BEHAVIOR Sex Pheromone Investigation of serpentina (Diptera: )

1 2 3 4 DAVID C. ROBACKER, MARTIN ALUJA, ALLARD A. COSSE´ , AND PATRIZIA SACCHETTI

Ann. Entomol. Soc. Am. 102(3): 560Ð566 (2009) ABSTRACT Attraction of virgin female Anastrepha serpentina Wiedemann (Diptera: Tephritidae) to the odor of calling males was demonstrated. This sex pheromone-mediated attraction occurred during the latter half of a 13-h photophase but not during the Þrst half of the day. Two major components of emissions of calling males, 2,5-dimethylpyrazine (DMP) and 3,6-dihydro-2,5-dimeth- ylpyrazine (DHDMP), and trimethylpyrazine (TMP), a minor component, were tested for phero- monal activity. DMP and TMP elicited antennal responses using electroantennogram assays, but no response was observed for DHDMP. DMP and combinations of DMP and TMP were not attractive in laboratory bioassays. Bioassays of DHDMP were complicated by its instability making puriÞcation of the compound unfeasible. Bioassays of DHDMP in unpuriÞed form were further complicated by the presence of reaction byproducts and unreacted reagent in crude preparations of the DHDMP. However, statistical analysis indicated that combinations of 100Ð500 ng of DHDMP in mixtures with DMP as the major component elicited attraction in laboratory bioassays with sexually active female ßies. Taking into account all of the data, we could not conclude that any of the chemicals emitted by males are pheromones, but based on female ßy responses to mixtures of DHDMP and DMP, this is the most likely scenario.

KEY WORDS trimethylpyrazine, 2,5-dimethylpyrazine, dihydropyrazine

Sapote fruit ßy, Anastrepha serpentina Wiedemann Most of what is known about the biology, ecology, (Diptera: Tephritidae), is placed within the serpentina and behavior of A. serpentina stems from studies con- group with 10 other species, including Anas- ducted in Mexico. Aluja et al. (1989) Þrst described trepha striata Schiner, Anastrepha bistrigata Bezzi, the sexual behavior of this species. Behavioral studies Anastrepha ornata Aldrich, and Anastrepha anomala under natural conditions involving mature mango Stone (Norrbom 2002). Based on the most recent trees surrounded by various other host and nonhost morphological and molecular Anastrepha phylogenies trees in southern Mexico revealed that males call sin- (McPheron et al. 2000, Norrbom et al. 2000), this gly or in groups (leks) from the underside of leaves species group is close to the fraterculus group, which and defend territories visited by females for the pur- contains some of the most economically important pose of mating (Aluja et al. 1989). Interestingly, Aluja species in the genus [e.g., Anastrepha fraterculus Wiede- et al. (1989) observed the occasional formation of mann, Anastrepha ludens (Loew), Anastrepha obliqua heterospeciÞc leks, composed of A. serpentina, A. lu- (Macquart), and Anastrepha suspensa (Loew)]. Of the dens, and A. obliqua males calling simultaneously. Call- eight economically important Anastrepha species (A. ing males perform a combination of behaviors, appar- fraterculus, Anastrepha grandis Macquart, A. ludens, A. ently to attract sexually active females (Aluja et al. obliqua, A. serpentina, Anastrepha sororcula Zucchi, 2000). These behaviors include wing-fanning, a rapid A. striata, and A. suspensa), A. sororcula and A. serpentina vibration of the wings that produces high frequency are the least studied (Aluja 1994). sounds. Sivinski et al. (1984) referred to similar wing fanning in A. suspensa as calling songs. Other behaviors performed by calling A. serpentina males are pufÞng of Use of a product brand in this work does not constitute an en- dorsement by the USDA, the Instituto de Ecologõ´a, or the Universita` the pleural areas of the abdomen, evagination of an degli Studi di Firenze. anal membrane (proctiger), and touching of the proc- 1 USDAÐARSÐCQFIR, Kika de la Garza Subtropical Agricultural tiger to leaf surfaces. The functions of these behaviors Research Center, 2413 E. Highway 83, Weslaco, TX 78596 (retired). have not been proven in A. serpentina. In various Current address: 830 Moon Lake Dr. N., Progreso Lakes, TX 78596. 