Site of synthesis of the haemolymph agglutinin of Melanoplus differentialis (Acrididae: Orthoptera)

J. InsecfPhysiol.Vol. 34, No. 12,PP. 1077-1085,1988 Printedin Great Britain.Al! rights reserved

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SITE OF SYNTHESIS OF THE HAEMOLYMPH AGGLUTININ OF MELANOPLUS DIFFERENTIALIS (ACRIDIDAE: ORTHOPTERA) BRAD STILES,ROGER S. BRADLEY,GWENDY S. STUARTand KENNETHD. HAPNER* Biochemistry Department and Montana Agricultural Experiment Station, Montana State University, Bozeman, MT 59717, U.S.A. (Receiued 1 February 1988; revised 13 April 1988) Abstract--Several species of Melunoplusgrasshoppers contain a haemolymph agglutinin exhibiting both D-galactosidic and D-glucosidic binding specificities. With the exception of the flesh Ay Sarcophaga peregrina, the site(s) of synthesis of insect agglutinins is uncertain. Primary cultures of M. differentialis fat body, haemocytes, ovary and testes were established. An ELBA assay demonstrated that the fat body, ovary and testes tissues released significant amounts of agglutinin into the culture medium over a 4-day period. Immunoprecipitated agglutinin from the culture medium was radiolabelled when the cultures were metabolically la.belled with [35S]methionine. Haemocyte culture medium and cell lysates of the 4 tissues did not contain detectable amounts of radiolabelled agglutinin. Attempts to alter the kinetics of agglutinin release from fat body cultures through addition of microbial cell wall components (lipopolysaccharide, peptidoglycan, zymosan, or laminarin) were unsuccessful. Key Word Index: Agglutinin, fat body, haemocytes, ELISA, Melanoplus,acrididae, primary culture,

biosynthesis

INTRODUCTION Agglutinins occur in most if not all insects and other invertebrates (GoId and Balding, 1975; Lis and Sharon, 1986), yet their function and regulation are still obscure. The ability of agglutinins to bind to carbohydrates has led many researchers to hypothesize that those found. in the haemolymph especially are involved in the re!cognition of foreign substances (e.g. pathogens) or degraded host tissue (reviews by Ratcliffe, 1985; Rowley et al., 1986). One approach to determining the possible involvement of agglutinins in insect immunity is to study the regulation of the synthesis of these proteins following challenge of the insect by injury or the introduction of microorganisms into the haemcoel. While Castro et al. (1987) specifically state that the Iectin of Hyafophora cecropia is not inducible, examples of the apparent induction of haemolymph agglutinin by wounding or microorganisms ‘have recently been reported in the flesh fly, Sarcop,baga peregrina (Komano et al., 1981; Komano et al., 1983; Takahashi et al., 1985; Takahashi et al., 1986), the velvetbean caterpillar, Anticarsia gemmatab’s (Pendland and Boucias, 1985), and the tobacco hornworm, Manduca sexta (Minnick et al., 1986). Induction studies are greatly enhanced if the site of synthesis of the protein is known. Definitive proof of the site of insect aggutinin synthesis, in this case fat body, has been presented for only one insect, S. peregrina (Komano et al., 1983). Other researchers have provided evidence suggesting that in the cockroach Leucophaea nzaderae (Amirante and Mazzalai, 1978) and the moth Hyafophora ce-

*To whom correspondence

should be addressed.

cropia (Yeaton, 1980) the haemocytes are the site of haemolyph agglutinin synthesis. Several species of Melanoplus grasshoppers have been shown to have an agglutinin in their haemolymph that demonstrates a D-galactosidic and D-glucosidic binding specificity (Jurenka et al., 1982; Hapner, 1983; Stebbins and Hapner, 1985). The agglutinin has been purified by affinity chromatography and shown to be a high-molecularweight protein aggregate (Stebbins and Hapner, 1985). The purpose of this study was to investigate the site of synthesis of the haemolymph agglutinin utilizing primary cultures of several tissues from M. dzferentialis in combination with metabolic radiolabelling of synthesized proteins. An ELISA assay specific for the agglutinin (Hapner, unpublished) has also been used to follow the release of agglutinin from various primary tissue cultures. Additionally, the effects of exposing fat body tissue to various microbial cell-wall components was determined by following the release of agglutinin from fat body into the culture medium.

