Temperature effects on the oxygen consumption during development of Aulocara elliotti (Thomas)
by Inez Ilene Laine
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
DOCTOR OF PHILOSOPHY in Zoology
Montana State University
© Copyright by Inez Ilene Laine (1966)
Abstract:
Oxygen consumption at various developmental stages of the grasshopper species, Aulocara elliotti
(Thos.), was measured at various temperatures. As the amount of embryonic tissue and the organization
of the embryo increased oxygen consumption rose at a steady rate until the time the egg went into
diapause. During diapause, oxygen consumption did not drop to a low level but continued at a fairly
high level. A histological comparison of pleuropodia of embryos that had been in diapause for different
lengths of time did npt show an increase in size of the cells of these embryonic structures. Biosynthesis
in these cells could not, therefore, contribute materially to the diapause oxygen consumption rate,
Variability in oxygen consumption was large in postdiapause developmental stages. It was also shown
that oxygen utilization-temperature relationships did not fit Arrhenius type equations at temperatures
between 9 and 20°C.
I j'T.'S
TEMPERATURE EFEECTS ON THE OXYGEN CONSUMPTION DURING DEVELOIMENT•OF
AULOCARA EDDIOTTI (THOMAS)
by
INEZ IDENE DAINE
A thesis submitted to the Graduate Faculty in partial
fulfillment of the requirements for ,the degree
of
DOCTOR OF PHILOSOPHY
in
Zoology
V '
Appro v e d ;
Head^yMajor Department
Cha^>frfan, 'Exam^n^ng Committee
Graduate Dean
MONTANA. STATE UNIVERSITY
Bozeman, Montana
August, 1966
iii
ACKNOWLEDGMENT
The counsel a n d ,assistance of my major advisor. Dr, James H, Pepper,
during the course of this research is gratefully acknowledged.
I would
like to offer special thanks to Dr. George Roemhild for his technical
advice and interest during the course of this study and for his criticism
of this manuscript.
I also thank Dr. Graeme Baker of the Chemistry
Department, and D r s . Eorman Anderson and P. D„ Skaar of the Zoology and
Entomology Department for critical reading of this manuscript and
Professor Ellsworth Hastings for obtaining experimental material and
for help in manuscript preparation.
This investigation was supported by a U,. 8. Department of Health,
Education,- and Welfare through a Title IV, HDEA Fellowship for which I
am'grateful.' I also thank the Office of the Montana State Entomologist
for financial assistance.
iv
TABLE OF CONTENTS
PAGE
V I T A .................. ................. .................................
ACKNOWLEDGMENT
..................
. . . . .
........................
ill
LIST OF TABLES . .................................... ■............ ..
LIST OF F I G U R E S ...................... ..
ABSTRACT . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . .
MATERIALS AND METHODS
RESULTS
.........
v
. .............. .............
.........
ii
vi
. . . . . . . . . . .
viii
. . . . . . . . . . . . . .
I
.................................... ..
IO
. ..............................................................
IT
HISTOLOGICAL-STUDY OF PLEUROPODIA OF AULOCARA ELLIOTTI IN DIAPAUSE .
'37
DISCUSSION AND CONCLUSIONS
48
SUMMARY
.........
. . . . . . . . . . . . . .
. . . . . . . . .
...........
LITERATURE CITED . . . . . . . . . . . . . . . .
.........
.
. . . . . . . . . .
66
. ..................
68
V
L I S T 'O F TABLES
PAGE
Table I,
Width of cells of pleupopodia of diapausing A. elliotti
embryos ............................... . . . . . . . . .
42
vi
.,LIST OF FIGURES
PAGE
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
I.
2.
3.
4.
5.
6.
7.
8.
9.
Respiration diver used to measure respiration of eggs
of. A." elliotti'............................. ............. .
13
Cartesian diver respirometer used to measure respiration
of eggs of A. elliotti . . . . . . .
......................
l4
Oxygen consumption at 90C of eggs of A. elliotti during
embryonic development
...................... ..
22
Oxygen consumption at l6°C of eggs of A. elliotti during
embryonic development
. . . . . . . . . . . . . . . . . .
22
Oxygen.consumption at 20°C of eggs of A. elliotti during
.................... ..
embryonic development
23
Oxygen consumption,at 27.2°C of eggs of A. elliotti during
embryonic development
..................
24
Oxygen consumption at 35°C of eggs of A. elliotti during
embryonic development
................................. . . .
25
Oxygen consumption at 27.2°C of diapausing A. elliotti eggs
of different ages
. ........................................
26
Q1Q for oxygen consumptipn of A. elliotti eggs for 9-l6°C
27
Fig. 1 0 .
Fig. 1 1 .
Fig. 1 2 .
Qjn for oxygen consumption of A. elliotti eggs for l6-2Q°C
interval
. .. . . . . . . . . . .. .. . .. . . . . . . . . ..
28
Q-]n for oxygen consumption of A. elliotti eggs for 20-27°C
IlxfcoiTVSll
O
O
*
0.0
•
6
•
9
Q
O
Q
Q
O
t
O
O Q
O
Q
Q
Q
O
O
29
Q1H for oxygen consumption of A. elliotti eggs for 27-35°C
interval
. . . . . . . . . .
9 . . . . . . . . . . . . .
.
30
Fig. 1 3 .. Arrhenius plot of oxygen consumption of stage I A. elliotti
31
Fig, l4.
Arrhenius plot of oxygen consumption of stage 15. A. elliotti
eggs
. . . . . . . . . . .
o
,
32
vii
Fig. 15.
Arrhenius plot of oxygen consumption of stage I 9 A. elliotti
eggs ............................. ..
33
Fig. 1 6 .
Arrhenius plot of oxygen consumption of stage 22 A: elliotti
eggs . .'............................................... .. . .
34
Fig. 17.
Arrhenius plot of oxygen consumption of stage 2k A. plljotti
e g g s ........... .. . . . . . . . . . .............
35
Fig. 1 8 .
\
Arrhenius plot of oxygen consumption of stage 26 A. elliotti
e g g s ................'.................................... .. .
36
Fig. 19.
Wid,th of cells of pleuropodia of liapausing A. elliotti
embryos of varying ages
.................................. . .
^5
Width of cellp of pleuropodia of diapausing A . -elliotti
embryos which have been exposed to cold for varying
......... .......................
lengths of time
k6
Width of cells of pleuropodia of diapausing A, elliotti. '
embryos kept at -25°C for varying lengths of time before
b e i n g •exposed tp cold
. . ......... . . . . . . . . . . .
kj
Fig. 20.
Fig. 21.
ABSTRACT
Oxygen consumption at various developmental stages of the grasshopper
species, Aulocara elliotti (Thos.), was measured at various temperatures.
As the amount of embryonic tissue and the organization of the embryo
increased,oxygen consumption rose at a steady rate until the time the egg
went into diapause.
During diapause, oxygen consumption did not drop to
a low level but continued at a fairly high level.
A histological compari­
son of pleuropodia of embryos that had been in diapause for different '■
lengths of time did not show an increase in size of the cells of these
embryonic structures. Biosynthesis in these- cells could not, therefore,
contribute materially to the diapause oxygen consumption -rate, Var i a ­
bility in oxygen consumption was large in postdiapause developmental
stages.
It was also shown that oxygen utilization-temperature relation­
ships did not fit Arrhenius type equations at temperatures between 9
and 20°C.
'
INTRODUCTION
The grasshopper species, Aalpcara elliotti, is widely distributed on
western rangelands (Pfadt, 19^9) and is one of the most damaging species
in Montana (Anderson and Wright, 1952).
Field studies on this species
show that year-to-year fluctuations in population numbers may be expected
but the causes of these fluctuations have,
in most instances^ not been
obvious even after carrying out detailed investigations on probable causes.
There is, however, recent research on mammals which indicates that
animal populations may be self regulating depending on population density
(Christian and Lemunyan,
1958j Christian, 1957J Christian and Davies, 1964;
Chitty, 1957 and 1964).
The possibility then exists that insects may be
self regulating in the same manner and that the failure to correlate
population changes in. A,, elljotti with environmental factors may lie
;
within the physiological variability of the insects themselves.
Such
physiological attributes might endow certain individuals or populations
with- outstanding ability to withstand certain types of stress while other
groups of insects would soon succumb under the same conditions.
There are
certain indications that this is the case since Hastings and Pepper (1964)
found differences in resistance to stresses of temperature extremes and
starvation between several populations of nymphs of A. elliottj.
In
addition, they found differences between groups within what might be
considered a single population indicating that there are subpapulations
within the larger one and these also differ in their response to ,stress.
The variability in resistance was found to be population dependent but no
Single factor could prove a reasonable explanation for this variability.
-2The authors have suggested segregation, physiological vigor or environ­
mental stress as possible explanations.
That populations may change in their characteristics has been shown
by V a n Horn (1963 ) who has shown a great variability in stages of
development of A. elliotti with a greater range of developmental stages
for a given age group in a 1959 laboratory reared population than in' one
reared in the same manner in i 9 6 0 .
A statistically significant difference in the number of retarded
embryos found in eggs of various wild populations in both diapause and
postdiapause stages of development was also found.
