The Search for a New Non-toxic Insecticide:

Glutamate Receptors and the Righting Reflex in Crickets

Laura B. Eichhorn

St. Andrew's Episcopal School, Ridgeland, MS 39157 and

Department of Pharmacology, University of Mississippi Medical Center, Jackson, MS 39216

[Laura B. Eichhorn received the Mississippi Junior Academy of Sciences Clyde Sheely Award for 1997. Special thanks to Mississippi Chemical Corporation in Yazoo City, Mississippi, for underwriting the publication of this research paper.]

With the ultimate goal of developing a non-toxic insecticide targeted at glutamate receptors in the insect nervous system, crickets were tested to determine which glutamate receptor subtype (quisqualic acid (QA) and NMDA (N-methyl-D-aspartate)) mediates the loss of righting reflex. It was hypothesized that the agonist QA, because of its permanent binding to the receptor site, would cause dose-dependent immobility (paralysis) in crickets and that the agonist NMDA would not. Righting reflex tests with weight-adjusted doses of QA at 1, 10, 30, 50, and 100 nmoles/µl showed a dose-dependent effect with an ED₅₀ of 10 nmoles/µl and an ED₉₅ of 50 nmoles/µl for all crickets (males plus females). N-methyl-D-aspartate tests at the maximal concentration of 1000 nmoles/µl showed no effect on the righting ability of the crickets. The QA hypotheses were correct and NMDA did not inhibit glutamate-mediated neurotransmission. Because the righting reflex is a function of locomotion that expresses muscle contraction, the quisqualic acid receptor is a receptor involved with glutamate neuromuscular transmission, the disruption of which leads to "natural" insect death. Quisqualic acid, therefore, shows promise as an effective insecticide and is non-toxic to humans. Additional study of glutamate receptors in insects and the most efficient delivery of QA as an insecticide is under way.

The goal of this long-term research effort is to develop a new non-toxic insecticide that will only affect an insect's neuromuscular system and cause no harm to humans or the environment. Most of today's typical insecticides are harmful poisons that not only kill insects but can kill plants, animals, and humans. Organophosphate insecticides, by far the most common agricultural insecticides, are toxic because they are very nonspecific, attacking all cholinergic nerve endings, including those in the human parasympathetic nervous system. While they kill insects, these compounds are very dangerous to humans and cause many acute symptoms in the ocular, respiratory, and cardiovascular systems. Without treatment, most human victims die of respiratory failure within 24 hours (Goodman and Gilman, 1980).

One important function of the neuromuscular system is mediation of locomotion. It has been shown that glutamate receptors facilitate locomotion in insects (Satelle, 1992). Glutamate is a messenger, or transmitter, in the insect neuromuscular system. Its release is stimulated by nerve impulses to the muscle. The glutamate molecules then attach to receptors, stimulating the muscle contraction. The glutamate then rapidly detaches from the receptor, thus ending the muscle contraction and freeing the receptor. There are four main known types of glutamate receptors, each defined by a specific chemical that binds to it: quisqualic acid, NMDA (N-methyl-D-aspartate), kainic acid, and ACPD (aminocylopentanedicarboxylate). Each of these compounds is a glutamate agonist (acts like glutamate). However, when QA binds to the glutamate receptor, it is permanently bound, causing a continuous stimulus and contraction of the muscle. This should immobilize (paralyze) the insect. Studies have yet to determine the precise site of glutamate action in the neuromuscular system, although it is reasonable to hypothesize that it is at the neuromuscular junction. This is similar to acetylcholine in humans, and a competitive inhibitor would be similar to curare (used to cause muscle relaxation) in people.

In previous research by this investigator (unpublished data, 1995), it was found that cockroach locomotion was a good assay for insect neuromuscular activity. It was also found that there was no difference in locomotion between different temperature levels, but cockroaches travelled more in the dark than in the light. This research on locomotion inspired further study of the insect neuromuscular system and how locomotion is specifically controlled in insects in order to find a chemical that would target this system and could be used as an insecticide. The insecticide would not kill the insect, but paralyze it by contracting the leg muscles, and the insect would die "naturally."

In this study, righting reflex of the cricket, an insect closely related to the cockroach, was used as the test function of locomotion. The loss of the righting reflex (the ability of a cricket to flip back over when placed on its back) indicates that the cricket has lost control of its muscular function. The goal of this research was to find which glutamate receptor subtype might cause the loss of the righting reflex in the cricket. Considering studies on other animals (Castle, Evans, and Kirkpatrick, 1984), it was hypothesized that QA in appropriate doses would cause immobility in the crickets and, if so, a dose-response curve could be constructed. It was also hypothesized that because NMDA is a traditional agonist (acts like glutamate), it would not cause immobility in the crickets.

