Molecular markers of phase transition in locusts - Wiley Online Library

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The advent of EST-databases of locusts (e.g. Kang et al., 2004) is a most encouraging novel development in physiological and behavioral locust research.
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Molecular markers of phase transition in locusts A R N O L D D E L O O F, I L S E C L A E Y S , G E RT S I M O N E T, P E T E R VERLEYEN, TIM VANDERSMISSEN, FILIP SAS and JURGEN HUYBRECHTS Laboratory for Developmental Physiology, Genomics and Proteomics, Zoological Institute, Leuven, Belgium Abstract The changes accompanying the transition from the gregarious to the solitary phase state in locusts are so drastic that for a long time these phases were considered as distinct species. It was Boris Uvarov who introduced the concept of polyphenism. Decades of research revealed that phase transition implies changes in morphometry, the color of the cuticle, behavior and several aspects of physiology. In particular, in the recent decade, quite a number of molecular studies have been undertaken to uncover phase-related differences. They resulted in novel insights into the role of corazonin, neuroparsins, some protease inhibitors, phenylacetonitrile and so on. The advent of EST-databases of locusts (e.g. Kang et al., 2004) is a most encouraging novel development in physiological and behavioral locust research. Yet, the answer to the most intriguing question, namely whether or not there is a primordial molecular inducer of phase transition, is probably not within reach in the very near future. Key words corazonin, Locusta, neuroparsin, pacifastin, phase polyphenism, phenylacetonitrile, Schistocerca DOI 10.1111/j.1744-7917.2006.00061.x

Introduction The transition from the solitary to the gregarious phase is a complex process (Simpson et al., 2006) that involves changes in morphometry, color of the cuticle (Fig. 1), hormonal balance, reproduction, longevity, and above all, behavior. Crowding is the trigger that initiates this transition. By brushing different parts of the body, Simpson et al. (2001) detected that sensory cells at the surface of the femur of the hind legs is the major site where crowding conditions are perceived. When under laboratory conditions hatchlings from eggs of solitary phase females make contact with each other for a few hours, the switch towards the gregarious phase is initiated. Very soon, within hours, the first signs of the blackish cuticular coloration, which is typical for the gregarious phase, becomes visible. This

Correspondence: Arnold De Loof, Laboratory for developmental Physiology, Genomics and Proteomics, Zoological Institute, Naamsestraat 59, 3000 Leuven, Belgium. Tel: +32 16 324260; fax: +32 16 323902; e-mail: [email protected]

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means that crowding elicits rapid changes in the physiology underlying phase transition. For physiologists, the most intriguing question is whether there is such a thing as a primordial molecular driver of phase transition. In this paper, we review the advances of recent years in this field.

Hemolymph proteins As early as 1921 for Locusta migratoria and in 1966 and 1977 for Schistocerca gregaria, Boris Uvarov suggested that phase polymorphism was based on differential phasespecific gene expression, which should reflect in the proteins present. By means of isoenzyme patterns it has been shown that aldolase and glycerine-3-phosphate-dehydrogenase activity differed in quantity between the phases of Locusta migratoria (Colgan, 1987). A comparable investigation to reveal phase-specific isoenzyme expression in Schistocerca gregaria failed (Wedekind-Hirschberger, 1998). With the introduction of 2D gel electrophoresis it became possible to generate hemolymph polypeptide maps. These maps from mature male S. gregaria locusts (solitary www.blackwellpublishing.com/ins 3

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and gregarious laboratory strains) revealed 20 differential spots. Seventeen were crowded-specific whereas three were solitary-specific. Field catches of solitary and gregarious S. gregaria showed the same phase-specific expression for these 20 polypeptide spots. Crowding of wild solitary animals repressed one of the solitary-specific, and expressed 14 of the gregarious-specific, polypeptide spots and this only after two generations. A rough estimate of the molecular weight and the isoelectric point are the only data available for these proteins; the true identity remains unknown (Wedekind-Hirschberger et al., 1999).

