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Vitellogenin Titres in Normal and Accelerated Maturation of Gregarious-Phase Schistocerca gregaria
Comp. Biochem. Physiol. Vol. 116B, No. 4, pp. 447–451, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0305-0491/97/$17.00 PII S0305-0491(96)00274-X
Vitellogenin Titres in Normal and Accelerated Maturation of Gregarious-Phase Schistocerca gregaria Hassane Mahamat,* Ahmed Hassanali* and Hans-Joerg Ferenz† *The International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya and †Insect Physiology Group, University of Oldenburg, Oldenburg, Federal Republic of Germany
ABSTRACT. Isolation of vitellogenin of the Schistocerca gregaria (Forskal) in its gregarious phase was achieved by a combination of gel permeation and anion exchange chromatography. Staining for carbohydrate and lipid moieties showed that the vitellogenin is a glycolipoprotein. The vitellogenin of S. gregaria has a native molecular weight of about 700 kDa. On SDS-PAGE, the protein showed nine apoproteins of about 124, 120, 105, 60, 59, 58, 57, 53 and 34 kD. Determination of the levels of vitellogenin by ELISA in the haemolymph of maturing females showed that those exposed to mature males from 1 to 2 days after ecdysis had increased levels of vitellogenin from day 10 (81.1 6 4.5). In contrast, females exposed to immature males or kept alone showed an increase (107.3 6 0.9 and 70.2 6 2.7) not until day 16 or later, respectively. These results are consistent with the accelerating effect of pheromonal emissions from mature males on the maturation of female S. gregaria. comp biochem physiol 116B;4:447–451, 1997. 1997 Elsevier Science Inc. KEY WORDS. Insects, orthoptera, locust, Schistocerca gregaria, haemolymph protein, vitellogenin, maturationaccelerating pheromone, primer pheromone, bioassay
INTRODUCTION Vitellogenin in most insects is a female-specific protein synthesised in extraovarial and fat body tissue from which it is released into the haemolymph before being selectively sequestered by the developing oocytes where it circulates freely (6,7). Production of mature eggs involves the incorporation of vitellogenins into developing oocytes as vitellin (3). The production and appearance of vitellogenin and its titre in the haemolymph provide an indication of the developmental and maturation status of the female insect (5). Highnam and Lusis (9) reported that Schistocerca gregaria Forskal (Orthoptera: Acrididae) females reared with mature males did not accumulate neurosecretory materials and maturation proceeded rapidly. Hill (10) observed that the haemolymph protein concentration of females reared without males remained low and the ovaries did not develop. However, when immature females were reared with mature males, the haemolymph protein concentration of those females rose rapidly and yolk deposition began in the terminal oocyte earlier than females reared without males (10). Recently, Mahamat et al. (14) confirmed the accelerating effect of mature males of the gregarious-phase S. gregaria, on the maturation of immature male and female conspecifics, first reported by Norris (17). Mahamat et al. (14) also Address reprint requests to: H. Mahamat, The International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya. Tel. XX254-2802501; Fax XX-254-2-803360; E-mail: [email protected]. Received 27 April 1996; accepted 2 October 1996.
confirmed a previous suggestion (13) that the accelerating effect is mediated by a pheromone and further demonstrated that this is associated with the volatiles from maturing and mature males. These volatiles are made up of a series of substituted benzene derivatives (14), which also function as the adult aggregation pheromone (24). The purpose of the present study was to determine if the accelerated maturation of female S. gregaria that occurs in the presence of mature male conspecifics is reflected in the haemolymph vitellogenin titres. Isolation, purification and characterisation of the vitellogenin was also performed because the properties of vitellogenin in S. gregaria has not been previously described. MATERIALS AND METHODS Insects Gregarious S. gregaria at the International Centre of Insect Physiology and Ecology colony used in this study originated from a stock colony maintained at the Desert Locust Control Organisation for Eastern Africa in Addis Ababa, Ethiopia. They were reared at a temperature of 30–35°C and 12: 12 hr light-dark cycle in a special room (4.5 3 4.5 m) that was well aerated by a duct system (10–15 air changes/hr) that maintained a negative pressure. Fresh sorghum shoot (Serena variety) and wheatbran were provided daily. Immature adults used in the study were 1–2 days old with respect to ecdysis. Sexually mature insects used as sources of the pheromone were over 4 weeks old with respect to ecdysis.
