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Changes in the morphogenetic response of Tenebrio molitor pupae to juvenile hormone in relation to age
x Insect Physiol., 1973,Vol.19,~~. 773to780.Pergamon Press. Printed inGreat Britain
CHANGES IN THE MORPHOGENETIC RESPONSE OF TENEBRIO MOLJTOR PUPAE TO JUVENILE HORMONE IN RELATION TO AGE* GUNDA
REDDY
and A. KR1SHNAKUMARA.N
Department of Biology, Marquette University, Milwaukee, Wisconsin 53233 (Received 24 August 1972) Abstract-Morphogenetic effect of juvenile hormone (JH) and its analogues, dodecyl methyl ether, ethyl trimethyl dodecadienoate and methylenedioxyphenoxy-6-epoxy-3-ethyl-7-methyl-2-nonene, on carefully timed Tenebrio pupae was determined. These results show that the response of pupal epidermal cells to JH varied with age during the first 48 hr after larval-pupal ecdysis. The pupae showed low morphogenetic response soon after pupal ecdysis but their response increased gradually until 18 hr. The response to JH decreased in pupae older than about 32 hr; and 48 hr old pupae were unresponsive to low doses of JH employed in this study. Age-related differences in the pattern of response of the individual body regions to JH were also observed. The synergistic effect of 1 pg of ecdysterone with these JH compounds was also tested in relation to the age of Tenebrio pupa. The results show that the synergistic effect of ecdysterone was generally limited to > 18 hr old pupae. This suggests that the physiological basis of the synergistic effect of ecdysterone may be the latter’s ability to synchronize epidermal cells. The significance of these observations in the analysis of time of action of juvenile hormone is discussed. INTRODUCTION
THE TENEBRIO pupal bioassay is widely used to identify and measure the morphogenetic potency of insect juvenile hormone (JH) and its synthetic analogues (KARLSONand NACHTIGALL,1961; BOWERSand THOMPSON,1963; R~~LLERet al., 196.5; ROSE et al., 1968). Routinely 0 to 24 hr old Tenebrio pupae were used for this assay. Our observation, that injection of 1 pg of ecdysterone simultaneous with the application of JH agent to a 8 rt:2 hr old Tenebrio pupa enhanced the morphogenetic effect of JH (REDDY and KRISHNAKUMARAN, 1972a), suggested that there may be age-related differences in the JH sensitivity of a Tenebrio pupa. Reasons for this belief are based on the fact that the synergistic effect of ecdysterone possibly resulted from its ability to synchronize epidermal cells and at the same time make them responsive to JH. Another reason for the synergistic effect of ecdysterone may be the innate juvenilizing property of ecdysterone (BOWERS, 1968; SCHNEIDERMAN et al., 1969; SOCHAand SEHNAL, 1972). In order to determine the * This research was supported by N.S.F. University funds.
Research Grant No. GB-19629 and Marquette 773
774
GUNDA FREDDY
AND
A.
KRISHNAKUMARAN
possible reasons for the synergistic action of ecdysterone on JH, we undertook a study of the morphogenetic response of Tenebrio pupae to low doses of JH in relation to the age of the pupa. First, we investigated the relative JH-sensitivity of Tenebrio pupae in relation to age because this may be important in the analysis of the correlation between cell division and morphogenetic effect of JH (CHASE, 1970; SQCHA and SEHNAL, 1972) as well as in the application of JH agents as insecticides. In addition, we also examined whether the synergistic effect of ecdysterone on JH analogues extends throughout the JH-sensitive period or is limited to younger pupae. The results reported in this communication show that morphogenetic response of Tenebrio pupae to low concentrations of JH varies with age of the pupa showing progressive increase in JH-sensitivity until their peak at 18 hr after pupal ecdysis. JH-sensitivity decreases after. about 32 hr of age. Furthermore, the synergistic effect of ecdysterone on the morphogenetic activity of JH agents is generally limited to a period preceding the period of peak JH-sensitivity suggesting that its synergistic effect may be due to synchronization of target cells. MATERIALS
AND METHODS
Natural JH (methyl 12,14-dihomojuvenate or methyl lo-epoxy 7-ethyl 3,11dimethyl 2,6_tridecadienoate, ROLLER et al., 1967) and its analogues methylenedioxyphenoxy 6-epoxy 3-ethyl 7-methyl 2-nonene (methylenedioxyphenoxy analogue) (BOWERS, 1969), dodecyl methyl ether, and ethyl 3,7,11_trimethyl 2,4_dodecadienoate (STAAL et aZ., 1971) (Fig. 1) were used in this study. The first two compounds were gifted by Hoffmann La Roche, Nutley, N.J. These were mixed isomers but were otherwise pure. Dodecyl methyl ether was kindly given by Professor H. A. Schneiderman and originally came from Hoffmann La Roche, Basel, Switzerland. Ethyl trimethyl dodecadienoate was supplied by Zoecon Corporation, Palo Alto, California. Ecdysterone (/3-ecdysone) was purchased from Rohto Pharmaceuticals, Osaka, Japan. JH and its analogues were dissolved in analytical grade acetone and were topically applied to the ventral side of the abdomen. Ecdysterone was dissolved in insect Ringer (EPHRUSSI and BEADLE, 1936) containing 1Oo/oethanol and was injected into pleural region of the second and third abdominal segments. The source of the Tenebrio pupae, methods of application of test chemicals, scoring scheme for the estimation of inhibition of metamorphosis, and the statistical analysis of the scores are similar to those reported earlier (REDDY and KRISHNAKUMARAN, 1972a). Pupae of uniform weight (110-120 mg) were collected every 2 hr and were aged at 30°C for the desired time. Thus, the true age of the pupae is + 1 hr. Tenebrio pupae that were 3, 6, 10, 14, 18, 24, 32, 40, or 48 hr old were treated with 0.1 pg of natural JH, O*Olpg of ethyl trimethyl dodecadienoate, 0.01 pg of methylenedioxyphenoxy analogue, or 80 pg of dodecyl methyl ether. After application of JH compounds the pupae were maintained at 30°C and 60 to 70% r.h. Under these conditions, acetone-treated controls emerged as adults in 6 to 6.5 days. The extent of retention of pupal characters was determined according
RESPONSE TO JH IN TENEBRIO
775
PUPAE
to the method described earlier. To determine the synergistic effect of ecdysterone, 1 pg of ecdysterone solution was injected into Tenebrio pupae before application of JH compounds while controls received 1~1 of Ringer solution.
