Identification of a juvenile hormone binding protein in the castes of the termite, Reticulitermes flavipes, by photoaffinity labeling

Insect Biochem.Vol. 21, No. 7, pp. 775-784, 1991 Printed in Great Britain. All rights reserved

0020-1790/91 $3.00+ 0.00 Copyright© 1991PergamonPress plc

IDENTIFICATION OF A JUVENILE HORMONE BINDING PROTEIN IN THE CASTES OF THE TERMITE, RETICULITERMES FLAVIPES, BY PHOTOAFFINITY LABELING B. MOSESOKOT-KOTBERand GLENND. PRESTWlCH* Department of Chemistry, State University of New York, Stony Brook, NY 11794-3400, U.S.A.

(Received 2 January 1991; revised and accepted 20 May 1991) Abstract--Hemolymph proteins of the Eastern subterranean termite, Reticulitermes flavipes (Isoptera, Rhinotermitidae, Rhinotermitinae) were examined from sterile and reproductive castes using native and denaturing polyacrylamide gel electrophoresis (PAGE). A high-mass protein (ca. 700 kDa) exhibited specific, JH III-displaceable photoaffinity labeling with [3H]EFDA, a diazoacetate analog of JH IlL This protein was present in each termite caste, and had the characteristics of a glycosylated lipoprotein, i.e. a lipophorin. The JH-binding subunit of this protein showed a molecular size of 230 kDa using SDS-PAGE. The differences in the hemolymph proteins present in the soldiers, workers, larvae, nymphs, and replacement reproductives of this rhinotermitid are discussed. Key Word Index: JH III; [3H]EFDA; photoaffinity labeling; lipophorin; caste differentiation; Eastern subterranean termite; Isoptera; Rhinotermitidae; hemolymph proteins

INTRODUCTION A unique system of post-embryonic development has evolved in termites, in which eggs hatch into identical first larval instars which subsequently undergo differentiation into various castes or morphs. Some larvae develop into reproductives, while others develop into soldiers and workers, the so-called sterile (neuter) castes. In Reticulitermes flavipes, the developmental scheme is similar to that described for R. lucifugus and R. santonensis by Biichli (1958). Thus, separation into reproductive and neuter castes begins with the second instar larvae. The reproductive line of development goes through six nymphal instars into imagoes (alates, primary reproductives, and replacement reproductives). The neuter line also goes through six worker molts, with gradual increases in size. Soldiers are formed from this line, either from fourth or fifth instar workers through an intermediate stage known as presoldier (Noirot, 1985). Juvenile hormones (JH) and pheromones appear to be central to the mechanisms involved in the differentiation of these castes (L/ischer, 1974). Indeed, Howard and Haverty (1978) demonstrated that R.flavipes workers can be induced to differentiate into soldiers using methoprene. Thus, it seemed reasonable to propose the existence of a specific, juvenile hormone binding protein (JHBP) in the hemolymph of these insects. Since the discovery of JHBPs (Whitmore and Gilbert, 1972; Trautmann, 1972), increasing attention has been focused on this class of proteins which are important for protection of JH from degradation (Hammock et al., 1975; Sanburg et aL, 1975), trans-

*Author for correspondence.

port of JH in the hemolymph (Gilbert et al., 1976), and delivery of JH to the target tissue (Goodman and Chang, 1985). Numerous reports have described these proteins from representative species (see reviews by Koeppe and Kovalick, 1986; Goodman 1990). It now appears that high affinity JHBPs may be categorized by molecular size, as suggested by King and Tobe (1988): high molecular mass JHBPs are found in orders other than Lepidoptera, while low molecular mass JHBPs predominate in the Lepidoptera. The high molecular mass JHBPs can be divided into the lipophorin type, e.g. those found in Periplaneta americana, Leptinotarsa decemlineata (de Kort and Koopmanschap, 1986, 1987), and Leucophaea maderae (Rayne and Koeppe, 1988), and the nonlipophorin type reported from Locusta migratoria (Peter et al., 1979; Stupp and Peter, 1984; de Bruijn et al., 1986; Koopmanschap and de Kort, 1988) and other related species. Attempts have been made to characterize the JHBPs of termites as well. The first report was made by Wyss-Hiiber (1981), working with Zootermopsis angusticollis and Macrotermes subhyalinus (now M. michaelseni), in which the general hemolymph protein compositions of different castes were analyzed by PAGE. More recently, preliminary results from this lab (Mohamed and Prestwich, 1988; Prestwich et al., 1987) indicated the existence of a high mass JHBP in the rhinotermitid R. flavipes which could be photoaffinity labeled with the JH III mimic [3H]EFDA. We now report detailed studies on the characterization of the R. flavipes JHBP from sterile and reproductive castes, the characterization of this JHBP as a glycosylated lipoprotein, and a detailed analysis of the major proteins detected in the hemolymph of each caste of this termite.