2 Instituto de Ecologõ´a, Asociacion Civil, Kilo´metro 2.5 Antigua species of Tephritidae, it has been demonstrated that Carretera a Coatepec 351, 91070 Xalapa, Veracruz, Me´xico. calling songs attract females, pheromones are pro- 3 Corresponding author: Crop Bioprotection Research, USDAÐ duced in various types of glands including some lo- ARS, National Center for Agricultural Utilization Research, 1815 N. cated in pleural areas of the abdomen, pheromones are University St., Peoria, IL 61604-3999. 4 Dipartimento di Biotecnologie Agrarie, Universita` degli Studi di emitted from the anal membranes, and touching of the Firenze, Via Maragliano, 77-50144, Firenze, Italy. proctiger to leaf surfaces serves to dispense phero- May 2009 ROBACKER ET AL.: SEX PHEROMONE OF A. serpentina 561 mone for more effective dispersal (Aluja et al. 2000, located at each end of the chamber, to allow easy and references therein). Furthermore, the male-pro- access to the chamberÕs interior. duced pheromones emitted during calling attract sex- Male emissions used as the test odor were produced ually active females. Castrejon-Gomez et al. (2007) by 20 sexually mature males (10Ð20 d old) in a two- also studied calling in this species and reported the neck 500-ml round-bottom ßask with ground-glass effects of age, time of day, and male density on calling joints. Compressed air entered the ßask through a by laboratory-reared and wild males. ground-glass Þtted gas inlet tube that extended into Recently, we began an investigation to identify the the ßask to within 2 cm of the ßask wall on the opposite sex pheromone of A. serpentina (Robacker et al. 2009). side. Flow rate was set to 200 ml/min using a ßow In that work, several chemicals emitted by calling controller (VICI Condyne, Valco Instruments Co., males were identiÞed. The chemicals were 2,5-dim- Inc., Houston, TX). Air exited the ßask through the ethylpyrazine (DMP), 3,6-dihydro-2,5-dimethylpyr- second neck via a ground-glassÐÞtted hose adapter. azine (DHDMP), and trimethylpyrazine (TMP). The This arrangement allowed the air to sweep through purpose of the current work was to demonstrate that the entire ßask before exiting. The glass tubes for sexually active females of this species are attracted to incoming and exiting air were loosely packed with a a male-produced pheromone and to determine piece of tissue to exclude ßies. The ßask was turned whether the previously identiÞed chemicals function upside down so that the incoming air tube emptied as sex pheromones. onto the upper wall of the ßask and air exited on the bottom. Calling ßies congregated on the upper wall Materials and Methods because a large green leaf was taped onto the top on the outside of the ßask to allow ßies to sit on the and Test Conditions. Except as noted, the “underside” of a leaf, a typical site for calling on trees work was conducted at the USDAÐARS laboratory in in Anastrepha (Aluja et al. 2000). Additional light was Weslaco, TX. All work was conducted with ßies from provided above the leaf with a 75-W incandescent a laboratory culture that originated from mamey fruit light bulb to create shadow on the underside of the (Pouteria sapota Jacq.) (Sapotaceae), a native host of leaf. the ßy, collected in Chiapas, Mexico, in 2000, and Emissions from the calling males were piped into reared on artiÞcial diet for at least 25 generations. the wind tunnel through a 30-cm length of polypro- were segregated by sex 2Ð3 d after eclosion. Males pylene tubing (1-cm internal diameter) connected to were put into Plexiglas cages (20.5 by 20.5 by 20.5 cm) the outlet hose adapter on the ßask containing the with screened tops. Females were put into aluminum- males. The tubing entered the wind tunnel chamber framed, aluminum-screened cages (30 by 30 by 30 cm) through a hole in the top of the wind tunnel and for testing of putative pheromone components. Fe- vented onto a plastic tree located directly below the males to be used in wind-tunnel bioassays were trans- ferred to 473-ml cardboard cartons with screen tops access opening on the upwind end of the chamber. just before testing. Cages were