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Insect hormones: Their effects on diapause and development of hymenoptera
Vol . 8, Part II, pp. 767-774, 1969. Life Sciences Printed in Great Britain .
Pergamon Press
INSECT HORMONES : THEIR EFFECTS ON DIAPAUSE AND DEVEIAPMENT OF HYlfENOPTERA* Catherine Hsiao and Ting H . Haiso Department of .Zoology, Utah State University, I.ogan, Utah
84321
(Received 17 March 1969; in final form 29 April 1969)
In insecte the immediate cause of Larval and pupal diapause is a lack of ecdysone, the growth and molting hormone . prothoracic glands which,
Since ecdysone ie secreted by the
in turn, are activated by the brain hormone,
diapause can be terminated by introducing either ecdysone or the brain hormone (1,2) .
Ecdyeone and its analogs have been shown to terminate pupal
diapause ~n Ispidoptera (3,4) and Diptera (5) .
Juvenile horaane has also
been demonstrated to have a prothoracotropic effect and thus terminate pupal diapause in Lepidoptera (6,7,8),
but not in Diptera (5) .
Juvenile hormone,
however, produces sarphogenetic effects on adult development of I.epidoptera and Diptera (5,6,9) .
In an early study, Church
(10) demonstrated that larval
diapause of the wheat atom sawfly, Cephus cinctus , was under hormonal control . However, no study has hitherto been made on the effect of synthetic or purified insect hormones on diapauaing Hymenoptera . Materials and Methods Our experiments were performed on two species of solitary bees which are important pollinators of alfalfa .
The alkali bee, Nomia melanderi
Cockerell, has 1 generation per year in Utah . inmobile prepupa in the soil .
It enters diapause as an
The leaf-cutter bee, Megachile rotundata (F .),
has 1 to 2 generations per year and enters diapause as a mobile mature larva inside a cell made of leaf materials . throughout the winter months .
*Paper No . 904,
Both species remain in diapause
The diapausing insects used in the present
Utah Agricultural Experiment Station
767
788
INSECT HORMONES
Vol. 8, No. 14
study were collected from the field in the fall and kept in cold storage (5 °C)
for about 2 months .
Neither species began development within 40 days
while being incubated at 26 .5 °C .
They were, therefore, still in a diapaueing
state . Synthetic d.ecdysone,
juvenile hormone (methyl trana , trans , cis-10-
epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate), three phytoecdysones (cyasterone, ecdysterone and inokosterone), sulfate and zinc acetate were tested .
d-Ecdysone, copper sulfate and zinc
acetate were dissolved in distilled water, and cholesterol and ~-sitoaterol in refined olive oil . solvents or carrier .
cholesterol, ~-sitoaterol, copper
phytoecdysones in 107 isopropanol
in 10% ethanol .
Juvenile hormone was carried
The control insects were treated with the corresponding Injections were made into the abdomen .
Depending on the
size of the insect, the amount of solution injected per individual varied from 0 .5 to 2 .0 ul .
After treatment,
the insects were kept in a chamber with a
photoperiod of 16 hours light and 8 hours darkness at 26 .5 ° C and 50% relative humidity .
They were observed twice daily to determine the stage of develop-
ment . Results and Discussion When diapause was terminated experimentally, the immobile prepupa of the alkali bee began to wriggle on the 5th day after injection . morphological changes could be observed at this time .
No noticeable
On the 7th day,
wriggling became more vigorous and pupation occurred 1 day later . stage lasted from 10 to 14 days, after which the =dult emerged . leaf-cutter bee, injection .
The pupal In the
the termination of diapause occurred on the 7th day after
This was indicated by the transformation of the wriggling larva
into an immobile prepupa .
Pupation occurred 3 to 5 days later and the adults
emerged in about 10 days . Synthetic a~ ecdysone, cyasterone, ecdysterone and inokoaterone were all effective in terminating diapause of the alkali bee (Table 1) . body weight of the injected prepupae was 140 mg .
