THE EFFECTS OF JUVENILE HORMONE
ON MITROCHONDRIAL METABOLISM IN THE__INDIAN MEAL
MOTH, Plodia interpunctella (HUBNER)

By
DONALD ELLIOTT FIRSTENBERG

A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF

THE UNIVERSITY OF FLORIDA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
1975

ACKNOWLEDGEMENTS

The author would like to express his deep appre-
ciation to the members of his committee for their guidance
and assistance in this study. Appreciation is expressed
to Dr. Herbert Ober lander; Dr. Harvey L. Coraroy; Dr. David
S. Anthony; Dr. James L. Nation, who served as Cochairman
of the Supervisory Committee; and, particularly, to Dr.
Donald L. Silhacek, who served as Chairman of the Super-
visory Comjnittee and under whose direction this study was
carried out, and whose encouragement, advice and interest
in this study provided a great deal of inspiration and
V7ho aided in developing and molding the author's rf^search
philosophy.

The amthor also v/ishes to express his appreciation
to Dr. W.G. Eden, Chairman of the Department of Entomology
and Nematology, for providing financial assistance during
the study and to the Insect Attractanfcs, Behavior and
Basic Biology Laboratory, Agriculture Research Service,
United States Department of Agriculture, for providing finan-
cial assistance and the facilities to carry out the neces-
sary research.

Finally, the author wishes to express his gratitude
to his wife, Ilene, for her help, patience and encourage-
ment throughout the course of this investigation.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ii

LIST OF TABLES V

LIST OF FIGURES vi

ABSTRACT viii

INTRODUCTION 1

LITERATURE REVIEW 3

Juvenile Hormone 7

Metabolic Effects of Juvenile Hormone 12

Mitochondrial Oxidations 15

Hormonal Control of Mitochondrial Oxidative

Activities 25

Conclusion 27

MATERIALS AND METHODS 29

Insect Rearing Methods 29

Test Diet Preparation 30

Selection of Insects 31

Isolation of Mitochondria ." 32

Polargraphic Enzyme Activity Determination 33

Paqe

Assay of NADH Dehydrogenase Activity 35

Mitochondrial Nitrogen Determinations .... 35

Assay of Mitochondrial Cytochromes 36

Assay of Mitochondrial Hemes 37

Assay of De Novo Heme Synthesis 39

Electron Microscope Studies 39

RESULTS 41

Electron Microscopy 41

Effects of In Vivo JH Treatment of Mito-
chondrial Enzyme Activity 41

Effects of In Vitro JH Treatment on Mito-
chondrial Enzyme Activities 51

Cytochrome Analyses 62

Assay of Mitochondrial Hemes 74

Assay of De^ Novo Synthesis 80

DISCUSSION 82

CONCLUSIONS 92

LITERATURE CITATIONS 94

BIOGRAPHICAL SKETCH 109

LIST OF TABLES

Table Page

A Comparison of Mitochondria in Midguts

of Control and JH-Fed Larvae 46

Rates of Pyruvate-Malate Oxidation in
Isolated Mitochondria from Plodia inter-
punctella treated in vivo with Juvenile
Hormone 48

Rates of Succinate Oxidation in Isolated
Mitochondria from Plodia interpunctella
treated in vivo with Juvenile Hormone 50

Effect of Juvenile Hormone on the Rates
of Substrate Oxidation by Isolated Mito-
chondria from Plodia interpunctella 52

Effect of Inhibitors and Aging on NADH
Oxidation by Mitochondria from Plodia
interpunctella 55

Comparison of Mitochondrial Cytochrome
Concentrations of 7-day-old JH-fed Larvae
which had undergone a Supernumerary Molt
with those from Larvae which had not
molted 69

Ratios of Cytochrome A+A3 , B and C Con-
centrations to Cytochrome A+A3 Concen-
tration for Mitochondria isolated from
Control and JH-treated Larvae 73

LIST OF FIGURES

Figure Page

The sequence of reactions in the elec-
tron transport chain (Hansford and
Sacktor, 1971) .

19

The sequence of reactions in the citrate
cycle showing the points of origin of
NADH and succinate dehydrogenase (SDH)
(Lehninger, 1965) . ^^

Electron micrograph of midgut tissue

from control larvae (magnification,

15,000 X) . ^^

Electron micrograph of midgut tissue

from JH-fed larvae (magnification,

15,000 X) . ^^

The relationship between NADH dehydro-
genase activity and time of aging of
isolated mitochondria from larvae of
Plodia interpunctella. ^^

The relationship of juvenile hormone

concentration to the percent inhibition

of NADH and pyruvate-malate oxidations. 59

Plot of (S)/V versus (S) ( (S) is the _
NADH concentration and V is the reaction
rate) . The substrate constant (Kg) , in-
hibitor constant (Ki) and the maximum

velocity of the reaction (V ) are

max ci

shown. "-^

The relationship of larval age and age
at initiation of JH treatment to the
mitochondrial cytochrome content. 65

The relationship of larval age to cyto-
chromes a, b and c concentration per
insect for JH-treated and control in-
sects. ^2

Figure Page

10 The relationship of larval age to
cytochromes a, b and c concentration

per gram tissue for JH-treated and ^^

control insects.

11 The relationship of larval age to
hemes a, b (protoheme) and c concen-
tration per insect for JH-treated and ^^
control insects.

12 The relationship of larval age to hemes
a, b (protoheme) and c concentration
per gram tissue for JH-treated and con-
trol insects.

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

THE EFFECTS OF JUVENILE HORT^ONE
ON MITOCHONDRlAL metabolism in THE INDIAN MEAL
MOTH, Plodia interpunctella (HUBNER)

By
Donald Elliott Firstenberg
March, 1975

Chairman: Donald L. Silhacek

Cochairman: James L. Nation

Major Department: Entomology and Nematology

Investigation into the effects of synthetic H.
cecropia juvenile hormone (methyl-10, ll-epoxy-7-ethyl-3 ,
11-dime thy 1- trans 2, trans 6-tridecadienoate) on mito-
chondrial metabolism in larvae of the Indian Meal moth,
Plodia interpunctella (Hiibner) revealed that juvenile hor-
mone affected citrate cycle oxidations, electron trans-
port, heme synthesis and cytochrome synthesis.

Juvenile hormone inhibited all NAD-linked oxida-
tions in the citrate cycle of isolated mitochondria. How-
ever, the f lavoprotein-linked oxidation of a-glycerophos-
phate was not affected by juvenile hormone while succinate
oxidation was stimulated.

NADH oxidation in vitro by aged and uncoupled mito-
chondria was also inhibited by juvenile hormone. Experiments

using ferricyanide to artificially accept electrons from
NADH dehydrogenase indicated that juvenile hormone pre-
vented electron transport at the nonheme iron level of
complex I in the electron transport chain. Further exper-
iments revealed that the inhibition was noncompetitive.
These results indicated that the inhibition of NAD-linked
oxidations in the citrate cycle was due to the effects
of juvenile hormone in the electron transport chain.

Inhibition of NAD-linked substrate oxidations in
the citrate cycle could result in an increase in cyto-
chrome synthesis by a mechanism involving synthesis of
malats from pyruvate and the reversal of reactions in the
citrate cycle. Inclusion of juvenile hormone in the diet
of the larvae resulted in an increase in the concentration
of mitochondrial cytochrome. However, this stimulation
was dependent upon some factor associated with the larval-
larval molt. The concentration of juvenile hormone at the
beginning of an instar affects the mitochondrial cytochrome
concentration v^7ithin that instar.

The results of the cytochrome analyses indicated
that the hypothetical mechanism of control of cytochrome
synthesis had a second level of control associated with the
molt. Since cytochromes are composed of heme and apopro-
tein, the effects of juvenile hormone on the synthesis of
just the heme portion of the molecule was investigated.
Inclusion of juvenile hormone in the diet stimulated de novo

heme synthesis, but this effect was immediate and there-
fore presumably direct.

The data suggest a metabolic mechanism for juve-
nile hormone controlling insect growth and development by
determining the maximum capacity of cellular energy produc-
tion. Energy production levels may be limited by the con-
centration of electron transport chain components.

INTRODUCTION

Insect growth and metamorphosis are characterized
by a series of sequential changes. These stepwise changes
are necessitated by limits imposed by the insect's rigid
exoskeleton. A number of reviews are available which dis-
cuss growth and give descriptions of development (Agrell,
1964; Whitten, 1968) , the physiology of grov/th and meta-
morphosis (Wigglesworth, 1954, 1957, 1959a, 1965) and the
histological and biochemical aspect of insect growth
(Williams, 1951; Wigglesworth, 1959b; Wyatt, 1968) .

The growth and development of insects are under
hormonal control. During the growth and development of
insects, characteristic changes occur in the quantity
(Silhacek, 1967), morphology (Brosomer et al. , 1963, Willis,
1966; V'Jatanabe and Williams, 1953), phospholipid composi-
tion (Silhacek, 1967) and oxidative activities (Silhacek,
1967; Brosomer et al . , 1963; Michejda, 1964) of mitochon-
dria. Some investigators have linked these changes in
mitochondrial metabolism with endocrine control. In an
earlier study, DeWilde and Stegwee (1958) demonstrated
that corpora allata, endocrine glands v;hich produce juvenile
hormone (JH) , exerted a direct effect on the respiration of

LeptijTotarGa decemlineata. A subsequent study (Stegwee,
19 60) revealed that a JH-active extract from Hyalophora
cecrqpia stimulated succinate oxidation in isolated mito-
chondria from L. decemlineata. Clarke and Baldwin (1960)
also concluded that JH preparations affected the oxida-
tive activity of isolated mitochondria from Locusta migra-
toria and Schistocerca gregaria. In contrast, the oxidative
activities of mitochondria isolated from allatectomized
L. migratoria (Minks, 1967) and Blaberus discoidalis
(Keeley, 19 71) were found to be similar to those of normal
insects. The recent identification and synthesis of juve-
nile hormone permitted the direct testing of the effects
of JH on isolated insect mitochondria in a defined medium.

It is evident that mitochondrial activities could
play a central role in the processes responsible for growth
and development in insects. However, our current knowl-
edge is very limited and much data is contradictory. More
inform.ation on the role of mitochondrial activities and juve-
nile hormone action and the relationship of these to other
aspects of metabolism is required. The purpose of the
present study is to elucidate the effects of juvenile hor-
mone on mitochondria in vitro and in vivo, to determine
the biochemical m.echanism of action of "JH in mitochondria
and to determine the significance of the primary action of
the hormone to other aspects of insect metabolism.

LITERATURE REVIEW

Hormonal Control of Growth and Development

The growth and development of insects is under hor-
monal control. The three hormones that appear to be impor-
tant in this developmental regulation are prothoracico-
tropic hormone (brain hormone) , ecdysone (molting hormone)
and juvenile hormone (Neotenin) . Prothoracicotropic hor-
mone is produced by the median neurosecretory cells in the
pars intercerebralis of the insect brain. It was shown
that the prothoracicotropic hormone has no direct effect
on m.olting but stimulates the prothoracic (ecdysial) glands
to secrete ecdysone (Fukuda, 1944). Ecdysone, v;hich ini-
tiates the molting process, is secreted shortly before each
m.olt and disappears shortly afteirward (Burdette, 1961;
Karlson and Shaaya, 1964; Shaaya and Karlson, 1965). The
crystalline hormone first prepared by Butenandt and Karl-
son (1954) was used in experiments on Chironomus tentans
by Clever and Karlson (1960) and Clever (1961, 1964, 1965).
These experiments indicated that injections of ecdysone in-
duce a pattern of puffing at chromosome loci identical to
that which occurs in normal metamorphosing last instar

larvae. The puffing of the chromosomes is thought to be
a mechanism of exposing the DNA which is normally covered
by protein and making it available to participate in RNA
synthesis. Beermann and Clever (19 6 4) found that the
puffs induced by ecdysone were associated with rapid RNA
synthesis. This resulted in the hypothesis (Karlson et
al., 1964) that ecdysone acts directly on the genome and
activates specific gene loci which results in the produc-
tion of specific messenger RNAs and, consequently, specific
proteins necessary for the physiological events of meta-
morphosis .

Kroeger (196 3a) proposed a second hypothesis that
ecdysone acts directly on the nuclear membrane causing an
altered ionic balance of sodium and potassium which causes
the activity described in the genome. Ito and Lowenstein
(1965) found that nuclear membrane permeability to ions
does change during development and that the changes in mem-
brane permeability could be induced by ecdysone. Lezzi
and Gilbert (1970) found that the chromosome puffs could
be induced by altering sodium and potassium concentrations
alone without ecdysone. However, work by Congote et al .

