THE INFLUENCE OF CERTAIN RESPIRATORY INHIBITORS AND OTHER. CHEMICALS ON THE INTEGRATED NETABOLISM OF THE GRAY GARDEN SLUG, DEROCERAS RETICULATUS (MULLER)

GRANT MELVIN WHITE

A THESIS

euthuitted to

OREGON STATh COLLEGE

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

June 1955 AP PRO VET) ;

Associate Professor of Entomology

In Charge of Major

Chairman of the Department of Entoo1or

o' Chairman of School Graduate Conrntittee

IJean of Graduate School

Date thesis is oresentect May 11, 1955

Typed by Mary Adams LCîNOWLEDGMENT

Gr&teful acknowledgment i made th Dr. Leon C. Terriere for hi constructive criticism and helpful guidance during both the experiment1 work and actual writing of the theiis, and to

Dr. Paul O. Ritcher, Chairman of the Department of Entomology, for the graduate research asbiztantship. I wish to exprese my thanks to Marvin F. Hill for translating German references nd his assistance in collecting specimens. TABLE OF CONTENTS

Page

Introduction ...... , . . . . i Mteria1 and Methode ...... 7 tesultß and Dicusion ...... ,., 1. icespiration ......

2 Role of Integument . . . . 5 ...... S.S 18

Conclusion ...... -...... 23

Suxrnary ...... 21.

Bibliography ...... 31

LIST OF FIGURES AND TÁBLFS

Figure

1. The Effect of Azide and Malonic Acid on Rate of Oxygen Consumption sos...... 2

2. The Effect of Arsenite, Fluoride and Urethane on RateofOxygenconanuiption...... , 26

3. The Effect of Metr;ldede on hate of Oxygen

Consumption ...... 27 4. Schenie to Summarize the Enzyme-Catalysed Steps in the Glycolytic Cycle, Tricarboxylic Jcid Cycle, and Cytochrorne System, rith the Pointe of Action of the

Inhibitory Chemicals ...... 55 55...... 28

Table

1. Toxicity of Parithion to Slugs ...... 29

2. Toxicity of Heptachior to Slugs ...... 30 THE INFLIJCE OF CERTAIN RESPIRATORY INHIBITORS AND OTHER CBE4ICALS ON THE INTEGRATED 4ETMsOLISM OF THE GRAY GkRDEN SLUG, DEROCERAS RETICULATUS (MULLER)

INTRODUCTION

The grey garden slug, Derocera reticulatus (Muller) , is of growiii economic concern in estern Oregon. It is a ßeriou8 pe8t to field and truck crops, sna1l fruits, ornamentals, and greenhouse plants, causing considerable losses. The versatile feeding habits, and adaptability of this organism to its environment render it non- susceptible to natural controls. Slugs E&re one of the most destruc- tive, persistent and difficult to control of all organisms.

The struggle to control slugs undoubtedly commenced when it was first recognized more than two centuries ago in England. Since then, it has increased in economic iniport&nce to the present time.

As early as 1920, serious studies were undertaken in the hopes of finding an adequate control for the slug. Control measures used were contact materials and irritants such as sulfur and sodium hydroxide; a mixture of carbolic acid, gasoline, and sulfur; nico-

tine; lead arsenate; paris green; copper sulfate; lemon oil; clove

(23, oil; and Bordeaux mixture pp.18-23) . Although no worthwhile results were obtained, these observations showed that the Bordeaux mixture possessed repeflant qualities. Lovett and Black (23, p.42) recognized the growing importance of this invertebrate and summarized

their attempted control as follows: They have comparatively few

atural enemies and are surprisingly resistant to poisons either internal or external. Control practices in common use against th8ects iid other nima1 pests are of little avail against them.

In an extensive experimental study of s1u toxicnts in 1931,

(25, pp.390-400) the dusting of copper sulfatte, c.lcium cranide an

calciui carbide upon the slug, resulted in n iediate kill. How-

ever, these compounds showed only a temporary control. A short

residual life or hytotox1city were disadvantages that limited their

usefulness.

These materials have been gradually discarded for a variety

of reasons Primarily because of the inadequate control obtained.

i4etaldehyde was introduced in slug control about 20 years

ago when it was accidentally discovered to be a slug attractent.

