Molting hormone titer changes and their significance during development of the colorado beetle, Leptinotarsa decemlineata

J. Insect Physiol.. 1976, Vol. 22, pp. 1257 to 1261. Pergamon Press. Printed in Great Britain.

MOLTING HORMONE TITER CHANGES AND THEIR SIGNIFICANCE DURING DEVELOPMENT OF THE COLORADO BEETLE, LEPTINO7’ARSA DECEMLINEATA T. H. HSIAO,* C. HSIAO,* and J. DE WILDE? *Department of Biology, Utah State University, Logan, Utah 84322, U.S.A. TDepartment of Entomology, Agricultural University, Wageningen, Netherlands (Received 17 February 1976; revised 23 March 1976)

Abstract-Molting hormone (MH) titer in whole animal extracts of Leptinotarsa decemlineata was determined by chemical extraction and the Musca test (1 MU = 3.5 ng ecdysterone) during the developmental span from newly-ecdysed fourth instar larva to an adult 3 days after eclosion. Within the 17-day period, 21 age groups were chosen to estimate the MH titer. Two peaks of MH titer were detected, one in the post-feeding larval stage and the other during the pupal and pharate adult stage. MH activity was first detected in 2-day-old post-feeding larvae, and reached a maximum of 23.5 MU/g tissue on the third day. It began to decline on 3.5 days, and fell to 5.5 MU/g tissue on 4.5 days, the time of larval-pupal ecdysis. In the pupal and pharate adult stage MH rose after the first day and increased to a maximum of 91.5 MU/g tissue on the third day. The titer again declined on the fourth day, and became undetectable one day before adult emergence and in adults 3 days after emergence. MH was demonstrated to be produced by isolated larval abdomens. A peak of 11.5 MU/g tissue was detected in 7-day post-ligation preparations. The titer decreased to 6.9 MU/g tissue in IO-day post-ligation preparations, which was the time of the ecdysis. The finding raises questions concerning the rale of MH synthesis by other tissues in relation to the function of the prothoracic glands during insect development.

INTRODUCI’ION

we have initiated a series of experiments to determine the MH titer changes during the period between last larval instar and adult. Bioassay with the Musca test was used in our work because of its convenience and availability at the time of the experiment. The relationships between the MH titers and developmental events are discussed.

CHANGESin the molting hormone (MH) titers during development of insects have now been determined for a number of species belonging to Lepidoptera (BURDETJZ:,1962; SHAAYAand KARLSON, 1965a; KAPLANIS et al., 1966a; HANAOKA and OHNISHI, 1974; BOLLENBACHERet al., 1975), Diptera (SHAAYA and KARLSON, 1965b; BARRIT and BIRT, 1970; OHTAIU and TAKAMATERIALS AND METHODS HASHI, 1972; BORST et al., 1974), Orthoptera (HOFFMANN et al., 1974; MORGAN et al., 1975), Hemiptera The Colorado beetles used in our experiment were (FEIR and WINKLER, 1969), Dictyoptera (MASNER et derived from a laboratory culture kept in the Entoal., 1975), and Odonata (SCHALLERet al., 1974). Such mology Department, Agricultural University, Wageninformation is vital to the understanding of endocrine ingen, The Netherlands. They were maintained in regulation and interaction. Because of vast differences potato foliage grown in a greenhouse. Rearing was in the time sequence of developmental events occuraccomplished in a rearing room maintained at ring in insects, MH titers obtained from one species 25°C &- 1°C and a photoperiod of 18 hr light and may not accurately represent the same event found 6 hr darkness. To obtain insects of a specific age, in another species. Consequently, MH titer deternewly-ecdysed individuals were sorted out from the mination for individual species is essential if critical culture and reared in an incubator. At a given time evaluation of titer changes and inference of its signifiinterval these insects were weighed and kept in a cance are required. freezer until extraction. To avoid contamination of Several recent studies (HSIAO and HSIAO, 1972, and plant materials in the sample, feeding larvae were unpublished data; DEWILDE, 1975) on endocrine relaremoved from food and starved for 4 to 6 hr before tionships of the Colorado beetle have indicated a processing. need for more precise knowledge of MH titer at speciMature larvae, upon cessation of feeding and initiafic time intervals during development in order to intion of digging behavior, were used for ligation experterpret the effects of experimental manipulation on iments. They were ligated with a thread between the endocrine systems. Since no information is currently metathorax and the abdomen. The anterior part was available on the MH titer of a coleopteran species, sliced off and discarded. The wound was sealed with 1257

