Insect Chemical Communication

Chemical communication involves the production and release of specific chemicals called semiochemicals by the emitter, and the detection and olfactory processing of these signals (see introduction) leading to appropriate behavioral responses in the receiver. In most insect species, chemical attraction is the major means of sexual recruitment (MOVIE requires RealPlayer or MS Media Player), with females in most (but not all) cases being the emitter and males being the receivers. In this case, these female-produced semiochemicals are referred to as sex pheromones. Semiochemicals (pheromones, kairomones, allomones, etc.) are invaluable tools in integrated pest management (IPM) programs. They may be used to either monitor populations (MOVIE requires RealPlayer or MS Media Player), thus assisting in treatment timing, or to reduce populations by mass trapping, lure-and-kill or mating disruption. Pheromone traps are widely used for monitoring insect populations. A number of lepidopteran species have been successfully controlled using sex pheromones in mating disruption strategies, whereas mass trapping and lure-and-kill are more popular with other groups of insects. The cornerstone of any semiochemical-based approach is a well-defined pheromone system. In moths, this system is largely formed by multiple constituents in a precise ratio.

Chemical Ecology Tools

Bioassay-based chemical ecology approaches generally lead to the full characterization of pheromone systems. There are a number of techniques that can be applied for the identification of pheromones and other semiochemicals. Initially, the pheromone has to be extracted. When a pheromone gland is known, the semiochemicals may be extracted by excising the gland and washing with organic solvents. Alternatively, airborne volatile compounds may be collected from the whole insect placed in an “aeration” chamber (like this). Typically, these crude extracts are fractionated, with the activity being monitored by a bioassay. This can be done in the laboratory using wind tunnels (see one here) or Y-olfactometers (MOVIE requires RealPlayer or MS Media Player). A short-cut bioassay is the hyphenated technique with a gas chromatography linked to an electroantennographic detector (GC-EAD) (see here). Basically, this is a biosensor in which an insect antenna is the sensing element. The chemical constituents of a crude extract or active fractions are separated by a capillary column in the GC, with the eluted peaks passing through the antennae connected to the biodetector (EAD). Matching the EAD peaks with those detected by a regular (flame-ionization) detector lead to the characterization of active peaks (here is an example). Largely, these EAD-active peaks are behaviorally active, but this has to be confirmed with a bioassay. This technique allows the identification of semiochemicals that appear even in trace amounts (as in here). Once the active EAD-peaks have been identified, the next step is the characterization of their chemical structures. The first steps are to analyze the compounds by gas chromatography-mass spectrometry and to interpret the data to determine their structures. Although the former is easier, the interpretation may be complex, particularly when the structure is novel. Data generated by infrared spectroscopy, chemical derivatization, nuclear magnetic resonance (NMR) and other techniques lead to a proposed chemical structure. The structure is confirmed by organic synthesis and comparison to the natural product. Activity is confirmed by indoor bioassays and field tests. Once synthetic attractants are available, they can then be formulated and applied for monitoring and/or controlling populations.

Our Research Programs

The Honorary Maeda-Duffey has both fundamental and applied research activities. The former is aimed at getting a better understanding of the molecular basis of insect olfaction. Using Bombyx mori as a model, the PI, his associates, and collaborators have unveiled the mechanism of binding and release of pheromones by pheromone-binding proteins (Wojtasek and Leal, 1999; Damberger et al., 2000;Leal, 2000; Sandler et al., 2000; Horst et al., 2001a, 2001b;Horst et al., 2001b has been selected for the Faculty of 1000). Structural studies are part of on-going collaborations with Professor Jon Clardy and Professor Kurt Wuthrich, Nobel Prize Laureate in Chemistry (2002). In addition to the structures of bombkyol-PBP complex and the acidic form of BmPBP, we have now determined the structure of the unliganded pheromone-binding protein from the silkworm moth at physiological pH. This basic form of the protein showed that even in the absence of a pheromone molecule, BmPBP at the pH of the sensillar lymph contains a large hydrophobic cavity with a sufficient volume to accommodate the natural ligand, bombykol (Lee et al., 2002). Recently, we have identified olfactory proteins from various species of scarab beetles (Deyu and Leal, 2002;Nikonov et al., 2002), a primitive termite, Zootermopsis nevadensis nevadensis (Ishida et al., 2002), the Argentine ant (Ishida et al., 2002), and females of a mosquito species, Culex quinquefasciatus (Ishida et al, 2002). The identification of the first mosquito odorant-binding protein is part of an effort by the PI’s team to develop a protein-based approach for the screening of mosquito attractants and repellents. This project is part of a special cooperation agreement with the USDA-ARS-Chemical Affecting Insect Behavior Laboratory (link is here) and is partially funded by the University of California Systemwide Mosquito Research Fund (link).

