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This is to certify that the

thesis entitled

Carbohydrate Involvement during DDT Poisoning of the

American Cockroach, Periplaneta americana'

presented by

Jeffrey Granett

has been accepted towards fulfillment
of the requirements for

 

Ph.D. degree in Entomology

   

hhfiupnmuun

Date May 19, 1971

 

0-7639

ABSTRACT

CARBOHYDRATE INVOLVEMENT DURING DDT POISONING OF THE
AMERICAN COCKROACH, PERIPLANETA AMERICANA

By

Jeffrey Granett

In DDT-treated American cockroaches, Periplaneta americana, L.
(Orthoptera:Blattidae), glycogen and trehalose concentrations were
lowered over a relatively short time Span corresponding to the period
from late hyperactivity to early prostration. Such a depletion corres-

ponded with a peak in 14

14

C02 evolution from cockroaches receiving glucose-
C injections. The depletion occuned even after supplemental injections
of up to 6 mg trehalose per cockroach were made, but was delayed by a
rise in temperature. Dieldrin and propoxur poisoning caused a similar
carbohydrate depletion. In DDT-poisoned cockroaches carbohydrate
depletion is proposed as a link between the effect of DDT on the nervous
system and the response by the other tissues of the insect to the
insecticide.

Serum from DDT-prostrate cockroaches injected into previously
untreated cockroaches caused an increase in the trehalose content. This
was not observed with serum from DDE-treated cockroaches. This hyper-
glycemic activity is present in serum mainly from the prostrate stages
of DDT poisoning. The factor is stable to heating and is stable at room

temperature for as long as four hours. The active fractions from

Jeffrey Granett

molecular sieve chromatography are distinct from the bulk of the serum

protein. The physiological significance of this factor is discussed.

CARBOHYDRATE INVOLVEMENT DURING DDT POISONING OF THE
AMERICAN COCKROACH, PERIPLANETA AMERICANA

By

Jeffrey Granett

A Thesis

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of
DOCTOR OF PHILOSOPHY

Department of Entomology

1971

A one-foot waterfall-
it too makes noises,
and at night is cool.

Issa

to my parents, Sandy and the future.

ACKNOWLEDGMENTS

I wish to express my sincere thanks to Dr. Norman C. Leeling, my
professor, who advised me and has had long-standing patience with me
and this work.

I wish to acknowledge the sequential financial assistance of a
Title IV, National Defense Education Act Fellowship, a National Science
Foundation Fellowship and an Entomology Department assistantship.

For the two years of happiness during which the work for this
thesis was done I wish to acknowledge the diligent cooperation of

my wife, Sandy.

iii

TABLE OF CONTENTS

LITERATURE REVIEW.. ..... ............................. .......... ......1
I. Trehalose in Insects ............................ . ...... .......l

A. Trehalose Levels. ............. . .......... . ..... . ..........
B. Functions of Trehalose... ...... ................... ..... ...
l. Trehalose Utilization during Insect Development.......

2. Trehalose and Insect Flight...........................

3. Circadian Fluctuations in Trehalose Levels.... ...... ..

4. Cut Absorption of Carbohydrates.......................

C. Trehalose Metabolism......................................
1. Synthesis and Breakdown of Trehalose..................

2. Regulation of Trehalose Levels........................

a. Feedback Control..................................

b. Hormonal Control....... .......... ......... ..... ...

PmWUWWNNNI—‘H

II. DDT........ ............. . ...... ..... ....................... ...

U1

A. Introduction ...... .................... ..... ...............5
B. Mode of Action of DDT............................. ...... ..6
l. Symptoms..............................................6
2. Action on Nerves..... ........ ...... ........ ...........7
3. Biochemical Changes during DDT Intoxication...........8
4. Neurohormonal Involvement in DDT poisoning.... ...... .10

PART I. Trehalose and Glycogen Depletion during DDT Poisoning of
American Cockroaches, Periplaneta americana.................12

I. Materials and MethodS........................ ....... .........13
II. Results... .................... .............. ......... . ....... 16
III. Discussion.................................. .......... .......27

PART II. A Hyperglycemic Factor in the Serum of DDT-Prostrate
American Cockroaches, Periplaneta americana...... ..... .....31

I. Materials and Methods........................... ..... . ....... 32
II. ResultSOOOOOOOOO00.0.0.........OOOQOOI...00.0.00000000000000034

III. Discussion............. ...... ..... ........ ......... ...... ....43

iv

Table of Contents (Continued) Page

AN OVERVIEWCCCOOOCOO000...... ..... O.

I. Interpretations of the Research... ...... . ..... ..............45

A. Experiments Concerning Carbohydrate Levels ........ ......45

B. Experiments Concerning the Hyperglycemic Factor.. ..... ..47
II. ExperimentSOOOO0.00.00.00.00.........OOOOOOOOOOOOOOOOO0.0.0048
. Lipid and Protein Levels ........... . ..... .... ........ ...48
. Additional Effects of the Factor.... ...... . ...... .......48
. ;3_Vitro Fat Body Bioassays..................... ..... ...49
. Chemical Characterization of the Factor.................49
Biological Characterization of the Factor...............49

NUCW>

III. Significance of these Studies...............................50

LIST OF REFERENCES........... ............ ...

LIST OF TABLES

Table Page

1. Trehalose Levels in the Hemolymph of Treated American
COCkrOaCheSooooooooooooo.0000.00000.000000ooooooooooooooooooooo0.017

2. Trehalose and Glycogen Levels after DDT and DDE Treatments of
American COCkroaChesoOOCOO0.00.00.00.00.0.00.00...00.0.0000000000018

3. Trehalose and Glycogen Levels in DDT- and DDE-treated
American Cockroaches after Trehalose Injections...................22

4. Trehalose and Glycogen Levels in Dieldrin- and Propoxur-
treated American COCkroaChes...00............OOOOOOOOOOOOOCOO.....28

5. Hyperglycemic Activity of Serum from DDT-prostrate Cockroaches
D11UtedW1th Ringer's saline.00.000.000.000...0.00.00.00.00000000040

vi

LIST OF FIGURES

Figure Page

1.

2.

Glycogen and Trehalose during DDT Treatment as Percent of
DDE-treated Control Cockroaches...................................20

The Effect of a Temperature Rise on Trehalose and Glycogen
Levels in DDT-treated American Cockroaches........................24

Difference Between 14CO Respired from DDT- and DDE-treated
American Cockroaches In acted with Glucose-14C as a Percent

Of TOtal DPM'S InjeCtedOOOOOOOOO...0.0.0....0.0.00.00000000000000026

The Level of Trehalose in Cockroaches Treated with Serum from
DDT-proStrate COCkroaCheSCOOOIOOOOO0..........OOOOO...O.O.O0.0.0.036

Hyperglycemic Activity from Serum of Cockroaches in Different
DDT P01soning StageSIOOOOOOOOOOOOOO......OOOOOOOOOOOOOOOOO0.00.00039

Sephadex Chromatography of the Hyperglycemia Factor...............42

vii

LITERATURE REVIEW

1. Trehalose in Insects

A. Trehalose Levels

Wyatt and Kalf (1957) reported the presence of trehalose in
representatives of five orders of insects. In four lepidopterous
species trehalose comprised 90% of the total hemolymph carbohydrates.
Concentrations in the plasma ranged from 306 mg percent (mg per 100 m1)
trehalose in the oriental silkworm larva, Bombyx mori, to 1398 mg percent
in the silk moth, Telea polyphemus. Evans and Dethier (1957) reported a
range of 200 to 3000 mg percent trehalose in the hemolymph of the blowfly,
Phormia regina. Steele (1963) found an average of 1070 mg percent
trehalose in the American cockroach, Periplaneta americana. Contrary
to the high level of trehalose present in most insects, Barlow and
House (1960) found that only 1 to 2 percent of the carbohydrates in the
hemolymph of the parasitic fly larvae, Agria affinis, was trehalose;
glucose accounted for 80% of the carbohydrates. Hansen (1964) found no
trehalose in the hemolymph of locusts, but detected high levels of
maltose, cellobiose, or other sugars depending on the diet.