The average
ot-Ecdysone was tested at
Vol. 8, No . 14
INSECT HORMONES
0 .05, 0 .125, 0 .25, 0 .50 and 1 .0 ug per prepupa.
769
A minimum dose of 0.125 ug
was required to cause most of the diapausing alkali bee prepupae to develop. At doses up to 1 .0 ug per prepupa, normal morphologically .
all the resulting pupae and adults were
The three phytoecdysones were injected with doses
ranging from 0.01 to 2 .0 ug per prepupa .
Table 1 shows that the minimum
Table 1 Minimum done of four ecdysonea required to induce normal development Ten prepupae were used of diapausing prepupae of the alkali bee . in each treatment Compound
Dose per prepupa (ug)
No . pupated within 14 days
No . remaining in diapause
at.ecdysone
~.125
8
2
Cyasterone
0 .016
LO
0
Ecdysterone
0.125
10
0
Inokosterone
0 .125
9
1
Diat . H20
1 ul
0
LO
10% isopropanol
1 ul
0
10
109 ethanol
1 ul
0
10
effective doses for inducing normal development were 0 .016 ug for Cyasterone and 0 .125 ug for ecdysterone and inokoeterone .
At doses above this level,
abnormal development of the treated pupae was observed with the phytoecdysones . This result indicated that the three phytoecdysones have a very narrow range of effective dose inducing normal development as compared to o~-ecdysone . Cyasterone was the most active compound tested .
It was effective at a dose
nearly 10 times leas than that of the other ecdysonea .
The order of
effectiveness of the four ecdysonea to the alkali bee is similar to that reported for the oak silkworm, Samia cynthia, by Williams (4) .
However,
on
the basis of unit dose per gram fresh weight, the alkali bee appears to be about 5 times more sensitive than S . cynthia to the ecdysonea .
The minimum
effective dose for ~ecdysone is estimated at 0.9 ug per gram for the alkali bee, whereas Williams reported a minimum effective dose of 5 ug per gram
INSECT HORMONES
770
Vol. 8, No. 14
for S . cynthia . Prepupae treated with excessive phytoecdysonea failed to shed their prepupal cuticle and subsequently died . showed a series of abnormal forma .
Upon dissection, these insects
In the least abnormal cases, pupae
developed with appendages having fewer segments than normal .
In the more
abnormal cases, a distinct but thin cuticle was secreted by the epidermis and However, moat of these
molting fluid was present between the two cuticles .
individuals did not differentiate and showed no pupal characters .
In some
individuals of this group (about 10X), pupal differentiation had occurred and resulted in the formation of four layers of cuticles .
These could be
recognized as the old prepupal, 2nd prepupal, pupal and untanned adult cutiIlea .
All of these four cuticles were deposited within 14 days .
extreme cases,
In the
prepupae became pinkish brwn in color about 3 days after
injection with the prepupal epidermis retracted from the cuticle . between vas filled with molting fluid .
The space
The retracted tissues did not form
a nev cuticle and no further differentiation was observed .
Aberrant development
caused by excessive amounts of ecdysones~have been reported in several instances (3,4,11) .
The arrested pupal differentiation observed in the
present study apparently was caused by an excessive dose of ecdysones supplied too early in the developmental process .
This resulted in precocious molting
and inhibited further developmental events . Diapausing larvae of the leaf-cutter bee were injected with ,(-ecdysone at doses of 0 .015, 0 .037, 0 .15, and 0 .3 ug per Larva (average body weight 50 mg) .
The diapause was terminated in all cases .
was found among the treated and control larvae .
Aowever, high mortality
For this reason, other
compounds vets not tested on thin insect . Cholesterol, ~-sitoaterol, copper sulfate and zinc acetate have been reported to induce development of brainless saturnöd pupae (2,12) .
These
compounds were tested to determine if they could also terminate diapause of the alkali bee .
