desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae), and fully identified. Its concentration is higher in crowd-reared (gregarious) animals than in.
Physiological Entomology (2003) 28, 39–45
Purification, isolation and search for possible functions of a phase-related 6080-Da peptide from the haemolymph of the desert locust, Schistocerca gregaria MAZIBUR M. RAHMAN, GEERT BAGGERMAN, M U R S H I D A B E G U M , A R N O L D D E L O O F and MICHAEL BREUER Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute, K. U. Leuven, Naamsestraat 59, B-3000. Leuven, Belgium
Abstract. A novel 6-kDa peptide has been isolated from the haemolymph of the desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae), and fully identified. Its concentration is higher in crowd-reared (gregarious) animals than in isolated-reared (solitarious) ones. Its concentration decreases progressively from generation to generation with solitarization of gregarious locusts. The peptide is also present in freshly laid eggs. The concentration in eggs is higher in those from crowd-reared locusts. It is likely that the peptide is transferred from the female’s haemolymph into the eggs because injection of the peptide into females before oviposition increases the amount of the 6-kDa peptide in the eggs. A two-step HPLC purification procedure for this peptide is described. It allowed several bioassays to test for a possible function. Although the peptide’s concentration in the haemolymph is high (0.1 mM), which suggests some physiological function, we have as yet not been able to identify a function. We hypothesize that the 6-kDa peptide may somehow play a role as a maternal factor in the determination of the phase-state of the offspring. Key words. Locusts, maternal factor, peptides, phase polymorphism, phase transition, RP-HPLC purification.
Introduction Unravelling the mechanisms of phase transition, the transition from the solitary to the gregarious phase and vice versa, in locusts has proved to be a challenge for insect physiologists because of the multitude of controlling factors involved. The major quest is to elucidate which signalling cascades are involved and how these result eventually in
Correspondence to: Mazibur M. Rahman, Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute, K. U. Leuven, Naamsestraat 59, Belgium. Tel.: þ32 16 324260; fax: þ32 16 323902; e-mail: mazibur.rahman@bio. kuleuven.ac.be
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2003 The Royal Entomological Society
changing the well-known morphometrical, behavioural, reproductive and other phase-related characteristics. The possible involvement of Juvenile Hormones (JH) and ecdysteroids in the regulation of phase transition in locusts was investigated by several authors (see reviews by Dale & Tobe, 1990; Pener & Yerushalmi, 1998). Ecdysteroid titres in hoppers were found not to differ significantly between the two phases. There is as yet no consensus about the significance of minor differences in titres that have been reported during the adult stage (Tawfik et al., 1996). Without doubt, JH induces certain solitary characteristics, such as the green coloration of solitarious hoppers (Pener & Yerushalmi, 1998). However, according to Pener (1991), JH is not the primary trigger of phase transition. Some neuropeptides are likely to be involved as well. In extracts of corpora cardiaca, differences in the relative
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40 M. M. Rahman et al. amounts of neuroparsin A, ovary maturating parsin, adipokinetic hormone (AKH) I and II and AKH-precursor-related peptide (Ayali et al., 1996a, 1996b) were found. However, direct effects of these peptides, or the reported differences in their amounts, on phase characteristics have not yet been demonstrated. On the other hand, the neuropeptide [His7]corazonin, which has recently been isolated from the corpora cardiaca (CC) of the locusts Locusta migratoria and Schistocerca gregaria (Tawfik et al., 1999), undoubtedly influences some phase-related traits such as the dark-pigmentation of the cuticle as well as morphometrics of several body parts (Hoste et al., 2002c). Yet, the presence or absence of [His7]corazonin does not fully explain the whole process of phase transition (Hoste et al., 2002b). We therefore continued our search for additional molecular markers that might be involved in phase polymorphism. Peptide profiles of the brain, CC and the haemolymph of both isolated- and crowd-reared locusts were compared using high-performance liquid