Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3744-3747, May 1991 Developmental Biology Epidermis as the source of ecdysone in an argasid tick (ecdysteroids/tissue culture/integument/Ornithodors parkeri) X. X. ZHU, J. H. OLIVER, JR.*, AND E. M. DOTSON Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, GA 30460-8056 Communicated by Wendell Roelofs, February 7, 1991 (received for review November 19, 1990)

ABSTRACT Various tissues excised from nymphs of the MATERIALS AND METHODS tick Ornithodoros parkeri at the time of epicuticle deposition were incubated in vitro. The medium from the incubation of Ticks (0. parkeri) were reared at 270C and 85% relative salivary glands, coxal glands, synganglion, testis, midgut, and humidity by standard techniques (10). RIA of ecdysteroids fat body associated with tracheal trunk showed little or no was carried out following established methods (11, 12). For each assay, a standard curve was prepared using ecdysone ecdysteroid immunoreactivity. Only medium from incubated England Nuclear). integument contained ecdysteroids. The following evidence (Sigma) and [23,24-3H]ecdysone (New The antiserum used was a generous gift from T. Ohtaki the source of ecdysone: (i) indicated that epidermal cells are (Kanazawa, Japan) and has a 2.5-fold higher sensitivity for when dorsal and/or ventral integuments were incubated sep- ecdysone than for 20-hydroxyecdysone when their 50% arately, both produced ecdysteroid immunoreactive material cross-reaction was compared. during the course of incubation. As compared with the ecdy- Ecdysteroid titer was highest during epicuticle deposition steroid content in the integument before incubation, the in third instar nymphal 0. parkeri (unpublished data), and amount of ecdysteroids produced after a 24-h incubation thus nymphs at this stage were used in all our experiments. increased 4- to 7-fold; (ii) enzymatic hydrolysis showed that To locate the possible site ofecdysteroid production, various neither highly polar ecdysteroid conjugates nor apolar conju- tissues were dissected out after removing the old cuticle. The gates were stored in the integument; (iii) histological and midgut contents were washed out and each tissue was rinsed scanning electron microscope observations demonstrated that twice separately in the incubation medium [GIBCO TC-199 these excised integuments consisted of newly deposited epicu- medium fortified with Ficoll (20 mg/ml)]. Like tissues from ticle and epidermis as well as some fat body cells; (iv) HPLC three nymphs were pooled and then incubated at 270C with RIA showed that the integument with associated fat body gentle shaking in 150 1.l of medium. After 24 h, 50 p1L of produced ecdysone and 20-hydroxyecdysone, while the integ- medium, in duplicate, was transferred to RIA tubes for assay ument produced only ecdysone after removing fat body. Pre- of ecdysteroid content. Results are expressed per 150 1Ad of sumably, ecdysone secreted by epidermis was converted into medium. 20-hydroxyecdysone by fat body. For HPLC analysis of the components active in the RIA assays, the incubation medium was passed through a C18 Sep Ticks are of considerable medical and economic importance. Pak cartridge (Waters), which was then rinsed with H20 and However, little is known about their neurohormonal regula- subsequently with 25% methanol (13). The immunoreactive tion of development and reproduction. So far, ecdysteroids material was eluted with pure methanol, concentrated, and are the only hormones that have been definitely identified (1). injected into a HPLC (Spectra-Physics; model 8800) In immature stages of argasid and ixodid ticks (Ornithodoros equipped with a programmable wavelength detector and an moubata and Amblyomma hebraeum), ecdysone and mainly SP 4270 integrator. A reversed-phase column (Keystone, 20-hydroxyecdysone are present, and their increasing and Bellefonte, PA; 15 cm, 4.6-mm i.d. packed with Lichrosorb higher titers parallel apolysis and epicuticle deposition (2, 3). RP-18; 5 ,um) with 50o methanol/water as a solvent and a In females of ixodid ticks (Boophilus microplus and A. flow rate of 800 ,ul/min were used. Fractions were collected hebraeum), a peak titer of either mainly free ecdysone or every 30 sec for 15 min and examined by RIA for the presence 20-hydroxyecdysone has been observed during oogenesis (4, of ecdysteroids. Ecdysone and 20-hydroxyecdysone (Sigma) in has been were used as references. After running the standards, the 5) and a possible role salivary gland degeneration and injector were rinsed and a blank contain- In addi- column sample suggested in Amblyomma americanum females (6). ing only the eluate was injected into HPLC to ensure no tion, ecdysteroid immunoreactive materials have been de- contamination of the column and injector. No immunoreac- tected in males of the ixodid tick Dermacentor variabilis (7). tive material was detected in the fractions of the blank run. The origin of ecdysteroids in ticks remains uncertain. In Before injecting the sample, an aliquot was assayed by RIA Amblyomma variegatum nymphs it was suggested, based on to determine the amount of immunoreactivity present. The in vitro tissue culture and the measurement of RIA-positive recovery of ecdysteroid immunoreactivity from fractions of material, that fat body is the site of ecdysteroid production the samples injected on the HPLC was >80%o. (8). However, this work has been criticized for not comparing To determine whether ecdysteroid conjugates were pres- ecdysteroid amounts before and after incubation (1). The ent, tissues were dissected from six nymphs during the same criticism exists of another report dealing with the fat deposition of epicuticle and were pooled for each sample. body as the site of ecdysteroid production in D. variabilis The dorsal and ventral integuments were extracted sepa- females (9). Here we describe experiments indicating epider- rately in methanol, dried, and then resuspended in H20 and mis as a source of ecdysone secretion in third instar nymphal loaded on a C18 Sep Pak cartridge. Each sample was sepa- Ornithodoros parkeri. rated into three fractions: 25% methanol eluate (containing polar products), 60o methanol eluate (free ecdysteroids), and 100% methanol eluate (apolar products) (13). Helix The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 3744 Downloaded by guest on September 30, 2021 Developmental Biology: Zhu et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3745

Table 1. Ecdysteroid immunoreactivity from medium incubated 500 1 A 24 h with various tissues 20 E Ecdysone Tissue n equivalent, pg 300 Midgut 6 0 Synganglia 4 0 E Salivary glands 4 0 100 Coxal glands 3 0 .*mmmmmm-. 1 II- Testis 6 8.6 ± 8.6 bO - Whole integument 6 359.3 ± 83.6 " 500- B Tissues from three nymphs were pooled and incubated in 150,ul of TC-199 medium for 24 h at 270C. After incubation, two 50-1I aliquots of medium were used directly for RIA. Data are expressed as the 300 - mean ± SEM immunoreactivity in 150 ;J of medium. pomatia enzymes (Sigma; type H-1) and pig liver esterase (Sigma) were used to hydrolyze polar and apolar products, 100 - respectively (14, 15). I Conventional histological paraffin sections (6 Am) of tis- 5 10 15 20 25 sues within or beneath the integument were double stained Fraction with Delafield hematoxylin and eosin (16) and examined by light microscopy. The inner surface was observed by scan- FIG. 1. HPLC RIA analysis of the ecdysteroids from the incu- ning electron microscopy (17). bation medium ofthe ventral integument with (A) and without (B) fat body. Retention times of markers: E, ecdysone; 20E, 