Lokensgard Research

Behavioral and Biochemical Analyses of Sex Identification Pheromones in Crickets

As one of the projects in a 2001 institutional proposal to the Merck Foundation, Professor Brad Rence of the Lawrence biology department and I outlined an ambitious interdisciplinary project to study the details of the mechanism by which House Crickets, Acheta domesticus, tell one sex from the other. Characteristically a male who is mature and ready to mate (an adult with a fully formed spermatophore in place) will show distinctly different behavioral reactions to encountering a male or a female. Typically he will sing aggressively to the male and will emit the courtship song to the female. Using behavioral choice and/or the behavioral methodology established by Rence and Loher (1977) for the Australian Field Cricket, several investigators have tried to elaborate the identification mechanism in the House Cricket. These studies (Otte and Cade, 1976; Hardy and Shaw, 1983; Tregenza and Wedell, 1997) resulted in some confusing and contradictory results which have been resolved in the studies we have conducted with Merck funding over the past three summers (Rence, et al., 2005). We now know that the sex identification pheromones are not volatile, that males and females have distinctive gas chromatographic profiles of cuticular extracts, that there are approximately ten compounds in the cuticular layer that differ somewhat between the sexes, that males will only respond appropriately to the pheromone if they possess a spermatophore, that the major site of perception is the antennae, and that the most probable site of production of the cuticular pheromones is in unique metanotal glands found in both males and females. We are now positioned to finish investigating the problem. We have proposed, in a new Merck/AAAS proposal, to expand and extend the behavioral and biochemical analyses of the putative sex identification pheromones of the House Cricket.

The focus of the biological aspect of the project will be to establish a clear, biologically relevant and realistic bioassay for testing the putative pheromonal cuticular extracts for their ability to elicit sex specific behavior in adult male test crickets which have spermatophores in place; and, hence, are fully sexually receptive. In previous studies the pheromones have been tested in Y-mazes (Otte and Cade, 1976; Hardy and Shaw, 1983), on severed antennae (Rence and Loher, 1977) or live crickets shoved into vials (Hardy and Shaw, 1983) manipulated by the investigator, and on dead crickets (Tregenza and Wedell, 1997). We feel that all of these methods are artificial and remove the testing of the putative pheromones from a real world biological context.

As an alternative we have proposed to use living juvenile crickets as test subjects. In their antennal fencing experiments Rence and Loher (1977) found that adult, sexually receptive Australian Field Cricket males responded neutrally to the touch of severed juvenile male and female antennae. We will use whole, living juveniles as the stimulus animals for our experiments. We will first simply observe the natural behavioral interactions between either a male or a female juvenile cricket and a sexually receptive adult male test subject. If we can substantiate the neutrality of juveniles, we will then begin to use them as substrates for whole body extracts from adult males or females; and then, subsequently, for specific molecules or complexes (ratios) of molecules. We believe that living, behaving juveniles provide the best possible biological stimulus for bioassaying the putative sex identification pheromones.

In addition to the bioassay development and usage we also propose several other biological extensions of the previous pheromone work. We will look more carefully at the presumed glandular site of pheromone production. We have excellent scanning electron microscopic views of the external surface of these glands, so the next step will be to begin examining them with sectioned specimens under the light microscope. This will allow further evidence for their glandular nature. We have shown that there is no electroantennogram response to anything volatile coming from either male or female house crickets. The next step here is to develop a methodology for applying the stimulus chemo-tactily to severed antennae while recording from them. The major stumbling block will be to standardize the tactile part of the stimulus from one application to the next. By attaching the stimulus delivery object (most likely a severed antenna with its natural pheromonal constituents or a wire upon which extracts have been placed) to a piezo-electric crystal and then applying known standard voltages to the crystal, we should be able to effect the same strength and duration of touch from one test to the next.

Analysis of the cuticular lipids has been done, to this point, primarily through gas chromatographic separation and mass spectrometric compound identification (GC-MS) of hexane or chloroform washings of whole or dissected animals. To identify the active compounds through the bioassays described above, we will need samples that have been fractionated by compound type and compound size. Initially, at least, we intend to follow Warthen et al. (1980), who separated saturated and unsaturated hydrocarbons by chromatography on silver nitrate impregnated columns (which selectively retain unsaturated materials) and separated linear from branched alkanes by selective adsorption of linear molecules in molecular sieves. We plan also to separate these fractions by carbon number, using preparative GC or HPLC (high performance liquid chromatography). As the bioassay procedure allows us to draw inferences concerning the compound(s) involved in the gender recognition, we will test those hypotheses with synthetic samples prepared with commercially available pure compounds (or pure samples we synthesize ourselves if the materials are not readily available).

Hardy, T.N. and Shaw, K.C. 1983 The role of chemoreception in sex recognition by male crickets, Acheta domesticus and Teleogryllus oceanicus. Physiol. Entomol. 8:151-166.

Otte, D. and Cade. W. 1976 On the role of olfaction in sexual and interspecies recognition in crickets (Acheta and Gryllus). Anim. Behav. 24:1-6.

Rence, B. and Loher, W. 1977 Contact chemoreceptive sex recognition in the male cricket, Teleogryllus commodus. Physiol. Entomol. 2:225-236.

Rence, B., Putterman, D., Lokensgard, J., and Wesenberg, A. 2005 Behavioral, anatomical, physiological and biochemical aspects of pheromonal sex recognition in the house cricket, Acheta domesticus (L.). Submitted for publication to Journal of Insect Behavior.

Tregenza, T. and Wedell, N. 1997 Definitive evidence for cuticular pheromones in a cricket. Anim. Behav. 54:979-984.

Warthen, J.D. and Uebel, E.C. 1980 Comparison of the unsaturated cuticular hydrocarbons of male and female house crickets, Acheta domesticus (L) Insect Biochem. 10:435-439.

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