provisioned with a The tree had 40 green leaves (2 by 5 cm) and seven protein/sugar mixture and additional sugar in a sep- fruit models (ranging from 2.5 to 4 cm in diameter, arate container, as well as water. Laboratory condi- colored green to yellow and yellow-orange). tions for holding and testing ßies were 24 Ϯ 2ЊC, 60 Ϯ To conduct a trial, a cardboard carton (473 ml) 20% RH, and a photoperiod of 13:11 (L:D) h that containing 20 sexually mature, virgin females (10Ð20 began at 0330 hours and ended at 1630 hours. Bioassays d old) was placed under the downwind opening. Flies were conducted in the same room where adult test were allowed 10 min to leave the carton and respond ßies were held. The room contained inlet and outlet to the odor, and then they were removed from the vents to bring new air into the room from outdoors and chamber. Upwind movement was recorded if ßies to remove air from the room to the outdoors. Com- passed a point two thirds of the distance from the plete air replacement in the room occurred at the rate release carton to the tree, and landing was recorded if of 8/h. Males were kept on the air-outlet side of the ßies alighted on the tree. Multiple upwind movements room and bioassays were conducted on the inlet and landings by the same female were recorded if ßies side. Overhead lighting was provided by ßuorescent left the upwind end of the chamber, landed some- “cool white” lights (F40CW, General Electric, Cleve- where in the downwind end, then returned to the land, OH). upwind end. Multiple recordings from the same fe- Attraction of Sexually Active Females to Male Emis- male were rare. Control trials were conducted using sions. Bioassays were conducted in a Plexiglas wind clean tubing and air that bypassed the ßask with males. tunnel with the dimensions of 0.3 by 0.3 by 1.2 m. Each Trials were conducted at two times during the day: end of the wind tunnel was screened to allow airßow. 6Ð8 h and 10Ð11 h after onset of photophase. This was The downwind end contained a bafße system to create done because previous research showed that emis- a uniform airßow through the chamber. Air was pulled sions by males are greater 9Ð11 h after onset of pho- through the chamber at 0.4 m/s by an exhaust fan tophase than 6Ð8 h after onset of photophase connected to the downwind end. Air exiting the cham- (Robacker et al. 2009). One replication of the exper- ber was directed into an exhaust hose and removed to iment included one trial with males and one control at the outdoors. The top of the chamber had two circular each of the two test times. Twelve replications were openings (12.8 cm in diameter) with Plexiglas covers, conducted, each on a different day. 562 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 3

Test Chemicals. DMP (99%) and TMP (99%) were (Ephrussi and Beadle 1936). GC efßuent was intro- obtained from Aldrich (Milwaukee, WI). DHDMP duced into the airstream for rapid delivery to the was synthesized by the method described previously antennal EAG preparation. The EAG responses were (Robacker et al. 2009). For bioassays of DHDMP, the ampliÞed (2,000ϫ) with an AC/DC UN-6 ampliÞer reaction product was used in unpuriÞed form because (Syntech). Responses were analyzed using a com- previous work demonstrated that DHDMP was very puter with an analog to digital conversion board labile and probably would not survive puriÞcation (IDAC, Syntech) and GC-EAD software (Syntech). methods (Robacker et al. 2009). The concentrations of Attractiveness Bioassays of Putative Pheromone DMP and DHDMP in the reaction product were de- Components. Attractiveness of putative pheromone termined by GC analysis within3hofthebioassays components was evaluated using cage-top bioassays using a DMP calibration curve. The analytical method similar to those used to evaluate sex pheromone com- was described in Robacker et al. (2009). The test ponents of A. ludens (Robacker and Hart 1985). solution of DHDMP was stored in a Ϫ20ЊC freezer Brießy, the bioassay was conducted by placing two until 5 min before bioassays. Þlter paper triangles (3 cm per side) containing 10 ␮l Electroantennogram Assays of