Group Title: effects of juvenile hormone on mitrochondrial sic metabolism in the Indian meal moth, Plodia interpunctella (Hubner) /
Title: The Effects of juvenile hormone on mitrochondrial sic metabolism in the Indian meal moth, Plodia interpunctella (Hubner)
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Title: The Effects of juvenile hormone on mitrochondrial sic metabolism in the Indian meal moth, Plodia interpunctella (Hubner)
Physical Description: x, 109 leaves : ill. ; 28 cm.
Language: English
Creator: Firstenberg, Donald Elliott, 1946-
Publication Date: 1975
Copyright Date: 1975
 Subjects
Subject: Moths   ( lcsh )
Insects -- Physiology   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1975.
Bibliography: Includes bibliographical references (leaves 94-108).
Statement of Responsibility: by Donald Elliott Firstenburg.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098671
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000425655
oclc - 38046388
notis - ACH4201

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THE EFFECTS OF JUVENILE HORMONE
ON MITOCHONDRIAL 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 Oberlander; Dr. Harvey L. Comroy; 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 Committee 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

who aided in developing and molding the author's research

philosophy.

The author also wishes 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 Attractants, 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 ......................................

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










Page


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


1 A Comparison of Mitochondria in Midguts
of Control and JH-Fed Larvae 46

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

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

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

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

6 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

7 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


1 The sequence of reactions in the elec-
tron transport chain (Hansford and
Sacktor, 1971). 19

2 The sequence of reactions in the citrate
cycle showing the points of origin of
NADH and succinate dehydrogenase (SDH)
(Lehninger, 1965). 22

3 Electron micrograph of midgut tissue
from control larvae (magnification,
15,000 X). 43

4 Electron micrograph of midgut tissue
from JH-fed larvae (magnification,
15,000 X). 45

5 The relationship between NADH dehydro-
genase activity and time of aging of
isolated mitochondria from larvae of
Plodia interpunctella. 55

6 The relationship of juvenile hormone
concentration to the percent inhibition
of NADH and pyruvate-malate oxidations. 59

7 Plot of (S)/V versus (S) ((S) is the
NADH concentration and V is the reaction
rate). The substrate constant (Ks), in-
hibitor constant (Ki) and the maximum
velocity of the reaction (V ) are
shown. max 61

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

9 The relationship of larval age to cyto-
chromes a, b and c concentration per
insect for JH-treated and control in-
sects. 68










Figure Page


10 The relationship of larval age to
cytochromes a, b and c concentration
per gram tissue for JH-treated and 72
control insects.

11 The relationship of larval age to
hemes a, b (protoheme) and c concen-
tration per insect for JH-treated and 76
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 HORMONE
ON MITOCHONDRIAL 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-dimethyl-trans 2, trans 6-tridecadienoate) on mito-

chondrial metabolism in larvae of the Indian Meal moth,

Plodia interpunctella (Hubner) 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 flavoprotein-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


viii











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

malate 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 within 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 growth 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; Watanabe 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 which produce juvenile

hormone (JH), exerted a direct effect on the respiration of










Leptinotarsa decemlineata. A subsequent study (Stegwee,

1960) revealed that a JH-active extract from Hyalophora

cecropia 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, 1971) 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 mitochondrialactivities 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

information on the role of mitochondrial activitiesand 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 mechanism 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 molting but stimulates the prothoracic (ecdysial) glands

to secrete ecdysone (Fukuda, 1944). Ecdysone, which ini-

tiates the molting process, is secreted shortly before each

molt and disappears shortly afterward (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 (1964) 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 (1963a) 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 JIH 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 (Hemimetabola)

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 Wiggles-

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 (1956) and a purified










preparation of juvenile hormone was 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; Rbller et al., 1965, 1969).

The amount of JI per abdomen in the adult male

H. cecropia moth has been determined to be between 6.0 pg

(Metzler et al., 1971) and 0.5 pg (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 JH was also found to have the greatest effect of the

eight possible isomers of juvenile hormone on several

species (Rbller and Dahm, 1968; Rose et al., 1968; Wester-

mann et al., 1969; Wigglesworth, 1969; Pfiffner, 1971;

Schweiter-Peyer, 1973). Several synthetic schemes have

been developed for the production of juvenile hormone

(R1ller and Dahm, 1968b; Berkoff, 1969; Findlay and Mac-

Kay, 1969).