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Haemolymph Collection Haemolymph from 14-day-old immature S. gregaria females ovariectomized when 3–5 days old was collected using the ‘‘flushing out’’ method (3). Flushing was carried out with ice-cold pH 7.5 buffer (14) containing 130 mM NaCl, 5 mM KCl, 1.9 mM NaH2PO4–H2O, 1.7 mM K2 HPO4, 10 mM EDTA and 0.02% NaN3 (15) with 1.0 nM diisopropylphosphofluoridate added as the protease inhibitor. A few crystals (5 mM) of phenylthiourea were also added to the haemolymph after sampling to prevent melanization. Purification of Vitellogenin The haemolymph sample was diluted with 50 mM Tris– HCl, pH 8.4, containing 0.4 M NaCl, 1 mM phenylmethylsulfonylfluoride and 1.5 mM glutathione. This diluted sample was centrifuged (29,000g, 15 min, 4°C) to remove the haemocytes and floating lipids. The supernatant solution was filtered through a 0.2-µm filter. The following purification procedure is based on the method described previously (4). Protein in the filtrate was separated using gel permeation chromatography on a Pharmacia Ultrogel A6 column (2.5 3 85 cm). Fractions (1 ml) containing the vitellogenin peak were pooled, dialysed repeatedly with four changes of 0.15 M sodium phosphate buffer (pH 6.3) and applied onto a DEAE-52 cellulose column (Whatman, 1.6 3 12 cm) equilibrated with the phosphate buffer. After washing the column to remove unbound proteins, bound proteins were eluted using a 0.1 M phosphate buffer (pH 6.3) containing 0.25 M KCl. The absorbance of protein in the fractions (1 ml) collected was monitored at 280 nm using a Beckman spectrophotometer (Beckman, Palo Alto, CA, U.S.A.). The fractions were dialised against locust saline overnight. Fractions containing vitellogenin were pooled, concentrated, aliquoted and kept at 270°C until use. Gel Electrophoresis Electrophoresis on SDS-PAGE was performed on a 4–15% gradient gel as described previously (12). The samples were separated at a constant current of 20 mA and at room temperature. Molecular weight standards from Bio-Rad (Richmond, CA, U.S.A.) were used for comparison. Non-denaturating gel electrophoresis was carried out on a 1.32–10% polyacrylamide gel with agarose for stacking as described previously (25). The gradient gels were cast using a gradient maker (BRL, Gaithersburg, MD, U.S.A.). Gels were stained for protein with Coomassie Brilliant Blue or silver nitrate (27). Presence of carbohydrates covalently bound to protein in the gels was tested with the periodic acid-Schiff staining reagent as reported previously (11). Proteins containing lipid were detected in gels by a previous method (16). Immunological Procedures Polyclonal antibodies against the purified vitellogenin (Fig. 2, lane 2) were raised in New Zealand white rabbits follow-
ing the method described by Osir et al. (22). Double radial immunodiffusion was carried out as described (20) to determine the presence or absence of vitellogenin in the haemolymph of the S. gregaria. An ELISA described originally (26) was carried out to determine the level of vitellogenin in female locusts. In each well of the microtiter plate was placed 100 µl of 1 : 50000 diluted haemolymph in coating buffer (50 mM phosphate pH 7.4), and the plate containing diluted hemolymph was kept at room temperature in a humid chamber overnight. The microtiter plate wells were then washed three times with 200 µl of washing buffer (137 mM NaCl, 15 mM KH2PO4, 2.7 mM KCl, and 7 mM Na2HPO4 , 0.5% gelatine, pH 7.4, 0.05% Tween 20) and dried on filter paper. Each well was then coated with 150 µl of a blocking solution (1% gelatine and 1% BSA in coating buffer) and incubated for 1.5 hr at 37°C. The wells were washed three times with washing buffer, dried and 100 µl of vitellogenin antibody (the first antibody) diluted 1 : 6000 in phosphate-buffered saline was added to each well followed by incubation for 2.5 hr at 37°C). Thereafter, the wells were washed three times with washing buffer and 100 µl of the second antibody (goat anti-rabbit IgG A 8025 from Sigma conjugated with alkaline phosphatase) diluted 1:9000 in washing buffer (pH 9.6) was added to each well and incubated for 1.5 hr followed by washing. Finally, 100 µl of the substrate containing 10 mg of p-nitrophenyl phosphate dissolved in 10 ml of substrate buffer (98% diethanolamine, 100 mg MgCl2 and 0.2 g NaN3 , pH 9.3) was added to each well and the reaction stopped