B. Functions of Trehalose

Trehalose, as the predominant carbohydrate in the hemolymph of
most insects, is comparable to glucose in mammalian systems and serves
as a mobile source of energy (Wyatt and Kalf, 1957; Chefurka, 1965).

Utilization of trehalose has been studied during various physiologic

2
activities. Discussed here will be 1) trehalose utilization during
development, 2) utilization in flight, 3) levels at different times
of day and 4) the effect of gut absorption on hemolymph trehalose
levels.

1. Trehalose Utilization during Insect Development

Between the pupal and adult stages of several silkworm Species
the trehalose level decreases by one-half (Wyatt and Ralf, 1957).
Candy and Kilby (1962) suggest that this drop may be a result of
chitin synthesis, although their proposed pathway does not necessarily
indicate this.

Diapause hormone injected into silkworm pupae enhances gg_ggyg
synthesis of trehalase (the enzyme hydrolyzing trehalose to glucose)
in pupal ovaries, resulting in higher ovarian glycogen levels
(Yamashita and Hasegawa, 1967). Similar effects are shown by pupae
injected with ecdysone or cholesterol subsequent to excision of the
supraeosophageal ganglia (Kobayashi gt 31., 1967).

2. Trehalose and Insect Flight

During flight the rate of glycogen decrease in the fat body of
Drosophila is directly proportional to the wing-beat frequency
(Williams $5.21,, 1943). Clegg and Evans (1961) extended this
correlation to the trehalose of the hemolymph with the observation
that high trehalose concentrations accompany high wing-beat frequencies.
They hypothesized that the source of this trehalose was fat body glycogen
and dietary monosaccharides.

3. Circadian Fluctuation in Trehalose Levels
Nowosielski and Patton (1964) reported peak hemolymph trehalose

concentrations in the house cricket,Gryllus domesticus,at 3 hours before

 

dawn (in a 12 hour day).
4. Gut Absorption of Carbohydrates
The absorption of various sugars through the gut and their in-
corporation into hemolymph trehalose and fat body glycogen was studied
in Bombyx mori by Horie (1960). Sugars which increased the trehalose
level also increased fat body glycogen. Treherne (1958a and b)
observed in the locusg,Schistocerca gregarigdthat sugars are mainly
absorbed by diffusion through the midgut caecae and to a lesser extent
through the ventriculus. Once in the hemolymph the sugars are rapidly
converted to trehalose, providing a steep glucose concentration gradient
between the hemolymph and the gut lumen for the absorption of dietary
glucose.
C. Trehalose Metabolism
1. Synthesis and Breakdown of Trehalose
Trehalose in insect hemolymph may be cleaved at the cell membrane
into two glucose moieties by trehalase. This enzyme has been isolated
and purified from a number of insects (Howden and Kilby, 1956; Friedman,
1960; Derr and Randall, 1966). In trehalose synthesis UDP-glucose
and glucose-6-ph03phate form trehalose-6-ph08phate which is then
hydrolyzed by trehalose phOSphate phosphatase to yield trehalose
(Chefurka, 1965).
2. Regulation of Trehalose Levels
Synthesis of trehalose and glycogen in the insect is competitive.
The balance of the two is under feedback and hormonal control.
a. Feedback Control
Excess trehalose inhibits the incorporation of glucose into

trehalose and stimulates its incorporation into glycogen in ig_vitro

fat body incubations (Murphy and Wyatt, 1965). It is believed

that trehalose is an allosteric inhibitor of trehalose phoSphate
synthetase, binding to a site on the enzyme that is separate from the
catalytic site. The inhibition can be eliminated by mild protein
denaturation.

Trehalose activation of glucose-6-phoSphate hydrolysis in fat body
extracts of the blowfly, Phormia regina, was demonstrated by Friedman
(1967a). Since glucose-6-phosphate is required for trehalose synthesis
the net affect is to inhibit trehalose synthesis.

b. Hormonal Control

Steele (1961) extracted a hyperglycemic factor from the corpus
cardiacum of the American cockroach, Periplaneta americana. When
injected into other cockroaches, this extract increased the hemolymph
trehalose 150 percent within 5 hours, increased inorganic phosPhate
release into the hemolymph and loweredthe amount of glycogen in the
fat body (Steele, 1963). Steele suggested that this hormone, thought
to be peptidyl in nature, affected the phosphorylase activity. Ralph
and McCarthy (1964) in similar experiments found that the hyperglycemic
factor was present in the corpus cardiacum, brain, corpus allatum, and
subeosophageal ganglion (in order of decreasing activity). Bowers and
Friedman (1963) noted a hyperglycemic hormone in the cockroach, Blaberus
discoidalis. Migliori Natalizi and Frontali (1966) isolated two such
factors from the corpus cardiacum of the American cockroach and
head homogenates of the honeybee, Apis mellifera. Migliori Natalizi
25,11. (1970) purified one from a corpus cardiacum extract from
American cockroaches using Sephadex column chromatography and demonstrated

trypsin sensitivity. Brown (1965) isolated two factors with similar

activity from an American cockroach extract by paper chromatography.

These factors were also trypsin sensitive and presumed to be low
molecular weight polypeptides.

Friedman (1967b) found that the hyperglycemic hormone was inactive
in blow flies fed ag_libitum, but was active if they were starved.
He suggested that the trehalose synthesis system normally works
at full capacity as governed by the feedback inhibition of trehalose
synthesis. When the trehalose concentration is low and there are
abundant sugars from the gut, these sugars are efficiently converted
into trehalose. During starvation, however, glycogen breakdown is
the rate-determining factor of trehalose formation; so Friedman
hypothesized that the hormone acts on glycogen breakdown. Wiens
and Gilbert (1967) studied the effect of the hyperglycemic hormone
on phoSphorylase activity and the respiration of igflyiggg fat body
preparations. They hypothesized several sites of hormone action,
including; a) an increase of phosPhofructokinase activity, b) inhibition
of glycolysis by pentose phosphate cycle acceleration, c) hexokinase

activation and d) trehalose-6-ph08phate synthetase activation.

II. DDT
A. Introduction
Although DDT was first synthesized in 1894 by Zeidler, its insec-
dcidal properties were not established until 1939 by P. Muller of the
Geigy Chemical Corporation. Widespread and continued use came after
World War 11 due to DDT's low cost, extremely wide Spectrum of insect-
icidal activity, stability and low acute mammalian toxicity. This

stability and DDT's lipid solubility leads to its accumulation in the

environment and storage in animal adipose tissue. In recent years, fear
of chronic poisoning by DDT has caused controversy and as a result it
has been banned for many uses in several states and countries.

However, the continued study of DDT, even though its use is
declining, is of more than academic interest. First, DDT is indis-
pensible in certain applications and in countries less technically
developed than the United States. It will be used in significant
quantities for a long time. Secondly, DDT and its metabolite DDE
are some of the most wideSpread environmental contaminants and will be
prevalent for many years even if the environmental input should be
halted immediately. Thirdly, the action of DDT in organisms appears
to affect basic animal processes. An understanding of DDT's action may
thus give information on these biological systems.

