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1 March 2015 Effects of male age and mating status on response to the female sex pheromone of Copitarsia decolora (Lepidoptera: Noctuidae)
Humberto Reyes, René Arzuffi, Norma Robledo
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The effects of male age and mating status on the behavioral response of Copitarsia decolora (Guenée) males to the glandular extract of the female sex pheromone were studied by conducting wind tunnel bioassays and electroantennography (EAG). To study the effect of age, 2–3, 4–5, 6–7, 8–9, 10–11 day old males were used to measure attraction in the wind tunnel. To study the effect of age on virgin male antenna depolarization, Individual males 3-, 6- and 9-days old were used. The effect of male mating status was studied with 4–6 day old males and subsequent pheromone response was measured after a period of 24 and 48 h. Both male age and mating status significantly affected male response to the female sex pheromone. In the wind tunnel, male attraction to the extract reached its maximum in males 4–7 days old and then decreased as moth age increased. The glandular extract provoked a significantly greater depolarization of the antennae of 6 day old males than in 3 and 9 day old males. In the wind tunnel, virgin males were more attracted to female extracts than mated males. Significantly greater depolarization was observed in the antennae of virgin than mated males.

Males moths use sex pheromones emitted by females to locate a mate. However, male response can be affected by numerous factors, including the physiological state of the insect (Anton et al. 2007; Evenden & Gries 2008) and experience (Anton et al. 2007). Pheromones are detected by olfactory receptor neurons (ORN) housed within cuticular sensilia mainly on the antennae (Keil 1999). The axons of ORNs project via the antennal nerve into the antennal lobe (AL). There they make synaptic contact with intrinsic AL neurons, the local interneurons, and with AL projection neurons, which transfer information to higher brain centers (de Belle & Kanzaki 1999). The AL consists of a species-specific number of glomeruli (Hansson & Anton 2000). Male moths have large numbers of olfactory receptor neurons tuned to single compounds of the sex pheromone produced by females, and large glomeruli, called the macroglomerular complex, are entirely dedicated to the processing signals pertaining to these sex pheromones (Hansson & Anton 2000).

For some moth species such as Plutella xylostella (L.) (Zhang et al. 2009), Agrotis ipsilon (Hufnagel) (Anton & Gadenne 1999; Gadenne & Anton 2000; Gadenne et al. 2001; Barrozo et al. 2010a, 2010b, 2011), Heliothis virescens (F.) and Heliothis subflexa (Guenée) (Soques et al. 2010) it has been demonstrated that age and mating status can modify olfactory reception and sexual pheromone processing in males. In these species, the male response to the female sex pheromone is regulated by hemolymph titers of juvenile hormone (JH) (Gadenne et al. 1993; Duportets et al. 1998; Anton & Gadenne 1999; Gadenne & Anton 2000), and by neuromodulators that act on ORNs and AL neurons (Anton et al. 2007).

Copitarsia decolora (Guenée) (Lepidoptera: Noctuidae) is an important pest of cruciferous crops in Mexico (Suarez-Vargas et al. 2006) and subject to quarantine in the United States (Venette & Gould 2006). Rojas et al. (2006) identified the sex pheromone of C. decolora and, although synthetic sex pheromones have been used as an alternative to synthetic insecticides (Rojas et al 2006; Muñiz-Reyes et al. 2007; Barrientos-Hernández et al. 2011; Diaz-Gomez et al. 2012), the effect of environmental (Dumont & McNeil 1992) and physiological factors on male response to the sex pheromone have not been studied. However several factors, including of age and physiological status of males, should be evaluated before the pheromone compounds can be used effectively for C. decolora management. The efficiency of traps baited with pheromone could be affected importantly by these 2 factors.

This study examined the effect of age and male mating status on male attraction and antennal responses to sex pheromone extracts of C. decolora females. Understanding the effect of the factors on olfactory guided behavior of C. decolora males will contributes to improving current pheromone-based methods for the management of this pest species.

Materials and Methods


Insects used in the bioassays were obtained from a C. decolora colony at the Centro de Desarrollo de Productos Bióticos, National Polytechnic Institute (IPN) in Yautepec, Morelos, Mexico. The insects were maintained at 25 ± 3 °C, 60 ± 5% RH and 12:12 h L:D, with the photophase and scotophase reversed with respect to the natural light cycle in order to allow pheromone extraction and bioassays during the day. Larvae were fed with a special artificial diet for lepidopterans (Cibrián-Tovar & Sugimoto 1992). Adults were fed with a 50% sucrose solution placed on cotton wool, which was replaced every 3rd day.


The gland that produces the sex pheromone, situated between the seventh and ninth abdominal segment (Rojas et al. 1995), was dissected from virgin 3–5 day old females that presented sexual calling behavior during the final third of the scotophase (Rojas et al. 2006). Sixty glands were placed in 2 mL glass vials containing 1 mL of dichloromethane as a solvent for 10 min. The supernatant was concentrated to 100 µL under a nitrogen flow. Each extract had a concentration of 3 female equivalents (FE) for each 5 µL. The gland extracts was stored at -4 °C until chemical analysis or use in the bioassays.


Two µL of extract were injected into a gas chromatograph (GC) (HP 6890) coupled with a mass spectrometer (MS) (HP 5972) (Agilent, USA). The samples were analysed using a non-polar HP 5MS column (30 m long, 250 µm internal diameter and 0.25 µm film thickness; Agilent Technologies, Santa Clara, California). The initial oven temperature was 60 °C, increasing 15 °C/min until it reached 280 °C. The carrier gas was helium at a constant flow of 1 mL/min. The injector temperature was 225 °C and the auxiliary was 280 °C