V a n Horn termed as
retarded any embryo in a single pod. that was several stages behind in
development as compared to the majority of the embryos in' that particular
pod.
All of the above factors tend to show differences among populations.
O n the other hand, Bunde (1965 ), who studied free amino, acids in
the developing egg, and Svoboda (1964), who studied lipids in the egg,
were not able to distinguish significant differences in levels of these
materials between populations.
The studies carried out on this species have had two main objectives;
first, an elucidation of the basic physiological processes within the
insect and second, a possible detection of differences in these processes
that might account for the differential ability of various populations to
survive stress.
Since temperature is an environmental factor to which the inhabitants
of an area are continually exposed and is usually considered to exert an
-3influence on the vital processes of the individual, a study' to try to
understand h o w it could influence a given population seemed, in order.
The effect of temperature on birth rate may be considered in two
parts; first,
its effect on the reproducing adult which would help
determine the number o f eggs laid by an adult and second, the effect on
the developmental stages where it would be an important factor in rate
of development and percentage of hatching.
It is with this latter area
that most of the present study is concerned.
Studies on a number of insects show that development can occur only
within a fairly narrow range of temperatures.
This range may be as great
as 8-31°C as. in the egg and larval stages of Oncppera (Madge, 1956) or
as narrow as from 30 - 39 OC as in the egg and larval stages of Damalina
ovis (Murray, 1956).
Above and below these figures there is complete
mortality (bursell, 1964).
It is important that the stage of development
be stated in any studies such as those cited above because the effects of
temperature have been shown to be different not only between species but
also between.developmental stages of a single species (Melvin,
1928;
Fink, 1925J Argo, 1939J Keister and Buck, 1961 ).
The fact that different stages in the life history may have a
different range or threshold of activity has led some investigators
(Lin, et. al., 1954; Hodson and Alrawy, 1956) to set down several types
of temperature thresholds that would bring about complete mortality.
In
the first type of lethal threshold, the temperature may bp sp low that no
development occurs and death eventually results.
In the second, type, the
_ll—
temperature may be too low for development to reach completion.
In a
third type, the embryo may have reached full development but the tempera­
ture is too low for hatching to occur.
Hodson a n d •Alrawy (1956 ) added
another threshold; hatching may occur but the temperature is too low for
survival.
Oxygen consumption is a commonly used measure of metabolic activity .
and is generally accepted as a measure of energy utilization in living
systems requiring aerobic conditions.
Variability i n metabolic rates due
to temperature and morphogeriic- changes have been extensively studied by
measuring changes in oxygen consumption (Fink, 1925; Melvin,
1928; Bodine,
1929; Keister and Buck, 19 S 1 ) .
Studies show that biological, systems differ from isolated chemical
reactions in their response to temperature (Keister and Buck, 1964).
1
The rate of a simple chemical reaction increases with rising temperature
b y a constant per cent per degree over a considerable range of temperatures.
The rate of increase over a 10°C interval is referred to as Q i q •
chemical reactions have a
Many
two but the Q^q of biological processes
varies widely and usually decreases at both the upper and lower ends of
the temperature r a n g e .
The oxygen consumption-temperature curve is thus
characterized by a gradual increase i n the. amount of oxygen consumed as
the temperature increases•until the upper lethal temperatures are
approached.
At this point,
and Buck, 1961 ; Richards,
it falls off abruptly (Birch, 1947J Keister
1964).
Keister and Buck (1964) found this
same type of curve in many enzymatic reactions studied.
I
“5When oxygen consumption, of a living system, is measured over a range
of temperatures, the values obtained are the result of many biochemical
reactions each of which may be affected differently by temperature changes.
Putter calculated the
for a variety of biological processes over a wide
range of temperatures and concluded that it was impossible to identify any
single reaction because the values were the result of a great complexity
of systems.
Although the temperature-oxygen.consumption curves cannot be
used to analyze specific metabolic reactions, they may be useful in com­
paring the responses of different organisms.or systems to changes in ■
temperature.
Argo (l939) compared the effects of temperature on- oxygen consumption
of cold-blooded animals and classified responses.according to their pattern
of change.
In some animals an increase in oxygen consumption was constant
for each unit rise in temperature.
In.others each unit rise in temperature
resulted in a decrease in the size of.the oxygen consumption increments.
The third type of response showed that oxygen consumption increased sharply
within certain temperature limits but within other limits there was little
or no increase.
This variable type of response is common, in insects.
A n unexplained
absence- in rise in oxygen.consumption with a rise in temperature has-been
reported by many investigators.
For example, Keister and Buck (1961 )
found no increase in the amount of oxygen consumed, by larvae of Phormia
regina w hen temperature was increased from 10 to 15°C.
Vernberg and
Meriney (1957) found no increase in oxygen consumption between 18 and
-6220 C and none between 27 and 30°C in Drosophila pupae.
Keister and Buck
(1964) noted that irregularities in respiration curves usually occur when
temperatures' are involved which are either considerably lower than or
immediately above the rearing temperatures but did not attach any signifi­
cance to these observations.
Oxygen consumption during embryonic development has been studied
with the eggs of many different insects.
In general, oxygen consumption
of an insect is closely adjusted to its energy demands.
Thepe should be
'
therefore,, a correlation between the rate of development and amount of
oxygen consumed.
Fink (1929 ) worked with the non-diapause eggs of
Leptinotarsa, Crioceris, Ootinis, and Popillia and Melvin (1928 ) worked
with.eggs of Tropaea luna, Platysamia cecropia, and iyrausta' sinsliei and
both reported a short formative period during which metabolism was lowest.
As development proceeded,, they found a regular increase in. oxygen con­
sumption until the time of hatching.
Agrell (1964) is of the opinion
that the reported low metabolism during the formative period is- a misin­
terpretation of the data.
He states that when Melvin's respiratory curves
are drawn on a logarithmic scale, there is no lag period but instead a
continuous increase in oxygen consumption from the time of laying until
hatching.
Burkholder (1934), working with M. differentialis eggs which have
a diapause, found a close relationship between developmental stages and
oxygen consumption.
In prediapause eggs oxygen use rose, gradually but
when diapause began, it dropped to a very low level.
With the resumption
-Tof development at the epd of diapause, there was an abrupt rise. . During
the postdiapause period, the curve showed a series of maxima in.oxygen
consumption.
The first maximum occurred during the early stages of
blastokinesis, the second in late blastokinesis when the embryonic dorsal
wall was completed,
and the third during the period of yolk engulfment.
Tuft (19^9) also found a relationship between the developmental stages
and oxygen consumption.in the non^diapause eggs, of Rhodnius prolixus.
Although Tuft tried to correlate this increase in oxygen consumption to
increased activity due to blastokinetic movements, Ag^ell (1964) stated
that Burkholder’s work shows that most of the increase was due to changes
in metabolic activity as a result of increased development.
The most extensive, study of oxygen consumption of grasshopper eggs
was done by Bodine and his associates from 1928 to 1953-
They classified
grasshopper-eggs on the basis of response to temperature. . According to
their classification (Bodine, 1929) Class I was made up of those eggs
that are normally subjected to low temperatures but will hatch if kept
at high temperatures.
These eggs show a pattern of respiration charac-
terized by a peak preceding diapause, a sharp drop at the beginning of
diapause, and an abrupt rise as development resumes.
Class II included
those eggs which are not normally exposed to low temperatures and will
not hatch if so exposed.
These embryos show a continuous rise in respi­
ration as development p r o c e e d s .
Class III included those eggs that will
hatch only if kept at. low temperatures for a period of time and show a
pattern of oxygen consumption similar to those in Class I.
■
-8• B o d i n e .and his co-workers worked principally on M. differentialis,
the eggs of which have a facultative diapause, apd therefore, belong to
Class I.
They found thgt oxygen consumption always followed the same
characteristic pattern during embryonic development irrespective of the
temperature at which the eggs were allowed to develop (Bodine, 1929)•
After the first initial fall in oxygen consumption at the beginning of
diapause, there was a continued slow decrease for a few days after which
it remained low throughout most of the remaining diapause period.
About
a week before any visible evidence of termination of diapause, oxygen
consumption began to increase gradually.
When morphological development ■
after diapause resumed, there was a great increase in oxygen consumption.
The rate then continued to increase during the postdiapause period'(Boell,
1935)« • Schneiderman and Williams (1953) recorded the same type of pattern
in diapause oxygen consumption of Platysamia cecropia pupae and suggested
that the biochemical mechanisms, that bring about the termination of dia­
pause began at least a week before morphological changes became evident.
The- action of various inhibitors and stimulants on Qxygen. consumption
was shown to depend on the stage of development of the egg (Bodine, 193^-J
1937).
For example, potassium cyanide and carbon monoxide reduced oxygen
consumption.of the developing egg to a minimum, but did not affect that' of
a diapausing egg.
Methylene blue stimulated the respiration of diapausing
eggs but did not affect active ones.
The insensitivity of diapause eggs
to carbon monoxide and cyanide is thought to result from a large excess
of cytochrome oxidase relative to cytochrome c during this stage
(Kurland
-9.and Schneiderman, 1959J Gilbert and Schnelderman^
1961 ).
