Effect of methoprene and 20-hydroxyecdysone on vitellogenin production in cultured fat bodies and backless explants from unfed female Dermacentor variabilis

Journal of Insect Physiology 45 (1999) 755–761www.elsevier.com/locate/ibmbjip

Effect of methoprene and 20-hydroxyecdysone on vitellogeninproduction in cultured fat bodies and backless explants from unfed

femaleDermacentor variabilis

Naby Sankhona, Timothy Lockeyb, Rosemarie C. Rosellc, Marjorie Rothschildd,Lewis Coonsd,*

a Science and Mathematics Division, Rust College, Holly Springs, MS 38635, USAb St Jude’s Children Research Hospital, Memphis, TN 38105, USA

c Department of Biology, University of St Thomas, Houston, TX 77006, USAd Department of Microbiology and Molecular Cell Sciences, University of Memphis, Memphis, TN 38152, USA

Received 25 August 1998; accepted 13 November 1998

Abstract

The effect of 20-hydroxyecdysone (20HE) and the juvenile hormone analogue methoprene (JHA) on vitellogenin (Vg) productionin fat body organ cultures and backless explants of unfed femaleDermacentor variabiliswas measured. An indirect double antibodyenzyme linked immunosorbent assay (ELISA) was developed using a monoclonal antibody that recognized a 98 kDa subunit ofVg and a Vg specific polyclonal antibody made against vitellin (Vn). Peak Vg titers in culture medium from fat body culturestreated with 0.1µM 20HE or 1 µM 20HE were 24 ng/ml and 20 ng/ml respectively. In culture medium from backless explantstreated with 0.1µM 20HE or 1 µM 20HE, peak Vg titers were 36 ng/ml and 26 ng/ml, respectively. JHA produced only a slightincrease in Vg titers that was statistically different from Vg titers produced by 20HE but was not statistically different from hormone-free controls. These results support the conclusion that Vg production in fat body trophocytes ofD. variabilis is regulated by 20HE. 1999 Elsevier Science Ltd. All rights reserved.

Keywords:Vitellogenin; 20-hydroxyecdysone; Methoprene; Fat body;Dermacentor variabilis

1. Introduction

Vitellogenesis is under hormonal control in ticks(Shanbaky and Khalil, 1975; Oliver and Dotson, 1993;Coons and Alberti, 1999). The major yolk protein vitel-lin (Vn), is a hemoglycolipoprotein (Chinzei, 1983;Chinzei et al., 1983; Rosell and Coons, 1991; James andOliver, 1997). It is synthesized outside the ovary as vitel-logenin (Vg), released into the hemolymph and trans-ported to the ovary where it is selectively taken up asVn (Chinzei and Yano, 1985; Rosell-Davis and Coons,1989a). The fat body is a site of Vg production in theixodid Dermacentor variabilis(Rosell-Davis and Coons,1989b; Rosell and Coons, 1992). Fat body trophocytes

* Corresponding author. Tel.:+1-901-678-2034; fax:+1-901-678-4457.E-mail address:[email protected] (L. Coons)

0022-1910/99/$ - see front matter. 1999 Elsevier Science Ltd. All rights reserved.PII: S0022-1910 (99)00054-2

from females but not from males undergo a dramaticchange during feeding, resulting in an ultrastructurecharacteristic of cells producing a protein secretory pro-duct (Coons et al., 1990).

Ecdysteroids, hormones known to be involved in theproduction of Vg in insects, occur in ticks (Delbecqueet al., 1978; Connat et al., 1984; Dees et al., 1984;Crosby et al., 1986; Whitehead et al., 1986; Zhu et al.,1991; James et al., 1997) where they control ixodid sali-vary gland degeneration (Harris and Kaufman, 1985;Lindsay and Kaufman, 1988; Kaufman, 1990, 1991a,b)and molting in the argasidOrnithodoros moubata(Germond et al., 1982).

The presence of a juvenile hormone (JH) or a juvenoidin several species of ixodids has been suggested usingindirect technics: anti-JH sera (Connat, 1987), the pres-ence of enzymes involved in JH catabolism (Venkateshet al., 1990), the presence of proteins in the hemolymphthat bind JH (Sonenshine et al., 1989; Lomas et al.,

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1996), and unidentified radiolabeled products from thesynganglion (Roe, 1992; Roe et al., 1993). These studiesonly suggest the presence of JH or a juvenoid, ecdystero-ids are the only hormones that have been unequivocallyidentified in ticks (Oliver and Dotson, 1993).