T. H. HSIAO,C. HSIAO,AND J. DEWILDE

1258

paraffin. The isolated abdomens were kept in a Petri dish lined with a piece of moist filter paper. At specific time intervals they were weighed and frozen. Houseflies were used in the bioassay of MH activity. The preparation of test insects followed the method described by STAAL (1967). Each untanned fly abdomen was injected with 2 ~1 of test solution and the molting hormone activity of each fraction was scored after 24-hr incubation at 25°C. Ecdysterone was used as a standard for comparison of MH activity. Twenty untanned housefly abdomens were used in each bioassay. A series of dilutions was made with each test extract. The dosages that gave 20 to 807; MH activity were chosen and replicates were made with these doses. The average from two bioassays of each dose was used to calculate the MH activity. Each Musca Unit (MU) corresponded to the dose required to obtain a 50% MH activity. The procedure for chemical extraction was similar to the method of KARLSONand SHAAYA(1964) with slight modification. Initial experiments showed that 20 g of insect tissue was adequate for chemical extraction and bioassay. Frozen insect materials were homogenized with 50 ml of methanol. After centrifugation the residue was reextracted twice with 60% methanol in water. The combined methanol extract was concentrated in vacua to dryness and then dissolved in 20 ml of water. Following centrifugation the water phase was extracted with 10 ml of n-butanol three times successively. The combined butanol extract was washed stepwise with 10 ml of each of the following: 1% sulfuric acid, 10% sodium bicarbonate, 5% acetic acid, water. The resulting butanol phase was concentrated to dryness, suspended in 20 ml of water, and then extracted with an equal volume of petroleum ether. The procedure was repeated once. The water extract was again concentrated to dryness, suspended in 1 ml of water, with 9 vol. of acetone added to facilitate precipitation. After centrifugation the precipitates were treated once in the same manner as described above. The acetone-soluble filtrates from the two precipitations were combined and concentrated to dryness. This fraction was further purified on a silica gel column (4 g) of 0.7 x 7 cm which was preconditioned with benzene. The sample was dissolved in 0.1 ml of methanol and then mixed with 0.9 ml of benzene. The mixture was applied to the column and eluted, first with 15 ml of benzene:methanol = 9:1, and then with 15 ml of benzene:methanol = 3: 1. The eluent from the last separation was concentrated to dryness and dissolved in 0.2 ml of water for bioassay. RESULTS Standardization of extraction procedure and bioassay

In the early phase of this study, several chemical extraction methods were tested to determine their efficiency for recovery of MH from insect tissues. The procedure of KARL~~N and SHAAYA(1964) was fol-

lowed initially with a sample of 20 g of housefly white puparia. Bioassay of the butanol extract with the Musca test showed a molting hormone activity of 40 MU per g of tissue. Extraction of a sample of 20 g of 3-day-old post-feeding larvae of the Colorado beetle using the same procedure yielded an extract that gave no MH activity. An examination of the treated fly abdomens showed that many individuals died before completion of the 24-hr scoring period. Dilution of the test solution 10 times still strongly irritated the test animals and showed high mortality. When ecdysterone was added to the Colorado beetle extract, the test result was also negative. indicating a complete inhibition of molting hormone activity by the extract. Experiments were then attempted to remove the inhibitory factors. The Colorado beetle extract was initially treated with acetone precipitation. This method removed a considerable amount of inhibitory compounds, but a further purification step with silica gel column and solvent elution resulted in a fraction that showed no inhibitory effects even when injected at 5 times the amount used in the original tests. To determine if the procedure of cold methanol extraction in Karlson and Shaaya’s method was adequate for Colorado beetle tissues, we also reextracted the residue portion with boiling methanol according to the procedure recently described by HANAOKAand OHNISHI(1974) who found additional MH activity from tissue residue of the silkworm. Our test results showed that the extraction with cold methanol recovered all the MH, whereas the hot methanol extract did not show additional MH activity. Therefore, in all subsequent experiments only cold extraction was employed. Because the results obtained in different replicates were consistent, we have not attempted to quantify the recovery rate in our extraction procedures. Changes in molting hormone titers during development