MATERIALSAND

METHODS

Insects Melanoplus d&erentialis grasshoppers were from a laboratory colony maintained by Dr J. Henry, Rangeland Insect Laboratory, USDA, ARS, Bozeman, MT. Antibodies

Agglutinin-specific monoclonal antibodies were prepared (described in detail elsewhere) by fusing spleen cells from immunized mice with mouse X63 1077

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BRAD STILESet al

myeloma cells. The resulting hybridoma cells were selected with hypoxanthine/thymidine-containing medium (Galfre and Milstein, 1981: Reading, 1982) and screened for specific antibody production. Hybridoma cell (clone F6C2 or F6B4) culture medium containing a low concentration of specific antibody was used for immunostaining, but for immunoprecipitation and ELISA assays pure immunoglobulin G was obtained from monoclonal antibody enriched ascitic tumour fluid. Approximately lo6 antibody-producing hybridoma cells were injected into BALB/C Byj mice (injected 14 days before with 0.5 ml pristane) and the fluid drained from the resulting ascitic tumours (Galfre and Milstein. 1981). Immunoglobulin G was purified from the ascitic tumour fluid using a DEAE Affi-Gel Blue column (Bruck et at., 1982). An agglutinin-specific rabbit polyclonal antibody was prepared using agglutinin which had been purified by affinity chromatography (Stebbins and Hapner, 1985) followed by polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose (see immunoblotting below) and the agglutinin band was cut out of the sheet, dissolved in dimethylsulphoxide, mixed with an equal volume of Freund’s complete adjuvant (Knudsen, 1985) and utilized for multiple intradermal injections into New Zealand white rabbits (Vaitukaitis, 1981). Rabbits were bled at weekly intervals until the antibody titre reached its peak (at 8 weeks) and the rabbits were exsanguinated by cardiac puncture. The serum was filtered through a 0.22 pm filter and stored frozen at - 40°C. The specificity of mono- and polyclonal antiagglutinin antibodies was confirmed by immunostaining Western blots (Towbin and Gordon, 1984) of M. differentialis haemolymph proteins separated by SDS-PAGE. Primary cultures Insects to be used for establishment of primary tissue cultures were surface sterilized by submersion in the following solutions for the designated times: 0.5% liquid detergent in 50% isopropyl alcohol (5 min), 2.6% NaOCl (lOmin), 5 changes (2 min each) of sterile distilled water (Goodwin, 1987). The insects were then blotted dry on sterile absorbent paper before dissection. Most experiments were repeated using fifth-instar and adult tissues. The agglutinin induction series included fifth-instar insects only. Haemocyte cultures were set up by cutting a foreleg and applying gentle pressure to expel the haemolyph which was allowed to drip into a 35 mm tissue culture dish (Falcon) containing 3 ml sterile Dulbecco’s phosphate-buffered saline (1.5 mM KH,PO,, 8 mM Na,HPO,, 2.7mM KCl, 200mM NaCl; pH 7.3. 400 mOsm) plus 1 mg/ml reduced glutathione (Sigma). Haemolymph from 4 to 6 fifth-instar insects was collected into each dish. The dish was swirled after each addition in order to dilute the haemolymph and was left undisturbed for 1 h to allow the haemocytes to attach to the bottom surface. The haemocyte monolayer was washed 3 times with Dulbecco’s phosphate-buffered saline and overlaid with 2.5 ml of medium. The medium. designated