775

776

B. Mosr~ OKoT-KOTBER and GLENN D. I~,KSTWICH MATERIALS AND METHODS

Chemicals Common laboratory reagents were purchased either from Fisher Scientific or Sigma Chemical Company; racemic JH II and JH III were also from Sigma. (7S)-Methoprene was a gift from Zoecon Research Institute. Radiolabeled JH analogs 10,11-epoxyfarnesyl diazoacetate ([3H]EFDA, specific activity= 14Ci/mmol), epoxyhomofarnesyl diazoacetate ([3H]EHDA, specific activity = 58 Ci/mmol) and methoprene diazoketone ([3H]MDK, specific activity= 80 Ci/mmol) were synthesized in this laboratory (Ujv~ry et al., 1990; Palli et al., 1990). OTFP was prepared in this laboratory by W.-s. Eng. Termite culture Colonies of the Eastern subterranean termite Reticuliterrues flavipes (Isoptera, Rhinotermitidae, Rhinotermitinae) weie collected in Stony Brook, Long Island, N.Y. in fallen tree limbs and stumps during summer months. These were kept in plastic shoe boxes on moist paper in a temperaturecontrolled cabinet (27°C, 60% r.h., 16:8 L:D photoperiod). After allowing the colonies to re-establish themselves (as indicated by trail formation and gallery building), different castes were sorted after tapping them out of wood pieces. Hemolymph preparation Hemolymph was collected from a required number of workers, soldiers, nymphs or reproductives by decapitation and the hemolymph which oozed out was collected by capillary action directly into a pipet tip containing an appropriate amount of the bleeding buffer. This buffer consisted of 100 mM NaC1, 10 mM Tris-HC1, 5 mM EDTA, pH 7.4, and was fortified with a cocktail of protease inhibitors from a commercial set (Bochringer Mannbeim Biochemica GmbH). The proteases included: antipain, 75 #g/ml; PMSF, 25 #g/mi; aprotinin, 12.5 #g/ml; bestatin, 12.5 #g/ml; chemostatin, 12.5 t~g/ml; E-64, 75 #g/m1; pepstatin, 12.5 #g/ml; leupeptin, 12.5 #g/ml; and phosphoramidon, 75/~g/ml. This method was found to be the most suitable for obtaining hemolymph from these termites, which may weigh as little as 1 or 2 nag. The pipet tip was kept on a cold-pack (4°C) during the whole procedure. Hemocytes were removed from the collected hemolymph by centrifugation (13,000 8, 5 min, 10°C). The supernatant was stored at -80°C. Larvae could not be bled effectively, and were gently homogenized on ice by a few strokes of a pestle in an Eppendorf tube containing the bleeding buffer. Homogenized larvae were first centrifuged at 13,0008 for 5min to remove the carcasses. The supernatant was collected while avoiding the lipid phase, and then re-centrifuged (13,000 8 , 4°C) for 30 min to remove the remaining debris. The clear yellowish supernatant was collected and stored at - 8 0 ° C until used. Routinely, 100 larvae collected randomly from all instars were homogenized in 200 #i of bleeding buffer. This gave sufficient protein (3-5#g protein per #1) for electrophoresis and photoaffinity labeling. Isolation of JHBP from workers Two procedures were used for JHBP isolation. First, preparative native PAGE was used to separate proteins in the hemolymph collected as described above. Gel electrophoresis was carried out according to a modification of the Laemmli (1970) method under nondenaturing conditions, i.e. without sodium dodeeyl sulfate (SDS) or 2-mercaptoethanol. Native polyacrylamide gradient slab gels ( 4 - 1 5 % ) of 1.5 mm thickness were cast with 3 0 stacking gels. Samples diluted in sample buffer were loaded onto the gels and electrophoresed at 70V for 17h at 8°C. After electrophoresis, a strip of gel was excised, briefly stained (5 min) with 0.2% Coomassie Brilliant Blue R-250 in