An enigmatic 6 kDa peptide in the hemolymph of S. gregaria By comparing the peptidome of the hemolymph of solitary and gregarious phase Schistocerca, a peptide of 6 080 Da was discovered that was present in higher concentrations (up to 0.1 mmol/L) in the gregarious condition than in the solitarious one. Its concentration decreases progressively from generation to generation with solitarization of gregarious locusts. The peptide is taken up by the eggs and it accumulates to higher concentrations in eggs from crowd-reared females than in the ones from isolated reared ones. Because of this accumulation, it has been hypothesized, but not experimentally proven, that the 6 kDa peptide may somehow play a role as a maternal factor in the determination of the phase state of the offspring (Rahman et al., 2002). Despite intensive research, no clear function could as yet be attributed to the 6 kDa peptide. It does not act as a protease inhibitor, neither as an antibacterial or antifungal agent. It has no effect on the coloration of the cuticle, neither does it influence egg production. The purified peptide was also tested in a bioassay for summer morph-producing hormone activity. Therefore, short-day pupae of the Asian comma butterfly, Polygonia c-aureum L., were injected. The 6 kDa peptide did not induce summer morph coloration (unpublished observations: Endo et al., 2005).

Density dependent maturation: yellow protein An interesting phenomenon is the yellow coloration of the cuticle of sexually mature males (Norris, 1954) (Fig. 1). This color shift only occurs in gregarious males and is totally absent in solitarious males. In 2001 it was discovered that a beta-carotene binding protein, calledYellow Protein , produced by the epidermal cells integrated in the cuticle was responsible for this coloration (Wybrandt & Andersen, 2001). Adult gregarious males, isolated imme-

diately after adult molt also do not turn yellow. This physiological process is centrally regulated since a braincorpora cardiaca extract is able to induce yellow coloration in isolated males. A bioassay in combination with molecular biology techniques has been developed and the search for the identity of this factor is initiated.

Neurotransmitters and neuromodulators The changes of neurotransmitters and neuromodulators in the central nervous system related to phase changes were studied in detail (Rogers et al., 2004). Among them were aspartate, glutamate, citrulline, glycine, arginine, taurine, γ -aminobutyric acid, octopamine, N-acetyldopamine, dopamine, tyramine, serotonin and acetylcholine. Longterm solitary and gregarious locusts (both adults and final instar nymphs, males and females) differed in 11 of the 13 sampled chemicals; only N-acetyldopamine and octopamine were not significantly different. Whereas acetylcholine, tyramine and citrulline decreased in solitarious animals, the others increased. Significant changes in the amounts of many of these chemicals were also seen within 24 hours of either the isolation of gregarious locusts or the crowding of solitary locusts. Thus, for some chemicals the magnitude of these rapid changes exceeded the long-term differences in amounts between the phases.

Classical insect hormones: juvenile hormone and ecdysteroids Since the 60s, it is known that juvenile hormone (JH) promotes the appearance of the solitary phase. Implantation of active corpora allata, or application of synthetic JH makes the cuticle turn greenish, a typical feature for solitary phase Schistocerca and Locusta. However, Pener (1991), Pener and Yerushalmi (1998), Breuer et al. (2003) and Hartfelder and Emlen (2005) conclude that, although a major player, JH is not the primary inducer of phase transition. The possibility exists that allatotropins and allatostatins, neuropeptides that at least in some insects control JH biosynthesis, are high in the hierarchy of hormonal control, but evidence is completely lacking. The peptides belonging to the allatostatin family of Schistocerca have been chemically identified (Veelaert et al., 1996). They promoted ovarian development but did not stimulate or inhibit JH biosynthesis of the model insect Diploptera. The other classical hormone, ecdysone, is produced mainly by the prothoracic glands. These are much better developed in the solitary phase than in the gregarious one, not only in the nymphal instars, but also in adults. Although the Insect Science 13, 3J12

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Fig. 1 Locusts display a vividly colored cuticle, which can differ enormously within the species. The extremes are shown in this figure. Gregarious males and females are pinkish immediately after adult molt, later on only males turn bright yellow upon maturation. The phases can also differ enormously in their body coloration, depending on the environmental conditions. Whereas solitary animals are green (cryptic coloration) their gregarious counterparts have a bright yellow-black color pattern (aposematic coloration). A form of albinism also occurs in the locust. They are lacking pigments in the cuticle and this results in pale, whitish or yellowish animals.