It seems that a woman residing in South Africa used Heta-fuel

(metaldehyde) for heating her curling tongs. Later, after throwing

the unused fuel out the window, she observed the assemblage of dead

slugs. Upon this discovery, ìietaldehyde was adopted in 1ng1and for

slug control (14, pp.l67-l&).

Since then, basic and applied research has evolved around

this material. In 1942 metaldehyde was observed by Barries and Well

(3, pp.56-6) to contribute in some unexplained way to the death of

the organism. In 194$, Thomas stated that metaldehyde exhibited

three physiological manifestations, "a mortally toxic effect produc-

Ing a transarency of the gut wall, an anaesthetic effect, and an irritating effect with. slime procíucedW (35, p.207).

Since the discovery of the iuolluscicidal properties of met- aldehyde, many baits have been manufactureu which incorporated this 3

riiaterial along with sorne heavy metal poison, usually arsenic (4, p.56)

(21, pp.321-322). The latest advance has been the development of

metaldehyde for u.se as a dust or spray (13, p.370). The high cost,

the formulation problems arising from its limited solubility, the

short residual life, and most important the inconsistent toxic

effect3, 1init rnet8ldehyde as a reliable molluscicioe for large

scale operation.

In spite of the rnany ootent materials available in our modern

arsenal of insecticides, the control of the slug ha not been achieved.

The failure of the many toxic materials to possess molluscicidal

properties m&y be due to three factors; the slug's haUt. and

ecology, fundamental biochemical differences, or the possession of

some protective feature such as the slime mechanism.

Thus the versatile feeding habite, the ability to be inactive

for long periods of time without food, and a prolific reproduction

capacity, are characteristics that favor the survival of the slug.

In addition, the activity of the slug being greatest during the wet weather would have an important influence on the ty)e of control measures.

A second possibility, given support by the failure of the organic phosphates, the chlorinated hydrocarbons, and other modern insecticides to control the sl, is that this organism, compared to other invertebrates, is different with respect to its metabolic systems. If this were true, its failure to be controlled by current pesticides would be understandable. Finally, the ability of these animals to cast and discard a

slime envelope, whenever irritated, could be a factor for preventing

a pesticide from reaching its site of action. If the production of

slime is a protective mechanism, it would help explain why current

insecticides are inadequate.

The present study was undertaken, from a comparative bio-

chemistry standpoint, to obtain knowledge of the interated metabolism

of the gray garden slug. If this was ascertained to be comparable with other organiains, a study was then to be made to determine whether

the integument of the slug includes a defense mechanism.

In recent years workers have emphasized the liKelihood that the primary part of drug action uon cells is explained mainly by their effect on cellular enzymes (i, p.74). Accompanying this thought, it is assumed that if a specific enzyme inhibitor effects a complex metabolic process, it can be concluded that the inhibited enzyme takes part in the process (17, pp.191-252).

Knowledge of the integrated metabolism of the slug was obtained in terms of its intermediate metabolism. To accomplish this study, the technique of respiratory inhibition was selected because it has been manifested to be instrumental in identifying specific intermediate enzyme systems. This technique involves the application of vmmrious chemicals that are known to interrupt cellular metabolic processes, and studying their effects upon the respiration of the slug. The uaritative measurement of the rate of oxygen con- suinption is considered to be a reliable index of the tota). metabolism of an organism (6, p.344).

The number of respirE tory inhibiting compounds, and the pos-

sible modes of action are quite nulrLerous. From practical standpoint,

compounds were selected on the basis of being the most desirable for

indicating the presence of the better known enzymatic systems such as the glycolytic, the tricarboxylic ada, arid the cytochrorne systems. It is obvious that the interpretation of a comparative bio- chemical relationship from a comparison of a chemical action on

various tyes of organisms is purely hypothetical. The differences

of selective action of chemicals among the same species is common knowledge. The objective of this research is not a study of specific metabolic processes, but to determine whether the gray garden slug is basically different from other organisms in its biochemical structure. It is felt that by studying the integrated ietabolism of the gray garden slug, a general picture of its cellular respiratory system will arise.

The rate at which a. chemical gains accas to its site of action depends largely upon the latent period between administration and onset of action. In many instances the latent period or question of absorption is influenced by the integument. It is therefore cori-

ceived that if the integument is a barrier, it will influence the dosage levels of chemicals when they are administered parenteraUy and topically.