RIL-14-S plus 5X PEGSALZ lipid supplement, was developed especially for grasshopper cell cultures by Dr R. Goodwin (1987), Rangeland Insect Laboratory, USDA, ARS, Bozeman. MT. The basal medium was supplemented with 0.15 mg/ml gentamicin, 0.5 or 1.0 mg/ml reduced glutathione and foetal bovine serum (15% final concentration). The cultures were incubated at 28°C. Surface-sterilized insects were aseptically dissected as follows in order to obtain fat body. ovarian and testicular tissues. First, the legs and wings (or wing pads) were cut off. Then the integument was cut down both the mid-dorsal and mid-ventral lines, from the cervix to the last abdominal segment. The cervix membrane was cut allowing each lateral body wall section (with attached muscles and sheets of fat body) to be pulled away from the gut, cut loose near the posterior end of the insect, and placed into a Petri sterile Dulbecco’s phosphatedish containing buffered saline (plus 1 mg/ml reduced glutathione). The ovarian or testicular tissue was removed from its attachment to the gut, washed twice with the buffered saline and 3-5 organs were placed into a 35 mm dish containing 2 ml supplemented RIL-14-S medium and incubated at 28°C. Before the fat body was removed from the lateral body wall sections, the sections were transferred through a series of 8 Dulbecco’s phosphate-buffered saline washes (in which they were swirled vigorously for 30 s) to help remove haemolymph proteins and haemocytes. Pieces of the fat body were then carefully pulled away from the body wall sections using forceps and a dissecting microscope. Fat body was deposited into a dish of the buffered saline, divided into roughly equal numbers of fragments and transferred by pipette into separate 35mm tissue culture dishes containing 2 ml supplemented RIL-14-S medium. Each dish contained fat body from 3 to 6 different insects. The fat body, ovary and testes cultures were incubated at 28°C on a rocking platform. In one series of experiments, we wished to observe the effects of various components on the release of agglutinin from fat body cultures. In these experiments 10 fifth-instar h4. dzflerentialis were injected 24 h before dissection with either 10 ~1 Dulbecco’s phosphate-buffered saline alone or 10 p/ of the saline containing 50 ~1 E. coli lipopolysaccharide (Sigma), 100 pg laminarin from Laminaria digitate (Sigma), 100 ~1g zymosan from Saccharonzyces cerevisiae (Sigma) or 100 pg Micrococcus luteus D50Al cell wall peptidoglycan preparation (gift from Dr P. Dunn, Purdue University, W. Lafayette, IN) (Dunn et a/., 1985). These materials were also added to the respective fat body culture dishes at a final concentration of lOO~g/ml (50flg/ml for lipopolysaccharide). ELISA assay for agglutinin To determine whether cultured tissues were releasing aggtutinin into the medium, small samples of the medium were taken at 12 or 24 h intervals from the duplicate or triplicate cultures. Complete details of the ELISA are to be described elsewhere. Briefly, the assay involves coating plastic microtitre plates with the agglutinin-specific mouse monoclonal

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Grasshopper agglutinin synthesis immunoglobuhn G, incubating medium samples from the tissue cultures in the wells for 4 h at 37°C followed by agglutininspecific rabbit polyclonal antibody, and finally by horseradish peroxidaseconjugated goat anti-rabbit antibody (Biorad). The amount of agglutinin was indirectly quantified by adding 0-phenylenediamine (4 mg/ml in McIlvain’s buffer: 1 M Na,HPO,, 0.5 M citric acid, pH 5.0, containing 1% urea peroxide) as enzyme substrate. The amount of coloured product released was determined with a Dynatech model MR 580 Microelisa Auto reader. The culture medium samples were added as a series of 2-fold dilutions and the results were expressed as endpoint dilution titers. Fresh RIL-14-S complete medium wa:; used as a control. Metabolic labelling and immunoprecipitation.

