breeding and genetic animal science

J. exp. Biol. (1978), 75. 123-132 123

Wih 3 figures

inted in Great Britain

L-CANAVANINE AND L-CANALINE ON THE NERVOUS

SYSTEM OF THE MOTH MANDUCA SEXTA (L.)

BY ANN E. KAMMER

Division of Biology, Kansas State University, Manhattan, Kansas 66506

D. L. DAHLMAN

Department of Entomology, University of Kentucky, Lexington, KY 40506

AND GERALD A. ROSENTHAL

T. H. Morgan School of Biological Sciences, University of Kentucky,

Lexington, KY 40506

(Received 1 November 1977)

SUMMARY

Injection of L-canavanine, a naturally occurring arginine analogue, and of

its metabolic derivative, L-canaline, induced almost continuous motor

activity in adult tobacco hornworms, Manduca sexta (L.). Initially the moths

flew normally, but after a time interval that depended both on the amino

acid and on the dose (1-45/miol/g fresh weight) the moths became disorientated

and muscle activity was less patterned. Canaline produced its

initial effects 12-30 min after injection, whereas activity in response to

canavanine began after a delay of i-z h. Canaline (derived from canavanine

by an arginase-mediated hydrolytic cleavage) is probably the biologically

active factor.

Canaline did not affect axonal conduction of action potentials nor the

activity of mechanoreceptors on the forewing. Canaline (22/miol/g fresh

weight) prolonged the postsynaptic potential of flight muscle fibres, but

after 20-40 min. the electrical activity of muscle fibres was normal. The

results show that canaline alters the activity of the central nervous system of

adult M. sexta, but its mode of action is unknown.

INTRODUCTION

Many plants accumulate secondary metabolites that may curtail the feeding activity

of phytophagous insects and other herbivores (Fraenkel, 1959, 1969; Ehrlich & Raven,

1965; Whittaker & Feeny, 1971; Feeny, 1975). The nature and efficacy of plant

chemical defences have been of keen interest to those studying herbivore-plant

interactions and plant resistance to various pests. Plant metabolites are also of interest

because these compounds may have pharmacological properties useful in research.

One such metabolite is L-canavanine, H2N—C(= NH)—NH—O—CH2—CH2—

CH(NH2)CO2H, a structural analogue of L-arginine produced by certain leguminous

plants (Rosenthal, 1977 a).

Previous studies on the mode of action of canavanine in insects have focused

primarily on developmental and biochemical effects. Severe and often fatal develop

mental abnormalities result when Manduca sexta or other larvae that normally do not

feed on canavanine-containing plants ingest diets supplemented with canavanine

(references in Dahlman & Rosenthal, 1976; Harry, Dror & Applebaum, 1976).

In the present study we examined the physiological effects of L-canavanine injected

into Manduca sexta (L.) (Lepidoptera: Sphingidae). L-canaline, H2N—0—CH2—

CH2—CH(NH2)CO2H, an ornithine analogue, was also studied, since it is a metabolic

derivative of canavanine and may be the biologically active factor.

MATERIALS AND METHODS

Manduca sexta were raised on agar-based artificial diet (modified after Yamamoto,

1969). Moths were used one day after pupal-adult ecdysis. L-Canaline and L-canavanine

were prepared according to the methods of Rosenthal (1973, 19776). L-

[guanidinooxy-34C]canavanine was prepared by enzymatic synthesis utilizing

L-canaline and L-[guanidino-14C]arginine after the method of Allende & Allende

(1964). At the start of an experiment the appropriate amino acid was dissolved in

physiological saline solution (pH 7-0) consisting of 25 mM-NaCl, 25 mM potassium

methanesulphonate, 4 mM-CaCl2, 33 mM-MgCl2 and 150 mM Tris methanesulphonate

(modified after Rheuben, 1972). For studies on intact moths, small volumes of the test

solution (ca. o-i ml/g fresh wt) were injected into an anterior abdominal segment.