(1969) indicates that ecdysone can stimulate RNA synthesis
in a preparation of isolated nuclei in the presence of ec-
dysone without sodium or potassium ions. Congote et al.

(1970) also show that in isolated nuclei the hormone- induced
RNA differs from RNA isolated from control insects.

The third hormone, juvenile hormone (JH) , is pro-
duced by the corpora allata. During development juvenile
hormone titer is minimal at each molt but increases rapid-
ly during the early part of the instar and decreases to-
ward the end of the instar (Gilbert and Schneiderman ,
1961b; Williams, 1963; Stephen and Gilbert, 1970). Juve-
nile hormone titer at the beginning of an instar is thought
to affect the phenotype which will occur after the next
molt (Wigglesworth, 1940; Clever, 1963; Kroeger, 1968),
and may also affect the sequence of morphological changes
in insect development (Gilbert, 1964). Juvenile hormone
is thought to affect genetic regulation in such a way as
to cause a larval- larval molt at high concentration, a
larval-pupal molt at lower concentrations and a pupal-adult
molt when it is absent (Bounhiol, 1938; Piepho, 1942, 1946,
1951; Fukuda, 1944; Nayar, 1954; Gilbert and Schneiderman,
1961b; Williams, 1961; Gilbert, 1963; Williams and Kafatos,
1971) . However, the specific response to JH is modified
by the ability of the specific tissues to react with JH
(Piepho, 1942; Wigglesworth, 1948; Gilbert and Schneider-
man, 1960).

Secretion of JH by the corpora allata may be under
the control of nervous stimulation from the brain (Luscher
and Englemann, 1960) . Juvenile hormone titer decreases
as larval age increases with the previously described
cyclic variation occurring during each instar (Fukuda,

1944; Piepho, 1950b, 1951, 1952; Rehm, 1951; Williams,
1961; Johnson and Hill, 1973). The decrease in JH titer
has been attributed to several different causes. One
hypothesis states that because larval volume increases
more rapidly than corpora allata volume there is a dilu-
tion of the hormone (Kaiser, 1949; Beljaeva, 1960; Novak
and Cervenkova, 1960; Novak and Slama, 1962). A second
hypothesis is that inactivation or degradation of JH be-
comes more intense in older larvae (Gilbert and Schneider-
man, 1960) . In support of the latter hypothesis, Weirich
et al. (1973) has found the activity of esterase (an en-
zyme which inactivates JH) is higher in the fifth-larval
instar in Manduca sexta than in the fourth-larval instar.

Williams and Kafatos (1971) have recently pro-
posed a model which predicts three separate sets of genes,
one set coding for larval characteristics, another set
coding for pupal characteristics and a third set coding
for adult characteristics. Only one of the three gene
sets could be active at any one time and the titer of juve-
nile hormone would determine which one was active.

Insect growth and development is characterized by
stepwise changes that are necessitated by limits imposed
by the insect's rigid exoskeleton. In order to increase
in size the insect must molt, shedding its exoskeleton and
reforming another of a larger size. Insects may develop
toward the adult form gradually or by distinct stages.

Insects that undergo gradual development (Heinimetabola)
have immature stages (larvae) that are morphologically
similar to the adult. Some structures show a gradual
progression from the immature to the imaginal form as the
molts progress. Immature insects that undergo complete
metamorphosis (Holometabola) undergo extensive morpholog-
ical alterations in transforming from the larvae to the
adult stages. In holometabolous insects the larval molts
permit increases in body size. An intermediate pupal
stage is then required for the transition of larva to
adult. During the pupal stage many larval tissues disap-
pear, others are modified and new tissues are formed. The
degree of tissue reorganization is dependent on the species
of insect. Many of the imaginal tissues are derived from
embryonic tissue (imaginal discs) which does not develop
until the pupal stage.

Juvenile Hormone

Juvenile hormone was first demonstrated by VJiggles-
worth (1935, 1940) who showed by the use of parabiosis ex-
periments that a substance carried in the hemolymph of
Rhodnius prolixus was responsible for the retention of
immature characteristics. An extract with strong juvenile
hormone activity was prepared from abdomens of adult male
Hyalophora cecropia moths by Williams (19 56) and a purified

preparation of juvenile hormone v/as obtained by Williams
and Law (1965) . Several other procedures have been de-
veloped for the isolation and purification of juvenile
hormone (Meyer and Ax, 1965; Roller et al. , 1965, 1969).

The amount of JH per abdomen in the adult male
H. cecropia moth has been determined to be between 6.0 yg
(Metzler e_t ajL. , 1971) and 0.5 yg (Meyer et al . , 1965).
Roller et al. (1967) identified the structure of juvenile
hormone as methyl-10, ll-epoxy-7-ethyl-3 , 11-dimethyl-
trans 2, trans 6-tridecadienoate . This structural isomer
of JK was also found to have the greatest effect of the
eight possible isom.ers of juvenile hormone on several
species (Roller and Dahm, 1968; Rose et al. , 1968; Wester-
mann e_t al., 1969; Wigglesworth, 1969; Pfiffner, 1971;
Schweiter-Poyer , 1973). Several synthetic schemes have
been developed for the production of juvenile hormone
(Roller and Dahm, 1968b; Berkoff, 1969; Findlay and Mac-
Kay, 19 69) .

Two other naturally occuring juvenile hormones
have been found. A second JH , methyl-10, ll-epoxy-3, 7,
ll-trimethyl-2, 6-dodecadienoate has been found in H.
cecropia by Meyer et al. (1968) . Judy et al. (1973)
found this JH along with a third juvenile hormone, methyl-
10, ll-epoxy-3, 7, ll-trimethyl-2, 6-tridecadienoate, in
Manduca sexta organ cultures. Schooley et a^^. (1973) ,
using corpora allata cultured from M. sexta, found that

the biosynthesis of these juvenile hormones proceeds by
a terpenoid pathway with biosynthesis of the carbon skele-
ton initiated through homomevalonate arising from propion-
ate and acetate.

Prior to the extraction of juvenile hormone from
H. cecropia, many investigators sought to correlate the
molecular structures of a variety of substances with juve-
nilizing activity. These studies quantitate JH activity
by correlating dosage to retention of juvenile morphological
characteristics. Wigglesworth (1969b) discussed the corre-
lation of 42 different compounds in relation to natural H.
cecropia juvenile hormone. Other juvenilizing agents which
have been studied include isoprenoid and straight chain al-
cohols (Bowers and Thomson, 1963; Schneiderman et al. , 1965) ,
other terpenoids (Schwarz et al . , 1970), aromatic terpenoid
ethers (Bowers, 1969; Kiouchi et al. , 1974), terpenoid
amines (Cruickshank and Palmere, 1971), acetals applied as
a vapor (McGovern et al. , 1971) and long chain fatty acids
including oleic and linoleic acids (Slama, 1961, 1962).
Although juvenilizing agents are generally nonspecific,
one juvenile hormone analog, p-(l, 5-dimethyl-hexyl) ben-
zoic acid v;as selective on Dysdercus sp. (Suchy et al. ,
1968) . Farnesol and its derivatives have been the subject
of many studies of JH activity (Karlson and Schmialek,
1959; Schmialek, 1961; Williams, 1961; Wigglesworth, 1961,
1962, 1963, 1969a, 1969b; Yamamoto and Jacobson, 1962;

10

Karlson, 1963; Schneiderman and Gilbert, 1964; Braun et
al. , 1968; Schv/arz et al . , 1969; Sonnet et aJ . , 1969).

A study conducted by Slade and Wilkinson (1973) in
which the enzymatic degradation of JH was quantitatively
determined in the presence or absence of JH analogs indi-
cated that the ana.-ogs prevented the inactivation of the
natural hormone. his study casts some doubt as to whether
the JH analogs have any juvenilizing effect or whether they
only have a synergistic effect with the natural hormone.

Many of the studies to determine the physiological
effects of juvenile hormone prior to the availability of
isolated or synthetic JH were done by removal or implanta-
tion of the corpora allata, the glands which produce the
hormone. It was shown that implantation of corpora allata
would cause supernumerary molts and extend the immature
life span of insects (Pflugf elder , 1937; Bodenstein, 1943;
Srivastava and Gilbert, 1969) . Other physiological effects
attributed to JH include enhancing the regenerative abili-
ties in Carausius morosus (Pf lugfelder , 1939) , induction
of green coloration in larvae of Locusta under environmental
conditions which would normally produce the brown, gregar-
ious phase (phase polymorphism) (Staal , 1959, 1961) and in-
fluencing determination of caste polymorphism in social
insects (Kaiser, 1955; Luscher, 1961; Rembold, 1974).
Juvenile hormone is required for the maturation of the fe-
male reproductive organs and oocytes in adult insects

11

(Ichikawa and Nishiitsutsuj i-Iwo, 1959; Williams, 1959;
Gilbert, 1964; Ilighnam, 1964; Wigglesworth , 1964; Postle-
thwait and Weiser, 197 3; Sroka and Gilbert, 19 72; Kamby-
sellis et al. , 1974). Although JH is required for egg
maturation, application of exogenous JH to insect eggs can
interrupt development (Riddiford and Williams, 1967; Riddi-
ford, 1970). Implantation of corpora allata was also found
to influence insect behavior in that it affects larval-lar-
val premolt cocoon spinning in Galleria mellonella (Piepho,
1950) and the hormone induced mating behavior in giant
supernumerary larvae or adultoids of Pyrrhocoris apterus
(Zdarek and Slaraa, 1968). Juvenile hormone also stimulates
sex pheromone production in Ips confusus (Borden et al. ,
1969) and Tonebrj^ molitor (Menon, 1970) .

Adult diapause in Leptinotarsa decemlineata appears
to be due to a deficiency in JH (DeWilde and Stegwee, 195 8;
DeWilde, 1959). However, larval diapause in Dia_traea
grandiosella appears to be induced by JH (Chippendale and
Yin, 1973; Yin and Chippendale, 1973, 1974). The physio-
logical functions of juvenile hormone have been reviewed
by Scharrer (1953) Gilbert and Schneiderman (1961) , Gilmour
(1961), Wigglesworth (1962, 1964), Gilbert (1964), Novak
(19 66) , and Slama et al_. (1974) .

12

Metabolic Effects of Juvenile Hormone^

Although DNA synthesis in immature tissues is
probably not stimulated by juvenile hormone (Novak, 1971) ,
in the adult insects of some species DNA synthesis must
be simulated by JH in order for the reproductive organs to
develop (Hodkova, 1974). In larval insects juvenile hor-
mone may play a role in determining which molecular species
of RNA are synthesized (Williams and Kafatos, 1971) , but
may not have any effect on the overall quantity of RNA
synthesized. Oberlander and Schneiderman (19 66) found no
stimulation of RNA synthesis in isolated pupal abdomens
which lacked prothoracic glands during JH treatment. This
was interpreted to mean that juvenile hormone does not have
a direct motabolic regulatory role except on the prothor-
acic glands. More recent studies have shown that JH did
stimulate RNA synthesis in silkworm wing disks (Patel and
Madhavan, 1969) and in isolated fat body cell nuclei (Con-
gote et al., 1969). However, in these systems simultaneous
application of JH and ecdysone did not result in stimulation
of RNA synthesis.

Because new proteins appear at different times
during insect development, the production of different spe-
cies of RNA must be under the control of JH . In some cases
the effect may be stimulatory while in others inhibitory
(Williams and Kafatos, 1971) . Experiments by Ilan et al.

13

(1970) suggest that the synthesis of new species of trans-
fer RNA and their activating enzymes (amino acyl-tRNA syn-
thetases) which also require RNA synthesis are under the
control of juvenile hormone. An increase in purine syn-
thesis as an effect of JH is reliant on RNA synthesis and
this increased purine synthesis results in a reduced uric
acid excretion (L'Helias, 1956).

Following allatectomy (removal of the corpora
allata) L'Helias (1956) found increases in uric acid ex-
cretion and free amino acids in the tissues with a decrease
in tissue protein in larvae of Dixippus morosus. Allatec-
tomy also caused an increase in hemolymph levels of amino
acid (iMinks, 19 67) . Drastic decreases in protein synthe-
sis were found in all tissues, following allatectomy by
Vandenberg (1963) . Patel and Madhavan (1969) found that
injection of JH induced protein synthesis.

The opposite effect was found for hemolymph pro-
tein concentrations in adult females. Allatectomy in adult
females caused an increase in hemolymph protein (Hill,
1962, 1963; Highnam and Hill, 1963; Slama, 1964; Minks,
1963) . This effect may be due to the continued synthesis
of vitellogenic proteins without incorporation into the
oocyte whose development is controlled by juvenile hormone.