with 50 µl of 3 N NaOH. The optical density of each well was measured with an ELISA plate reader at 410 nm. Effect of Mature Males on Vitellogenin Titres in Immature Females Four replicates/experimental sets of newly moulted (1–2 day old) immature female insects (referred to as recipients) were exposed to the same number of mature (group A) or immature males (group B) and immature females not exposed to males (group C) (referred to as sources of stimuli) in cubic aluminum cages (15 cm3 ). The recipient insects were bled between the hind led and the thorax on the day the experiment started and thereafter on days 3, 5, 8, 10, 12, 14, 16 and 18. At each bleeding, 10 µl of haemolymph was collected, diluted in 90 µl of Mordue buffer (15) and kept at 220°C until used. The data obtained were analysed using ANOVA (SAS Institute, Cary, NC). RESULTS Isolation and Properties of Vitellogenin A two-step procedure as described previously (4) was used to purify vitellogenin from S. gregaria female locusts. In the first step, haemolymph proteins from ovariectomized locusts were separated by gel permeation chromatography. One ma-
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FIG. 1. Chromatogram of protein absorbance (at 280 nm) in
a haemolymph sample of female S. gregaria after separation by gel permeation (A) and anion-exchange (B) column chromatography. Arrows indicate vitellogenin (Vg) containing fractions. FIG. 2. Non-denaturing PAGE (1.32–10%) of isolated vitel-
jor peak (arrowed) and two smaller peaks were detected (Fig. 1A). The fractions corresponding to these peaks were separately pooled and concentrated to smaller volumes (5 ml). In the second step, the concentrated sample derived from the major peak was applied onto an anion-exchange column and another major peak (arrowed, Fig. 1B) was obtained. This anion-exchange fractions containing vitellogenin were pooled, concentrated and aliquoted in Eppendorf tubes and kept at 270°C until used. Non-denaturing polyacrylamide gel electrophoresis of the major anion-exchange peak sample (Fig. 1B) gave two protein bands (Fig. 2), a major band at about 700 kDa corresponding to the purified vitellogenin and a smaller band at about 680 kDa. The smaller band may be a proteolytic product of the major band or simply a smear that appeared due to the high molecular weight of the protein (Fig. 2). SDSPAGE (Fig. 3, lane 2) of major peak from the anion exchange chromatography containing vitellogenin yielded nine distinct apoproteins of about 124, 120, 105, 60, 59, 58, 57, 53 and 34 kDa. The presence of covalently bound carbohydrates and lipids are shown in Fig. 3, lanes 3 and 4, respectively. Two apoproteins of about 124 and 120 kDa gave positive tests for the two moieties (Fig. 3, lanes 3 and 4).
logenin. Lane 1, molecular weight marker; lane 2, isolated vitellogenin.
Immunological Studies Ouchterlony tests (Fig. 4) showed precipitation lines for the isolated vitellogenin and haemolymph samples from mature S. gregaria females and ovariectomized females but not for mature male haemolymph. A standard curve on the basis of known amounts of vitel-
FIG. 3. SDS-PAGE (4–15%) of isolated vitellogenin. Lane 1,
molecular weight marker; lane 2, isolated vitellogenin; lane 3, vitellogenin stained for carbohydrates; lane 4, vitellogenin stained for lipids.
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FIG. 6. Haemolymph vitellogenin concentration (ng/ ml) of
immature female S. gregaria; group A (exposed to immature males), group B (exposed to mature males) and group C (not exposed to males).
FIG. 4. Double radial immunodiffusion on 1% agarose. V,
anti-vitellogenin antibody; 1, mature female, 2, ovariectomized female; 3, mature male haemolymph.
logenin was developed (Fig. 5) and used to quantify the vitellogenin level in locust haemolymph with ELISA. A 1 : 100 dilution of mature female haemolymph contained 80 ng of vitellogenin. In contrast a 1:10 dilution of mature male haemolymph has less than 0.1 ng of vitellogenin showing that this protein is specific to the former. Vitellogenin Titres of Maturing Females Exposed to Different Sources of Stimuli Titres of vitellogenin in haemolymph from immature female locusts exposed to mature males (group B) increased (81.1 6 4.5) from day 10 and reached a plateau (192.7 6
FIG. 5. ELISA-generated standard curve of vitellogenin con-
centration (ng/ ml) based on different titrations.
