J. exp. Biol. 175, 45–59 (1993) 45 GENERATION OF ... · Adult locusts (Locusta migratoria) and embryos were obtained from the Insektarium Dr Frieshammer, Jaderberg. Day E1 was the

45J. exp. Biol. 175, 45–59 (1993)Printed in Great Britain © The Company of Biologists Limited 1993

*To whom reprint requests should be addressed.

Key words: olfaction, insects, monoclonal antibody, locust, Locusta migratoria.

GENERATION OF MONOCLONAL ANTIBODIES DETECTINGSPECIFIC EPITOPES IN LOCUST ANTENNAE

J. STROTMANN, I. BOEKHOFF, S. GÖGGERLE and H. BREER*

University of Stuttgart-Hohenheim, Institute of Zoophysiology, 7000 Stuttgart 70,Germany

Accepted 15 October 1992

Summary1. Following a tissue-specific screening paradigm, monoclonal antibodies have been

generated that interact with distinct subpopulations of cells in locust antennae.2. Antigens were identified as high molecular weight components.3. Immunoreactivity was not detectable during embryonic development, but rapidly

appeared within a few hours of hatching.4. The time course of antigen expression in antennal cells could be followed in situ as

well as invitro.5. Expression of monoclonal antibody B14/6D2-like immunoreactivity was prevented

by blocking protein synthesis with cycloheximide.

Introduction

Volatile semiochemicals detected by the highly sensitive chemosensory receptor cellsin the antennae play an important role in regulating the behaviour of insects; they provideinformation on the location of food, hosts, mates or oviposition sites and may initiatespecific physiological and behavioural transformations. In the desert locust, olfactionplays a central role in the detection of food sources (Greenwood and Chapman, 1984) andis thought to be involved in maturation and social interactions (Amerasinghe, 1978;Gillet, 1983). Approaches to controlling locust plagues by interfering with this importantsensory system have been considered for quite a while, particularly as these methods aresupposed to be environmentally safe, target-specific and do not lead to the resistance seenwith pesticides (Ferenz, 1990). An essential prerequisite for developing sound strategiesfor manipulating the chemosensory system is a more detailed understanding of theprimary mechanisms that enable locusts to perceive airborne chemical cues.Unfortunately, investigations on insect olfaction have been restricted to a few species,mainly to aspects of pheromone detection in moths (Kaissling, 1986). The antennae of thelocust (Locusta migratoria) consist of about 20 segments equipped with several thousandsensilla (Boeckh, 1967), which can be classified into trichoid, basiconic and coeloconictypes (Altner et al. 1981). Electrophysiological recordings have demonstrated that most

of these sensillum types have an olfactory function (Boeckh, 1967; Kafka, 1970).However, at present there is very little information on the molecular machinery whichenables the receptor cells in locust antennae to detect odorous molecules and to transducethe chemical stimulus into neuronal signals.

One way of investigating the specificity of olfactory receptor cells is by using specific,site-directed agents. As such ligands are not available per se, hybridoma technology canbe employed to generate monoclonal antibodies (mAbs) against molecules that areselectively expressed in one particular tissue but not in others, following a differentialscreening paradigm (Köhler and Milstein, 1975). The availability of specific mAbsallows the application of analytical approaches disclosing the cellular localization, themolecular identity and ultimately even the function of an antigenic protein. Attempts toraise mAbs against olfactory receptor cells from vertebrates and insects have recentlybeen made in several laboratories (Hempstead and Morgan, 1985; Hishinuma et al. 1988;Anholt et al. 1990; Strotmann and Breer, 1991).

Here we report the generation of two monoclonal antibodies which react with antennalcells of Locusta migratoria.

Materials and methods

Adult locusts (Locusta migratoria) and embryos were obtained from the InsektariumDr Frieshammer, Jaderberg. Day E1 was the day of oviposition, hatching was on day E13at 28˚C. Balb/c mice were supplied by the Zentralinstitut für Versuchstierzucht(Hannover).