Specific proteins under juvenile hormone control
include vitellogenic proteins (Englemann, 1969) , which may
be the same proteins found by Minks (1967) in L. migratoria.

14

tRNA acylases and cuticular proteins (Ilan et al^. , 1970)
and esterases (Whitmore et al. , 1972; Weirich et al . ,
1973) . The induction of esterases is apparently a mech-
anism to deactivate the JH which can be degraded by hydroly-
sis of the methyl ester, hydrolysis of the epoxide group
or conjugation with polar groups (Ajarai and Riddiford,
1971; Siddall, et al. , 1971; Slade and Zibitt, 1971a, 1971b;
White, 1972) . Another protein which binds and transports
juvenile hormone (Trautmann, 197 2; Whitmore and Gilbert,
1972; Emmerich and Hartmann, 1973) apparently secves to
protect the hormone from degradation (Ferkovich, personal
communication) .

Both lipid and carbohydrate content undergo changes
after the; removal or implantation of corpora allata. In
larvae of Dixxipus allatectomy caused an increase in gly-
cogen concentration (L'Helias, 1955, 1964) and Sehnal and
Slama (1956) and Sehnal {1971) found that implantation of
corpora allata into last instar G. mellonella prevented
accumulation of lipids but glycogen content increased.
Studies relating the rate of lipid synthesis to JH during
development (Stephen and Gilbert, 1969, 1970) indicate
that high juvenile hormone titer may serve to inhibit lipid
synthesis.

The metabolic effects of JH on adult insects is
apparently due to stimulation of the reproductive system.
These effects include stimulation of DNA synthesis in

15

developing ovarian tissue (Hodkova, 1974) , increasing
lipid and glycogen synthesis (Minks, 1967; Liu, 1973)
and stimulation of incorporation of yolk precursors into
oocytes (Janda and Slama, 1965; Minks, 1967; Lanzrein,
1974) .

A recent review by Slama et al. (19 74) comprehen-
sively discussed the morphological, physiological, and bio-
chemical effects of the neuroendocrine system and the chem-
istry of juvenile hormone and other insect growth regulators
in insects. Other recent surveys of the effects and chem-
istry of JH have been made by Novak (19 66) , Wyatt (196 8) ,
Slama (1971) and Pfiffner (1971) .

Mitochondrial Oxidations

Mitochondria are subcellular organelles composed
of two membranes, an outer relatively smooth membrane and
an inner membrane which has many invaginations, called
cristae (Lehninger, 1965) , Mitochondria have some degree
of autonomy from the control of the cell nucleus in that
they can synthesize some of their own proteins under the
direction of mitochondrial DNA (Beatie, 1971) and the bio-
genesis of mitochondria doesn't appear -to be controlled
by the nucleus (Ashwell and Work, 1970) .

Insect flight muscle mitochondria have been studied
because of an enormous increase in oxygen consumption above

16

the basal rate in actively flying insects (Davis and
Fraenkel, 1940). In the flight muscles of Phormia re-

Q

gina there are approximately 1.1 x 10 mitochondria per
mg tissue v/hich comprise 40% of the total muscle mass
(Levenbook and Williams, 1956) . Lehninger (1970) has cal-
culated that active flight muscle mitochondria may have
10-tiraes the inner membrane surface area of less active
mammalian mitochondria -

There is a space between the inner and outer mem-
branes and another space internal to and bounded by the
inner membrane (matrix) resulting in four enzymatically
distinct compartments; the outer membrane, the intermem-
brane space, the inner membrane and the matrix. Enzymes
associated with the outer membrane include monoamine
oxidase and rotenone-insensitive KADH : cytochrome c reduc-
tase (Reed and Sacktor, 1971) . Adenylate kinase is lo-
cated in the intermembrane space (Reed and Sacktor,
1971) . The inner membrane is thought to contain the en-
zymes for coupled ATP synthesis (Hansford and Sacktor,
1971) , f lavoprotein-linked a-glycerophosphate dehydro-
genase (Zebe and McShan, 1957; Reed and Sacktor, 1971) ,
succinic dehydrogenase (Greville et^ a_l. , 1965) , proline
dehydrogenase (Brosomer and Veerabhadrappa , 19 65; Sacktor
and Childress, 1967) , trehalase (Reed and Sacktor, 1971) ,
a-keto acid dehydrogenases and the enzymes of the elec-
tron transport chain (Hansford and Sacktor, 1971).

17

Enzymes found as soluble matrix proteins include citrate
synthetase (Beenakkers et a]^. , 1967), NAD and NAD) -
linked isocitrate dehydrogenases (Goebell and Klingenberg,
1963, 1972), malate dehydrogenase (Delbruck et_ aJ . , 1959;
Reed and Sacktor, 1971) , alanine and aspartate aminotrans-
ferases (Brosomer et al. , 1963) , 3-hydroxylacyl-CoA de-
hydrogenase (Beenakkers et aA. , 1967) and palmitoyl and
carnitine ace tyltransf erase (Beenakkers and Henderson,
1967; Beenakkers et al. , 1967; Childress et al . , 1967).

Oxidative phosphorylation is defined as the addi-
tion of a terminal phosphate group to ADP to form ATP as
a result of: reactions coupled to the electron transport
chain (Figure 1) . The significance is that elec-
trons derived from the oxidation of organic acids are
passed to the electron transport chain which results
finally in the reduction of oxygen to form water and the
production of ATP, a form of chemically utilizable energy.
The existence of oxidative phosphorylation in insects was
first confirmed by Sacktor (1954) and Lewis and Slater
(1954) . Oxidative phosphorylation in insect mitochondria
has been found to be coupled to the oxidation of pyruvate,
citrate cycle intermediates (Sacktor, 1954; Gregg et. al . ,
1960; Birt, 1961), a-glycerophosphate (Sacktor and Cochran,
1958; Van Der Bergh and Slater, 1960), amino acids (Rees,
1954; Sacktor and Childress, 1967) and fatty acids (Meyer
et al., 1960; Beenakkers, 1963, 1965; Beenakkers and

Figure 1. The sequence of reactions in the electron

transport chain (Hansford and Sacktor, 1971)

19

SUCCINATE

SUCCINATE
DEHYDROGENASE

NADH

NADH
DEHYDROGENASE

NONHEME IRON

NONHEME IRON

COENZYME Q

CYTOCHROME b

CYTOCHROME c

CYTOCHROME c

CYTOCHROMES a + a.

H^O

20

Klinaenberq, 1964; Beenakkers and Henderson, 1967).

The conditions necessary for oxidative phosphory-
lation have been discussed by Sacktor (1953) . Free fatty
acids may uncouple oxidative phosphorylation (Wojtczak and
Wojtczak, 1960; Wojtczak, et al^. , 1968) but serum albumin may
afford some functional and structural protection.

The electron transport chain accepts electrons from
NADH and various flavoprotein dehydrogenases. NADH is
formed in the oxidation of pyruvate to acetyl-CoA, the oxi-
dation of L-6 -hydroxy lacy 1-CoA to L-3-ketoacyl-CoA in the
B -oxidation of fatty acids and in several oxidative reac-
tions of tJio citrate cycle (Kreb's cycle, tricarboxylic
acid cycle) (Figure 2) . The introduction of electrons into
the electron transport chain through reduced flavoprotein
dehydrogenases is the result of the oxidations of succi-
nate, a-glycerophosphate and fatty acyl-CoA. Electrons
from NADH enter electron transport at NADH dehydrogenase
and those from flavoprotein dehydrogenases enter at coen-
zyme Q. The rate of substrate oxidation depends upon the
permeability of the mitochondrial membrane to the substrate
(Childress and Sacktor, 1966; Tulp et al. , 1971) which ap-
pears to be facilitated by substrate ion translocators
(Tulp and Van Dam, 1969; Tulp et al. , 1971).

Mitochondrial enzyme activities as well as mito-
chondrial mass changed as development proceeded from one
week prior to the imaginal molt to one week after in

Figure 2. The sequence of reactions in the citrate cycle
showing the points of origin of NADH and suc-
cinate dehydrogenase (SDH) (Lehninger, 1965).

Acetyl~CoA

22

CITRATE

OXALACETATE

->- NADH

MALATE

FUMARATE

cis-ACONITATE

NADH-^

ISOCITRATE

NADH-H

->-C0,

a-KETOGLUTARATE

-V SDH (FADH)

■>-C0,

'SUCCINATE -e

-SUCCINYL-CoA

23

L. miqraborla (Brosomer et al. , 1963). Michejda (1964)
found similar results in developing flight muscles of H.
cecropia. In adult flight muscle mitochondrial size in-
creases as a function of age directly after adult emergence
in Drosophila funebris and Phormia regina (Watanabe and
Williams, 1953). Silhacek (1966) found changes in oxygen
consumption correlated to age in the last two larvel instars
of G. mellonella. Similar effects have been found in iso-
lated mitochondria from Plodia interpunctella (Silhacek
et al., 1974) .

Leenders and Berendes (1972) and Leenders and Beck-
ers (1972) found that inhibition of respiratory enzymes
which increased the metabolic demand for the enzymes can
activate certain gene loci in polytene chromosomes. The
same effect is seen in dinitrophenol-treated larvae or
larvae recovering from anaerobiosis (Berendes et al^. ,
1965; Van Brenzel, 1966; Ashburner, 1970).

Keilin (1925) studied 40 species of insects and
found light absorption bands in flight muscle. Three of
the bands at approximately 605, 563, and 550 nm were desig-
nated to correspond to three hemochromagens , cytochromes
a, b and c, respectively. Williams (1951) found high con-
centrations of the cytochromes in flight muscle and Sacktor
(19 5 3) found that within the muscle the cytochromes were
restricted to the mitochondria. Individual cytochromes
ranged from 0.5 to 1.5 ymoles/g protein in flight muscle

24

of several insects (Barron and Tahmisian, 1948; Levenbook
and Williams, 1956; Chance and Sacktor, 1958; Bucher and
Klingenberg, 1958; Klingenberg and Bucher, 1959; Stegwee
and van Kammen-Wertheim, 1962; Slack and Bursell, 1972).
The ratios of cytochromes are approximately 1:1.5:0.5
(a:c:b) (Chance and Sacktor, 1958; Bucher and Klingenberg,
1958; Stegwee and van Kammen-Wertheim, 1962).

Very little work has been done on the biosynthesis
of cytochromes in insects. Soslau et al. (1971) found that
exogenous 6 -aminolevulinic acid would stimulate the synthe-
sis of heme, a precursor of cytochrome. Hamdy et al.
(1974) found incorporation of ^'^C-glycine into heme. These
studies indicated that heme biosynthesis proceeded as de-
scribed for vertebrate systems (Burnham, 1969) . Chan and
Margoliash (1966) demonstrated the de novo synthesis of
cytochrome c in Samia cynthia. Stimulation of the synthe-
sis of cytochrome c was not inhibited by actinomycin D
indicating that the stimulation of apoprotein synthesis
was not a direct effect on the genome (Soslau et al. ,
1971; Williams et al. , 1972) .

Mitochondrial metabolism in insects has been re-
viewed by Sacktor (1965, 1970, 1974) and Hansford and Sack-
tor (1971).

25

Hormonal Control of Mitochondrial
Oxidative Activities

Implantation of corpora allata increased the oxy-
gen consumption of the recipient insect. The degree of
stimulation was directly proportional to the activity of
the implanted gland (Novak et al. , 1959, 1962; Slaraa and
Hrubesova, 1963) . Allatectomy produced a decrease in oxy-
gen consumption (Thomsen, 1949), however, these effects
were weak and temporary. Others found that JH stimulated
oxygen consumption only in female insects but had no ef-
fect on males (DeWilde and Stegwee, 19 58; Sagesser, 196 0;
Novak and Slama, 1962; Slama, 1964). This effect on fe-
males has been attributed to the ability of JH to stimulate
developmeTil: of the female reproductive system and not due
to a direct stimulation of respiratory activity (Pflug-
felder, 1952; Thomsen, 1955).