Studies indirectly measuring the effect of ecdysteroidsand juvenoids on reproductive processes in ticks haveproduced conflicting results. Two studies conclude thatecdysteroids inhibit vitellogenesis inOrnithodoros mou-bata (Connat et al., 1983a; Connat et al., 1986). But apositive correlation occurs between ecdysteroids andvarious aspects of reproduction in some ixodid ticks. InBoophilus microplus, a peak titer of mostly free ecdy-sone or 20HE occurs during oogenesis (Wigglesworth etal., 1985); and inDermacentor variabilis, ecdysteroidlevels increase during feeding and oviposition (Dees etal., 1984). Several studies have suggested that JH stimu-lates vitellogenesis, as measured by the size of the eggmass (Bassal and Roshdy, 1974; Pound and Oliver,1979; Connat et al., 1983b). Yet no role could be estab-lished for JH or an ecdysteroid in egg development ofthe ixodid Amblyomma hebraeumas measured by yolkproduction (Lunke and Kaufman, 1993). Differencesbetween the reproductive physiology of soft and hardticks has been reviewed by Oliver and Dotson (1993).

In this study, our objective was to test the effect of20-hydroxyecdysone (20HE) and the JH analoguemethoprene (JHA) on the production of Vg by culturedfat bodies and backless explants of unfed femaleDermacentor variabilisusing an indirect double anti-body enzyme linked immunosorbent assay (ELISA). Anorgan-culturing technique coupled with a sensitiveenzyme immunoassay procedure, such as an indirectdouble antibody ELISA, is essential for demonstratingthe direct relationship of a hormone and its target tissue(Ma et al., 1984, 1986, 1987).

2. Materials and methods

2.1. Preparation of antibody

Polyclonal rabbit anti-Vn was prepared against pur-ified Vn and tested for specificity to Vg as previouslydescribed (Rosell-Davis and Coons, 1989b; Rosell andCoons, 1992). Monoclonal antibody (mAb) was madeagainst the purified Vn as described by Marion et al.(1982), at the Hybridoma facility, Molecular ResourceCenter, University of Tennessee, Memphis.

A protein G agarose affinity column (BoerhringerMannheim) was used to purify the mAb from crudeascites fluid (Chao et al., 1985). Ascites fluid from clone2.1 was diluted 1:5 in 0.01 M phosphate buffer saline(PBS) pH 7.5 and centrifuged at 10,000 rpm for 5minutes to remove cells. Two ml of diluted ascites fluidwas applied to the protein G affinity column and incu-

Fig. 1. SDS–PAGE 7.5–15% gradient gel of the purified monoclonalantibody IgG under denaturing, reducing conditions. Coomassie Brilli-ant Blue staining. Lane 1, low molecular weight standards; lane 2,purified IgG; lane 3, crude ascites fluid.

bated for one hour to allow sufficient binding. The col-umn was washed with PBS to remove non-bound pro-tein. Immunoglobulin (IgG) was eluted with 0.2 Mglycine (pH 2.8) neutralized with NaOH, and concen-trated to 1 mg/ml using immersible-CX ultrafilters(Millipore). The purity of the immunoglobulin was veri-fied by electrophoresis on a 7.5–15% polyacrylamidegradient gel under denaturing and reducing conditions(Chao et al., 1985). Two bands with molecular weightsof 50 kDa and 30 kDa, respectively, were present on the

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gel (Fig. 1). These bands represent the heavy and lightchains of the IgG molecule. The purified monoclonalIgG was used in the ELISA assays.

For Western blots, proteins were transferred frompolyacrylamide gels to nitrocellulose membranes (NCM)with a Bio-Rad Trans-Blot cell (Rosell-Davis and Coons,1989a). NCM was incubated in TBST (1% BSA and 1%goat serum in tris-buffered saline plus 0.005% Tween20) (Sigma). The NCM was then incubated with mAb(hybridoma supernatant) diluted 1:500 in antibodydilution buffer (ADB: 0.05% BSA, 1% goat serum inTBST). The NCM was washed twice in ADB and incu-bated in a 1:9000 dilution of biotin-labelled goat anti-mouse IgG. After three washes in ADB, the NCM wasincubated with streptavidin alkaline phosphatase(1:12,000), washed three times in tris-buffered salineplus 0.0005% Tween 20 (TBS), and incubated with 5-bromo-4-chloro-3-indolyl phosphatep-nitroblue tetrazo-lium chloride until a satisfactory signal was visible. Inwestern blots of purified Vn, the mAb recognized a sin-gle subunit with an approximate molecular weight of 98kDa (Fig. 2). When used in a western blot against hemo-lymph of ovipositingDermacentor variabilis, the mAbrecognized two major polypeptides with molecularweights of 135 kDa and 98 kDa respectively (Fig. 3).The larger subunit may be a precursor of the 98 kDapolypeptide or may have similar antigenic epitopes.

2.2. Indirect double antibody ELISA

The ELISA we designed used a protein-A purifiedrabbit polyclonal anti-Vn IgG as the primary antibodyand protein G purified mAb from clone 2.1 as the sec-ondary antibody. Biotin conjugated goat anti-mouse IgGStreptavidin alkaline phosphatase was the tertiary anti-body used to visualize the reaction.