Initial experiments were conducted to determine the timing of different developmental events under the rearing conditions used. The feeding stage of the fourth instar larvae required 3.5 days (Fig. 1). The larvae, upon cessation of feeding, initiated the behavior of digging into soil for pupation. We have observed that the majority of these post-feeding larvae begin the wandering and digging behavior during the early part of the photophase. Therefore, by the use of a modified fraction collector, larvae of the same physiological condition could be sorted out with precision for our experiments. In the soil the postfeeding larvae remained quiescent. The larval-pupal ecdysis occurred in about 4.5 days, and the pupal and pharate adult stage required 6 days. On the basis of the above developmental time requirements, insect materials used for MH titer determination were selected initially at the onset of each developmental event; i.e. newly-ecdysed fourth instar larvae, postfeeding larvae of the wandering and digging stage, and newly-ecdysed pupae; and at intervals of 24 hr

Molting hormone titer changes of I... decemlineata

I.259

Day 4th

instar larva

Pupa and pharate adult

Adult

Fig. 1. Changes in molting hormone titer during development of the Colorado beetle. Solid line indicates average MH activity. Dotted line indicates mean fresh weight of insects.

after each event. Additional age intervals of 12 hr were selected in the region where MH activity was detected. With the exception of the feeding stage, where only one determination was made, three replicates of insect samples were extracted and assayed in each age group. Figure 1 summarizes the changes in MH activity during the course of development. The solid curve represents the average of all replicates obtained from each age group. Individual determinations were also included as points to show the variations among samples. The dotted .curve shows the average fresh weight of the insects used in our determination. Because of the difference in weight of different age groups, the number of insects used in the extraction varied from 500 larvae of newly-ecdysed fourth instar to about 130 larvae of the post-feeding stage. The amount of fresh tissue used was approximately 20 g in all samples regardless of the number of insects required. The size of insects also varied slightly between batches, depending upon the rearing conditions and quality of food supplies. Therefore, it was more practical to calculate our data in terms of MU per g of fresh tissue rather than the number of insects in the samples. In order to establish the minimum detectable MH activity in insect tissues, in one experiment 50 g of post-feeding larvae of the wandering stage were extracted and the resulting extract was dissolved in 50 ~1 of water. Injection of 2 4 of such extract to each Musca preparation yielded negative results, indicating that the amount of MH present in the tissue was less than 0.5 MU/g. This figure was used as the lowest limit of MH recovery in our tests. In our bioassay with ecdysterone as a standard, we calculated 1 MU equal to 3.5 ng of this compound, based on about 50 bioassays, with 20 Musca preparations