In order determine if the cultured tissues were synthesizing agglutinin, I_-[~‘S]methionine (Amersham) was added to the medium immediately after the cultures were set up 1.0 give a final concentration of 15-30pCi/ml. After 2 days (haemocytes) or 4 days (fat body, ovary and testes) the medium was removed from the cultures, a.nd EDTA (to inactive lectin binding activity) and phenylmethane sulphonyl fluoride (PMSF) were added to give final concentrations of 5 and 1 mM, respectively. Aliquots (5 ~1) of the medium were placed onto glass microfibre filters and precipitated with trichloroacetic acid to determine the amount of incorporation of the radiolabel into released proteins. Lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% sodium deoxycholate, 0.5% Triton X-100, pH 7.6; 0.1 ml per ml medium) modified from Dhadialla and Wyatt (1983) and approx 70 pg of mouse monoclonal antibody were added to the remaining culture medium. The solutions were incubated 1 h at 37°C and then overnight at 4°C. The remaining cells or tissues were washed once with Dulbecco’s phosphate-buffered saline and then suspended in 1 ml of lysis buffer (PMSF and EDTA added to give 1 and 5 mM final concentrations, respectively). Cell lysis was induced by rapid vortexing (30 s), sonication (in a Branson 80W ultrasonic bath, 60 s) and additional vortexing (30 s). The cell lysates were allowed to sit at 4°C for 20 min and then centrifuged at 13,000 g and 4°C for 20 min (Yee et al., 1983). The supernamnts were collected and 5 ml of phosphate-buffered saline (5 mM KH,PO,, 150mM NaCI; pH 7.2) and approx 70 ng of monoclonal antibody were added and the mixtures incubated at 37°C for 1 h and then 4°C overnight. Approximately 80~1 of hydrated (in phosphatebuffered saline) Prol:ein A Sepharose beads (Sigma) were added to the medium samples and the cell lysates and the mixtures were incubated at 4°C for 2 h with constant gentle mixing (Schultz et al., 1979). The protein A beads were pelleted by centrifugation and then washed successively with two changes Tris-saline (150 mM Tris, 500 mM NaCI, pH 7.5). 2 changes Tris-saline plus 0.5% Tween 20. 2 changes Tri-saline-Tween plus 5 mM EDTA, and once with Tri-saline. Laemmli (1970) non-reducing sodium dodecyl sulphate sample buffer (100 ~1) was added to the pelleted beads and the samples heated at 100°C for 3 min in order to solubilize the antigen-antibody

complexes. The samples were stored frozen at - 20°C until analyzed by polyacrylamide gel electrophoresis. Polyacrylamide gel immunoblotting.

electrophoresis

(PAGE),

and

Samples of M. dtrerentialis haemolymph, affinity column purified agglutinin and immunoprecipitated culture medium and cell lysates were electrophoresed according to Laemmli (1970) on 1.5 mm thick polyacrylamide gels (4% stacking, 7.5% separating gel) at 4 W per gel until the samples had entered the separating gel and 7 W per gel thereafter. After electrophoresis, the separated proteins were transferred to a sheet of nitrocellulose (Western blot) by the method of Towbin et al., (1979) using a Hoeffer transphor electrophoresis cell. The nitrocellulose blots of separated proteins were blocked by incubation for 2 h in Tris-salineTween buffer (0.15 M Tris, 0.5 M NaCl, 0.05% Tween 20, pH 7.5) and then incubated overnight at 22°C in buffer containing a 1: 1 dilution in Trisaline-Tween buffer of anti-agglutinin monoclonal antibody hybridoma supernatant medium. The nitrocellulose sheets were washed 3 times with Tri-saline-Tween buffer (10 min each) and then incubated in the same buffer containing goat antimouse horseradish peroxidase conjugated antibody (Biorad, used at l/3000 dilution) for 90 min at room temperature. The Western blots were washed 3 times with Tri-saline_Tween buffer (10 min each), once with Tri-saline buffer, and then incubated in substrate solution (25 ml Tris-saline buffer plus 12.5 ~1 30% H,O, and 5ml of a 3 mg/ml solution of 4-chloro-1-naphthol in methanol) for 1 h at room temperature. The immunostained blots were photographed wet using an O(G) filter. Autoradiograms of [35S] methionine labelled immunoprecipitates on dried Western blots were produced by exposing the blots to Kodak X-Omat X-ray film at -40°C.

RESULTS Agglutinin released from primary tissue cultures

Typical haemocyte cultures consisted of monolayers of cells approx. 75% confluent. The other cultures were set up with approximately equal portions of tissue mass from 3 to 6 grasshoppers with the following approximate average wet weights per insect: fat body, 27 mg; ovary, 10 mg; testes, 26 mg. Samples of the culture medium from the primary tissue cultures were taken at 12 or 24 h intervals and assayed by ELISA for the presence of agglutinin. Figure 1 shows a set of typical results for each of the tissues tested in this study. The haemocytes appeared to release only minimal amounts of agglutinin into the medium during 80 h. These cultures began to deteriorate after approx 48 h, as the cells rounded up and started to detach. The cultures containing fat body, ovarian and testicular tissues were all found to release increasing amounts of agglutinin (Fig. I) regardless of whether the tissues were obtained from fifth-instar or adult grasshoppers. The rise in the agglutinin titres appeared to peak after about 4 days incubation. We