For the ELISA, primary antibody was diluted to a finalconcentration of 1µg/ml in the assay. Ninety-six wellmicrotiter plates (Falcon) were coated with 100µl of theprimary antibody and allowed to stand overnight at 4°C.Wells were washed three times with TBST. Unoccupiedprotein binding sites were blocked by incubating with1% BSA for 2 hours at 37°C. After washing with TBS,diluted antigen (purified Vn, or unknown aliquots fromexperimental procedures) was added to each well (100µl/well) and the microtiter plates were incubated for 20hours at 4°C.

Following incubation, plates were washed three timeswith TBST. The secondary antibody was diluted1:20,000 in ADB, 100µl were added to each well, andmicrotiter plates were incubated for 4 hours at 37°C. Fol-lowing incubation, the microtiter wells were washed asdescribed above. Biotinylated goat anti-mouse IgG wasdiluted 1:5000 in ADB containing 0.05% BSA and 1%goat serum, and 100µl were added to each well.Microtiter plates were incubated at room temperature for

Fig. 2. SDS–PAGE 5–15% gradient gel of the purified Vn underdenaturing, reducing conditions. Coomassie Brilliant Blue staining.Lane 1, high molecular weight standards; lane 2, purified Vn; lane 3,Western blot of the purified Vn probed with antibody from hybridomacell line designated clone 2.1.

1 hour. After washing as described above, streptavidinalkaline phosphatase diluted at 1:1000 was added to eachwell and microtiter plates were incubated for 30 minutes.Following five washings of TBST, 100µl of p-nitro-phenyl phosphate (1 mg/ml) in 10% diethanolaminebuffer (pH 9.6) was added to each well and plates wereincubated for 1 hour. The reaction was stopped byadding 50µl of 1 N sulfuric acid to each well. Resultswere read at 405 nm on Bio-Tek Instruments ELS 309Microplate Autoreader.

Various known concentrations of purified Vn wereused to generate a standard curve. The assay was linearup to 700 ng/ml of the purified Vn and that portion of thestandard curve was used to quantitate Vg in the ELISA.

In order to show that nonspecific binding of nonre-lated proteins did not occur with the mAb or with thepolyclonal antibody, the following were omitted in a ser-ies of control wells: antigen, polyclonal antibody, mAb

758 N. Sankhon et al. / Journal of Insect Physiology 45 (1999) 755–761

Fig. 3. SDS–PAGE 5–15% gradient gel of the purified Vn underdenaturing, reducing conditions. Coomassie Brilliant Blue staining.Lane 1, high molecular weight standards; lane 2, hemolymph fromovipositing ticks; lane 3, western blot of hemolymph from ovipositingDermacentor variabilisprobed with mAb from hybridoma cell linedesignated clone 2.1

and conjugate. Other antibody specificity controls per-formed were a substitution of mAb with normal goatserum or conjugate. The background reading was lessthan or equal to 0.05 OD in these tests.

2.3. Fat body organ and backless explant culturetechniques

Unfed female ticks were immobilized in paraffin(Coons et al., 1989) then flooded with culture mediumconsisting of equal parts of Eagle’s Minimum EssentialMedium (MEM) with Hank’s BSS and Leibovitz’s L-15medium supplemented with 20% fetal calf serum, 10%tryptose phosphate broth, 0.1% BSA (fraction V), 100U/ml penicillin, 100µg/ml streptomycin sulfate, and 40mg/ml neomycin sulfate. The upper integument was

removed from each tick and the fat body dissected fromaround the large tracheal trunks and around the ovaryand transferred to microtiter plate wells. Backlessexplants were ticks with the upper integument removed.Each well contained fat bodies from nine ticks and 60µl of culture medium, or one backless explant coveredwith 60 µl of culture medium. A single aliquot of tissueculture medium (60µl) was taken periodically for Vgdetermination, and replaced with 60µl of fresh culturemedium.

Groups of nine fat bodies or individual backlessexplants were incubated for 42 hours at 25°C in mediumcontaining either 1µM or 0.1 µM of 20HE or JHA.20HE (Sigma) and JHA (Zoecon Corporation) were dis-solved in 70% ethanol to produce stock solutions (1mg/ml). Stock solutions were diluted to 1µM and 0.1µM working solutions in sterile culture medium prior touse. Sixty microliters of medium were removed every 3hours from each culture dish and Vg concentration wasdetermined using the ELISA assay. Control wells con-sisted of fat bodies in hormone free culture medium.Experiments were replicated three times.

2.4. Statistical method

A Duncan range test was used to determine statisticaldifferences between the four study conditions.