used in each bioassay. This figure is in agreement with that of KAPLANISet al. (1966b) and STAAL(1967). As indicated in Fig. 1, MH activity was not detectable during the feeding stage of the fourth instar larvae and on the first day after the larvae dug into the soil. MH activity (average 5 MU/g) was detected in 2-day-old post-feeding larvae, and reached a peak in those 3 days old (average 23.7 MU/g). It began to decline in 3.5-day-old post-feeding larvae, and fell to a low level of 5.5 MU/g at the time of larval-pupal ecdysis. In the pupal and pharate adult stage MH activity rose after the first day and increased noticeably on the second day. It reached a peak at 2.5 to 3 days (average 91.5 MU/g). The titer again declined on the fourth day, and became undetectable one day before adult emergence. In the adult stage, l-day-old specimens of mixed sexes showed no detectable MH activity. When grouped separately. neither males nor females 3 days old produced any detectable amounts of MH in their tissues. Molting hormone production in isolated abdomens The ability of isolated abdomens of the Colorado beetle larvae to molt successively into pupal and adult forms was observed in ligation experiments (Hsiao and Hsiao, unpublished data). Whether or not the isolated abdomen actually produced MH to cause ecdysis was the focus of the present investigation. In our experiments, isolated abdomens of post-feeding larvae of 1, 4, 7, and 10 days after ligation were collected and extracted according to methods used for MH titer determination. The use of these different age groups was based on the fact that the average time from ligation to ecdysis of the isolated abdomen required 10 days. A time interval of 3 days was used in our experiments. As summarized in Table 1, MH was not detectable in l-day-old and 4-day-old iso-

1260

T. H. HSIAO, C. HSIAO.AND J. DE WILDE

Table 1. Molting hormone production by isolated abdomen of post-feeding larvae of the Colorado beetle Days after ligation 1 1 4 4 7 I 10

No. abdomen

wt. of sample

used

k)

153 150 154 137 157 158 159

20.572 19.797 20.253 17.127 20.740 20.414 20.054

MH activity in MU/g tissue Undetected* Undetected* Undetected* Undetected* 11.8 11.1 6.9

* Less than 1 MU/g tissue. lated abdomens, but was found to have an average of 11.5 MU/g tissue in 7-day-old samples. At the time of ecdysis (lo-day-old sample), the MH titer decreased to 6.9 MU/g tissue. Because of the time-consuming requirements of this experiment, we have not attempted to determine the peak of MH titer during the development of the isolated abdomens. We would expect the MH production of the isolated abdomen to be much lower than in normal insects, as shown in Fig. 1. Some of the reasons for a low MH production by the isolated abdomens are the absence of the prothoracic glands, low oxygen supply, and the slower body-fluid circulation. Our findings prove conclusively that MH can be produced by tissues other than the prothoracic glands and that the amount of MH produced by tissues is sufficient to cause ecdysis of the isolated abdomen. DISCUSSION The MH titers obtained during larval and pupal stages of the Colorado beetle resemble those found in other endopterygote insects. Two MH titer peaks were detected, one in the post-feeding larval stage and the other during the pupal and pharate adult stage. The initial increase in MH in the post-feeding larva occurred about 2 days after the larva dug into the soil. Before this time, MH was undetectable. Previously, HSIAOand HSIAO(1972) found that the length of time required from the digging into the soil to larval-pupal ecdysis could be delayed considerably by not allowing the larvae to dig into the soil. This was accomplished by suspending the larvae individually on a thread. However, by the injection of u-ecdysone, it was possible to avoid such a delay in development time of suspended larvae. The lack of MH in the early phase of the post-feeding larval stage, as shown in this study, supports our earlier assumption that the secretion of MH is associated with the behavioral event of digging into the soil. MH was detected in a noticeable amount (5.5 MU/g tissue) in newly-ecdysed pupae, indicating that a low rate of MH synthesis persisted throughout the larval-pupal ecdysis. The question of how the MH