"Studies on oxygen consumption of the developing postdiapause egg o f ■
Ao elliotti have been made by Roemhild (1961 ).
He found that the p o s t ­
diapause pattern, of oxygen, consumption is, in general, the same as that
found for other species. . Several different oxygen consumption rate
changes were noted, however,
5 to 35°C.
For' example,
as the eggs were progressively warmed from
in the vicinity of IO0C he found that as the
temperature was raised,, there was np corresponding increase in oxygen
consumption but this could be modified by the length of acclimatization.
It was proposed that this reversal might be due to enzyme systems being
activated at or near this temperature.
The present investigation.extends the above study to determine the
effects of temperature over the entire period of development. ,
MATERIALS M D METHODS
The eggs used in this study were collected from a population of
grasshoppers maintained in the greenhouse.
These grasshoppers were
collected as nymphs' from a wild population and fifteen pairs were placed
in each of five lucite-walled cages.
The one square foot circular bottom
contained a removable nine inch aluminum pan filled with soil in which
the females could oviposit.
Three vials of western wheatgrass (Agropyron
smithii), the p r e f e r r e d .food plant of this insect (Anderson, I 962 , Efadt,
19^9); were set into holes cut in the bottom of each cage.
Fresh food
was supplied and eggs were collected during the cooler morning ,hours of
each day while the grasshoppers were relatively inactive and had not yet
begun to oviposit.
The egg p o d s .were placed in a vertical position in.
vials filled with, soil of the same type as that' in which the eggs were
laid and then, stored at 25°C.
V an Horn (1963 ) found that after forty days
at 25°C most of the eggs of this species entered diapause.
The only
successful method found to terminate diapause in this species is prolonged
exposure to low temperatures; -therefore,
on the fortieth day all eggs not
used in prediapause studies were transferred to
9°C, Each week all eggs
were watered lightly to prevent de-siMoatloa^
All measurements taken during the course of this study were done on
the whole e g g ,apd thus include the oxygen consumption of the embryo plus
the yolk.
Bodine and Boell (1936 ) studied respiration in both whole eggs
and embryos of M. differentialis and Roemhild (1961 , 1965 b) studied yhole
egg and embryo respiration in A. elliotti.
The isolated embryos of dia-
pausing M. differentialis were reported to respire at approximately the
same rate as the intact egg but this was not true of A. elliotti.
Bodine
-11and Boellj, however,
stressed that the data obtained for oxygen consumption
of the whole egg represented a complete system.
Since the objective of
this study was to obtain basic data on the oxygen consumption of whole
developing eggs, the amount of o x y g e n 'consumed by isolated embryos was
not relevant and therefore not measured.
When V a n Horn (1963 ) studied the embryonic development of A. elliotti
she encountered a wide variation in the morphological development of
embryos of a given age so she divided the period of development into 27
stages based on distinguishable morphological differences rather than age.
According to her data, stages l-l 8 represent prediapause development.
Stage
I usually occurs about the eighth or ninth day after laying when the embryo
is a germinal disc lying on the surface of the yolk at the posterior end.
Stage 2 may be found from as early as the eighth day until about a week
later,
depending on the rate of development of a particular egg.
At this
time, the embryo has elongated toward the anterior end of the egg.
Development and differentiation continue until at stage l 8 the external
morphology is nearly completed and the first evidence of red pigmentation
occurs along the. lateral margins of the eyes.
Most of the embryos of this
species enter an obligatory diapause at stage 19 which is about 40 dgys
after laying.
distinguished.
This is also the first stage at which the sexes can be easily
Stage 20 represents the beginning of the postdiapause
development and at this stage the embryo begins to revolve around the
posterior pole of the egg.
embryo continues to develop.
Blastokinesis is completed at stage 23. and the
Stage 27 represents the definitive embryo.
All the embryos used in this study were staged according to this criteria.
•
-12Every stage of development is not represented in this study b e c a u s e
of difficulty in getting the embryos, at the right time as prediapause
development is quite rapid.
Many methods have been used for respiration measurements but Roemhild
(1961 ) found the Cartesian diver micro-respirometer most satisfactory
because' of its sensitivity and accuracy.
study.
It was therefore used in this
The Cartesian diver respirometer was first described by Linderstrom.
Lang (1937 ) and Linderstrom-Lang and Click (1937)«
This method operates on the principle that pressure is transmitted
equally to all parts of a confined fluid.
In this respirometer the
confined fluid is "the air in the manometer and the flotation media which
is contained in the flotation vessels.
The volume of the confined fluid
and air- and thus its pressure is controlled by means of a manometer.
A
small hollow tube sehved as the respiratory vessel or diver (see Fig. l ) .
The grasshopper egg,, a carbon dioxide' absorbent,
and an air bubble were
confined.in the diver b y means of an oil seal.
The flotation vessels used were modified Thunberg tubes with ground
glass stoppers and side arms (Fig. 2).
They were filled with flotation
media, 11 R lithium, chloride (RoemhiId, 1961 ), to a level that was m a i n ­
tained constant at all times.
The vessels w e r e .connected'to a-manometer
filled with B r o d i e ’s solution (Umbreif, e t . al., 1957).
Connecting tubes
and flotation vessels were immersed in a constant temporature bath.
Temperature control was critical since the confined gases must be main­
tained at a constant volume. - The temperature was maintained, at + .05°C
—
A.
B.
C.
D.
E.
13 —
Oil seal
Air bubble
Filter paper saturated with 20$ KOH
Egg
Tail of diver
Figure I.
Respiration diver used to measure respiration of eggs of
A. elliotti.
-
A.
B.
C.
D.
E.
F.
Cr.
H.
I.
J.
14 -
Manometer tube
Equalizing cock
Manifold
Stopcocks
Flotation media (ll W lithium chloride)
Flotation chambers
Respiration divers
Fine adjustment
Coarse adjustment
Ground glass joint
c
A
C
E
I
Figure 2 .
I
Cartesian diver respirometer used to measure respiration of
eggs of A. elliotti.
K
of the desired temperature for all'readings.
respiration was measured were
The temperatures at which
9°, l 6 °, 20°, 27.2°, and 35°C.
The divers used.in this study were made of pyrex glass as described
by Linderstrom-Lang (1937) and Holter (1943).
Before each use they were
cleaned with chromic acid cleaning solution, washed with distilled water,
and rinsed with 100$- alcohol. . The interior surface of the necks of the
divers were treated with paraffin dissolved in toluene and divers were
;
then inverted over an air jet to remove the organic solvent.
The waxed
surfaces were then polished to prevent creeping, of the solutions used.
A single egg was placed in the bottom of each diver' and a piece of
pleated filter paper saturated with 20 $ potassium, hydroxide to absorb
carbon dioxide was inserted into the heck.
A seal ma,de from equal
portions of heavy mineral oil and light Hevastan grease (Roejuhild, 1961 )
was used to seal the neck.
Each diver was carefully lowered into the
flotation vessel and air was added or subtracted with a micro-pipette
until the density of the diver was adjusted so that it would just settle
to the bott o m of the flotation vessel.
The vessels were then attached
to the manometer tube and placed into the constant temperature bath.
All stopcocks were left open to prevent any increase in pressure of
the gas when- the manifold was inserted into the ground glass joint.
The
system was allowed to equilibrate for at least thirty minutes before any
readings were taken.
. After equilibration the manometer fluid was adjusted to a standard
point at which it was kept between measurements.
All stopcocks except
■-l6the one on the first flotation vessel were closed and the manometer was
used to reduce the pressure over the flotation medium and thereby increase
the volume of the diver until it rose to a reference point previously
etched on the flotation, vessel.
This point was intermediate between the
bottom and top of the fluid where the diver was at its position of
maximum, sensitivity (Kirk, 1950) „
The manometer reading, time, and
•temperature were carefully recorded and the manometer fluid returned to
its standard point,
causing the diver to sink to the bottom of the vessel.
The stopcock of the first vessel was then closed and the second one opened.
The procedure w a s repeated for each of the remaining four divers.
At least
four separate readings were made on each diver at about 8 minute intervals.
As the eggs used oxygen and potassium hydroxide absorbed the carbon
dioxide produced, the volume of the gas within the diver decreased.
This
caused an increase in the specific gravity of the diver. .At each successive
reading, as the divers were adjusted to the original reference -point, the
pressure exerted ■by the manometer fluid on the confined air was, therefore,
decreased.
Thus the manometer reading was lower each t i m e .
were plotted to check their consistency.
metrically identical,
These readings
Since the divers were not g e o ­
each had to be calibrated separately to determine
its total gas volume (Kirk, 1950)«
This method enabled the measurement
of the amount of oxygen consumed by a single egg.
RESULTS
The oxygen consumption from four days + one day after Laying until
hatching was measured and the data recorded in Figures 3-7.
These
figures include the data for all five temperatures utilized in the study.
A comparison of Figures 3-7 shows that,
in general, the higher the
temperature, the greater the amount of oxygen consumption.
This is true
of all stages hut the difference, is greater in the older stages.