Cell culture media were from GIBCO (RPMI 1640) and Serva (Schneider’sDrosophila medium and basal medium Eagle [BME]). 5+4 medium was made from fiveparts of Schneider’s Drosophila medium and four parts of BME. Calf sera and cell cultureadditives were purchased from GIBCO, as were complete and incomplete Freund’sadjuvants. Polyethylene glycol (PEG) 500 was supplied by Roth. Culture plates andenzyme-linked immunosorbent assay (ELISA) plates were obtained from Nunc.Nitrocellulose sheets (0.45 mm) were obtained from Schleicher and Schuell.Tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse Ig wasobtained from Sigma. Fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse Igcame from Amersham. Alkaline-phosphatase-conjugated goat anti-mouse Ig was fromPromega, horseradish peroxidase (HRP)-conjugated goat anti-mouse was from BioRad.All other reagents were obtained in the purest form commercially available.

Preparation of crude membranes

Animals were anaesthetized on ice and muscle tissue, head and thoracic ganglia andantennae were dissected and collected at 4˚C. Crude membranes were prepared asdescribed by Breer et al. (1985). Tissues were homogenized in 50mmol l21 Tris/HCl,pH6.8, 150mmol l21 NaCl, 5mmol l21 EDTA, 3mmol l21 EGTA, 0.1mmol l21

phenylmethylsulphonyl fluoride (PMSF) and centrifuged for 10min at 1000 g. Thesupernatant (S1) was centrifuged at 27000 g for 10min. The pellet (P2) was resuspended

46 J. STROTMANN AND OTHERS

in 10mmol l21 Tris/HCl, pH7.4, 150mmol l21 NaCl and stored at 270˚C. Proteinconcentration was determined according to Bradford (1976).

Generation of monoclonal antibodies

Female 6-week-old Balb/c mice received intraperitoneal injections of 100 mg ofantennal protein emulsified in complete Freund’s adjuvant and two similar injections withincomplete Freund’s adjuvant at 2-week intervals. Three days after the last immunization,the spleen was removed and the spleen cells were fused to P3-X63 Ag8.653 myelomacells (Kearney et al. 1979) by a standard fusion protocol (Galfre and Milstein, 1981)using PEG 500 as the fusion reagent. Cells were plated in microtitre wells inhypoxanthine, aminopterin and thymidine (HAT) selection medium with 10% foetal calfserum at 37˚C in a humidified atmosphere containing 5% CO2. Antibody-producingclones were identified by ELISA and subcloned twice by limiting dilutions.

Enzyme-linked immunosorbent assay (ELISA)

ELISA plates were coated overnight at 4˚C with 100ng protein per well in 50mmol l21

NaHCO3, pH9.5. Unbound material was washed out with 10mmol l21 Tris/HCl, pH7.6,with 150mmol l21 NaCl (TBS). After preincubation with TBS containing 1% bovineserum albumin (BSA), culture supernatants were incubated in the plates for 1h, followedby HRP-conjugated goat anti-mouse Ig (1:7500 in TBS). Each step was followed by threeTBS washes. Antibody binding was visualized with 0.005% 3,5,39,59-tetramethylbenzidine and 0.003% H2O2 in 100mmol l21 sodium acetate/citrate buffer;substrate turnover was stopped after 10min with 2mol l21 H2SO4; absorbance wasmeasured at 450nm with a Dynatech microplate reader MR700.

Gel electrophoresis and Western blot analysis

Samples of membrane preparations were diluted 1:1 in sample buffer (125mmol l21

Tris/HCl, pH6.8, 20% glycerol, 10% 2-mercaptoethanol, 2% SDS) to a final proteinconcentration of 1 mg ml21. Samples were heated in a boiling water bath for 3min andloaded onto 12.5% gels using the Laemmli buffer system (Laemmli, 1970). Proteins weretransferred onto nitrocellulose according to Towbin et al. (1979). Non-specific bindingsites were blocked by incubation of blots with 1% BSA in 10mmol l21 Tris/HCl, pH8.0,150mmol l21 NaCl, 0.05% Tween 20 (TBST). An incubation with culture supernatantsfor 1h was followed by three TBST washes of 15min. Blots were subsequently incubatedwith alkaline-phosphatase-conjugated goat anti-mouse Ig (1:7500) and washed asdescribed above. Antibody binding was visualized with 0.015% nitro-blue tetrazoliumand 0.007% 5-bromo-4-chloro-3-indolylphosphate in 100mmol l21 Tris/HCl, pH9.5,100mmol l21 NaCl, 5mmol l21 MgCl2. Substrate turnover was stopped by addition of20mmol l21 Tris/HCl, pH8.0, 5mmol l21 EDTA.