The evaluation of any defense mechanism is bsed on the percent mortality resulting after a given compound is administerea parenterally 6 and toçlcally. Representative compounas with established toxic doses

from the organic phosphate and chlorinated rdrocarbon ;roups were

chosen for this study.

The information so obtained from these studies should be of

value to those engaged in developing methods for controUing the gray

garden slug. MATERIALS AND ÌliTHODS

In the present study, the rn&noxnetric rnethoct was used fox' nieasuring the integrated iaethbolisrn of the gr&y garden slug. This method enb1es the respirtory rate of the orgnisrn to be measured in teis of oxygen consumption. The effect of respiratory inhibitora can then be studied tr applying them to the organism c.nd noting the effect ori oxygen uptake.

Measurements of oxygen consumption 'were made with a respiro- meter of the warburg constnt-vo1ume type described r Urabreit et al

(36, pp.1-ió). This app&ratus consists of a suill flask att&ched in a closed system to a manometer. The slug is placed in the flask and the inhibitory compound is placed in a side arm out of contact with the slug. .s the slug consumes the oxygen in the flask the correspond- ing pressure change is measured by the manometer. Tipping the flaa introduces the inhìbitor and any effects on oxygen uptake can be noted by the manometer readings.

The specimens were obtained locally from the E tomolo- farm .nd were identified as Deroceras reticulatus (i4uller) . In the labor- atory these slugs were kept in sïaLl groups in covered jars containing a piece of glass wool saturated with water for înaintaining a humid atmosphere. They were stored at a temperture of 150 C . The animals wore retained u. to 4 days, at which time fresh secimens were obtained. They were starved for 24 hours prior to the beginning of the experi-. mente. The organisms were weighed before the test ori a holler-Smith precision balance. After this, the volume of the slug was calculated 8

usina the water displacement method.

Experiuents were conducted in a constant temperature bath

adjusted to 18°tO.l° C. This was found to be an ideal temperature

for normal slug activity.

The control slugs were exposed to 0.4 ml. of water at the

seme time that the treated slugs were exposed to the material being

testec . This eliminated drowning as a cause of respiratory inhibition.

To determine wnether the observed results were due to osmotic phenoxuex

sodium chloride solution, at a concentration ecual to that of the most

concentrated inhibito compound, was ubstLtuted for the water con-

trois during the studies with sodium fluoride.

The test compounds were dissolved in water and all were used

at the sanie volumes, 0e4 flu. The pli was adjusted to pH 6-7 with

sodium hydroxide. Preliminary experiments were conducted to deter- mine the inhibitory concentration of the test compounds. The con

pounös selected for study and the molar concentrations at which they were used vere as foflows: malonic acid 3.3 L; ethyl carbonate

(urethane) 0.3 M.; sodium azide 0.]. M.; sodium fLuoride 0.1 '1.;

sodium arserìite 0.1 í., and rnethldehycie 3.00014 M.

The solutions were added to the side arias of the flass before they were connected with the manometers. Exposure of the slug's to the solu.tions could then be accomplished without openin' the system, thereby allowing a constant measurement of oxygen uptake. To assure proper contact with the test media, the slus were confined on the bottom of the flasc by placing a doughnut shaped plastic screen over them. Exposure consisted of tipping the test material into the bottom of the flask, sixty minutes after the oxygen uptake readings were started. The sixty ninute recording of respiration rato prior to exposure to the inhibitors indicated whether the s1us were reípiring normally.

.A 20 percent potasiuin hyuroxide solution ws used for absor ing the expired carbon dioxide, thus neutra1izin its effect on the gs pressure within the system.

Readings of the pressure changas were taken every 30 minutea.

These figures were then converted to m.tcroliters of oxygen as described by- IJinbreit (3S, pp.57-60).

Each experiment ineLded two controls and four treated slua.

At least two exerinents were performed for each of the compounds being studied. At the end of the series of tet, the verage normal respiratory rate, based on a unit of weight of untreated slugs, was determined. With the data obtained from preliminary experimente on normal slugs, this average value included data from 50 slugs. The resiration curves of the treated slugs were thus compared with a

"normal" respiration obtained from several animals over a 60 day

)eriod while each day' s run also included controls which indicated whether the apparatus was functioning properly and whether the test animals were normal.

