Proc. Natl. Acad. Sci. USAVol. 88, pp. 3744-3747, May 1991Developmental 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. DOTSONInstitute 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 thetick Ornithodoros parkeri at the time of epicuticle depositionwere incubated in vitro. The medium from the incubation ofsalivary glands, coxal glands, synganglion, testis, midgut, andfat body associated with tracheal trunk showed little or noecdysteroid immunoreactivity. Only medium from incubatedintegument contained ecdysteroids. The following evidenceindicated that epidermal cells are the source of ecdysone: (i)when dorsal and/or ventral integuments were incubated sep-arately, both produced ecdysteroid immunoreactive materialduring the course of incubation. As compared with the ecdy-steroid content in the integument before incubation, theamount of ecdysteroids produced after a 24-h incubationincreased 4- to 7-fold; (ii) enzymatic hydrolysis showed thatneither highly polar ecdysteroid conjugates nor apolar conju-gates were stored in the integument; (iii) histological andscanning electron microscope observations demonstrated thatthese excised integuments consisted of newly deposited epicu-ticle and epidermis as well as some fat body cells; (iv) HPLCRIA showed that the integument with associated fat bodyproduced ecdysone and 20-hydroxyecdysone, while the integ-ument produced only ecdysone after removing fat body. Pre-sumably, ecdysone secreted by epidermis was converted into20-hydroxyecdysone by fat body.

Ticks are of considerable medical and economic importance.However, little is known about their neurohormonal regula-tion of development and reproduction. So far, ecdysteroidsare the only hormones that have been definitely identified (1).In immature stages of argasid and ixodid ticks (Ornithodorosmoubata and Amblyomma hebraeum), ecdysone and mainly20-hydroxyecdysone are present, and their increasing andhigher titers parallel apolysis and epicuticle deposition (2, 3).In females of ixodid ticks (Boophilus microplus and A.hebraeum), a peak titer of either mainly free ecdysone or20-hydroxyecdysone has been observed during oogenesis (4,5) and a possible role in salivary gland degeneration has beensuggested in Amblyomma americanum females (6). In addi-tion, ecdysteroid immunoreactive materials have been de-tected in males of the ixodid tick Dermacentor variabilis (7).The origin of ecdysteroids in ticks remains uncertain. In

Amblyomma variegatum nymphs it was suggested, based onin vitro tissue culture and the measurement of RIA-positivematerial, that fat body is the site of ecdysteroid production(8). However, this work has been criticized for not comparingecdysteroid amounts before and after incubation (1). Thesame criticism exists of another report dealing with the fatbody as the site of ecdysteroid production in D. variabilisfemales (9). Here we describe experiments indicating epider-mis as a source of ecdysone secretion in third instar nymphalOrnithodoros parkeri.

MATERIALS AND METHODS

Ticks (0. parkeri) were reared at 270C and 85% relativehumidity by standard techniques (10). RIA of ecdysteroidswas carried out following established methods (11, 12). Foreach assay, a standard curve was prepared using ecdysone(Sigma) and [23,24-3H]ecdysone (New England Nuclear).The antiserum used was a generous gift from T. Ohtaki(Kanazawa, Japan) and has a 2.5-fold higher sensitivity forecdysone than for 20-hydroxyecdysone when their 50%cross-reaction was compared.

Ecdysteroid titer was highest during epicuticle depositionin third instar nymphal 0. parkeri (unpublished data), andthus nymphs at this stage were used in all our experiments.To locate the possible site of ecdysteroid production, varioustissues were dissected out after removing the old cuticle. Themidgut contents were washed out and each tissue was rinsedtwice separately in the incubation medium [GIBCO TC-199medium fortified with Ficoll (20 mg/ml)]. Like tissues fromthree nymphs were pooled and then incubated at 270C withgentle shaking in 150 1.l of medium. After 24 h, 50 p1L ofmedium, in duplicate, was transferred to RIA tubes for assayof ecdysteroid content. Results are expressed per 150 1Ad ofmedium.For HPLC analysis of the components active in the RIA

assays, the incubation medium was passed through a C18 SepPak cartridge (Waters), which was then rinsed with H20 andsubsequently with 25% methanol (13). The immunoreactivematerial was eluted with pure methanol, concentrated, andinjected into a HPLC (Spectra-Physics; model 8800)equipped with a programmable wavelength detector and anSP 4270 integrator. A reversed-phase column (Keystone,Bellefonte, PA; 15 cm, 4.6-mm i.d. packed with LichrosorbRP-18; 5 ,um) with 50o methanol/water as a solvent and a

flow rate of 800 ,ul/min were used. Fractions were collectedevery 30 sec for 15 min and examined by RIA for the presenceof ecdysteroids. Ecdysone and 20-hydroxyecdysone (Sigma)were used as references. After running the standards, thecolumn and injector were rinsed and a blank sample contain-ing only the eluate was injected into HPLC to ensure no

contamination of the column and injector. No immunoreac-tive material was detected in the fractions of the blank run.