Putative Pheromone of test-chemical solution (T papers) and two papers Components. puparia were shipped to the USDAÐ containing 10 ␮l of control solution (C papers) on the ARS laboratory in Peoria, IL, for electroantennogram top of an cage (30 by 30 by 30 cm). Test mixtures (EAG) studies. Puparia were irradiated with gamma contained DMP, TMP, DHDMP reaction product, or rays (Cobalt 60; 40 grays) before shipping because of combinations of the three. quarantine laws. Volatiles from calling male ßies were Various amounts of the chemicals and chemical collected by solid-phase microextraction (SPME) combinations were applied to Þlter papers in a series with a polydimethylsiloxane coated Þber (100-␮m of bioassays (Table 1). DMP and TMP were prepared coating) (Supelco, Inc., Bellefonte, PA). Collections in methanol solution. DHDMP reaction product con- were made by exposing the Þber for 20 min in an tained oximinoacetone, tetrahydrofuran, and hexane airstream at 10 ml/min exiting a 125-ml ßask contain- (used for dilution). Control solvent was methanol for ing 15 sexually mature males (18 d old). The air was DMP and DMP ϩ TMP assays. Control solutions were puriÞed through Super-Q (Alltech Associates, Inc., either hexane or a mixture of oximinoacetone, tetra- DeerÞeld, IL) before entering the ßask. Collections hydrofuran, and hexane for assays of DHDMP reac- were made 8Ð10 h into the 13-h photophase. tion product. Control solutions were a mixture of ox- Volatiles from males were Þrst analyzed by coupled iminoacetone, tetrahydrofuran, and methanol for gas chromatography mass spectrometry (GC-MS) to assays of DHDMP reaction product ϩ TMP. verify identiÞcation of putative pheromone compo- The Þlter papers were raised 5 mm above the cage nents before performing EAG assays. For GC-MS, a top by using plastic rings to ensure that responses were GC (model 6890, HewlettÐPackard Company, San olfactory and not contact chemoreception. Each bio- Fernando, CA) equipped with a mass selective detec- assay cage contained 60Ð100 (depending on availabil- tor (model 5973, HewlettÐPackard) (70 eV) was used. ity) virgin females of ages 12Ð16 d old. The number of Volatiles samples were injected by insertion of the ßies beneath each Þlter paper was counted once each SPME Þber into an injector Þtted with an SPME- min for 10 min after application of the test materials to optimized glass liner (Supelco, Inc.) in splitless mode the papers. Bioassays were conducted 8Ð10 h after at 150ЊC. The analytical column was a DB-1 (15 m, 0.25 onset of photophase to coincide with maximum emis- mm i.d., 1.0-␮m Þlm) (Agilent Technologies, Inc., sion of volatiles by calling males. Santa Clara, CA). Linear velocity of helium carrier gas Statistical Analyses. Numbers of females that ßew was 49 cm/s. Temperature was programmed from 40 upwind and landed on the tree in wind-tunnel assays to 280ЊCat10ЊC/min. of attraction of females to male odor were tested by EAG assays were performed using an antennal EAG analysis of variance (ANOVA). Effects of treatments, preparation as a GC detector (EAD). Flame ionization i.e., the four combinations of time of day and odor detection was conducted simultaneously by splitting source, and test days were partitioned out of the total column efßuent between a ßame ionization detector sum of squares using a randomized complete block (FID) and the antennal preparation. The GC, column, design. and conditions were as described above. The temper- Total counts of ßies beneath C papers were sub- ature of the GC-EAD interface was set at 125ЊCto tracted from counts beneath T papers for each cage- reduce thermal degradation of the DHDMP. Other top bioassay and the differences were analyzed by than interface temperature, GC-EAD analyses were paired t-tests (Snedecor and Cochran 1967) to deter- performed by methods described by Cosse´ and Bartelt mine whether attractiveness of each chemical or com- (2000). Brießy, a glass pipet silver-grounding elec- bination of chemicals differed from the controls. For trode was inserted into the back of an excised head of 38 bioassays that contained between 100 and 500 ng of a sexually mature