Methyl
IO-epoxy 7-ethyl 3, I I-dimethyl 2,6+ridecadienoate (Methyl I2,14-dihomojuvenate)
Ethyl
3,7,
It-trimethyldodeca
2,4-dienoate
Methylenedioxyphenoxy 6-epoxy 3-ethyl 7-methyl (Methylenedioxyphenoxy analogue)
Z-nonene
-0’ Dodecyl
methyl
ether
FIG. 1. Structuralformulae of the JH compounds used in the study.
RESULTS
Sensitivity toJH
in relation to age of pupa
The results graphically presented in Figs. 2 to 5 show that the morphogenetic effect of JH on Tenebrio pupa varies with age. Eighteen hour old Tenebrio pupae showed the highest morphogenetic response to a given dose of JH and the three analogues used in this study. In younger pupae (3 f 1 hr and 6 + 1 hr old) the same dose of JH compound caused a significantly lower morphogenetic response than in the 18 hr old pupae. With all the compounds tested, 48 hr old pupae showed no inhibition of metamorphosis. However, there were differences between the different JH compounds in regard to the period during which a maximal response was obtained. While dodecyl methyl ether (Fig. 5) and methylenedioxyphenoxy analogue (Fig. 4) elicited near peak level response from 18, 24, and 32 hr old pupae, natural JH at the dose tested (Fig. 2) failed to evoke any response in 24 and 32 hr old pupae. Ethyl trimethyl dodecadienoate was intermediate in effect. In response to this
776
GUNDA REDDY AND A. KRISHNAKUMAFCAN
compound, 24 and 32 hr old pupae also showed morphogenetic however lower than that noticed in 18 hr old pupae (Fig. 3).
response which was
Natural JH
0
12
4s
36
24 Age,
hr
FIG. 2. The response to 0.1 pg of natural JH. FIGS. 2 to 5. Effect of age and 1 pg of ecdysterone on the morphogenetic response of Tenebrio pupae to juvenile hormone compounds. JH activity is measured in Tenebrio score on a scale of 0 to 5. 0, Normal adult; 5, perfect second pupa except for the eyes. At least 40 pupae were used for each point and the standard deviations for some of the scores are also shown. A, JH compound alone; 0, JH compound + 1~1 of insect Ringer; 0, JH compound + 1 pg of ecdysterone in 1~1 of insect Ringer.
Ethyl
trimethyl
dadecadienoate
Age,
hr
FIG. 3. The response to 0.01 pg of ethyl trimethyl dodecadienoate.
Temporal pattern of inhibition of metamorphosis in Tenebrio
pupa
The pupal characters retained by JH-treated pupae varied depending upon the age of pupa at the time of application of hormone. While application of hormone to 6 hr old pupae inhibited the appearance.of adult cuticle in the thorax, abdomen and head, in 18 hr old pupae metamorphosis of the head and thoracic cuticle was
RESPONSE TO JH IN
TENEBRIO
777
PUPAE
Methylenedioxyphenoxy onologue
I 6
0
Age,
FIG.
, 36
I 16
‘d--_--4 46
hr
4. The response to 0.01 ,ug of methylenedioxyphenoxy analogue. 5
4
F I/’ .I
Dodecyl
ether
“\_
Age,
FIG. 5.
methyl
\I
hr
The response to 80 pg of dodecyl methyl ether.
rarely inhibited. In 32 hr old pupae, even the abdomen failed to show any pupal patches although urogomphi and gin traps may be persistent. Essentially similar results were obtained when higher doses (1 or 10 pg) of natural JH were used. Application of 10 lug of natural JH to 18 hr old pupae failed to elicit a pupal cuticle in thorax and head while application of even low doses (O-5 pg) may induce deposition of pupal cuticle in the thorax in 8 hr old pupae. These data are of importance in assessing the association between cell division and the time of action of JH (CHASE, 1970; SOCHA and SEHNAL, 1972) and hence are briefly mentioned. Synergistic
efSect of ecdystemne
h relation to age of pupa
The data presented in Figs. 2 to 5 also show that injection of 1 ,ug of ecdysterone increases the morphogenetic effect of JH agents in younger ( > 14 hr) pupae. This synergistic effect is most pronounced in 3 and 6 hr old pupae. Furthermore, there were differences in the degree of synergism exhibited with the different analogues The morphogenetic effect elicited by 0.1 pg confirming our earlier observations.
