments were conducted. In the first, we monitored female attraction to (1) freshly killed flies, or (2) paper discs that contained hexane extracts from varying treatments. In these tests, females were sighted more often (1) near GRO-exposed than non-exposed males (even when the males were visually concealed) and (2) near extracts from GRO-exposed than non-exposed males. These findings suggest a ‘perfume effect’, whereby female mate choice is mediated by olfactory differences. In the second set, we compared (1) mate choice between intact females and females from which both antennae had been surgically removed, and (2) mating success between intact males and males from which both antennae had been surgically removed before GRO exposure. Intact females preferred GRO-exposed males, whereas females lacking both antennae rarely mated and showed no preference between GRO-exposed and non-exposed males. In the opposite treatment (intact females but surgically altered males), GRO-exposed males lacking both antennae mated as frequently as GRO-exposed intact males. These data suggest that female choice was dependent on olfactory perception of male odor but that male mating success did not depend on olfactory perception of GRO aroma, suggesting, in turn, that GRO conferred a mating advantage through an external phenomenon (possibly alteration of cuticular scent) rather than through internal processing (pheromone synthesis).     

Response and Adaptation to Analogs of Disparlure by Specialist Antennal Receptor Neurons of Gypsy Moth, Lymantria dispar

Reception and adaptation of antennal receptors in response to (+)-disparlure [(+)-D], the sex attractant pheromone of the gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae)], its enantiomer, (?)-D, and analogs of disparlure in which the epoxide group is replaced with various three-membered rings [(±)-cyclopropyl (2HC), (±)-difluorocyclopropyl (2FC), (±)-dichlorocyclopropyl (2ClC), and (±)-aziridinyl (AZ), (+)-AZ, and (?)-AZ] as well as a mixture of 1,1-epoxy analogs were investigated using electroantennograms. Antennal receptors of male moths were more responsive (5 μg stimulus load) to (+)-D than to (?)-D; while responses to each of the analogs were significantly, less and did not differ from each other. Antennal receptors of female moths did not respond significantly to any of the compounds at this stimulus load. Dose–response curves revealed greater sensitivity of male antennae to (+)-D than (?)-D. Among the cyclopropane analogs and 1,1-epoxy mixture (1,1E), the order of sensitivity was: 2FC > 2ClC > 2HC > 1,1E. Antennae were least sensitive to the aziridinyl analogs; however, (+)-AZ was more active than (?)-AZ, while an intermediate response was elicited by the racemate. Males exposed to (+)-D adapted to (+)-D, while responses to (+)-D were not significantly affected by exposure to (?)-D, 2FC or (+)-AZ. After exposure to hexylacetate, males showed adaptation to (+)-D or (?)-D, but not to the other odorants. Preliminary field data correlate well with knowledge obtained in our electrophysiological studies and demonstrate the usefulness of such studies. These results indicate that novel approaches to the design and synthesis of potential agonists of disparlure receptors are necessary before substantial effects on the activity of disparlure are realized.     

Effects of sex pheromone on locomotion in the male American cockroach,Periplaneta americana

The locomotory activities of individual males ofPeriplaneta americana in a circular arena 2.5 m in diameter were investigated during the portion of the photocycle in which the cockroaches are most active. Control animals ran at an average speed of 10.86 cm/sec but remained immobile 68 % of the time. Pauses in locomotion occurred frequently and at fairly regular intervals (x=0.88 sec). Males showed a strong tendency to remain near the sides of the arena. Sex pheromone presented in the center of the arena produced a reduction in immobility time and a slight increase in running speed. The frequency of pauses decreased, and the time between pauses became less regular. The proportion of time spent near the sides of the arena was greatly reduced also.     

Attraction of female fathead minnows,Pimephales promelas,to chemical stimuli from breeding males

Female fathead minnows,Pimephales promelas, were attracted to water that had contained conspecific males in breeding condition. The attraction was particularly strong in the morning and occurred in both females with mature gonads and gonadally regressed females. Females were also attracted to water that had contained other females but this attraction was weaker than the attraction to breeding males and tended to occur in afternoon trials. When offered a choice, females preferred breeding male water over regressed male water or female water. Swarming behavior, in which females formed a very active group near the water inlet, occurred primarily in test locations receiving water from breeding males. Our results indicate that breeding maleP. promelas produce water-borne chemical stimuli that attract females and that females distinguish breeding male stimuli from female or regressed-male waterborne stimuli.     