50: 10:40 (methanol: acetic acid: water), and destained for about 5 min in 50% methanol and 10% acetic acid. This was aligned against the rest of the gels to use as a guide to localize the protein band of interest. The area containing the required protein was then sliced out and small pieces introduced into gel sample tubes for micro-electroelution using Centrilutor (Amicon) at 8°C following the manufacturer's instructions. Electroeluted protein was concentrated using Centricon 30 (Amicon) and the protein was then stored at -80°C. Since preliminary results showed that the JHBP in its denatured state migrated like lipophorins described from other species, a second procedure was employed. Thus, a KBr gradient ultracentrifugation was carried out as described by Shapiro et al. (1984) with minor modifications. Hemolymph was collected from 730 workers by centrifuging decapitated animals in bleeding buffer (13,000 g, 5 min, 8°C) to remove the carcasses: next, the supernatant was re-centrifuged 13,000 g, 4°C, 30 min). The supernatant was mixed with a solution of KBr giving a final KBr concentration of 41%, transferred to Beckman polyaUomer centrifuge tubes, and carefully overlayered with an equal volume of 150 mM NaCI. Samples were then ultracentrifuged using a Beckman Model L5-50 Ultracentrifuge with an SW 50.1 swinging bucket rotor at 200,000 8, for 24 h at 8-10°C. Each sample was fractionated with a Pipetman by successive removal of 200-#1 aliquots from the top of the gradient solution. Each fraction was diluted to 1 ml with the bleeding buffer, and aliquots were taken for protein concentration determinations using the BCA protein assay (Pierce) with bovine serum albumin (BSA) as a standard. The bulk of each sample was concentrated using Centricon 30 units and stored at - 80°C.

Photoaffinity labeling Photoaffinity labeling was carried out by modifications of the techniques described by Prestwich et al. (1984). The larval extract and hemolymph samples from workers, soldiers, nymphs and reproductives, and the JHBP preparations were diluted with a buffer containing 10raM Tris-HC1 and 10 mM KCI, pH 7.4 with I mM 3-octylthiol,l,l-trifluoro-2-propanone (OTFP) as an inhibitor of JH esterase. Aliquots (50/~1) were then pipeted in pairs into 750-#1 quartz tubes which had been previously coated with 2% solution of polyethylene glycol (PEG) 20,000 (Fluka), washed, and dried at 60°C. One tube in each set received an appropriate amount of an ethanolic solution of the competing ligand, unlabeled JH III (for[aH]EFDA), JH II (for[3H]EHDA) or methoprene (for[3H]MDK), while the other tube received an equal volume of ethanol alone. Each tube was then vortexed and preincubated in ice for 1 h. An ethanolic solution of either [3H]EFDA, [3H]EHDA, or [3H]MDK was then added to both tubes of each pair, and the samples were vortexed and incubated on ice for an additional 2 h. The samples were finally vortexed and photolysed for 30--45 s using a Rayonet photoreactor fitted with six 254nm u.v. lamps. These were then frozen at - 80°C or in liquid nitrogen and lyophilized, reconstituted in appropriate sample buffer and electrophoresed accordingly. Typically 200 nM of [3H]EFDA, 25 nM of [3H]EHDA or 25 nM of [3H]MDK were used for photolabeling and competed with 100-fold excess JH III, 250-fold excess JH II or 500-fold excess methoprene, respectively. The final concentration of ethanol in the protein solution was always below 5%. Gel electrophoresis and fluorography Samples were run on vertical SDS-gradient polyacrylamide slab gels (4-12.5% acrylamide, 0.75 mm thickness) using a Tris-Gly buffer system (Laemmli, 1970). High molecular weight cold or [~4C]methylated proteins (Amersham) were used as molecular weight markers. Gels were routinely electrophoresed at 70V at 8°C for about 16h.