Before injecting the sample, an aliquot was assayed by RIAto determine the amount of immunoreactivity present. Therecovery of ecdysteroid immunoreactivity from fractions ofthe samples injected on the HPLC was >80%o.To determine whether ecdysteroid conjugates were pres-

ent, tissues were dissected from six nymphs during thedeposition of epicuticle and were pooled for each sample.The dorsal and ventral integuments were extracted sepa-rately in methanol, dried, and then resuspended in H20 andloaded on a C18 Sep Pak cartridge. Each sample was sepa-rated into three fractions: 25% methanol eluate (containingpolar products), 60o methanol eluate (free ecdysteroids),and 100% methanol eluate (apolar products) (13). Helix

*To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 88 (1991) 3745

Table 1. Ecdysteroid immunoreactivity from medium incubated24 h with various tissues

EcdysoneTissue n equivalent, pg

Midgut 6 0Synganglia 4 0Salivary glands 4 0Coxal glands 3 0Testis 6 8.6 ± 8.6Whole integument 6 359.3 ± 83.6

Tissues from three nymphs were pooled and incubated in 150,ul ofTC-199 medium for 24 h at 270C. After incubation, two 50-1I aliquotsof medium were used directly for RIA. Data are expressed as themean ± 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,respectively (14, 15).Conventional histological paraffin sections (6 Am) of tis-

sues within or beneath the integument were double stainedwith Delafield hematoxylin and eosin (16) and examined bylight microscopy. The inner surface was observed by scan-ning electron microscopy (17).

RESULTSSynganglion, salivary glands, coxal glands, and midgut pro-duced no ecdysteroids. In addition, the testis did not appearto produce RIA-positive material (in only one of six incuba-tions was a measurable but low level of ecdysteroid immuno-reactivity detected). Only the integument showed clearlypositive activity (Table 1). Since the integument contained fatbody cells associated with tracheae, we carefully separatedthese cells with the tracheal trunk and the synganglion fromthe ventral integument, incubated them separately, and com-pared their activity of ecdysteroid production in vitro. Theresults (Table 2) clearly show that the integument producedsignificantly higher immunoreactivity than fat body (t test; P< 0.01).To characterize ecdysteroids produced by the integument

in vitro, the combined media from 9 incubations of ventralintegument containing fat body and 13 incubations of ventralintegument without fat body were analyzed by HPLC RIA.Tissues from three nymphs were used in each incubation. Inthe case of the integument with fat body, the RIA activitymigrated as ecdysone and predominantly 20-hydroxyecdy-sone (Fig. 1A). In the case ofthe integument without fat body,the amount of 20-hydroxyecdysone was reduced to back-ground level and only ecdysone was present (Fig. 1B). Thesedata suggest that the ecdysone produced by the integumentis converted into 20-hydroxyecdysone by fat body.

Production of ecdysone by the integument during in vitroincubation appears to occur. However, the possibility existsthat the hormone is stored in the tissue and is released duringincubation or that stored conjugated ecdysteroids were setfree during incubation. To resolve these questions, we firstcompared separately the ecdysteroid content in dorsal andventral integument before and after incubation. The average

Table 2. Ecdysteroid immunoreactivity from medium incubated24 h with ventral integument and fat body

Ecdysonen equivalent, pg

Fat body 5 22.3 ± 14.9Ventral integument 5 153.9 ± 35.7

500 1 A

300

100

bO -"

500-

300 -

100 -

20 E

. . 1E

II-*mmmmmm-

B

5 10 15Fraction

I20 25

FIG. 1. HPLC RIA analysis of the ecdysteroids from the incu-bation medium of the ventral integument with (A) and without (B) fatbody. Retention times of markers: E, ecdysone; 20E, 20-hydroxy-ecdysone. Fractions of 400 ,ul (0.5 min) were collected and areexpressed in pg ecdysone equivalents.

amount of ecdysteroid immunoreactive materials in the me-dium after incubation was around 115 pg per incubationmixture, whereas the quantity of ecdysteroids in extracts ofthese two parts of the integument prior to incubation wasmuch lower (Table 3). Indeed, ecdysteroid production in-creased almost 4-fold in the dorsal integument and >7-fold inthe ventral integument after 24 h of incubation.To determine whether stored conjugates were present in

the integument, the immunoreactivity of 25% and 100%methanol fractions of extracts of the dorsal and ventralinteguments were measured before and after treatment withHelix enzymes and porcine esterase, respectively. No im-munoreactive highly polar or apolar products were found andno ecdysteroids were released by hydrolysis of apolar prod-ucts. After subtracting the background (-91 pg) due to theimmunoreactivity of the Helix juice itself, we found noecdysteroids released by hydrolysis of highly polar products(Table 4).Having demonstrated that the integument does secrete

ecdysone during in vitro incubation, we then examined theintegument and found that it consists of newly formedepicuticle, epidermis, and some fat body (Fig. 2).