For
example,- in stage I, the oxygen consumption was .0046 microliters per
egg per minute- at 9°C and ..0l4$ at 35°^.
This is almost a 300$ increase.
At stage 15, the rise was -from. .0059 at 9o0 to .0223 at 35° or about a
400$ increase.
At stage 27, there was about a 500$ increase.
A gradual rise in oxygen consumption from the time of laying- until
diapause is noticed at all temperatures utilized. , It is not a- consistent
rise from stage to stage but there is an overall rise during this p r e ­
diapause period.
Although this effect is evident at all five temperatures,
it is greatest at the higher temperatures. ,At 27.2 and 35°C, the peak
in oxygen consumption was at stage l4.
27.2°C.
This is especially evident at
Figure 6 shows that the oxygen consumption at this stage was
almost twice that of the stages preceding or following it.
None of the data in this study showed a sharp decrease in oxygen
consumption when the embryos entered diapause and no great increase was
observed when the embryos resumed development after diapause was terminated.
A number of measurements were taken throughout the period of diapause
to determine the level of oxygen consumption during this period.
Eggs
used for these measurements were kept at 9 °C, a temperature found to
.-,18break diapause, and periodically pemoved to 27„2°C for measurements„
Although the usual temperature at which eggs in the laboratory tfere held
during development was 25°C,.
water bath used.
from cold.
2J.2°C proved to be easier to maintain in the
Measurements were started within one hour after-removal
The results of these tests are shown in Figure 8 .
This graph
shows that there was a decrease of about -50 $ in the oxygen consumption
but it was not found at all times during this period.
The lowest amount
of oxygen consumed w a s .found for embryos which had been in diapause for
50 days but a higher rate was found in eggs which had ,been in diapause
for 60 days. . Eighty days after the beginning, of diapause, the oxygen
consumption had decreased again.
From this point, oxygen consumption
rose until at the time development resumed, the egg was consuming oxygen
at a rate slightly greater than that just before it entered diapause.
After measuring oxygen consumption of these diapausing eggs, they
were placed on moist filter- paper in Petri dishes and observed for a
period of several days.
This was. to insure that postdiapause oxygen
consumption was not m e a s u r e d .
The eggs continued to -appear normal and
no development was seen thus indicating that they were still in diapause.
Postdiapause eggs showed greater variability in oxygen consumption
from stage to stage at all temperatures utilized than did the prediapause
eggs.
The patterns at all five temperatures were similar in that the
greatest oxygen consumption, occurred at stage 25 when dorsal closure was
completed.
Eggs at all temperatures showed an increase in oxygen con­
sumption at stages 20 or 21 during blastokinesi s .
Figures. 9-3-2 show the Q^g values for the temperature intervals
studied.
Because the temperatures were not ten degrees apart, v a n ’t
H o f f 1S equation, in the form I q g .Q 10 = IOZt 1 - tg log H 2 Zk 1 was used to
determine Q 1Q „
In this formula, kg is the rate of respiration at tempera­
ture tg and H 1 is the rate at t^ (Giese, 1962 ).
The bars in Figures 9-12 have as a base a Q^q of I.
A Q 10 of. I
represents no change in oxygen consumption while a Q1Q of less than I
means there was a decrease in the amount of oxygen consumed as the
temperature increased.
In, these graphs,
a Q^ q of less than I.is shown
below the base line in order to emphasize the fact that there was a
decrease in oxygen consumption as the temperature was raised, instead of
the expected increase.
These graphs do not show absolute oxygen consumption values but
show the change in oxygen consumption between the consecutive temperatures
tested.
It is seen in Figures. 9 and 10 that there were a number of stages
in which an increase in temperature resulted, in no change or in a decrease
in the amount of oxygen used.
The data for the 9-l6°C range in prediapause
eggs is not complete due to technical difficulties.
In three of the five
stages studied, the oxygen consumption decreased as the temperature
increased from 9 to l 6 °C.
In the l6-20°C range, four of. the ten prediapause stages studied
showed a decrease in oxygen consumption as the temperature increased
while in five stages only a slight increase in activity was found.
Only
in the stage immediately preceding the. diapause period was there a sharp
■
“
20
“
increiase in oxygen consumption when the temperature increased.
The Q 10
in the temperature interval for the stage l 8 embryos was 4 ,7 5 .
These graphs do not necessarily show the optimum temperature for
development but they do show the range in which the oxygen consumptipn
was increased the greatest amount.
Comparing the graphs,
all stages did not have their highest Q
it is seen that
in the same temperature range.
The highest Q 10 found for stage I and 2 was in the 20 to 27° interval
while in stage 4 the highest Q 10 was between 2 7 ,and 35°C.
Stage 6 embryos
had almost the same Q 10 in the 20-27° and 27-35° intervals.
12, the highest Q 10 was between 27 and 35°C.
In stages Q-
A very high Q 10 was found ■
between 20 and 27°C for stage 13 and l4 but the-pattern 1 changed again
because the greatest increase in oxygen consumption for stage 15 and l 6
was between 27 and 35°C.
For stage l 8 until hatching, the greatest in­
crease was between l 6 and 20 °C although there was n o t ■much differenqe in
Q 10 between the l6-20°C interval and 20-27°C interval at stage 26 and 27«
Figures 13-18 are Arrhenius plots for various stages throughout the
developmental period.
The rate of oxygen consumption for prediapause
stage I and 15, diapause stage IQ, and postdiapause stages 22 , 24, and
26 are presented in these g r a p h s .
In these graphs, the Iog 10 of the
rate of oxygen consumption is plotted against the reciprocal of the
absolute temperature.
These plots show that a straight line relation­
ship is not always found.
A fairly straight line relationship is found
in stages 15 and 24 but in most of the others, at least one point is where
this correlation is absent.
In stage I, the point corresponding to the
oxygen consumption at 20°C is far below the line.
At stage 19, it is
the point representing the rate at l 6 °C that is below and this is also
true at stage 2 2 » ' The pattern of the stage 26 embryo,g is almost a
straight line but there is a slight deviation at 9°C.
-
22 -
Prediapause
Oxygen consumption, microliters per egg per minute
Postdiapause
STAGE OF DEVELOPMENT
Figure 3.
Figure 4.
Oxygen consumption at 9°C of eggs of A. elliotti during
embryonic development.
o
Oxygen consumption at l6 C of eggs of A. elliotti during
embryonic development.
Oxygen consumption, microliters per egg per minute
—
23 —
Prediapause
-pOstdiapause
STAGE OF DEVELOPMENT
Figure 5*
Oxygen consumption at 20°C of eggs of A. elliotti during
embryonic development.
Oxygen consumption, microliters per egg per minute
—
24 —
Prediapause
Postdiapause
STACrJi u p DEVELOPiVMiT
Figure 6.
Oxygen consumption at 27.2 C of eggs of A. elliotti during
embryonic development.
-
25-
Postdiapause
Oxygen consumption, microliters per egg per minute
Prediapause
STAGE OF DEVELOiMENT
Figure
rJ.
Oxygen consumption at 35°C of eggs of A. elliotti during
embryonic development.
26“
Oxygen consumption, microliters per egg per minute
—
)
40
DAYS
STAGE
.figure 8 .
50
AT
18
Oxygen consumption at
19
60
70
80
90
9°C
20
2J.2°C of diapausing A. elliotti
eggs of different ages.
(Total age = 40 + days at 9°C).
27-
STAGE OF DEVELOPMENT
Figure 9.
Q10 for oxygen consumption of A. elliotti eggs for 9-l6°C
interval.
-
Figure 10.
28-
Q 10 for oxygen consumption of A. elliotti eggs for l6-20°C
interval.
-
29-
STAGE OF DEVELOPMENT
Figure 11.
Q-^ for oxygen consumption of A. elliotti eggs for 20-27°C
interval.
-30
-
STAGE OF DEVELOPMENT
Figure 12.
Q-^q for oxygen consumption of A. elliotti eggs for 27-35°C
interval.
31 —
DEGREES CENTIGRADE
30
20
x
Figure 13.
IO
IO
Arrhenius plot of oxygen consumption of stage I A. elliotti
eggs.
-32
-
DEGREES CENTIGRADE
I / T x I O'
Figure l4.
Arrhenius plot of oxygen consumption of stage 15 A. elliotti
eggs.
-
33 -
DEGREES CENTIGRADE
Figure
15
.
Arrhenius plot of oxygen consumption of stage
eggs.
19
A. elliotti
-
34-
DEGR E E S CENTIGRADE
I/T
Figure l6.
x
10
Arrhenius plot of oxygen cons u m p t i o n of stage 22 A. elliotti
eggs.
-
35 -
DEGREES
30
Figure 17.
centigrade
20
Arrhenius plot of oxygen consumption of stage
eggs.
10
2b A. elliotti
—
36 —
D E G R E E S CENTIGRADE
Figure l8.
Arrhenius plot of oxygen consumption of stage 26 A. elliotti
eggs.
HISTOLOGICAL ■S T U D Y .OF PLEUROPODIA OP AULOCARA ELLIOTTI IR. DIAPAUSE
In the p r e v i o u s .section,
it was noted that the oxygon consumption, of
A. elliotti eggs did not drop to a low level during diapause.
This would
indicate that, although most development seems to be at a standstill)
some
activity which requires oxygen does continue.