Cholesterol and F-sitosterol were tested between 0.1 and L .0
Vol . 8, No. 14
INSECT HORMONES
ug per prepupa .
771
Copper sulfate and zinc acetate were injected in amounts of
20 to 500 ug per prepupa . pause of the alkali bee .
None of these compounds terminated prepupal diaCopper sulfate was toxic above 100 ug per prepupa:
Synthetic juvenile hormone (,T$) injected alone did not provoke development of diapausing prepupae of the alkali bee (Table 2) .
When it wa
injected simultaneously with ecdysone, prepupae molted to normal pupae,
but
subsequently developed into a aeries of papal-adult intermediates which were unable to abed their papal cuticle .
In this respect, the effects of JH and
ecdysone are clearly distinguishable in the alkali bee since JH exerts no effect on the papal morphogenesis even when applied at extremely high doses . Table 2 The effect of juvenile hormone (JH) alone and in combination with .4 ecdysone on the development of diapauaing prepupae of the alkali bee . Five individuals were used in each treatment . Dose per prepupa (ug)
No . pupated within 14 days
No . remaining in diapause
Adult development
JN + ecdysone 0 .3 + 0.25
5
0
Adults with papal cuticle at the site of injection
0 .6 + 0.25
5
0
Papal-adult intermediates
1 .2 + 0.25
5
0
Intermediates with 2nd papal cuticle
0 .3
0
5
0.6
0
5
1 .2
0
5
Olive oil 1 ul
0
5
Olive oil 1 ul + ecdysone(0 .25)
5
0
JN alone
Normal adults
The degree of abnormality produced by JH could be determined through the transparent papal cuticle .
The first sign of a juvenilizing effect was
772
INSECT HORMONES
Vol. 8, No. 14
indicated by abnormal pigmentation of the outer edge of the compound eyes . In contrast, the compound eyes of the normal pupae were an even pink color .
Ab-
normal eye pigmentation as a result of JH treatment has also been reported in Lepidoptera(13) and Diptera(5) . 5,00'7 Tenebrio Units(8)),
At a dose of 0 .3 ug JH per prepupa (1 ug
the eye pigmentation was nearly normal . Most of the
pupae developed into adults with only a patch of typical pupal cuticle visible at the site of injection .
At 0 .6 ug JH per prepupa, half of the outer edges of
the eyes pigmented and the head was tanned with small patches of pupal cuticle at the base of the antennae and around the eyes . The antennae were partially tanned and the thorax was tanned only at the dorsum .
The legs were tanned ex-
cept for a large patch of pupal cuticle on the femur .
The abdomen remained in
pupal form except for small patches of irregularly distributed adult cuticle . At a dose of 1 .2 ug JH per prepupa,
all the insects had two pupal molts
in the sequence of a pupal-pupal molt and the normal pupal-adult molt . lower dose of~JH, only one pupal-adult molt was observed . intermediates obtained with
At a
The pupal-adult
1 .2 ug JH showed more adult characters than those
trèated with 0 .6 ug JH per prepupa . These intermediates exhibited a Limited degree of eye pigmentation, which occurred only as a narrow line on the outer edges of the eyes .
Antennae were partially tanned .
The thorax and abdomen
were mostly in the adult form and the cuticle was tanned with numerous hairs . Pupal characters were visible only near
the sutures and intersegmental
membranes . In the normal
co~~rse of adult development of the alkali bee,
the
initiation of the cuticular tanning process occurred on the 10th day after pupation and adult ecdysis took place on the~l4th day .
Individuals treated
with JH at 0 .3 and '1 .6 ug showed cuticular tanning on the 7th day after pupation, or about 3 days earlier than in siormal development .
With an
injection of 1 .2 ug JH, the extra pupal-pupal molt completed on the 7th day after pupation and adult cuticular tanning occurred on the 10th day as for normal development .
The present finding suggests that JH not only preserved
Vol. 8, No. 14
773
INSECT HORMONES
the papal characters but also greatly accelerates adult development .