778
GUN~A REDDYANDA. KRISHNAKUMARAN
of natural JH (Fig. 2), 0.01 pg of ethyl trimethyl dodecadienoate (Fig. 3), and 80 ,ug of dodecyl methyl ether (Fig. 5) was enhanced four- to sixfold by 1 pg of ecdysterone in 6 hr old pupae. Ecdysterone enhancement of the morphogenetic effect by the methylenedioxyphenoxy analogue (Fig. 4) was only marginal. Ecdysterone had no synergistic effect in 18 hr or older Tenebrio pupae with any of the JH analogues tested. The natural JH (Fig. 2), however, was exceptional in that the synergistic effect of ecdysterone was observed even in 32 hr old pupae. DISCUSSION Response toJEl in relation to age of pupa The results reported in this paper show that response to JH in Tenebrio pupae varies with age. Given a particular concentration of JH, 18 hr old pupae showed a higher morphogenetic response than younger pupae. Morphogenetic response to JH in Tenebrio pupae was low immediately after pupal ecdysis and gradually reached a peak at about 18 hr. Pupae remained highly responsive to JH up to about 32 hr and thereafter showed reduced morphogenetic response. Although it is not possible to equate morphogenetic response with JH-sensitivity of target cells, the results show an overall age-related change in the response of a pupa to JH. The reasons for the observed differences in response to JH may be several. Possibly, there is a true JH-refractory period followed by a JH-sensitive period in the Tenebrio pupal epidermal cell (see below). A second possibility is that these apparent differences in morphogenetic response are correlated with the rate of JH metabolism. If the rate of JH metabolism in younger pupae were high, the effective concentration of the hormone at the target cells would be reduced and thus result in a lower morphogenetic response. On the other hand, a low rate of JH metabolism in 18 to 32 hr old pupae results in a higher effective dose of the hormone and high morphogenetic response. Such a view is supported by the observations of SOCHA and SEHNAL (1972) who showed a more or less uniform morphogenetic response in 0 to 24 hr old Tenebrio pupae on application of 10 pg of JH in 1~1 of olive oil. In view of the fact that JH persists longer on application in oils the observations of SOCHAand SEHNALmay not reflect true differences in the sensitivity to JH (WIGGLESWORTH, 1963; KRISHNAKUMARANand SCHNEIDERMAN,1965). A third possible explanation for the low morphogenetic response of 3 to 10 hr old pupae may be the presence of an inhibitory factor(s) or the absence of some as yet unidentified factor(s) needed for the effective function of JH. Since the present data do not have a bearing on this, these alternative explanations can remain only as speculations. Whatever may be the reasons for the low morphogenetic response of > 14 hr old Tenebrio pupae, it can be corrected by simultaneous application of 1 ,ug of ecdysterone. In the presence of ecdysterone, morphogenetic response of 6 hr old pupae to JH was similar to 18 hr old pupae. Possibly this is associated with ecdysterone’s ability to initiate adult development (WILLIAMS, 1968) ; a fact hardly surprising, because, the expression of the morphogenetic effect of JH is possible only in the context of simultaneous action of ecdysterone (see SCHNEIDEF~MAN and GILBERT,
RESPONSE
TO
JH
IN
TENEBFLIO PUPAE
779
1964). The fact that ecdysterone can render the young pupae as responsive to JH as 18 hr old pupae, suggests that there may be true age-related differences in the JH-sensitivity. Further, the fact that the synergistic effect of ecdysterone is limited to younger pupae also suggests that the low morphogenetic response of these pupae is the result of their cells not being at the peak JH-sensitivity. Thus, we would like to suggest that genuine age-related changes in JH-sensitivity of Tenebrio pupal epidermal cells occur during the first 40 hr of the post-ecdysial period. The changes in JH-sensitivity of Tenebrio pupal epidermal cells appear to correspond to the time of onset of DNA synthesis in these cells. The detailed studies of CHASE (1970) an d our observations (KRISHNAKUMARAN,GRANGER, and SCHNEIDERMAN,unpublished observations) show that little or no DNA synthesis occurs in pupal epidermal cells during the first 12 hr after larval-pupal ecdysis. This corresponds to the period of low morphogenetic response to JH. Furthermore, the head region is the first to engage in DNA synthesis (see CHASE, 1970) and it is also the region that loses its sensitivity to JH first. In addition, DNA synthesis in abdomen is completed by 48 hr, by which time the pupa becomes unresponsive to JH. Thus, the time of action of JH appears to correspond to the period of DNA synthesis (see also SOCHAand SEHNAL,1972). Such an interpretation is in agreement with the present view that reprogramming of a genome usually coincides with the time of DNA replication (see EBERT, 1968). Synergistic activity of ecdysterone The results reported here confirm our preliminary observations on the synergistic activity of ecdysterone (REDDY and KRISHNAKUMARAN, 1972a). Furthermore, these data also show that the synergistic action of ecdysterone is limited to pupae that have not reached a state of peak morphogenetic response suggesting that synergistic activity of ecdysterone is associated with its ability to synchronize epidermal cells and initiate the pupal-adult apolysis. The present data also confirm our preliminary observations on the differences in the degree of synergism exhibited with different JH analogues. Reasons for these differences are not clear. Ecdysterone showed the least synergistic effect with methylenedioxyphenoxy analogue, a compound which was shown to be more stable than natural JH in Tenebrio pupae (REDDY and KRISHNAKUMARAN,1972b). This suggests that the low synergistic action may be correlated with its stability: a compound that is stable persists longer and will be available when the target cells Thus, ecdysterone which synchronizes the target cells and become sensitive. induces them to be responsive earlier than normal may not have a high synergistic effect. Synergistic effects of ecdysterone with natural JH even in pupae at their peak sensitive period was anomalous when compared to the other JH agents used in this study. Reasons for this anomaly are unknown. This may also be related to differences in the metabolic stability and the length of persistence. Assuming that natural JH is more rapidly metabolized than the other analogues, one may suggest that ecdysterone increases the morphogenetic response to natural JH even in pupae
780
GUNDA
FCEDDY
AND A.