The role of the integument of the slug iii its resIstance to pesticides was staciied by comparing the toxic doses of parathion and heptachior when administered parentally and topically, respectively. This work was complicated by the selection of suitable o1vents for the materials. A solvent was reuuired which wa non-toxic &nd non-. irritating to the slug and which would dißsoive sufficient atiounta of the toxicants to i11ow 1are doses to be given in small volumes. It wa concLtded after testing several solvents that a comnion solvent meeting all the specifications wa not available. it wa found that

8usçeniou1& 8uitable for injection could be made by diluting alcoholic solutions of these coiipounds with water containing a wetting agent. This solvent sstern was irritating when topically applied, causing immediate production of slime. This reaction was avoided by forniulat- ing the parathion as a water suspension and the heptachior as a n- hexane solution for the topical application. Neither of these solventa caused abnormal aliìe production.

Dosages were administered on a body-weight basis, using volumes of 0.01 to 0.02 ini. for injections, and 3.0]. to O.O3 ini. for the topical treatment. The desired quantity of parathion or heptachior was injected into the loser pleural abdominal region, half-way between the sDiracle and the caudal region.

The injections were made by means of a micrometer-driven standard 0.25 ml. tuberculin syringe, through a 27 gauge needle.

Topical treatments were made with a blunt pointed 27 gauge needle.

The syringe was calibrated by weighing the mercury delivered.

The desired dosages were given to individual slugs in groups of five. The experiments were repeated two to five times on differ- ent days with different organisms. Following the treatments, the :i.i

slug5 were held in Petri dishes moistened -ith water to maintain humidity. After 24 hours the percent mortality was calculated. 12

RESULTS AND DISCUSSION

The ineffectiveness of insecticides and fungicides when

adathi8tered to the s1u, $uggest th&t this invertebrate may utilize

a physiological process that is not characteristic of other or&nisme.

If there is a variation in sieh a furidaraental rocess as cellular

oxidation or glycolysis, it should be possible to demonstrate this deviation by the use of chemicals known to effect these processes.

The following experiments were desied to determine whether the iuetibo1isin of the slug comprises unusual metabolic processes.

Respiration. Five mettboiic poisons were selected on the

biis of their known action on some part of the glycolytic or term-

thai oxidative yøteni of vs.rious organisms . The glycoly tic system

constitutes a universal mechanism by which carthkrdrates are degraded

to yield energy and pynwic acid. Likewise, the tricarboiylic acid

cycle arid the cytochrome systera, by which the resultant pyruvic acid

Is further metabolized to carbon dioxide and water, appear to be present in all living organistas.

The first compound to be tested for its inhibitory action on

the respiration of the slug was sodium arsenite. Lxamp1es of arsenic

toxicity to respiratory metabolism are widespread. Schmitt sind Skow

(30, pp.?ll-?19) found that arsenite inhibits the respiration of medullated frog nerves. This compound was a10 shown to decrease the oxygen consunption of codling moth larvae, cockroaches, and Colorado potato-beetle larvae (9, pp.75-276).

It is generally thought tbt the site of action of arsenic is 13

the tricarbolic acid cycle, where it interferes with the conversion

of ketoglutaric acid into (2, p.441). Results obtained

by Voegtlin and Dyer (37, p.334) led theja to believe that this c1em-

leal inhibits the activity of biological systems by reacting with the

functional thiol groups on enzymes. This conclusion is substantiated

by Stocicen and Thompson (33, pp.529-535) and arron et al (5, pp.221-

The exoeriaental data show that when the s1u was exoed to

a 0.1 molar solution of sodiu.a arsenite, its respiration w&s inhibited.

With the inmediate application of arsenic there ws a marked deereaße

in rate of oxygen consumption, stablizing after &3 minutes with a

gradual decrease thereafter (Fig. 2) . The average ogen uptake at

the breaking point was 204 ul./gxn./hr. The oxygen uptJce, 15 minutes after the arsenite exposure, was observed to be 183 pl./gxn./hr. The q1axiIJ1u!n oxygen uptake was 212 j.xl./gi./hr. before treatment and 34 u1./

gmnr, 150 minutes after treatment.

Considering this inhibitory response in conjunction with the citea evidence, that arsenic aet on the tricarboxylic acid cycle, would indicate that the slug contains euch a system.