In her'studies. V a n Horn (1963 ) removed, eggs from cold and observed
that some developed, as expected but others,
although they appeared normal,
failed to undergo blastokinesis and remained in diapause-,
To find any
differences between the embryos that failed, to develop normally a pd an
embryo just entering diapause,
she did a histological comparison of
day diapause embryos and older diapause -embryos.
Uo
The 40 day embryos were'
just entering the diapause period while the older ones had been exposed to
and removed from low temperatures but had failed to develop.
W h e n serially-sectioned diapause embryos were observed,' it was found
that mitosis was
infrequent in all specimens.but there was evidence that
certain processes .of conversion and synthesis continued.
As stated
earlier, there was an.increase in size of gonad cells, pleuropodial glands,
and fat body.
There was also a depletion of cytoplasm from the o e n o c y t e '
ce l l s . ' The change;
In the pleuropodial cells was the subject of the .
present study.
. Pleuropodia are true embryonic organs because they either are dis­
carded or degenerate at the time of.hatching.
Although present in .most
insects, they are very small or wanting in a few orders.
These .organs ■
arise as ectodermal thickenings on the first abdominal segment but
development may differ in different orders (Hussey, I 926 ).
Many theories
-
38-
as-to their function have been proposed.
Mopt of these were based on
morphological evidence until glifer (.1937 , 1938 ) did a study on. their
morphology,
development,
jand function in the grasshopper Melanoplus
differenti a l i s .
In M. differentialis, the pleuropodia first appeared on the eleventh,
day of development.
This species of grasshopper entered diapause on the
twenty-firpt day of development but mitotic division in the pleuropodial
cells stopped on the eighteenth day.
not fully developed.
'When mitosis cea,sed, the cells were
After diapause, the glands were observed to increase
in size rapidly until they reached their maximum size a. few days after
blastokinesis.
before hatching.
They remained constant in size until three or four days
They then began to decrease in.size until at the time of
hatching they appeared very shrunken.
The newly hatched insects shed the
shrunken glands along with the chitinpus exoskeleton.
Ligation studies' (Slifer, 1937) and histological studies on the
appearance and disappearance of secretion granules within the pleuropodial
cells furnished evidence for the theory that,
at least in M. differentialis,
these glands secreted an enzyme which dissolved.the white cuticle of the
egg so that the insect could escapp more easily at hatching.
The pleuropodia. of 40 day old A. elliotti were comparable in a p ­
pearance to those of .21 day old M. differentialis embryos.
The nuclei of
the cells lay between the periphery and the center of the cells and the
thickness of these cells in the center of the organ was 20 /3- .
When V an
Horn (1963 ) compared 229, 259 , and 309 day old diapause embryos to 40 day
-
old embryos,
the cells.
39-
she found striking differences in the size and appearance of
The pleupopodia of the 229 day diapa,using embryos resembled
those usually found.in postdiapause stage 22 A. elliotti embryos; the
cells were 40'yU_ thick and had twice as much cytoplasm as' cells from the
Uo
day emb r y o .. The pleurppodial cells of 259 day embryos were SOytx
thick.
The 309 day embryos had pleupopodial cells which were larger than
those of 259 day embryos and resembled those of post-blastokinesis. M.
differentialis 33 day old embryos (Slifer, 1938).
were aligned at the inner surface.
T h e .nuclei of the cells
Increase in cytoplasm indicates that
certain activity had continued during, diapause.
Va n horn suggested that
some anabolic activities might have started due to low temperatures to.
which all the older embryos had been exposed but the mechanisms for
initiating blastokinesis had not been activated.
Differences in the
pleuropodial cells in embryos which had been exposed to- cold and those'
that had received no cold were studied over a period of 177 days by the
present author to determine if cold is necessary'fbr the development of
these ce l l s . .
,
All the eggs used in this study were greenhouse eggs collected July
26, 1964.
old.
At the time of collection, the eggs were less than 24 hours
The egg pods were stored in.a vertical position in individual vials
filled with soil of the same type as that in which the eggs were laid.
The eggs were kept at 25°C for 40 days, the approximate time they.enter
diapause, and then were divided into three groups.
to 25°, another to 4°, and the last to 9°C.
One group was exposed
All eggs were watered lightly
-
40
-
to prevent dessication.
At two-week intervals, two pods from each group were taken from
storage apd the eggs dissected out.
Two' eggs from each group were fixed
immediately in alcoholic B o u i n 1s fixative and the rest of the eggs were
placed on moist filter paper in Petri dishes and incubated at 25°C.
After five days, two more eggs were fixed and this process again repeated
after five mope days.
Only eggs still in diapause were fixed.
Toward the
end of tbe study period, eggs that would remain in diapause after being
removed from the cold and yet appeared normal were difficult to o b t a i n .so
the study was terminated.
After fixation,of the whole egg, the embryo was dissected o u t 'and
embedded in Paraplast (m.p. 56-57°C)..
at
The embryo was serially sectioned
6 - 8 , stained with Harr i s’ Hematoxylin and counterstained with eosin
(Humason, 1962 )„
The.width of.four cells near the center of the gland was
measured, and the average width recorded in Table I.
In an attempt to find if and When an increase in size of the cells
took place, the results were graphed in three different ways.
The width ■
of the cells versus the total age in days is plotted in Figure 1 9 . - Figure
20 shows a plot of the width of the cells versus dyys at low temperatures.
Figure 21 shows the width of the cells plotted against the number of days
at 25°C before exposure to cold.
All three graphs and Table I as well show that there was a wide
variation in the size of the pleurppodial cells.
from 20
to 4 2 u.
.
The cells sizes ranged
with n o ‘pattern to this variation.
Because pf the wide
range in sizes at all ages,
it is impossible to determine if these cells
require cold in order to develop„
The major difficulty in this study was that all of the eggs taken
from the cold either died or began developing.
None of the eggs stayed
healthy and in diapause after removal■from the cold as the eggs used in
V a n H o r n ’s study did.,
Therefore, none of the eggs used in the present
study were as old as those in the former study.
This, study did not show any large amount of biosynthesis going on
during diapause which could explain the high rate of oxygen consumption
during the period in A. elliptti eggs.
Table I
Width of cells of pleuropodia of diapa,using A. elliotti embryos
Wo. of days
at 2^0
Wo. of days
at room temp.
60
0
.0
•4o
20
0
80
.0
4o
4o
80
Total age
of eggs
Width of
in /<*
60
3 3 .2
60
3 3 .2
80
24.0
0
80
38.0
0
5
-85
3 0 .0
80
0
5
8-5
4o
40
5
85
34.0
4g
4o
-12
■92
42.0
4o
4o
12
92
30.0
4o-
4o
12
92 .
48.0
100
0
0
100
34.0
4o
60
0
100
38.0
4o
60
0
100
3 3 .2
105
0
0
105.
20.0
4o
60
-5
105'
2 0 .0
0 '
■
'
'
'
34.0
-42-
.
Wo. of days
at law temp.
Table -I (continued)
' Width of cells of pleuropodia of diapausing A. elliotti embryos
H o . of days
at 25 ° -
Ho, of days
at low t^emp.
H o . of days
at room temp. .
Total age
of eggs
Width of cells
in Zv-
4o
60
5
105
2 0 .0
4o
60
5 .
105
" 2 6 .0
115
0
115
41.5
115
0
0
115
2 4 .9
115
0
0
115
3 0 .0
40
60
'15
115
2 6 ,0
4o
60
30
’130
2 2 .0
120
0
0
120
3 4 .0
4o
80
0
120
"24.0
136
0
'0
136
3 2 .0
4o
80
16
136
26.0
143
0
0
143
24.0
4o ■
103
.0
143
24.0
4q
103
0
143
2 6 .0
l48
0
0
148
4o.o
148
0
■0
148
30.Q
' O
Table I (continued)
Width of cells of pleuropodia of diapausing A. elliotti embryos
N o . of days
at 25 °
40
153
'
' 153
• 172
40 -
Ho. of days
at room temp.
Total age
of eggs
103
5
l48
42.0
0
0
153
3 6 .0
.0
O
153
3 4 .0
o
0
172
3 8 .0
132
0
172
3 2 .0
H o . of days
at low temp.
,
■
Width of
in
177
0
■o
177
36.0
177
0
0
177
3 4 .0
4o
132
5
177
2 6 .0
50
K
X
40
/
Width of cells
<
x
X
x
*
x
x
5
xx
*
*
/
30
x
X
*
x
<
X
x
20
X
X
y
A
X
*
-
X
60
8*0
100
TOTAL AGE H
Figure 19.
120
140
DAYS
Width of cells of pleuropodia of diapausing A. elliotti
embryos of varying ages.
I _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I___
160
180
50
/
I
t
x
A
Width of cells
X
i
%
/
I
X
O
X
X
X
x
/
/
X
t
-x
o
Xy
20
40
60
80
Too
DAYS AT COLD
Figure 20.