Whether
or not the precocious development of adult characters results from the direct effect of JH or the indirect stimulation of the prothoracic glands by JH is not clear .
The fact that JH alone did not terminate the prepupal diapause of the
alkali bee suggests that the prothoracic glands are not responsive at this stage .
It is, however, possible that in the papal stage the prothoracic
glands are sensitive to stimulation by JH . The differences in the effects of the ecdysones and JH in the diapausing alkali bee were rather striking .
Excessive ecdysone profoundly affected the
papal morphogenesis, whereas JH only influenced the adult morphogenesis . However,
the period in which the bee was susceptible to these two hormones
was similar as demonstrated by injection of excessive quantities of the ecdysones (2 .0 ug ecdysterone or 1 .0 ug cyasterone) or JH (1 .2 ug) into newly molted pupae .
Neither hormone affected the subsequent adult morphogenesis,
indicating that the critical period was between the initiation of breaking diapause and pupation .
In this respect,
the present finding is similar to
that reported by Williams for S . Cynthia (4) where excessive doses of ecdysone and JH could not induce morphogenesic effects more than 60 hours after initiation of adult development . The present study demonstrated for the first time that ecdysones and synthetic juvenile hormone have profound effects on the larval and prepupal diapause and adult differentiation of Hymenoptera .
Like Diptera,
the two
diapausing hymenopteran species investigated show a high degree of specificity to true insect hormones and not to non-specific substances .
The fact that
the alkali bee responded to a very low dose of the ecdysones tested suggests that it is an ideal insect species for the assay of ecdysones and for the study of interactions of different insect hormones . Summary Prepupal diapause of the alkali bee, Nomia melanderi , was terminated by injection of ~l-ecdysone and three phytoecdysones (cyasterone,
ecdysterone
774
INSECT HORMONFS
and inokoaterone) .
Yol. 8, No. 14
Synthetic juvenile hormone, cholesterol, a-sitosterol,
copper sulfate and ainc acetate had no effect . injected simultaneously with .C-ecdysone,
When juvenile hormone was
the diapausing prepupae molted into
normal pupae, but later developed into papal-adult intermediates .
.L-Edysone
also terminated the larval diapause of the leaf-cutter bee, Mestachile rotundata . Ac knowledAmenta We are grateful to Prof . H. Röller of Texas A ~ M University for samples of synthetic juvenile hormone,
to Prof . T. Takemoto of Japan for samples of
3 phytoecdysonea, to Dr . J. Siddall of Zoecon Corporation for samples of synthetic ,~ecdysone and to Drs. G. E. Bohart and W. A . Brindley of our department for a supply of leaf-cutter and alkali bees . References 1.
W. R. Harvey, Annu . Rev . Entomol .
7, 57 (1962) .
2.
H. A. Schneiderman and L. I . Gilbert,
3.
M. Robayaehi, T. Takemoto, S . Ogawa and N. Nishimoto, J . Insect Physiol . 13, 1395 (1967) .
4.
C. M. Williams, Biol . Bull .
5.
G. Fraenkel and C . Heiao, J . Insect Physiol . 14, 707 (1968) .
6.
C. M. Williams, Biol . Bull . 116, 323 (1959) .
7.
L. I . Gilbert and H. A . Schneidermau, Nature 184,
8.
H. R311er and H. A . Dahin, Recent Prortr . Hormones Rea . 24, 651 (1968) .
9.
U. S . Srivaetava and L. I. Gilbert, Science , 161,
Science , 143, 325 (1964) .
134, 344 (1968) .
171 (1959) .
61 (1968) .
10 .
N. S . Church, Caned . J . Zool . ~, 339 (1955) .
11 .
K. Madhavan and A. A . Schneideruran, J . Insect Physiol . 14, 777 (1968) .
12 .
C. M. Williams, In I~secta and PhyeioloSV , P . 133, Ed . J. W. L . Beamen t and J. E . Treherne, Slsevier, American, New York (1968) .