Further evidence for the presence of the tricarboxylic acid cycle is established by a study of malonic acid. i4alonic acid acts as a competitive inhibitor of succinic dehydrogerase thua preventing succinic acid breakdown to fiaric acid (23, pp.693-694). Ma.lonic acid was invaluable in establishing the cycle theory

(19, pp.445-455). Succinic acid has been shown to be an active 14

mnetabolite in cells of very diverse types, ranging from bacteria to

those of inaimnals. Malonic acid was found to decre&se the cellular

metabolism of these aniinala (27, pp.117-127).

When the slug was exposed to a 3.1 molar 8olution of malonic

acid it underwent definite decrease in oxygen uptake (Fig. i).

The average respiratory rate at this point was 131 Ltl./gm./hr.

Fifteen minutee after treatment, the rate decreased to 104 pl./gm./

hr. The ogen uptake was 3 pi./gm./hr., 120 minutes after the

slug had come into contact with hlalonic acic&, indicating a steady

decrease in oxygen consumption.

The rnalonate inhibition of respiration obtained with the

experiments on the slug, would indicate the participation of euccinic

dekrdrogenase in the biological processes occurring in the gray

garden slug. Rees (29, pp.47-43) has demonstrated in the snail,

a closely related species, a functional tricarboiilic acid cycle.

Attention is now focused on a compound which has been eatab-

lished as an inhibitor of the glycolytic system. xperiments per-

formed by various workers, leave little doubt that sodium fluoride

is such a compound. The foundations of our present knowledge of

fluoride inhibition of the assimilatory processes were laid by

Warburg and Christian who conclucied that fluoride inactivates the enzyme, enolase (3d, pp.38h.-421) (39, p.590). This key enzyme is essential in the conversion of enol-phoephopyruvic acid from 2- phosphoglyceric acid. It is thought that fluoride acts on enolase by the displacement of inanesiura which is necessary for its action. 15

The position enola5e occupies in the hypothetical metabolic scheme is

shown In Fig. 4.

The evidence obtained (Fig. 2) indicates that fluoride is an

active inhibitor of s1u respiration. The respiratory depression

for sodiun fluoride ws not as marked &s the otberß. The averge

before treatment wa 15 p1./gn./hr. Fifteen minutes after trethent oxygen consuiaption ws 140 u1./giu./hr. and 180 minutes later it ws

35 u1./gii./hr. This study may indicate the existence of a glycolytic system within the slug.

A discussion of the fo11owin! two compounds, urethane and

sodium azide, substantiate the probable presence of a cytochrome

system or terminal oxidative system. Urethane is classed s narcotic.

Narcotics inhibit the activity of the dehydrogenasas arid hence, the reduction of oxidized cytochronie (2, pp.17&-177). This compound has been deiuonstrated to be inhibitory to oxidation processes in rat tissue, nerve tissue, heart muscle, grasshopper embryos, and bacteria

(S, pp.9-13)(l0, pp.159-172)(22, p.2l9-239)(26, pp.241-250)(31, pp.

51-54).

The effect of urethane when the 1ug is exposed to a 0.3 molar aolution is similar to that obtained with other organisms. Before treatment, the slugs were observed to be respiring at an average of

171 pl./gm./hr. Immediately after the exposure the reduction in respiratory rate was quite sharp, indicating a marked inhibition

(Fig. 2). Fifteen minutes after treatment the rate was hr. and 150 minutes after exposure was recorded to be 40 u.1./gni./hr. 16

In direct contrast to the narcotic type of action, sodium aiäe

inhibits the oxidation of th cytochrozaes b the cytochrouie oxidase

system in a manner similar to that of potassium cyanide (16, p.165).

Since the studies of Keilin, a later contribution augget that azide

dissociates phosphorylation from glycolysis thus preventin cellular

utilization of the metabolic energy derived from the glycolytic cycle

(32, pp.99-103).

Sodium azide has oroven toxic to a great variety of organisms

including vertebrates, insect iarv&e, crstacea, woriis, ciliates,

moulds, and bacteria. The lethal concentration axd time for kill

v&ried from 0.5 percent requiring 3 hours, to 0.1 percent requiring

20 or more hours (17, pp.312-339).

The effect of sodium azide on the slug is indicated in Fig. 1.