titer is controlled at this stage is not entirely clear, but the possibility of MH excretion during the ecdysis can be eliminated since in an extraction prepared from log of larval exuviae we were unable to detect MH activity. The amount of MH found in the pupal and pharate adult stage of the Colorado beetle was several times greater than the amount detected in the post-feeding larval stage. This pattern of MH titers was also reported in several lepidopteran species (e.g. Bombyx mori, Manduca sexta) but not in dipteran species (e.g. Calliphora erythrocephala, Sarcophaga peregrina). The higher amount of MH synthesis in the pupa and pharate adult stage reflects a greater need of MH during this stage of development. The presence of one MH peak in the pupa and pharate adult stage of L. decemlineata is similar to those reported in 2 dipteran species, C. erythrocephala (SHAAYA and KARLSON, 1965b) and S. peregrina (OHTAKI and TAKAHASHI,1972). but is different from the two distinct peaks found in the lepidopteran species, B. mri (KARLSONand SHAAYA, 1965a; HANAOKA and OHNISHI,1974). As recently shown by HANAOKA and OHNISHI(1974), the second MH peak in the pupa and pharate adult stage of B. nwri is found only in the female and not in the male or ovarectomized female, indicating that the second MH titer is associated with ovarian development. Since in the Colorado beetle and the blowflies ovarian development occurs after adult emergence, the absence of a second MH peak in the pupa and pharate adult stage of these species seems to be reasonable. In this connection it is interesting to note that MH was detected in adult females of Aedes aegypti (SCHLAEGER et al., 1974), and the source of the hormone was the ovary (HAGEDORN et al., 1975). However, we were unable to detect MH in 3-day post-eclosion females of the Colorado beetle (Fig. 1). In this species the ovarian development is nearly completed by the third day of emerged adult life and egg laying started by the fourth or fifth day. It appears that MH production may not be essential for ovarian activity in this species. The importance of juvenile hormone in the reproduction of the Colorado beetle is well documented (DE WILDE and DE LOOF, 1973). The significance of MH synthesis by the isolated larval abdomen of the Colorado beetle has been discussed briefly by HSIAO et al. (1975). Several additional findings are worth mentioning. As indicated in Table 1, the titer of MH shows a characteristic increase by the seventh day and declines on the tenth day, which is the time of ecdysis. This pattern of MH titer is not different from the titer of normal insects, as shown in Fig. 1. The much smaller quantity of MH produced by the isolated abdomen as compared with the normal insects can be explained by the fact that the tissues of the former, especially the internal organs, were less differentiated and circulation was much reduced. However, the amount of MH synthesis inside the abdomen is sufficient for the development

Molting hormone titer changes of L. decemlineata of the epidermal tissues. Our observation that a pupal abdomen obtained by ligation of a larval abdomen can develop into an adult abdomen suggests that MH synthesis is resumed in the pupal and pharate adult stage of such a preparation. Whether or not the MH titer would be similar to that of the normal insect remains to be demonstrated. Information on the MH titers of insects is essential for interpreting the regulatory mechanisms of various developmental events in insects. As demonstrated in our studies, the knowledge of MH titers of the Colorado beetle is crucial to our subsequent finding of MH synthesis in the isolated larval abdomen of this species. In the light of this finding, many questions remain to be answered concerning the roles of MH synthesis by tissues in relation to the function of the prothoracic glands during insect development. Acknowledgements-This work was supported in part by a research fellowship (to T.H.H.) from Agricultural Univer-

sity, Wageningen, Netherlands. REFERENCES BARRITL. C. and BIRT L. M. (1970) Prothoracic gland hormone in the sheep blowfly, Lucilia cuprina. J. Insect Physiol. 16, 671-677. B~LLENBACHER W. E., VEDECKIS W. V., GILBERTL. I., and O'CONNOR J. D. (1975) Ecdysone titers and prothoracic

gland activity during the larval-pupal development of Manduca sexta. Deuel. Biol. 44, 4653. BORSTD. W., BOLLENBACHER W. E., O’CONNORJ. D., KING D. S., and FRISTROM J. W. (1974) Ecdysone levels during metamorphosis of Drosophila melanogaster. De&. Biol. 39, 308-3 16.

BURDETTEW. (1962) Changes in titer of ecdysone in Bomhyx mori during metamorphosis. Science, Wash. 135, 432.

FEIR D. and WINKLERG. (1969) Ecdysone titres in the last larva and adult stages of the milkweed bug. J. Insect Physiol. 15, 899-904.

HAGEWRN H. H., O'CONNOR J. D., FUCHS M. S., SAGE B., SCHLAEGER D. A., and BOHMM. K. (1975) The ovary as a source of cc-ecdysone in an adult mosquito. Pro;. nat. Acad. Sci. U.S.A. 72. 3255-3259.