Sehnal and Slama (1966) implanted corpora allata
into larvae of G. me Hone 11a and monitored the oxygen
consumption of the intact insects through supernumerary
larval instars. They concluded that the stimulatory ef-
fect on oxygen consumption is indirect and due to the
increased mass of the insects. Clarke and Baldwin (1960)
found that extracts of corpora allata stimulated oxygen
consumption in tissue homogenates of L. migratoria but
depressed oxygen consumption under the same conditions in
Schistocerca gregaria. Stegwee (1960) found that oxygen

26

consumption by mitochondria isolated from diapausing L.
decemlineata were stimulated by juvenile hormone. DeWilde
(1961) confirmed this in vitro and found similar effects
in vivo with implanted corpora allata. Both used sodium
succinate as a substrate for in vitro mitochondrial oxida-
tions. Stegwee (19 59) has demonstrated that adult diapause
is due to a deficiency in JH secretion. Diapause is char-
acterized by low rates of oxygen consumption and very low
cytochrome concentrations (Shappirio and Williams, 1957a,
1957b) . This was detected in pupae of H. cecropia which
undergo a pupal diapause. Cytochrome concentrations de-
clined rapidly within hours of the larval-pupal molt. In
contrast, nondiapausing pupae show little decline in cyto-
chrome contents.

Minks (1967) found no effect on oxygen consumption
of mitochondria isolated from flight muscles following al-
latectomy or implantation of corpora allata into adult
L. migratoria. In addition, extracts or corpora allata
had no effect on isolated mitochondria. However, an effect
on oxidative phosphorylation was detected when "cecropia
oil" V7as added to the mitochondria. At concentrations
approaching 10~^ (v/v) and above pyruvate-malate oxidation
was inhibited and P/0 ratio decreased (Minks, 1967).
Keeley (1970, 1971) also found hormonal control of insect
mitochondrial oxidative activity, but the control was
exerted by the corpora cardiaca and no effect was noted

27

after allatectomy in adult Blaberus discoidalis . He found
that cardiatectoray resulted in reduced oxygen consumption
and lowered cytochrome c reductase and cytochrome oxidase
activities. However, the addition of corpora cardiaca to
isolated mitochondria had no effect (Keeley, 1971; Keeley
and Wadill, 1971). Treatment of cardiatectomized insects
in vivo with an extract of the corpora cardiaca for several
days resulted in restoring oxidative enzyme activities
(Keeley and Wadill, 1971). The effects of cardiatectomy
were duplic^"lted by severing the nerves from the brain to
the corpora cardiaca. The factor appears to be a poly-
peptide and appears to be produced in the brain and released
by the corpora cardiaca (Keeley and Wadill, 1971). Keeley
(19 72) found that the corpora cardiaca hormone is a control-
ling factor in the biogensis of mitochondria in the adult
cockroach.

Conclusion

The preceding literature review has dealt primarily
with the endocrine control of growth and development, the
physiological and biochemical effects of juvenile hormone
and the biochemistry of insect mitochondria. It is evident
that mitochondrial activities could play a central role in
the processes responsible for growth and development in
insects. However, our current knowledge is very limited

28

and much data is contradictory. More information on the
role of mitochondrial activity and juvenile hormone action
and the relationship of these to other aspects of metabo-
lism is required.

MATERIALS AND METHODS

Insect Rearing Methods

Successive generations of the Indian-naeal luoth,
Plodia interpunctella , were reared by the standardized
method of Silhacek and Miller (1972) ina2.4mx2.4inx
2.4 m walk-in incubator (American Instrument Company,
Silver Springs, Md.) maintained at 30° C + 1/2°C and 70%
R.H. + 1%. A photoperiod consishing of sixteen hours
light and eight hours dark was supplied by four Westing-
house high output daylight lamps (Westinghouse , 9 6tl2)
controlled by an electric time clock.

For egg collection, 23-day-old adult insects were
first anesthetized with carbon dioxide and placed in a
container with an 18-mesh screened bottom. The container
of moths was returned to the incubator and set on a piece
of black construction paper at the onset of the dark pe-
riod. The eggs which accumulated on the construction
paper were removed after one hour and cleaned. Forty- four
mg of cleaned eggs and 450 g of loosely packed growth
medium were placed in each of twelve 12.7 cm x 17.8 cm x
10.2 cm polystyrene plastic pans with plastic lids having

29

30

a 6.4 cm diameter screened hole. The eggs were thoroughly
mixed into the rearing medium and the pans were then in-
cubated as previously described for the required time.

The medium for rearing P. interpunctella was essen-
tially the same as that described by Silhacek and Miller
(1972) . Medixim components were refrigerated at 4°C to
prevent insect contamination. The medium was prepared by
mixing 620 g ground Gaines Dog Pellets, 255 g ground rolled
oats, 1,665 g white corn meal, 1,480 g whole wheat flour,
160 g wheat germ and 325 g of brewers yeast. The dry com-
ponents were first mixed and then a mixture of 1,000 g
glycerol and 900 g of honey was added. The fresh medium
was placed in sealed containers and allowed to stand 24
hours. It was then ground in a Viking hammer mill to give
a particle size which passed through an American Standard
8-mesh sieve and stored at room temperature until it was
used.

Test Diet Preparation

Test diets were prepared by adding 0.5 ml of an
acetone (Mallinckrodt , A.R.) solution of the hormone to
7 00 mg of the mixture of dry food components. The ace-
tone was removed under a stream of dry nitrogen while
stirring in order to uniformly distribute the hormone
throughout the food. Three hundred milligrams of

31

honey : glycerol (1:1 v/v) were then thoroughly mixed into
each 700-rag portion of test food. Acetone solutions of
0.2 and 1.0 mg/ml of Cecropia JH (methyl cis-10, 11-epoxy-
7-ethyl-3, 11-dimethyl-trans , trans-2, 6-tridecadienoate)
v/ere used to provide final hormone concentrations of 0.10
and 0.50 mg/g of food, respectively. After adding the
honey : glycerol the diets were routinely held at room tem-
perature overnight and stirred the next day before use to
break any clumps of food.

Selection of Insects

Small amounts of food containing larvae were
placed on a brown paper towel and larvae were removed
v;ith mosquito forceps. Fourth- and fifth-instar larvae
could be recognized by the relative sizes of the head
capsules .

Larval ages are presented as -2, -1, 0, +1, +2,
+3, +4 and +7 days with respect to days prior to (minus)
or following (plus) the molt into the fifth larval instar
which occurred just prior to collection of 0-day insects.
Minus-two-day larvae were early-f our-instar larvae approx-
imately two days prior to the larval- larval molt. For
in vivo JH treatment, -2-, 0- and +2-day larvae were ob-
tained from pans which were seeded with eggs eight, ten
and twelve days earlier, respectively. As they were

32

collected, two hundred to one-thousand larvae were placed
on approximately 100 g of test diet in a 12.5 cm x 6.6 cm
X 6.6 cm polystyrene plastic pan with a plastic lid having
a 5.7 cm screened hole. The pans containing JH diet and
larvae were then placed in the incubator at standard rear-
ing conditions.

After holding on test diets for appropriate inter-
vals, the insects were collected from the test diets and
used for mitochondrial isolation. Control insects were
collected from stock culture pans.

Isolation of Mitochondria

Mitochondria were isolated by differential centri-
fugation in a refrigerated centrifuge fitted with a 3.6 x
spindle speed attachment (Model PR-6, International Equip-
ment Co., Needham, Mass. Rotor #859) (Firstenberg and Sil-
hacek, 1973) . Larvae were placed in a small breaker on
ice prior to mitochondrial isolation. One to two grams
of larvae were required for isolation of mitochondria.
A 5% homogenate was prepared by grinding a group of test
insects of known weight in a ground glass homogenizer with
an isolation medium containing 0.5 M recrystallized man-
nitol, 10~^M triethanolamine (TEA), 10 M EDTA and suf-
ficient HCl to give a pH of 7.4 (Silhacek, 1967).

33

Mitochondria are defined as that fraction which
sedimented betv/een 500 x g for 10 minutes and 600 x g^
for 10 minutes. Mitochondria were resuspended in fresh
isolation medium and resedimented to provide a twice-
washed mitochondrial pellet. Mitochondria were then re-
suspended in a volume equal to the original tissue weight
(1,0 ml/g tissue) in a suspension mediiom consisting of
0.45M mannitol, 10"\ MgSO^ , 6 x 10~\ ATP (pH 7.4 with
TEA) and 5 mg/ml bovine serum albumin (BSA) . All pro-
cedures during isolation were carried out at 0-4°C.

Polarographic Enzyme Activity Determinations

Mitochondrial oxidative activities v;ere determined
using an oxygraph (Model KM, Gilson Medical Electronics
Inc., Middleton, Wise.) equipped with a vibrating platinum
electrode emd a jacketed cell maintained at 30°C to deter-
mine oxygen uptake. The basic incubation medium contained
600 ymoles recrystallized mannitol, 15 umoles MgSO^ , 6
ymoles ADP and 0.25 mg cytochrome c in 1.55 ml of water.
To this medium was added either 0 . 5 ml BSA (25 mg/ml) or
0.5 ml BSA-JH (BSA containing 250 yg Cecropia JH (a mix-
ture of isomers of methyl-10, ll-epoxy77-ethyl-3 , 11-di-
methyl-2, 6-tridecadienoate/ml) . The BSA-JH was prepared
by mixing 25 yl of JH solution (10 mg JH/ml of acetone)
per 25 mg of dry BSA, drying under nitrogen and making up

34

to volume (1.0 ml) with water. Lower concentrations of
JH in the cell were obtained by substituting equivalent
volumes of BSA for BSA-JH. One-tenth of a ml of mito-
chondrial suspension was then added to the cell and al-
lowed to preincubate for 5 minutes. After the preincuba-
tion period 30 ymoles of each substrate being tested in
a total volume of 0.2 ml were added to the cell contents.
The substrate systems used were: pyruvate-malate , pyru-
vate-glutamate, pyruvate, malate, glutamate, a-keto-
glutarate, succinate, a-glycerophosphate , ascorbate and
NADH. Oxygen consumption was monitored for 3 to 6 min-
utes. After the addition of 50 ymoles of inorganic phos-
phate (P.) in 0.05 ml (pH adjusted to 7.8 with TEA), a
second rate of oxygen consumption was measured. Respira-
tory control ratios (RC) V7ere calculated by dividing the
rate of oxygen consumption after addition of P^ by the

rate prior to the addition.

-3

One-tenth milliliter volumes of 10 M antimycm

_ -) _ c

A, 10 M oligomycin and 1.5 x 10 M rotenone were used
in ethanol solution to inhibit mitochondrial reactions
in some experiments. The effects of ethanol were deter-
mined prior to the use of inhibitors.

Three experiments consisting of duplicate enzyme
assays were run with different mitochondrial preparations,
For some experiments it was necessary to maximize the
permeability of the mitochondrial membranes to NADH by

35

aging the mitochondrial suspension at 30°C for 45 minutes
prior to storage on ice for experimentation.

Assay of NADH Dehydrogenase Activity

NADH dehydrogenase was assayed in a system using

ferricyanide as an electron acceptor. For the assay 0,1

-3 -2

ml volumes of 10 M antimycin A, 0 . 5M P., 5 x 10 M

K_Fe (CN) ,"^"^, 0.04M NADH and aged mitochondria were added
to 0.5 ml BSA or BSA-JH plus 1.55 ml basic incubation
medium in a 3.0 ml cuvette. The rate of ferricyanide re-
duction at 30°C v^^as monitored at 420 nm on a Gilford re-
cording spectophotometer (Model 2000, Gilford Laboratories
Inc., Oberlin, Ohio) with a Beckman monochromator (Model
DU, Beckman Instruments Inc., Fullerton, Calif.). The
initial rate of decrease in the optical density (O.D.)
is a measure of the NADH dehydrogenase activity.

Mitochondrial Nitrogen Determinations

Mitochondrial nitrogen content was determined by
micro-Nesslerization (Minari and Zilversmit, 1962). Du-
plicate 0.05 and 0.10 ml samples of each mitochondrial
preparation and 0.10 ml samples of the .suspension medium
were put into 50 ml Nessler's tubes. One milliliter of
dilute sulfuric acid (1:4, H2SO^:H20) was added to each
tube. Tubes were covered with parafilm and stored at

36

-18 °C for later determination. Tubes were warmed to room
temperature and placed in a Kjeldahl digestion apparatus
to char organic residue. Two to three drops of H^O^ v/ere
then added to the tubes and the contents were allowed to
reflux until all of the peroxide was removed. After cool-
ing, approximately 35 ml of water was added to each tube.
After mixing, 7.5 ml of Nessler's reagent (Sigma Chemical
Co., St. Louis, Mo.) was added, the volume adjusted to
50 ml with water, and the contents were mixed by inversion,
The O.D. at 490 nm was read against a blank on a Bausch
and Lomb colorimeter (Spectronic 20, Bausch and Lomb Inc.,
Rochester, N.Y.). The O.D. for the suspension medium was
subtracted from the mitochondrial sample O.D.s and the re-
sulting value was compared to a standard curve to give
nitrogen concentrations of the mitochondrial suspensions.
The standard curve was prepared each day Nessler's tests
were run from duplicate tubes containing 0, 150 and 300
mg nitrogen (0.0, 0.75' and 1.5 ml of a 571.4 yg/ml
NH NO^ solution, respectively) .