3.0) after day 16 postexposure (Fig. 6). The groupings of females exposed to immature males (group A) or female conspecifics (group C) showed no increase in their vitellogenin titres up to day 16 postexposure. At day 16 postexposure, a significant increase was observed in the level of haemolymph vitellogenin (107.3 6 0.9) from immature females exposed to males (group A) that were initially immature. By day 18 postexposure, the vitellogenin titres (202.0 6 2.8) in the group A females had caught up with those in group B (200.6 6 2.3). Group C females showed the longest delay in the buildup of vitellogenin titres (104.1 6 2.4 at day 18) relative to the other two groups. DISCUSSION The vitellogenin of S. gregaria appears to be a glycolipoprotein with an estimated apparent native molecular weight of about 700 kDa. SDS-PAGE gave nine apoproteins with a combined mass of about 670 kDa, which is consistent with the estimated size of the native protein. Previous studies reported smaller sizes for the native vitellogenins of other insects. For example, native Locusta migratoria R & F (Orthoptera: Acrididae) vitellogenin was estimated to have a molecular weight of about 550 kDa (2), that of Tenebrio molitor Linne´ (Coleoptera:Tenebrionidae) to be 460 kDa (8) and that of Manduca sexta Johansen (Lepidoptera: sphingidae) to be 500 kDa (20). The relatively large size of S. gregaria vitellogenin is interesting, although this may well be due to a different electrophoretic procedure (25) used in this study that significantly expands the useful fractionation range of native PAGE. Vilim and Krajickova (25) used their procedure to separate large aggregating proteanglycans with molecular weights ranging from 1–4 3 106 Da. It is possible that previous determinations of the molecular weights of vitellogenins from other insects may be underestimated through fragmentation or non-penetration of the native
Vitellogenin Titres
protein into the gel. Re-examination of these vitellogenins will help resolve the question. In the present study, young females exposed to mature males were first to increase their vitellogenin titres, whereas those not in contact with males were the last to demonstrate any increase in their vitellogenin titre (Fig. 6). These results are consistent with the maturation-accelerating effect of the male-produced adult aggregation pheromone (14,24). Recently, Assad et al. (1) confirmed that the nymphal aggregation pheromone of S. gregaria also acts as a maturation retardant of young adult conspecifics. Thus, the adult gregarious population is sequentially exposed to two opposite effects. First, development of early fledgers is retarded by the nymphal pheromone until most nymphs have moulted into adults. Second, adult development is accelerated by the effect of the adult pheromone on young adults. It has been suggested that these sequential effects result in maturation synchrony of the entire adult population (14,19). Maturation synchrony constitutes an important attribute of gregarious phase locusts, because it results in simultaneous mating of the sexes and facilitates communal oviposition critical for spatial and temporal cohesiveness of the progeny (14,19,23).
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9. 10. 11. 12. 13. 14.
15. 16. 17. 18.
We are thankful to the Scientific Advisory Committee members Professors J. Borden, P. Haskell and K. Slessor for helpful discussions. We thank Dr. E. Osir for reading the manuscript. This work was supported by funds coordinated by the International Fund for Agricultural Development through the Consultative Group on Locust Research and a fellowship from the German Academic Exchange to H. Mahamat.
References 1. Assad, Y.O.H.; Hassanali, A.; Torto, B.; Mahamat, H.; Bashir, N.H.H.; Bashir, S.E. Effects of fifth instar nymphs on maturation of immature adults of the desert locust Schistocerca gregaria (Forskal) (Orthoptera: Acrididae). J. Chem. Ecol. 5;1997. 2. Chen, T.T.; Strahlendorf, P.; Wyatt, G.R. Vitellin and vitellogenin from locusts (Locusta migratoria). Properties and post translocational modification in the fat body. J. Biol. Chem. 253:5325–5331;1978. 3. Chino, H.; Hirayama, Y.; Kiyomoto, Y.; Downer, R.G.; Takahashi, K. Spontaneous aggregation of locust lipophorin during haemolymph collection. Insect Biochem. 17:89–97;1987. 4. Chinzei, Y.; Chino, H.; Wyatt, G.R. Purification and properties of vitellogenin and vitellin from Locusta migratoria. Insect Biochem. 11:1–7;1981. 5. Chinzei, Y.; Wyatt, G.R. Vitellogenin titre in haemolymph of Locusta migratoria in normal adults, after ovariectomy and in response to methoprene. J. Insect Physiol. 31:441–445; 1985. 6. Engelmann, F. Insect vitellogenin: Identification, biosynthesis and role in vitellogenesis. Adv. Insect Physiol. 14:49–108; 1979. 7. Hagedorn, H.H.; Kunkel, J.G. Vitellogenin and vitellin in insects. Annu. Rev. Ent. 24:475–505;1979. 8. Harnisch, D.G.; White, B.N. Insect vitellins identification,