Behavioural observations were made on moths placed in individual containers after

injection. Moths receiving either canavanine or saline were observed every 15 min,

and moths receiving canaline were inspected every 5 min. Each moth was observed for

1 min at the stated intervals, and the percentage of this observation period spent in

large-amplitude wing movements was measured. The length of time after injection,

during which a moth spent at least 50% of an observation period making largeamplitude

wing movements, will be referred to as 'duration of 50% flight'.

Extracellular muscle potentials were recorded by means of fine copper wires

inserted into the flight muscles of a moth or pharate adult waxed to a support. After

recording of the normal motor pattern had been obtained, the animal was injected

with the test solution, as described above.

Intracellular potentials were recorded from the dorsal longitudinal muscle, which

was exposed by dissecting away the overlying ventral cuticle and muscles. The nerve

to this muscle (dlj^; Eaton, 1971I was stimulated via a glass suction electrode. Saline

containing canaline sufficient to give 22 /imo\/g body weight was added to the space

around the thoracic ganglia and ventral aspect of the muscle. In other experiments the

activity of a large sensory nerve that supplies the forewing (nerve II Nib; Eaton, 1974)

was recorded by means of a suction electrode. These moths were injected in the

abdomen with 22 /imol canaline/g and then dissected quickly to expose the nerve.

Arginase activity was determined as follows. Individual, frozen insects (adults or

pharate adults) were ground for 30 sec in a Sorvall Omni-mixer with 25 ml of 100 mM

glycylglycine buffer (pH 76) containing 2 mM-MnCl2 and saturated with phenylthiourea

at 4 °C. After expressing the resulting slurry through cheesecloth, the

homogenate was clarified further by centrifugation at 19000 g for 15 min. One ml of

insect homogenate, supplemented with 4 mg of Sigma type III urease. was placed in

each of eight 25 ml Erlenmeyer flasks. Each flask was sealed with a rubber septu^

Canavanine and canaline effects on flight in M. sexta 125

supporting a plastic centre well containing 4 drops of Hydroxide of Hyamine, and

placed at 37 °C for 45 min to metal-activate the arginase. The enzyme assay was

initiated by injecting 1 ml of 100 mM-L-[guanidinooxy-14C]canavanine (3750

cpm/^mol) into each of the sealed flasks. After 10, 20 and 40 min the reaction was

terminated in duplicate flasks by injecting 2 ml of 2 N-HC1. Sixty min later the septum

was removed and the evolved 14CO2 was determined by placing the plastic centre well

into 10 ml of Bray's scintillation fluid and assaying the radioactivity of the Hydroxide

of Hyamine by liquid scintillation spectroscopy (Rosenthal, 1970). Duplicate zerotime

samples served as the controls. Homogenate protein was determined by the

method of Lowry et al. (1951). A unit of arginase is that amount of enzyme that forms

1 /^mol canaline/min under the described assay conditions. Specific activity is defined

as units/mg soluble protein.

Table 1. Responses of Manduca sexta adults to injections of canavanine or

canaline {data are means ± S.E.)

2 2

90

1

11

2 2

12

12

10

5

56

2 0

I 2 ± I

102 + 6

I22± 12

88+11

12+ 1

11 + 1

2'I +O-2

2'2±O-2

I-9 + O-2

I-6±O2

I'2±0'2

I-2 + O-2

10-8+ I-O

I-2 ±0-2

0-0 +I-I

5 EXB75

If the effects of canavanine were mediated by canaline, then M. sexta must have an

arginase able to produce the required canaline. Arginase activity, determined under

assay conditions that included an initial canavanine concentration of 50 mil, produced

canaline at a rate of 4-6 /imo\ • B min-1 (Table 2).

Table 2. Arginase activity of individual Manduca sexta

Pharate adult 8—12 h 5'86 OO488 + O'OOZ9

preeclosion

Adult 4-14 o-O434±o-ooi7

* Each value is the mean of three determinations conducted with a single insect. A unit of arginase is

that amount of enzyme that forms i /imol canaline/min under the assay conditions described in the

text. Specific activity is defined as arginase units/mg soluble protein.

Recordings of muscle potentials from tethered moths injected with either canavanine

or canaline (22 and 45 /imo\/g) initially showed a normal flight-motor pattern.