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glands remain present and well-developed in solitary adults, Tawfik et al. (1997) found no conclusive evidence of a sustained ecdysone production in adults. The general trend is that the ecdysteroid concentration is higher in the hemolymph of isolated-reared locusts, but that it is substantially higher in eggs of crowd-reared females than in those of isolated-reared ones (Tawfik et al., 1999a). At this moment, the prevailing idea is that ecdysteroids are involved, but that the exact role of the prothoracic glands in solitary locusts needs further investigation. The neuropeptide that controls ecdysone biosynthesis (PTTH) has not yet been identified in locusts.

The neuropeptide corazonin It took until 1999 before the first neuropeptide that definitely is involved in phase transition, namely corazonin, was identified by Tawfik et al. (1999b). This was made possible by the discovery by Seiji Tanaka of an albino mutant of Locusta migratoria originating from Okinawa island (Fig. 2). This mutant lacked a factor from the braincorpora cardiaca complex necessary for melanization of the cuticle. Tanaka demonstrated this by showing that

Fig. 2 Crowd-reared Locusta migratoria 5th instar hoppers of the albino strain from Okinawa Island, control (top) and corazonininjected (below). The locusts were injected on day 0, 2 and 4 after the moult to the 4th instar with 2 µL soya oil (control) or 1 nmol of [His7]-corazonin dissolved in 2 µL soya oil.

implantation of corpora cardiaca from wild-type samples resulted in melanization of the cuticle after the next molt (Tanaka & Pener, 1994). This bioassay was used to monitor the purification of this factor from several thousands of dissected corpora cardiaca from Schistocerca gregaria and Locusta migratoria. In both species, the factor turned out to be an undecapeptide: pGln-Thr-Phe-Gln-Tyr-Ser-HisGly-Trp-Thr-Asn-amide. This sequence appeared identical to that of corazonin, a peptide that had been isolated before by Veenstra (1989) in a totally different physiological context, namely as a neuropeptide that potently accelerates heartbeat in the American cockroach, Periplaneta americana. The finding that corazonin is causal to melanization of the darkening of the cuticle, incited several laboratories to investigate if, perhaps, it would act as the primordial hormonal inducer of phase transition. If that would be the case, it should also affect all other important parameters of phase transition. It was found that injection of corazonin into isolated-reared S. gregaria nymphs changes their morphometrics towards more gregarious values (Hoste et al., 2002a). These results were confirmed and similar conclusions were made for Locusta migratoria (Tanaka et al., 2002a; Maeno et al., 2004). Furthermore, injections with corazonin of isolated-reared S. gregaria fails to induce the changes in behavior that typically accompany gregarization (Hoste et al., 2002a). This, however, was somewhat different in L. migratoria, where treatment of albinos with corazonin changed part of their behavior towards that of normal phenotypes. This observation was linked to the corazonin-induced pigment migration in the eyes of the albinos (Hoste et al., 2003). On the other hand, this albino strain, which lacks corazonin, is not necessarily solitary (Hoste et al., 2002b). Thus, the complete absence of corazonin is not sufficient to bring about the solitary state. In other words, this neuropeptide cannot be the only primordial inducer of phase transition. In addition, both the sequence and distribution pattern of corazonin are highly conserved among insect species and orders, but apparently not its function. Corazonin was actually identified in all insect species investigated (except for beetles); hence, also in species without polymorphism. To date, only two other corazonin isoforms are detected: [Arg7]-corazonin in Schistocerca americana (Veenstra, 1991); Diptera and Lepidoptera; and [Thr4, His7]-corazonin in the honey bee Apis mellifera (Verleyen et al., 2006). Immunohistochemical studies showed that the distribution of corazonin or corazonin-like substances in the central nervous system (CNS) is similar in the studied insects. In all cases, a group of neurons in the pars lateralis of the protocerebrum, project axons towards the ipsilateral retrocerebral neurohemal organs (Roller et al., 2003). Despite the conserved sequence and distribution of Insect Science 13, 3J12

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corazonin, its function(s) seems less conserved. First of all, the heart-accelerating effect of corazonin appeared largely restricted to Periplaneta americana (Predel et al., 1994). Furthermore, corazonin was associated with other functions, such as the reduction of the spinning rate in the silkworm, B. mori (Tanaka et al., 2002b). Recently, corazonin was shown to stimulate the release of pre-ecdysis and ecdysistriggering hormones from the Inka cells of Manduca sexta, thereby inducing precocious pre-ecdysis and ecdysis behaviors (Kim et al., 2004). Thus, corazonin plays many functions and is certainly a key player in phase transition, but not the primordial inducer. Nevertheless, this conclusion stimulated the search for other hormonal players, in particular neuropeptides.