KRIS~AKIJMARAN
at peak periods of JH sensitivity by synchronizing the JH target cells or increasing the half life of JH by an, as yet, unknown mechanism. AcknowZedgements-Our sincere thanks are due to Drs. ELLENM. RASCHand KESTUTIS TAUTWDASfor critical reading of the manuscript and help with the English. Thanks are due to Professor H. A. SCHNEIDERMAN of the University of California, Irvine, Dr. J. C. BAUERNFEIND of Hoffmann La Roche, Nutley, N.J., and Dr. J. B. SIDDALLof Zoecon Corporation, Palo Alto, California, for generously providing us with the juvenile hormone compounds. REFERENCES BOWERSW. S. (1968) Juvenile hormone: activity of natural and synthetic synergists. Science, Wash. 161, 895-897. BOTNERS W. S. (1969) Juvenile hormone: activity of aromatic terpenoid ethers. Science, Wash. 164, 323-325. BOWERSW. S. and THOMPSONM. J. (1963) Juvenile hormone activity : effects of isoprenoid and straight-chain alcohols in insects. Science, Wash. 142, 1469-1470. CHASEA. M. (1970) Effects of antibiotics on epidermal metamorphosis and nucleic acid synthesis in Tenebrio molitor. J. Insect Physiol. 16, 865-884. EBERTJ. D. (1968) Levels of control: a useful frame of perception, Curr. Topics Dev. Biol. 3, xv-xxv. EPHRUSSIB. and BEADLEG. W. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218-225. KAFZSONP. and NACHTIGALL M. (1961) Ein biologischer Test zur quantitativen Bestimmung der Juvenilhormon-Aktivitgt von Insektenextrakten. r. Insect Physiol. 7, 210-215. KRISHNAKUMARAN A. and SCHNEIDERMAN H. A. (1965) Prothoracotropic activity of compounds that mimic juvenile hormone. J. Insect Physiol. 11, 1517-1532. REDDY G. and KRISHNAKUMARAN A. (1972a) Synergistic effect of ecdysterone on morphogenetic activity of juvenile hormone analogues. Life Sci. 11, 781-792. REDDY G. and KRISHNAKUMARAN A. (1972b) Relationship between morphogenetic activity and metabolic stability of insect juvenile hormone analogues. J. lizsect Physiol. 18, 2019-2028 R~~LLERH., BJERKEJ. S., and MCSHAN W. H. (1965) Juvenile hormone-I. Methods of purification and isolation. J. Insect Physiol. 11, 1185-1197. RBLLER H., DAHM K. H., SWEELYC. C., and TROST B. M. (1967) The structure of the juvenile hormone. Angew. Chem. 6,179-180. ROSE M., WE~TERMANN J., TRAUTMANNH., and SCHMIALEKP. (1968) Juvenile Hormone Wirkung bei Tenebrio molitor L. in Abhangigkeit von der Konzentration der hormonalen Substanz. 2. Naturf. 23b, 1245-1248. SCHNEIDERMAN H. A. and GILBERT L. I. (1964) Control of growth and development in insects. Science, Wash. 143, 325-333. SCHNEIDERMAN H. A., KF~SHNAKUMARAN A., BRYANTP. J., and SEHNALF. (1969) Endocrinological and genetic strategies in insect control. Proc. Symp. Potentials Crop Protec. pp. 14-25. SOCHA R. and SEHNALF. (1972) Inhibition of adult development in Tenebrio molitor by insect hormones and antibiotics. J. Insect Physiol. 18, 317-337. STAAL G. B., HEM~ICKC. A., and SIDDALLJ. B. (1971) A novel juvenile hormone analog with possibilities for insect control. Abst. ent. Sot. Am. No. 106. WIGGLESWORTH V. B. (1963) The juvenile hormone effect of farnesol and some related compounds: quantitative experiments. J. Insect Physiol. 9, 105-119. WILLIAMS C. M. (1968) Ecdysone and ecdysone-analogues: their assay and action on diapausing pupae of the Cynthia silkworm. Biol. Bull., Woods Hole 134, 344-355.