Both treated and saline-injected moths produced a motor pattern characterized by

(1) one or a pair of muscle potentials in each motor unit during each wing stroke, (2)

elevator and depressor muscles excited alternately, and (3) a wingstroke period of

40-55 msec (Fig. 1A-C). Subsequently the wingstroke period was longer, and bursts

of potentials were produced. Later the motor output was more irregular and the,

Canavanine and canaline effects on flight in M. sexta 127

—L

40 ms

The possibility that sensory input to the flight pattern generator was altered by

canaline was evaluated by measuring the electrical activity of a large sensory nerve

containing axons of mechanoreceptors located on the forewing (A. E. Kammer &

G. F. Athey, in preparation). Both canaline-treated (22,Mmol/g) and untreated wing

nerves were active tonically and also responded with a brief burst of impulses when

the wing was touched (Fig. 2). Wing nerve activity persisted apparently unchanged

after the thoracic muscles of the dissected preparations exhibited the continuous

rhythmic contractions induced b) canaline treatment. Furthermore, «tivity continued beyond the time required for intact, canaline- tmreeactehda nomreocthesp totor

come unresponsive to stimuli.

5-2

To evaluate the possibility that canaline was not carried to the mechanoreceptors on

the wing, moths were injected with 113indium. After 20 min the radioactivity was

assayed by placing a Geiger-Muller tube near the wing. The 113indium had spread

throughout the wing, suggesting that circulation of haemolymph was adequate to

transport compounds from the abdomen throughout the wing.

The possibility that canaline altered muscle membrane potentials or synaptic

transmission was evaluated by recording intracellularly from the dorsal longitudinal

muscle. Synaptic transmission at these neuromuscular junctions is probably mediated

by glutamate (Rheuben, 1974). The resting potential of the muscle fibres was

unaffected by 22 /imo\ canaline/g. Within 2-10 min after the application of canaline,

the duration of the postsynaptic potential was 2-5 msec longer than normal (Fig. 3).

In affected fibres the amplitude of the muscle potential ranged from 55 to 80 mV, but

more spikes failed to overshoot o than in untreated fibres. The effect on the duration

of the postsynaptic potential was transitory. In some preparations the fibres recovered

quickly (Fig. 3C), but in other preparations a prolonged postsynaptic response was

observed for 20-40 min. After this time most fibres gave normal postsynaptic

responses. These results indicated that canaline did not excite muscle contraction

directly, although it influenced synaptic transmission. They also provided further

evidence that canaline did not interfere with axonal conduction.

Since canaline could affect the CNS by blocking the action of an inhibitory transmitter

such as y-aminobutyric acid (GABA), the effects of picrotoxin, a known

antagonist of GABA, were evaluated. Moths injected with 50 fig picrotoxin/g

(8 moths) or 100 fig picrotoxin/g (3 moths) behaved normally. Four of five moths

receiving 200 fig picrotoxin/g body weight became active and flew within 4-5 min

Canavanine and canaline effects on flight in M. sexta 129

c

Pharate moths produce a flight motor pattern like that of adults (Kammer &

Rheuben, 1976); however, 8-12 h before eclosion the thoracic muscles are rarely

active (Kammer & Kinnamon, 1977). Nine pharate moths in this inactive stage were

injected with 17 fin\o\ canavanine/g and two with 22/tmol/g. Of these 11 animals,

2 produced a flight motor pattern, 2 produced brief bursts of unpatterned potentials,

and 7 (including the higher dose) were unaffected. Seven pharate adults were injected

with 22/imol canaline/g; 6 exhibited a flight motor pattern and the 7th produced

unpatterned potentials. It is not known why the majority of the treated pharate moths

were unaffected by canavanine whereas adult moths were susceptible to canavanine at

these concentrations. These results cannot be rationalized in terms of a decreased

arginase activity of the pharate moth (Table 2).

DISCUSSION

The results show that an injection of canaline or canavanine initially activates the

flight pattern-generator in M. sexta and subsequently causes less patterned but

continual motor activity. In the following discussion possible mechanisms of action of

these amino acids are considered.