chromatography (HPLC) (Clynen et al., 2002; Rahman et al., 2002b). Some pronounced differences could be detected. Significantly, a major chromatographic peak in extracts of haemolymph from crowd-reared adults was found to be only minor in extracts of isolated-reared individuals. This peptide has been identified as a novel peptide with a molecular mass of 6080 Da. Edman degradation in combination with enzymatic fragmentation and Q-Tof mass spectrometry revealed the full sequence: DNADEDTICVAADNKFYLYANSLKLYTCYNQLPKVYVVKPKSQCRSSLSDCPTS (Rahman et al., 2002b). In the present article, we further document differences in concentration of 6-kDa peptide between the crowd- and isolated-reared S. gregaria and its presence in eggs, and we describe a simple purification procedure of this 54aa peptide
Materials and methods Crowded and isolated-reared insects The desert locust Schistocerca gregaria (Forskal) (Orthoptera: Acrididae) was raised under crowded (gregarious) and under isolated (solitarious) conditions. The crowded animals were kept with 500–1000 newly emerged hoppers per cage (40 32 48 cm) in our laboratory at 32 1 C in a light–dark, 13 : 11 h photocycle. They were fed fresh cabbage leaves supplemented with dry oat flakes. The humidity in the cages was not regulated and depended only on the food supply. To raise animals showing solitary features, freshly laid eggs of crowd-reared adults were separated and washed carefully with insect saline solution to remove the foam factor as described by Rahman et al. (2002a). The hatchlings (F1) were kept individually in containers (8.5 9.5 14 cm) for their whole development, and the locusts were bred further in isolation for four consecutive generations under comparable diurnal temperature and light conditions as the crowd-reared locusts (for details see Hoste et al., 2002a). #
Collection and preparation of samples of haemolymph The haemolymph of mature adults, reared under crowded and isolated conditions, was collected in glass capillaries from a small puncture made in the coxal membrane of the hind leg. Twenty microlitres was taken per animal and mixed immediately with 200 mL of an extraction medium containing methanol, water and acetic acid (90 : 9 : 1 v/v/v). The samples were then centrifuged at 13 000 r.p.m. for 5 min at 4 C. The supernatant was removed and combined with 400 mL of 0.1% aqueous trifluoroacetic acid (TFA). The methanol was evaporated in a speedvac. The aqueous solution was then extracted with 200 mL of nhexane to remove the bulk of lipids. The remaining traces of solvents were evaporated in vacuo and the watery layer was again diluted with 0.1% TFA, filtered through a Millipore PVDF filter (0.45 mm pore size) and used for HPLC analysis. To purify a sufficient amount of the 6-kDa peptide for bioassays, larger quantities of haemolymph were collected from a batch of adults and extracted as described above. Following de-lipidation, the sample was prepurified on a Megabond Elute C18 cartridge (Varian) that had been activated with 90% CH3CN containing 0.1% TFA in MilliQ water and afterwards rinsed with aqueous 0.1% TFA. The cartridges were eluted with 50% CH3CN in 0.1% aqueous TFA.
HPLC analysis and purification HPLC analysis was performed on a Beckman HPLC system (Programmable Solvent Module 126 Connected with a Diode Array Detector Module 168) with a m-Bondapak C18 steel column (2 300 mm, 125 A˚, 10 mm, Waters). The gradient used was 0.1% (v/v) aqueous TFA (solvent A) for 10 min followed by a linear increase to 50% CH3CN containing 0.1% TFA (solvent B) for 30 min at a flow rate of 0.5 mL min1. Analysis was repeated with haemolymph samples from at least 10 locusts per rearing condition. For mass purification of the 6-kDa peptide, bulk collected haemolymph extracts were loaded on a preparative DeltaPak C18 column (25 200 mm, 100 A˚, 15 mm, Waters) and eluted with 100% A for 10 min, followed by a linear gradient to 50% B in 60 min at a flow rate of 6 mL min1. Fractions of the selected peak were further purified on a SymmetryPrep C8 column (7.8 300 mm, 100 A˚, 7 mm) in a gradient adjusted to the corresponding peaks: 100% A for 10 min, linear increase to 20% B in 5 min followed by a further increase to 35% B in 65 min at a flow of 1.7 mL min1. The absorbance was monitored at 214 and 280 nm, and fractions were collected at intervals of 1 min with an automatic fraction collector (Fig. 1). The purity of the collected fractions was verified by mass spectrometry and amino acid sequencing as described by Rahman et al. (2002b).