Width of cells of pleuropodia of diapausing A. elliotti
embryos which have been exposed to cold for varying lengths
of time.
Hh)
T^O
50
X
*
x
X
>
*
30
v
X
X
A
^
X
*
Width of cells
*
Z
X
X
K
$
X
X
20
X
X
Vo
so
g^5
Too
r&o
140
DAYS AT 25°C
Figure 21.
Width of cells of pleuropodia of diapausing A. elliotti
embryos kept at 25°C for varying lengths of time before
exposure to cold.
iAo
DISCUSSION AND CONCLUSIONS
The work carried out by the author was designed to answer two
questions.
First, as basic information concerning this species,
it was
desirable to know the normal oxygen consumption pattern of the eggs-duping
their development.
Second,
information was needed regarding the effect
of temperature on oxygen consumption of the eggs at various stages during
development.
In comparing the pattern of oxygen consumption during the develop­
mental period of A. elliotti with that found in other insects which undergo
diapause, both differences and similarities were found.
Oxygen consumption
of eggs increased in accordance with other species tested from the time
of laying until diapause.
During this period the amount Qf embryonic
tissue and the organization of the embryos increased so an increase in
oxygen consumption would be expected.
the higher temperatures used.
The increase was most evident at
This could be attributed to the greater
number of enzymes present and being activated at the higher temperatures
in the older embryos as compared to the younger ones.
One Qf the major' ways in which eggs of A. elliotti differed was that
oxygen consumption during diapause did not drop down to the levels found
in other species.
For example, the U-shaped curve observed for this
period in eggs of Melanpplus differentialis (Bodine, 1929) and eggs qf
Gryllus pennsyIvanicus (Rakshpal, 1962 ) was absent.
Instead of the very
sudden drop reported for these species, there was only a slight decrease
in oxygen consumption of A. elliotti. • Although the oxygen consumption of
eggs which had been in' diapause:/forIVdryihg: dehgtli^^oftimepwas::m%as,ufed,
only a slightly lower rate was found.
Diapause is usually characterized
by an extraordinarily low and stable metabolic rate X Keister and Buck,
1964).
When Bodine (1929 ) measured the oxygen consumption of M.
differentialis eggs, he found they had a very low oxygen consumption rate
during diapause and this rate prevailed regardless of the temperature of
development.
Burkholder (193^) alsti worked on diapausing M. differentialis
i
embryos and observed a cessation of growth and development and a low
respiratory rate.
abruptly.
W h e n diapause was terminated, ■oxygen consumption rose
The oxygen consumption rate of diapausing Gryllus pennsyI v a n i c u s .
.
eggs fell t o about one-ninth that of the eggs just before they went into
diapause.
Ppstdiapause.embryos took three days to reach a level comparable
to the maximum rate found in the prediapause period (Rakshpal, 1962 ).
In
postdiapause A. elliotti, oxygen consumption v a ried considerably between
stages but was in most' cases above the maximum found in prediapause d e ­
velopment.
The highest point at all temperatures was at stage 25 when
dorsal closure was completed.
It has usually been taken for granted that the amount of pxygen
consumed is a fairly good measure of the rate of utilization of available
energy substrates.
Harvey (1962 ) stated,
"These demands can be analyzed
in three components— those associated with,morphogenesis, those associated
with maintenance, and those associated with function.
The oxygen con­
sumption at any stage would then be a resultant of all three."
Energy
requirements for growth are much higher than for tissue maintenance
(Lees, 1955)•
The low oxygen consumption found in mpst diapausing insects
-
50
-
is attributed to energy demands associated only with tissue maintenance
although workers have found low levels of biosynthetic processes continue
For example, Wyatt (1958 ) found a low level of incorpo­
during diapause.
ration of labeled inorganic phosphate into the RNA of wing epithelium of
H. cecropia arid Demjanowski,
et. al. (1952) found labeled methionine
incorporated slowly into the blood and tissue proteins of diapausing
Antheraea pernyi G-Iierin.
Much of the evidence for diapause biosynthesis has been obtained by
injecting labeled substances into an insect and then measuring their
incorporation.
A complicating factor, however,
is that Harvey and
Williams (1961 ) found that oxygen consumption of H. cecropia and other
saturniid insects, increased following, integumentary injury.
was in direct proportion to the size of the wound.
The increase
Until recently, no
criterion had been found to distinguish increased oxygen consumption.due
to development from that due to injury.
Bowers and Williams (1964) found
that although oxygen consumption increased in both cases in H. cecropia,
DNA synthesis in the epidermal tissues of the developing pupa increased
but it did not in the- injured o n e s .
Not even extensive injury qould
stimulate mitosis op DNA synthesis in tissues not directly involved in
closing, a w o u n d .
They suggested that this information could be used to
determine what type of activity was being studied when a substance was
injected into a diapausing insect.
Low oxygen consumption.such as that encountered d u r i n g ,diapause does
not necessarily mean that metabolism is at a low level.
■
The possibility
,i
-51exists that the energy demands of the systems may be supplied by alternate
anaerobic .pathways.
Chino (1957) found a sudden disappearance of glycogen
with the onset of diapause and a sudden reappearance at diapause termi­
nation.
Moreover, he found that the glycogen originally present was nearly
,
1
all converted ipto sorbitol and glycerol as soon as diapause began.
The
formation of such trihydric alcohols may act as hydrogen acceptors to
furnish energy needs.
Roemhild
(1966)
found an almost
10
fold increase
in osmotic pressure in A. elliotti eggs from the beginning of diapause
until about a month later when pressure again came back to normal.
This
increase took place in eggs regardless of whether or not they were exposed
to low temperatures.
This rise in osmotic pressure was attributed by
Roemhild to an increase in the amount of glycerol produced by the embryo
and diffusing through the membrane into the yolk.
Gilmour
(1965)
points
out that stored glycogen is converted into glycerol and sorbitol because
anaerobic mechanisms play an important role in diapause respiration.
Chino
(i960)
suggested that this anaerobic condition was due to an actual
blocking of the electron transport system but his studies did not show
this to be true.
Whatever the energy source, Telfer and Williams
(i960)
state that there are at least three endergonic processes which occur in
diapausing lepidopteran pupae--penetration of the cells b y amino acids,
incorporation of the amino acids into proteins,
into the blood.
and secretion of protein
Maintaining the stable conditions of the cells usually
requires active transport and this would also require energy (Harvey,
1962).
If anaerobic conditions exist in the diapausing egg, oxygen con­
sumption is not a true measure of activity of this stage.
-52. In addition: to physiological processes such as that producing
glycerol and sorbitol,
evidence for -other changes in the diapausing egg
was brought to.light when Van.Horn (1963 ) did a histological.comparison
of A. elljbtti embryos which had been in diapause for varying lengths of
time.
These eggs had been put into the cold for a period of time and
then:removecl to.25°C.
The majority of the eggs treated in this way
finished.development and hatched.
A few, however, did not hatch and
these V a n Horn.used for comparison. •• She found differences in these '
embryos which had been in.diapause fqr varying lengths of time and these
differences indicated.some diapause activity.
For example,
she found an
increase in the size' of gonad cells> cells of the pleuropodial glands,and., fat body.
Svoboda (1964) studied the fatty acid content of the egg
and could not confirm the report of deposition.of fatty material during
diapause but neither did his findings conflict with her studies,
He
stated that small portions of lipid material could have been transferred
from yolk to embryo during diapause although he found no qualitative or
quantitative changes in the lipid classes during this period.
The present
author studied the increase in size of the cells of the pleuropodial glands
•during diapause but could neither confirm nor disprove V a n H o r n ’s findings
on these cells.
Although no increase in size was found,in the embryos
studied, none-had attained the"age of those used by V a n Horn,
V an H o r n ’s
findings indicate that some processes besides those of maintenance continue
during the diapause period of A. elliptti.
These processes would require .'
energy but that energy could be aerobic or, as suggested by the increase
-53in glycerol during this period, anaerobic.
If it is the latter, we could
not measure it b y the oxygen consumption.method.
The increase in oxygen consumption which occurs when A. elliottj
eggs were subjected to higher temperatures agrees with observations made
on most other insects.
Rogers (1929 ) found an increase in oxygen use
correlated with an increase in temperature but only jip to a certain l e v e l .
Beyond this point, oxygen consumption dropped abruptly and she suggested
the temperature at which the drop occurred was the limit of normal physi­
ological activity of eggs of Melanoplus differentialis, the insect with
which she worked.
The same type of pattern has been found for many,
enzymatic reactions .(Keister and Buck, 1964).
actions,
For most enzymatic r e ­
a Q-^q of 2 is found at the lower temperatures b u t .falls
gradually as the temperature is raised until the ratio is I,
higher temperatures it assumes a fractional v a l u e .
At even
This was shown to
be the result of the favorable effect of temperature on the rate of
reaction being counterbalanced by t h e •denaturation of the enzyme at the
higher temperatures (Gortner and Gortner, 1953).
Different interpretations of the results of studies on temperature
effects on respiration have been made.
The three most commonly referred
to are those of Krogh, van't Hoff, a n d .Arrhenius,' Both of the latter two
believed that the effect of temperature on physiological processes follows
the same laws as for an isolated chemical process. . For instance, .van't
.