13 .
C . M. Williams, Biol . Bull . 121,
572 (1961) .
Pergamon Press
INSECT HORMONES : THEIR EFFECTS ON DIAPAUSE AND DEVEIAPMENT OF HYlfENOPTERA* Catherine Hsiao and Ting H . Haiso Department of .Zoology, Utah State University, I.ogan, Utah
84321
(Received 17 March 1969; in final form 29 April 1969)
In insecte the immediate cause of Larval and pupal diapause is a lack of ecdysone, the growth and molting hormone . prothoracic glands which,
Since ecdysone ie secreted by the
in turn, are activated by the brain hormone,
diapause can be terminated by introducing either ecdysone or the brain hormone (1,2) .
Ecdyeone and its analogs have been shown to terminate pupal
diapause ~n Ispidoptera (3,4) and Diptera (5) .
Juvenile horaane has also
been demonstrated to have a prothoracotropic effect and thus terminate pupal diapause in Lepidoptera (6,7,8),
but not in Diptera (5) .
Juvenile hormone,
however, produces sarphogenetic effects on adult development of I.epidoptera and Diptera (5,6,9) .
In an early study, Church
(10) demonstrated that larval
diapause of the wheat atom sawfly, Cephus cinctus , was under hormonal control . However, no study has hitherto been made on the effect of synthetic or purified insect hormones on diapauaing Hymenoptera . Materials and Methods Our experiments were performed on two species of solitary bees which are important pollinators of alfalfa .
The alkali bee, Nomia melanderi
Cockerell, has 1 generation per year in Utah . inmobile prepupa in the soil .
It enters diapause as an
The leaf-cutter bee, Megachile rotundata (F .),
has 1 to 2 generations per year and enters diapause as a mobile mature larva inside a cell made of leaf materials . throughout the winter months .
*Paper No . 904,
Both species remain in diapause
The diapausing insects used in the present
Utah Agricultural Experiment Station
767
788
INSECT HORMONES
Vol. 8, No. 14
study were collected from the field in the fall and kept in cold storage (5 °C)
for about 2 months .
Neither species began development within 40 days
while being incubated at 26 .5 °C .
They were, therefore, still in a diapaueing
state . Synthetic d.ecdysone,
juvenile hormone (methyl trana , trans , cis-10-
epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate), three phytoecdysones (cyasterone, ecdysterone and inokosterone), sulfate and zinc acetate were tested .
d-Ecdysone, copper sulfate and zinc
acetate were dissolved in distilled water, and cholesterol and ~-sitoaterol in refined olive oil . solvents or carrier .
cholesterol, ~-sitoaterol, copper
phytoecdysones in 107 isopropanol
in 10% ethanol .
Juvenile hormone was carried
The control insects were treated with the corresponding Injections were made into the abdomen .
Depending on the
size of the insect, the amount of solution injected per individual varied from 0 .5 to 2 .0 ul .
After treatment,
the insects were kept in a chamber with a
photoperiod of 16 hours light and 8 hours darkness at 26 .5 ° C and 50% relative humidity .
They were observed twice daily to determine the stage of develop-
ment . Results and Discussion When diapause was terminated experimentally, the immobile prepupa of the alkali bee began to wriggle on the 5th day after injection . morphological changes could be observed at this time .
No noticeable
On the 7th day,
wriggling became more vigorous and pupation occurred 1 day later . stage lasted from 10 to 14 days, after which the =dult emerged . leaf-cutter bee, injection .
The pupal In the
the termination of diapause occurred on the 7th day after
This was indicated by the transformation of the wriggling larva
into an immobile prepupa .
Pupation occurred 3 to 5 days later and the adults
emerged in about 10 days . Synthetic a~ ecdysone, cyasterone, ecdysterone and inokoaterone were all effective in terminating diapause of the alkali bee (Table 1) . body weight of the injected prepupae was 140 mg .