B. Mode of Action of DDT
1. Symptoms

The acute toxicity of DDT to insects is attributed to the
chemical's action on the nervous system reflected in the behavior
of the poisoned insects. The abnormal neuronal activity results in
tremors, hyperactivity, ataxia and eventual paralysis. Roeder and
Weiant (1946) found that the sensory nerves were the most sensitive
part of the nervous system to DDT. They also described the high-
frequency trains of spike potentials caused by DDT in the axons of
cockroach legs.

Increases in respiration rates due to DDT were described by
Ludwig (1946), Lord (1949) and Harvey and Brown (1951). Also, weight
loss and water loss occurred with DDT poisoning (Ludwig, 1946) and

heart beat frequency was affected (Patel and Cutkomp, 1967).

2. Action on Nerves

The mode of action of DDT on nerves has been studied intensively.
O'Brien (1967) summarizes some of these studies. Several areas of
DDT's action have been further explored since O'Brien's review.

The hypothesis of the charge-transfer complex of DDT with nerve
proteins first described by O'Brien and Matsumura (1964) was essentially
refuted by Hatanaka g£.gl. (1967). They found that binding of DDT
was non-Specific and occurred with rat liver, muscle and brain in
addition to cockroach nerve cord. In addition, the relatively
non-toxic surfactant Triton X-100 mimicked the tissue binding effect.
Brunnert and Matsumura (1969) revived the charge-transfer complex
hypothesis on the basis of competition studies between DDE and DDT.

A small portion (6%) of a 5 nmole DDT solution was bound to a site
in the synaptic junction different from the DDE binding site.

Some of the above work was done with rat brain fractions.

Similar preparations were used also for studies with ATPase. Matsumura
and Patil (1969) reported that DDT inhibited the Na+, K+, Mg2+-adenosine
triphOSphatase found in a fraction of rat brain. The inhibition by

DDT was about 1000 times higher than that by DDE. In addition, the
inhibition was greater at low temperatures, a phenomenon resembling

the negative temperature coefficient of DDT toxicity. However, the
validity of this work has been questioned by Akera g£_gl. (1971)

who found high inhibition with DDE as well as with DDT. They suggested
that the contradictory results were due to the low enzyme activity

in the preparations used in the earlier work.

3. Biochemical Changes during DDT Intoxication

Tobias £5 11. (1946) noted that free acetylcholine in the nervous
system of DDT-prostrate flies and cockroaches increased 200 percent
while the bound acetylcholine decreased. However, the acetylcholine-
forming enzymes were unaffected. Lewis (1953) substantiated the higher
acetylcholine levels in DDT-prostrate house flies. In 1960 Lewis
gtflgl. found that this rise in acetylcholine was similar to that
observed under physically-induced prostration. The magnitude of the
acetylcholine increase could be correlated with the degree of neuro-
muscular activity before prostration. They suggested that the rise was
due to release of bound acetylcholine from the axons. The rise could
not be attributed to increased activity of the acetylcholine synthe-
sizing enzymes, choline acetylase or acetylkinase (Rothschild and
Howden, 1961). Interpreting data of Sternburg and Hewitt (1962),
Winteringham (1966) concluded that DDT poisoning increases acetylcholine
turnover in the insect ventral nerve.

The high concentrations of amino acids in insect hemolymph lend
themselves nicely to monitoring for possible changes resulting from
insecticide poisoning. Winteringham (1958) reported accumulation of
glutamine in the hemolymph, arising possibly through transamination.
Cline and Pearce (1963) found that DDT interfered primarily with
proline, formate and glycine metabolism in house flies. A greater

14C was converted into uric acid and

percentage of injected formate-
allantoin than into proline in DDT-poisoned insects. They noted
that a carbamate insecticide did not affect formate metabolism. Corrigan

and Kearns (1963) found that injected proline-14C was oxidized to carbon

dioxide three times faster in DDT-poisoned than in control American

cockroaches. Also it was metabolized to glutamine-14

C which Wintering-
ham (1958) had suggested might be an ammonia trap for amino acid
oxidation. Corrigan and Kearns also suggested that the demand for
oxidizable carbon shifted metabolism to proline. In 1966 Cline and
Pearce confirmed the drop in proline by radiotracer studies with house
flies. In addition they found a decrease in radiolabelled trehalose
after glucose-14C injections in DDT-treated insects as compared to
solvent-treated controls. Patel $5.11. (1968) surveyed all the amino
acids and found that their average concentrations decreased 22.7
percent in the ovaries of DDT-poisoned susceptible house flies as
opposed to a 5.5 percent increase in poisoned resistant flies.

Several papers have reported a drop in carbohydrates during DDT
poisoning in insects. This was reviewed in the introduction to Part I
of this thesis. There has also been considerable work on intermediary
energy metabolism during DDT poisoning. Winteringham _£.gl. (1960)
reported significant breakdown of about 20% of the thoracic ATP in
DDT-poisoned flies. The ATP drop and prostrate symptomology was
reversed by injection of glucose. Sparing the insect hypermotor
activity during DDT poisoning by the use of anesthesia did not sustain
the ATP level. With DDT poisoning they also saw a drop in L-t-glycero-
phosphate and phospholipids at prostration.

Agosin gt 31. (1961 and 1963) studied the influence of DDT on
intermediary carbohydrate metabolism in Triatoma infestans. They
found that DDT (as well as non-toxic DDE) inhibited anaerobic glycolytic
pyruvate production by cell-free preparations. DDT enhanced incorporatflnn

of glucose into carbon dioxide, glycogen and fatty acids while DDE did

not. Glucose oxidation via the pentose phOSphate pathway amounted to

10

77 percent in DDT-treated insects compared to 22 percent in normal
insects. Plapp (1970) confirmed this increase in the pentose pathway
in house flies. However, Ela £5 31, (1970) found no increase in the
pentose phoSphate pathway over the glycolytic pathway with DDT poisoning
in cockroaches.

In Triatoma DDT increased the NADP level, but not the NADP/NADPH
ratio, possibly because of increased NAD-kinase. The increased NAD-
kinase is thought to be related to detoxification of DDT and resistance
(Ilevicky g£_§l,, 1964). Increased glutathione turnover (glutathione
is necessary for activity of DDT-dehydrochlorinase, a detoxifying
enzyme) and increased protein synthesis are also related to DDT
poisoning in resistant house flies (Agosin 95.31., 1966).

4. Neurohormonal Involvement in DDT Poisoning

Sternburg and Kearns (1952) found that the hemolymph of DDT-
poisoned cockroaches, when injected into normal insects, produced
DDT poisoning symptoms. Since the injected hemolymph did not contain
sufficient DDT to cause the symptoms, they concluded that the DDT had
induced production of a neurotoxic factor. Using electrophysiological
preparations, this toxin was found to cause multiple firing in ganglia
and sensory nerve fibers. It was unstable in the hemolymph, but was
dialyzable and stable in the dialysate (Shankland and Kearns, 1959).
Furthermore, it was found that body stress, such as physical immobil-
ization, forced movement or electrical stimulation produced similar
(although not necessarily identical) substances which also caused
DDT-like symptoms and paralysis in unpoisoned insects (Beament, 1958;
Heslop and Ray, 1959). Cook t al. (1969) proposed that their

Factor S derived from nerve cords and heads of normal and stressed

11

American cockroaches may be a neurohormone, transmitter or modulator
substance and may be identical to the neurotoxin of Sternburg _£__1,
(1959). The production and release of this type of substance was
observed histochemically in the corpus cardiacum (Hodgson and Geldiay,
1959). With parabiosis experiments, Colhoun (1960) showed that the
toxin was not the primary cause of the DDT-induced death.