CHANGES IN THE MORPHOGENETIC RESPONSE OF TENEBRIO MOLJTOR PUPAE TO JUVENILE HORMONE IN RELATION TO AGE* GUNDA
REDDY
and A. KR1SHNAKUMARA.N
Department of Biology, Marquette University, Milwaukee, Wisconsin 53233 (Received 24 August 1972) Abstract-Morphogenetic effect of juvenile hormone (JH) and its analogues, dodecyl methyl ether, ethyl trimethyl dodecadienoate and methylenedioxyphenoxy-6-epoxy-3-ethyl-7-methyl-2-nonene, on carefully timed Tenebrio pupae was determined. These results show that the response of pupal epidermal cells to JH varied with age during the first 48 hr after larval-pupal ecdysis. The pupae showed low morphogenetic response soon after pupal ecdysis but their response increased gradually until 18 hr. The response to JH decreased in pupae older than about 32 hr; and 48 hr old pupae were unresponsive to low doses of JH employed in this study. Age-related differences in the pattern of response of the individual body regions to JH were also observed. The synergistic effect of 1 pg of ecdysterone with these JH compounds was also tested in relation to the age of Tenebrio pupa. The results show that the synergistic effect of ecdysterone was generally limited to > 18 hr old pupae. This suggests that the physiological basis of the synergistic effect of ecdysterone may be the latter’s ability to synchronize epidermal cells. The significance of these observations in the analysis of time of action of juvenile hormone is discussed. INTRODUCTION
THE TENEBRIO pupal bioassay is widely used to identify and measure the morphogenetic potency of insect juvenile hormone (JH) and its synthetic analogues (KARLSONand NACHTIGALL,1961; BOWERSand THOMPSON,1963; R~~LLERet al., 196.5; ROSE et al., 1968). Routinely 0 to 24 hr old Tenebrio pupae were used for this assay. Our observation, that injection of 1 pg of ecdysterone simultaneous with the application of JH agent to a 8 rt:2 hr old Tenebrio pupa enhanced the morphogenetic effect of JH (REDDY and KRISHNAKUMARAN, 1972a), suggested that there may be age-related differences in the JH sensitivity of a Tenebrio pupa. Reasons for this belief are based on the fact that the synergistic effect of ecdysterone possibly resulted from its ability to synchronize epidermal cells and at the same time make them responsive to JH. Another reason for the synergistic effect of ecdysterone may be the innate juvenilizing property of ecdysterone (BOWERS, 1968; SCHNEIDERMAN et al., 1969; SOCHAand SEHNAL, 1972). In order to determine the * This research was supported by N.S.F. University funds.
Research Grant No. GB-19629 and Marquette 773
774
GUNDA FREDDY
AND
A.
KRISHNAKUMARAN
possible reasons for the synergistic action of ecdysterone on JH, we undertook a study of the morphogenetic response of Tenebrio pupae to low doses of JH in relation to the age of the pupa. First, we investigated the relative JH-sensitivity of Tenebrio pupae in relation to age because this may be important in the analysis of the correlation between cell division and morphogenetic effect of JH (CHASE, 1970; SQCHA and SEHNAL, 1972) as well as in the application of JH agents as insecticides. In addition, we also examined whether the synergistic effect of ecdysterone on JH analogues extends throughout the JH-sensitive period or is limited to younger pupae. The results reported in this communication show that morphogenetic response of Tenebrio pupae to low concentrations of JH varies with age of the pupa showing progressive increase in JH-sensitivity until their peak at 18 hr after pupal ecdysis. JH-sensitivity decreases after. about 32 hr of age. Furthermore, the synergistic effect of ecdysterone on the morphogenetic activity of JH agents is generally limited to a period preceding the period of peak JH-sensitivity suggesting that its synergistic effect may be due to synchronization of target cells. MATERIALS
AND METHODS
Natural JH (methyl 12,14-dihomojuvenate or methyl lo-epoxy 7-ethyl 3,11dimethyl 2,6_tridecadienoate, ROLLER et al., 1967) and its analogues methylenedioxyphenoxy 6-epoxy 3-ethyl 7-methyl 2-nonene (methylenedioxyphenoxy analogue) (BOWERS, 1969), dodecyl methyl ether, and ethyl 3,7,11_trimethyl 2,4_dodecadienoate (STAAL et aZ., 1971) (Fig. 1) were used in this study. The first two compounds were gifted by Hoffmann La Roche, Nutley, N.J. These were mixed isomers but were otherwise pure. Dodecyl methyl ether was kindly given by Professor H. A. Schneiderman and originally came from Hoffmann La Roche, Basel, Switzerland. Ethyl trimethyl dodecadienoate was supplied by Zoecon Corporation, Palo Alto, California. Ecdysterone (/3-ecdysone) was purchased from Rohto Pharmaceuticals, Osaka, Japan. JH and its analogues were dissolved in analytical grade acetone and were topically applied to the ventral side of the abdomen. Ecdysterone was dissolved in insect Ringer (EPHRUSSI and BEADLE, 1936) containing 1Oo/oethanol and was injected into pleural region of the second and third abdominal segments. The source of the Tenebrio pupae, methods of application of test chemicals, scoring scheme for the estimation of inhibition of metamorphosis, and the statistical analysis of the scores are similar to those reported earlier (REDDY and KRISHNAKUMARAN, 1972a). Pupae of uniform weight (110-120 mg) were collected every 2 hr and were aged at 30°C for the desired time. Thus, the true age of the pupae is + 1 hr. Tenebrio pupae that were 3, 6, 10, 14, 18, 24, 32, 40, or 48 hr old were treated with 0.1 pg of natural JH, O*Olpg of ethyl trimethyl dodecadienoate, 0.01 pg of methylenedioxyphenoxy analogue, or 80 pg of dodecyl methyl ether. After application of JH compounds the pupae were maintained at 30°C and 60 to 70% r.h. Under these conditions, acetone-treated controls emerged as adults in 6 to 6.5 days. The extent of retention of pupal characters was determined according
RESPONSE TO JH IN TENEBRIO
775
PUPAE
to the method described earlier. To determine the synergistic effect of ecdysterone, 1 pg of ecdysterone solution was injected into Tenebrio pupae before application of JH compounds while controls received 1~1 of Ringer solution.