The average
ot-Ecdysone was tested at
Vol. 8, No . 14
INSECT HORMONES
0 .05, 0 .125, 0 .25, 0 .50 and 1 .0 ug per prepupa.
769
A minimum dose of 0.125 ug
was required to cause most of the diapausing alkali bee prepupae to develop. At doses up to 1 .0 ug per prepupa, normal morphologically .
all the resulting pupae and adults were
The three phytoecdysones were injected with doses
ranging from 0.01 to 2 .0 ug per prepupa .
Table 1 shows that the minimum
Table 1 Minimum done of four ecdysonea required to induce normal development Ten prepupae were used of diapausing prepupae of the alkali bee . in each treatment Compound
Dose per prepupa (ug)
No . pupated within 14 days
No . remaining in diapause
at.ecdysone
~.125
8
2
Cyasterone
0 .016
LO
0
Ecdysterone
0.125
10
0
Inokosterone
0 .125
9
1
Diat . H20
1 ul
0
LO
10% isopropanol
1 ul
0
10
109 ethanol
1 ul
0
10
effective doses for inducing normal development were 0 .016 ug for Cyasterone and 0 .125 ug for ecdysterone and inokoeterone .
At doses above this level,
abnormal development of the treated pupae was observed with the phytoecdysones . This result indicated that the three phytoecdysones have a very narrow range of effective dose inducing normal development as compared to o~-ecdysone . Cyasterone was the most active compound tested .
It was effective at a dose
nearly 10 times leas than that of the other ecdysonea .
The order of
effectiveness of the four ecdysonea to the alkali bee is similar to that reported for the oak silkworm, Samia cynthia, by Williams (4) .
However,
on
the basis of unit dose per gram fresh weight, the alkali bee appears to be about 5 times more sensitive than S . cynthia to the ecdysonea .
The minimum
effective dose for ~ecdysone is estimated at 0.9 ug per gram for the alkali bee, whereas Williams reported a minimum effective dose of 5 ug per gram
INSECT HORMONES
770
Vol. 8, No. 14
for S . cynthia . Prepupae treated with excessive phytoecdysonea failed to shed their prepupal cuticle and subsequently died . showed a series of abnormal forma .
Upon dissection, these insects
In the least abnormal cases, pupae
developed with appendages having fewer segments than normal .
In the more
abnormal cases, a distinct but thin cuticle was secreted by the epidermis and However, moat of these
molting fluid was present between the two cuticles .
individuals did not differentiate and showed no pupal characters .
In some
individuals of this group (about 10X), pupal differentiation had occurred and resulted in the formation of four layers of cuticles .
These could be
recognized as the old prepupal, 2nd prepupal, pupal and untanned adult cutiIlea .
All of these four cuticles were deposited within 14 days .
extreme cases,
In the
prepupae became pinkish brwn in color about 3 days after
injection with the prepupal epidermis retracted from the cuticle . between vas filled with molting fluid .
The space
The retracted tissues did not form
a nev cuticle and no further differentiation was observed .
Aberrant development
caused by excessive amounts of ecdysones~have been reported in several instances (3,4,11) .
The arrested pupal differentiation observed in the
present study apparently was caused by an excessive dose of ecdysones supplied too early in the developmental process .
This resulted in precocious molting
and inhibited further developmental events . Diapausing larvae of the leaf-cutter bee were injected with ,(-ecdysone at doses of 0 .015, 0 .037, 0 .15, and 0 .3 ug per Larva (average body weight 50 mg) .
The diapause was terminated in all cases .
was found among the treated and control larvae .
Aowever, high mortality
For this reason, other
compounds vets not tested on thin insect . Cholesterol, ~-sitoaterol, copper sulfate and zinc acetate have been reported to induce development of brainless saturnöd pupae (2,12) .
These
compounds were tested to determine if they could also terminate diapause of the alkali bee .
Cholesterol and F-sitosterol were tested between 0.1 and L .0
Vol . 8, No. 14
INSECT HORMONES
ug per prepupa .
