INTRODUCTION

As documented previously, the higher plant nonprotein amino acid L-canavanine is a potent arginine antimetabolite that is typically insecticidal to non-adapted insects (Rosenthal, 1982; Rosenthal and Bell, 1979). For example, terminal instar larvae of the tobacco hornworm, Manduca sexta [Sphingidae], reared on an agar-based diet supplemented with 2.5 mM canavanine, produced pupae and adults that exhibited striking developmental aberrations.

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In marked contrast, larvae of the tobacco budworm, Heliothis virescens [Noctuidae], a highly destructive agricultural pest (Lincoln, 1972) reared on canavanine-containing diet had an LC₅₀ value for this arginine antagonist of 300 mM (Berge et al., 1986).

This LC₅₀ corresponds to 53,000 ppm wet diet weight or nearly 40% on a dry weight basis (Berge et al., 1986). While the pupae that emerged from these canavanine-treated larvae were depauperate, they lacked discernible developmental aberrations. A group of five larvae sustained on a staggering 500 mM canavanine-containing diet survived for 9 days before the first larval dead was recorded.

Heliothis virescens resistance to this arginine antagonist was also demonstrated by parenteral injection of canavanine: the LD₅₀ for this nonprotein amino acid in M. sexta was 1.0 g kg-1 fresh body weight as compared to 10.7 g kg-1 for H. virescens (Berge et al., 1986). Heliothis virescens might tolerate elevated levels of dietary canavanine because of its ability to excrete this potentially toxic allelochemical. Analysis of the larval fecal matter, produced by terminal instar larvae from feeding on 150 mM canavanine-containing diet, disclosed that only 0.6% of the consumed canavanine was eliminated by this excretory route. Examination of the body fluids and tissues established the lack of significant sequestration of canavanine within the larval body (Berge and Rosenthal, 1990). Thus, H. virescens actively metabolized canavanine; the t_1/2 for canavanine clearance from the hemolymph was 135 min (Berge and Rosenthal, 1990).

LARVAL ABILITY TO TOLERATE CANAVANINE

Biochemical studies were conducted to determine if larval ability to tolerate canavanine resulted from its constitutive metabolism, or alternatively was induced in response to dietary consumption of canavanine (Berge and Rosenthal, 1990). In one study, the t_1/2 for canavanine clearance of parenterally injected L-[guanidinooxy-¹⁴C]canavanine, in larvae maintained on a 150 mM canavanine-containing diet for 72 h, was 138 min. This is exactly the same value found for control insects that were not exposed previously to canavanine prior to determining the t_1/2 value for canavanine clearance.

In another approach to resolving this question, H. virescens larvae were provided sufficient cycloheximide to inhibit nearly 80% of the L-[³H]leucine-labeled protein formation observed in control animals. Such cycloheximide-treated insects cleared canavanine with a t_1/2 equal to 121 min; once again this value was identical in control animals (Berge and Rosenthal, 1990). Electrophoretic analysis of hemolymph proteins of larvae reared on 150 mM canavanine-containing diet was identical to that obtained from larvae raised on control diet.

IN SUMMARY, NONE OF THE DESCRIBED EXPERIMENTS PROVIDED EVIDENCE FOR SYNTHESIS OF A UNIQUE PROTEIN BY CANAVANINE-TREATED INSECTS. THIS RESULT SUPPORTED THE VIEW THAT H. VIRESCENS DREW UPON A PREEXISTING, CONSTITUTIVE ENZYME TO CATABOLIZE CANAVANINE.