MATERIALS AND METHODS

A control vehicle (later to be used to dilute and inject the test compounds) was developed and tested. This "insect saline" solution [mM concentration in dionized water: NaCl 140, KCl 5, NaHCO₃ 4, MgCl₂ 1, CaCl₂ 0.75, HEPES 5] approximates the naturally occurring fluid within the cricket body and was injected into 20 crickets to verify that this saline and the injection procedure had no effect on the righting reflex in the subject crickets. Five microliters of the saline was injected into the cricket lower abdomen with a 10 µl tuberculin microsyringe. Quisqualic acid (Sigma Chemical) was then diluted to concentrations of 1, 10, 30, 50, and 100 nmoles/µl in "insect saline." At each concentration, 10 male and 10 female crickets were tested. N-methyl-D-aspartate (Sigma Chemical), known to be less potent, was then prepared at an approximately proportionate concentration of 1000 nmoles/µl in "insect saline." Results were expressed as percent crickets immobilized at each dose and a semi-log plot was created for the dose-response curve in order to define ED₅₀ and ED₉₅ values. Then "probits" (arbitrary probability units of an all-or-none pharmacologic response related to area under a normalized curve, employing a scale of about 2.5­7.5) were calculated to allow a linear plot of the resultant dose-response curve. The UMC Pharmacologic Calculation System, Version 4.0, was used for t-tests and linear regression testing of the male vs. female subsets of crickets. P < .05 was considered statistically significant.

The specific protocol for each test was: healthy adult crickets (Ridgeland, MS, Cricket Farm) were selected at random from large batches and weighed on an electronic balance. These crickets were then tested for initial righting ability before injection. Quisqualic acid or NMDA solution volumes were 1 µl/gram insect weight further diluted in the 10 µl syringe in all cases to a total injection volume of 5 µl, which was then deposited in the center of the lower abdomen. The crickets were allowed to run freely for 5 minutes in the test box and were then given three righting reflex tests of 1 minute each. Failure to return to a normal standing position during all three 1-minute tests was considered "immobility" (paralysis). All crickets were sacrificed (immersion in alcohol) following completion of testing.

RESULTS

Quisqualic acid induced cricket immobilization at all concentrations tested. As the concentration of QA increased, the percent immobilized response also increased (Table 1). While there was a suggestion in the data that males are more susceptible than females with male response of 100% beginning at the 30 nmoles/µl concentration and female response of 100% only at the 100 nmoles/µl concentration, there was no statistically significant difference between males and females by t-test comparison. The ED₅₀ for QA (males + females combined) was 10 nmoles/µl and the ED₉₅ was 50 nmoles/µl. A probit plot of the dose-response curve is shown in Figure 1. N-methyl-D-aspartate, even at a maximum dose of 1000 nmoles/µl, does not induce any cricket immobilization (n=20, 0 % immobilized: no effect seen).

Table 1. Percent Crickets Immobilized with Varying Concentrations of Quisqualic Acid.

DISCUSSION

Quisqualic acid immobilized the crickets at doses within the test range that was, in part, suggested by previous work by other investigators (Castle, Evans, and Kirkpatrick, 1984). Therefore, these results support the hypotheses.

The NMDA clearly had no effect compared to quisqualic acid, even at a much higher concentration. It does not inhibit glutamate-mediated neurotransmission that controls locomotion expressed as the righting reflex. Because the end point was loss of righting reflex and NMDA had no effect, these data do not allow analysis of the role of the NMDA receptor in the insect neuromuscular system.

The righting reflex is a function of insect locomotion that involves muscle contraction. The QA glutamate receptor is a receptor involved with glutamate neuromuscular impulse transmission that specifically influences locomotion, which is further indicated by the results of this study. Immobilization of an insect for a sufficient period of time will eventually kill it. Quisqualic acid binds permanently to the glutamate receptor. Therefore, QA can be an effective insecticide specifically targeting the glutamate receptor, in sharp contrast to the non-specific generalized cholinergic (acetylcholine mediated) toxicity of organophosphate insecticides. Quisqualic acid should be non-toxic to humans because it does not influence cholinergic transmission, the main mediator in humans.

The suggestion that male crickets may be more susceptible to QA than female crickets (though not statistically significant) is interesting from an insect physiology and endocrinology standpoint. If a significant difference could ever be shown, this may influence the strategy of actually administering such an insecticide.

With these findings about QA, work will continue to develop a practical insecticide. An attempt to verify that QA acts at the peripheral neuromuscular junctions rather than in the central nervous system is underway and involves a study preparation including application of the study compounds directly to the hyperneural muscle of the cockroach, the main muscle of locomotion. Testing of QA antagonists such as Joro spider toxin to test effects on the righting reflex, the action of QA shown in these results, and the production of glutamate is also planned. Eventually, the practicality of a delivery system for a commercial preparation of an actual insecticide containing QA that will work on cockroaches as well as crickets and other members of that insect family, will be considered. The lack of toxicity to humans is anticipated to encourage and support eventual commercial development.

ACKNOWLEDGEMENTS

The author thanks: Robin W. Rockhold, Ph.D., Professor of Pharmacology at the University of Mississippi Medical Center, for his assistance as a mentor, his support, and making available his lab for this research; the science faculty at St. Andrew's Episcopal School, headed by Mrs. D. Chambliss, for inspiration and encouragement; and, lastly, the author's parents for their support and all of their valuable help.

LITERATURE CITED

Castle, F., R.H. Evans, and J. Kirkpatric. 1984. A comparison of the effect of kainate and some related amino acids on locomotor activity in cockroaches and electrical activity recorded from locust ventral nerve cord. Journal of Comparative Biochemical Physiology. 77C:399­402.

Goodman, L.S., A.G. Gilman, and A. Gilman. 1980. The pharmacological basis of therapeutics. Macmillan, NY, pp.100­116.

Sattelle, D.B. 1992. Receptors for L-glutamate and GABA in the nervous system of an insect (Periplaneta americana). Journal of Comparative Biochemical Physiology. 103C:429­438.