Neuropeptides from the pars intercerebralis: neuroparsins and insulin The pars intercerebralis (PI) of the insect brain is the functional equivalent of the hypothalamus of vertebrates. Both are major controlling centers. In the insect forebrain, the PI contains the perikarya of secretory neurons that project towards the storage lobes of the corpus cardiacum (CCs) via the nervi corporis cardiaci (NCCI). In the CCs, the neurosecretory material is stored until it is released into the hemolymph. Peptide factors originating from the PI and released via the CCs are called parsins . The best-known subfamily is theneuroparsins. Neuroparsins were initially isolated from corpora cardiaca (CC) of the migratory locust, Locusta migratoria. With 12 cysteine residues, they belong to the most cysteine-rich neurohormones. These factors appear as monomers with six intramolecular disulfide bridges (Bourême et al., 1987; Girardie et al., 1989; Hietter et al., 1991). They display multiple biological activities, including anti-juvenile (Girardie et al., 1987), anti-diuretic (Fournier & Girardie, 1988) and neuritogenic effects (Vanhems et al., 1990). More recently, Claeys et al. (2003) have discovered additional members of the neuroparsin family in the desert locust (Scg-NPP1J4; Schistocerca gregaria neuroparsin precursors 1J4), as well as in a variety of other arthropod species (Janssen et al., 2001; Claeys et al., 2003). Multiple sequence comparisons of these arthropod neuroparsins indicated that they all share a characteristic pattern of positionally conserved cysteine residues. Most interestingly, the characteristic features of these peptides are strikingly similar to these of vertebrate insulin-like growth factor binding proteins (IGFBP). At least two independent reports refer to a possible functional relationship between neuroparsin-like and insulin-like peptides in insects. Vanhems et al. (1990) observed that both neuroparsin and Insect Science 13, 3J12

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insulin-stimulated neurite outgrowth in cultures of locust embryonic neurons, and these effects were synergistically improved by addition of 20E to the medium. In mosquitoes, ovarian ecdysteroidogenesis can be induced by OEH (ovarian ecdysteroidogenic hormone), an NP-like peptide, as well as by insulin (Brown et al., 1998; Graf et al., 1997), and both hormones appear to act via the conserved insulin signaling pathway (Riehle & Brown, 1999). Furthermore, an in-depth analysis of the transcript levels of the neuroparsin precursor genes in several tissues and in different developmental stages in both gregarious and solitary animals revealed interesting results (Claeys et al., 2005; Claeys et al. in preparation). In gregarious locusts, Scg-NPP1 and Scg-NPP2 mRNAs remained restricted to the brain. Moreover, the Scg-NPP2 mRNA levels were upregulated in the adult male and female brains at the moment of the onset of sexual maturation, whereas for Scg-NPP1 more-or-less constant mRNA levels were detected. Surprisingly, Scg-NPP1 and Scg-NPP2 transcripts were observed in fat body tissue of solitary counterparts, where an increase of both mRNAs coincided with the first appearance of immature oocytes in the ovariole terminals and the yellow coloration of the testis. Scg-NPP3 and Scg-NPP4 transcripts were not only found in gregarious and solitary locust CNS, but also in several peripheral tissues, the fat body being the most important source of Scg-NPP3 and Scg-NPP4 mRNAs. In gregarious female fat body, a sudden increase of both transcripts was observed around day 6 after adult emergence, whereas solitary females displayed no clear regulation of these neuroparsin transcripts. The phenomenon of phase-transition implies important alterations in locust reproductive physiology. For instance, female desert locusts of the solitary phase are characterized by a slower sexual maturation, but a higher fecundity. Reproductive phase differences probably demand specific regulations. The very remarkable spatial and temporal differences between Scg-NPP transcript levels in gregarious and solitary locusts are indicative of the existence of such phase-specific regulatory conditions. In solitary female locust fat bodies, the presence and regulation of ScgNPP1 and Scg-NPP2 mRNAs sharply contrasts with the absence of regulation for Scg-NPP3 and Scg-NPP4 mRNAs, whereas in gregarious animals an almost exactly opposite situation occurs. In brief, Scg-NPP transcript levels appear to be fine-tuned during adult locust life in a reproductionand phase-dependent manner (Fig. 3).