Methyl
IO-epoxy 7-ethyl 3, I I-dimethyl 2,6+ridecadienoate (Methyl I2,14-dihomojuvenate)
Ethyl
3,7,
It-trimethyldodeca
2,4-dienoate
Methylenedioxyphenoxy 6-epoxy 3-ethyl 7-methyl (Methylenedioxyphenoxy analogue)
Z-nonene
-0’ Dodecyl
methyl
ether
FIG. 1. Structuralformulae of the JH compounds used in the study.
RESULTS
Sensitivity toJH
in relation to age of pupa
The results graphically presented in Figs. 2 to 5 show that the morphogenetic effect of JH on Tenebrio pupa varies with age. Eighteen hour old Tenebrio pupae showed the highest morphogenetic response to a given dose of JH and the three analogues used in this study. In younger pupae (3 f 1 hr and 6 + 1 hr old) the same dose of JH compound caused a significantly lower morphogenetic response than in the 18 hr old pupae. With all the compounds tested, 48 hr old pupae showed no inhibition of metamorphosis. However, there were differences between the different JH compounds in regard to the period during which a maximal response was obtained. While dodecyl methyl ether (Fig. 5) and methylenedioxyphenoxy analogue (Fig. 4) elicited near peak level response from 18, 24, and 32 hr old pupae, natural JH at the dose tested (Fig. 2) failed to evoke any response in 24 and 32 hr old pupae. Ethyl trimethyl dodecadienoate was intermediate in effect. In response to this
776
GUNDA REDDY AND A. KRISHNAKUMAFCAN
compound, 24 and 32 hr old pupae also showed morphogenetic however lower than that noticed in 18 hr old pupae (Fig. 3).
response which was
Natural JH
0
12
4s
36
24 Age,
hr
FIG. 2. The response to 0.1 pg of natural JH. FIGS. 2 to 5. Effect of age and 1 pg of ecdysterone on the morphogenetic response of Tenebrio pupae to juvenile hormone compounds. JH activity is measured in Tenebrio score on a scale of 0 to 5. 0, Normal adult; 5, perfect second pupa except for the eyes. At least 40 pupae were used for each point and the standard deviations for some of the scores are also shown. A, JH compound alone; 0, JH compound + 1~1 of insect Ringer; 0, JH compound + 1 pg of ecdysterone in 1~1 of insect Ringer.
Ethyl
trimethyl
dadecadienoate
Age,
hr
FIG. 3. The response to 0.01 pg of ethyl trimethyl dodecadienoate.
Temporal pattern of inhibition of metamorphosis in Tenebrio
pupa
The pupal characters retained by JH-treated pupae varied depending upon the age of pupa at the time of application of hormone. While application of hormone to 6 hr old pupae inhibited the appearance.of adult cuticle in the thorax, abdomen and head, in 18 hr old pupae metamorphosis of the head and thoracic cuticle was
RESPONSE TO JH IN
TENEBRIO
777
PUPAE
Methylenedioxyphenoxy onologue
I 6
0
Age,
FIG.
, 36
I 16
‘d--_--4 46
hr
4. The response to 0.01 ,ug of methylenedioxyphenoxy analogue. 5
4
F I/’ .I
Dodecyl
ether
“\_
Age,
FIG. 5.
methyl
\I
hr
The response to 80 pg of dodecyl methyl ether.
rarely inhibited. In 32 hr old pupae, even the abdomen failed to show any pupal patches although urogomphi and gin traps may be persistent. Essentially similar results were obtained when higher doses (1 or 10 pg) of natural JH were used. Application of 10 lug of natural JH to 18 hr old pupae failed to elicit a pupal cuticle in thorax and head while application of even low doses (O-5 pg) may induce deposition of pupal cuticle in the thorax in 8 hr old pupae. These data are of importance in assessing the association between cell division and the time of action of JH (CHASE, 1970; SOCHA and SEHNAL, 1972) and hence are briefly mentioned. Synergistic
efSect of ecdystemne
h relation to age of pupa
The data presented in Figs. 2 to 5 also show that injection of 1 ,ug of ecdysterone increases the morphogenetic effect of JH agents in younger ( > 14 hr) pupae. This synergistic effect is most pronounced in 3 and 6 hr old pupae. Furthermore, there were differences in the degree of synergism exhibited with the different analogues The morphogenetic effect elicited by 0.1 pg confirming our earlier observations.