R. Hovipes (0) +

L

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+

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N

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(b)

R

+

L

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$ --

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kDa 669

440 232

140

67

Native

4-15

%

Fig. l. (a) Coomassie Blue-stained proteins and (b) fluorogram of hemolymph proteins (40-50 #g protein per lane) of various castes of R. flavipes separated on 4-15% gradient native PAGE. [3H]EFDAPhotoaffinity labeled samples from larvae (L), workers (W), soldiers (S), nymphs (N), and replacement reproductives (R) in the absence ( - ) or presence ( + ) of excess JH III were used for each sample. An arrow (JHBP) shows the band which specifically photolabels with [3H]EFDA and is protected with 100-fold JH III.

R.

Havipes (a)

L

W

S

N

(b)

R

L

W

S

N

R --

kOa

JHBP 200

97

69

46

30 22 14

SDS 4-12%

Fig. 2. (a) Coomassie Blue-stained proteins and (b) fluorogram of hemolymph proteins of various castes of R. flavipes separated on 4-12% SDS-PAGE. [3H]EFDA-Photoaffinity labeled samples are labeled as designated in Fig. 1. An arrow (JHBP) indicates the band which specifically photolabels with the [3H]EFDA. 777

"1-

R. flav/pes (o)

Protein

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97 69

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Fig. 3. Cross competition of radioligands and competing hormones: (a) representative Coomassie Blue-stained protein pattern and (b~l) fluorograms of nymph and worker hemolymph samples (15-20 # g of protein per lane). Samples were photoatfinity labeled with (b) [3H]EFDA, (c) [3H]EHDA, or (d) [3H]MDK. Competitors were absent from lanes marked with ( - ); competitors used in lanes marked ( + ) were JH III (Wl and NI), JH II (W2 and N2) and (7S)-methoprene (W3 and N3).

778

R. flov/pes {G)

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occurring JHBPs: (a) Coomassie Blue-stained pattern and

labeled

samples

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Samples include: hemolymph of nymphs (N), 40 #g; workers (W), 40/tg; storage proteins (SP), 15 #g; JHBP isolated by KBr gradient ultracentrifugation (Lpc), 18/ag and JHBP isolated by native-PAGE (LPG), 24/~g of protein loaded on each lane. Photoatfinity labeling was performed in the absence ( - ) or presence ( + ) of excess JH III. R. f t avipes (o)

N +

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%

Fig. 5. Identity of subunits of isolated JHBP and naturally occurring protein. (a) Coomassie Blue-stained proteins and (b) fluorogram of [3H]EFDA photoaffinity labeled proteins separated on 4-12% SDSPAGE: hemolymph of nymph (N), 24#g; worker hemolymph (W) 20#g; storage protein (SP), 9 #g; JHBP isolated by KBr gradient ultracentrifugation (Lpc), 9/~g; and JHBP isolated by native PAGE (LPC), 10#g. 779

Reticukitermes ( e ) Coomessie N

W

( b ) Sudon bl ock BP

SP

N

W

(c)FITC-ConA BP

SP

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W

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-~00

97 69

46

30 22 14

SDS 4 - 1 5 %

Fig. 6 JHBP is a lipophorin. Staining properties of hemolymph proteins from nymphs and workers and of proteins isolated from worker hemolymph and separated by SDS-PAGE: Nymphs (N), 35 #g; workers (W), 50 # g; JHBP (BP), 30 # g; storage protein (SP), 30 p g. After electrophoresis, separate gels were stained with (a) Coomassie Blue, (b) Sudan Black B for lipids, or (c) FITC-Con A for mannose-rich oligosaccharide moieties.