female ßy held by a micromanipu- DHDMP, ANOVA was conducted to test whether lator (WPI, Sarasota, FL) in a stream of puriÞed, attractiveness of chemical combinations with DMP/ humidiÞed air (20 ml/s). A glass pipet silver-recording DHDMP ratios Ͼ1 differed from attractiveness of probe (Syntech, Hilversum, The Netherlands) was combinations with DMP/DHDMP ratios Ͻ1. Each placed in contact with the distal end of one antenna. data point for this ANOVA was calculated as the ratio Both pipettes were Þlled with Beadle-Ephrussi saline of the sum of the counts of ßies at T papers to the sum May 2009 ROBACKER ET AL.: SEX PHEROMONE OF A. serpentina 563

Table 1. Mean counts of sexually mature, virgin females of A. serpentina at filter papers (T) with putative pheromone components vs. papers (C) with control solutions in cage-top bioassays

Test mixture Ca Replicatesb TC tc 100 ng of DMP Met 8 5.5 2.0 1.70 500 ng of DMP Met 8 8.5 7.1 0.70 50 ng of DMP ϩ 5 ng of TMP Met 10 15.8 12.9 1.66 100 ng of DMP ϩ 10 ng of TMP Met 10 26.7 26.0 0.13 500 ng of DMP ϩ 50 ng of TMP Met 10 22.8 23.9 Ϫ0.37 RP (75 ng of DMP, 100 ng of DHDMP) OxIA 4 6.5 10.5 Ϫ1.63 RP (500 ng of DMP, 100 ng of DHDMP) OxIA 4 8.2 5.8 0.58 RP (100 ng of DMP, 200 ng of DHDMP) OxIA 4 5.2 9.0 Ϫ4.4* RP (400 ng of DMP, 200 ng of DHDMP) OxIA 10 5.2 3.9 1.7 RP (400 ng of DMP, 500 ng of DHDMP) OxIA 4 18.2 36.0 Ϫ3.1 RP (1.2 ␮g of DMP, 700 ng of DHDMP) OxIA 4 18.2 31.0 Ϫ2.0 RP (500 ng of DMP, 200 ng of DHDMP) Hex 4 29.2 5.5 4.2* RP (300 ng of DMP, 500 ng of DHDMP) Hex 8 17.8 5.4 6.3** RP (100 ␮g of DMP, 2 ␮g of DHDMP) Hex 4 7.2 8.2 Ϫ0.37 RP (3 ␮g of DMP, 4 ␮g of DHDMP) Hex 4 9.5 6.5 0.44 RP (40 ␮g of DMP, 35 ␮g of DHDMP) Hex 4 5.8 3.5 1.5 RP (100 ng of DMP, 150 ng of DHDMP) ϩ 20 ng of TMP OxIA 10 21.8 29.2 Ϫ3.2* RP (200 ng of DMP, 150 ng of DHDMP) ϩ 20 ng of TMP OxIA 10 18.5 27.7 Ϫ3.1*

RP, reaction product with DHDMP and DMP; Met, methanol; Hex, hexane; OxIA, mixture of oximinoacetone, tetrahydrofuran, and either hexane or methanol to match amounts in DHDMP test mixture. a Solvent or mixture applied to C papers. b Number of bioassays conducted. c Paired t tests (Snedecor and Cochran 1967) of counts at T vs. C; *, P Ͻ 0.05; **, P Ͻ 0.01. of the counts at C papers for each bioassay, trans- Antennae responded to the same three by-products in formed by square root to normalize variance. Effect of a standard solution (Fig. 2C). the control mixture on T/C count ratios was parti- Attractiveness Bioassays of Putative Pheromone tioned out of the total sum of squares. Components. DMP and DMP/TMP combinations in All ANOVAs were conducted using SuperANOVA methanol solution were not signiÞcantly more attrac- (Abacus Concepts, Inc. 1989). Means separations tive than methanol solvent blanks to sexually active were done by Fisher protected least signiÞcant dif- female ßies (Table 1). Crude reaction product con- ference (LSD) method (Snedecor and Cochran taining DMP and DHDMP, with or without added 1967). TMP, was not signiÞcantly more attractive than sol- vent blanks that contained oximinoacetone, and in three cases it was signiÞcantly less attractive than the Results blanks with oximionacetone. Crude reaction product containing DMP and DHDMP was signiÞcantly more Attraction of Sexually Active Females to Male Emis- attractive than hexane solvent blanks in two cases in sions. Females made signiÞcantly more upwind ßights (F ϭ 9.9; df ϭ 3, 33; P Ͻ 0.0001) and landings on the tree (F ϭ 6.7; df ϭ 3, 33; P Ͻ 0.01) during the period 10Ð11 h after onset of photophase when emissions from males were piped into the wind tunnel than when clean air was piped into the tunnel (Fig. 1). Emissions from males did not affect upwind move- ments and landings on the tree during the period 6Ð8 h after onset of photophase. EAG Assays of Putative Pheromone Components. Results of one tandem GC-EAD assay of male emis- sions are shown in Fig. 2A. Only small antennal re- sponses were observed to DMP. Antennal responses to TMP were Ϸ5ϫ larger than to DMP on a unit amount basis. No response to DHDMP was observed in any of the 17 assays that were made from calling males. GC/ EAD analysis of the crude reaction mixture of syn- thetic DHDMP showed antennal activity to several synthetic by-products (DMP, diethyl oxalate, TMP) Fig. 1. Attraction of virgin female A. serpentina to male (Fig. 2B). No response to DHDMP was evident al- emissions at 6Ð8 h and 10Ð11 h after onset of photophase. though a small response could have been hidden by Different letters above bars within a series (upwind move- the large positive deßection during the antennal/soft- ments or landings on tree) indicate signiÞcant differences ware recovery phase after response to diethyl oxalate. (P ϭ 0.05). 