HANAOKAK. and OHNISHI’E.(1974) Changes in ecdysone titre during pupal-adult development in the silkworm, Bombyx mori. J. Insect Physiol. 20, 2375-2384.

HOFFMANNJ. A., KCIOLMAN J., KARL.~~NP., and JOLY P. (1974) Molting hormone titer and metabolic fate of injected ecdysone during the fifth larval instar and in adults of Locusta migratoria (Orthoptera). Gen. camp. Endocr. 22, 90-97.

1261

HSIAOT. H. and HSIAOC. (1972) Behavioural control of ecdysone release during pupation of the Colorado potato beetle. Abstracts 14th int Cong. Em., p. 176. HSIAOT. H., HSIAOC., and DE WILDE J. (1975) Moulting hormone production in the isolated larval abdomen of the Colorado beetle. Nature, Lond. 255, 727-728. KAPLANISJ. N., THOMP~C~N M. J., YAMAMOTO R. T., ROBBINDW. E., and LOULOUDES S. J. (1966a) Ecdysones from the pupa of the tobacco hornworm, Manduca sexta (Johannson). Steroids 8, 605623. KAPLANISJ. N., TABOR,L. A., THOMPSON M. J., ROBBINS W. E., and SHOR~NOT. J. (1966b) Assay for ecdysone (molting hormone) activity using the house fly, Musca domestica L. Steroids 8, 6255631. KARLXJN P. und SHAAYAE. (1964) Der Ecdysontiter wiihrend der insektenentwicklung--1. Eine methode sur Bestimmung des Ecdysongehalts. J. Insect Physiol. 10, 797-804.

MASNER P., HANGARTNERW., and SUCHY M. (1975) Reduced titres of ecdysone following juvenile hormone treatment in the German cockroach, Blattella germanica. J. Insect Physiol. 21, 1755-1762.

MORGANE. D., WOODBRIDGE A. P., and ELLISP. E. (1975) Studies on the moulting hormones of the desert locust, Schistocerca gregaria. J. Insect Physiol. 20, 1-15.

OHTAKIT. and TAKAHASHIM. (1972) Induction and termination of pupal diapause in relation to the change of ecdysone titer in the fleshfly, Sarcophaga peregrina. Jap. J. med. Sci. Biol. 25, 369-376.

SCHALLERF., ANDRIE~J. C., MOUZE M.. et DEFOS~EZA. (1974) Nouveaux aspects du contrble hormonal du cycle biologique des Odonates: recherches sur la larve d’Aeshna cyanea (Miiller) (Anisoptera: Aesnidae). Odonatologica 3, 49-62.

SCHLAEGER D. A., FUCHS M. S., MORTONS., and KANG S. H. (1974) Ecdvsone mediated stimulation of Dooa decarboxylase activity and its relation to ovarian development in Aedes aegypti. J. Cell Biol. 61, 454465. SHAAYAE. und KARLSONP. (1965a) Der Ecdysontiter wahrend der Insektenentwicklung-IV. Die Entwicklung der Lepidopteren Bombyx mori L. und Cerura uinula L. Deuel. Biol. 11, 424432. SHAAYAE. und KARLSONP. (1965b) Der Ecdysontiter wlhrend der Insektenentwicklung-II. Die Postembryonale Entwicklung der Schmeissfliege Calliphora eryf~rocep~a~u Meig. J. Insect Physiol. 11, 65-69. STAALG. B. (1967) Plants as a source of insect hormones. Proc. K. Ned. Akad. Wet. (Cl , 70. 409418. DE WILDEJ. (1975) An endocrine view of metamorphosis, polymorphism, and diapause in insects. Am. Zoo!. 15

(Suppl. l), 13-27. DE WILDEJ. and DE L~CIFA. (1973) Reproduction-endocrine control. In The Physiology of Insecta (Ed. by ROCKSTER‘IM.) 1, 97-157, Academic Press.