780

Termite JH binding proteins Native gels were run under the same conditions as above but without the reducing agents and using high molecular mass protein markers from Pharmacia for native molecular size determinations. After electrophoresis, gels were stained for at least 2 h and destalned as mentioned above for I h, then in 30% methanol and 10% acetic acid for l-2h. Gels were photographed before processing for fluorography as described by Skinner and Griswold (1983) and miniaturization as described by Mohamed et al. (1989). Briefly, after destaining, the gels were soaked in glacial acetic for 5 rain to remove traces of water and impregnated with 15% 2,5-diphenyloxazole (PPO) in glacial acetic acid on an orbital shaker for 1 h. The PPO was precipitated in the gel with distilled water for about 2-5 min, and the gel was miniaturized in 50% aqueous PEG 2000 solution for about 30rain. The shrunken gels were dried on 3 MM Whatman chromatography paper using a slab gel dryer and exposed on pre-flashed Kodak X-ray film (XOMAT XAR-5) at -80°C for 2-7days to obtain fluorograms. Staining for carbohydrates and lipids Carbohydrate and lipid moieties were visualized after separation of proteins on SDS-PAGE. Periodic acid-Schiff reagent (PAS) was used to detect general carbohydrates, and the fluorescent lectin FITC-Con A was employed to detect mannose-rich carbohydrate chains (Furlan et al., 1979). Lipoproteins were detected with Sudan Black B. Molecular size determination Molecular sizes of the proteins were computed from log plots of standard molecular mass markers vs their Rf values. The following molecular mass markers were used for SDS-PAGE standards: myosin (200 kDa), phosphorylase b (97.4kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30kDa), trypsin inhibitor (21.5 kDa) and lysozyme (41.3) obtained from Amersham. The standards for native molecular mass were obtained from Pharmacia: thyroglobulin (669kDa), ferritin (440kDa), catalase (232kDa), lactate dehydrogenase (140 kDa) and BSA (67 kDa). RESULTS Juvenile hormone binding proteins in different castes Juvenile hormone binding proteins were revealed by using [3H]EFDA as a specific photoaffinity label for the JH III binding site and by using two other photoaffinity label-competitor systems to probe for JH homolog and analog specificity (Prestwich et al., 1984; Prestwich, 1987; Prestwich, 1991a,b). The results for the native JHBPs are presented in Fig. 1. The protein patterns [Fig. l(a)] show that most castes of R.flavipes share the same major hemolymph proteins; in addition, the fluorogram [Fig. l(b)] shows that each caste also has a single high-mass protein band which is specifically photolabeled by [3H]EFDA. This protein, which has an apparent molecular size of 700 kDa, has the same electrophoretic mobility in each caste and shows complete displacement of the [3H]EFDA photolabeling by 100-fold excess of unlabeled JH III. No other proteins exhibit specific or nonspecific labeling. When photoaffinity-labeled hemolymph or larval extracts were electrophoresed under denaturing (SDS) conditions (Fig. 2), the fluorograms showed a major protein band at 230 kDa which was photolabeled specifically in each of the castes examined [Fig. 2(b)]. Occasionally, minor bands of molecular

781

weights of about 200 and 170 kDa were also photolabeled, particularly in the worker samples. In order to ascertain the specificity and efficiency of photolabeling, [~H]EHDA and [3H]MDK were also used as photoaffinity labels in addition to [3H]EFDA (Fig, 3). Three separate experiments, one for each radioligand, were performed with worker and nymphal hemolymph; in each experiment, the radioligand was subjected to preincubation in the presence (or absence) of an excess of JH III, JH II, or methoprene. The protein banding pattern for the SDS-PAGE gels is shown in Fig 3(a) for comparison for each of the fluorograms. Figure 3(b) shows that [3H]EFDA efficiently photolabels the 230 kDa band, and that labeling was displaced by 100-fold concentrations o f J H III, JH II and methoprene. In contrast, while [3H]EHDA [Fig. 3(c)] and [3H]MDK [Fig. 3(d)] also photolabeled the same 230 kDa JHBP subunit with high efficiency, they could not be displaced effectively by any of the labeled JH homologs or by methoprene. To determine the nature of the putative JHBP, two methods were used to isolate the protein: preparative native gel eiectrophoresis and KBr gradient ultracentrifugation. As illustrated in Fig 4, the proteins isolated by either method showed essentially identical electrophoretic mobility in a native gel, and showed only minor changes relative to the putative JHBP band in the crude hemolymph. Purified protein samples appeared to be homogeneous [Fig. 4(a)], and both preparations were specifically photolabeled with [3H]EFDA [Fig. 4(b)]. A purified fraction of what may be an abundant storage protein (see below) co-migrated with a lower JHBP subunit which was not photoaffinity labeled [Fig. 4(b)]. Since the isolated protein showed JH III-competable labeling by [3H]EFDA, it can be considered to be the true JHBP. Photolabeled, isolated JHBP was then subjected to electrophoresis under denaturing conditions. This binding protein is composed of two prominent subunits [Fig. 5(a)]. While the 230 kDa subunit was photolabeled with [3H]EFDA [Fig. 5(b)], the 76-kDa subunit showed no labeling. Occasionally, a minor photoaffinity-labeied band at 170 kDa was observed to be present in the JHBP isolated from worker hemolymph by preparative PAGE; however, this band was absent from the KBr gradient-isolated JHBP and is probably an artifact [Fig. 5(b)]. Some aspects of chemical properties of the JHBP were determined by post-electrophoresis staining techniques (Fig. 6). The JHBP stained positive for carbohydrates by the PAS method (data not shown) and for lipids with Sudan Black B [Fig. 6(b)]. Man-