564 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 3

Fig. 2. Simultaneously recorded gas chromatogram (FID) and EAD of female A. serpentina antenna to SPME-collected volatiles from calling male A. serpentina adults (A); crude synthetic sample of 3,6-dihydro-2,5-dimethylpyrazine (B); and synthetic standards (C). Numerals denote DMP (1), DHDMP (2), TMP (3), and diethyl oxalate (4). which the test amounts of these two chemicals were papers but was most evident when hexane was the between 200 and 500 ng. In these cases, it was later control solvent because oximinoacetone controls shown that the attractiveness of the crude product greatly masked the attractiveness of the DMP/DH- probably was mostly because of unreacted oximino- DMP combinations. ANOVA indicated that there was acetone in the mixture. This was determined by testing a signiÞcant difference in attractiveness depending on oximinoacetone solutions in cage-top bioassays. the DMP/DHDMP ratio (F ϭ 13.9; df ϭ 1, 35; P Ͻ Visual examination of the data in Table 1 led to the 0.001). observation that the attractiveness of reaction product seemed to be related to ratios and amounts of DMP to Discussion DHDMP in the product. Daily mean T/C response ratios for the 38 bioassays of DHDMP crude reaction Results demonstrated that chemicals emitted by product with amounts of DHDMP between 100 and calling males are attractive to sexually active females 500 ng are shown in Table 2. T/C ratios were consis- during assays conducted between 10 and 11 h after tently greater on test days in which the ratio of DMP/ onset of assays (Fig. 1). Therefore, chemicals emitted DHDMP in the test solution was Ͼ1 than on days by males function as a sex pheromone. No attraction when the DMP/DHDMP ratio was Ͻ1. This effect to male emissions was observed during assays con- occurred regardless of the solvent used on the control ducted 6Ð8 h into the photophase. Previously, we May 2009 ROBACKER ET AL.: SEX PHEROMONE OF A. serpentina 565

Table 2. Ratios of sexually mature, virgin females of A. serpentina at filter papers (T) with test mixtures containing various amounts of DHDMP and DMP vs. papers (C) with control solutions in cage-top bioassays

Test day Test Mixture DMP/DHDMPa Cb Replicatesc T/Cd 22 March 300 ng of DMP, 500 ng of DHDMP 0.6 Hex 4 6.24 23 March 300 ng of DMP, 500 ng of DHDMP 0.6 Hex 4 7.39 24 March 500 ng of DMP, 200 ng of DHDMP 2.5 Hex 4 10.00 17 April 75 ng of DMP, 100 ng of DHDMP 0.75 OxIA 4 0.65 18 April 500 ng of DMP, 100 ng of DHDMP 5.0 OxIA 4 3.77 9 May 100 ng of DMP, 200 ng of DHDMP 0.50 OxIA 4 0.56 10 May 400 ng of DMP, 200 ng of DHDMP 2.0 OxIA 4 2.05 12 May 400 ng of DMP, 200 ng of DHDMP 2.0 OxIA 6 1.59 29 Nov. 400 ng of DMP, 500 ng of DHDMP 0.80 OxIA 4 0.53

Hex, hexane; OxIA, mixture of oximinoacetone, tetrahydrofuran, and hexane to match amounts in DHDMP test mixture. a Ratio of DMP to DHDMP in test mixture. b Solvent or mixture applied to C papers. c Number of bioassays conducted on test day. d Mean of the sum of counts at T papers divided by the sum of counts at C papers for each bioassay, over all replications conducted on test day. showed that emission by males (same laboratory strain product. This is supported by the fact that DHDMP is as used in the current work) of DMP and DHDMP the chemical emitted in the largest amount when peaked 9Ð11 h after onset of photophase (Robacker et males call (Robacker et al. 2009). If this is the case, al. 2009). Emissions of these two chemicals were sig- then DMP may not actually be emitted by males. This niÞcantly lower 