Hoff thought that the Q-^q for both types of processes would be about 2.
This was found to be true for most- chemical reactions but Arrhenius found
-54Q10 values above 2„
He theorized that the rate of reaction is dependent
on the number of molecules possessing a given energy of activation.
A
straight line should therefore be obtained when the log of the reaction
velocity is plotted against the reciprocal of the absolute temperature.
Krogh did not agree and found that the increment in rate was proportional
to the increment in ,temperature.
Under these ■c o n d i t i o n s a straight line
should be obtained if the rate of oxygen consumption is plotted against
temperature (Rogers, 1929)=
Different, workers interpret their work in different w a y s .
For
example, Birch (1947) working with Calandra and Rhizopertha, Edwards
(1946) with .Musca domestica and Melanotus, and Dirken (1922) with
Periplaneta, found their results supported. Kr o g h ts theory while Heller
(1930 ) interpreted his results as two,
lines.
sometimes three,
intersecting
The strange part is that Heller also interpreted the results
found by Buddenbrock and Rohr (1923 ) as two intersecting lines while the
authors themselves interpreted the results as agreeing with Kr o g h *s
theory.
Although some workers have tried to interpret the temperature-
oxygen consumption relationship as a simple mathematical formula,
Richards (1957) says that any algebraic expression that fits this relation­
ship would be fortuitous.
Putter (1914) pointed out that to try to identify
the processes in a living system by Q1Q values would be impossible because
they m ay be the resultant of opposing forces. ■ The results of this study,
as will be discussed further in this section,
show the futility of applying
any simple equation or constant to the temperature rate of oxygen consumption
r
■-55relationship even within the so-called "physiological range of
temperatures."
A rise in the rate of oxygen consumption with an increase in tempera­
ture was present in all stages but was especially evident in the later
stages.
This difference may be attributed to the greater relative mass of
active enzymes in the late stages as compared to those in earlier, less
developed embryos.
The greater the amount of enzymes, the greater the
activity and therefore, the greater the rate of oxygen consumption at the
higher .temperatures.
At the lower temperatures,
enzyme systems are slowed
down very much and drop to a low level at all stages.
Another source of difference between younger and older eggs would be
the difference in degree of muscular activity.
In an.early egg there is
no muscular movement but such movement is quite evident in later stages.
M u s c u l a r ■activity would be greater at higher temperatures and could account
for the greater difference between the rate of oxygen consumption at lower
and higher temperatures.
In the earlier stages, there would be an increase
in the rate of oxygen consumption due to an increase in basal metabolism
but in the later stages there would be an. increase due to the increase, in
basal metabolism plus an additional increase due to enhanced muscular
activity at higher temperatures.
The patterns found at various stages when oxygen.consumption was
plotted, against temperature did not remain the same during the entire
developmental period.
Also, the temperature interval for the highest Q^ q
was not found in ^he same temperature range for all stages of development.
-56Richards (1964) found that the Q-^q for oxygen consumption in Oncopeltus
eggs was 3 .2 -3 .8 during development hut dropped to 2 .5 after the larvae
hatched.
The values found in-this study were higher and had a much
greater range during the developmental period.
unusually high because Scholander, et. al.
They were not, however,
(1953 ) found Q 10 values up to
50 in-the 0° to -5°C range for the larvae of an arctic species of
Chironbmus.
'Whether the highest Q j_q value found is the optimum temperature
for development is speculative because it is not always the temperature at
which the greatest oxygen consumption takes place but is the temperature
interval in which the rate increased the greatest a m ount.
greatest amount of oxygen is consumed at a temperature,
development is fastest at this temperature.
Even if the
it does not mean -
For example,. in a number of
studies reported by Keister and Buck (1964), the rate of oxygen consumption
times length of time for development was used to determine the total
oxygen consumption for the developmental period.
Some of the insects
studied showed a nearly constant total oxygen consumption over a range of
temperatures but many show a minimal amount at some temperature within the
physiological range.
This, is seldom the higher temperature where the
oxygen consumption rate is greatest but often corresponds to environmental
conditions of the inse c t „
In other words, there seems to be an optimum
temperature for development which corresponds to that temperature at which
total oxygen consumption is least.
Although morphogenesis may increase
s o m e ■at higher temperatures, total oxygen consumption is greater.
According
to the statement b y Harvey (1962 ) referred to earlier, energy is used for
-
maintepance and function also.
57-
There is little that can he termed function
in eggs but at higher temperatures all reactions are faster and these in­
clude the b r e a k d o ™ of enzymes and other cellular materials.
The cost of
maintenance may thus be greater at the higher temperature and account for
at least some of the extra oxygen consumed.
Keister and Buck, however,
point out that the temperatures at which minimum oxygen consumption is
found may not be optimum because they are not necessarily the ones at
which development is most rapid and mortality lowest.
Also to be taken
into consideration when applying laboratory data to field studies is the
observation b y Richards and Suanraksa (1962 ) that eggs exposed to fluctu­
ating temperatures develop more rapidly and have a lower total oxygen
uptake than eggs exposed to a constant temperature.
Roemhild •(1965 a)
reported that the average daily fluctuation during September in the
Bozeman area at one- inch, the depth at which the egg pods of A. elliotti
are found, was 6 ° to 25°C.
The highest Qpg for prediapause stages was found in the 20-27°C and
in the 27-35°C r a n g e s .
tween 16 and 20°C.
The highest Qpg for postdiapause stages was b e ­
To find if this is important for the developing egg,
a study of soil temperatures during pre- and postdiapause periods would
have to be made to determine any correlation.
The maximum oxygen consumption in the prediapause developmental
pattern at 27°C was found in stage l4.
A
of over 8 was found for
the 20- 27 ° C interval for this stage and
a,
of almost 5 for stage 13
.in this interval.
According to V a n Horn (1963 ) many changes take place
-58in these stages.
Some of these, are differentiation of the neuroendocrine
system, the differentiation and development of the oenocyte cells,,origin
of the optic ganglia,
and many other changes.
These activities not only
increased the oxygen consumption very mubh but the high Q10' indicates that
they are very temperature dependent with the greatest increase in oxygen
consumption.occurring as the temperature rose from 20 to 2T°C.
The Q^ q for the temperature interval l6-20°C for stages 18-27 ranges
from 2.5 at stage 2 3 .all the way to 8 .9 at stage 22.
This would indicate
that the oxygen consumption of these stages was. also very temperature
dependent.
.
The highest Q-^q values were in the diapause stage 19 embryos
and those embryos going through blast©kinesis.
Temperature is thought to
play an important role in initiating development after diapause and is
probably also very important for rapid postdiapause development.
These
data show that the greatest change in oxygen consumption, is in these
stages but we can only speculate on its effects on development.
The eggs
used in this study were held at a postdiapause temperature of 25°C.
There was not much difference in Q]_q values for stages 26 and 27
between 16-20° and 20-27°C but some of the oxygen consumption in these
last stages of development is probably due to increased muscular movements
of the embryos which can be seen during this time (Roemhild, 1961 ).
In the 9-16° and l6-20°C intervals,
several,stages showed a decrease
in oxygen consumption as t h e temperature increased.
This decrease in
oxygen consumption with an increase in temperature in part of the tempera­
ture range would be an example of the third type of response in oxygen
-59consmnption due to temperature changes according to A r g o ’s (1939)
classification which was mentioned earlier■and has been reported in many
insects.
When V e r n b e r g :-and Meriney (1957) found two plateaus,
one on
either side of the rearing temperature, they suggested,acclimatization
might be involved.
But such plateaus are not always near the rearing
temperatures for all species.
For example, Edwards and Gonsdlez (1952)
found that oxygen consumption, of the coffee borer was higher at both 30 °
and 40° than at 35°C.
Richards (1959) observed that this phenomenon was •
found, in eggs of Oncopeltus at a point critical for hatching and Bukett
(1962 ) found it at a point critical for spinning in Galleria larvae.
When. Keistdr and Buck (1961 ) found a plateau in Phormia regina larvae
between 10 and 15°C, they did not give any theories of their own to explain.'.this phenomenon but did discount the acclimatization theory of
Vernberg and M e r i n e y .
In Bull o ck’s (1954) review of acclimatization- of
poikliotherms to temperature, he noted that usually the effect was a shift
in the respiration-temperature curve rather than a change in the slope
of the curve.
It should be noted, however, that he did find a change in
slope as well in.a few poikliotherms but this was not reported in the
insects,he mentioned.
Roemhild (1961 ) found a. decrease, in the oxygen use in the regionotif
IO0C in the postdiapause A., elliotti eggs and stated that in the range of
temperatures he utilized (5 -35 °C), a number of different enzyme systems
could be activated or ina'ctivated.
A Q^ q of less than I was not found at the higher temperatures
although a decrease in oxygen consumption could be expected when lethal
-6otemperatures were reached.
Lethal 'temperatures were not used in the
present study.
When a decrease in oxygen consumption with increasing temperature
is found in an organism,, it indicates that the Arrhenius equation can
not be used to predict oxygen consumption at a given temperature.