There was a iarked inhibition of respiration almost imedi8te1y after

exoosure to 0.1 molar solution, with a progressive decrease in rate

of oxygen consumption occurring over a period of 150 £ìthiute. The

oxygen uptake before treatient ws an average of 141 1./m./hr. The

oxygen rate 15 minutes after exposure to azide was 103 ul./gin./hr.

Since urethane and azide Inhibit respiratory metabolism by

interfering with the cytochrone sy stem , we might conclude that the metabolism of the slug includes this system.

Inasruch as the compounds discussed thua fir are water aoluble,

it was considered possible that the observed effects were due to a

disarrangement of osmotic relationships. This wa investigted by

treating control slugs with a sodium chloride solution instead of 17 water. No respiratory ffect were noted and it was concluded that o$mo$i5 ws not a causative factor for the resultant inhibition.

With the establiehinent of the above evidence, it wa& consiered worthwhile to study the effect of ietaldehyde on the respiration of the s1u. The administration of a 1.4 x iO molar solution of met- aldehyde, which was about 1000 times less concentrated than previously tested compounds, produced a decrease in respiratory rate.

The rate of oxygen consumption decreased ediate1y after treatment but ws not shsrp. In some cases the res4ratory rate rturned to normal in about two hours indicating that the effect vai transitory. The respiratory rates of these animals were plotted separately as indicated in Fig. 3. The reinining slugs showed a continual decrease in respiration. It is interesting that despite these differences in respiratory effects, all of the metaldehyde treated oranisms died within 12 hours.

At the present time the mechanism and sIte of action of metaldehyde are not known. Its action has received occasional comment, but no large scale systemic study has developed. Aside froxíi Urag and Vincefit (11, pp.392-406) only two other investigatore appear to have stiiuied metaldehyde from a hysio1ogical standpoint

(7, pp.1-20). Their work with frogs revealed that metaldehyde, injected at a concentration of 1:5000 into the ventral lymph sac,

caused a characteristic syndrome, this beinL in the order of occur- renco; hyperexcitability, convulsions, paralysis, and death.

Metaldehyde is practically insoluble in vater. It is a 18

po1yir of acetJLdehyde (four acet1dehyde molecules) anc i depoly-

merized to acetaldohye in the presence of strong suifLiric ada. In

view of this, it might be suspected that cetaldehyde is the toxic

a gent.

Acetaldebyde is water soluble. This compound has been meni-

fested to be a normal constituent in cellular metabolism of several

types of organisms. It being a decomposition product of pyrmivic acid,

it is therefore nontoxic (15, pp.69-4)(23, pp.550-555)(34, pp.91-lOS).

This may explain why acetaldehyde was inactive when aciininistered to

the slug by Cragg and Vincent (11, pp.392-406). They administered

acetaldehyde in concentrations 100 times that of metaldehyde with no

apparent toxic action. In addition, they compared the lethil effects

of metaldehyde when athuinistered by the injected, topical, and oral methods. They found that the greatest toxicity to the slug resulted

from the tooical application of metaldehyde.

The slug has a specific gravity of approximately 1.0360,

indicating a probable high water percentage within ita tissues. It

is conceivmble that the comparative insolubility of mnetaldehyde may

cause it to be precipitated within the cells. This woula indicate

that riietaldehyde is not depolynierized when the initial association

is with the epidermal cells.

Further study of the problem might profitably be pursued experiiaentally with greater attentioLi focused on the site of action of metaldehyde.

Role of thejntewnent. comparison of the percent norta1itie 19 when parathion and heptachior are administered parenterally and topi- ca3.ly to the slug is given in Tables i and 2. It can be seen that parathion injected at a rate of 250 mg./kg. was lethal to ail of the slugs. The LD50 appears to lie between 150 and 200 mg./kg. This figure is about 50 tiine8 that found when parathion is applied to insects and about 20 times the figure usually given for the IO to vertebrate.

Table 1 also shows that oses as high &s O0 mg./kg., applied topically, produce little or no mortality. The results obtained in these experiments were erratic it it is obvious that from a practical standpoint parathion would be useless as a contact poison for slugs.

The low order of toxicity of parathion to the slug is surQria- iri: in view of the highly toxic nature of this niateria]. to many other animals. There seems to be at least three possible explanations for this lack of toxicity. Metcalf and March (24, pp.72l-72a) have shown that organic phosphates which contain the P0 group are more toxic to insects than those containing the pS group. They postulate that the latter type of compound, as represented bir parathion must be converted in VIVO into the more toxic form before it can exert its full lethal effect. In the case at hand we might be dea1in with an organism which does not possess the enzymes necessary for this conversion.

