Diverse methods have been used to sample insect semiochemicals. Sampling methods can differ in efficiency and affinity and this can introduce significant biases when interpreting biological patterns. We compare common methods used to sample tephritid fruit fly rectal gland volatiles (‘pheromones’), focusing on Queensland fruit fly, Bactrocera tryoni. Solvents of different polarity, n-hexane, dichloromethane and ethanol, were compared using intact and crushed glands. Polydimethylsiloxane, polydimethylsiloxane/divinylbenzene and polyacrylate were compared as adsorbents for solid phase microextraction. Tenax-GR and Porapak Q were compared as adsorbents for dynamic headspace sampling. Along with compounds previously reported for B. tryoni, we detected five previously unreported compounds in males, and three in females. Dichloromethane extracted more amides while there was no significant difference between the three solvents in extraction of spiroacetals except for (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane for which n-hexane extracted higher amount than both dichloromethane and ethanol. Ethanol failed to contain many of the more volatile compounds. Crushed rectal gland samples provided higher concentrations of extracted compounds than intact rectal gland samples, but no compounds were missed in intact samples. Of solid phase microextraction fibers, polyacrylate had low affinity for spiroacetals, ethyl isobutyrate and ethyl-2-methylbutanoate. Polydimethylsiloxane was more efficient for spiroacetals while type of fiber did not affect the amounts of amides and esters. In dynamic headspace sampling, Porapak was more efficient for ethyl isobutyrate and spiroacetals, while Tenax was more efficient for other esters and amides, and sampling time was a critical factor. Biases that can be introduced by sampling methods are important considerations when collecting and interpreting insect semiochemical profiles.
The banana fruit fly, Bactrocera musae (Tryon) (Diptera: Tephritidae), is an economically important pest endemic to Australia and mainland Papua New Guinea. The chemistry of its rectal glands, and the volatiles emitted during periods of sexual activity, has not been previously reported. Using gas chromatography–mass spectrometry (GC-MS), we find that male rectal glands contain ethyl butanoate, N-(3-methylbutyl) acetamide, ethyl laurate and ethyl myristate, with ethyl butanoate as the major compound in both rectal gland and headspace volatile emissions. Female rectal glands contain four major compounds, ethyl laurate, ethyl myristate, ethyl palmitate and (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, as well as 11 minor compounds. For both male and female B. musae, all compounds found in the headspace were also present in the rectal gland extracts, suggesting that the rectal gland is the main source of the headspace volatiles. Gas chromatography–electroantennography (GC-EAD) of rectal gland extracts confirms that male antennae respond to male-produced ethyl laurate and female-produced (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, while female antennae respond to male-produced ethyl butanoate but no female-produced compounds. This is an important step in understanding the volatiles involved in the chemical communication of B. musae, their functional significance, and potential application.
Bactrocera frauenfeldi (Schiner) (Diptera: Tephritidae) is a polyphagous fruit fly pest species that is endemic to Papua New Guinea and has become established in several Pacific Islands and Australia. Despite its economic importance for many crops and the key role of chemical-mediated sexual communication in the reproductive biology of tephritid fruit flies, as well as the potential application of pheromones as attractants, there have been no studies investigating the identity or activity of rectal gland secretions or emission profiles of this species. The present study (1) identifies the chemical profile of volatile compounds produced in rectal glands and released by B. frauenfeldi, (2) investigates which of the volatile compounds elicit an electroantennographic or electropalpographic response, and (3) investigates the potential function of glandular emissions as mate-attracting sex pheromones. Rectal gland extracts and headspace collections from sexually mature males and females of B. frauenfeldi were analysed by gas chromatography-mass spectrometry. Male rectal glands contained (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro [5.5]undecane as a major component and (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane as a moderate component. Minor components included palmitoleic acid, palmitic acid, and ethyl oleate. In contrast, female rectal glands contained (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and ethyl laurate as major components, ethyl myristate and ethyl palmitoleate as moderate components, and 18 minor compounds including amides, esters, and spiroacetals. Although fewer compounds were detected from the headspace collections of both males and females than from the gland extractions, most of the abundant chemicals in the rectal gland extracts were also detected in the headspace collections. Gas chromatography coupled electroantennographic detection found responses to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane from the antennae of both male and female B. frauenfeldi. Responses to (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane were elicited from the antennae of females but not males. The two spiroacetals also elicited electropalpographic responses from both male and female B. frauenfeldi. Ethyl caprate and methyl laurate, found in female rectal glands, elicited responses in female antennae and palps, respectively. Y-maze bioassays showed that females were attracted to the volatiles from male rectal glands but males were not. Neither males nor females were attracted to the volatiles from female rectal glands. Our findings suggest (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane as components of a sex-attracting pheromone in B. frauenfeldi.
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