Attraction of Male European Tarnished Plant Bug, <Emphasis Type="Italic">Lygus rugulipennis</Emphasis> to Components of the Female Sex Pheromone in the Field

Previous work showed that females of the European tarnished plant bug, Lygus rugulipennis Poppius (Heteroptera: Miridae), produced three chemicals, hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-4-oxo-2-hexenal, and that these were suspected to be components of the female sex pheromone. In field experiments, traps baited with blends of these chemicals dispensed from polyethylene vials and sachets failed to catch significant numbers of males. Here, we report more recent field experiments in which the chemicals were released from glass microcapillary tubes. A blend of hexyl butyrate and (E)-4-oxo-2-hexenal was significantly attractive to male L. rugulipennis. In addition, whereas the mixture of all three components attracted fewer L. rugulipennis males, this tertiary blend captured significantly greater numbers of males of the congeneric species Lygus pratensis than the binary mixture. The possible reasons for the success of the microcapillaries compared with other dispensers are discussed.     

Laboratory and field tests with the synthetic sex pheromone of threeMatsucoccus 1 pine bast scales

Matsunone, (2E,4E)-4.6,10,12-tetramethyl-2,4-tridecadien-7-one, is the primary sex pheromone ofMatsucoccus resinosae, M. thunbergianae, andM. matsumurae. The synthetic compound was tested in the laboratory and in field tests with all three species, and significantly more males responded to synthetic matsuone than to controls. The attraction of synthetic matsuone to males in field tests was not significantly different from that of virgin females at the same matsuone release rate. An analog of matsuone previously shown to be biologically active was also tested in the laboratory withM. resinosae andM. thunbergianae, and field tested withM. resinosae. The active analog required a dose approximately 100 times greater than matsuone to yield maximum laboratory bioassay response, and in field tests, attraction was not significantly different from controls at doses at which matsuone was significantly attractive.Homoptera: Coccoidea: Margarodidae.Deceased.     

Reproductive Character Displacement in Lymantria monacha from Northern Japan?

Our objective was to test the hypothesis that the pheromone blend and/or diel periodicity of pheromonal communication differ in populations of the nun moth, Lymantria monacha(Lepidoptera: Lymantriidae), from eastern Asia (northern Honshu, Japan) and Central Europe (Bohemia, Czech Republic). Coupled gas chromatographic–electroantennographic detection (GC-EAD) analyses of pheromone gland extract of female L. monachafrom Japan confirmed the presence of compounds previously identified in pheromone extracts of L. monachafrom Bohemia, as follows: (Z)-7-octadecene, 2-methyl-(Z)-7-octadecene (2me-Z7–18Hy), cis-7,8-epoxy-octadecane (monachalure), and cis-7,8-epoxy-2-methyloctadecane (disparlure). Field experiments in Honshu suggested that (+)-monachalure is the major pheromone component of L. monacha. 2me-Z7–18Hy significantly enhanced attractiveness of (+)-monachalure. Addition of (+)-disparlure to (+)-monachalure plus 2me-Z7–18Hy in Honshu and Bohemia increased attractiveness of lures by 1.2 and 20 times, respectively, indicating that (+)-disparlure is of least and most significance in the respective L. monachapopulations. Moreover, capture of male L. monachain pheromone-baited traps between 18:00 and 24:00 hr in Bohemia and 2:00 and 5:00 hr in Honshu revealed a markedly different diel periodicity of pheromonal communication. Pheromonal communication late at night and use of (+)-monachalure, rather than (+)-disparlure, as the major pheromone component by L. monachain Honshu may have resulted from interspecific competition with coseasonal L. fumida, which uses the early night for pheromonal communication and (+)-disparlure as major pheromone component. Whether communication channel divergence of L. monachain Honshu indeed constitutes a case of reproductive character displacement is difficult to prove. The evolution of such divergence in sympatricpopulations of L. fumidaand L. monachawould have to be demonstrated.     

Laboratory and field evidence for maleproduced aggregation pheromone inRhynchophorus cruentatus (F.) (Coleoptera: Curculionidae)

Laboratory and field assays were conducted to determine if palmetto weevil,Rhynchophorus cruentatus (F.), adults produce an aggregation pheromone. Attraction of females in a Y-tube olfactometer to conspecific males was greater than to clean air. Male and female attraction to conspecific male volatiles combined with host-palm,Sabal palmetto (Walter), volatiles was greater than to host-palm volatiles alone. Similarly, more weevils were caught in the field in traps baited with conspecific males plus host-palm tissue than in similar traps baited with only males, or palm tissue, or females, or females plus palm tissue. These results suggest thatR. cruentatus males produce an aggregation pheromone(s) that is highly attractive to conspecific adults of both sexes when combined with host-palm volatiles. This study is an important step towards understanding the chemical ecology ofR. cruentatus.     

