Peptide differential display: a novel approach for phase transition in locusts

Comparative Biochemistry and Physiology Part B 132 (2002) 107–115

Peptide differential display: a novel approach for phase transition in locusts夞 Elke Clynen*, Dirk Stubbe, Arnold De Loof, Liliane Schoofs Laboratory for Developmental Physiology and Molecular Biology, K.U. Leuven, Leuven, Belgium Received 27 January 2001; received in revised form 1 May 2001; accepted 2 May 2001

Abstract Today, the question of the physiological cause of phase transition, the transition from the solitary to the gregarious phase, in locusts remains unanswered. We hereby present a novel approach by which we have attempted to determine whether different phases express or release different peptides in similar physiological conditions. For this purpose, a peptidomic analysis of the corpora cardiaca and hemolymph of crowded and isolated locusts of Schistocerca gregaria was performed using high performance liquid chromatography and matrix-assisted laser desorption ionisation time of flight mass spectrometry. A comparison between the two conditions reveals differences in the number and amount of peptides present in the corpora cardiaca and the hemolymph. Further research will have to identify these phase specific differences and their role in locust phase polymorphism. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Corpora cardiaca; Hemolymph; HPLC; MALDI-TOF MS; Neuropeptides; Peptidomics; Phase polymorphism; Schistocerca gregaria

1. Introduction The desert locust, Schistocerca gregaria, is one of the most feared locusts, causing periodic devastating swarms. In the northern half of the African continent, such swarms, which can contain hunAbbreviations: AKH, adipokinetic hormone; APRP, AKHprecursor related peptide; AUFS, absorption unit full scale; CC, corpora cardiaca; HPLC, High performance liquid chromatography; MALDI-TOF MS, Matrix-assisted laser desorptionyionisation time of flight mass spectrometry; OMP, ovary maturating parsin; SGPI, Schistocerca gregaria protease inhibitor; TFA, trifluoroacetic acid. 夞 This paper was submitted as part of the proceedings of the 20th Conference of European Comparative Endocrinologists, organized under the auspices of the European Society of Comparative Endocrinology, held in Faro, Portugal, 5–9 September 2000. *Corresponding author. Zoological Institute, Naamsestraat 59, B-3000 Leuven, Belgium. Tel.: q32-16-324260; fax: q 32-16-323902. E-mail address: [email protected] (E. Clynen).

dreds of millions of voracious individuals, occur with an average interval of 10–15 years. Despite several decades of research, the most enigmatic issue in locust physiology is still phase transition, or the transition from the solitary to the gregarious phase (Uvarov, 1966, 1977). Locust populations often persist for years in semi-arid regions in the solitary phase, until environmental factors bring solitary locusts together against their behavioural predisposition to avoid each other ¨ (Bouaıchi et al., 1996; Collett et al., 1998) and close-range stimuli (principally mechanical, but also visual, olfactory and perhaps contact chemical) change behaviour and produce grouping ¨ (Roessingh et al., 1998; Hagele and Simpson, 2000). The colour, morphometry, reproduction and behaviour differ between the two phases. In between the extreme solitary and gregarious phases, various intermediate states occur. Hence, the name ‘density-dependent continuous phase poly-