2003 The Royal Entomological Society, Physiological Entomology, 28, 39–45
A phase-related peptide from the desert locust
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Haemolymph + Extraction solution (1 + 10)
Vortex and centrifuge
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Analytical purposes µ-BondaPak C18 Gradient to 50% CH3CN + 0.1% TFA in 30 min
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SymmetryPrep C8 Gradient to 20% CH3CN + 0.1% TFA in 5 min, then to 35% CH3CN + 0.1% TFA in 65 min Fig. 1. Overview on the purification strategy of the 6-kDa peptide.
Presence of 6-kDa peptide in the eggs Preliminary experiments have shown that the 6-kDa peptide could be also found in freshly laid eggs of S. gregaria females. To check if the peptide is maternally transmitted, isolated-reared females were injected twice with 120 mg peptide in 5 mL of insect saline just after they laid their first batch of eggs. The first injection was given on the day of egg laying and the second on the next day. The tip of the Hamilton syringe was introduced at the ventro-lateral side (alternating left/right) behind the first abdominal segment. Control females were injected with insect saline only. Twenty-five freshly laid eggs of the second egg pod from the treated and untreated females were collected and immediately homogenized in 2 mL of extraction medium containing methanol, water and acetic acid (90 : 9 : 1 v/v/v). For comparison, extracts were also made from eggs of a second egg pod of crowd-reared S. gregaria. The samples were then centrifuged at 13 000 r.p.m. for 5 min. The supernatant was collected and the pellets were re-extracted with extraction medium. All supernatants were pooled per condition. The methanol was evaporated (SpeedVac Concentrator, Savant) and the remaining aqueous residue was re-extracted with
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ethyl acetate and n-hexane to remove the bulk of lipids. The organic layer was decanted and the aqueous solution was evaporated in vacuo. Subsequently, the extract was purified on Megabond Elute C18 cartridges (Varian VA, Analytichem). Cartridges were activated with 90% CH3CN in MilliQ water containing 0.1% TFA and rinsed with 0.1% TFA in MilliQ water. Samples were eluted in 50% CH3CN in MilliQ water containing 0.1% TFA. The acetonitrile was removed by evaporation in vacuo. The remaining aqueous solution was filtered in the same way as mentioned above and used for HPLC analysis. Analysis of the egg extracts (four replications per condition) was performed on a m-Bondapak C18 column as described for the haemolymph. Five egg equivalents were loaded on the column for each run.
Results Chromatographic analysis of haemolymph samples Extracts of haemolymph samples of individual crowdreared S. gregaria were analysed by HPLC. On the
2003 The Royal Entomological Society, Physiological Entomology, 28, 39–45
42 M. M. Rahman et al.
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Fig. 2. Typical HPLC profile of an acidic methanolic extract of haemolymph from adult crowd-reared Schistocerca gregaria. Arrow indicates the 6-kDa peptide.
C18 column used (m-Bondapak) the 6-kDa peptides eluted as the major peak with a retention time of 31 min (Fig. 2). The peak was much smaller in extracts of haemolymph from isolated-reared locusts (Fig. 3). Furthermore, the peak decreased more or less progressively during
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successive generations of isolated breeding, whereas peaks eluting between 21 and 23 min generally increased with successive generations (Fig. 3). The peptide profiles were quite reproducible within samples of the same rearing condition.
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Fig. 3. Typical HPLC profiles of acidic methanolic extracts of haemolymph from adults from successive generations of isolated-reared Schistocerca gregaria. Arrows indicate the 6-kDa peptide.
2003 The Royal Entomological Society, Physiological Entomology, 28, 39–45
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Fig. 4. Purification of 6-kDa peptide from haemolymph of S. gregaria by reversed-phase high performance chromatography (RP-HPLC): (a) first purification step on a DeltaPak C18 column; (b) second purification step on a SymmetryPrep C8 column (for details see text). Arrows indicate the 6-kDa peptide.