Assay of Mitochondrial Cytochrom.es

Mitochondrial cytochrome concentrations were de-
termined using a variation of the Chance and V7illiams
method (1955) . Isolated mitochondria were suspended in
approximately 10 ml of suspension medium and 1 . 0 ml ali-
quots were placed in each of two 1.0 cm light path, 1.0

37

ml black wall cuvettes. Analysis was accomplished with

a two-wavelength double beam scanning spectrophotometer

(model 356, Perkin Elmer Corp., Norwalk, Conn.) operated

in the split beam mode. A baseline was established by

scanning the cuvettes from 650 nm to 500 nm. A difference

spectrum was then obtained by adding 10 yl 10 M

K Fe(CN) "^^ to the reference cuvette to fully oxidize the
3 6

sample and 10 yl 0.05 M Na2S20^ to the sample cuvette in
order to fully reduce the sample. The cuvettes were then
rescanned from 650 nm to 500 nm and maxima at 605 nm, 564
nm and 551 nm corresponding to cytochromes a, b and c,
respectively were revealed. Corresponding minima occurred
at 630 nm, 575 nm and 540 nm. Subtraction of minimal
O.D.s from maximal O.D.s yielded a measure of cytochrome
concentration. Millimolar extinction coefficients for
cytochromes a, b, and c are 16.0, 20.0, and 19.0, re-
spectively (Chance and Williams, 1955) .

Assay of Mitochondrial Hemes

Mitochondrial hemes were extracted from mitochon-
• dria and analyzed using the method of Reiske (1963) . In
this procedure mitochondria were extracted with acetone,
centrifuged at 6000 x g for 15 minutes (International
PR-6 centrifuge, rotor #859) , extracted with chloroform-
methanol (2:1 v/v) , and then with acetone to remove lipids.
The supernatants were discarded. Three extractions with

acetone-HCl (2.5 ml 36% KCl in 100 ml acetone) removed
hemes a and b from the protein precipitate and left heme
c in the precipitate. The precipitate was dissolved in
approximately 10 ml alkaline pyridine (50 ml 1 . OM NaOH
in 50 ml pyridine) and a difference spectrum for heme c
was obtained as described for cytochrome analysis. The
pooled extracts from the acetone-HCl extractions were
flash evaporated to near dryness in a rotary vacuum evap-
orator at 0°C and alkaline pyridine was added. All pro-
cedures during the isolation of hemes were carried out
at 0-4 °C in subdued light. A difference spectrum for heme
b was obtained as described earlier. A difference spec-
trum for heme a was obtained by scanning dithionite re-
duced alkaline pyridine extract against water. Maxima
of 587 nm, 556 nm, and 550 nm were obtained for hemes
a, b and c, respectively. Millimolar extinction coeffi-
cients for hemes a, b, and c are 24.0, 30.0, and 19.1,
respectively (Reiske, 1953) .

A quantity of hemoglobin from a hemolysate of
human blood was determined spectrophotometrically by the
method of Hainline (1958) and was used as a standard to
determine extraction efficiency. Admixing hemoglobin to
a mitochondrial suspension (100 mg hemoglobin/ml mito-
chondria yielded about a 50% recovery of the added heme) .
Quantitative mitrochondrial heme determinations were ad-
justed for extraction efficiency.

39

Assay of De Novo Heme Synthesis

De novo synthesis of hemes was determined iso-
topically. Early-fourth- in star insects 2 days before the
last larval-larval molt were placed on either a control
or JH-treated diet prepared to give a uniform distribu-
tion of 2-''"'^C-glycine (specific activity in the diet 2.0
yCi/g diet) . The 2-"*" C- glycine (Schwarz Mann Radiochem-
icals, Orangeburg, N.Y.) was added to the diet in acetone
solution. The acetone was removed by drying under a stream
of nitrogen. Insects were removed from the diet after
four days, and mitochondria were isolated and hemes a and
b were extracted from the mitochondria. The hemes were
solubilized in soluene 100 (Packard Instrument Co.) and
were assayed by scintillation counting in a toluene based
scintillation fluid in a refrigerated scintillation coun-
ter (model Tri-Carb #3003, Packard Instrument Co., Downers
Grove, 111.). The difference in radioactivity between the
control and JH-treated tests was proportional to the quan-
tity of JH-stimulated de^ novo heme synthesis.

Electron Microscope Studies

Early-fifth-instar larvae placed on either a con-
trol diet or a 0.1 mg/g JH test diet for four days were
dissected and their midguts were removed. The midguts
were fixed with glutaraldehyde and osmium tetroxide

40

(Venable and Coggeshall, 1965) . The fixation required
sequential dehydration in ethanol-water solutions of
increasing concentrations and finally acetone. Embedding
was in an Epon-Araldite mixture (Mollenhauer , 1965).
Sectioning was performed on a Sorvall ultramicrotome
(model MT-2B, Ivan Sorvall Inc., Norwalk, Conn.). Only
silver or gray sections were placed on the 200 mesh grids.
Specimens were viewed on a Hitachi electron microscope
(model HU-125E, Hitachi Ltd., Tokyo, Japan). Photomicro-
graphs were analyzed for mitochondrial size, number, shape,
dispersion, surface aroei, volume and general condition.

RESULTS

Electron Microscopy

As a basis for subsequent biochemical studies, I
examined the ultrastructure of midgut mitochondria from
control and JH-fed larvae. Midgut mitochondria of control
larvae were relatively small, roughly spherical and were
concentrated on the hemocoel side of the midgut (Figure
3) . In JH-fed larvae (Figure 4) the mitochondria were
more irregular, twice as large as control mitochondria
and there were only half as many as in the control. Also,
they were more dispersed through the cytoplasm than mito-
chondria in midguts of control larvae (Table 1) . Mito-
chondria in control larvae had a higher surface area to
volume ratio than mitochondria in JH-fed larvae. How-
ever, the total mitochondrial volum.e within the midguts
were essentially equal for JH-fed and control larvae.

Effects of In Vivo JH Treatment on Enzyme Activities
of Mitochondria Isolated from JH-fed Insects'

These cytological differences in mitochondria
suggested altered metabolism. Experiments to test this
hypothesis established that the rate of pyruvate-malate

41

Figure 3. Electron micrograph of midgut tissue from
control larvae (magnification, 15,000 X) .

4 3

Figure 4. Electron micrograph of midgut tissue from JH-
fed Icirvae (magnification, 15,000 X).

45

'M

^9: '

46

Table 1. A Comparison of Mitochondria in Midguts of
Control and JH-Fed Larvae

JH-Treated Control

o -3 -3

Mean volume (um ) 7.8 x 10 3.0 x 10

2

Mean surface area (ym ) .203 .093

Surface area to volume ratio 23.66 34.96

Mean concentration in midgut

columnar epithelium cells

(mitochondria/vim3) * .522 1.33

Per cent of mitochondria within

10 ym of the midgut wall

(hemocoel) 48.4 62.1

*Mitochondria v/ere counted in one sagittal section from
each of three midgut columnar epithelium cells.

47

oxidation by mitochondria isolated from +4-day larvae
treated in vivo for four days with JH (treatment ini-
tiated on 0-day) was higher than the rate by mitochon-
dria isolated from the same age control larvae (Table
2) . Rates of pyruvate-malate oxidation by mitochondria
isolated from +4-day control larvae and +4-day JH-treated
larvae were lower than that of 0-day control larvae. The
rate of pyruvate-malate oxidation by mitochondria isolated
from larvae fed a diet containing 0 . 5 mg JH/g for 4 days
was not significantly different from the rate observed
for mitochondria from larvae fed a diet containing 0.1
mg JH/g for 4 days. The +4-day control larvae may not
offer a good comparison with JH-treated insects because
these insects had stopped feeding while the JH-treated
insects were still actively feeding (Firstenberg and Sil-
hacek, in press) . Mitochondria from insects fed on the
diet containing 0 . 1 mg JH/g for 7 days oxidized pyruvate-
malate at a lower rate than those from insects similarly
treated for 4 days. Mitochondria from insects fed on 0.5
mg/g diet for 7 days oxidized pyruvate-malate at a lower
rate than mitochondria from larvae fed on the 0.1 mg/g
diet for 7 days. In all cases mitochondria from larvae
treated in vivo with JH were inhibited more by JH treat-
ment (1.77 x 10~ M) in vitro than control insects. In
addition, mitochondria from JH-treated larvae had slightly
lower respiratory control ratio values than mitochondria

48

Table 2. Rates of Pyruvate-Malate Oxidation in Isolated
Mitochondria from Plodia interpunctella treated
in vivo with Juvenile Hormone

Duration of in vivo
Hormone Treatment*

Days

Hormone Mean

Concentration Additions Q

MG/G

O2 m

Mean

0 (Control)
4 (Control)

0.1

0.5

0.1

0.5

None

335.6**

5.27

JH

11.5

0.74

None

87.1

2.37

JH

3.3

-

None

171.0

3.92

JH

1.8

-

None

163.9

4.42

JH

0.8

-

None

104.0

3.91

JH

1.6

-

None

62.4

4.25

JH

0.5

-

*Treatment began with 0-day larvae
**Values represent duplicate runs on each of three
replicates
***Units of Q, I . are yatoms oxygen consumed/hour/mg
2 ^ '

mitochondrial nitrogen.

49

from the 0-day control larvae but higher values than from
+4-day control larvae. Since control insects stopped feed-
ing by the 4th day and pupated by the 7th day there were
no suitable mitochondria to serve as controls.

The results of the tests for succinate oxidation
by mitochondria (Table 3) indicated that there was a small
decrease in their ability to oxidize succinate in +4-day
control larvae as compared to 0-day control larvae. Mito-
chondria isolated from both 0-day and +4-day control lar-
vae had higher rates of succinate oxidation when treated
with JH (1.77 X 10~ M) in vitro. Mitochondria isolated
from larvae fed on either the 0.1 mg/g or the 0.5 mg/g
JH diets for 4 days had greater rates of succinate oxida-
tion than either of the control mitochondrial preparations,
Mitochondria from insects fed the 0.1 mg/g JH diet for 7
days had a lower rate of succinate oxidation than those
mitochondria from larvae fed for 4 days on the same diet.
Mitochondria from larvae fed on the 0.5 mg/g JH diet for
7 days had an even lower rate of succinate oxidation.

None of the mitochondrial preparations from larvae treated

-4
with JH responded appreciably to JH (1.77 x 10 M) in

vitro. These results indicated that the mitochondria in

JH-treated larvae differ from the controls in their
c£ipacity to be stimulated by JH in vitro. The only appar-
ent effect of in vitro JH treatment of isolated mitochon-
dria from JH-treated larvae was a small decrease in

50

Table 3. Reites of Succinate Oxidation in Isolated
Mitochondria from Plodia interpunctella
treated in vivo with Juvenile Hormone

Duration of
in vivo
treatment*

Days

Hormone
Concentration Additions

MG/G

Mean

Mean

^0^{l^f** ^-^^

0 (Control)
4 (Control)

0.1

0.5

0.1

0.5

None

121

4**

1

23

JH

160

8

1

20

None

94

1

1

39

JH

130

9

1

29

None

157

1

1

43

JH

137

4

1

12

None

136

6

1

62

JH

137

.7

1

14

None

110

.0

1

.59

JH

119

.9

1

.08

None

61

.6

1

.36

JH

53

.2

1

.04

*Treatment began with 0-day larvae
**Values represent duplicate runs on each of three
replicates
***Units of Q„ , > are yatoms oxygen consumed/hour/mg

mitochondrial nitrogen,

51

respiratory control ratios when succinate was used as the
substrate. The +4-day controls in the succinate tests
had the same deficiencies as noted in the pyruvate-malate
experiments. More recent results indicated that the stim-
ulation of succinate oxidation in mitochondria from +4-day
control larvae by JH treatment in vitro may be erroneous
and that the capacity of JH to stimulate succinate oxida-
tion may be lost as a consequence of larvae aging (Silhacek
and Kohl, unpublished data).

Effects of In Vitro JH Treatment on
Mitochondrial Enzyme Activities

The iji vivo data demonstrated an inhibitory effect
by JH on both pyruvate-malate and succinate oxidation.
The inhibition of pyruvate-malate oxidation and the stimu-
lation of succinate oxidation by JH in vitro in mitochon-
dria isolated from untreated larvae suggested that JH may
have a direct effect on mitochondrial metabolism. This
was tested by determining the oxidative activities of iso-
lated mitochondria v/ith various citrate cycle intermediates
in the presence and absence of JH.