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purification and characterisation from eight orders. J. Exp. Zool. 220:1–10;1981. Highnam, K.C.; Lusis, O. The influence of mature males on the neurosecretory control of ovarian development in the Schistocerca gregaria. J. Micr. Sci. 103:201–215;1962. Hill, L. Neurosecretory control of haemolymph protein concentration during ovarian development in the desert locust. J. Insect Physiol. 8:609–619;1962. Kapitany, R.A.; Zebrowski, E.J. A higher resolution PAS stain for polyacrylamide gel. Analyt. Biochem. 56:361–369;1984. Laemmli, U.K. Cleavage of structural protein during the assembly of the heads of bacteriophage T4. Nature 227:680– 685;1970. Loher, W. The chemical acceleration of the maturation process and its hormonal control in the male of the desert locust. Proc. R. Soc. Lond. (B) 153:380–397;1960. Mahamat, H.; Hassanali, A.; Odongo, H.; Torto, B.; El Bashir, E. S. Studies on the maturation-accelerating pheromone of the desert locust Schistocerca gregaria (Orthoptera: Acrididae). Chemoecology 4:159–154;1993. Mordue, W.; Goldsworthy, G.L. The physiological effect of CC extracts in locusts. Gen. Com. Endocr. 12:360–369;1969. Narayan, K.A. Electrophoresis Methods for Separation of Serum Lipoproteins. Champaign, IL: The American Oil Chemists Society; 1975. Norris, M.J. Sexual maturation in the desert locust (Schistocerca gregaria) Forskal with special reference to the effects of grouping. Anti-Locust Bull. 28:1–44;1954. Norris, M.J. Laboratory experiments on gregarious behaviour in ovipositing females of the desert locust Schistocerca gregaria (Forskal). Entomol. Exp. Appl. 6:269–363;1963. Norris, M.J. Accelerating and inhibiting effects of crowding on sexual maturation in two species of locusts. Nature 103: 874–875;1964. Ouchterlony, O. Antigen-antibody reaction in gels. In: Handbook of Immunodifusion and Immunoelectrophoresis. Ann Arbor, MI: Ann Arbor Science Publishers; 1958. Osir, E.O.; Wells, M.A.; Law, J.H. Studies on vitellogenin from the tabacco hornworm Manduca sexta. Arch. Insect Biochem. Physiol. 3:217–233;1986. Osir, E.O.; Labongo, L.V.; Unithan, G.C. A high molecular weight diapause-associated protein from stem borer, Busseola fusca: purification and properties. Arch. Insect Biochem. Physiol. 11:173–187;1989. Popov, G.B. Ecological studies on oviposition by swarms of the desert locust Schistocerca gregaria (Forskal). Entomol. Exp. Appl. 6:279–303;1958. Torto, B.; Obeng-Ofori, D.; Njagi, P. N. G.; Hassanali, A.; Amiani, H. Aggregation pheromone system of adult gregarious desert locus, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae). J. Chem. Ecol. 20:1749 –1762;1994. Vilim, V.; Krajickova, J. Electrophoretic separation of large proteoglycans in large-pore polyacrylamide gradient gels (1.32–10.0%) and a one step procedure for simultaneous staining of proteins and proteoglycans. Anal. Biochem. 197: 34–39;1991. Voller, A.; Bidweel, D.E.; Bartlett, A. The Enzyme linked immunosorbent assay (ELISA). Alexandria, VA: Dynatech Laboratory; 1979. Wray, W.; Boulikas, T.; Wray, V.P.; Hancock, R. Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118: 197–203;1981.
ISSN 0305-0491/97/$17.00 PII S0305-0491(96)00274-X
Vitellogenin Titres in Normal and Accelerated Maturation of Gregarious-Phase Schistocerca gregaria Hassane Mahamat,* Ahmed Hassanali* and Hans-Joerg Ferenz† *The International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya and †Insect Physiology Group, University of Oldenburg, Oldenburg, Federal Republic of Germany
ABSTRACT. Isolation of vitellogenin of the Schistocerca gregaria (Forskal) in its gregarious phase was achieved by a combination of gel permeation and anion exchange chromatography. Staining for carbohydrate and lipid moieties showed that the vitellogenin is a glycolipoprotein. The vitellogenin of S. gregaria has a native molecular weight of about 700 kDa. On SDS-PAGE, the protein showed nine apoproteins of about 124, 120, 105, 60, 59, 58, 57, 53 and 34 kD. Determination of the levels of vitellogenin by ELISA in the haemolymph of maturing females showed that those exposed to mature males from 1 to 2 days after ecdysis had increased levels of vitellogenin from day 10 (81.1 6 4.5). In contrast, females exposed to immature males or kept alone showed an increase (107.3 6 0.9 and 70.2 6 2.7) not until day 16 or later, respectively. These results are consistent with the accelerating effect of pheromonal emissions from mature males on the maturation of female S. gregaria. comp biochem physiol 116B;4:447–451, 1997. 1997 Elsevier Science Inc. KEY WORDS. Insects, orthoptera, locust, Schistocerca gregaria, haemolymph protein, vitellogenin, maturationaccelerating pheromone, primer pheromone, bioassay
INTRODUCTION Vitellogenin in most insects is a female-specific protein synthesised in extraovarial and fat body tissue from which it is released into the haemolymph before being selectively sequestered by the developing oocytes where it circulates freely (6,7). Production of mature eggs involves the incorporation of vitellogenins into developing oocytes as vitellin (3). The production and appearance of vitellogenin and its titre in the haemolymph provide an indication of the developmental and maturation status of the female insect (5). Highnam and Lusis (9) reported that Schistocerca gregaria Forskal (Orthoptera: Acrididae) females reared with mature males did not accumulate neurosecretory materials and maturation proceeded rapidly. Hill (10) observed that the haemolymph protein concentration of females reared without males remained low and the ovaries did not develop. However, when immature females were reared with mature males, the haemolymph protein concentration of those females rose rapidly and yolk deposition began in the terminal oocyte earlier than females reared without males (10). Recently, Mahamat et al. (14) confirmed the accelerating effect of mature males of the gregarious-phase S. gregaria, on the maturation of immature male and female conspecifics, first reported by Norris (17). Mahamat et al. (14) also Address reprint requests to: H. Mahamat, The International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya. Tel. XX254-2802501; Fax XX-254-2-803360; E-mail: [email protected]. Received 27 April 1996; accepted 2 October 1996.