Isolation and identification of the toxins are difficult because
of the small quantities present in the insects. Sternburg (1960)
tried to solve this problem by using crayfish which are considerably
larger than insects and produce similar toxins. He found that the
toxin was not a known neurohumoral agent or a DDT metabolite.
Hawkins and Sternburg (1964) partially identified it as an aromatic
amine, possibly an ester. Patel and Cutkomp (1968) established
that the substance was fluorescent and therefore easily detectable.
It was not produced by insects treated with certain organophOSphate
insecticides and therefore is not a dying tissue response. Davey
(1963) found that enforced activity of cockroaches also released a
cardiac stimulator in the hemolymph. The literature on such toxins

is reviewed by Sternburg (1960 and 1963).

PART I

Trehalose and Glycogen Depletion during DDT Poisoning of
American Cockroaches, Periplaneta americana

Carbohydrate levels in DDT-poisoned insects were investigated
during the initial research on this insecticide. Ludwig (1946) showed
a drop in glycogen and glucose in insects during DDT poisoning.

Merrill g£.§l, (1946) did further work on the carbohydrate depletion
including experiments using anesthesia and glucose injections as
possible antidotes to the insecticide's activity. They concluded that
the depletion of carbohydrates did not play a primary role in the action
of DDT. However, their paper did not include complete data on symptom-
ology of the insects. Winteringham (1956) similarly did not report
symptomology in full, although he agreed that the carbohydrate drop

was not a primary cause of death. Heslop and Ray (1959) suggested that
a biochemical lesion was the cause of the decrease in energy compounds.

Cline and Pearce (1966) found that trehalose-14C,

14

the major metabolite
of injected glucose- C, was greatly reduced in DDT-treated NAIDM flies.
This paper presents detailed data on carbohydrate depletion
during DDT poisoning and some evidence for the cause of this depletion.
Although it may not be the primary cause of death, the carbohydrate
depletion must be considered significant to the understanding of what
happens biochemically during the dying process of insects and may

directly or indirectly be associated with the other factors character-

izing the DDT poisoning syndrome.

12

13

I. Materials and Methods

Adult male American cockroaches, Periplanteta americana (L.)
maintained under a daily 16 hr photophase were the experimental animals.
They were given Purina Dog Chow® and water £51 libitum. The cockroaches
were treated with the following insecticides and analogs: p,p'DDT
(99%, City Chemical Corp., New York, N. Y.); p,p'DDE, 98%, propoxur
(97%, o-isopropoxyphenyl methylcarbamate [Baygodgy, Pesticide Repository,
USPHS, Pesticide Research Laboratory, Perrine, Fla.); and dieldrin
(99+%, Shell Chemical Co., New York, N. Y.). Treatments were topical
on the ventral portion of the abdomen in 10 or 20 ’11 acetone as stated
in each experiment. After treatment the cockroaches were kept in
battery jars with pressboard partitions. No food was available, but
humidity was maintained with a damp paper towel.

In experiments with DDT the cockroaches were sampled at times
determined by symptoms the DDT-treated groups showed. Terminology
used in this paper to describe the stages of poisoning was modified from
Heslop and Ray (1959). Initial tremors denotes the first stage of
poisoning when the insect is slightly ataxic and shows slight tremors.
During the tremors stage, all appendages are in continual nervous
motion. In the hyperactivity stage the insect exhibits continual
walking and running, with occasional convulsions. The late hyperactilg
stage indicates a slowing, with the insect having little control over
its movements. Tremors are continuous. During the prostrate stage the
insect cannotright itself, but has continual tremors. In the late
prostrate stage all movements have ceased except for occasional, but
slight, twitching. Except for the experiments where cockroaches were

sampled hourly, the stages of hyperactivity and late prostration were

14

found to be convenient for the sampling of groups for carbohydrate
determinations. At 24°C with 50-100 Pg DDT per cockroach, typical
hyperactivity symptoms appeared 3-5 hr after treatment and prostration
occurnxibetween 8 and 12 hr after treatment.

Hemolymph for trehalose assays was obtained from the cockroaches
by cutting appendages (antennae, tarsi, cerci, Styli and phallomeres)
and collecting with a capillary pipette the droplets formed. Samples
were immediately frozen with dry ice. The hemolymph was lyOphilized
and then suSpended in 2 ml 60% ethanol. The anthrone colorimetric
test for trehalose was run according to Wyatt and Kalf (1957) and the
results presented as mg trehalose per mg dry hemolymph. These qual-
itative and quantitative determinations were confirmed by formation
of the trimethylsilyl derivatives (Sweeley g£_gl., 1963) and analysis
by GLC using a 6 ft x k in. stainless steel column of 3% OV-l on 100/120
mesh Gas Chrom Q, isothermally at 220°C with 40 m1/min helium flow and
flame ionization detection. Cellobiose was used as an internal standard
and the peaks were quantitated by peak height ratios. This column resohed
derivatives of cellobiose, maltose and trehalose from each other
and from derivatives of monosaccharides for identification purposes.

In addition, trehalose was distinguishable from either cellobiose or
maltose alone because of its single peak.

Glycogen and trehalose were extracted from homogenates of whole
cockroaches. Groups of 3-7 cockroaches were homogenized 15 min in a
Lourdes Model MM-l homogenizer in 15 ml 10% trichloroacetic acid. The
homogenates were centrifuged at 10,000 g for 15 min, the pellet resus-
pended in 5 ml water and the centrifugation repeated. Glycogen was

precipitated from a 2 m1 aliquot of the combined Supernatant

15

1 ml saturated Na2304 and 13 ml 95% ethanol. The preparation was kept
at -20°C overnight, the tubes centrifuged in a clinical deskrtop cen-
uifuge and the supernatant containing the mono- and disaccharides
removed. The precipitate containing glycogen was resuspended in 10 ml
95% ethanol, kept at -20°C for 2 hr and centrifuged again. The ethanol
precipitate from the 2 centrifugations was dried and resuSpended in 2 ml
water. Glycogen was determined by an anthrone test (Morris, 1948).
Standards were prepared containing glycogen and Na2SO4 equal to that

in the sample tubes. Trehalose was determined in the combined ethanol
supernatants. A 2 m1 aliquot was dried and the trichloroacetic acid
removed by rinsing twice with 10 ml anhydrous diethyl ether. The
samples were then derivatized and chromatographed as described previously.

In one set of experiments, trehalose was injected into DDT-treated
and control cockroaches. A series of one, two or three injections of
trehalose (2 mg/20 P1 water per injection) was made at 3 hr intervals
beginning with the first hyperactive symptoms shown in the DDT-treated
groups. Carbohydrate levels were determined at late prostration.

A set of experiments was run in which DDT- and DDE-treated
cockroaches were held initially at 18°C. When the DDT-treated group
showed late hyperactive symptoms, groups from both treatments were
placed at 33°C until late prostration symptoms were shown by the
DDT-treated groups. At intervals, samples were taken and carbohydrate
levels determined as described previously.

A series of reapirometry studies measured 14C02 after the injection
of 20 P1 of uniformly labelled glucose-14C (0'0255.PCi/0’5 mg glucose
per 20 Pl water per cockroach). The respired 14C02 was bubbled through

10 ml of monoethanolamine:ethyleneglycol monomethyl ether (1:2, v/v) and

16

collected hourly. In these experiments, 10 cockroaches were used per
treatment of DDT or DDE. The insects were treated with the chemicals
2 hr after the glucose injections.