Parathion is toxic to organisms by virtue of inactivating esterase enzymes that are associated with the chemical transmission of nerve impulses. Essentially, this concept holds that nerve impulses effect responses through the liberation of a chemical substance that acts as the local exciting agent. This mediator is subsequently destroyed r a specific enzyme.

The high dosage of parathion necessary to elicit a response would indicate that the physiological nervous system possessed by the slug may be different structurally. Namely, the chemical mediator and its antagonistic enzyme may be different with respect to the

characteristic ester and esterase type ro1ecules exiatin in other nervous systems.

Correlated with this reusonin, is the fact that the inhibit.-

ing reaction by organic phosphates is proportional to their ease of hydrolysis (1, pp.473-476). Parathion is effective when given in suf-

ficient concentrations. It is feasible that the slug cont&ins an enzyme which can drolyze parathion a1rnot as rapidly as the normal

substrates. If this were the case, larr concentrations of parathion would be reuired for toxicity.

TÌio toxicity of hept&chlor to the slug is similar to that obtained when this chemical is adniinistered to other organisms.

Table 2 shows that the mortality exceeded 90 percent when the slugs were injecteû with a dose of 100 mg./kg. The median lethal dose is indicated between 50 and 100 mg./kg. This figure is about five times that found when heptachlor is applied to insects. On the other hand, there is no appreciable difference between the median lethal dose quoted for vertebrates and that found for the slug.

Table 2 also shows that heptachior applied topically in a dose of 50 mg./kg. resulted in a 30 percent kill. Here again the reultø 2].

are erratic. There is no appreciable increase in percent ortaiity

as the ciose is increased. A dosage eight tine th.t for 30 percent

kill, was lethal to only 40 percent of the slugs.

Particularly interesting, la the fact, that when parathion and

heptachior were injected, the toxicity of parathion vas not as great

as that of heptachior. However, experiiental evidence iniicates a

basic correlation between the injection and topical effects of these

chemicals when aftninistered to the slug. Îeither parathion nor

heptachior was effective in terrns of producing a maxiura kill when

applied topically. The imortaxt aspect is that these insecticides

once within the body cavity will kill the slug.

The erratic mortality achieved when parathion and heptachior were applied topically ay be related to an inconsistent systeiric

concentration. There i a possibility that the causative factor for

this inconsistency ìs inadequat absorption due to the formation of

slie. ¡ccoìnpenylng the topical administration of these foreign xnaterials, there was a copious secretion of thick, white, nonvicouß

slime. The sliming was severe, esc;ecially with the parathion treated

1ugs. It was observed thct injection seemed to reduce any unfavor- able reaction, auch as sliming.

In support of this obsertion it was noted that when slugß vare treated topically with the pure solvents, hexane, or the water and wetting agent mixture, there was only a slight amount of thin, watery type of slLiìe formed, disappearing shortly. However, with the subsequent application of the solutions containing the active 22

ingredients, the ch&racteristic thick 8lime would precipitate immedi- ately into a f im adherent Once this formed, the slug would crawl from under lt.

These observ.tions in conjunction with the results obtLinod with the topical applications would suggest that the slime, the integ- ument, or both are vita], to the welfare of the slug. 23

CONCLUSION

In conc1uion the results obtained in this atady indic te that the slug is not esentia11y different from other organisms in regard to basic metabolic processes. ifl widition, the integument of the slug appears to constitute a protective barrier, probably r virtue of the slime production. 24

The integrated rnetathlism of the cray garden slug Deroceras reticulatus (Muller), has teen studied by utilizing the respiratory inhibition tecbniqu.e. This technique consisted of testing the effects of the etabo1ic poisons, arsenite, inalonic acid, fluoride, uretherie cnd azicte upon the rate of oien conuinption of the slug.

In addition, iiietaldehyde wa tested for its effect on respiratory metabolism.

Evidence ha been obtained which indicates that the general metabolic processes of the slug compare with those found in other orgnisnis.

Parathion nd heptachior were employed to deteiine whether the inteunient of the slug constitutes a defense mechanism. The injection of these two chemicals Ìs been shon to produce toxicity.