Several observations support the hyposthesis that the effects of canavanine result

primarily from the action of canaline. (1) Both amino acids produce essentially the

same sequence of behavioural effects. (2) Except at the highest doses, the effects of

canavanine do not occur until 1-2 h after injection, whereas canaline acts within

15 min. Although the in vivo formation of canaline from canavanine was not determined,

it is reasonable to propose that arginase activity in the adult moth is sufficient

to produce adequate canaline during the observed time delay. (3) Canavanine-treated

moths exhibit the initial response of coordinated large-amplitude flight movements for

a longer time than do the canaline-treated moths; the flight behaviour of the latter

soon degenerates to quivering. If canaline is slowly produced from canavanine, the

consequences of a canavanine injection will be delayed.

The results suggest that canaline alters the activity of central neurones and thus

causes the production of continuous motor output. The central processing of sensory

input is also impaired, as indicated by three observations: (1) the moths become

unresponsive to tactile stimulation, although the activity of wing mechanoreceptors

appears normal; (2) the tarsal reflex that normally inhibits flight is inoperative; (3) the

ability to remain upright is lost.

The mode of action of canaline on the CNS is not known. It may alter synaptic

transmission, since it prolongs the postsynaptic potential at the neuromuscular

junction. However, the transitory nature of this effect is puzzling, particularly in

comparison with the long-lasting effects of canaline on the CNS. Furthermore, the

efficacy of canaline is difficult to explain by comparison of its structure with the

structure of putative neurotransmitters. Possible transmitter substances include

acetylcholine, GABA, glutamic acid, dopamine, noradrenaline, and 5-hydroxytryptamine

(Lunt, 1975). Another neuroactive amino acid is L-leucine, which blocks the

activity of isolated abdominal nerve cords of Pmplaneta americana (Tashiro,

Taniguchi & Eto, 1972). A high dose of picrotoxin, an antagonist of GABA, produces

behavioural changes similar to those caused by canaline, but the effects are transitory,

possibly because the picrotoxin is metabolized rapidly. In contrast, the effects of

canaline on the CNS are more prolonged than the effects of either picrotoxin on the

CNS or canaline on the neuromuscular junction.

Another possible explanation for the results is that canavanine or canaline may act

non-specifically or indirectly. For example, nicotine stimulates the release of factors

that facilitate initially, and then depress, transmission from the cereal nerves to the

giant fibres in Periplaneta americana (Flattum & Sternberg, 1970 a, b). Non-specific

Canavanine and canaline effects on flight in M. sexta 131

depression of neural activity by canaline may first affect inhibitory pathways, allowing

the flight pattern generator to become active. Subsequently, canaline would depress

other neurones, impairing coordination and reducing the number of active motor

units. Alternatively, the active factor may influence the neurones indirectly by altering

properties of the blood-brain barrier that actively regulates cation concentrations

around the neurones in the CNS (Treherne, 1974, 1976). These speculations await

testing by direct examination of the central nervous system.

We thank Sue Kinnamon, Terry Vogan, Patti Hobson and George Athey for their

assistance with this research. We also thank Dr L. L. Boyarsky for his valuable

criticism of the manuscript. Kansas M. sexta were provided by Dr Karl Kramer and

his associates at the U.S.D.A. Grain Marketing Research Laboratory, Manhattan.

Supported by N.S.F. Grant BNS75-18569 (to A.E.K.) and by funds to G. A. R. from

the National Institutes of Health (AM-17322), N.S.F. Grant GMS-75-19770, the

Research Committee of the University of Kentucky, and N.I.H. Biomedical Support

grant no. 5-SO5-RRO7114-08. This is publication number 77-7-83 of the Kentucky

Agricultural Experiment Station, Lexington.

REFERENCES

ALLENDE, C. C. & ALLENDE, J. E. (1964). Pruification and substrate specificity of arginyl-ribonucleic

acid synthetase from rat liver. J. biol, Chem. 239, 1102—1106.

DAHLMAN, D. L. & ROSENTHAL, G. A. (1976). Further studies of the effect of L-canavanine on the

tobacco hornworm, Manduca sexta. J. Insect Physiol. 22, 265—271.

EHRLICH, P. R., & RAVEN, P. H. (1965). Butterflies and plants: a study in coevolution. Evolution 18,

586-608.