Gel filtration

The membrane pellet from adult locust antennae was resuspended in 30mmoll21 Tris,pH8.0, 100mmol l21 NaCl, 2mmol l21 EDTA, 2mmol l21 EGTA, 0.1mmol l21 PMSFand 0.5% CHAPS and proteins were solubilized for 1h at 4˚C. After a centrifugation at

47Antibodies to locust antennal cells

27000 g for 15min, CHAPS in the supernatant was reduced to 0.05% using PD-10columns (Pharmacia). Gel filtration was performed using a Pharmacia FPLC andSephacryl S200 as column material with a constant flow rate of 0.5mlmin21. Absorbanceat 280 nm was monitored with a single-path UV-1 monitor; 2ml fractions were collectedand dotted onto nitrocellulose. Immunoreactive fractions were visualized as described forWestern blots.

Epitope analysis

ELISA plates were coated with 100ng protein per cup as described above. After threewashes with TBS/gelatine, the cups were incubated with 0.01% trypsin in 10mmol l21

Tris/HCl, pH7.4, 150mmol l21 NaCl for 30min at 37˚C. Subsequently, the plates werewashed three times with TBS/gelatine followed by the ELISA protocol described above.All solutions used subsequently were supplemented with 0.01% soybean trypsininhibitor. Control experiments were performed with trypsin preincubated with 0.1 %trypsin inhibitor.

Alternatively, samples were incubated with 0.001% proteinase K in 20mmol l21

Tris/HCl, pH7.8, 0.1% CaCl2 for 30min at 37˚C. After three washes in TBS/gelatine,the ELISA protocol described above was followed.

Immunohistochemistry

Cryostat sections

Immunohistochemical examination was carried out on cryostat sections of antennaefrom adult locusts. Specimens were cut into small segments and fixed for 4h byimmersion in 4% paraformaldehyde in phosphate-buffered saline (PBS), pH7.4, at 4˚C.After three rinses in PBS, they were transferred for 24h to ice-cold 30% sucrose in PBS.Fixed and cryoprotected tissues were embedded in Tissue Tec (Miles Inc.) and rapidlyfrozen by immersion in isopentane (280˚C). Sections (10 mm) were cut on a Reichertand Jung cryostat model 2800 E, thaw-mounted on chrome-alum/gelatin-coated slidesand air-dried for 30min. Sections were treated with 0.1% Triton X-100 in PBS for 2minand additional binding sites were blocked with 1% BSA in PBS for 30min.

For double-labelling experiments, mAb B14/6D2 was purified from culturesupernatant by caprylic acid precipitation according to Reik et al. (1987) and conjugatedto FITC according to The and Feltkamp (1970). Sections were sequentially incubatedwith mAb S1/5D5 followed by TRITC-conjugated goat anti-mouse Ig (1:400) and FITC-conjugated mAb B14/6D2 for 1h each. Each of the steps was followed by three PBSwashes for 5min. Sections were mounted in Citifluor (Amersham) to retard fading offluorescence during microscopy and were examined under a Zeiss epifluorescencemicroscope.

Cell dissociation

Antennal cells from adult and embryonic locusts were prepared by cutting off anantenna, opening it with a razor blade and carefully scraping out the cells. The cells of tenantennae were collected in 1ml of 5+4 medium and carefully dissociated with a Pasteurpipette. The preparation was filtered through a 100 mm gauze and centrifuged for 5min at


1000 g. The pellet was resuspended in 5+4 medium and the cells were plated in 3.5cmculture dishes. They were covered with 5+4 medium and kept at 27˚C. Forimmunohistochemical examination, cells were allowed to attach, the culture supernatantwas aspirated and the cells were fixed with 4% paraformaldehyde in PBS for 10min atroom temperature. Visualization of antibody binding was performed as described fortissue sections.