Two other naturally occurring juvenile hormones

have been found. A second JH, methyl-10, 11-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 al. (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 I.

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 tcrpenoids (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 was 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;











Karlson, 1963; Schneiderman and Gilbert, 1964; Braun et

al., 1968; Schwarz et al., 1969; Sonnet et al., 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 analogs 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 (Pflugfelder, 1937; Bodenstein, 1943;

Srivastava and Gilbert, 1969). Other physiological effects

attributed to JH include enhancing the regenerative abili-

ties in Carausius morosus (Pflugfelder, 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










(Ichikawa and Nishiitsutsuji-Iwo, 1959; Willians, 1959;

Gilbert, 1964; Highnam, 1964; Wigglesworth, 1964; Postle-

thwait and Weiser, 1973; Sroka and Gilbert, 1972; 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 Slama, 1968). Juvenile hormone also stimulates

sex pheromone production in Ips confusus (Borden et al.,

1969) and Tenebrio molitor (Menon, 1970).

Adult diapause in Leptinotarsa decemlineata appears

to be due to a deficiency in JH (DeWilde and Stegwee, 1958;

DeWilde, 1959). However, larval diapause in Diatraea

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

(1966), and Slama et al. (1974).










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 forthe 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 (1966) 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 metabolic 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.










(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'Ilelias (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 (Minks, 1967). 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,











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 (Ajami and Riddiford,

1971; Siddall, et al., 1971; Slade and Zibitt, 1971a, 1971b;

White, 1972). Another protein which binds and transports

juvenile hormone (Trautmann, 1972; Whitmore and Gilbert,

1972; Emmerich and Hartmann, 1973) apparently serves 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'Ilelias, 1955, 1964) and Sehnal and

Slama (1966) 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











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. (1974) 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 Jll have been made by Novak (1966), Wyatt (1968),

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











the basal rate in actively flying insects (Davis and

Fraenkel, 1940). In the flight muscles of Phormia re-

gina there are approximately 1.1 x 10 mitochondria per

mg tissue which comprise 40% of the total muscle mass

(Levenbook and Williams, 1956). Lehninger (1970) has cal-

culated that active flight muscle mitochondria may have

10-times 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 NADH: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), flavoprotein-linked a-glycerophosphate dehydro-

genase (Zebe and McShan, 1957; Reed and Sacktor, 1971),

succinic dehydrogenase (Greville et al., 1965), proline

dehydrogenase (Brosomer and Veerabhadrappa, 1965; Sacktor

and Childress, 1967), trehalase (Reed and Sacktor, 1971),

--keto acid dehydrogenases and the enzymes of the elec-

tron transport chain (Hansford and Sacktor, 1971).











Enzymes found as soluble matrix proteins include citrate

synthetase (Beenakkers et al., 1967), NAD and NAD)-

linked isocitrate dehydrogenases (Goebell and Klingenberg,

1963, 1972), malate dehydrogenase (Delbruck et al., 1959;

Reed and Sacktor, 1971), alanine and aspartate aminotrans-

ferases (Brosomer et al., 1963), 3-hydroxylacyl-CoA de-

hydrogenase (Beenakkers et al., 1967) and palmitoyl and

carnitine acetyltransferase (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 (HIansford and Sacktor, 1971).












SUCCINATE NADH

I I
SUCCINATE NADH
DEHYDROGENASE DEHYDROGENASE
I 1
NONHEME IRON NONHEME IRON

COENZYME Q
1
CYTOCHROME b

CYTOCHROME c

CYTOCHROME c

CYTOCHROMES a + a3

02 H20










Klincenberg, 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-B-hydroxylacyl-CoA to L-B-ketoacyl-CoA in the

8-oxidation of fatty acids and in several oxidative reac-

tions of the 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




CITRATE



OXALACETATE cis-ACONITATE


-----NADH NADH



MALATE ISOCITRATE



NADH CO2


FUMARATE a-KETOGLUTARATE


SSDH (FADH) / CO2


SUCCINATE SUCCINYL-CoA










L. migratoria (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

(1953) found that within the muscle the cytochromes were

restricted to the mitochondria. Individual cytochromes

ranged from 0.5 to 1.5 pmoles/g protein in flight muscle











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.
14
(1974) found incorporation of 1C-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).