778
GUN~A REDDYANDA. KRISHNAKUMARAN
of natural JH (Fig. 2), 0.01 pg of ethyl trimethyl dodecadienoate (Fig. 3), and 80 ,ug of dodecyl methyl ether (Fig. 5) was enhanced four- to sixfold by 1 pg of ecdysterone in 6 hr old pupae. Ecdysterone enhancement of the morphogenetic effect by the methylenedioxyphenoxy analogue (Fig. 4) was only marginal. Ecdysterone had no synergistic effect in 18 hr or older Tenebrio pupae with any of the JH analogues tested. The natural JH (Fig. 2), however, was exceptional in that the synergistic effect of ecdysterone was observed even in 32 hr old pupae. DISCUSSION Response toJEl in relation to age of pupa The results reported in this paper show that response to JH in Tenebrio pupae varies with age. Given a particular concentration of JH, 18 hr old pupae showed a higher morphogenetic response than younger pupae. Morphogenetic response to JH in Tenebrio pupae was low immediately after pupal ecdysis and gradually reached a peak at about 18 hr. Pupae remained highly responsive to JH up to about 32 hr and thereafter showed reduced morphogenetic response. Although it is not possible to equate morphogenetic response with JH-sensitivity of target cells, the results show an overall age-related change in the response of a pupa to JH. The reasons for the observed differences in response to JH may be several. Possibly, there is a true JH-refractory period followed by a JH-sensitive period in the Tenebrio pupal epidermal cell (see below). A second possibility is that these apparent differences in morphogenetic response are correlated with the rate of JH metabolism. If the rate of JH metabolism in younger pupae were high, the effective concentration of the hormone at the target cells would be reduced and thus result in a lower morphogenetic response. On the other hand, a low rate of JH metabolism in 18 to 32 hr old pupae results in a higher effective dose of the hormone and high morphogenetic response. Such a view is supported by the observations of SOCHA and SEHNAL (1972) who showed a more or less uniform morphogenetic response in 0 to 24 hr old Tenebrio pupae on application of 10 pg of JH in 1~1 of olive oil. In view of the fact that JH persists longer on application in oils the observations of SOCHAand SEHNALmay not reflect true differences in the sensitivity to JH (WIGGLESWORTH, 1963; KRISHNAKUMARANand SCHNEIDERMAN,1965). A third possible explanation for the low morphogenetic response of 3 to 10 hr old pupae may be the presence of an inhibitory factor(s) or the absence of some as yet unidentified factor(s) needed for the effective function of JH. Since the present data do not have a bearing on this, these alternative explanations can remain only as speculations. Whatever may be the reasons for the low morphogenetic response of > 14 hr old Tenebrio pupae, it can be corrected by simultaneous application of 1 ,ug of ecdysterone. In the presence of ecdysterone, morphogenetic response of 6 hr old pupae to JH was similar to 18 hr old pupae. Possibly this is associated with ecdysterone’s ability to initiate adult development (WILLIAMS, 1968) ; a fact hardly surprising, because, the expression of the morphogenetic effect of JH is possible only in the context of simultaneous action of ecdysterone (see SCHNEIDEF~MAN and GILBERT,
RESPONSE
TO
JH
IN
TENEBFLIO PUPAE
779
1964). The fact that ecdysterone can render the young pupae as responsive to JH as 18 hr old pupae, suggests that there may be true age-related differences in the JH-sensitivity. Further, the fact that the synergistic effect of ecdysterone is limited to younger pupae also suggests that the low morphogenetic response of these pupae is the result of their cells not being at the peak JH-sensitivity. Thus, we would like to suggest that genuine age-related changes in JH-sensitivity of Tenebrio pupal epidermal cells occur during the first 40 hr of the post-ecdysial period. The changes in JH-sensitivity of Tenebrio pupal epidermal cells appear to correspond to the time of onset of DNA synthesis in these cells. The detailed studies of CHASE (1970) an d our observations (KRISHNAKUMARAN,GRANGER, and SCHNEIDERMAN,unpublished observations) show that little or no DNA synthesis occurs in pupal epidermal cells during the first 12 hr after larval-pupal ecdysis. This corresponds to the period of low morphogenetic response to JH. Furthermore, the head region is the first to engage in DNA synthesis (see CHASE, 1970) and it is also the region that loses its sensitivity to JH first. In addition, DNA synthesis in abdomen is completed by 48 hr, by which time the pupa becomes unresponsive to JH. Thus, the time of action of JH appears to correspond to the period of DNA synthesis (see also SOCHAand SEHNAL,1972). Such an interpretation is in agreement with the present view that reprogramming of a genome usually coincides with the time of DNA replication (see EBERT, 1968). Synergistic activity of ecdysterone The results reported here confirm our preliminary observations on the synergistic activity of ecdysterone (REDDY and KRISHNAKUMARAN, 1972a). Furthermore, these data also show that the synergistic action of ecdysterone is limited to pupae that have not reached a state of peak morphogenetic response suggesting that synergistic activity of ecdysterone is associated with its ability to synchronize epidermal cells and initiate the pupal-adult apolysis. The present data also confirm our preliminary observations on the differences in the degree of synergism exhibited with different JH analogues. Reasons for these differences are not clear. Ecdysterone showed the least synergistic effect with methylenedioxyphenoxy analogue, a compound which was shown to be more stable than natural JH in Tenebrio pupae (REDDY and KRISHNAKUMARAN,1972b). This suggests that the low synergistic action may be correlated with its stability: a compound that is stable persists longer and will be available when the target cells Thus, ecdysterone which synchronizes the target cells and become sensitive. induces them to be responsive earlier than normal may not have a high synergistic effect. Synergistic effects of ecdysterone with natural JH even in pupae at their peak sensitive period was anomalous when compared to the other JH agents used in this study. Reasons for this anomaly are unknown. This may also be related to differences in the metabolic stability and the length of persistence. Assuming that natural JH is more rapidly metabolized than the other analogues, one may suggest that ecdysterone increases the morphogenetic response to natural JH even in pupae
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AND A.