Two other insecticides, dieldrin and propoxur, were used in some
experiments. Acetone was used as the solvent and for the control
treatments. Trehalose and glycogen were determined as described

previously when the poisoned insects reached a prostrate stage.

II. Results

At DDT-induced late prostration, the trehalose in the hemolymph
had decreased 7-8 fold compared to that of cockroaches treated with
DDE or controls sampled simultaneously (Table 1). All treatments
showed higher trehalose values at the hyperactive stage than the
controls with no acetone treatment.

Table 2 shows comparable results for homogenates of whole cock-
roaches treated with DDT, DDE and acetone. Both trehalose and glycogen
were depleted in the DDT-treated cockroaches at late prostration. In
these cases, the levels in DDE-treated insects were essentially the
same as those for the acetone-treated controls. Because of this
similarity, further tests used only the DDE-treated cockroaches as
controls. The hyperactive cockroaches in these experiments showed
no difference in the carbohydrates among treatments.

Figure 1 depicts the depletion of the carbohydrates with time and
the symptomology of the cockroaches treated with 50 Pg DDT or DDE
in 10 P1 acetone/cockroach at 23°C. Each value represents 3-5 groups
of 5 cockroaches each and was calculated by dividing the values for the

DDT treatments by the DDE values and plotting as percent versus time.

17

Table l. Trehalose levels in the hemolymph of treated American cockroaches.

 

 

 

 

mg trehalose/mg dry hemolymphI: S.E.b
Treatmenta Hyperactive Late prostrate
stagec stage
DDT 0.17 i 0.03 0.02 i 0.01
DDE .16 j; .02 .12 i .02
Acetone .17 i .02 .12 i .02
Control .11 i .01 .10 i .01

 

8Treatments were 48 Pg DDT or DDE/10 P1 acetone or 10 ’11 acetone
alone topically per cockroach. The control was without any treatment.
Temperature was 25°C.

bEach value represents 5-7 groups of 5 cockroaches each.

c
Stage refers to the time these symptoms were shown by DDT-

treated cockroaches.

18

Table 2. Trehalose and glycogen levels after DDT and DDE treatments
of American cockroaches.

 

 

 

 

 

Trehalose Glycogen
(mg/g wet weight i S.E.)b (mg/g wet weight 1 S.E.)b
Treatmenta
Hyperactivec Late prostratec Hyperactivec Late prostratec
DDT 3.1 i 0.27 0.15 i 0.07 3.7 i 0.32 0.17 i 0.02
DDE 3.1 i .24 2.80: .27 3.4: .32 3.40: .31
Acetone 2.8 i .23 2.70 i .24 3.6 i .45 2.90 i .65

 

aTreatments were 50 lg DDT or DDE/10,11 acetone or 10 P1 acetone alone
per cockroach. Temperature was 23°C.

bEach value represents 3-7 groups of 5 cockroaches each.

CStage refers to the time these symptoms were shown by DDT-

treated cockroaches.

19

Figure l. Glycogen and trehalose during DDT treatment as percent of
DDE-treated control cockroaches. Vertical lines indicate

half standard errors.

 

 

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-x- Glycogen

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21

The greatest decrease in carbohydrate levels occurred during the late
hyperactive stage, although some occurred before and after this
stage.

The results for experiments in which trehalose was injected are
shown in Table 3. The values are from cockroaches taken when the
DDT-treated groups exhibited late prostrate symptoms 22 hr after treat-
ment. Although injections did not appear to reverse the DDT symptoms,
the time to death was prolonged. Treated cockroaches showed little
adverse reaponse to the repetitive injections other than slowed activity.
In all the DDT-treated groups, the carbohydrates were similarly depleted.
In the control groups, the injected trehalose tended to accumulate or
was metabolized to glycogen.

A series of experiments determined the effect of temperature on
carbohydrate depletion. Cockroaches were treated topically with
100 Pg DDT or DDE in 20.P1 acetone per cockroach. They were moved
from an 18 to a 33°C chamber when the DDT-treated groups were in the
hyperactive to late hyperactive stages. The DDT symptoms appeared to
diminish as evidenced by a decrease in the jerking motions and tremors;
however, the late prostrate stage still occurred by 22 hr. Neither
trehalose nor glycogen was replenished during the reversal of symptoms
(Figure 2). The 8, 10 and 12 hr values in Figure 2 are not significantly
different from each other. Each value in this figure represents 3-5
groups of 5 cockroaches each.

Carbon dioxide collection in 3 experiments showed an average of
70.3% (i;2.1% S.E.) of injected glucose-14C recovered by late prostration
as 14002 from DDT-treated cockroaches. This average compared to 34.6%

(i 5.1% S.E.) for the DDE-treated insects. Figure 3 shows the hourly

22

Table 3. Trehalose and glycogen levels in DDT- and DDE-treated American
cockroaches after trehalose injections.

 

 

 

 

 

Trehalose Glycogen
(mg/g wet weight i S.E.)b (mg/g wet weight;i S.E.)b
No.
in' a c c C C
jections DDE DDT DDE DDT
0 2.8 i 0.34 0.14 i 0.08 2.5 i 0.56 0.17 i 0.04
l 2.9 1; .30 .25 i .04 3.5 3; .40 .19 i .01
2 4.2 i .21 .18 j; .06 2.9 i .36 .17 i .03
3 4.3 i .18 .16 i .03 4.1 i .29 .14 i .01

 

82 mg trehalose/20 P1 water were injected at 3 hr intervals begin-
ning at the hyperactive stage (5 hr). Samples were taken at late
prostration, 22 hr.

b

Each value represents 3-6 groups of 5 cockroaches each.

c100 Pg DDT or DDE/10 Pl acetone per cockroach. Temperature was 18°C.

23

Figure 2. The effect of a temperature rise on trehalose and glycogen
levels in DDT-treated American cockroaches. Values are

presented as a percent of the DDE-treated controls. Vertical

lines represent half standard errors.

24

its as:

 

 

 

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25

Figure 3. Difference between 14COZ reSpired from DDT- and DDE-treated
American cockroaches injected with glucose-14C as a percent

of total dpm's injected. Values were calculated by the

formula:
(DPM 14002 from DDT-) __ (DPM 14(:02 from DDE-
rtreated_;nsects treated insects X 100%

 

total DPM injected

DPM (%)

muguw'flmw

26

 

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27

recovery of 14

C02 from one such experiment in which groups of 10 cock-
roaches were treated with 100 Pg DDT or DDE in 20.P1 acetone per cock-
roach. Similar curves with 2 maxima were obtained with replicate runs
for groups of cockroaches as well as with individual cockroaches per
respirometer flask. One peak occurred during the hyperactive to late
hyperactive stages; the second occurred while the insects were prostrate.
Dieldrin and propoxur were used to determine whether the depletion
of carbohydrates was specific to DDT or also could be attributed to
insecticides with different modes of action. Table 4 shows the results
of experiments with these insecticides. Dieldrin treatment caused
depleted carbohydrate levels very similar to the results with DDT.
With propoxur, the trehalose level was reduced by a factor of only
2 but the glycogen level was down 20 fold. Because glycogen and

trehalose are presumably interchangeable, the trehalose level probably

would have been lower had more time elapsed before its determination.