771
Copper sulfate and zinc acetate were injected in amounts of
20 to 500 ug per prepupa . pause of the alkali bee .
None of these compounds terminated prepupal diaCopper sulfate was toxic above 100 ug per prepupa:
Synthetic juvenile hormone (,T$) injected alone did not provoke development of diapausing prepupae of the alkali bee (Table 2) .
When it wa
injected simultaneously with ecdysone, prepupae molted to normal pupae,
but
subsequently developed into a aeries of papal-adult intermediates which were unable to abed their papal cuticle .
In this respect, the effects of JH and
ecdysone are clearly distinguishable in the alkali bee since JH exerts no effect on the papal morphogenesis even when applied at extremely high doses . Table 2 The effect of juvenile hormone (JH) alone and in combination with .4 ecdysone on the development of diapauaing prepupae of the alkali bee . Five individuals were used in each treatment . Dose per prepupa (ug)
No . pupated within 14 days
No . remaining in diapause
Adult development
JN + ecdysone 0 .3 + 0.25
5
0
Adults with papal cuticle at the site of injection
0 .6 + 0.25
5
0
Papal-adult intermediates
1 .2 + 0.25
5
0
Intermediates with 2nd papal cuticle
0 .3
0
5
0.6
0
5
1 .2
0
5
Olive oil 1 ul
0
5
Olive oil 1 ul + ecdysone(0 .25)
5
0
JN alone
Normal adults
The degree of abnormality produced by JH could be determined through the transparent papal cuticle .
The first sign of a juvenilizing effect was
772
INSECT HORMONES
Vol. 8, No. 14
indicated by abnormal pigmentation of the outer edge of the compound eyes . In contrast, the compound eyes of the normal pupae were an even pink color .
Ab-
normal eye pigmentation as a result of JH treatment has also been reported in Lepidoptera(13) and Diptera(5) . 5,00'7 Tenebrio Units(8)),
At a dose of 0 .3 ug JH per prepupa (1 ug
the eye pigmentation was nearly normal . Most of the
pupae developed into adults with only a patch of typical pupal cuticle visible at the site of injection .
At 0 .6 ug JH per prepupa, half of the outer edges of
the eyes pigmented and the head was tanned with small patches of pupal cuticle at the base of the antennae and around the eyes . The antennae were partially tanned and the thorax was tanned only at the dorsum .
The legs were tanned ex-
cept for a large patch of pupal cuticle on the femur .
The abdomen remained in
pupal form except for small patches of irregularly distributed adult cuticle . At a dose of 1 .2 ug JH per prepupa,
all the insects had two pupal molts
in the sequence of a pupal-pupal molt and the normal pupal-adult molt . lower dose of~JH, only one pupal-adult molt was observed . intermediates obtained with
At a
The pupal-adult
1 .2 ug JH showed more adult characters than those
trèated with 0 .6 ug JH per prepupa . These intermediates exhibited a Limited degree of eye pigmentation, which occurred only as a narrow line on the outer edges of the eyes .
Antennae were partially tanned .
The thorax and abdomen
were mostly in the adult form and the cuticle was tanned with numerous hairs . Pupal characters were visible only near
the sutures and intersegmental
membranes . In the normal
co~~rse of adult development of the alkali bee,
the
initiation of the cuticular tanning process occurred on the 10th day after pupation and adult ecdysis took place on the~l4th day .
Individuals treated
with JH at 0 .3 and '1 .6 ug showed cuticular tanning on the 7th day after pupation, or about 3 days earlier than in siormal development .
With an
injection of 1 .2 ug JH, the extra pupal-pupal molt completed on the 7th day after pupation and adult cuticular tanning occurred on the 10th day as for normal development .
The present finding suggests that JH not only preserved
Vol. 8, No. 14
773
INSECT HORMONES
the papal characters but also greatly accelerates adult development .
Whether
or not the precocious development of adult characters results from the direct effect of JH or the indirect stimulation of the prothoracic glands by JH is not clear .