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at peak periods of JH sensitivity by synchronizing the JH target cells or increasing the half life of JH by an, as yet, unknown mechanism. AcknowZedgements-Our sincere thanks are due to Drs. ELLENM. RASCHand KESTUTIS TAUTWDASfor critical reading of the manuscript and help with the English. Thanks are due to Professor H. A. SCHNEIDERMAN of the University of California, Irvine, Dr. J. C. BAUERNFEIND of Hoffmann La Roche, Nutley, N.J., and Dr. J. B. SIDDALLof Zoecon Corporation, Palo Alto, California, for generously providing us with the juvenile hormone compounds. REFERENCES BOWERSW. S. (1968) Juvenile hormone: activity of natural and synthetic synergists. Science, Wash. 161, 895-897. BOTNERS W. S. (1969) Juvenile hormone: activity of aromatic terpenoid ethers. Science, Wash. 164, 323-325. BOWERSW. S. and THOMPSONM. J. (1963) Juvenile hormone activity : effects of isoprenoid and straight-chain alcohols in insects. Science, Wash. 142, 1469-1470. CHASEA. M. (1970) Effects of antibiotics on epidermal metamorphosis and nucleic acid synthesis in Tenebrio molitor. J. Insect Physiol. 16, 865-884. EBERTJ. D. (1968) Levels of control: a useful frame of perception, Curr. Topics Dev. Biol. 3, xv-xxv. EPHRUSSIB. and BEADLEG. W. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218-225. KAFZSONP. and NACHTIGALL M. (1961) Ein biologischer Test zur quantitativen Bestimmung der Juvenilhormon-Aktivitgt von Insektenextrakten. r. Insect Physiol. 7, 210-215. KRISHNAKUMARAN A. and SCHNEIDERMAN H. A. (1965) Prothoracotropic activity of compounds that mimic juvenile hormone. J. Insect Physiol. 11, 1517-1532. REDDY G. and KRISHNAKUMARAN A. (1972a) Synergistic effect of ecdysterone on morphogenetic activity of juvenile hormone analogues. Life Sci. 11, 781-792. REDDY G. and KRISHNAKUMARAN A. (1972b) Relationship between morphogenetic activity and metabolic stability of insect juvenile hormone analogues. J. lizsect Physiol. 18, 2019-2028 R~~LLERH., BJERKEJ. S., and MCSHAN W. H. (1965) Juvenile hormone-I. Methods of purification and isolation. J. Insect Physiol. 11, 1185-1197. RBLLER H., DAHM K. H., SWEELYC. C., and TROST B. M. (1967) The structure of the juvenile hormone. Angew. Chem. 6,179-180. ROSE M., WE~TERMANN J., TRAUTMANNH., and SCHMIALEKP. (1968) Juvenile Hormone Wirkung bei Tenebrio molitor L. in Abhangigkeit von der Konzentration der hormonalen Substanz. 2. Naturf. 23b, 1245-1248. SCHNEIDERMAN H. A. and GILBERT L. I. (1964) Control of growth and development in insects. Science, Wash. 143, 325-333. SCHNEIDERMAN H. A., KF~SHNAKUMARAN A., BRYANTP. J., and SEHNALF. (1969) Endocrinological and genetic strategies in insect control. Proc. Symp. Potentials Crop Protec. pp. 14-25. SOCHA R. and SEHNALF. (1972) Inhibition of adult development in Tenebrio molitor by insect hormones and antibiotics. J. Insect Physiol. 18, 317-337. STAAL G. B., HEM~ICKC. A., and SIDDALLJ. B. (1971) A novel juvenile hormone analog with possibilities for insect control. Abst. ent. Sot. Am. No. 106. WIGGLESWORTH V. B. (1963) The juvenile hormone effect of farnesol and some related compounds: quantitative experiments. J. Insect Physiol. 9, 105-119. WILLIAMS C. M. (1968) Ecdysone and ecdysone-analogues: their assay and action on diapausing pupae of the Cynthia silkworm. Biol. Bull., Woods Hole 134, 344-355.