20-hydroxy- ecdysone. Fractions of 400 ,ul (0.5 min) were collected and are RESULTS expressed in pg ecdysone equivalents. Synganglion, salivary glands, coxal glands, and midgut pro- amount of ecdysteroid immunoreactive materials in the me- duced no ecdysteroids. In addition, the testis did not appear dium after incubation was around 115 pg per incubation to produce RIA-positive material (in only one of six incuba- mixture, whereas the quantity of ecdysteroids in extracts of tions was a measurable but low level ofecdysteroid immuno- these two parts of the integument prior to incubation was reactivity detected). Only the integument showed clearly much lower (Table 3). Indeed, ecdysteroid production in- positive activity (Table 1). Since the integument contained fat creased almost 4-fold in the dorsal integument and >7-fold in body cells associated with tracheae, we carefully separated the ventral integument after 24 h of incubation. these cells with the tracheal trunk and the synganglion from To determine whether stored conjugates were present in the ventral integument, incubated them separately, and com- the integument, the immunoreactivity of 25% and 100% pared their activity of ecdysteroid production in vitro. The methanol fractions of extracts of the dorsal and ventral results (Table 2) clearly show that the integument produced integuments were measured before and after treatment with significantly higher immunoreactivity than fat body (t test; P Helix enzymes and porcine esterase, respectively. No im- < 0.01). munoreactive highly polar or apolar products were found and To characterize ecdysteroids produced by the integument no ecdysteroids were released by hydrolysis of apolar prod- in vitro, the combined media from 9 incubations of ventral ucts. After subtracting the background (-91 pg) due to the integument containing fat body and 13 incubations of ventral immunoreactivity of the Helix juice itself, we found no integument without fat body were analyzed by HPLC RIA. ecdysteroids released by hydrolysis of highly polar products Tissues from three nymphs were used in each incubation. In (Table 4). the case of the integument with fat body, the RIA activity Having demonstrated that the integument does secrete migrated as ecdysone and predominantly 20-hydroxyecdy- ecdysone during in vitro incubation, we then examined the sone (Fig. 1A). In the case ofthe integument without fat body, integument and found that it consists of newly formed the amount of 20-hydroxyecdysone was reduced to back- epicuticle, epidermis, and some fat body (Fig. 2). ground level and only ecdysone was present (Fig. 1B). These data suggest that the ecdysone produced by the integument is converted into 20-hydroxyecdysone by fat body. DISCUSSION Production of ecdysone by the integument during in vitro Our data demonstrate the site of ecdysone secretion in ticks. incubation appears to occur. However, the possibility exists The net increase in the ecdysteroid content during a 24-h that the hormone is stored in the tissue and is released during incubation of the integument (Table 3) and the analysis of incubation or that stored conjugated ecdysteroids were set those ecdysteroids by HPLC RIA (Fig. 1) indicate that the free during incubation. To resolve these questions, we first epidermis of 0. parkeri nymphs synthesizes ecdysone but not compared separately the ecdysteroid content in dorsal and 20-hydroxyecdysone and other ecdysteroids. The predomi- ventral integument before and after incubation. The average Table 3. Quantity of ecdysteroids before and after incubation (24 Table 2. Ecdysteroid immunoreactivity from medium incubated h) of dorsal and ventral integument 24 h with ventral integument and fat body DI VI Ecdysone n equivalent, pg Tissue before incubation 30.3 ± 5.9 16.0 ± 2.6 Medium after incubation 114.6 ± 14.8 115.9 ± 14.6 Fat body 5 22.3 ± 14.9 Ventral integument 5 153.9 ± 35.7 Ecdysteroid content is expressed in pg ecdysone equivalents. Data are the means ± SEM of five replicates for dorsal integument (DI) Fat body fraction contained tracheae, synganglion, and a part of and ventral integument (VI) before incubation and ofeight replicates muscle that was separated from ventral integument. Conditions of for each after incubation. Each value represents the tissue from three incubation were the same as in Table 1. Data are the means ± SEM. ticks. Downloaded by guest on September 30, 2021 3746 Developmental Biology: Zhu et al. Proc. NatL Acad. Sci. USA 88 (1991) Table 4. Ecdysteroid contents of polar and apolar fractions and integument extracts Highly polar products Apolar products Before After Before After Tissue hydrolysis hydrolysis hydrolysis hydrolysis Dorsal integument 0 97.1 ± 5.9 0 0 Ventral integument 0 95.0 ± 7.3 0 0 Control (with enzyme only) 90.5 ± 10.4 0 Ecdysteroid content is expressed in pg ecdysone equivalents. Highly polar products were hydrolyzed with H. pomatia enzymes and apolar products were hydrolyzed with esterase. Data are the means + SEM of three replicates, except for controls, which are duplicates. Each value represents the tissue from the equivalent of three ticks. nant sources of ecdysteroids are the prothoracic (or corre- hormone. If the same is true in ticks, the probable pathway sponding) glands in insect larvae (18-21) and the ovary in of ecdysteroids in immature stages is that epidermis secretes adult insects (21, 22). However, there is a growing amount of ecdysone, which is subsequently converted into 20- evidence showing the existence of alternative sites of ecdy- hydroxyecdysone by fat body or other peripheral tissue(s). steroid production-e.g., oenocytes, epidermis, and testis Then 20-hydroxyecdysone stimulates the epidermis and ini- (23). Epidermis as a source of ecdysone has been clearly tiates the molting process. demonstrated in pupae of the beetle Tenebrio molitor in The idea that the epidermis may serve as both a source and which the prothoracic glands have degenerated (23, 24). a target of ecdysteroids in insects is just beginning to be Prothoracic glands and epidermis are both of ectodermal accepted (23). These autocrine (cell secretes hormone that origin, and ticks may represent an example of animals that has specific action on the cell itself) and paracrine (cell periodically molt but do not possess specific molting glands. secretes hormones of which the neighboring cells are the Oenocytes also are reported to be a possible source of target) mechanisms are better known in vertebrates and ecdysteroids in T. molitor (25). However, no oenocytes could increasing numbers of examples exist in the literature. Del- be found either within or beneath the epidermis of 0. parkeri becque et al. (23) suggest that perhaps autocrine or paracrine nymphs. ecdysteroid interactions may represent a more primitive Some reports show that ecdysteroids function as a molting system than the endocrine control of molting represented stimulant in ticks and that both ecdysone and 20-hydroxy- in most insects. Since ticks, and especially argasid ticks, ecdysone are present in immature stages of argasid (2) and retain many ixodid ticks (3). HPLC RIA analysis of the extract from 0. evolutionarily primitive traits (26), perhaps parkeri nymphs during epicuticle deposition also shows the such a mechanism of molt regulation may be common in presence of these two ecdysteroids (unpublished data). The ticks. finding that the integument alone produces ecdysone but that We thank Prof. T. Ohtaki (Kanazawa University, Kanazawa, in combination with fat body it produces ecdysone and Japan) for his generous gift of ecdysone antiserum and Prof. S. 20-hydroxyecdysone (Fig. 1) strongly suggests that fat body McKeever (Georgia Southern University) for his kind help in per- cells are one of the sites for conversion of ecdysone to forming the scanning electron microscopy. Thanks also to Martha 20-hydroxyecdysone. In most insect species that have been Joiner for editorial assistance. The research was partially supported investigated, 20-hydroxyecdysone is biologically more active by National Institute of Allergy and Infectious Diseases Grant than ecdysone and is thought to be the actual molting AI-09556. ~~~ 2