3. Results

In 20HE treated cultures of fat body and backlessexplants, Vg concentration increased gradually, reacheda peak after 27 hours of incubation in culture medium,and then declined (Figs. 4 and 5). The peak Vg titer in

Fig. 4. Fat body organ cultures, treated with 20HE and JHA. Errorbars represent standard deviation of the mean (SD). Duncan rangeresults are shown below graph. A treatment underlined by the samebar was not significantly different (P,0.05).

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Fig. 5. Backless tick explants treated with 20HE and the JHA. Errorbars represent standard deviation of the mean (SD). Duncan rangeresults are shown below graph. A treatment underlined by the samebar was not significantly different (P,0.05).

the fat body culture medium treated with 1µM 20HEwas 20 ng/ml and 24 ng/ml when treated with 0.1µM20HE (Fig. 4). The peak Vg titer in culture medium frombackless explants treated with 1µM 20HE was 26 ng/mland 36 ng/ml when treated with 0.1µM 20 HE (Fig. 5).A small, constant titer of Vg occurred in hormone freeculture medium of fat body and backless explants (Figs.4 and 5). It ranged from a high of 10 ng/ml in culturemedium from fat body cultures to a high of 9 ng/ml inculture medium from backless explants. This titer ismost likely due to a small amount of Vn carried overfrom the egg (Rosell-Davis and Coons, 1989a).

In JHA treated cultures of fat body and backlessexplants, Vg concentration rose only slightly from thatfound in hormone free culture medium (Figs. 4 and 5).In both cultures of fat body treated with JHA and back-less tick explants, the peak Vg titer in the culturemedium was 13 ng/ml.

The Vg titers from untreated (hormone free) andmethoprene treated fat body cultures (Fig. 4) and back-less explants (Fig. 5) did not statistically differ, but werestatistically significant from the 20HE treatments in eachstudy (Duncan range test,P,0.05). The two treatmentlevels of 20HE were not statistically different in the cul-tured fat body study (Duncan range test,P.0.05) (Fig.4), but were different from one another in the backlessexplant study (Duncan range test,P,0.05) (Fig. 5). Inthis study, the smaller treatment level of 20HE produceda greater Vg titer than the larger treatment level. Thiseffect could be due to receptor de-sensitization at thehigher treatment level of ecdysteroid.

4. Discussion

In this study we directly measured the effect of 20HEand JHA on the production of Vg. Our results showedthat 20HE by itself elicited synthesis and release of Vgin cultures of fat body and in backless explants. We alsoshowed that the effect of JHA on the production of Vgdid not statistically differ from untreated controls in thesame experimental systems. Vg titers did however, riseslightly higher than Vg titers from hormone free culturemedium in JHA treated cultures of fat body and backlessexplants. This increase occurs earlier than the peak Vgtiter in 20HE treated fat body cultures and backlessexplants, which suggests that JHA treatment may resultin a slightly increased release of stored Vn carried overfrom the egg.

Other studies using soft ticks that directly measuredthe effect of juvenile hormones on Vg synthesis hadsimilar results to ours. Topical application or injectionof Juvenile hormones (JHI, JHII, JHIII) and JHA(methoprene) failed to induce Vg synthesis in the softticksOrnithodoros moubata, andO. parkerias measuredby in vivo labeling and fluorography (Chinzei et al.,1991; Taylor et al., 1991). In these two studies, resultsare independent of dose or solvents used to deliver JH’sor JHA.

The existence of JH in ticks has been questioned bythe results of another study. No JHI, JHII, JHIII, JHIIIbisepoxide, methyl farnesoate or farnesol could be foundin different organs systems and different developmentalstages ofDermacentor variabilisor Ornithodoros park-eri as measured by radiobiosynthesis, chemical andenzymatic digestion or mass spectrometry (Neese et al.,in press). Nor was there any juvenilizing activity asso-ciated with tick extracts using the Galleria bioassay.

Our results support the conclusion that Vg synthesisin hard ticks is at least in part regulated by 20HE. Ifso, attention should now be directed towards the peptidefactor(s) that regulate ecdysteroid production. A recentstudy demonstrated that a neuropeptide from the syngan-glion of a hard tick stimulates ecdysteroid productionin femaleAmblyomma hebraeum(Lomas et al., 1997).Presumably, the increased ecdysteroids initiate Vg pro-duction. Another study showed that a peptide from thesynganglion of a soft tick stimulates Vg production bythe fat body (Chinzei et al., 1992), but it is not knownif it acts through an ecdysteroid. Much work remains toelucidate the endocrine interactions that control repro-duction in ticks.

Acknowledgements

The authors thank Dr. M. Ma for suggestions indesigning the ELISA, and Dr. John Sauer for helpfuldiscussions of our results. We thank Dr. Ruben Kaufman

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for critically reading the manuscript, and Mrs. LoriMontgomery for technical assistance. We thank the twoanonymous reviewers for their helpful comments.

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