EATON, J. L. (1971). Morphology of the head and thorax of the adult tobacco hornworm, Manduca sexta

(Lepidoptera: Sphingidae). I. Skeleton and muscles. Ann. ent. Soc. Am. 64, 437-445.

EATON, J. L. (1974). Nervous system of the head and thorax of the adult tobacco hornworm, Manduca

sexta (Lepidoptera: Sphingidae). Int. jf. Insect. Morph Embryol. 3, 47-66.

FEENY, P. P. (1975). Biochemical coevolution between plants and their insect herbivores. In Coevolution

of Animals and Plants (ed. L. E. Gilbert and P. H. Raven), pp. 3-19. University of Texas Press,

Austin.

FLATTUM, R. F., & STERNBURC, J. G. (1970a). Action of nicotine on neural synaptic transmission in the

American cockroach. J. econ. Entomol. 63, 67^70.

FLATTUM, R. F., & STERNBURG, J. G. (19706). Release of a synaptically active material by nicotine in the

central nervous system of the American cockroach. J. econ. Entomol. 63, 67-70.

FRAENKEL, G. S. (1959). The raison d'etre of secondary plant substances. Science, N. Y. 129, 1466—1470.

FRAENKEL, G. S. (1969). Evaluation of our thoughts on secondary plant substances. Ent. exp. & Appl.

12, 473-486.

HARRY, P., DROR, Y. & APPLEBAUM, S. W. (1976). Arginase activity in Tribolium castaneum and the

effect of canavanine. Insect Biockem. 6, 273-279.

KAMMER, A. E. & KINNAMON, S. C. (1977). Patterned muscle activity during eclosion in the hawkmoth

Manduca sexta. J. comp. Physiol. 114, 313-326.

KAMMER, A. E., & RHEUBEN, M. B. (1976). Adult motor patterns produced by moth pupae during

development. J. exp. Biol. 65, 65-84.

Folin phenol reagent. J. biol. Chem. 193, 265-275.

LUNT, G. G. (1975). Synaptic transmission in insects. In Insect Biochemistry and Function (ed. D. J.

Candy and B. A. Kilby), pp. 283-306. London: Chapman and Hall.

RHEUBEN, M. B. (1972). The resting potential of moth muscle fiber. J. Physiol. 225, 529-554.

RHEUBEN, M. B. (1974). The permeability of the 'synaptic complex' of moth neuromuscular junctions.

Physiologist 17, 388.

ROSENTHAL, G. A. (1970). Investigation of canavanine biochemistry in the jack bean plant, Canavalia

ensiformis (L.) DC. I. Canavanine utilization in the developing plant. PL Physiol. 46, 273-276.

ROSENTHAL, G. A. (1973). The preparation and colorimetric analysis of L-canaline. Analyt. Biochem. 51,

354-361.

132 ANN E. KAMMER, D. L. DAHLMAN AND G. A. ROSENTHAL

ROSENTHAL, G. A. (1977 a). The biological effects and mode of action of L-canavanine, a structural

analogue of L-arginine. Q. Rev. Biol. 52, 155-178.

ROSENTHAL, G. A. (19776). Preparation and colorimetric assay of L-canavanine. Analyt. Biochem. 77,

147-151.

TASHIRO, S., TANIGUCHI, E. & ETO, M. (1972). L-Leucine: a neuroactive substance in insects. Science,

Lond. 175, 448-449.

TREHERNE, J. E. (1974). The environment and function of insect nerve cells. In Insect Neurobiology

(ed. J. E. Treherne), pp. 187-244. Amsterdam: North-Holland.

TREHERNE, J. E. (1976). Extracellular cation regulation in the insect central nervous system. In Perspectives

in Experimental Biology. Vol. 1. Zoology (ed. P. S. Davies), pp. 323-330. Oxford: Pergamon Press.

WHITTAKER, R. H. & FEENY, P. P. (1971). Allelochemics: chemical interactions between species.

Science, N. Y. 171, 757-770.

YAMAMOTO, R. T. (1969). Mass rearing of the tobacco hornworm. II. Larval rearing and pupation.

J. econ. Entomol. 62, 1427-1431.