Odor-oriented navigation in insects, be it a male moth flying towards a pheromone source (a female advertising her readiness to mate) or a female mosquito seeking a blood meal from a human host, requires both the fast delivery of semiochemicals to their olfactory receptors and their inactivation on a millisecond timescale (soon after the signal is delivered). Unlike the mechanism of pheromone binding and release (see introduction), the molecular basis of signal inactivation in insects is under considerable debate. The cloning and characterization of the first putative insect odorant-degrading enzyme (Ishida and Leal, 2002) is part of the effort by the PI’s team to test the hypothesis that chemical signals are inactivated by an enzymatic process.

Contribution to the Agricultural Experiment Station

The fundamental research activities in the Honorary Maeda-Duffey lab may lead us to the long-term goal of understanding how animals perceive and orient in the environment. A full understanding of the molecular basis of signal processing and inactivation may pave the way for the development of novel strategies for controlling insect pest populations based on manipulation of the olfactory system.

On the other hand, the mid- and short-term goals of our applied research activities are the discovery of attractants (pheromones and other semiochemicals) and development of new formulations to be used in IPM programs. We focus both on species that are economically important to California agriculture and invasive species, with the potential to threaten California agriculture. For example, we have been screening possible attractants from plants preferred by females of the glassy winged sharpshooter (Homalodisca coagulata) (GWSS) for oviposition. These plant-derived volatile compounds are identified by gas chromatography with a mass spectrometer detector, synthesized, and tested in the field in collaboration with Dr. Frank Zalom. This project may lead to improved trap systems for monitoring population levels of the GWSS.

The PI has identified pheromones and other semiochemicals from a number of species, including scarab beetles, true bugs, cerambicids, moths, etc. Many formulations are now commercially available for monitoring populations (MOVIE requires RealPlayer or MS Media Player)of these economically important species. Recently, the PI and his collaborators in Brazil and in Japan have identified the sex pheromone of the citrus fruit borer, Ecdytolopha aurantiana (Leal et al., 2002; Bento et al., 2002), which causes orange growers in Brazil an estimated 50 million dollars in crop losses annually. The availability of a pheromone system for monitoring population levels of the citrus fruit borer has allowed a dramatic reduction in the number of sprays, with consequent decrease in losses (both economic and environmental).

Although these results may not have immediate application to California agriculture, these pre-emptive actions are highly desired since the availability of early detection is essential for eradication programs in the event of the citrus fruit borer becoming an invasive species.

Another AES-related project is aimed at the development of a protein-based high throughput system for the screening of mosquito repellents. User-friendly, better repellents would reduce the chances of human virus infection by deterring mosquito feeding. These new products would benefit farmers and the public in general.

References

• Damberger, F., Nikonova, L., Horst, R., Peng, G.H., Leal, W.S. and Wuthrich, K. (2000). NMR characterization of a pH-dependent equilibrium between two folded solution conformations of the pheromone-binding protein from Bombyx mori. Protein Science, 9, 1038-1041.
• Horst, R., Damberger, F., Peng, G., Nikonova, L. Leal, W. S. and Wüthrich, K (2001a). NMR assignment of the A form of the pheromone-binding protein of Bombyx mori.” J. Biomol. NMR, 19, 79-80.
• Horst, R., Damberger, F., Luginbuhl, P., Guntert, P., Peng, G., Nikonova, L., Leal, W.S. and Wuthrich, K. (2001b). NMR structure reveals intramolecular regulation mechanism for pheromone binding and release. Proc. Natl. Acad. Sci. USA, 98, 14374-14379.
• Ishida, Y., Chiang, V. P., Haverty, M. I. and Leal, W. S. (2002). Odorant-binding proteins from a primitive termite. J. Chem. Ecol., 28, 1887-1893.
• Leal, W.S. (2000). Duality monomer-dimer of the pheromone-binding protein from Bombyx mori. Biochem. Biophys. Res. Commun., 268, 521-529.
• Sandler, B.H., Nikonova, L., Leal, W.S. and Clardy, J. (2000). Sexual attraction in the silkworm moth: structure of the pheromone-binding protein-bombykol complex. Chem. Biol., 7, 143-151.
• Wojtasek, H. and Leal, W.S. (1999). Conformational change in the pheromone-binding protein from Bombyx mori induced by pH and by interaction with membranes. J. Biol. Chem., 274, 30950-30956.