BEHAVIOR Sex Pheromone Investigation of Anastrepha serpentina (Diptera: Tephritidae) 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- species 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 Insects 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. Flies 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).