III. Discussion

The depletion of glycogen and trehalose occurred almost simul-
taneously and over a relatively short time period during and just
after the peak in activity of the DDT-poisoned insects. The depletion
was complete by the late prostrate stage, the time when the insect's
tremors stopped. The largest amount of 14C02 respired from the glucose-
14C injections also came at, and just after the peak in activity and
corresponds fairly well with the carbohydrate depletion. The correlations

of symptomology, 14

C02 respiration and the carbohydrate depletion could
suggest that the carbohydrate depletion is primarily a result of the

higher amounts of energy used by the insect's muscles during hyper-

28

Table 4. Trehalose and glycogen levels in dieldrin- and propoxur-
treated American cockroaches.

 

 

 

 

 

Trehalose Glycogen
(mg/g wet weight'i S.E.)a (mg/g wet weight‘i S.E.)a
Insecticide Acetone Insecticide Acetone
Dieldrinb 0.25 i 0.07 2.0 i 0.13 0.14 i- 0.01 3.9 :3; 0.29
Propoxurc 1.20 i .09 2.3 i .10 .21 i .03 4.1 i .45

 

8Each value represents 4-6 groups of 5 cockroaches each.

bTreatment was with 80 P8 dieldrin/20 Pl acetone per cockroach,
topically. Acetone treatment was with 20 Pl acetone. Samples were taken
at 24 hr while cockroaches were prostrate, but exhibiting strong tremors.

cTreatment was with 5 ’lg propoxur/S ’11 acetone. Samples were
taken at 5% hr while cockroaches were prostrate, but exhibiting

strong tremors.

29

activity. However, the possibility of some additional unSpecified
means of carbohydrate utilization, as Winteringham (1956) suggested,
was not ruled out.

The experiment with temperature variations supports the hypothesis
that carbohydrate depletion may result from increased muscular activity.
When the hyperactive symptoms were lessened by a higher temperature,
depletion was delayed possibly because a decreased level of muscular
activity results in a slower utilization of the carbohydrates.

Merrill ggwal. (1946) in their experiments with anesthesia used time
after poisoning rather than symptomology as a criterion for making
analyses. This accounts for their results showing high carbohydrate
levels with the poisoned, anesthetized insects, which were probably
in early stages of DDT symptomology, instead of the depleted levels
shown in our work.

The trehalose injection experiments were performed to determine
if the insects could be maintained and DDT symptoms reversed by
replacing the carbohydrates lost during poisoning. Although symptoms
were reversed somewhat, the insects could not be maintained alive at
the trehalose levels used. This experiment also sought to determine
whether it was possible to reach the late prostrate stage without
a complete depletion of carbohydrates. If it were possible to separate
late prostration from carbohydrate depletion, the hypothesis that
carbohydrate depletion was necessary for DDT prostration would be
diSproved. The carbohydrate levels did drop indicating that the
depletion may be necessary for the insects to reach the late prostrate
stage. Up to 6 mg trehalose were administered and the entire amount

used by the time of late prostration. An important distinction here

30

is between the prostrate and late prostrate designations. During the
prostrate stage the insect may be highly active though too uncoordinated
to run; however, in the late prostrate stage most activity has ceased.
Carbohydrate levels seem to reflect these stages. During the prostrate
stagethere still may be some carbohydrates present; in the late
prostrate stage the depletion is essentially complete.

From these observations and information in the literature, a
hypothetical route of DDT poisoning can be drawn. DDT binds with nerve
tissue and causes trains of impulses (Roeder and Weiant, 1946; Brunnert
and Matsumura, 1969). These trains cause the insect to become hyper-
active and the insect becomes increasingly uncoordinated. The hyper-
activity depletes the carbohydrate reserves. As the carbohydrate
level passes below some threshold concentration the insect ceases to
move as a result of the lack of an oxidizable carbon source. Such a
sequence would be supported by the reported carbohydrate levels after
prostration with dieldrin and propoxur. These insecticides also cause
hyperactivity even though their mode of action is different than that
of DDT.

This sequence would not be in conflict with data on the influence
of DDT poisoning on amino acid levels (Winteringham, 1958; Corrigan
and Kearns, 1958; Patel and Cutkomp, 1968). Here stress on the
carbohydrate reserves might cause the insect to mobilize the amino
acids for use as carbon sources (Corrigan and Kearns, 1963).

In conclusion, this sequence would suggest that although carbo-
hydrate depletion is not the cause of DDT's effect on the nervous
system, it is a link between the effect of DDT on the nervous system

andthe death of the other tissues in the poisoned insect.

PART II

A Hyperglycemic Factor in the Serum of DDT-prostrate
American Cockroaches, Periplaneta americana

Although the primary action of DDT in insects involves disruption
of nervous tissue, there has been considerable Speculation as to other
possible lesions (See O'Brien, 1967). Sternburg and Kearns (1952)
described a neurotoxin other than the poisoning agent, which was
produced by cockroaches after DDT-induced prostration. The structure,
mode of action and function of this neurotoxin has not been elucidated
(Sternburg, 1963). Cook _£_§l, (1969) suggested that their "Factor S,"
which has properties similar to the neurotoxin and is released during
stress, may be a neurohormone, a transmitter or a modulator substance.
A hormone has been found in cockroaches which causes nerve trains
similar to those produced by the neurotoxin and the Factor 8 and
was described by Migliori Natalizi ggflgl. (1970).

The possibility that the factors described above may
influence carbohydrate levels suggested the research described in
this paper. Steele (1961, 1963) and Migliori Natalizi and Frontali
(1966) have described hormones from cockroaches that increase trehalose
levels in the hemolymph. This paper presents data showing the presence
of a hyperglycemic factor in the hemolymph of DDT-prostrate cockroaches.
Some characteristics of this factor are described and its possible

significance discussed.

31

32

I. Materials and Methods

Adult male American cockroaches, Periplaneta americana, L.
maintained under a daily 16 hr photophase were used as experimental
animals. They were given Purina Dog Chov® and water 2d libitum.
Treatments of p,p'DDT (99%, City Chemical Corp., New York, N. Y.)
and p,p'DDE (98%, Pesticide Repository, USPHS, Pesticide Research
Laboratory, Perrine, Fla.) were topically administered to the ventral
portion of the abdomen in 10 ’11 acetone at 25°C. Doses were 100 Pg
DDT or DDE per insect. After treatment the cockroaches were kept
in covered battery jars with pressboard partitions. No food was
available but humidity was maintained by a damp paper towel.

Procedures for collection of cockroach hemolymph for bioassay
were as previously described in Part I. The terminology used for the
various stages of DDT poisoning were also as before. Prostration
occurred about 20 hr after poisoning under the test conditions. Samples
of hemolymph were immediately frozen with dry ice, stored at -20°C
and used within 2 days.

After defrosting, the hemolymph serum was separated from cellular
debris with a microsyringe. The serum was either diluted with a Ringer's
salinea (Maddrell, 1968) for use directly in the bioassay or lyophilized
for fractionation by molecular Sieve chromatography. The lyophilized
serum for this chromatography was resuspended in a minimal volume of
0.1 M acetic acid and placed on a 0.9 x 22 cm Sephadex G-25 column

with Dextran 2000 dye to determine the void volume. The serum components

 

aThe Ringer's saline used consisted of 9.82 g NaCl, 0.77 g KCl, 0.5 g
CaC12, 0.18 g NaHCO3, 0.10 g NaH2P04 in 1 liter distilled water.

33

were eluted with 0.1 M acetic acid and collected in 2 ml fractions.
These fractions were lyophilized and resuspended in Ringer's saline
for bioassay.

The bioassay procedure consisted of the injection of 10.P1 of the
diluted serum or reconstituted Sephadex eluant into carbon dioxide
anesthetized cockroaches. Injections were made through the integument
between the second and third abdominal sternites on the left side
of the midline. Two hours after injection the insects were again
immobilized with carbon dioxide, frozen on dry ice and stored at
-20°C until trehalose assays were performed within 4 days.