confirmed a previous suggestion (13) that the accelerating effect is mediated by a pheromone and further demonstrated that this is associated with the volatiles from maturing and mature males. These volatiles are made up of a series of substituted benzene derivatives (14), which also function as the adult aggregation pheromone (24). The purpose of the present study was to determine if the accelerated maturation of female S. gregaria that occurs in the presence of mature male conspecifics is reflected in the haemolymph vitellogenin titres. Isolation, purification and characterisation of the vitellogenin was also performed because the properties of vitellogenin in S. gregaria has not been previously described. MATERIALS AND METHODS Insects Gregarious S. gregaria at the International Centre of Insect Physiology and Ecology colony used in this study originated from a stock colony maintained at the Desert Locust Control Organisation for Eastern Africa in Addis Ababa, Ethiopia. They were reared at a temperature of 30–35°C and 12: 12 hr light-dark cycle in a special room (4.5 3 4.5 m) that was well aerated by a duct system (10–15 air changes/hr) that maintained a negative pressure. Fresh sorghum shoot (Serena variety) and wheatbran were provided daily. Immature adults used in the study were 1–2 days old with respect to ecdysis. Sexually mature insects used as sources of the pheromone were over 4 weeks old with respect to ecdysis.
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Haemolymph Collection Haemolymph from 14-day-old immature S. gregaria females ovariectomized when 3–5 days old was collected using the ‘‘flushing out’’ method (3). Flushing was carried out with ice-cold pH 7.5 buffer (14) containing 130 mM NaCl, 5 mM KCl, 1.9 mM NaH2PO4–H2O, 1.7 mM K2 HPO4, 10 mM EDTA and 0.02% NaN3 (15) with 1.0 nM diisopropylphosphofluoridate added as the protease inhibitor. A few crystals (5 mM) of phenylthiourea were also added to the haemolymph after sampling to prevent melanization. Purification of Vitellogenin The haemolymph sample was diluted with 50 mM Tris– HCl, pH 8.4, containing 0.4 M NaCl, 1 mM phenylmethylsulfonylfluoride and 1.5 mM glutathione. This diluted sample was centrifuged (29,000g, 15 min, 4°C) to remove the haemocytes and floating lipids. The supernatant solution was filtered through a 0.2-µm filter. The following purification procedure is based on the method described previously (4). Protein in the filtrate was separated using gel permeation chromatography on a Pharmacia Ultrogel A6 column (2.5 3 85 cm). Fractions (1 ml) containing the vitellogenin peak were pooled, dialysed repeatedly with four changes of 0.15 M sodium phosphate buffer (pH 6.3) and applied onto a DEAE-52 cellulose column (Whatman, 1.6 3 12 cm) equilibrated with the phosphate buffer. After washing the column to remove unbound proteins, bound proteins were eluted using a 0.1 M phosphate buffer (pH 6.3) containing 0.25 M KCl. The absorbance of protein in the fractions (1 ml) collected was monitored at 280 nm using a Beckman spectrophotometer (Beckman, Palo Alto, CA, U.S.A.). The fractions were dialised against locust saline overnight. Fractions containing vitellogenin were pooled, concentrated, aliquoted and kept at 270°C until use. Gel Electrophoresis Electrophoresis on SDS-PAGE was performed on a 4–15% gradient gel as described previously (12). The samples were separated at a constant current of 20 mA and at room temperature. Molecular weight standards from Bio-Rad (Richmond, CA, U.S.A.) were used for comparison. Non-denaturating gel electrophoresis was carried out on a 1.32–10% polyacrylamide gel with agarose for stacking as described previously (25). The gradient gels were cast using a gradient maker (BRL, Gaithersburg, MD, U.S.A.). Gels were stained for protein with Coomassie Brilliant Blue or silver nitrate (27). Presence of carbohydrates covalently bound to protein in the gels was tested with the periodic acid-Schiff staining reagent as reported previously (11). Proteins containing lipid were detected in gels by a previous method (16). Immunological Procedures Polyclonal antibodies against the purified vitellogenin (Fig. 2, lane 2) were raised in New Zealand white rabbits follow-