Inhibition of protein biosynthesis

To inhibit protein biosynthesis, the left antenna from hatching locusts was cut off andtransferred to 5+4 medium supplemented with 100 mmol l21 cycloheximide for 6h; theright antenna from the same animal served as a control and was incubated in 5+4 mediumwithout additives. After that period, immunochemical examination of dissociated cellswith mAb B14/6D2 was carried out as described above.

Results

Fusion and antibody selection

Hybridoma cell lines were produced from the fusion of a myeloma cell line (P3-X63Ag8.653) with spleen cells from mice immunized with membrane preparations fromlocust antennae. Antibodies secreted by hybridoma cells were monitored for reactivitywith membrane preparations from antennae as well as from nervous and muscle tissueusing ELISA assays. This differential screening approach led to the identification ofantibodies that displayed distinct immunoreactivity with antennal preparations but onlylimited or no reactivity with membranes from ganglia and muscle tissue. The monoclonalantibody (mAb) designated S1/5D5 showed particularly high binding to antennal andganglionic membrane proteins, whereas the mAb designated B14/6D2 specificallyreacted with antennal membranes (Fig. 1). Therefore, these antibodies were chosen forfurther investigation. Both mAbs were found to be of the IgG1 subtype.

Distribution of immunoreactivity in locust antennae

In phase-contrast images from longitudinal sections of antennal segments themulticellular organization of sensory organelles, as already described for othergrasshopper species, is evident (Fig. 2). Slifer et al. (1959) have found that the number ofsensory neurones associated with an insect sensillum varies from a few to more than 50.Indirect immunofluorescence analyses on cryostat sections were performed to explore thetopochemical localization of the antigens in locust antennae. The distribution ofimmunoreactivity for both antibodies in longitudinal sections of segments from locustantennae is shown in Fig. 3. By application of a double labelling technique the reactivesites for both antibodies were visualized in the same section. It is immediately obviousthat the antennal nerve is heavily labelled by the S1/5D5 antibody (Fig. 3A), whereas theB14/6D2 antibody does not react with it (Fig. 3B). In the sense organs, the surface ofseveral large cell bodies, which are probably receptor neurons, is labelled by the S1/5D5antibody. In contrast, the immunoreactivity of the B14/6D2 antibody is distributed


between these cell clusters. Control experiments were performed using non-relevantantibodies of the same subtype (IgG1); these antibodies showed no reaction.

Characterization of the antigens

Biochemical analyses were performed to characterize the antennal antigens. Membraneproteins from adult locusts were separated by SDS–PAGE, transferred onto nitrocelluloseand probed for immunoreactivity. As can be seen in Fig. 4, the monoclonal antibodyS1/5D5 strongly stained a protein band with an apparent Mr of 70000; the weakly stainedband at Mr 25000 is due to an unspecific reaction of the second antibody. The B14/6D2antigen could not be detected on Western blots. Following a size-dependent separation ofantennal membrane proteins by gel filtration on Sephacryl S200, the B14/6D2immunoreactivity was detected in the void volume (Fig. 5), suggesting that the B14/6D2epitope is located on a large molecule. The antigens were further characterized by theirsensitivity to various treatments. As documented in Table 1, the S1/5D5 antigen was verysensitive to protease treatment (trypsin and proteinase K); the B14/6D2 reactivitydisappeared upon treatment of tissue sections with various detergents (Deoxycholic acid,Tween 20) or fixatives (glutaraldehyde).

Labelling of dissociated antennal cells

To circumvent the well-known problems of characterizing antennal cells protected bythe tough and impermeable cuticle (Slifer et al. 1959), clusters of cell bodies wereisolated from split antennal segments. The cells were mechanically dissociated and keptalive in primary cultures for several days. As can be seen in Fig. 6, in a suspension ofcultured cells from adult locust antennae the surface of some cell somata is labelled by the


0.6

0.5

0.4

0.3

0.2

0.1

0Hybridoma clones

AntennaeGangliaMuscle

S1/5D5 B14/6D2

Fig. 1. Immunoreactivity of hybridoma supernatants with membrane preparations fromdifferent locust tissues. ELISA plates were coated with 100ng protein per well; reactivity wasvisualized using HRP-conjugated goat anti-mouse Ig. Arrows indicate clones (B14/6D2 andS1/5D5) that were analyzed further.