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morphism’ was introduced (Pener and Yerushalmi, 1998). Several hypotheses on the physiological cause of the phase transition have been proposed. Juvenile hormone affects certain phase characteristics, such as the green solitary colour, but is probably not the primary physiological factor that induces the solitary phase (reviewed by Pener and Yerushalmi, 1998). Other researchers emphasise the importance of ecdysteroids in phase transition. Although phase specific differences in ecdysteroid titres have been found, their role in phase transition remains unclear (Tawfik et al., 1996, 1997). Another team provided evidence that the population density of the parents influences the behaviour and colour of the progeny (Islam et al., 1994). The progeny of parents that were grouped during the reproduction period or during oviposition behave the same as those of parents that live ¨ continuously under crowded conditions (Bouaıchi et al., 1995). Others have demonstrated the presence of a maternal gregarising factor in the accessory glands and in the foam plugs of the egg pods of gregarious females. Solitary females do not produce this factor. The identity of this factor is ¨ as yet unknown (McCaffery et al., 1998; Hagele et al., 2000). In a recent study, the technique of two-dimensional-gel electrophoresis was used to generate hemolymph protein maps from Schistocerca gregaria. Three solitary-specific and 17 crowded-specific spots were found, providing evidence that a number of hemolymph proteins are expressed and repressed, respectively, in relation to phase (Wedekind-Hirschberger et al., 1999). Because pigmentation is one of the differential characteristics between the solitary and gregarious phase, the dark-inducing hormone wHis7xcorazonin is likely to be one of the many factors that is differentially released in the two phases (Tawfik et al., 1999; Baggerman et al., 2001). Assuming that the environmental and social conditions involved in phase transition are integrated by the brain and translated in the different use of neuronal messengers playing a role as neurotransmitter, neuromodulator or neurohormone, we started a comparative study on the neuropeptide profile of the corpora cardiaca (CC) and hemolymph of solitary and gregarious locusts. More than 60 neuropeptides have already been identified in the model insects Locusta migratoria and Schistocerca gregaria and the CC are very important neuro-endocrine organs, involved in

brain-associated neurosecretion (Schoofs et al., 1997; Veelaert et al., 1998). Ayali and co-workers used electrophoretic, chromatographic and immunological techniques to study differences in the three major neuropeptides of the CC of isolated vs. crowded locusts of Locusta migratoria. For these three neuropeptides, quantitative differences were found: the AKHprecursor related peptide (APRP) was markedly higher in the CC of isolated locusts, while the amount of neuroparsins and ovary maturating parsin (OMP) was higher in the CC of crowded animals, the latter only in completely mature locusts (Ayali et al., 1996a). In our study, peptide profiles were compared using microflow high performance liquid chromatography (HPLC) combined with matrix-assisted laser desorptionyionisation time of flight mass spectrometry (MALDI-TOF MS). 2. Materials and methods 2.1. Animals Schistocerca gregaria was reared under crowded conditions— representing the gregarious phase — and under isolated conditions — representing the solitary phase. The crowded animals were kept with 100–200 animals in cages of 38=38=38 cm under controlled temperature (32 8C), a photoperiod of 13 h and relative humidity between 40 and 60%. They were fed daily with fresh cabbages and oatmeal. The isolated animals were raised in transparent containers of 8.5=7.5=14 cm in a separate room, but under similar conditions. For first generation solitary animals, the eggs of crowdedreared females were used. The egg foam was washed away and the eggs were separated. For the next generations, the eggs of the solitary females were used and placed separately after hatching. Fourth-generation solitary-reared locusts were used for the experiments. Morphometric standards were used according to Pener (1991). 2.2. Preparation of corpora cardiaca and hemolymph extracts The CC of 15 male and 15 female crowded and isolated adult locusts, respectively, were dissected in a Schistocerca ringer (8.77 gyl NaCl, 0.19 gyl CaCl2, 0.75 gyl KCl, 0.41 gyl MgCl2, 0.34 gyl NaHCO3, 30.81 gyl sucrose, 1.89 gyl trehalose,

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pH 7.2) and placed in a methanolywateryacetic acid (90:9:1, vyvyv) solution on ice. After sonication and centrifugation, the pellet was discarded and 100 ml aqueous trifluoroacetic acid (TFA) (0.1%) was added to the supernatant. The methanol was evaporated (SpeedVac Concentrator, Savant) and the remaining aqueous residue was re-extracted with ethyl acetate and n-hexane to remove the bulk of the lipids. The organic solvent layer was decanted and the aqueous solution was put in a vacuum centrifuge (SpeedVac Concentrator, Savant) to remove the remnant of organic solvent. The remaining aqueous solution was filtered (0.22 mm, Millex-GV, Millipore). From the same animals, 10 ml of hemolymph was taken. Extracts were made and treated the same way as the extracts of the CC. 2.3. Chromatographic separation Column and operating conditions for HPLC on a Beckman Programmable Solvent Module 126 connected to a Diode Array Detector Module 168 (Gold System) were as follows: Waters Symmetry C18 (4.6=250 mm) column, solvent A: 0.1% TFA in water; solvent B: 50% acetonitrile in 0.1% aqueous TFA. Column elution conditions: 100% A for 10 min, linear gradient to 100% B in 60 min, 100% B for 10 min, flow rate 1 mlymin, detector range: 1 absorption unit full scale (AUFS). Absorbance was recorded simultaneously at 214 nm and 280 nm and fractions were collected manually.