ing the method described by Osir et al. (22). Double radial immunodiffusion was carried out as described (20) to determine the presence or absence of vitellogenin in the haemolymph of the S. gregaria. An ELISA described originally (26) was carried out to determine the level of vitellogenin in female locusts. In each well of the microtiter plate was placed 100 µl of 1 : 50000 diluted haemolymph in coating buffer (50 mM phosphate pH 7.4), and the plate containing diluted hemolymph was kept at room temperature in a humid chamber overnight. The microtiter plate wells were then washed three times with 200 µl of washing buffer (137 mM NaCl, 15 mM KH2PO4, 2.7 mM KCl, and 7 mM Na2HPO4 , 0.5% gelatine, pH 7.4, 0.05% Tween 20) and dried on filter paper. Each well was then coated with 150 µl of a blocking solution (1% gelatine and 1% BSA in coating buffer) and incubated for 1.5 hr at 37°C. The wells were washed three times with washing buffer, dried and 100 µl of vitellogenin antibody (the first antibody) diluted 1 : 6000 in phosphate-buffered saline was added to each well followed by incubation for 2.5 hr at 37°C). Thereafter, the wells were washed three times with washing buffer and 100 µl of the second antibody (goat anti-rabbit IgG A 8025 from Sigma conjugated with alkaline phosphatase) diluted 1:9000 in washing buffer (pH 9.6) was added to each well and incubated for 1.5 hr followed by washing. Finally, 100 µl of the substrate containing 10 mg of p-nitrophenyl phosphate dissolved in 10 ml of substrate buffer (98% diethanolamine, 100 mg MgCl2 and 0.2 g NaN3 , pH 9.3) was added to each well and the reaction stopped with 50 µl of 3 N NaOH. The optical density of each well was measured with an ELISA plate reader at 410 nm. Effect of Mature Males on Vitellogenin Titres in Immature Females Four replicates/experimental sets of newly moulted (1–2 day old) immature female insects (referred to as recipients) were exposed to the same number of mature (group A) or immature males (group B) and immature females not exposed to males (group C) (referred to as sources of stimuli) in cubic aluminum cages (15 cm3 ). The recipient insects were bled between the hind led and the thorax on the day the experiment started and thereafter on days 3, 5, 8, 10, 12, 14, 16 and 18. At each bleeding, 10 µl of haemolymph was collected, diluted in 90 µl of Mordue buffer (15) and kept at 220°C until used. The data obtained were analysed using ANOVA (SAS Institute, Cary, NC). RESULTS Isolation and Properties of Vitellogenin A two-step procedure as described previously (4) was used to purify vitellogenin from S. gregaria female locusts. In the first step, haemolymph proteins from ovariectomized locusts were separated by gel permeation chromatography. One ma-
Vitellogenin Titres
449
FIG. 1. Chromatogram of protein absorbance (at 280 nm) in
a haemolymph sample of female S. gregaria after separation by gel permeation (A) and anion-exchange (B) column chromatography. Arrows indicate vitellogenin (Vg) containing fractions. FIG. 2. Non-denaturing PAGE (1.32–10%) of isolated vitel-
jor peak (arrowed) and two smaller peaks were detected (Fig. 1A). The fractions corresponding to these peaks were separately pooled and concentrated to smaller volumes (5 ml). In the second step, the concentrated sample derived from the major peak was applied onto an anion-exchange column and another major peak (arrowed, Fig. 1B) was obtained. This anion-exchange fractions containing vitellogenin were pooled, concentrated and aliquoted in Eppendorf tubes and kept at 270°C until used. Non-denaturing polyacrylamide gel electrophoresis of the major anion-exchange peak sample (Fig. 1B) gave two protein bands (Fig. 2), a major band at about 700 kDa corresponding to the purified vitellogenin and a smaller band at about 680 kDa. The smaller band may be a proteolytic product of the major band or simply a smear that appeared due to the high molecular weight of the protein (Fig. 2). SDSPAGE (Fig. 3, lane 2) of major peak from the anion exchange chromatography containing vitellogenin yielded nine distinct apoproteins of about 124, 120, 105, 60, 59, 58, 57, 53 and 34 kDa. The presence of covalently bound carbohydrates and lipids are shown in Fig. 3, lanes 3 and 4, respectively. Two apoproteins of about 124 and 120 kDa gave positive tests for the two moieties (Fig. 3, lanes 3 and 4).
logenin. Lane 1, molecular weight marker; lane 2, isolated vitellogenin.