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Culture induction experiments

In one set of experiments, we tested whether the presence of certain microbial surface of cell wall components would alter the kinetics of agglutinin release from fat body cultures. Laminarin, zymosan, lipapolysaccharide and D50A 1 peptidoglycan were added to the medium in duplicate or triplicate cultures derived from insects previously injected with the same substances. Control cultures were derived from insects injected with Dulbecco’s phosphate-buffered saline alone. Figure 2 illustrates the release of aggluI I I I 0 20 40 60 80 100 120 140 tinin from the various cultures over 34 days, as Sample time (h) determined by ELBA assay. With the possible excepcultures, none of the Fig. 1.Release of agglutinin from primary cultures of tissues tion of the laminarin-treated from M. dzfirenfialis as determined by an ELISA assay. microbial components appeared to stimulate an inEach plot represents one typical culture dish from one of crease in the release of agglutinin from the fat body. the three experiments performed. G-0, haemocytes; Laminarin may have caused a faster initial release of n--A, ovarian tissue; l -•, testicular tissue; agglutinin from the tissue. but after 48 h there was no A--A, fat body. difference between the treated and control cultures. In the case of D50Al-treated fat body cultures. there generally observed melanization in the fat body cul- was an apparent inhibition in the release of agglutitures at this time also, which may indicate that these nin. This was probably in response to the fact that cultures were beginning to deteriorate in some fashthese treated cultures exhibited significant amounts of ion. When similar fat body or ovarian cultures were medium melanization from 48 h onward. In addition, incubated in the presence of 10 PI/ml of the protein when the fat body was initially dissected from the synthesis inhibitor cycloheximide the agglutinin titres D50A 1-injected grasshoppers, numerous small, melain the medium were reduced approx 2-fold or 8-fold notic spots were observed in the tissue. respectively (data not shown), suggesting that at least Metabolic labelling studies. a portion of the released agglutinin was synthesized in these tissues. The agglutinin that was released into In order to demonstrate synthesis of agglutinin by the medium in the presence of cycloheximide may the various tissues, the cultures were incubated with represent pre-existing protein that was stored in the r’s] methionine and the agglutinin evaluated for tissues at the time they were dissected out of the radiolabel incorporation. Primary tissue cultures insect. were incubated with radiolabelled methionine for 2 (haemocytes) or 4 (all other tissues) days. Small aliquots of the medium were removed at various times and TCA precipitated in order to determine whether the tissues were incorporating the label into newly synthesized proteins and releasing these proteins into the culture medium. Table 1 presents a set of typical results for the various tissue cultures. All the tissues released radiolabelled proteins into the medium throughout the duration of the experiments, although the haemocytes released comparatively little label. When cultures of haemocytes, fat body and LPS ovary were maintained in the presence of 10 pg/ml of cycloheximide, in addition to the radiolabel, the amount of TCA-precipitable labelled material released from the cultures was decreased by l&30%, 3&40% and 70-80% respectively. Culture medium from radiolabelled fat body, I. IIIIlll 11 1 I ovarian and testicular tissues was immuno20 40 60 80 100 120 140 O 20 40 60 80 0 precipitated with agglutinin-specific antibody. FolTime 1h 1 lowing SDS-PAGE and transfer to nitrocellulose, the

_ii / b+-

r

Fig. 2. Effect of various microbial surface and cell wall components of the release of agglutinin by fat body primary cultures. Each plot represents the average titre values of duplicate or triplicate cultures with the vertical lines illustrating the range of titre observed. The endpoint dilution values were transformed by computing the log base 2 of the inverse factor before plotting on the y-axis. Fat body for the treated cultures were dissected from insects which were injected 24 h previously with the corresponding test material. The treated cultures contained lOOpg/ml of the test materials (except hpopolysaccharide which was 50 fig/ml). O-0, control cultures; O-0, treated cultures. D50Al = M. lureus cell wall peptidoglycan.

Table 1. Incorporation of [“S] methionrne into TCA-precipitable proteins released by in aim tissue cultures

Time (h) 24 48 12 96

TCA precipitable counts ( x IO’/min)* ~__~ ~-. ~_____ Fat body Testes ovary Haemocytes 21.2 36.5 60.5 71.5

2.8 3.1 4.9 8.3

5.0 5.1 6.9 10.2

6.2 8.5 -

*Represents a Spl aliquot taken from 2.0 to 2.5 ml of culture medium.