Inhibition of pyruvate-malate, pyruvate-glutamate,
malate, a-ketoglutarate , and glutamate oxidations occurred
in mitochondria isolated from 6 to 8 mg larvae treated in
vitro with 1.77 x 10~ M JH (Table 4) . Ascorbate and a-
glycerophosphate oxidations were not affected, while

52

Table 4

TOffect of Juvenile Hormone on the Rates of
Substrate Oxidation by Isolated Mitochondria
from Plodia interpunctella

Q

02(N)**

Substrate

Additions
None JH'

Pyruvate -Ma late

Pyruvate -Glutamate

Ma late

a-Ketoglutarate

Glutamate

Succinate

a- Glycerophosphate

Ascorbate

335.6
303.2

55.7
100.0

64.1
121.4
314.4

69.9

11.5

20.6

7.6

23.2

10.2

160.8

295.7

72.0

~4
*JH concentration during incubation = 1.77 x 10 M
**Units of Q„ /„> are y atoms oxygen consumed/hour/mg mito-

chondrial nitrogen.

53

succinate oxidation was stimulated. It was noted that only
those substrates requiring NAD as a cof actor for oxidation
were inhibited. These results indicated that the inhibi-
tion probably occurred in the NADH dehydrogenase complex,
complex I, of the mitochondrial electron transport chain.

NADH was used as a substrate to localize the site
of JH action in the electron transport chain. Fresh mito-
chondria oxidized NADH very slowly due to low permeability
of the mitochondrial membrane to NADH. The mitochondrial
membrane was rendered permeable to NADH by aging the mito-
chondria at 30°C. Mitochondria aged for 45 minutes and
then placed on ice gave almost maximal NADH dehydrogenase
activity which was realtively stable for 2.0 hours (Figure
5) . Mitochondria aged less than 45 minutes did not reach
maximal oxidative activity. Those aged more than 45 minutes
were unstable with a marked decrease in oxidative activity
occurring between 100 and 12 0 minutes of aging.

Aged mitochondria were less susceptible than fresh
mitochondria to inhibition by oligomycin, an inhibitor of
oxidative phosphorylation (2 4.1% versus 71.0%), indicating
that aging uncoupled oxidative phosphorylation (Table 5) .
Since JH v;as effective inhibiting NADH oxidation in both
fresh and aged mitochondria then it must be acting in the
electron transport chain.

Inhibition of pyruvate-malate oxidation in fresh
mitochondria and inhibition of NADH oxidation in aged

1

(tJ

0)

O H

to

+J

(I)

frt

-H

+J

C

S

U

Q)

c

tp^

3

O

(1)

Ci

U

4J

M

ri

(rt

fl)

>irH

-»-)

Si

0

a

0)

w

■H

^

•H

m

K ^

-H

P

O Ti

<;

o

^

tP>H

a

CM

c

•rH

(1)

trm-i

CI)

m

O

^

4-1

m

fl)

G)

()

ra

rQ

>

fl)

M

ae

fd

■H

■H

rH

^

4->

CQ

b

C Tl

C)

0

C

Sh

-rH

(0 M-i

-p

res

>i

05

■H

-P

-H

0)

•H

>H

M

>T1

-H

c

fl)

+J

C)

^

O ^

H

(IS

u

U

Cn
■H

P4

o < <5

Z UJ kiJ '

O O O iflZ

u < < * o

f
f.

JT .^t'

Cf

>

Cf

*^

"^/.

(jnoM/ M Sui/^ suiojeh) AIIAIIDV aSWN330daAH3a

HQVN

56

Table 5. Effect of Inhibitors and Aging on NADH Oxidation
l)y Mitochondria from Plodia interpunctella

Mitochondrial Qq (v[^i./

Aging Time (min) Inhibitor 2

0 None 107.2

30 None 288.0

45 None 414.5

60 None 448.6

0 Oligomycin— 31.1

45 Oligomycin 315.0

0 JH-/ 55.9

45 JH 120.7

45 Oligomycin + JH 43.3

45 Rotenone -'^ 21.5

—Oligomycin concentration during incubation = 4.10 x 10 M

2/ -4

— JH concentration during incubation = 1.77 x 10 M

3/ -8

—Rotenone concentration during incubation = 6.25 x 10 M

4^/Units of Q„ , , are yatoms oxygen consumed/hour/mg
mitochondrial nitrogen.

57

mitochondria were related to the concentration of JH in
the sarae wfiy (Figure 6) . Minimal inhibition was detected

by a JH concentration of approximately 3 x 10 M. Maxi-

-4
mal inhibition v/as reached by 1.41 x 10 M JH . A plot of

NADH concentration divided by reaction rate versus sub-
strate concentration at JH concentrations of 0, 7.1 x

-5 -4 -4

10 M, 1.06 X 10 M and 1.77 x 10 M (Figure 7) revealed

that NADH oxidation was noncompetitively inhibited (K. =

0.10 6 9 mM) .

These results indicated that the inhibition defi-
nitely did occur in the electron transport chain. Since
electrons from succinate and a-glycerophosphate oxidations
enter the electron transport chain at coenzyme Q and since
these oxidations v/ere not inhibited by JH, then the inhi-
bition must occur between NADH and coenzyme Q in the elec-
tron transport chain.

Sodium ferricyanide was used as an electron accep-
tor from NADH dehydrogenase to assay the activity of NADH

-3

dehydrogenase. The rate of reduction of Fe (CN) , by aged

mitochondria was not significantly altered by the presence

of 1.77 X 10~ M JH. When JH was present at a rate of 742.6

-3
ymoles Fe (CN) , /hour per mg mitochondrxal nxtrogen was

obtained compared to 762.8 ymoles/hour "per mg mitochondrial
nitrogen when JH was absent. These rates accounted for
92% of the oxygen uptake observed in polarographic experi-
ments and indicated that juvenile hormone did not inhibit

Figure 6. The relationship of juvenile hormone con-
centration to the percent inhibition of
NADH and pyruvate-malate oxidations.

100

59

Pyr-Mal

*. 50

c

0)
w

km

4)

Q.

0

0.0

NADH

0.1
JH Concentration (mM)

0.2

1

c;

o

Q)

0

D ^ 4J

■rH

tH

c

4J

ffi

(U

u

P

-p

(0

fl^

•

m

Q)

2

'aj

0

u

<U

■p

u

Q)

^

m

x;

■P

u

S-l
0

+j

w

c

-P M-l

■^^

o

-H

0

■H X!

,-^

-P

-r1

>l

10

o x;

-p

~-'

tti

c

■H

^ —

0)

■H

O
0

,-^

K

.H

w

0)

^^

0)

^

w>

-P

^

w

g

3

w

3

m

■rH

-p

i^

s^

d

■H

QJ

>

rO

X

c

>

+J

fC

&

'd

w

g

o

>

a

C

x\

\

(C

0

Q)

U)

o x:

CO

c

■P

Q)

o

CD

M

-H

-P n3

rO

m

-p

m

C

0

(d

M

TCi

^-^

M

+J

X

-p

-P

to

^■^

(3

0

C X!

-rH

e

rH

(U

^ « >

^

u

CO

~

~-'

Q)

tn

-H

61

62

at the love 1 of NADH dehydrogenase. This experiment local-
ized the inhibition by JH to the nonheme iron protein re-
gion of the electron transport chain (Figure 1) . The
mechanism of this inhibition was not investigated.

Cytochrome Analyses

Mitochondria isolated from larvae treated in vivo
with JH were red to purple, while mitochondria from con-
trol insects were tan. Such coloration may be the result
of differences in cytochrome content. Therefore, I in-
vestigated the cytochrome content of mitochondria from
control and JH-fed larvae.

Newly molted 5th-instar larvae (0-day) had a mito-
chondrial cytochrome content of approximately 88.7 pmoles/
insect. The cytochrome content increased to 158.2 pmoles/
insect in +4-day larvae. When 0-day larvae were fed JH
for 4 days the m.itochondrial cytochrome content increased
to approximately 418.6 pmoles/insect. Preliminary experi-
ments indicated that the amount of stimulated cytochrome
synthesis was identical with either 0.1 mg/g or 0.5 mg/g
JH diet so all subsequent tests utilized the 0.1 mg/g
diet. Mitochondrial cytochrome concentration (nmoles/g
tissue) was higher in 0-day insects than in older larvae.

These observations conclusively demonstrated that
JH participated in the stimulation of cytochrome synthesis.

63

but did iiol: oliminate the possibility that some other
event associated with the molt v/as also necessary. 0-,
-2- and -1-2-day larvae were placed on Jll-treated diet,
removed at one day intervals and assayed for mitochondrial
cytochrome. The mitochondrial cytochrome content (pmoles/
insect) of both the control and JH-fed larvae increased
throughout the test (Figure 8) . However, juvenile hormone
treatment did not stimulate cytochrome synthesis until
after the last larval-larval molt. At +l-day the JH-fed
larvae had a significantly higher cytochrome content than
the controls. The mitochondrial cytochrome contents for
larvae which were placed on JH diet at -2- days were iden-
tical to those for larvae which were placed on JH diet
at 0-days. Compared with comparably aged control insects,
the tvro JH-fed larval groups showed approximately a 40%
increase over controls in mitochondrial cytochrome content
(pmoles/insect) by +l-days, a 113% increase by +2-days,
a 200% increase by +3-days with no additional increase
]3y +4-days. The mitochondrial cytochrome content of the
larvae which were placed on JH diet at -f2-days did not
differ significantly from the controls. These results
indicated that JH stimulated cytochrome synthesis, but
the stimulation was dependent upon some event associated
with the molt. Figure 9 shows a comparison of the changes
in mitochondrial cytochrome contents (pmoles/insect) from
-2- to -t-4-days of larvae fed JH starting at -2-days and
of control larvae. Cytochrome synthesis in control larvae

+)

rt

0)

0

fr4J

nj

•H

T)

c

(U

Iti

^

Q)

tn 0

•

fO

-p

rH

-p

fl)

fO

a

+j

>

QJ

a

U

h

()

(d

+J

c;

rH

tri

0)

0)

m

M

R

O

+J

o
u

OiE ^

-rH

1-1

o

-C

o

U)

in

-p

C

O

>1

C)

C)

■H

SH

+J

C)

rH

(0

-H

rri

iH

+J

•H

(1)

frt

U

V^

-H

TJ

A->

a

(U

•rH

()

x:

d x^

H

•H

U

■H

65

: o V

/

/'

./'

*

0

<

<

c<

in

6

(pdSUf SdjOUJU)

INaiNOD IWOUHDOIAD

66

procoedoil from -2- to +4-days with most of the synthesis
occurrinq bc^fore +l-day. The mitochondrial cytochrome con-
tent of Jll-fed larvae also increased throughout the test
period at a rate of 6 times faster after the molt. This
resulted in a total cytochrome content per insect 3 times
greater in JH-fed as control insects by +4-days.

Figure 9 shows cytochrome concentrations as nmoles/g
tissue. Cytochrome concentration in control larvae was
highest in -2-day larvae, decreased in -1-day larvae, in-
creased in 0-day larvae and then decreased to the end of
the experiment (+4-days) . Again there was no difference
in cytochrome content of JH-fed larvae and control larvae
until after the last larval-larval molt. After the molt
cytochrome concentrations of the controls dropped sharply
while that of the JH-fed larvae remained high and dropped
more slowly. Approximately 30% of the larvae fed JH diet
had undergone a supernumerary molt by the seventh day on
the diet (treatment began with 0-day larvae) . The larvae
which molted an additional time had a higher mitochondrial
cytochrome concentration than non-molting JH-fed larvae
(Table 6) .