Analysis of the various body organs of the larvae demonstrated that canavanine catabolism occurred solely in the gut. In particular, the fat body, the rough insectan equivalent of the mammalian liver and a major detoxification organ, did not possess significant canavanine-degrading ability. Moreover,

CANAVANINE CATABOLISM

To unravel the metabolic disposition of canavanine, the larvae were provided L-[guanidinooxy-¹⁴C]canavanine. The principal degradation product of radiolabeled canavanine was [¹⁴C]guanidine(Berge and Rosenthal, 1991). Subsequent in vivo studies employing L-[1,2,3,4-¹⁴C]canavanine identified L-[1,2,3,4-¹⁴C]homoserine as the preponderant radiolabeled catabolite. This finding immediately suggested the following pathway in which the ¹⁴carbon atom of the guanidinooxy moiety of canavanine was transferred to guanidine:

Other experiments with larvae of H. virescens led to the discovery of a gut enzyme--a larval reductase able to catalyze an NADH-dependent reduction of hydroxyguanidine to guanidine (reference). This finding created the intriguing possibility that canavanine was catabolized initially to homoserine and hydroxyguanidine rather than guanidine by a novel hydrolase able to cleave the O-N bond of the guanidinooxy moiety of the substrate.

While This metabolic ability has been observed in a soil-borne Pseudomonas (reference), the responsible enzyme was not isolated nor has this metabolic capacity been described from an eukaryotic organism.

THE EXPERIMENTAL EVIDENCE INDICATED TWO ENZYMES WERE FUNCTIONING IN CONCERT TO METABOLIZE CANAVANINE. THE FIRST REACTION EMPLOYED A NOVEL HYDROLASE THAT DIRECTED THE FORMATION OF HOMOSERINE AND HYDROXYGUANIDINE FROM CANAVANIE. IN THE SECOND REACTION, HYDROXYGUANIDINE WAS REDUCED TO GUANIDINE.

CANAVANINE HYDROLASE

The existence of canavanine hydrolase (CH), an enzyme able to cleave an oxygen-nitrogen bond, would be an important finding since this enzyme is the only protein known to demonstrate this ability. As such it would represents a new type of hydrolase-- one that acts on oxygen-nitrogen bonds (EC 3.13.1.1).

The search for this enzyme in the gut of larval H. virescens culminated in the isolation of a homogeneous enzyme that mediated an irreversible hydrolysis of L-canavanine to L-homoserine and hydroxyguanidine. Canavanine hydrolase (EC 3.13.1.1) exhibited a high affinity for canavanine as judged by the apparent K_m value of 1.1 mM for canavanine. The turnover number for this reaction was 21.1 mmol min^-1 mmol^-1. Canavanine hydrolase also exhibited a high degree of specificity for L-canavanine as it could not function effectively with either L-2-amino-5-(guanidinooxy)pentanoate or L-2-amino-3-(guanidinooxy)propionate, the higher or lower homolog, respectively of L-canavanine nor its methyl ester. Canavanine derivatives such as L-canaline and O-ureido-L-homoserine were not metabolized significantly by canavanine hydrolase.

Literature cited

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Berge, M. A. and Rosenthal, G. A. (1991) Metabolism of L-canavanine and L-canaline in the tobacco budworm, Heliothis virescens [Noctuidae]. Chem. Res. Toxicology 4, 237-240.

Berge, M. A., Rosenthal, G. A. and Dahlman, D. L. (1986) Tobacco budworm, Heliothis virescens [Noctuidae] resistance to L-canavanine, a protective allelochemical. Pestic. Biochem. Physiol. 25, 319-326.

Brazzel, J. R., Newsom, L. D., Roussel, J. S., Lincoln, C. and Williams, F. J. (1953) Bollworm and tobacco budworm as cotton pests in Louisiana and Arkansas. La. Agric. Exp. Stn. Tech. Bull. 482, 47 pp. Fitt, G. P. (1989) The ecology of Heliothis species in relation to agroecosystems. Ann. Rev. Entomol. 34, 17-52.

Lincoln, C. (1972) Distribution, abundance and control of Heliothis spp. in cotton and other host plants., Oklahoma Agricultural Experimental Station, Oklahoma State University, Stillwater, OK.

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Rosenthal, G.A., and Bell, E.A. (1979) In: Herbivores: Their Interaction with Secondary Plant Metabolites (Rosenthal, G.A., and Janzen, D.H. eds.) pp. 353-385, Academic Press, San Diego, CA.

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