nose-rich oligosaccharide residuesmoiety testedpositive with FITC-Con A [Fig. 6(c)]. Major hemolymph proteins in different castes Major hemolymph proteins form various castes were resolved by native PAGE followed by Coomassie Blue staining. There were basically three groups of protein bands [Fig. l(a)]. The lowest relative mobility group has been already described above and includes primarily the JHBP. This protein was found to be the most abundant single protein in workers, soldiers and "replacement reproductives as judged by the intensity of the Coomassie Blue

782

B. MOSESOKOT-KOTBERand GLENND. PRESTWICH

staining. Nymphs and larvae had relatively lower amounts of this JHBP relative to other hemolymph proteins. The second most conspicuous set of native protein bands migrated as doublets, one with a relative molecular weight of about 450 kDa and the other 440 kDa. Larvae, workers, and soldiers had relatively more of the 450-kDa protein than the 440-kDa protein. In contrast, nymphs showed the opposite pattern and replacement reproductives had predominantly only the 440-kDa protein. The third set of major hemolymph proteins appeared in the gel as a doublet with apparent molecular weights of about 110 and 100kDa in larvae, workers, and soldiers, and at 120 and 110 kDa in the nymphs and replacement reproductives. Although this doublet was prominent in most castes, it was barely detectable in the nymph samples. Profiles of total hemolymph proteins under denaturing conditions are given in Fig. 2(a). While these complex patterns are less informative, it is evident that numerous minor bands appear with a major one at 230 kDa. Major storage protein subunits occur between 70 and 90 kDa. DISCUSSION

The identification of JHBP in the hemolymph of R. flavipes castes has been realized using the technique of [3H]EFDA photoaffinity labeling (see Prestwich, 1987). This termite JHBP was characterized both in its native state as well as in a dissociated form. Hemolymph of each sterile and reproductive caste contained an apparently identical JHBP, as determined by native and SDS-PAGE of photoaffinitylabeled proteins. The JHBP is the slowest-migrating major hemolymph protein, with an estimated molecular size of about 700 kDa. This molecular weight seems to vary slightly depending on the method used for isolation. JHBP isolated by ultracentrifugation seems to migrate slightly faster under the same electrophoretic conditions indicating some slight modification due to the KBr gradients. Engelmann (pers. commun.) also noted such an alteration in the mobility of this protein isolated by the two methods from the hemolymph of Leucophaea maderae. Under denaturing conditions, the JHBP dissociated into two apoproteins with estimated molecular sizes of 230 and 76 kDa. Only the 230-kDa apoprotein was photoaffinity labeled. High molecular mass JHBPs have been reported from tissues in a number of other insect species, including the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata (de Kort et al., 1983), the hemolymph and ovaries of the cockroach Leucophaea maderae (Kovalick and Koeppe, 1983; Koeppe et al., 1984), the hemolymph of the American cockroach Periplaneta americana and the locust Locusta migratoria (de Kort et al., 1984), the hemolymph and ovaries of the flesh fly Sarcophaga bullata (Van Mellaert et al., 1985), the larval bemolymph of Drosophila melanogaster (Shemshedini and Wilson, 1988), the hemolymph of adult females of the cockroach Diploptera punctata (King and Tobe, 1988), and the hemolymph of the grasshopper Melanoplus bivittatus (Jefferies and Roberts, 1990). Some of these