S1/5D5 antibody; staining with the B14/6D2 antibody was detected in a differentsubpopulation of cells (Fig. 6B). Thus, these cytological observations confirm the resultsobtained in the histological approaches (Fig. 3).


2A

B

Fig. 2. (A,B) Phase-contrast micrographs of longitudinal cryostat sections of antennalsegments from adult locust. Basiconic (one arrowhead) and coeloconic sensilla (twoarrowheads) are visible. The multicellular organization of the sensory organules is obvious;clusters of cells are associated with each sensillum. Scale bars, 10 mm.

Expression of antigens during development

In a quantitative analysis, antennal membrane preparations from differentdevelopmental stages were assayed for immunoreactivity. The data in Fig. 7A indicatethat B14/6D2 immunoreactivity was very low during embryogenesis; significantimmunoreactivity appeared just before hatching, whereas S1/5D5 reactivity was alreadydetectable in the earliest stages investigated. The level of B14/6D2 reactivity increasedfurther during maturation; in adult antennae, the binding of antibodies was about fivetimes that seen around hatching.

The ontogenetic development of B14/6D2 immunoreactivity could also bedemonstrated at the cellular level. As can be seen in Fig. 7B, 1h after hatching a faint


Fig. 3. (A,B,C) Double labelling experiment with mAbs S1/5D5 and B14/6D2 on cryostatsections of adult locust antenna. S1/5D5 (A) was visualized with TRITC-conjugated goat anti-mouse Ig. B14/6D2 was visualized by direct conjugation with FITC (B); staining of distinctsubpopulations of antennal cells is visible. In A, small arrowheads point to putative receptorcells and the large arrowhead indicates the antennal nerve. In B, small arrowheads point toputative supporting cells. In C, a schematic representation of the section is shown; co,coeloconic sensillum; cu, cuticle; n, antennal nerve; re, cluster of putative receptor cells; su,putative supporting cells. Scale bar, 10 mm.

Mr × 10−3

4Fig. 4. Western blot analysis of antennal membrane proteins with mAbs B14/6D2 (A) andS1/5D5 (B). Proteins were separated on 12.5% polyacrylamide gels and transferred ontonitrocellulose. Immunoreactive bands were visualized using alkaline-phosphatase-conjugatedgoat anti-mouse Ig. Relative molecular mass markers are as indicated.

94

67

43

30

A B

immunofluorescence first appeared; 6h after hatching the surfaces of several distinctsomata in an isolated cell cluster were heavily labelled.

Antigen expression

To explore whether the sudden appearance of B14/6D2 immunoreactivity was due tothe exposure of epitopes on preformed molecules or whether it depended on the synthesis


Table 1. Characterization of antigens

S1/5D5 B14/6D2

Relative molecular mass 70×103* >150×103†Membrane protein + +Epitope sensitivity

Proteases + −Detergents − +Glutaraldehyde − +

Localization Putative receptor Putative supportingcells cells

*Western blot; †gel filtration.

Fig. 6. (A,B,C). Dissociated antennal cells incubated with mAb S1/5D5 (A) and B14/6D2 (B);immunoreactivity was visualized as described in Fig. 3. Different subpopulations of cells arelabelled with the two antibodies. A phase-contrast micrograph of the same field of view isshown in C; arrowheads point to S1/5D5-reactive cells. Scale bar, 10 mm.

0.8

0.6

0.4

0.2

05 10 15 20 25 30 35 40 45

Fraction number

Voidvolume

Mr 67×103

Mr 20×103

Fig. 5. Gel filtration of a crude detergent extract of antennal membrane proteins. The extractwas applied to a Sephacryl S200 gel filtration column (2.5cm360cm, equilibrated in30mmol l21 Tris/HCl, pH8.0, 100mmol l21 NaCl, 0.05% CHAPS, 2mmol l21 EDTA,2 mmol l21 EGTA); 2ml fractions were collected at a flow rate of 0.5mlmin21 and assayedfor B14/6D2 immunoreactivity. The column was calibrated using the following markerproteins: catalase (Mr 2323103), b-galactosidase (Mr 1163103), BSA (Mr 673103),chymotrypsin (Mr 253103) and ribonuclease (Mr 13.73103). Ordinate indicates absorbance at280nm; immunoreactive fractions are indicated by the box.

of new proteins, the expression of immunoreactivity was monitored on antennal cells inculture. Fig. 8 demonstrates that the level of antigen in membrane preparations fromantennae before hatching increases more than fivefold after incubation for 6h in 5+4medium, while in the presence of cycloheximide, which efficiently blocks proteinsynthesis, no significant increase in immunoreactivity was observed. These resultsindicate that the developmental pattern of epitope appearance is sustained underexperimental conditions and requires expression of new proteins.