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; Slama 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, 1958; Sagesser, 1960;

Novak and Slama, 1962; Slama, 1964). This effect on fe-

males has been attributed to the ability of JH to stimulate

development 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. mellonella 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











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 (1959) 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 cecropiaa

oil" was added to the mitochondria. At concentrations

approaching 10-5 (v/v) and above pyruvate-malate oxidation

was inhibited and P/O ratio decreased (Minks, 1967).

Keeley (1970, 1971) also found hormonal control of insect

mitochondrial oxidative activity, but the control was

exerted by the corpora cardiac and no effect was noted











after allatectomy in adult Blaberus discoidalis. He found

that cardiatectomy resulted in reduced oxygen consumption

and lowered cytochrome c reductase and cytochrome oxidase

activities. However, the addition of corpora cardiac to

isolated mitochondria had no effect (Keeley, 1971; Keeley

and Wadill, 1971). Treatment of cardiatectomized insects

in vivo with an extract of the corpora cardiac for several

days resulted in restoring oxidative enzyme activities

(Keeley and Wadill, 1971). The effects of cardiatectomy

were duplicated by severing the nerves from the brain to

the corpora cardiac. The factor appears to be a poly-

peptide and appears to be produced in the brain and released

by the corpora cardiac (Keeley and Wadill, 1971). Keeley

(1972) found that the corpora cardiac 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 growt-hand 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-meal moth,

Plodia interpunctella, were reared by the standardized

method of Silhacek and Miller (1972) in a 2.4 m x 2.4 m x

2.4 m walk-in incubator (American Instrument Company,

Silver Springs, Md.) maintained at 300 C + 1/20C and 70%

R.H. + 1%. A photoperiod consisting of sixteen hours

light and eight hours dark was supplied by four Westing-

house high output daylight lamps (Westinghouse, 96t12)

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











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). Medium components were refrigerated at 4C 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 unHil 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

700 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











honey:glycerol (1:1 v/v) were then thoroughly mixed into

each 700-mg 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)

were 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

with 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-four-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











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 HC1 to give a pHl of 7.4 (Silhacek, 1967).











Mitochondria are defined as that fraction which

sedimented between 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 medium consisting of

0.45M mannitol, 10 MMgSO4, 6 x 10 M ATP (pH 7.4 with

TEA) and 5 mg/ml bovine serum albumin (BSA). All pro-

cedures during isolation were carried out at 0-40C.



Polarographic Enzyme Activity Determinations


Mitochondrial oxidative activities were determined

using an oxygraph (Model KM, Gilson Medical Electronics

Inc., Middleton, Wisc.) equipped with a vibrating platinum

electrode and a jacketed cell maintained at 30C to deter-

mine oxygen uptake. The basic incubation medium contained

600 pmoles recrystallized mannitol, 15 pmoles MgSO4, 6

moles 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 pg Cecropia JH (a mix-

ture of isomers of methyl-10, ll-epoxy-7-ethyl-3, 11-di-

methyl-2, 6-tridecadienoate/ml). The BSA-JH was prepared

by mixing 25 pl of JH solution (10 mg JH/ml of acetone)

per 25 mg of dry BSA, drying under nitrogen and making up











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 moles 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 moles 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) were calculated by dividing the

rate of oxygen consumption after addition of P. by the
1

rate prior to the addition.
-3
One-tenth milliliter volumes of 10 M antimycin

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











aging the mitochondrial suspension at 30C 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

ml volumes of 10-3M antimycin A, 0.5M Pi, 5 x 10-2

K3Fe(CN) 3 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 30C was 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, H2SO4:H20) was added to each

tube. Tubes were covered with parafilm and stored at











-18C 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 11202 were

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 pg/ml

NH4NO3 solution, respectively).


Assay of Mitochondrial Cytochromes


Mitochondrial cytochrome concentrations were de-

termined using a variation of the Chance and Williams

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











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 l1 10-1M

KFe(CN)3 to the reference cuvette to fully oxidize the
K3Fe(CN)6
sample and 10 p1 0.05 M Na2S204 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-IIC (2.5 ml 36% HC1 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-HClextractions were

flash evaporated to near dryness in a rotary vacuum evap-

orator at 0C and alkaline pyridine was added. All pro-

cedures during the isolation of hemes were carried out

at 0-4C 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, 1963).