FIG. 2. (A) Histological section of ventral integument during the deposition of epicuticle (ep). ed, Epidermal cells; oc, old cuticle; fb, fat body. (Bar = 20 ,um.) (B) Scanning electron micrograph of the inner surface of the ventral integument showing newly forming epicuticle. (Bar = 40 ,m.) Downloaded by guest on September 30, 2021 Developmental Biology: Zhu et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3747 1. Diehl, P. A., Connat, J.-L. & Dotson, E. (1986) in Morphology Int. J. Invertebr. Reprod. Dev. 8, 1-13. andBehavioral Biology ofTicks, eds. Sauer, J. R. & Hair, J. A. 15. Zhu, X. X., Gfeller, H. & Lanzrein, B. (1983) J. Insect Physiol. (Horwood, Chichester, U.K.), pp. 165-193. 29, 225-235. 2. Germond, J.-E., Diehl, P. A. & Morici, M. (1982) Gen. Comp. 16. Humason, G. L. (1979) Animal Tissue Techniques (Freeman, Endocrinol. 46, 255-266. San Francisco). 3. Diehl, P. A., Germond, J. E. & Morici, M. (1982) Rev. Suisse 17. McKeever, S., Wright, M. C. & Hagan, D. V. (1988) Ann. Zool. 89, 859-868. Entomol. Soc. Am. 81, 332-341. 4. Wigglesworth, K. P., Lewis, D. & Rees, H. H. (1985) Arch. 18. Chino, H., Sakurai, S., Ohtaki, T., Ikekawa, N., Insect Biochem. Physiol. 2, 39-54. Miyazaki, H., 5. Connat, J.-L., Diehl, P. A., Gfeller, H. & Morici, M. (1985) Int. Ishibashi, H. & Abuki, H. (1974) Science 183, 529-530. J. Invert. Reprod. Dev. 8, 103-116. 19. King, D. S., Bollenbacher, W. E., Borst, D. W., Vedeckis, 6. Lindsay, P. J. & Kaufman, W. R. (1988) J. Insect Physiol. 34, W. V., O'Connor, J. D., Ittycheriah, P. I. & Gilbert, L. 1. 351-359. (1974) Proc. Natl. Acad. Sci. USA 71, 793-796. 7. Dees, W. H., Sonenshine, D. E. & Breidling, E. (1984) J. Med. 20. Rees, H. H. (1985) in Comprehensive Insect Physiology, Bio- Entomol. 21, 514-523. chemistry and Pharmacology, eds. Kerkut, G. A. & Gilbert, 8. Ellis, B. J. & Obenchain, F. D. (1984) in Acarology VI, eds. L. I. (Pergamon, Oxford), Vol. 7, pp. 249-293. Griffiths, D. A. & Bowman, C. E. (Horwood, Chichester, 21. Koolman, J. (1990) Zool. Sci. 7, 563-580. U.K.), Vol. 1, pp. 400-404. 22. Hagedorn, H. H., O'Connor, J. D., Fuchs, M. S., Sage, B., 9. Schriefer, M. E., Beveridge, M., Sonenshine, D. E., Homsher, Schlaeger, D. A. & Bohm, M. K. (1975) Proc. Natl. Acad. Sci. P. J., Carson, K. A. & Weidman, C. S. (1987) J. Med. Ento- USA 72, 3244-3259. mol. 24, 295-302. 23. Delbecque, J.-P., Weidner, K. & Hoffmann, K. H. (1990) 10. Pound, J. M., Oliver, J. H., Jr., & Andrews, R. H. (1984) J. Invertebr. Reprod. Dev. 18, 29-42. Parasitol. 70, 279-284. 24. Delachambre, M. T., Besson, A., Quennedey, A. & Delbec- 11. Borst, D. W. & O'Connor, J. D. (1974) Steroids 24, 637-656. que, J.-P. (1984) in Biosynthesis, Metabolism and Mode of 12. Horn, D. H. S., Wilkie, J. S., Sage, B. & O'Connor, J. D. Action of Invertebrate Hormones, eds. Hoffmann, J. & Por- (1976) J. Insect Physiol. 21, 901-905. chet, M. (Springer, Berlin), pp. 245-254. 13. Lafont, R., Pennetier, J., Andriajafintrimo, M., Claret, J., 25. Romer, F., Emmerich, H. & Nowock, J. (1974) Steroids 24, Modde, J. F. & Blais, C. (1982) J. Chromatogr. 236, 137-149. 637-656. 14. Diehl, P. A., Connat, J.-L., Girault, J. P. & Lafont, R. (1985) 26. Oliver, J. H., Jr. (1989) Annu. Rev. Ecol. Syst. 20, 397-430. Downloaded by guest on September 30, 2021