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tion and signalynoise ratio was obtained. The results of 10–20 shots were averaged to obtain the final spectrum. 3. Results 3.1. Chromatographic analysis of the hemolymph Chromatographic patterns of the hemolymph of 2-day-old adult locusts were compared between the crowded and isolated conditions. The absorption patterns at 214 nm could easily be compared (Figs. 1 and 2). Only a single differential peak could be detected. In both male and female, a peak eluted at 48 min in the solitary condition, which was absent in the gregarious condition. Mass analysis of the peak showed several myz signals that were also present in the corresponding gregarious fraction. A few ion peaks, corresponding with masses of 5312, 5455, 5562, 5731, 5837 and 5983 Da, however, were only present in the solitary fraction (Fig. 3). Furthermore, there were some quantitative differences between the two conditions. The peaks eluting at 50 and 72 min are clearly larger in the gregarious condition. In the gregarious males, the 50-min peak is more than four times as large as in the solitary males. Mass analysis showed ion masses of 6075 Da for the 50-min peak and 6821 and 10114 Da for the 72-min peak (Fig. 4). We also observed that the absorption pattern at 214 nm of the solitary females is slightly different to that of the gregarious females, and the solitary and gregarious males in the area of 24–28 min.

2.4. Mass determination 3.2. Chromatographic analysis of the CC An aliquot (1y2 for the small peaks and 1y5 for the larger peaks) of the fractions was concentrated and subjected to MALDI-TOF mass analysis. The concentrated sample was mixed with 0.5 ml of a 50-mM solution of a-cyano-4-hydroxycinnamic acid in acetonitrileyethanolyTFA (50:49.9:0.1, vyvyv) and applied on a multisample target. This mixture was air-dried, and the target was then introduced in the instrument, a VG Tofspec (Micromass, UK), equipped with a N2laser (337 nm) and operating with continuous extraction. The samples were measured either in the linear (acceleration voltage 24 kV) or in the reflectron mode (acceleration voltage 24 kV, reflectron voltage 28.8 kV). In either case, the laser energy was reduced until an optimal resolu-

At 214 nm wavelength, there was one major difference between the profile of the CC of the gregarious and the solitary male 2-day-old adult locusts (Fig. 5). At 25 min, a peak elutes in the solitary phase, whereas in the corresponding chromatographic profile of the gregarious phase, this 25-min peak was absent. The same difference occurs in the profile of the females (Fig. 6). This peak did not absorb at 280 nm. MALDI-TOF mass analysis revealed a mass of 3795 Da (Fig. 7). The masses of the other prominent peaks of the chromatogram of the CC were also determined (Fig. 8). The peak eluting at 50 min contains, among other things, the masses of the sodiated and potassiated ionic forms of the adipokinetic hormone-I

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Fig. 1. HPLC profile (214 nm) of the hemolymph of 15 male 2-day-old gregarious and solitary Schistocerca gregaria. The differential peak is marked.

(AKH-I) (1181.5 Da and 1197.8 Da). The peak eluting at 51 min contains several masses, including AKH-II (sodiated form: 956.7 Da; potassiated form 972.5 Da) and a mass of 6078 Da, which probably corresponds to the 6075-Da peak in the 50-min fraction of the hemolymph, as such a mass error is not unusual for a MALDI-TOF instrument operating with continuous extraction. The peaks eluting at 67 and 71 min correspond, respectively, to the theoretical masses of the AKH-precursor related peptides 1 and 2 (observed ion masses 3125.1 and 3129.5 Da; theoretical masses 3126.5 Da for APRP-1 and 3129.6 Da for APRP-2). The relative quantitative differences of these peaks for different ages and sexes are summarised in Table 1. The absorption pattern at 214 nm of the gregarious females slightly differs from the other three in the region between 22 and 28 min. 4. Discussion One of the main questions in locust physiology is still how changes in environmental factors and