Cytochrome a, b and c concentrations (pmoles/
insect) of JH-treated and control larvae are shown in
Figure 9. In mitochondria from control larvae cytochrome
a + a-, concentration increased 10-fold, cytochrome b

(0

(I)

g

1

O X

U I-)

Xi

o

u

o

0

+J IH

>1

o

-p

0

o

0)

4J

CO

(1)

•H

tn

01

>H

•

a)

in

rH

a.-p

rfl

CJ

>

c

Q)

Vh

C)

tn

n1

•H

c

iH

-P

fd

-H

m

M

rH

o

■P

o

C

M

04 cu

-p

-H

C)

c

^

C

O

to

()

O

c

C)

C)

Tl

•H

C)

C

4J

to

m T3

.H

a T)

0)

fO

(I)

U

-p

X!

ta

0)

0)

;c

-

M

H

(0

■p

0

•H

68

f-m-irmmm!

fr

w u

e o

u u

Hnn

^^r-^

liiitltltililililili^B^JiiHH

- «

(pasuj/saioiud) NOIiVMlNaONOO 3W0)IH301A3

69

'd

T3

rH 0

a

0 W

A->

1 0

fd

>ix;

G)

0)

fO +J

::5

S-l

T!

w

+j

1 ^

tn

1

r^ 4J

-H

m

•iH

4-1

1-3

in S:

O

Cn

+J

\

W M

CO

C 0

o

0 s

rH

■H

0

rH

•+J >i

g

'^

(0 M

c

u

M fd

4->

+J u

c

c o

o

0) g

u

O 3

c c

o u

u o

a

(U ID

g w

o

"XJ

S-l (C

CD

^

4J

U 0)

ro

0 C nci

Q)

4-1 0

<U

M

>i Cn+J

4J

4-1

U M

,H

U

1

(U

0

0

n^

H no

g

w

Id

n3 C

c

•H 3

4->

•r^

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o

\

'O T3

c

U3

C fd

QJ

ox:^

rH

^

fd

0

u ^ x;

g

0 o

a,

r-4

4-1 -H ^ 1

0

■H ^

u

M

S 5

H

+J

x;

r,

U-) 0)

^

0

O (3

u

>

Q)

C M

rO

0 Hi

>

in ^q

U

-iH

rd

U Ti

^q

ta Q)

Ch^

g

B 1

0

0 K

U

U t^ M-l 1

0)

g

u

^4

rd

^

o

x:

CIJ

QJ

0)

()

g

g

g

4J

o

CJ

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M

h

54

u

x:

^

^

CJ

u

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C)

o

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d

4J

+J

4-)

4J

>i

>1

>t

O

u

u

u

H

70

concentration increased 4-fold and cytochrome c concentra-
tion increased 3-fold during the period from -2- to +4-
days. In Jll-treated larvae the corresponding increases
were 30-fold for cytochrome a + a^ , 11-fold for cytochrome
b and 4- fold for cytochrome c.

Cytochromes a + a^, b and c concentrations (nmoles/
g tissue) are shown in Figure 10. The concentration of
the individual cytochromes followed the same general trend
as noted for total cytochrome content. There was no dif-
ference between control and JH-fed insects in cytochromes
a + a^ and c concentrations prior to the last larval-larval
molt but after the molt the concentrations of these cyto-
chromes decreased rapidly in control larvae while the con-
centration was maintained at a level 3-times higher in JH-
treated larvae. On the other hand, cytochrome c concen-
tration was essentially identical in control and JH-treated
larvae throughout the experiment. The concentrations of
cytochromes a + a-, b and c were higher in insects which
under\-/ent a supernumerary molt than in corresponding non-
molting larvae (Table 6) .

The ratios of cytochrome b to cytochromes a + a^
decreased throughout the experiment and were essentially
the same for control and JH-fed insects (Table 7) . The
ratio of cytochrome c to cytochromes a + a^ decreased
throughout the experiment in both control and JH-fed in-
sects but the decrease in the ratio was greater in JH-fed

w

(1)

!-l

g

0

n

IH

u

^

0)

C)

3

()

CO

+J

w

>i-H

U

-p

0

Fi

-p

,

(1)

tn w

in

+J

fi5

5-1

0

0)

(U

.H

Qa W

fO

a

>

c

•H

J-i

0

trt

•H

H

rH

-P

0

n3

M

IH

!-<

-P

o

+J

d

C

0

a <u

u

•H

U

4::

f:; Ti

CO

0

c

a

0

(fl

0

-rH

U T)

m ^3 -P

0) (C 0)

M

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j:^

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CI)

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4::

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72

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73

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td

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tn

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0

u

5-1

jC

C)

U U-<

o

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>i o

C)

•H

■P

m

lu

o

M

+J

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C 0)

O

Q) (l!

-rH

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4J

C ^1

a

o fd

Pi U yA

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c;

(Tl

>

M

trt

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0)

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-P

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(d

fi)

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(d

■P

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in o 00 r- ■<3* ^

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r- in in -^ -=r

+ +

74

insects. The greater decrease in this ratio may be at-
tributed to loss of cytochrome c from the mitochondria
during isolation because cytochrome c is not bound to
the mitochondrial membranes as tightly as the other cyto-
chromes .

Assay of Mitochondrial Hemes

Because the stimulation of cytochrome synthesis
v;as dependent on both JH and some factor associated with
the last larval-larval molt, it was decided to determine
whether the two controls were exerted at the same site or
at different sites in the cytochrome synthetic pathway.
Since cytochromes are composed of heme and apoprotein,
the effects of JH on the synthesis of just the heme por-
tion of the molecule was investigated.

The total mitochondrial heme concentration (pmoles/
insect) increased 4-fold from -2- to +4-days in control
larvae. It increased 14-fold in insects fed on a JH diet
(0.1 mg/g JH) . A difference in heme concentration between
controls and JH-treated larvae v/as observed within one day
after initiating JH treatment 2 days before the molt into
the fifth instar (Figure 11) . Heme content of JH-fed in-
sects increased 63% above the control value within the
first day of the test and had doubled the control value by
the time of the last larval-larval molt (0-day) . The

^-p

a u

(U

en CO

<D C

e-H

(!)

x; M

•

(1)

tfi

0 a-p

-p

C)

c

0)

(U o

w

Cn-H

c

a 4-1

•H

(IS

M M

r-\

a P

o

> c

M

>-: 0)

•P

(0 O

d

rH a

()

o

D

m u

0

Ti

u

C

a,

(li

■H ^

^ c

-■0

m fO

Ci)

C

-P

o -.

rd

•H (U

0)

■p e

U

f3 (U

-P

rH ^

1

CD 0

K

U -P

I-)

O

QJ S-i

S-l

^ &4 0

tH --

m

76

"'•^^^^L

yg^jT^rx

nm

I

(pasuj s3|oiud) N0liVUlN33N03 3M3H

77

increase approached 2 30% by the termination of the experi-
ment at +4-days. The concentration per insect increased
linearly after a short lag bewteen -2- and -1-days in both
control and JH-fed insects.

Heme concentrations per gram tissue for control
larvae decreased initially but rose to a peak just after
the molt (Figure 12) . It decreased to a low level in
+2-day larvae and remained stable to the conclusion of
the experiment. Heme concentrations of the JH-fed larvae
increased immediately reaching a peak in 0-day larvae and
then decreased to a level 2- fold the control concentration
until the termination of the experiment at +4-days.

Concentrations of hemes a, b and c per insect
are shown in Figure 11. In control insects heme a increased
2.5-fold, heme b increased 12-fold and heme c increased 2-
fold during the period from -2- to +4-days. In JH-fed lar-
vae the corresponding increases were 12.5-fold for heme a,
50-fold for henie b and 6-fold for heme c. Hemes b and c
were synthesized at a linear rate after a short lag be-
tween -2- and -1-days. However, the rate of heme a synthe-
sis was higher in younger larvae and decreased toward the
end of the experiment.

In control larvae the ratios of concentrations of
heme a to heme b to heme c at the beginning of the experi-
ment were 1.00:0.49:1.25 but by the end of the experiment
the ratios were 1.00:1.36:0.70. The same ratios at the

I

Xi en

-H

-+>

a

g

to rt
Q) U

to
tn4J
U

-H

d

O H
O

(0
>

4-)

(13 -P

M C

+J O

C O
<D

U "Xi

a c

O rj

m u

O 'O

U QJ

a, +J

•H Ti fd

^ C 0)

W (0 n

C -P
O -- I

-H QJ K

4-> e "^

rH ^ ^

(U o o

M 4-> M-l

o

CU M (D
H — CO

0)
-H

7 9

M 5

(»nss!» 8/sa|ouiu) N0I1V)I1N33N03 3IIII3H

80

end of the experiment in JH-fed larvae were 1.00:1.89:0.63
indicating a shift in heme synthesis toward heme b and away
from hemes a and c.

Assay of De Novo Synthesis of Hemes

I could not determine from the heme analyses whether
the increase in mitochondrial hemes was due to de novo syn-
thesis, to a sequestering of hemes by mitochondria or to
a partial synthesis from sequestered intermediates in heme
synthesis. Incorporation of a carbon-14 labeled glycine
was used to determine if de novo synthesis of hemes did oc-
cur.

14
Insects placed on a medium containing 2- C-glycine

(2.0 uCi/g diet) from -2- to +2-days incorporated the
■'"^C-label into hemes. During that period control larvae
incorporated 213 cpm/hr. /insect into 6.9 nmoles of hemes
a and b. The corresponding values for JH-fed larvae were
1,14 0 cpm/hr. /insect incorporated into 2 3.9 nmoles hemes
a and b per insect. Com.paring the rates of heme synthe-
sis indicates that JH-fed larvae accumulated 3.46-times
as much heme as control larvae during the period from -2-
to +2-days. During the same period, the incorporation of
radiolabeled glycine into hemes was 3.58-times greater in
JH-fed larvae than in the controls. The similarity of
these two ratios indicated that hemes a and b were

synthesized de novo in both control and JH-fed larvae and
that the sequestering of preformed hemes or heme precursors
did not play an important role in the increased concentra-
tion of mitochondrial heme.

DISCUSSION

The results presented in this dissertation estab-
lish that juvenile hormone (JH) acts directly on the mito-
chondria of Indian meal moth larvae. Juvenile hormone
affects citrate cycle oxidations, electron transport, heme
synthesis and cytochrome synthesis. Juvenile horm.one inhi-
bits electron transport by the nonheme iron protein in com-
plex I of the m.itochondria which results in lov/er oxidation
rates for citrate cycle interxnedxates requiring NAD as a
cofactor for oxidation. Minks (1967) found that relatively

high concentrations of a crude extract of Hyalophora cecro-

-3

pi a, a potent source of JH, (10 %) depressed pyruvate-

malate oxidation in mitochondria from adult Locusta migra-
toria. The effect was very weak and was attributed to
"toxic effects" at high concentrations of hormone. Firsten-
berg and Silhacek (unpublished observations) have shown that
inhibition by JH of oxidation of NAD-linked substrates in
isolated mitochondria did not change throughout the last
larval instar. Silhacek and Kohl (unpublished observations)
have also shown that the response to H. cecropia JH (JH-1,
Roller et al. , 1967) is greater than the response to the
other two known insect juvenile hormones (JH-II, Meyer

82

83

et al,. , 3 96B, and JH-III, Judy et al . , 1973) indicating
that the inhibition is probably physiological and not
pharmacological. It may be noted that several substances
with insecticidal properties, rotenone , amytal and pieri-
cidin A, also inhibit electron transport by the noneheme
iron protein (Hatefi, 1968; Horgan and Singer, 1968). How-
ever, none of the other effects of JH on isolated mitochon-
dria reported in this dissertation have been reported for
rotenone, amytal or piericidin A.

This dissertation also establishes that juvenile
hormone stimulates succinate oxidation in isolated mito-
chondria from P. interpunctella larvae. Clarke and Baldwin
(19 60) noted stimulated succinate oxidation with mitochon-
dria isolated from adult L. migratoria when incubated with
preparations of corpora allata. However, the JH stimula-
tion of succinate oxidation did not occur when the same
procedure was done with larvae of Schistocerca gregaria and,
in fact, a slight inhibition was noted. Their experiments
were unreplicated experiments and the effects were weak.
DeWilde and Stegwee (1958) demonstrated that removal of
the corpora allata from diapausing Leptinotarsa decem-
lineata adults resulted in reduced succinate dehydrogenase
activity when oxygen consumption was measured in tissue
homogenates. DeWilde (1959) subsequently demonstrated
that both corpora allata and extracts of Hyalophora cecro-
pia stimulated succinate oxidation in tissue homogenates

84

from diapiausing L. decemlineata. Stegwee (19 60) confirmed
results of c^^rlier studies by treating isolated thoracic
muscle mitochondria from L. decemlineata with H. cecropia
extract. Keeley (1970, 1972, 1973) and Keeley and
Wadill (1971) found that neither allatectomy nor corpora
allata extract affected succinate oxidation in isolated
mitochondria from adult Dlaberus discoidalis fat body.