6Ð8 h into the photophase. These hypothesis is weakened because of the apparent lack results suggest that DMP and DHDMP are possible sex of antennal response to DHDMP. The second hypoth- pheromone components because their peak emission esis is that DMP is the pheromone to which females in previous studies coincided with attraction of fe- respond when the DHDMP precursor breaks down males to males in the current work. Emission of TMP into DMP. This is supported only by small EAGs to did not correlate well with calling, but its emission was DMP but not by behavioral assays. A third hypothesis higher during the middle of the day than at the be- is that both chemicals act together as the pheromone. ginning or end of the photophase, suggesting that it This is supported by the bioassay results in which also may be a pheromone component. speciÞc combinations of these two chemicals elicited Despite the strong correlation of calling and emis- signiÞcant attraction from sexually active females. In sion of DMP and DHDMP, it is not clear that any of this scenario, the chemicals synergize each other even the chemicals we identiÞed from male emissions have though each has little effect by itself. Also, the chem- any pheromone activity. EAGs of these chemicals did icals only have an attractive effect when perceived by not indicate strong antennal responses to DMP and ßies in speciÞc amounts and ratios, a very common possibly no response at all to the chemical (DHDMP) phenomenon in pheromone biology (Tumlinson et al. that males emit in the greatest amount (Robacker et 1982). Unfortunately, complicating factors such as al. 2009). These results seem to indicate that these two strong attraction to oximoacetone and low actual ßy chemicals are not pheromones. However, it is possible counts in the assays make the results unconvincing. A that irradiation of the ßies used in EAG assays may fourth hypothesis is that neither of these chemicals is have attenuated their responses to DMP and DHDMP. a pheromone component. This idea is supported by Although we know of no data testing effects of irra- both poor EAG and attraction data. diation on EAGs, in previous studies, irradiation was The last hypothesis that neither DMP nor DHDMP shown to diminish behavioral responses of A. ludens to is a pheromone component seems unlikely because pheromone (Moreno et al. 1991), and ßy age was emissions of both of these chemicals correlated shown to affect EAG responses of A. suspensa to food strongly with calling behavior. However, another pos- odors (Kendra et al. 2005). Given these results, de- sibility is that the actual pheromone emitted by the structive effects of irradiation on ßy antennal recep- males is neither DHDMP nor DMP but an even more tion seem possible. labile compound that spontaneously breaks down into In addition to the lack of EAG responses to DHDMP, DHDMP and Þnally DMP shortly after emission. As behavioral assays were ambiguous because possible such, our method results in identiÞcation of DHDMP attractiveness of DHDMP/DMP combinations was and DMP because the actual pheromone breaks down masked by strong attractiveness of oximinoacetone into these compounds during the analysis. This would present in the test material. PuriÞcation of the DHDMP also explain the lack of EAG response to DHDMP. reaction product to remove oximinoacetone was not Finally, there is the question of the status of TMP. attempted because of the instability of DHDMP This compound is emitted in low amounts by calling (Robacker et al. 2009). males and elicits a strong EAG response. However, Our results suggest several different hypotheses re- sexually active females were not attracted to it, and the garding the roles of DHDMP and DMP. One is that addition of TMP to mixtures of DMP and DHDMP did DHDMP is a pheromone and DMP is its breakdown not result in increased attraction. 566 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 3