Trehalose in homogenates of whole cockroaches was assayed by GLC
as described previously, except that 11% QF-l +-0V-l7 (Applied
Science Laboratories, College Park, Penn.) as the stationary phase
on 80/100 mesh Gas Chrom Q was used in some assays yielding results
similar to those with the OV-l column.

Heat stability experiments were conducted with the diluted serum.
Treatments of the serum ranging from 3 minutes at 60°C to 5 minutes at
100°C were used. Bioassays for hyperglycemic activity in the samples
were conducted as described above.

The content of DDT in the serum that was bioassayed was determined
by GLC. A 0.5 ml sample of serum was extracted twice with 5 m1 of
hexane and the combined extracts evaporated to 0.5 ml. DDT was quanti-
tated isothermally at 230°C using a 6 ft x 1/8 in. stainless steel
column of 3% QF-l + OV-l7 on 60/80 mesh Gas Chrom Q. Helium flow was
maintained at 30 m1/minute and an electron capture detector was used.
The peaks were quantitated by comparing peak heights with those of

standards.

34

Protein determinations were made on Sephadex eluants using the

Lowry method (Lowry g£_gl., 1951).

II. Results

The Serum of hemolymph from DDT-treated cockroaches at late
prostration, when injected into previously untreated cockroaches, was
found to increase trehalose levels. Serum from DDE-treated cockroaches
had no such hyperglycemic activity. The peak of the trehalose increase
was observed 1-3 hr after injection with some activity as long as 7 hr
after injection (Figure 4). Each trehalose value represents determinations
on 6-8 replicates of 4-5 cockroaches each. The control trehalose
values fluctuated around 2.2 mg trehalose per g wet weight. The
differences between the absolute values of the controls in each
experiment is due to variations in the cockroach colony. Insects judged
healthy and from a single cage were used in each experiment.

From two determinations the amount of DDT in the serum injected
for the assays averaged 0.025 Pg per insect (about 0.03 ppm). No
metabolites other than DDE were present. When this amount of DDT
was added to serum from DDE-treated insects and this mixture Subsequently
injected into previously untreated cockroaches, no rise in trehalose
was detected. The DDT plus serum injections resulted in a trehalose
level of 1.64 mg trehalose/g cockroach wet weight (i;0.12 S.E.) 2 hr
after injection. The serum injected controls had a level of 1.75 mg
trehalose/g wet weight (1 0.12 S.E.) at 2 hr. These values represent
the average of two experiments with 4 groups of 5 cockroaches in each.

Serum from cockroaches at early prostrate and late prostrate

Stages of DDT poisoning diaplayed hyperglycemic activity, whereas serum

35

Figure 4. The level of trehalose in cockroaches treated with serum from
DDT-prostrate cockroaches. Values are expressed as a percent
of controls injected with serum from DDE-treated cockroaches.

Vertical lines indicate standard errors.

36

 

 

 

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37

from cockroaches at the tremors or hyperactivity stages showed little
significant activity (Figure 5). Controls were injected with serum from
DDE-treated insects. Each value represents averages of two experiments
with 4 groups of 5 cockroaches each.

The stability of the hyperglycemic factor to temperature was
assayed. The activity of serum left at room temperature averaged 96%
that of normal serum for up to 4 hr, but had been completely lost by
24 hr. The activity withstood severe heat treatment. After heating at
60°C for 3 minutes an average activity of 93% remained. Heating to
75°C for 5 minutes similarly left 99% of the activity. However, heating
at 100°C for 5 minutes left only 69%. In these experiments Ringer's
saline injections were used as controls. Values represent averages
from 2-3 experiments with 3-4 groups of 5 cockroaches each.

Increasing dilutions of the active serum produced a graded decline
in the trehalose increase in injected cockroaches. It appears that
the active hemolymph diluted 1:16 with Ringer's saline produced no
hyperglycemic response in the injected cockroaches. Ringer's saline
injections were used as controls (Table 5).

The active factor was fractionated from the serum using a Sephadex
G-15 column. The most active portions of the eluant, as seen in Figure
6, are the fractions between 13 and 17 ml with a possible later peak
at the fraction at 25 ml. Protein determinations of individual fractions
showed that the bulk of the protein is eluted before the fractions
containing the hyperglycemic activity. This experiment was done
twice. In this representative run the bioassay values represent 3

groups of 4-5 cockroaches each.

38

Figure 5. Hyperglycemic activity from serum of cockroaches in different
DDT poisoning stages. Trehalose levels in cockroaches
injected with serum from DDT-treated (-x-) and DDE-treated
(-o-) cockroaches. Poisoning stages refer to symptoms
shown by DDT-treated groups. Vertical lines indicate standard

errors .

39

 

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-o- DDE Sara

 

 

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40

Table 5. Hyperglycemic activity of serum from DDT-prostrate cockroaches
diluted with Ringer's saline.

 

 

 

Dilution parts Trehalose
serum:saline (% of control)8 1 S.E.
1:2 149 i 16
1:4 140 i 20
1:8 123 i 10
1:16 102 i 10

 

8Values are the increase in trehalose as a percentage of saline
injected controls. Values represent 3 experiments each with 3-4

groups of 4-5 insects.

41

Figure 6. Sephadex chromatography of the hyperglycemic factor.
A 0.9 x 22 cm Sephadex G-15 column was used and eluted in
2 ml fractions with 0.1 M acetic acid. The void volume was
5 ml. Activity of the eluant (-x-) was detected with a
cockroach bioassay. The dashed line is the trehalose level
of the saline injected controls. Protein levels (~o-) were

determined by the Lowry method.

42

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43

III. Discussion

The hyperglycemic activity of serum from DDT-prostrate cockroaches
is due to a substance other than DDT or some metabolite of DDT. This
factor appears analogous to the neurotoxin of Sternburg g£_§l, (1959)
in that it is found mainly in prostrate stages of poisoning and is
heat stable. The heat stability of the factor and the elution from
Sephadex indicate that the factor is probably not a protein. The
hyperglycemic hormone described by Migliori Natalizi g£_gl, (1970)
had a similar elution pattern from Sephadex G-15. Their hormone was
thought to be a peptide due to its trypsin sensitivity.

The physiologic nature of this hyperglycemic factor is open to
Speculation. First, the factor may be a product of tissue breakdown
occurring in the late stages of DDT poisoning. The fat body tissues
in cockroaches during the late stages of poisoning appear emptied and
disrupted. Release of some pharmacologically active substance likely
could occur.

A second possibility is that the factor may be a hyperglycemic
hormone released into the hemolymph during DDT prostration. The release
may be stimulated by the low levels of trehalose in the hemolymph or,
alternatively, release may be stimulated directly by the action
of DDT on the glands producing the hormone. These glands are
probably associated with the nervous system and might be stimulated
by the action of DDT on the nervous system. If the release were indeed
effected by DDT, a variety of other hormones might also be released
into the hemolymph.

A third possibility is that the factor does not affect carbo-

hydrate metabolism directly, but causes the release of a hyperglycemic

‘—-—-—‘ F gnaw—"lb." .‘

44

hormone 13 vivo. For example, the factor could exert its action by
affecting nerve tissue such as the neurohormonal glands. Such an
action would be similar to that of the neurotoxin of Sternburg and

Kearns (1952).