Immunological Studies Ouchterlony tests (Fig. 4) showed precipitation lines for the isolated vitellogenin and haemolymph samples from mature S. gregaria females and ovariectomized females but not for mature male haemolymph. A standard curve on the basis of known amounts of vitel-
FIG. 3. SDS-PAGE (4–15%) of isolated vitellogenin. Lane 1,
molecular weight marker; lane 2, isolated vitellogenin; lane 3, vitellogenin stained for carbohydrates; lane 4, vitellogenin stained for lipids.
H. Mahamat et al.
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FIG. 6. Haemolymph vitellogenin concentration (ng/ ml) of
immature female S. gregaria; group A (exposed to immature males), group B (exposed to mature males) and group C (not exposed to males).
FIG. 4. Double radial immunodiffusion on 1% agarose. V,
anti-vitellogenin antibody; 1, mature female, 2, ovariectomized female; 3, mature male haemolymph.
logenin was developed (Fig. 5) and used to quantify the vitellogenin level in locust haemolymph with ELISA. A 1 : 100 dilution of mature female haemolymph contained 80 ng of vitellogenin. In contrast a 1:10 dilution of mature male haemolymph has less than 0.1 ng of vitellogenin showing that this protein is specific to the former. Vitellogenin Titres of Maturing Females Exposed to Different Sources of Stimuli Titres of vitellogenin in haemolymph from immature female locusts exposed to mature males (group B) increased (81.1 6 4.5) from day 10 and reached a plateau (192.7 6
FIG. 5. ELISA-generated standard curve of vitellogenin con-
centration (ng/ ml) based on different titrations.
3.0) after day 16 postexposure (Fig. 6). The groupings of females exposed to immature males (group A) or female conspecifics (group C) showed no increase in their vitellogenin titres up to day 16 postexposure. At day 16 postexposure, a significant increase was observed in the level of haemolymph vitellogenin (107.3 6 0.9) from immature females exposed to males (group A) that were initially immature. By day 18 postexposure, the vitellogenin titres (202.0 6 2.8) in the group A females had caught up with those in group B (200.6 6 2.3). Group C females showed the longest delay in the buildup of vitellogenin titres (104.1 6 2.4 at day 18) relative to the other two groups. DISCUSSION The vitellogenin of S. gregaria appears to be a glycolipoprotein with an estimated apparent native molecular weight of about 700 kDa. SDS-PAGE gave nine apoproteins with a combined mass of about 670 kDa, which is consistent with the estimated size of the native protein. Previous studies reported smaller sizes for the native vitellogenins of other insects. For example, native Locusta migratoria R & F (Orthoptera: Acrididae) vitellogenin was estimated to have a molecular weight of about 550 kDa (2), that of Tenebrio molitor Linne´ (Coleoptera:Tenebrionidae) to be 460 kDa (8) and that of Manduca sexta Johansen (Lepidoptera: sphingidae) to be 500 kDa (20). The relatively large size of S. gregaria vitellogenin is interesting, although this may well be due to a different electrophoretic procedure (25) used in this study that significantly expands the useful fractionation range of native PAGE. Vilim and Krajickova (25) used their procedure to separate large aggregating proteanglycans with molecular weights ranging from 1–4 3 106 Da. It is possible that previous determinations of the molecular weights of vitellogenins from other insects may be underestimated through fragmentation or non-penetration of the native
Vitellogenin Titres
protein into the gel. Re-examination of these vitellogenins will help resolve the question. In the present study, young females exposed to mature males were first to increase their vitellogenin titres, whereas those not in contact with males were the last to demonstrate any increase in their vitellogenin titre (Fig. 6). These results are consistent with the maturation-accelerating effect of the male-produced adult aggregation pheromone (14,24). Recently, Assad et al. (1) confirmed that the nymphal aggregation pheromone of S. gregaria also acts as a maturation retardant of young adult conspecifics. Thus, the adult gregarious population is sequentially exposed to two opposite effects. First, development of early fledgers is retarded by the nymphal pheromone until most nymphs have moulted into adults. Second, adult development is accelerated by the effect of the adult pheromone on young adults. It has been suggested that these sequential effects result in maturation synchrony of the entire adult population (14,19). Maturation synchrony constitutes an important attribute of gregarious phase locusts, because it results in simultaneous mating of the sexes and facilitates communal oviposition critical for spatial and temporal cohesiveness of the progeny (14,19,23).
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9. 10. 11. 12. 13. 14.
15. 16. 17. 18.
We are thankful to the Scientific Advisory Committee members Professors J. Borden, P. Haskell and K. Slessor for helpful discussions. We thank Dr. E. Osir for reading the manuscript. This work was supported by funds coordinated by the International Fund for Agricultural Development through the Consultative Group on Locust Research and a fellowship from the German Academic Exchange to H. Mahamat.
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