DISCUSSIONOur data demonstrate the site of ecdysone secretion in ticks.The net increase in the ecdysteroid content during a 24-hincubation of the integument (Table 3) and the analysis ofthose ecdysteroids by HPLC RIA (Fig. 1) indicate that theepidermis of0. parkeri nymphs synthesizes ecdysone but not20-hydroxyecdysone and other ecdysteroids. The predomi-

Table 3. Quantity of ecdysteroids before and after incubation (24h) of dorsal and ventral integument

DI VI

Tissue before incubation 30.3 ± 5.9 16.0 ± 2.6Medium after incubation 114.6 ± 14.8 115.9 ± 14.6Ecdysteroid content is expressed in pg ecdysone equivalents. Data

are the means ± SEM of five replicates for dorsal integument (DI)and ventral integument (VI) before incubation and of eight replicatesfor each after incubation. Each value represents the tissue from threeticks.

Fat body fraction contained tracheae, synganglion, and a part ofmuscle that was separated from ventral integument. Conditions ofincubation were the same as in Table 1. Data are the means ± SEM.

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Table 4. Ecdysteroid contents of polar and apolar fractions and integument extractsHighly polar products Apolar products

Before After Before AfterTissue hydrolysis hydrolysis hydrolysis hydrolysis

Dorsal integument 0 97.1 ± 5.9 0 0Ventral integument 0 95.0 ± 7.3 0 0Control (with enzyme only) 90.5 ± 10.4 0

Ecdysteroid content is expressed in pg ecdysone equivalents. Highly polar products were hydrolyzedwith 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 tissuefrom the equivalent of three ticks.

nant sources of ecdysteroids are the prothoracic (or corre-sponding) glands in insect larvae (18-21) and the ovary inadult insects (21, 22). However, there is a growing amount ofevidence showing the existence of alternative sites of ecdy-steroid production-e.g., oenocytes, epidermis, and testis(23). Epidermis as a source of ecdysone has been clearlydemonstrated in pupae of the beetle Tenebrio molitor inwhich the prothoracic glands have degenerated (23, 24).Prothoracic glands and epidermis are both of ectodermalorigin, and ticks may represent an example of animals thatperiodically molt but do not possess specific molting glands.Oenocytes also are reported to be a possible source ofecdysteroids in T. molitor (25). However, no oenocytes couldbe found either within or beneath the epidermis of 0. parkerinymphs.Some reports show that ecdysteroids function as a molting

stimulant in ticks and that both ecdysone and 20-hydroxy-ecdysone are present in immature stages of argasid (2) andixodid ticks (3). HPLC RIA analysis of the extract from 0.parkeri nymphs during epicuticle deposition also shows thepresence of these two ecdysteroids (unpublished data). Thefinding that the integument alone produces ecdysone but thatin combination with fat body it produces ecdysone and20-hydroxyecdysone (Fig. 1) strongly suggests that fat bodycells are one of the sites for conversion of ecdysone to20-hydroxyecdysone. In most insect species that have beeninvestigated, 20-hydroxyecdysone is biologically more activethan ecdysone and is thought to be the actual molting

~~~ 2

hormone. If the same is true in ticks, the probable pathwayof ecdysteroids in immature stages is that epidermis secretesecdysone, which is subsequently converted into 20-hydroxyecdysone by fat body or other peripheral tissue(s).Then 20-hydroxyecdysone stimulates the epidermis and ini-tiates the molting process.The idea that the epidermis may serve as both a source and

a target of ecdysteroids in insects is just beginning to beaccepted (23). These autocrine (cell secretes hormone thathas specific action on the cell itself) and paracrine (cellsecretes hormones of which the neighboring cells are thetarget) mechanisms are better known in vertebrates andincreasing numbers of examples exist in the literature. Del-becque et al. (23) suggest that perhaps autocrine or paracrineecdysteroid interactions may represent a more primitivesystem than the endocrine control of molting representedin most insects. Since ticks, and especially argasid ticks,retain many evolutionarily primitive traits (26), perhapssuch a mechanism of molt regulation may be common inticks.

We thank Prof. T. Ohtaki (Kanazawa University, Kanazawa,Japan) for his generous gift of ecdysone antiserum and Prof. S.McKeever (Georgia Southern University) for his kind help in per-forming the scanning electron microscopy. Thanks also to MarthaJoiner for editorial assistance. The research was partially supportedby National Institute of Allergy and Infectious Diseases GrantAI-09556.

FIG. 2. (A) Histological section of ventral integument during the deposition of epicuticle (ep). ed, Epidermal cells; oc, old cuticle; fb, fatbody. (Bar = 20 ,um.) (B) Scanning electron micrograph of the inner surface of the ventral integument showing newly forming epicuticle. (Bar= 40 ,m.)

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