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.










Assay of De Novo Heme Synthesis


De novo synthesis of hemes was determined iso-

topically. Early-fourth-instar 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

pCi/g diet). The 2- C-glycine (Schwarz Mann Radiochem-

icals, Orangeburg, N.Y.) was added to the diet in acetone

solution. The acetonewas 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, Ill.). 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










(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 area, volume and general condition.
















REI]LTTTS


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 volume 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


































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















































































pr~



'Alp

































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


















































?IA,.S


_
rL
'Vc~iii~

~E~Z~ c.,












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


JH-Treated


Control


Mean volume (uWm3) 7.8 x 10-3 3.0 x 10-3

Mean surface area (im2) .203 .093

Surface area to volume ratio 23.66 34.96

Mean concentration in midgut
columnar epithelium cells
(mitochondria/Vm3)* .522 1.33

Per cent of mitochondria within
10 Vm of the midgut wall
(hemocoel) 48.4 62.1


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











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-
-4
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












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



Duration of in vivo Hormone Mean Mean
Hormone Treatment* Concentration Additions Q0 (* R.C.
Days MG/G


0 (Control) 0 None 335.6** 5.27
JH 11.5 0.74

4 (Control) 0 None 87.1 2.37
JH 3.3

4 0.1 None 171.0 3.92
JH 1.8

4 0.5 None 163.9 4.42
JH 0.8

7 0.1 None 104.0 3.91
JH 1.6 -

7 0.5 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 Q0 are atoms oxygen consumed/hour/mg
mitochondrial nitrogen.
mitochondrial nitrogen.











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
-4
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 104M) in

vitro. These results indicated that the mitochondria in

JH-treated larvae differ from the controls in their

capacity 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











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



Duration of Hormone Mean Mean
in vivo Concentration Additions
treatment* Q02 (N** R.C.
Days MG/G


0 (Control) 0 None 121.4** 1.23
JH 160.8 1.20

4 (Control) 0 None 94.1 1.39
JH 130.9 1.29

4 0.1 None 157.1 1.43
JH 137.4 1.12

4 0.5 None 136.6 1.62
JH 137.7 1.14

7 0.1 None 110.0 1.59
JH 119.9 1.08

7 0.5 None 61.6 1.36
JH 53.2 1.04


*Treatment began with 0-day
**Values represent duplicate
replicates
***Units of Q0 (N) are atoms

mitochondrial nitrogen.


larvae
runs on each of three

oxygen consumed/hour/mg










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 in 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 with 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
-4
vitro with 1.77 x 10 M JH (Table 4). Ascorbate and a-

glycerophosphate oxidations were not affected, while











Table 4. Effect of Juvenile Hormone on the Rates of
Substrate Oxidation by Isolated Mitochondria
from Plodia interpunctella


02(N)**

Additions
Substrate None JH*


Pyruvate-Malate 335.6 11.5
Pyruvate-Glutamate 303.2 20.6
Malate 55.7 7.6
a-Ketoglutarate 100.0 23.2
Glutamate 64.1 10.2
Succinate 121.4 160.8
a-Glycerophosphate 314.4 295.7
Ascorbate 69.9 72.0

-4
*JH concentration during incubation = 1.77 x 10 M
**Units of Q0 (N) are patoms oxygen consumed/hour/mg mito-

chondrial nitrogen.












succinat~ oxidation was stimulated. It was noted that only

those substrates requiring NAD as a cofactor 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 300C. 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 120 minutes of aging.

Aged mitochondria were less susceptible than fresh

mitochondria to inhibition by oligomycin, an inhibitor of

oxidative phosphorylation (24.1% versus 71.0%), indicating

that aging uncoupled oxidative phosphorylation (Table 5).

Since JH was 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
























Figure 5. The relationship between NADH dehydrogenase
activity and time of aging of isolated mito-
chondria from larvae of Plodia interpunctella.











CONTROL
- *1:'. .-.-.. AGED AT 30
4 1e111111. AGED AT 3C
.. 45 MINUTES
/**.,* ON ICE



". . . .
/ t
~I





/ \




IL, .4-"-"t'


.4.
.4.
.4.
.4.
.4'.