:joweveI the toxicity of oarathion was not a great as that of heptachior. both of these compounds have been shown to be relatively ineffective as contact poisons to the slug.

The production of slime has been observed to be correlated with the inadequacy of the topical application. 200- EXPOSURE CONTROL I - I ---.' 180- + o-- ---. - 160-

C, o--

ow 'I0O-

MALONIC ACID

I I i I i I i I i 120 150 180 210 TIME-MINUTES FIQ. I. THE EFFECT OF AZIOE AiO MALONIC ACID '3 ON RATE 0F OXYGEN CONSUMPTI3N. \ CONTROL /

i... - - -.- - \ - - - - N :\ \

I L) C. N - -

URETHAN

L I 1 I I 210 TIME-MINUTES rio. 2. DIE EFFECT or AROENITE, rLuoRIOE,Ar40 URET-IANE ON RATE 0F OXYGEN CONUWTI ON. EXPOSURE

CONTROL

o -----. ------/

--.\ - -

METALDEHYDE

- - -

I i I i I i 120 150 180 20 TIME-MINUTES FIG. 3. THE EFFECT OF ?.ETALDEHYOE ON ) RATE OF OXVOEIJ CO«UUPTION. SLYCOGEN + PHOSPHATEGLUCOEHPHO8PHATE

SLUCOSE -4- AlP -GLUCOS6PHO8PMATC

FR UC T OS ..CPH OS PHATE FRUCT OSETPHOS PHArE

IPH OS PHOD I1 YO ROX Y AC ETO NE

3-PHOS PHOQi.ÇCERALDEHYOE

,3-oi PHOP1.OGLYCERALOEHYOC

,3-OIP11OSPIOGLYCERIC ACID

3-PHoSPHLYCCrC ACID

2.PHOSPHLVÇERIC ACID

J (ENOLASE+ MG ) - CNOL-PHOSPkOPYRUVIC ACID CARBOXYLASE)

ACETALDEHYDEs-0O2 PYRUVIC ACID - PHOSPHOiIC ACIDLACTIC ACID -CO2

OXALOACETIC ACID CISACONITIC ACIDZCITRIC ACID

MALIC ACID

FUMARIC ACjD

-JCCINIC ACID ETOGLUTARIC ACID

NATE

(2H) f ARE?JIT

(H) LJSTRATEDEHYOPOGENASE OIOIZO FECYTCHROME OXIDASE + 1120 f URETANE1 CYTOCHRcNIE NAN3I C/\( sUDTRAEoEHYOROGENAsE R:DJCED FEO2-ÇVTOCHROE OXIDASE

FE CYTOCHROME OXIDASE

FIo.4. SCHE TO SUMARIZE THE ENZYME- CATALYS STEPS IN THE SI_YCOLYTIC CYCLE, TRICARBOXYLIC ACID CYCLE, & CYTOCHROME SYSTEM, WITH THE POINTS OF ACTION OF THE INHIBITORY CHEICALS.

L 29

TABLE 1. TOXICITI 0F FARATFOEON TO SLUGS

BY INJECTION

Dose (sig.. per kg. Number of Sluga iverage body-weight) Treated Killed Percent Kill

2 10 0 0

4 10 0 0

8 10 0 0

32 10 0 0

64 10 0 0

100 20 3 15

150 20 7 35

200 22 10 77

250 15 15 100

300 10 10 100

Control (0.02 1.) 22 0 0

BY TOPICJL APPLICATION

Dose (ing. per g. Number of Slugß Average body-weight) Treated Killed Percent Kil].

150 5 0 0

200 25 4 16

250 10 0 0

300 10 0 0

400 20 0 0

800 20 1 5

Control (0.04 ml.) 20 0 0 30

TABLE 2. TOXICITY OF EE?TACBLOR TO SLUGS

BI INJECTION

Dose (md. er . Number of S1u verge body-weight) Treated Killed Percent Kill

10 15 2 13

30 15 3 20

50 25 10 44

loo 25 24 96

150 10 10 100

200 5 5 100

400 10 10 100

Control (0.02 ini.) 40 2 5

BY TOPIC.L APPLICÍTION

Lose per kg. Number of Slu8 Lverade body-weight) ireated Killed Percent Kill

50 10 3 30

100 20 5 25

200 O 1 3

400 20 a 40

Control (0.04 ml.) 40 0 0 31

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