Interpretations of previous studies are difficult
because the adult tissues studied may not be targets of
JH action, JH degradation may occur in crude tissue prep-
arations and the active principle in crude hormone prep-
arations may not be JH. Early studies on the effects of
corpora allata or juvenile hormone indicated that JH
stimulation of oxygen consumption in adult insects was
due to the stimulation of ovarian development. Since
that time investigators have placed their research empha-
sis on either male or ovariectomized female adult insect,
thereby avoiding tissues which are sensitive to juvenile
hormone. The overall result of this emphasis was that JH
was tested on mitochondria from tissues or insects in
which no function for JH is known. The exception to this
is the research on diapausing adult L. decemlineata in
which diapavise appears to be a result of deficiency in JH
secretion. Recent work by Silhacek and Kohl (unpublished
observations) indicates that stimulation of succinate de-
hydrogenase is dependent on larval age. Their experiments

demonstratod that the ability of JH to stimulate succinate
dehydrogenase is lost in older larvae. These results em-
phasize the need to study juvenile hormone effects in tis-
sues v;hich are responsive to JH .

The present study utilized a twice-washed mitochon-
drial preparation isolated from larval tissues which are re-
sponsive to juvenile hormone and a chemically defined juve-
nile hormone preparation. The titer of juvenile hormone in
P. interpunctella has not been determined and it is not
known whether the concentrations of JH used in this study
are physiological; however, the JH concentrations used are
similar to those determined in vivo in H. cecropia adults by
Meyer et al . (1965, 1968) and Bieber et al. (1972).

The results of in vivo JH treatment on mitochon-
drial enzyme activities presented in this dissertation are
inconclusive because the controls stopped feeding. How-
ever, the results do not disagree with the effects of in
vitro JH treatment of isolated mitochondria.

The evidence presented here shows that heme synthe-
sis, a topic having received little attention in insects,
is immediately and therefore presumably directly stimulated
by juvenile hormone. Since the occurrence of hemes is
general in nature a similar biosynthetic pathway is assumed.
Shemin and his coworkers (1952, 1953), Dresel and Falk
(1956a, 1956b, 1956c) , Granick and Mauzerall (1958) ,
Mauzerall and Granick (19 58) and Granick (1958) have

86

studied porphyrin and heme biosynthesis in duck and chick-
en erythrocytes. They found that the initial reaction in
heme biosynthesis is associated with citrate cycle enzymes
in mitochondria and involves an enzymatic condensation of
succinyl CoA and glycine to form 6-am.inolevulinic acid.
The next series of reactions involving cytosol enzymes
begins with the condensation of two molecules of 6-amino-
levulinic acid to form porphobilinogen which condenses to
form coproporphyrinogen, a cyclic tetrapyrrole . The third
enzyme group located in the mitochondria converts copropor-
phyrinogen to protoporphyrin. The final enzyme in heme
biosynthesis, ferrochelatase , is located on the inner sur-
face of the mitochondrial inner membrane and participates
in the insertion of iron into the protoporphyrin ring
(Jones and Jones, 1969). Hamdy et al. (1973) have demon-
strated incorporation of radiolabeled glycine and succinate
into hemes in a tick, Dermacentor andcrsoni , lending sup-
port to the occurrence of this pathway in arthropods. The

14
incorporation of C-glycine into hemes m the present

study indicates that the mechanism of heme biosynthesis

in P. interpunctella is similar to that found in duck and

chicken erythrocytes and in D. andersoni .

Two mechanisms are known for controlling the early

reactions of heme biosynthesis. Granick (1966) proposed

that induction of 6-aminolevulinic acid synthetase controls

heme synthesis in chickens by resulting in increased levels

of (S-aniinolovulinic acid which disturbs the steady-state
equilibrium to favor increased heme synthesis. Granick
(1966) demonstrated that 6-aminolevulinic acid synthetase
is inducible by several substances. Muthukrishnan et al .
(19 72) have shown that a mold, Neurospora crassa, the second en-
zyme in heme biosynthesis, 6-aminolevulinic acid dehydrase,
is inducable by iron. Its induction results in increased
heme biosynthesis. A further level of control in the heme
biosynthetic pathway involves end-product feedback inhibi-
tion. Muthukrishnan et al^. (1972) and Burnham and Las-
celles (1962) have demonstrated that 6-aminolevulinic acid
dehydrase is inhibited by coproporphyrinogen III. Burnham
and Lascelles (1962) , Scholnick et aJ. (1971) and V.'hiting
and Elliott (1972) demonstrated that 6-aminolevulinic acid
synthetase is inhibited by hemin.

In P. interpunctella the increase in heme synthe-
sis may be a result of the effects of JH on electron trans-
port and succinate dehydrogenase. As a result of my ex-
periments, I have proposed tliat inhibition of complex I
of electron transport could affect the citrate cycle and
related metabolism which would result in the production of
succinyl-CoA by a conversion of pyruvate to malatc and a
reversal of the citrate cycle (Silhacek, Firstenberg and
Kohl, in press). Malic enzyme, the enzyme v/hich catalyzes
the conversion of pyruvate to malate is active during the
early part of the last larval instar in P. interpunctella

(Silliacek, unpublished data) v/hen juvenile hormone titer
is thought to be high. Another explanation is that JH
could induce either 6-aminolevulinic acid synthetase or
dehydrase resulting in increased heme synthesis. One or
both of these mechanisms may contribute to the stimulation
of heme synthesis in P. interpunctella larvae. High con-
centrations of the end-product, hematin, could exert a
feedback inhibition on succinate dehydrogenase (Keilin
and Hartree, 1947) and/or 6-aminolevulinic acid synthe-
tase (Burnheim and Lascelles, 1963) .

Another possible function of the juvenile hormone
inhibition in electron transport is to provide a reducing

environment for conversion of ferric (Fe ) to ferrous ion

+2

(Fe ) by increasing intramitochondrial concentrations of

NADH. Barnes et al. (19 72) confirmed that the conversion

+ 3 +2
of Fe to Fe is facilitated by NADH. Porra and Jones

(1963) earlier determined that the enzyme, ferrochelatase,
which participates in the insertion of iron into porphyrins,
utilizes iron only in the ferrous form.

The synthesis of cytochromes depends on the coor-
dination of heme and cytochrome apoprotein syntheses. My
results indicate that cytochrome apoprotein synthesis is
stimulated by JH. This stimulation depends upon an event
associated v;ith larval molting. Therefore, the stimulation
of cytochrome apoprotein synthesis may be due in part to
ecdysone. Indeed, Patel and Madhavan (1969) have

89

demonstraLed a general stimulation of protein synthesis
associated with ecdysone titer in imaginal wing disks of
Samia cynbhia ricini. The same study also indicated that
JH had a stimulatory effect on protein synthesis. How-
ever, ecdysone is not the only factor that may be involved.
Recently, Keeley and Wadill (1971) found a corpora cardiaca
factor which stimulates cytochrome oxidase activity. Se-
cretion of this corpora cardiaca factor could also be the
event associated with the molt which stimulates cytochrome
synthesis .

Soslau et al. (1971) found that cytochrome c syn-
thesis was stimulated in developing adult Antheraea poly-
phcmus following injection of pupae 24 hours earlier with
6 -aminolevulinic acid. It was also found that actinomycin
D an inhibitor of RNA synthesis, had no effect on the stim-
ulation of cytochrome c synthesis indicating formation of
the m.essenger RNA for cytochrome c apoprotein prior to the
administration of the inhibitor.

An interesting correlation occurs in the case of
diapause. DeWilde (1959) demonstrated that diapause was
a result of JH deficiency in L. decemlineata. If diapause
v/ere caused by the lack of JH , one would expect diapausing
insects to have low cytochrome concentrations. This is
exactly what was found in induced diapausing Antheraea
pernyi by Shappirio (1965) who also found that non-dia-
pausing insects had little decrease in cytochrome content.

90

The current information (Kiese et^ al. , 1958;
Sinclair e_t al., 1967) indicates that hemes a and c are
the product of a conversion from heme b. The data pre-
sented in this dissertation indicates that this mechanism
is probably operative in P. interpunctella since the ratios
of the individual hemes follow the same pattern in JH-fed
larvae as in control insects. This result would be ex-
pected if heme b were the precursor of the other two hemes
and the enzymes controlling the conversion were unaffected
by JH.

It must be emphasized that my experiments do not
preclude a direct interaction of juvenile hormone with the
genome. However, the results do indicate that JH can af-
fect development under certain conditions by altering meta-
bolic reactions v/ithout interacting directly with the
genome .

My results also indicate that the titer of juvenile
hormone at the beginning of an instar can affect the metab-
olism of the insect within the same instar. This is con-
trary to the classical view which states that JH titer
within an instar will control the morphology (Wigglesworth,
1940; Clever, 1963) and metabolism (Kroeger, 1968) of the
insect in the following instar.

Since cytochrome levels are regulated by JH titer
and cytochrome concentrations could be rate limiting in
energy (ATP) production, juvenile hormone can control

91

cellular enetrgy levels. It is therefore possible that
JH, through the mechanisms presented in this dissertation,
determines the levels of metabolic processes in develop-
ing insect tissues.

These experiments provide a basis for a hypothe-
sis of metabolic control of insect development. I hope
that the information provided can be useful in designing
further experiments to elucidate the metabolic mechanisms
of insect hormones during growth and development.

CONCLUSIONS

It can be concluded from the results presented in
this dissertation that juvenile hormone directly inhibits
the NADH dehydrogenase complex of the electron transport
chain and stimulates succinate dehydrogenase activity in
mitochondria from larvae of Plodia interpunctella . Juve-
nile horm.one also stimulates the de novo synthesis of
hemes. However, the incorporation of hem.e into cytochrome
requires the intervention of the molt, suggesting the par-
ticipation oJ ecdysone in cytochrome apoprotein synthesis
as a second level of control.

The increase in heme synthesis may be a result of
the inhibition of NAD-linked oxidation and stimulation of
succinate dehydrogenase activity. The inhibition of NAD-
linked oxidatir: "J could result in the production of
succinyl-CoA from m.:;late. The malate is generated by con-
verting pyruvate to malate in the cytosol and reversing
the citrate cycle (Silhacek, Firstenberg and Kohl, in
press) . An increase in succinyl-CoA pool size could re-
sult in an increase in heme synthesis by altering the
steady state equilibrium of the reactions in the heme bio-
synthetic pathway to favor the formation of hema^s. In

92

93

addition, the inhibition of NADH oxidation in electron
transport could provide an intramitochondrial reducing
environment favorable for the insertion of iron into por-
phyrin to form heme.

Juvenile hormone could exert its metabolic effects
without an interaction with the genome. However, it is
possible that cytochrome synthesis is mediated by ecdysone
interacting with the genome. Juvenile horm.one through its
effects on mitochondrial metabolism could regulate the in-
tensity of metabolism by controlling maximum levels of
intracellular cytochromes. Therefore, I speculate that
the main function of juvenile hormone is to establish the
upper limit for mitochondrial oxidations in developing
insect tissues.

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BIOGRAPHICAL SKETCH

Donald Elliott Firstenberg was born September 13,
1946 at New Brunswick, New Jersey. He is married to the
former Iletxe Lois Sager. They have one child, Michael
Harrison Firstenberg.

Donald Elliott Firstenberg graduated from Scotch
Plains-Fanwood High School in June, 1964. In May, 1968,
he received the degree of Bachelor of Science with a major
in biochemistry from Rutgers University. In September,
1968, he enrolled in the Graduate School of the University
of Florida. From January, 1969, until December, 1974, he
worked as a graduate assistant in the Department of Ento-
mology and Nematology while pursuing his work toward the
degrees of Master of Science and Doctor of Philosophy.
In August, 1971, he received the degree of Master of
Science.

Donald Elliot Firstenberg is a member of the Ento-
mological Society of America, The American Chemical Society
and Phi Sigma National Biological Honorary Society.

109

I certify that I have read this study and that
in my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor
of Philosophy.

Donald L. Silhacek, Chairman
Assistant Professor, USDA
Insect Attractants, Behavior, and
Basic Biology Research Laboratory

I certify that I have read this study and that
in my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertat:ion for the degree of Doctor
of Philosophy. J

Jewries L. Nation, Co-<?hairman
Professor of Entomolcogy

I certify that I have read this study and that
in my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor
of Philosophy.

Harvey L. Cromroy
Professor of Entomology

I certify that I have read this study and that
in my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor
of Philosophy.

d,L/i ('^L^A.^.C

Herbert Oberlander
Associate Professor, USDA
Insect Attractants, Behavior, and
Basic Biology Research Laboratory

I certify that I have read this study and that
in my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor
of Philosophy.

p^ ^C>^-^-^-^

4

De

CUl^

avid S . Anthony
Professor of Botany

This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate Council,
and was accepted as partial fulfillment of the require-
ments for the degree of Doctor of Philosophy.

March, 1975

Agriculture

Dean, Graduate School

UNIVERSITY OF FLORIDA

3 1262 08553 0565