Arrhenius reported that by using the equation, log
2.3 x..1 . 98 x
= E (T2 -T1 )/
TjT2 ) he could predict E , the energy of activation for a
chemical reaction.
This could be calculated directly by using this
equation and substituting the reaction rates measured at several different
temperatures (Gortner an<^ Gortner, 1953).
In biological work, yK_
is
substituted for E and is termed the temperature characteristic.
E or yAC could also be found from plotting the logarithm of the
velocity against l/T.
is not always true.
This plot should result in a straight line.
This
It has been found that many enzyme systems do not
show a straight line but approximate two straight lines meeting.at an
angle.
Crozier (1924) found the same phenomenon in many plants and
animals, not only for oxygen consumption but for other physiological
activities as well.
Orr (1925 ) found this pattern in the rate of oxygen
consumption, in Drosophila p u p a e .
Richards (1957); on the other hand,
obtained a straight line when he plotted the oxygen consumption of eggs
of Melanoplus differentialis, Ostrinia nubilalis,
Culex pipens,
and Musca
domestica and the larvae of Tribolium confusum but he only measured oxygen
consumption down to 15°C.
When oxygen consumption of A - elliotti eggs
was plotted in this way, neither a straight line nor two intersecting
-61 -/
lines were found for all stages.
In a few stages plotted,
a relatively
straight line was obtained but in most cases one point fell below the line
This was usually a point at which a-Q 1 Q--Of toss •
'than: Ijihailfbeen -iIourid./
The Q1Q values for A. elliotti show a decrease in rate of oxygen con­
sumption with increasing temperatures.
insects but theories vary as to why.
This, as noted,
is found in many
The theory of activation and inacti­
v a t i o n of the many enzyme systems could explain this but would be very
difficult to test.
Even though.it might be possible to find the optimum
conditions for-each system,
it would be very difficult to prove they act
in the same way in the egg and in v i t r o .
More work, not only on the
optimum conditions for each enzyme but also for the way the different
systems interact would be helpful to test the line of reasoning that
activation, and inactivation of the different enzyme systems is the answer
to why oxygen consumption decreases as temperature increases, in part of
the temperature range.
The fact that this decrease in rate was found in
different parts of the temperature range for different‘developmental
stages can be explained
by this theory because enzSymes can change
quantitatively and qualitatively during the developmental period (Allen,
1940I Bodine,
e t . al«, 1954J Bodine and Boell, 1935J Carlson, 194l).
W h e n studying changes.in biochemical reactions, we are not, however,
dealing w i t h just temperature effects. . We must also consider other rate
limiting mechanisms.
Inhibitors,
substrate concentration, pH, and thermo­
dynamics may all effect the rate of reaction.
For example,
if we have a
reaction product acting as a feedback inhibitor, the reaction rate would
-62be slowed down.
It has been found that p H may be changed by temperature
>.
during diapause and this could have an effect on the rate.
Although the
Arrhenius plots are quite close to straight line plots, perhaps that point
at which there is a deviation might be a part of the temperature range
where one of these other rate limiting factors is m o r e :important.
V a n Horn (1963 ) also found another way in which the diapause eggs
of A. elliotti differ from those of other species studied.
For example,
Slifer (19^-6) found that the diapause egg of M. differentialis could be
induced to develop if treated with xylol, toluol, and other wax solvents.
Van.Horn was unsuccessful in breaking diapause in A. elliotti by use of
any of these substances.
In fact, sh e •found the only successful method
for terminating d i a p a u s e ■in this species was prolonged exposure to low
temperatures.
Because of the differences found between M. differentialis
and A., elliotti eggs,. Roemhild (1965 b) carried out several experiments to
further compare the two egg t y p e s .
As a result of these experiments, he
found a difference in the rate of o x y g e n .consumption of the embryo and
yolk isolated from the eggs as compared to the whole egg.
Both prediapause
and postdiapause whole eggs had.a higher respiration rate than the isolated
embryo-yolk but the whole egg had a.lower .rate in the diapausing egg.
Re­
ducing materials in the egg were highest during the diapause stage.
Although it was shown.in the present study that there is a slight decrease
in respiration when.the egg enters diapause, the highest oxygen utilization
for isolated diapause and postdiapause embryos was in diapause yolk
(Roemhild,
1965 b).
Roemhild suggested, therefore, that the maintenance of
-63the diapause state was due to properties of the serosal membrane sepa­
rating the embryo and yolk.
Studies have shown that hydrogen ions'' and
other materials occur-in considerably different concentrations on the t w o ■
sides of the m e m brane„
Using this model,, it is assumed that the membrane
wo u l d be impermeable to the easily oxidized materials but when the membrane
was broken at diapause termination, thq embryo could utilize the materials
from, the yolk.
Some support for this idea is given by the f a c t .that the
materials used by- Slifer (1948) to terminate diapause in M. differentlalis
eggs are capable of changing permeability.
The lack of decrease in oxygen
consumption between l 6 and I1O 0 C-indicates that membrane permeability seems
to have been affected by low temperatures.
This could be a direct tempera­
ture effectj that is, the permeability of the membrane is increased at low temperatures but more likely it is due to an indirect effect on active
transport systems which maintain the differences in materials on either
side of the m e m brane„
The last possibility appears most probable: since
it has usually been found that the rate of entry of compounds into a cell
is increased by an increase in temperature (G-iese, 1962 ).
Active -transr
port which maintains differences on the two sides of the membrane may
be slowed down by low temperatures.
This would allow substrate materials
to diffuse, across the serosal'membrane into the embryo compartment. .The
entry and use of these easily oxidized substrates could explain the higher
than expected rate of oxygen use at IO0C .(Roemhild, 1961 ) and the very
s l i g h t ,increase between 9 and. l6 °C found by the present author in dia­
pause eggs of A. elliotti.
-64Because of the differences found, the effects of inhibitors and
stimulants on diapausing eggs of A. elliotti would be interesting to
compare to findings of other researchers.
Bodine (1932, 1934) found
the oxygen consumption of idapausing- M.. differentialis was insensitive
to cyanide and carbon monoxide but this is not true of a l l .insects.
I
Those insects which have a high respiration rate during diapause are
not usually cyanide resistant.
Both diapausing Gasterophilus intesinalis
Be Geer (Levenbook, 1951) and Leptinotarsa decemlineata (Lees, 1959) are
killed.by cyanide.
These comparisons show different conditions exist in
different insects.
The differences in diapause oxygen consumption and termination in
different insects, along with the fact that diapause occurs in different
stages in different insects and can be either facultative or
obligatory,
seem to support Lees (1955) theory that the diapause mechanism is not
the same in all insects.
He believes that it may even differ between
species within the same g e n u s .
He suggests that these differences indi­
cate that diapause has evolved independently in -many cases... Andrewartha
(1952) does not agree with Lees because he believes that diapause, is
essentially the same in all insects.
This study has shown that A. elliotti eggs do not follow the usual
pattern of oxygen consumption in response to temperature during the
period of development.
They differ from the usual, pattern of most other
insects which have a diapause in that there is no sudden large drop in
the amount of oxygen utilized when this species enter obligatory diapause.
It -was also observed that at lower temperatures, the response of oxygen
consumption to temperature increases is not a.straight line; therefore,
no equation or constant can be used to predict the oxygen consumption.at
a given t emperature.
There was a correlation in amount of oxygen utilized
a n d .development of the embr y o .
In some of the developmental stages.where
embryological studies (Van Horn, 1963 ). have shown an.increase in this
activity,, an increase in oxygen consumption was also f o u n d .
however,
More work,
is needed before the optimum temperature for development of
A.- elliotti is known.
SUMMARY
Oxygen.consumption at various stages during the embryonic
development of A. elliotti was measured at various temperatures.
All-
measurements were done with a Cartesian diver micro-respirometer.
The
QlO for each temperature interval was calculated, using v a n ’t Hoff's
equatio'n.
Oxygen consumption during the prediapause stages was fairly .
constant from stage to stage for all temperatures utilized.
At the
higher temperatures, there was a peak in oxygen.consumption at develop­
mental stage l4, a time when histological studies have shown much
activity to be going on.
'
There was no great decrease in the amount of oxygen consumed when
the egg entered diapause and oxygen consumption remained at about the
same level throughout the diapause period.
Postdiapause oxygen.consumption varied from stage to stage but the
pattern for this-period was the same for all temperatures.
The peak
in oxygen.consumption was at developmental stage 25 when dorsal closure
was completed.
The highest Qpo for most prediapause eggs was in the 20-27° and
27-35°C- intervals.
The highest Q^ q for all stages beginning with the
one preceding diapause until hatching was in the.l6-20°C interval.
A number of stages showed the same oxygen consumption at 9 and l 6 °
while other stages used the same amount of oxygen at l 6 and 20°C.
-67A histological comparison of the pleuropodial glands of embryos
which had been in diapause for various lengths of time and which had
received.different time exposures to cold was made to determine any
change in the size of the cells during this period when little apparent
development is going on.
Uo change could be found; therefore, very
little energy-utilizing biosynthesis was going on. in the pleuropodial
glands during diapap.se.
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