population density influence the nervous and endocrine systems allowing the choice of the solitary– gregarious pathway (Hardie and Lees, 1985). In the present study, we present a novel approach to this problem. Based on the hypothesis that external stimuli involved in phase transition could affect neuropeptide expression, we searched for differences in peptide profiles of the CC and the hemolymph of solitary and gregarious locusts. Combining HPLC techniques with MALDI-TOF MS, we were able to reveal a differential display on the peptide level, which is more related to a physiological effect than the classical mRNA differential display. A comparison of the absorption pattern of the hemolymph indicates that there are some peptides, which are differentially released. This is the case for the 48-min peak that was solitary-specific. Several masses were detected in this fraction. Further purification will be necessary to determine their identity. Other peptides differed in quantity. This was the case for the 6075-Da peak, which was present in a large amount. Edman degradation will have to point out the identity of this putative novel peptide, as the 6075-Da mass

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Fig. 2. HPLC profile (214 nm) of the hemolymph of 15 female 2-day-old gregarious and solitary Schistocerca gregaria. The differential peak is marked.

Fig. 3. MALDI-TOF mass spectra (linear mode) of the differential peak eluting at 48 min in the HPLC profile of the hemolymph. The upper panel presents the solitary condition and the lower panel the gregarious condition. The differences between the two conditions are marked.

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Fig. 4. MALDI-TOF mass spectra of the peaks eluting at 50 min (upper panel; reflectron mode) and 72 min (lower panel; linear mode) in the HPLC profile of the hemolymph of 2-dayold adult solitary and gregarious locusts.

does not correspond to a calculated mass of a known locust peptide. This will also need to be done for the 72-min peak that revealed two masses (6821 and 10 114 Da) of unknown identity. Our results also clearly demonstrated that the CC differentially express at least one peptide. Since the same data were obtained both for males and females, the differential peak is likely to be specific for the phase and can not be attributed to a difference in physiological condition. The mass of this differential peak (3795 Da) matches to the theoretical mass of the Schistocerca gregaria protease inhibitor-2 (SGPI-2). The serine protease inhibitors (SGPI-1-5) were isolated and identified previously in the ovaries of the desert locust (Hamdaoui et al., 1998). Recently, we demonstrated the presence of the related Locusta migratoria chymotrypsin inhibitors (LMCI-I and LMCI-II) in the CC and hemolymph of the migratory locust, Locusta migratoria (Clynen et al., 2001). Here, we provide evidence that the serine protease inhib-

itor SGPI-2 is present in the CC of the desert locust and may play a role in phase transition as it is differentially expressed. Only in the 22–28min area, could a difference in peak profile be noticed between males and females in the same phase. As in the remaining part of the profile the pattern was similar, these differences may be related to gender differences. Ayali and co-workers previously demonstrated by HPLC that the APRP content of the CC for 12–16-day old males is higher in the isolated condition than the crowded condition (Ayali et al., 1996a). This is confirmed by our results. However, they also showed by polyacrylamide gel electrophoresis and Western blot analysis a higher amount of APRP in the CC of solitary locusts of other ages (5–9 and 22–31 days old) in both sexes. This is in conflict with our results where we find higher peaks of APRP in the crowded locusts of 8 and 25 days old. The AKH-content of the CC was also described by Ayali and colleagues to be higher in isolated adult males between 12 and 19 days old. The ratio of AKH-IyAKH-II was shown to be higher in the crowded condition (Ayali et al., 1996b). All these results are in agreement with our findings. In 25–30-day-old adults they found no significant difference between the crowded and the isolated condition. We found the same results for the females, but a slightly higher AKH-peak in the crowded condition for the males. The differences found between our results and those of Ayali could be species-specific as they worked with Locusta migratoria and our experiments were done on Schistocerca gregaria. From our results, we can also deduce that the relative amounts of AKH and APRP between solitary and gregarious locusts vary with age and depend on the gender. Our results prove that the peptidomic approach is very useful to screen for phase related differential peptides. Whether the observed phenotypic heterogeneity, with respect to the (neuro)peptide level, contributes to the physiological cascade that leads to phase transition, or represents a consequence, remains to be elucidated. We are well aware of the fact that so far, although many peptides have been identified in the locust nervous system, our approach suffers from the limited sequence information available. If a genomicyproteomic database would be available, as is the case for Drosophila, our approach would be very powerful. However, using nanoflow LC-MS it is now possible to detect and