Ol,


TIME (hours)


oC
IC FOR
THCN PLACED


r
~z











Table 5. Effect of Inhibitors and Aging on NADH Oxidation
by Mitochondria from Plodia interpunctella



Mitochondrial Q (N4/
Aging Time (min) Inhibitor 02

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 JH2/ 55.9
45 JH 120.7
45 Oligomycin + JH 43.3
45 Rotenone 3/ 21.5


/-Oligomycin concentration during incubation = 4.10 x 10-5
2/ -4
-/JH concentration during incubation = 1.77 x 10 M
3-8
-Rotenone concentration during incubation = 6.25 x 10-8 M
4/Units of Q0 (N) are atoms oxygen consumed/hour/mg

mitochondrial nitrogen.











mitochondria were related to the concentration of JH in

the same way (Figure 6). Minimal inhibition was detected
-5
by a JII concentration of approximately 3 x 10 M. Maxi-
-4
mal inhibition was 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

10-5M, 1.06 x 10 M and 1.77 x 10-4M (Figure 7) revealed

that NADH oxidation was noncompetitively inhibited (Ki =

0.1069 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 were 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)6 by aged

mitochondria was not significantly altered by the presence

of 1.77 x 104 M JH. When JH was present at a rate of 742.6
-3
pmoles Fe(CN) 6-/hour per mg mitochondrial nitrogen was

obtained compared to 762.8 gmoles/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


Pyr- Mal




-i-U

c 'NADH

4. I


C /
so
CI
2I





oI
50 -
c /I
9 I

0 / g













0.0 0.1 0.2

JH Concentration (mM)





























Figure 7. Plot of (S)/V versus (S) ((S) is the NADH con-
centration and V is the reaction rate). The
substrate constant (Ks), inhibitor constant
(Ki) and the maximum velocity of the reaction
(Vmax) are shown.
max



























Ks : 2.20




Vmax 259.35




Ki : .1069 mM


10.0 15.0


I =(.113)


I= (0)











at the liev L 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 JHI-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 mitochondrial 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,










but did not eliminate the possibility that some other

event associated with the molt was also necessary. 0-,

-2- and +2-day larvae were placed on JH-treated diet,

removed at one day intervals and assayed for mitochondrial

cytochrome. The mitochondrial cytochrome content polese/

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 two 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

by +4-days. The mitochondrial cytochrome content of the

larvae which were placed on JH diet at +2-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 +4-days of larvae fed JH starting at -2-days and

of control larvae. Cytochrome synthesis in control larvae


























Figure 8. The relationship of larval age and age at
initiation of JH treatment to the mito-
chondrial cytochrome content.











0.5











I-
z
Z
IAl

0
Z
O
V 0.25


0
I-




0
u
OJ


AGE JH TREATMENT INITIATED


2
............ CO TROL
CONTROL


I.0
14
J*


-2 O +2 +4

LARVAL AGE (days)












proceeded from -2- to +4-days with most of the synthesis

occurring before +l-day. The mitochondrial cytochrome con-

tent of JHl-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 + a3 concentration increased 10-fold, cytochrome b



























Figure 9. The relationship of larval age to cytochromes
a, b and c concentration per insect for JH-
treated and control insects.

















O CYTOCHROME a

O CYTOCHROME b

O -CYTOCHOME

- CONTROL
SJH-TREATED


-2 -1


?

.1
i
P

z

C
2


a
I
I-




z
C



U
0
C
0
I-
5-
u






0


I-


ri

I

I"'


0 .1 *2
LARVAL AGE (days)


Em.


Ii
\ii ~iiii

lii I

















I:
U


*+3


-r^
i i



It


age


::::::
::::::
~
~









Table 6. 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


pmoles/insect nmoles/g tissue
control JH-treated control JH-treated


Cytochrome a 157.9 255.5 5.46 7.18

Cytochrome b 23.4 61.6 0.81 1.73

Cytochrome c 45.4 83.0 1.57 2.33


Total Cytochrome 226.7 400.1 7.84 11.24











concentration increased 4-fold and cytochrome c concentra-

tion increased 3-fold during the period from -2- to +4-

days. In JII-treated larvae the corresponding increases

were 30-fold for cytochrome a + a3, 11-fold for cytochrome

b and 4-fold for cytochrome c.

Cytochromes a + a3, 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 + a3, b and c were higher in insects which

underwent a supernumerary molt than in corresponding non-

molting larvae (Table 6).