HEM

FBI

FE-C

ov

AG

FE2

TE

Fig. 3. 1mmunosta:ned Western blot and autoradiogram overlay of immunoprecipitated culture medium from [“S] methionine labelled haemocyte, fat body, ovarian and testicular tissue cultures. The samples were subjected to SDS-PAGE electrophoresis and the proteins were blotted onto nitrocellulose. The blots were immunostained with rabbit polyclonal (left 7 lanes) or mouse monoclonal anti-agglutinin antibody (right 5 lanes) followed by the appropriate horseradish peroxidase labelled goat anti-immunoglobulin G conjugate. The nitroceliulose sheets were later dried and exposed to X-ray film. The left-hand lane (labelled I) of each of the culture samples represents the immunostained blot and the adjacent lane (labelled A) represents the autoradiogram overlay of the same blot. HEM, haemolymph from M. differentialis; FBI, FB2, different fat body cultures; FB-C. fat body cultured in the presence of IOpg/ml cycloheximide; OV, ovarian tissue; AG, purified M. drfirentialis agglutinin; TE, testicular tissue. The arrowheads indicate the agglutinin and j5S band when visually detectable.

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Grasshopper agglutinin synthesis separated proteins were immunostained as shown in Fig. 3. Purified agglutinin and whole haemolymph were similarly treated. A distinct band was observed in the lanes from tb: fat body, ovary and testes culture medium which. corresponded to the position of the purified agglutinin. The additional bands observed in some of the immunostained sample lanes on the Western blots, (i.e. sample TE, lane I; sample FB2, lane I, Fig. 3.) represent the original mouse monoclonal immunoglobulin G antibody used in the immunoprecipitation, its subunits and some degradation products derived from it. Similar immunoglobulin G-derived bands were observed in control experiments after immunoprecitation of culture medium only with monoclonal antibody or nonimmune mouse immunoglobulin G (not shown). Autoradiogram overlays of these same Western blots (Fig. 3) were found to ex:hibit a major labelled protein band that corresponded to that of the immunostained agglutinin band confirming their identity (i.e. samples FBI, lane A; OV, lane A; FB2, lane A). The autoradiographic film was oriented directly over the Western blot, with matchlmg register marks, in order to confirm superimposition of the antigenic and “S-1abelled band. The agglutinin bands from the ovarian and testicular culture immunoprecipitates were generally fainter than the band from fat body cultures, on both the immunostained Western blots and the autoradiographs suggesting that relatively less agglutinin was derived from these tissues. Some poorly visible, but reproducible faint bands in Fig. 3 are indicated with an arrow. The immunoprecipitated haemocyte medium and the cell lysates from the haemocyte, fat body, ovarian and testicular tissue cultures did not exhibit an antigenic band on the immunostained Western blots (data not shown). Autoradiograms of these blots did detect a number of radiolabelled protein bands in the immunoprecipitated cell lysate samples, though not in the predicted position of the agglutinin. These non-specifically precipitated proteins may have had a natural affinity for the immune complex, the protein A- Sepharose beads, or may have been incompletely solubilized by the lysis buffer and then deposited on the surface of the Sepharose beads. It therefore appears that under these in vitro culture conditions newly synthesized agglutinin is not stored in the tissues to any large extent, and furthermore, haemocytes do not synthesize aggutinin in amounts detectable by these procedures. DISCUSSION

In order determine the site or sites of agglutinin synthesis in the grasshopper M. d@erentialis primary in vitro cultures of four different tissues were established. These included haemocytes and fat body, which have been suggested to be the site of agglutinin synthesis in other insects (Komano et al., 1983; Amirante and Mazzalai, 1978; and Yeaton, 1980) and ovarian and testicular tissues. Preliminary experiments indicated that the culture medium used in these experiments inhibited red blood cell agglutination, so this test could not be used to assay for agglutinin. With the availability of a specific ELISA we were able to follow the release of agglutinin from these primary