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Fig. 5. HPLC profile (214 nm) of the CC of 15 male 2-day-old gregarious and solitary Schistocerca gregaria.

Fig. 6. HPLC profile (214 nm) of the CC of 15 female 2-day-old gregarious and solitary Schistocerca gregaria.

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E. Clynen et al. / Comparative Biochemistry and Physiology Part B 132 (2002) 107–115 Table 1 Comparison of the AKH-I, AKH-II, APRP-1 and APRP-2 peaks and the ratios AKH-IyAKH-II and APRP-1yAPRP-2 between the CC of crowded and isolated male and female adults of different ages

AKH-I AKH-II AKH-IyAKH-II APRP-1 APRP-2 APRP-1yAPRP-2 Fig. 7. MALDI-TOF mass spectrum (reflectron mode) of the peak eluting at 25 min in the HPLC profile of the CC of 2day-old adult solitary locusts.

2-day old

8-day old

16-day old

25-day old

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乆

么

乆

么

乆

么

乆

i i c i i c

c i c c i c

c i c c c c

s s s c c c

i i c i i c

* * * * * *

c c c c c c

s s s * * *

Legend: c: higher in the crowded condition; i: higher in the isolated condition; s: equal in crowded and isolated conditions; *: no data available.

Fig. 8. MALDI-TOF mass spectra (reflectron mode) of the peaks eluting at respectively 50, 51, 67 and 71 min in the HPLC profile of the CC of solitary and gregarious locusts, representing respectively AKH-I, AKH-II, APRP-1 and APRP-2.

identify peptides using only 1y10th of a single CC (Baggerman et al., 2001). This innovative technique will enable us to look at individual differences. Using isotope-coded affinity tags, it is now also possible to quantify relative peptide levels in different conditions (Gygi et al., 1999). All these techniques will help us in the future to further analyse the differential expression of peptides and hopefully to come to an understand-

ing towards the physiological aspect of phase transition. Acknowledgments This project is sponsored by the Research Foundation of the K.U. Leuven (GOAy2000y04). Elke Clynen benefits from a scholarship from the Flemish Science Foundation (FWO).

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References Ayali, A., Pener, M.P., Girardie, J., 1996a. Comparative study of neuropeptides from the corpora cardiaca of solitary and gregarious Locusta. Arch. Insect Biochem. Physiol. 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. Physiol. Entomol. 21, 167–172. Baggerman, G., Clynen, E., Mazibur, R., Veelaert, D., Breuer, M., De Loof, A., Tanaka, S., Schoofs, L., 2001. Mass spectrometric evidence for the deficiency in the dark color inducing hormone, wHis7x-corazonin, in an albino strain of Locusta migratoria as well as for its presence in solitary S. gregaria. Arch. Insect Biochem. Physiol. 47, 129–138. ¨ Bouaıchi, A., Roessingh, P., Simpson, S.J., 1995. An analysis of the behavioural effects of crowding and re-isolation on solitary-reared adult desert locusts (Schistocerca gregaria) and their offspring. Physiol. Entomol. 20, 199–208. ¨ Bouaıchi, A., Simpson, S.J., Roessingh, P., 1996. The influence of environmental microstructure on the behavioural phase state and distribution of the desert locust Schistocerca gregaria. Physiol. Entomol. 21, 247–256. Clynen, E., Baggerman, G., Veelaert, D., Cerstiaens, A., Van der Horst, D., Harthoorn, L., Derua, R., Waelkens, E., De Loof, A., Schoofs, L., 2001. Peptidomics of the pars intercerebralis-corpus cardiacum complex of the migratory locust, Locusta migratoria. Eur. J. Biochem. 268, 1929–1939. Collett, M., Despland, E., Simpson, S.J., Krakauer, D.C., 1998. Spatial scales of desert locust gregarization. Proc. Natl. Acad. Sci. USA 95, 13052–13055. Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H., Aebersold, R., 1999. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999. ¨ ¨ Hagele, B.F., Oag, V., Bouaıchi, A., McCaffery, A.R., Simpson, S.J., 2000. The role of female accessory glands in maternal inheritance of phase in the desert locust Schistocerca gregaria. J. Insect Physiol. 46, 275–280. ¨ Hagele, B.F., Simpson, S.J., 2000. The influence of mechanical, visual and contact chemical stimulation on the behavioural phase state of solitarious desert locusts (Schistocerca gregaria). J. Insect Physiol. 46, 1295–1301. Hamdaoui, A., Wataleb, S., Devreese, B., Chiou, S.J., 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 Lett. 422, 74–78.