Thet complex of the mouse: Chemical characterization by urinary volatile profiles

Urine samples from C3H congenic house mice (Mus domesticus) differing only at thet complex were examined by capillary gas chromatography to assess variations in the volatile components that may cause olfactory discrimination between animals bearingt lethal and+(wild-type) haplotypes. Urine was collected from 192 males and females varying in age from 1 to 9 months. C3H congenic mice that have the same genetic background at all loci but differed in theirt complex genotypes: +/ +, +/t_w1,T/t _w1, T/+ were used. No urinary volatiles were unique to thet complex. However, significant differences amongt complex genotypes and among ages occurred for concentrations of 12 male volatiles and four female volatiles. Usually young males (1–2 months of age) had significantly higher concentrations of cyclic enol ethers and ketones than older males (4–9 months of age). Moreover, some urinary volatiles (cyclic enol ethers, one ketone, dehydrobrevicomin, and thiazoline) were excreted in the urine of T/+and/orT/t males in significantly higher concentration than in the urine of +/+ males. Age andt complex genotype influences on the urinary volatiles in females were observed for four ketones. Gas chromatography of urinary components has the potential to be used in field studies of thet complex because the two t complex genotypes found in wild populations, +/+ and +/t, had significant differences in concentration for two males volatiles and three female volatiles.We dedicate this paper to the memory of Dorothea Bennett.On leave from the Environmental Protection Institute, Ministry of Light Industry, Beijing, China.A.E.M.B. supported in part by NSF BSR 8909172.     

Field Tests of Synthetic Sex Pheromone of the Apple Leafminer Moth, Phyllonorycter ringoniella

The apple leafminer moth, Phyllonorycter ringoniella, is becoming a more serious insect pest on apple trees with four to five generations a year in Korea. In order to devise a forecasting method for more accurate estimation of their numbers and development timing, the sex attractant was studied. Various ratios, from 10:0 to 0:10, of the two components, (Z)-10-tetradecenyl acetate (Z10–14:Ac) and (E,Z)-4,10-tetradecadienyl acetate (E4,Z10–14:Ac), identified from the sex pheromone gland (Jung and Boo, 1997), were tested for attractivity in terms of behavioral response (taxis, approach, and landing) against P. ringoniella males in a wind tunnel. The lure with Z10–14:Ac/E4,Z10–14:Ac in a ratio of 4:6 elicited the highest response in two (taxis and approach) measurement categories. For eliciting landing behavior, the two blends of 5:5 and 4:6 were best. The single component, Z10–14:Ac, elicited taxis behavior, but a combination of two chemicals was needed for eliciting all three behaviors. In the field, male attraction to various lure mixtures in Pherocon IC traps was usually greater than attraction to virgin females. The best field activity was in the lure baited with a 4:6 ratio of Z10–14:Ac and E4,Z10–14:Ac. Similar results were obtained from tests conducted in a net house. This optimum ratio for attracting P. ringoniella males in Korea is different from those reported in Japan (10:3) or China (7:3 to 6:4). The isomer E10–14:Ac neither improved nor depressed the number of catches when added at up to 10% of the total mixture to lures of the two components in the 4:6 ratio. The attractivity of the lures increased with higher amounts of the pheromones, up to 10 g in the wind-tunnel experiment and 5 mg in the apple orchard. The number of males captured was not significantly different among traps installed at 0.3, 1.5, or 2 m above the ground, or among wing, delta, or water traps. A rubber septum dispenser impregnated with 1 mg of the 4:6 mixture maintained its field attractivity for up to eight weeks.     

(Z)-10-Heptadecen-2-one and other 2-ketones in the aggregation pheromone blend ofDrosophila martensis,D. buzzatii,andD. serido

(Z)-10-Heptadecen-2-one was identified as the major aggregation pheromone component inDrosophila martensis, D. buzzatii, andD. serido. Aliphatic 2-ketones were not detected in newly eclosed males or in virgin females of any age. The amount of (Z)-10-heptadecen-2-one in the ejaculatory bulb and on the cuticle of males increased with age. Only trace quantities of (Z)-10-heptadecen-2-one were transferred to the female during mating. 2-Tridecanone was found in the ejaculatory bulb and on the cuticle of maleD. martensis andD. buzzatii, but notD. serido. The amount of 2-tridecanone increased with age. Unexpectedly,2-tridecanone inhibited the aggregation activity of (Z)-10-heptadecen-2-one inD. buzzatii. InD. martensis, 2-tridecanone was significantly less attractive than controls but did not decrease the attractiveness of one fly equivalent of (Z)-10-heptadecen-2-one.D. serido males did not have any 2-tridecanone and the activity of (Z)-10-heptadecen-2-one was not diminished inD. serido by the presence of 2-tridecanone.