have been identified as lipophorins (de Kort and Koopmanschap, 1987, 1989; Kocppe et aL, 1988; Rayne and Koeppe, 1988). Since in the present study, the native molecular weight of the JHBP falls within the range of molecular weights (500-800kDa) characteristic of lipophorins as reported by Shapiro et al. (1988), and since the isolated JHBP dissociates during SDS-PAGE into basically two apoproteins as discussed above, we propose that the R. flavipes JHBP belongs to this class of bemolymph proteins. This hypothesis is further substantiated by lipid staining and carbohydrate detection, suggesting that the JHBP has both lipid and mannose-rieh oligosaccharide moieties known to be characteristic of lipophorins (Beenakkers et al., 1985). Using photoaffinity labeling, it was evident that JH was specifically bound by the higher molecular mass apoprotein (ca. 230 kDa) while the second major sub-unit (76 kDa) showed no affinity for [3H]EFDA. This suggests that the JH binding site is located on the larger sub-unit of the native protein. Similar observations were made by Koeppe et al. (1984) and by Rayne and Koeppe (1988) while analyzing the JHBP of Leucophaea maderae, and by de Kort and Koopmanschap (1989) for the JHBP of P. americana. The occasionally-observed minor bands of about 200 and 170 kDa which are photolabeled with [3H]EFDA and show JH III competition, may indicate some modification of the major binding component as was suggest for Leucophaea maderae (Koeppe et al., 1988). Apparently, the modified components also retain the binding sites. We commonly screen hemolymph samples from insects, crustaceans, and other arthropods with a variety of photoaffinity label-competitor combinations (Prestwich, 1991a,b). In this study, the other two radioligands used to verify the specificity and efficacy of the photoaffinity labeling were [3H]EHDA, and analog of JH II, and [3H]MDK, and analog of methoprene. These two radioligands have much higher specific activity than [aH]EFDA, and can therefore be employed at lower concentrations. Moreover, these two photolabels have been used to identify nuclear JH receptors in Manduca sexta epidermal cells (Palli et al., 1990). With the R. flavipes JHBP, photoaffinity labeling by these two iigands occurred specifically on the 230-kDa subunit of the 700 kDa protein. However, even a 250-fold excess of unlabeled JH III failed to competitively displace the photolabeling of the protein by [3H]EHDA. Likewise, even a 500-fold excess of methoprene failed to displace photolabeling by [3H]MDK. This suggests that the affinity for the more lipophilic homologs is greater than the affinity for JH III. This proposal is consistent with observation made by Gilbert and Chino (1974) that lipophorin may be the major carrier of hydrophobic products. Since Meyer et al. (1977) and Greenberg and Tobe (1985) have demonstrated that JH III is the natural JH homolog in termites, it is reasonable to expect that [3H]EFDA binds more specifically to the binding protein than either of the other two ligands. The most striking feature in the hemolymph protein profile, as revealed by Coomassie Blue staining, was that there were significant differences among the different castes in the relative proportions of the

Termite JH binding proteins various major proteins. For example, there was scarcely any detectable amount of a 450-kDa protein in the replacement reproductive, whereas it was the major fraction in this banding region in the other castes. This may suggest that 430-kDa protein is equivalent to the larval specific protein described in the larvae of Blattella germanica (Kunkel and Lawler, 1974) and in Blatta orientalis (Duhamel and Kenkel, 1983) which disappears after adult molt. Such a hypothesis is quite reasonable, given the evolutionary and physiological kinship between termites and cockroaches. The prothoracic glands are retained in the soldiers an workers of termites, and thus these castes are developmentally immature like the nymphs and larvae (Noirot, 1969). Since these glands degenerate in the replacement reproductives, these individuals can be considered adults. This maturity is consistent with the physiology and active reproduction by imagoes, in contrast to the inactive sex organs in larvae, workers, soldiers, and nymphs (Weesner, 1969). Still, the abundant 440-kDa protein is retained in all castes. Since these two proteins have native molecular weights close to 500 kDa, and since they are among the major hemolymph proteins, it is tempting to suggest that they are the storage proteins of termites, equivalent to those found in the well studied holometabolous insects (Levenbook, 1985). The third group of hemolymph major proteins (100-120 kDa doublets) remain unidentified and will require further investigation to determine their nature and function in the various castes. These proteins are particularly abundant in the replacement reproductives, suggesting a role in vitellogenesis or some other aspect of egg production. Work is in progress on the molecular cloning and tissue-specific expression of this JH-specific lipophorin in R. flavipes. In addition, we are investigating its role in the regulation of caste differentiation. Comparison of the R. flavipes JHBP to those of other termite species and to cockroach JHBPs will be presented subsequently. Acknowledgements--We thank the National Science Foundation for grants CHE-8809588 and DCB-8812322 in support of this research. The advice of Dr Mirza A. Mohamed and information on his unpublished results obtained prior to this work are gratefully acknowledged.

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