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Concentrations in the haemolymph The average concentration of the 6-kDa peptide in haemolymph of crowd-reared adults was estimated from the HPLC-runs and was found to be around 80 mg per 100 mL haemolymph which represents a concentration of 0.1 mM.
An improved purification procedure Because the 6-kDa peptide elutes from the analytical C18 column in a peak that is well separated from other peaks, and mass spectrometry showed that this peak was rather pure, we tried to scale up the method making use of preparative columns. Batches of 10 mL of haemolymph were collected and extracted with acidic methanolic solution. After prepurification of the haemolymph extracts on a Megabond Elute column, the extracts were first separated on a preparative DeltaPak C18 column. The 6-kDa peptide eluted in 43% CH3CN with a retention time of 62 min (Fig. 4a). Mass spectrometry showed, however, that the selected fraction was not pure. To guarantee full purity, this fraction was re-chromatographed on a SymmetryPrep C8 column (Fig. 4b). The resulting fraction containing the 6-kDa peptide eluted in 31% CH3CN as a single homogeneous peak at 52 min. Thus, the 6-kDa peptide can be #
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isolated and purified in bulk using a two-step HPLC procedure.
Maternal transmission Acidic methanolic extracts of freshly laid egg samples were analysed by reverse-phase HPLC on a m-Bondapak C18 column in a manner similar to that used with the haemolymph samples. The peak of the 6-kDa peptide was higher in eggs from crowd-reared females than from isolated-reared ones (Fig. 5). Interestingly, injection of 2 120 mg of the 6-kDa peptide into isolated-reared solitarious females after they laid their first batch of eggs resulted in an increase of the 6-kDa peptide in the eggs of the next batch, whereas in eggs from isolated-reared females treated with saline solution this peak continued to be small. This suggests that the 6-kDa peptide is transmitted maternally (Fig. 5).
Discussion The HPLC profile of acidic methanolic extracts of haemolymph samples of locusts reared under different conditions revealed some striking differences. The peak of the 6-kDa peptide was high in the haemolymph samples of crowd-reared
2003 The Royal Entomological Society, Physiological Entomology, 28, 39–45
44 M. M. Rahman et al. 0.3
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sis of a 54aa peptide is difficult and also costly. Since the peptide is present in quite high amounts in the haemolymph of crowd-reared adult locusts (see Rahman et al., 2002b), we searched for a rapid purification procedure with high yield. A two HPLC column procedure yielded the peptide in pure form. Per 100 mL of extracted haemolymph, the overall yield is 80 mg. A series of bioassays was carried out with the 6-kDa peptide. It was injected in hoppers to verify if it has a direct effect on the morphometrical and behavioural phase state. No relevant shift could be detected. We have also studied the potential of this peptide in inhibiting chymotrypsin and ACE activity since protease inhibitors (such as SGPI-2, see above) apparently could play a role in phase transition. Again, the results were negative. We finally tested also for antimicrobial activity but also here the peptide showed no activity (Rahman et al., 2002b). We then investigated whether the peptide is transferred from the female into the eggs. The concentration was found to be small in eggs of isolated-reared females and higher in those of crowd-reared females. This corresponds to the proportions found in the haemolymph of the two phases. Artificial increase of the haemolymph concentration by injection in isolated-reared females during the days before oviposition also increased the amount of the peptide traced in the eggs. This finding suggests that the 6-kDa peptide may act as a maternal factor and may perhaps play a role in the determination of the phase-state of the offspring.
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Acknowledgements 0.1
Mazibur Rahman is grateful to the Science Foundation of the K. U. Leuven for providing a scholarship and to the Bangladesh Atomic Energy Commission for granting him study leave. This project was supported by an NFWO-grant (G.0105.01) and a GOA-grant (GOA/2000/04) from the research council of the K. U. Leuven.