Whatever eventually proves to be the correct in- Ephrussi, B., and G. W. Beadle. 1936. A technique of trans- terpretation, our data present a novel challenge to plantation for Drosophila. Am. Nat. 70: 218Ð225. current thinking about pheromones that merits fur- Kendra, P. E., W. S. Montgomery, D. M. Mateo, H. Puche, ther examination and experimentation. N. D. Epsky, and R. R. Heath. 2005. Effect of age on EAG response and attraction of female Anastrepha suspensa (Diptera: Tephritidae) to ammonia and carbon dioxide. Acknowledgments Environ. Entomol. 34: 584Ð590. McPheron, B. A., H. Y. Han, J. G. Silva, and A. L. Norrbom. D.C.R. thanks Maura Rodriguez (USDAÐARS, Weslaco, 2000. Phylogeny of the genera Anastrepha and Toxot- TX) for technical assistance. M.A. thanks Benno Graf and rypana (: Toxotrypanini) based upon 16S Jo¨rg Samietz (Forschungsanstalt Agroscope Changins- rRNA mitochondrial DNA sequences, pp. 343Ð361. In M. Wa¨denswil ACW, Nyon, Switzerland) for providing ideal Aluja and A. L. Norrbom [eds.], Fruit ßies (Tephritidae): working conditions to Þnish writing this paper. Portions of phylogeny and evolution of behavior. CRC, Boca Ra- this work conducted in Mexico were Þnancially supported by ton, FL. INECOL, and by grants to M.A. from the Mexican Consejo Moreno, D. S., M. Sanchez, D. C. Robacker, and J. Worley. Nacional de Ciencia y Tecnologõ´a (CONACyT grant 46846-Q 1991. Mating competitiveness of irradiated Mexican fruit [2004Ð2008]) and the Mexican Campan˜ a Nacional Contra ßy (Diptera: Tephritidae). J. Econ. Entomol. 84: 1227Ð Moscas de la Fruta (Secretarõ´a de Agricultura, Ganaderõ´a, 1234. Desarrollo Rural y PescaÐInstituto Interamericano de Coop- Norrbom, A. L. 2002. A revision of the Anastrepha serpen- eracio´n para la Agricultura (SAGARPA-IICA). M.A. also tina species group (Diptera: Tephritidae). Proc. Entomol. acknowledges support from CONACyT through a Sabbatical Soc. Wash. 104: 390Ð436. Year Fellowship (Ref. 79449). Norrbom, A. L., R. A. Zucchi, and V. Hernandez-Ortiz. 2000. Phylogeny of the genera Anastrepha and Toxotrypana References Cited (Trypetinae: Toxotrypanini) based on morphology, pp. 299Ð342. In M. Aluja and A. L. Norrbom [eds.], Fruit ßies Abacus Concepts, Inc. 1989. SuperANOVA. Abacus Con- (Tephritidae): phylogeny and evolution of behavior. cepts, Inc., Berkeley, CA. CRC, Boca Raton, FL. Aluja, M. 1994. Bionomics and management of Anastrepha. Robacker, D. C., and W. G. Hart. 1985. (Z)-3-Nonenol, Annu. Rev. Entomol. 39: 155Ð178. (Z,Z)-3,6-nonadienol, and (S,S)-(Ϫ)-epianastrephin: Aluja, M., M. Cabrera, J. Guillen, H. Celedonio-Hurtado, and male-produced pheromones of the Mexican fruit ßy. En- F. Ayora. 1989. Behaviour of Anastrepha ludens, A. obli- tomol. Exp. Appl. 39: 103Ð108. qua and A. serpentina (Diptera: Tephritidae) on a wild Robacker, D. C., M. Aluja, R. J. Bartelt, and J. Patt. 2009. mango tree (Mangifera indica) harbouring three McPhail IdentiÞcation of chemicals emitted by calling males of the traps. Insect Sci. Appl. 10: 309Ð318. sapote fruit ßy, Anastrepha serpentina (Diptera: Tephriti- Aluja, M., J. Pinero, I. Jacome, F. Diaz-Fleischer, and J. dae). J. Chem. Ecol. (in press). Sivinski. 2000. Behavior of ßies in the genus Anastrepha Sivinski, J., T. Burk, and J. C. Webb. 1984. Acoustic court- (Trypetinae: Toxotrypanini), pp. 375Ð406. In M. Aluja ship signals in the Caribbean fruit ßy, Anastrepha suspensa and A. L. Norrbom [eds.], Fruit ßies (Tephritidae): phy- (Loew). Anim. Behav. 32: 1011Ð1016. logeny and evolution of behavior. CRC, Boca Raton, FL. Snedecor, G. W., and W. G. Cochran. 1967. Statistical meth- Castrejon-Gomez, V. B., S. Lascares, E. A. Malo, J. Toledo, ods. The Iowa State University Press, Ames, IA. and J. C. Rojas. 2007. Calling behavior of mass-reared Tumlinson, J. H., R. R. Heath, and P.E.A. Teal. 1982. Anal- and wild Anastrepha serpentina (Diptera: Tephritidae). J. ysis of chemical communication systems of Lepidoptera, Econ. Entomol. 100: 1173Ð1179. pp. 1Ð25. In B. A. Leonhardt and M. Beroza [eds.], Insect Cosse´, A. A., and R. J. Bartelt. 2000. Male-produced aggre- pheromone technology: chemistry and applications. gation pheromone of Colopterus truncatus: structure, American Chemical Society, Washington, DC. electrophysiological and behavioral activity. J. Chem. Ecol. 26: 1735Ð1748. Received 6 January 2009; accepted 6 March 2009.