AN OVERVIEW

I. Interpretations of the Research
A. Experiments Concerning Carbohydrate Levels

This research has demonstrated that in an insect treated with DDT,
control over carbohydrate metabolism is upset during the dying process.
This was reported in the first paper (Part I) as the increased
utilization of trehalose and glycogen and the depletion of these
carbohydrates by the time of prostration. Although circumstantial
evidence (intense physical activity and rapid 14002 respiration
from glucose-14C) suggests that these carbohydrates were primarily
oxidized to carbon dioxide, more evidence would be needed for proof
that this is indeed the main source of carbon dioxide and that the
higher oxidation is of significance in DDT poisoning.

Tracer studies in which other metabolites are checked would
lend significance. For example, what part does amino acid and lipid
oxidation play in the increased respiration? Until the extent to
which these other possible carbon sources contribute to the increased
carbon dioxide production is determined, the significance of the
carbohydrate depletion cannot be fully appreciated.

The second problem presented, though not completely solved in
the first paper, is the existence or non-existence of a cause-effect
relationship in the role of the carbohydrate depletion in DDT poisoning.

Experiments other than the ones done could be attempted to substantiate

45

46

the interpretation of this paper that there is a cause-effect relation-
ship, but in a complex system such as a whole organism such a relation-
ship is almost impossible to prove. For example, if large doses of
DDT were used and produced, hypothetically, a high carbohydrate level
in the prostrate cockroach, this could be interpreted to mean that

DDT affected the insect differently than the lower doses used in

the real experiments. It could be argued that instead of the absence
of the cause-effect relationship, the high carbohydrate levels really
indicate a qualitative change in the mode of action of DDT at the
higher dose, rather than the desired quantitative change.

Merrill §£_gl, (1946) stated that the changes in the carbohydrate
levels are not significant in the poisoning process. Arguments that
question the procedures used can cast doubt upon this conclusion.

They found that if the cockroaches were anesthetized the drop in carbo-
hydrates did not occur. As discussed earlier, no mention was made of
the symptoms the poisoned animals were exhibiting when they came out

of anesthesia. However, if we assume that the insects did show prostrate
symptomology, it could be argued that the experiment is not significant
because the role of the anesthetic itself in producing the prostration
is not known; there could be a synergistic or antagonistic effect
between the anesthetic and the insecticide. Gatfield e£_gl, (1966)
demonstrated that phenobarbital anesthesia in mice is accompanied by

a reduced utilization of glycogen and glucose in the brain. Such

an antagonistic affect might occur with insects.

In essence, then, the chicken-egg (which-came-first) relationship
is very difficult to prove or diSprove. The experimental manipulations

used to demonstrate it may themselves change the relationship being shown.

47

B. Experiments Concerning the Hyperglycemic Factor

The presence of a hyperglycemic factor is demonstrated fairly
conclusively in the experiments. The rise in trehalose is not due
to the absence of a substance or a dilution of the trehalose by injection
of the agent, since saline injections produce no rise in the trehalose.
The factor is probably organic (not an inorganic ion such as P042. or
Ca2+) since it is deactivated by 24 hr at room temperature and to
some extent by boiling for 5 minutes. Of course, direct experimentation
into the chemical nature of the factor would give conclusive answers.

The possibility that the factor is a hormone is not discounted
by the evidence. The factor is eluted from the Sephadex column in
about the same fraction, proportionately, as the hormone studied by
Migliori Natalizi $5.31, (1970). The fact that the factor is not
present in large quantities during the tremors or hyperactive stages
of DDT poisoning is also amenable to the hormone hypothesis. Maddrell
(1964) in studying the diuretic hormone of cockroaches found that it
was degraded while it exerted its activity on the malpighian tubules.
In DDT poisoning the hyperglycemic hormone can only exert its activity
in the early stages since in the prostrate Stages no glycogen is
available for conversion to trehalose. If degradation of the hormone
is similar to that of the diuretic hormone an accumulation in the
hemolymph might be expected only at the prostrate stages.

An understanding of the role of this hyperglycemic factor in DDT
poisoning is not possible without more information. The discussion
above treats this factor as a side effect of DDT poisoning. There is

at present no evidence supporting this view or contradicting it. More

48

about the site of activity of this factor must be known before placing

it in the sequence of events in DDT poisoning.

11. Experiments
Several areas of uncertainty were mentioned in the previous section.
Research in this area could profitably go in these and other directions.
A. Lipid and Protein Levels
In order to understand the significance of the changes in carbo-
hydrates during DDT poisoning, the levels of lipids and proteins in
the insect hemolymph at various stages of DDT poisoning are important.
Total lipids would be important since they too could be used as a
source of energy and might reflect the presence of other hormone-like
factors in the hemolymph. Levels of phospholipid precursors would
also give an indication of changes in lipid metabolism.
Differing protein levels would either indicate the activity of
a hormone-like factor or be an indication of tissue breakdown.
B. Additional Effects of the Factor
A Spectrum of the effects this factor has on the insect would be
of significance in understanding its role. First, does it increase
phOSphate levels and carbon dioxide production ig_yiyg? The hyper-
glycemic hormone of Steele (1963) increases phOSphate. Whether the
factor does would give an indication as to its equivalence to the
hormone. Second, how does the factor affect the utilization of
carbohydrates? Does it increase the metabolism of glucose-14C and
what are the ratios of C-6 versus the C-1 incorporation into carbon
dioxide? These types of studies wOuld necessitate purification of the

factor and an attempt to separate the various activities.

49

C. I_n_ M Fat Body Bioassays

Does the factor directly affect the fat body or work on other
systems i§_!iyg? The factor could be incubated with fat body prep-
arations. Increased release of trehalose by the fat bodies would
indicate a direct effect of the factor on the fat body and reduce
the possibility that some additional factor must act as an inter-
mediate ig_yiyg or that the new trehalose is not produced in the fat
body, but released from muscles or hemolymph glycoproteins.

D. Chemical Characterization of the Factor

Additional experiments could characterize the factor chemically.
For example, ion exchange chromatography, trypsin sensitivity tests
and various tests for active groups could be done. As an adjunct
to these tests, utilization of whole cockroach homogenates rather
than the hemolymph as a source of the factor should be attempted,
Since this would allow for the preparation of larger quantities
of the factor.

E. Biological Characterization of the Factor

Is the factor released during physical stress or poisoning with
other insecticides? This fact is important for comparison to the
neurotoxins as discussed previously.

Similarly, is the factor active on nerve preparations? Since
the neurotoxin is also present in the hemolymph of DDT-prostrate cock-
roaches this type of experimentation would also entail an attempt to
separate the two types of activity. A tentative identification of the
factor as being the same as the neurotoxin and/or the hyperglycemic
hormone could be made by their elution patterns from Sephadex columns

or other types of chromatography.

50

III. Significance of these Studies

The significance of this work possibly lies in two areas. First
it will produce a better understanding of how insects, and possibly
animals in general, reSpond to stresses. From the work on the neuro-
toxins, DDT appears to be a nonSpecific stressing agent on the insects.
Possibly study of this factor and others will indicate that they too
are released as a result of many types of stress. An understanding of
how an animal reacts to a stress is basic to understanding the methods
by which an organism controls its basic functions.

The second area of significance is an outgrowth of the possibility
that nonSpecific stress is an important part of DDT poisoning. In the
past, screening programs for potential insecticidal chemicals have
used mortality as the main criterion for selecting candidate insec-
dxides. Since insects in general do not differ grossly from other forms
of life biochemically, the resulting insecticides were also biocides.
If indeed this factor is part of a hormone system and it is significant
in the chain of events leading to mortality, the possibility exists
that these studies will be useful in devising insect-Specific screening

programs for insecticides.

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