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Fig. 5. Typical HPLC profiles of extracts of eggs laid by crowdreared (a), isolated-reared (b), and 6-kDa peptide-treated isolatedreared (c) S. gregaria females. Arrows indicate the 6-kDa peptide.
adults but decreased when the animals were solitarized and reared individually. It can therefore be suggested that the 6-kDa peptide is phase related. The differences in haemolymph titres could indeed be correlated with the morphometrical phase state of the locusts (Rahman et al., 2002b). Some other peaks, eluting about 10 min earlier than the 6-kDa peptide, generally increased with successive generations. One of these peaks has already been purified and is identical to the serine protease inhibitor SGPI-2 (Rahman et al., 2002b). This 3794-Da peptide was at first isolated from the ovaries of S. gregaria (Hamdaoui et al., 1998). To uncover a possible function of the 6-kDa peptide, it has to be made available in sufficient amounts. The synthe#
References Ayali, A., Pener, M.P. & Girardie, J. (1996a) Comparative study of neuropeptides from the corpora cardiaca of solitary and gregarious Locusta. Archives of Insect Biochemistry and Physiology, 31, 439–450. Ayali, A., Pener, M.P., Sowa, S.M. & Keeley, L.L. (1996b) Adipokinetic hormone content of the corpora cardiaca in gregarious and solitary migratory locusts. Physiological Entomology, 21, 167–172. Clynen, E., Stubbe, D., De Loof, A. & Schoofs, L. (2002) Peptide differential display: a novel approach for phase transition in locusts. Comparative Biochemistry and Physiology, 132, 107–115. Dale, J.F. & Tobe, S.S. (1990) The endocrine basis of locust phase polymorphism. Biology of Grasshoppers (ed. by R. F. Chapman & A. Joern), pp. 393–414. Wiley, New York.
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A phase-related peptide from the desert locust Hamdaoui, A., Wataleb, B., Devreese, B., Chiou, S., Vanden Broeck, J., Van Beeumen, J., De Loof, A. & Schoofs, L. (1998) Purification and characterization of a group of five novel peptide serine protease inhibitors from ovaries of the desert locust, Schistocerca gregaria. FEBS Letters, 422, 74–78. Hoste, B., Luyten, L., Claeys, I., Clynen, E., Rahman, M.M., De Loof, A. & Breuer, M. (2002a) An improved breeding method for solitarious locusts. Entomologia Experimentalis et Applicata, 104, 281–288. Hoste, B., Simpson, S.J., Tanaka, S., De Loof, A. & Breuer, M.A. (2002b) A comparison of phase related shifts in behavior and morphometrics of an albino strain, deficient in [7His]-corazonin, and a normally colored Locusta migratoria strain. Journal of Insect Physiology, 48, 791–801. Hoste, B., Simpson, S.J., Tanaka, S., De Loof, A. & Breuer, M.A. (2002c) Effects of [His7]-corazonin on the phase state of isolatedreared (solitarious) desert locusts, Schistocerca gregaria. Journal of Insect Physiology, 48, 981–990. Pener, M.P. (1991) Locust phase polymorphism and its endocrine relations. Advances in Insect Physiology, 23, 1–79. Pener, M.P. & Yerushalmi, Y. (1998) The physiology of locust phase polymorphism: an update. Journal of Insect Physiology, 44, 365–377.
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Rahman, M.M., Hoste, B., De Loof, A. & Breuer, M. (2002a) Developmental effect of egg pod foam in Schistocerca gregaria. Entomologia Generalis, 26, 161–172. Rahman, M.M., Vanden Bosch, L., Baggerman, G., Clynen, E., Hens, K., Hoste, B., Meylaers, K. & Vercammen, T. (2002b) Search for peptidic molecular markers in hemolymph of crowd(gregarious) and isolated-reared (solitary) desert locusts, Schistocerca gregaria. Peptides, 23, 1907–1914. Tawfik, A.I., Mat’hova, A., Sehnal, F. & Ismail, S.H. (1996) Haemolymph ecdysteroids in the solitary and gregarious larvae of Schistocerca gregaria. Archives of Insect Biochemistry and Physiology, 31, 427–438. Tawfik, I.A., Tanaka, S., De Loof, A., Schoofs, L., Baggerman, G., Waelkens, E., Derua, R. & Milner, Y. (1999) Identification of the gregarization-asscociated dark-pigmentotropin in locusts through an albino mutant. Proceedings of the National Academy of Sciences USA, 96, 7083–7087.
Accepted 14 January 2003
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