The ratios of cytochrome b to cytochromes a + a3

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 + a3 decreased

throughout the experiment in both control and JH-fed in-

sects but the decrease in the ratio was greater in JH-fed

























Figure 10. The relationship of larval age to cytochromes
a, b and c concentration per gram tissue for
JH-treated and control insects.




































HI ,, i i i..


iijjii

iiiiii
iiiiiiiiijijf
:::::::

i:i:i:i
I


i




.:II



:I


I
I I:
~i iiii:6
i iiii
i iii

I


I
I
'i
I


iiiiii


kiiii.


El...


I 0 +1 +2
LARVAL AGE (days)


Em.


tI
Iii

I :::f
:::i r
Si iii~





+3 +


SCYTOCHROME a

O CYTOCHROME b

O CYTOCHROME c
CONTROL
I1N- TREATED


iiii
:1iil




iiii

I
i


-L


ml-


+2
LARVAL AGE (days)












Table 7. Ratios of Cytochrome A+A3, B and C Concentrations to Cytochrome A+A3
Concentration for Mitochondria isolated from Control and JH-treated
Larvae


Larval Age Control Larvae JH-Treated Larvae
Days Cyt. a+a3 Cyt. b Cyt. c Cyt. a+a3 Cyt. b Cyt. c


-2 1.0 0.97 1.50 --- -- ---
-1 1.0 0.76 1.01 1.0 0.72 0.96
0 1.0 0.59 0.80 1.0 0.66 0.87
+1 1.0 0.58 0.75 1.0 0.51 0.45
+2 1.0 0.47 0.49 1.0 0.44 0.34
+3 1.0 0.41 0.44 1,0 0.36 0.33
+4 1.0 0.36 0.40 1.0 0.35 0.22











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

was dependent on both JII 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 was observed within one day

after initiating JH treatment 2 days before the molt into

the fifth instar (Figure 11). Ieme 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




























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


Figure 11.














HEME a

E HEME b

SCHEME c
CONTROL
r-a- IJH-TREATED


LARVAL AGE (days)










increase approached 230% 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 hemie 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




























Figure 12. The relationship of larval age to hemes a, b
(protoheme) and c concentration per gram tis-
sue for JH-treated and control insects.












HEME a

SHEME b
O HEME c
- CONTROL
S_-. JH-TREATED


LARVAL AGE (days)










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
14
1C-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

1,140 cpm/hr./insect incorporated into 23.9 nmoles hemes

a and b per insect. Comparing 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




81





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 hormone inhi-

bits electron transport by the nonheme iron protein in com-

plex I of the mitochondria which results in lower oxidation

rates for citrate cycle intermediates requiring NAD as a

cofactor for oxidation. Minks (1967) found that relatively

high concentrations of a crude extract of Hyalophora cecro-
-3
pia, 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











et al., 1968, 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

(1960) 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










from diapausing L. decemlineata. Stegwee (1960) confirmed

results of earlier 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 Blaberus 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 eithermaleorovariectcmized 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 diapause 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










demonstrated 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 which 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 (1958) and Granick (1958) have










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-aminolevulinic 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 andersoni, lending sup-

port to the occurrence of this pathway in arthropods. The
14
incorporation of 1C-glycine into hemes in 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 6-aminolevulinic 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.

(1972)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 al. (1971) and Whiting

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 that 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 malate and a

reversal of the citrate cycle (Silhacek, Firstenberg and

Kohl, in press). Malic enzyme, the enzyme which catalyzes

the conversion of pyruvate to malate is active during the

early part of the last larval instar in P. interpunctella











(Silhacek, unpublished data) when 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 (Burnham 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+3) to ferrous ion

(Fe 2) by increasing intramitochondrial concentrations of

NADH. Barnes et al. (1972) confirmed that the conversion
+3 +2
of Fe+3 to Fe+2 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 with larval molting. Therefore, the stimulation

of cytochrome apoprotein synthesis may be due in part to

ecdysone. Indeed, Patel and Madhavan (1969) have










demonstrated a general stimulation of protein synthesis

associated with ecdysone titer in imaginal wing disks of

Samia cynthia 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 cardiac

factor which stimulates cytochrome oxidase activity. Se-

cretion of this corpora cardiac 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-

phemus 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 messenger 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

were 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.











The current information (Kiese et al., 1958;

Sinclair et 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 without 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




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