Discussion

Following a differential screening paradigm, two monoclonal antibodies weregenerated which selectively interact with the surfaces of distinct populations of antennalcells from adult grasshoppers (Locusta migratoria). One of the antibodies (S1/5D5)recognizes a 703103 Mr constituent on the surface of putative antennal receptor cells thatis also present in the axons of the antennal nerve, whereas the B14/6D2 antigen wasidentified on the surface of cells that are possibly supporting cells. Both antigens may beconsidered as markers of specific cells in locust antennae in situ as well as in vitro.

mAb S1/5D5 may be used to identify isolated receptor cells. The identification ofreceptor neurons in vitro is an important step towards a precise evaluation of thephysiological mechanisms underlying the specific recognition and transduction processesin these highly specialized cells. Electrophysiological studies on identified receptor cells(Wegener et al. 1992a,b) will help to explore the sensory modality of isolated antennal


Day 8 Day 10 Day12 Day 13(hatching)

Adult

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0

AB14/6D2 S1/5D5

Fig. 7. (A) Immunoreactivity of mAb B14/6D2 with antennal membrane proteins fromdifferent developmental stages. ELISA plates were coated with 100ng protein per well andincubated with culture supernatant. Reactivity was visualized using HRP-conjugated goatanti-mouse Ig. Absorbance was measured at 450nm. (B) Immunoreactivity of dissociatedantennal cells from different developmental stages with mAb B14/6D2. Immunoreactive cellswere visualized with FITC-conjugated sheep anti-mouse Ig. Staining is first visible about 1 hafter hatching and reaches an intensity comparable to that seen in adults within 6h. (i) Cellsfrom antennae of hatching locusts; (ii) cells from locusts 1h after hatching; (iii) cells fromlocusts 6h after hatching. Scale bar, 10mm. Phase-contrast micrographs are shown on the left.


Bi

Bii

Biii

Fig. 7

cells and contribute to elucidating whether chemosensory receptor cells from locust arefine-tuned to detect specific pheromones, as has been described for several moth species(Kaissling, 1986), or broadly tuned for recognizing general odours relevant to adultlocust.

The sudden appearance of the B14/6D2 antigen around the time of hatching, the phaseof development when the sensitivity of antennal cells to odorous molecules first appears,suggests that it may represent a functionally important element of the chemosensorymachinery involved in odorant detection. This idea is strengthened by the observationthat the appearance of this epitope requires the synthesis of proteins and is not due to amodification (e.g. glycosylation or phosphorylation) of pre-exisiting molecules. Theappearance of immunoreactivity in a critical phase of differentiation apparently reflectsthe expression of new gene products; mAb B14/6D2 may thus be a valuable tool forfollowing the cellular and functional differentiation of antennal cells during development.

In summary, the monoclonal antibodies generated in this study provide the firstmarkers of antennal cells from locusts. They may be invaluable tools in future studies toelucidate the cellular and molecular mechanisms in odour recognition and chemo-electrical signal transduction in insects.

This work was supported by EC programme ‘Science and Technology forDevelopment’ and by the Land Baden-Württemberg as part of the cooperationprogramme between Hohenheim and Hebrew University.

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Fig. 8. Effect of cycloheximide on the expression of the B14/6D2 antigen in locusts during the6 h after hatching. (A) Immunoreactivity of embryonic antennae. Antennal cells wereincubated for 6 h in vitro in 5+4 medium (B) and supplemented with 100 mmol l21

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Documents

J. exp. Biol. 175, 45–59 (1993) 45 GENERATION OF ... · Adult locusts (Locusta migratoria) and embryos were obtained from the Insektarium Dr Frieshammer, Jaderberg. Day E1 was the