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Hardie, J., Lees, A.D., 1985. Endocrine control of polymorphism and polyphenism. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 8, Endocrinology II. Oxford Pergamon Press, pp. 441–490. Islam, M.S., Roessingh, P., Simpson, S.J., McCaffery, A.R., 1994. Parental effects on the behaviour and colouration of nymphs of the desert locust Schistocerca gregaria. J. Insect Physiol. 40, 173–181. McCaffery, A.R., Simpson, S.J., Islam, M.S., Roessingh, P., 1998. A gregarizing factor present in the egg pod foam of the desert locust Schistocerca gregaria. J. Exp. Biol. 201, 347–363. Pener, M.P., 1991. Locust phase polymorphism and its endocrine relations. Adv. Insect Physiol. 23, 1–79. Pener, M.P., Yerushalmi, Y., 1998. Mini review: the physiology of locust phase polymorphism: an update. J. Insect Physiol. 44, 365–377. ¨ Roessingh, P., Bouaıchi, A., Simpson, S.J., 1998. Effects of sensory stimuli on the behavioural phase state of the desert locusts, Schistocerca gregaria. J. Insect Physiol. 44, 883–893. Schoofs, L., Veelaert, D., De Loof, A., 1997. Peptides in locusts, Locusta migratoria and Schistocerca gregaria. Peptides 18, 145–156. Tawfik, A.I., Mat’hova, A., Sehnal, F., Ismail, S.H., 1996. Haemolymph ecdysteroids in the solitary and gregarious larvae of Schistocerca gregaria. Arch. Insect Biochem. Physiol. 31, 427–438. Tawfik, A.I., Tanaka, S., De Loof, A., Schoofs, L., Baggerman, G., Waelkens, E., Derua, R., Milner, Y., Yerushalmi, Y., Pener, M.P., 1999. Identification of the gregarization-associated dark-pigmentotropin in locusts through an albino mutant. Proc. Natl. Acad. Sci. USA 96, 7083–7087. Tawfik, A.I., Vedrova, A., Li, W., Sehnal, F., Obeng-Ofori, D., 1997. Haemolymph ecdysteroids and the prothoracic glands in the solitary and gregarious adults of Schistocerca gregaria. J. Insect. Physiol. 43, 485–493. Uvarov, B.P., 1966. Grasshoppers and Locusts. A Handbook of General Acridology, vol. 1. Cambridge University Press, London. Uvarov, B.P., 1977. Grasshoppers and Locusts. A Handbook of General Acridology, vol. 2. Centre for Overseas Pest Research, London. Veelaert, D., Schoofs, L., De Loof, A., 1998. Peptidergic control of the corpus cardiacum–corpora allata complex of locusts. Internat. Rev. Cytol. 182, 249–302. Wedekind-Hirschberger, S., Sickold, S., Dorn, A., 1999. Expression of phase-specific haemolymph polypeptides in a laboratory strain and field catches of Schistocerca gregaria. J. Insect Physiol. 45, 1097–1103.