The fact that JH alone did not terminate the prepupal diapause of the
alkali bee suggests that the prothoracic glands are not responsive at this stage .
It is, however, possible that in the papal stage the prothoracic
glands are sensitive to stimulation by JH . The differences in the effects of the ecdysones and JH in the diapausing alkali bee were rather striking .
Excessive ecdysone profoundly affected the
papal morphogenesis, whereas JH only influenced the adult morphogenesis . However,
the period in which the bee was susceptible to these two hormones
was similar as demonstrated by injection of excessive quantities of the ecdysones (2 .0 ug ecdysterone or 1 .0 ug cyasterone) or JH (1 .2 ug) into newly molted pupae .
Neither hormone affected the subsequent adult morphogenesis,
indicating that the critical period was between the initiation of breaking diapause and pupation .
In this respect,
the present finding is similar to
that reported by Williams for S . Cynthia (4) where excessive doses of ecdysone and JH could not induce morphogenesic effects more than 60 hours after initiation of adult development . The present study demonstrated for the first time that ecdysones and synthetic juvenile hormone have profound effects on the larval and prepupal diapause and adult differentiation of Hymenoptera .
Like Diptera,
the two
diapausing hymenopteran species investigated show a high degree of specificity to true insect hormones and not to non-specific substances .
The fact that
the alkali bee responded to a very low dose of the ecdysones tested suggests that it is an ideal insect species for the assay of ecdysones and for the study of interactions of different insect hormones . Summary Prepupal diapause of the alkali bee, Nomia melanderi , was terminated by injection of ~l-ecdysone and three phytoecdysones (cyasterone,
ecdysterone
774
INSECT HORMONFS
and inokoaterone) .
Yol. 8, No. 14
Synthetic juvenile hormone, cholesterol, a-sitosterol,
copper sulfate and ainc acetate had no effect . injected simultaneously with .C-ecdysone,
When juvenile hormone was
the diapausing prepupae molted into
normal pupae, but later developed into papal-adult intermediates .
.L-Edysone
also terminated the larval diapause of the leaf-cutter bee, Mestachile rotundata . Ac knowledAmenta We are grateful to Prof . H. Röller of Texas A ~ M University for samples of synthetic juvenile hormone,
to Prof . T. Takemoto of Japan for samples of
3 phytoecdysonea, to Dr . J. Siddall of Zoecon Corporation for samples of synthetic ,~ecdysone and to Drs. G. E. Bohart and W. A . Brindley of our department for a supply of leaf-cutter and alkali bees . References 1.
W. R. Harvey, Annu . Rev . Entomol .
7, 57 (1962) .
2.
H. A. Schneiderman and L. I . Gilbert,
3.
M. Robayaehi, T. Takemoto, S . Ogawa and N. Nishimoto, J . Insect Physiol . 13, 1395 (1967) .
4.
C. M. Williams, Biol . Bull .
5.
G. Fraenkel and C . Heiao, J . Insect Physiol . 14, 707 (1968) .
6.
C. M. Williams, Biol . Bull . 116, 323 (1959) .
7.
L. I . Gilbert and H. A . Schneidermau, Nature 184,
8.
H. R311er and H. A . Dahin, Recent Prortr . Hormones Rea . 24, 651 (1968) .
9.
U. S . Srivaetava and L. I. Gilbert, Science , 161,
Science , 143, 325 (1964) .
134, 344 (1968) .
171 (1959) .
61 (1968) .
10 .
N. S . Church, Caned . J . Zool . ~, 339 (1955) .
11 .
K. Madhavan and A. A . Schneideruran, J . Insect Physiol . 14, 777 (1968) .
12 .
C. M. Williams, In I~secta and PhyeioloSV , P . 133, Ed . J. W. L . Beamen t and J. E . Treherne, Slsevier, American, New York (1968) .
13 .
C . M. Williams, Biol . Bull . 121,
572 (1961) .