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tissue cultures. Fat body, ovarian and testicular tissue cultures were found to release increasing amounts of agglutinin into the culture medium, while the haemocytes released barely detectable amounts (Fig. 1). In the case of fat body and ovarian tissue cultures the presence of the protein synthesis inhibitor cycloheximide reduced but did not halt the release of agglutinin. This observation suggested that both preexisting stored agglutinin and newly synthesized protein were being released by the tissues. Radiolabelled methionine was added to the tissue cultures in order to determine whether a portion of the agglutinin released by these cultures represented newly synthesized protein. Because the ELISA data demonstrated that the titre of agglutinin in the medium continued to rise over a period a 4 days the metabolic labelling experiments were allowed to continue for this same length of time (instability of the haemocyte cultures restricted labelling to 2 days). The agglutinin released from the fat body, ovarian and testicular tissue cultures was immunoprecipated with an agglutinin-specific monoclonal antibody and was found to contain radiolabelled methionine. No radiolabelled agglutinin could be detected in the medium from the haemocytes or from the cell lysates of any of the 4 tissues. It appears certain that the fat body is capable of synthesizing the agglutinin. Fat body removed from insects generally contains a low number of contaminating cells such as haemocytes, oenocytes and Malpighian cells, but these are not considered to represent a significant source of metabolic activity (Keeley, 1985). In addition, our observations with larger numbers of haemocytes alone in culture show that these cells are not capable of synthesizing detectable amounts of agglutinin under the culture conditions described, even though haemocytes do have detectable levels of asssociated agglutinin (unpublished observations). In the case of the ovarian and testicular tissue cultures, there is some question as to the identity of the cell synthesizing the agglutinin. These tissues were impossible to obtain completely free of small adhering fragments of fat body, although this contaminating tissues represented only a few per cent of the total weight of tissue in the cultures. It is known, however, that fat body from different locations within the insect may exhibit significantly different levels of synthetic activity (Keely, 1985; Dean et al., 1985). It is conceivable that the small amounts of radiolabelled agglutinin released by the ovarian and testicular cultures originated from the highly active, contaminating, fat body cells. As mentioned earlier, three examples of definite or apparent induction (meaning increased levels of synthesis) of agglutinin have been described in insects as determined by synthesis of m-RNA (Takahashi et al., 1986) or increased haemagglutination titres in haemolymph samples (Penland and Boucias, 1985; Minnick, et al., 1986). In all of these holometabolous insects the agglutinin levels were initially low or undetectable. In the case of the Melanoplus grasshoppers studied so far, the haemolymph exhibits rather high levels of haemagglutinin activity (Hapner, 1983) which do not seem to fluctuate significantly after wounding or following injection of bacteria (unpublished observations). When several microbial sur-

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face or cell wall components, known to be immune system inducers in various other vertebrate and invertebrate organisms, were injected into grasshoppers and the fat body cultures from these insects were incubated with more of the same materials, we did not observe the induction of increased agglutinin release. Only laminarin stimulated an increase in the rate of agglutinin release compared to control cultures, but this effect was transitory, occurring only during the first 24 h of the experiment. Because some of the other experiments described suggested that the fat body may have a significant amount of stored agglutinin, we cannot say that an increased rate of release from the treated cultures represents the induction of synthesis, but rather only a modulation of the rate of release from the tissue. The immunoprecipitation procedure developed here was comparatively inefficient at removing soluble agglutinin as determined by immunoprecipitating haemolymph of high haemagglutination titre. This may reflect the occurrence of agglutinin as large molecular weight aggregates that likely have (masking) molecules associated with them (Stebbins and Hapner, 1985). This qualitative nature of the immunoprecipitation procedure makes it of doubtful use (in our hands) for quantitative comparisons of the rate of agglutinin synthesis in primary tissue cultures. Thus far no evidence for the induction of agglutinin in hemimetabolous insects has been found in our studies involving grasshoppers. A similar conclusion regarding cockroach agglutinin has been reported by Kubo and Natori (1987).We believe that the most practical future approach to the study of the regulation of synthesis of this protein in grasshoppers would involve the specific detection of transcribed m-RNA encoding the agglutinin protein. Acknowledgemenr-This research was supported by NSF grant DCB-8510097 and is published as contribution No. 2104 from the Montana Agricultural Experiment Station.

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Galfre G. and Milstein C. (198I) Preparation

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