The pheromone phenylacetonitrile In the early 90s it was discovered that only volatile substances (pheromones) of mature males were able to evoke an

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Fig. 3 A schematic overview of Scg-NPP transcripts in both gregarious and solitary adult desert locusts. (A) In the solitary phase, Scg-NPP1 and Scg-NPP2 are more abundantly expressed than in gregarious counterparts. Both precursor transcripts were even detected in the fat body of solitarious animals. (B) On the other hand, Scg-NPP3 and Scg-NPP mRNAs are more abundant in gregarious animals than in solitary ones.

aggregation stimulus to which both sexes were equally responsive (Obeng-Ofori et al., 1994). An important molecule within this bouquet of pheromones acting as a cohesion factor is phenylacetonitrile or PAN (Torto et al., 1994). The main production and release sites are the wings and legs (Seidelmann et al., 2003). Although these aggregation effects were generally accepted, its contribution to swarm cohesion is debatable at the moment. PAN turned out to be a courtship inhibition pheromone (Seidelmann & Ferenz, 2002) preventing wasteful homosexual activity and serving as an important function to avoid sperm competition. Most recently, the behavioral response of desert locust individuals to PAN has been investigated in a dynamic Y-T-olfactometer. Here, PAN evoked a repellent effect even at very low concentrations (Seidelmann et al., 2005).

Protease inhibitors of the pacifastin family Like in all animals, the blood of insects contains high concentrations of protease inhibitors. Recent transcript

profiling studies have revealed a phase-dependent expression of four pacifastin-related serine protease inhibitor precursors (SGPP-1J4) in the desert locust, Schistocerca gregaria (Simonet et al., 2004, 2005). The SGPP-2-transcript was found to be more abundant in brains from crowdreared locust as compared to solitary ones. Furthermore, SGPP-1J4-transcript levels were consistently higher in fat body of male gregarious desert locusts than in their solitary counterparts. In fat body of females on the other hand, SGPP-mRNAs were less abundant than in males (except for the SGPP-1 transcript) and phase-dependent differences were less apparent than in males (Fig. 4). Moreover, based on an HPLC-analysis of hemolymph and corpora cardiaca extracts, the SGPI-2 peptide, encoded by the SGPP-1-precursor, was found to be more abundant in isolated-reared than in crowded-reared desert locusts (Clynen et al., 2002; Rahman et al., 2002). Altogether, these data indicate that both serine proteases and their inhibitors, in particular pacifastin-related peptides, are expressed in a phase-dependent manner. Our results are in line with the data from the EST-library of Locusta by Kang et al. (2004) who found that the unigene cluster, coding for the serine protease inhibitor precursor LMPP-2, was differentially expressed in a phase- and tissue-dependent manner.

EST libraries of locusts Recently, the gene expression correlated to the phase changes of the migratory locust (Locusta migratoria) was analyzed (Kang et al., 2004). Therefore 76 012 ESTs were generated from the whole body and specified organs (head, hind legs and midgut) from the two phases. By comparing 12 161 unigene clusters, in total 532 genes were identified as (putatively) phase-related. Most of the annotated gene classes were down-regulated in the gregarious phase in comparison to the solitary form. In the head, results were rather complicated; whereas several housekeeping gene categories were down-regulated, others including those encoding (serine) peptidases, were up-regulated. Kang and colleagues suggested that there are specific regulatory activities in nerve cells during phase transition, most likely controlled through hormonal signals that govern the transition of morphological plasticity. Before the publication of this paper by Kang et al. our laboratory already initiated an EST project concerning the central regulation of phase transition in the desert locust, Schistocerca gregaria. This locust can also manifest itself as two opposite phases. We started tissue collection simultaneously from both phases (laboratory strains, regularly sampling from 3rd instar to mature adults). Four mRNA samples (solitary vs. gregarious locusts and head ganglia vs. thoracic and abdominal Insect Science 13, 3J12

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Fig. 4 Schematic overview of SGPP-1J4 transcript levels in different tissues from isolated- and crowd-reared desert locusts (adapted from Simonet et al., 2005). Using real-time RT-PCR, transcript levels of the S. gregaria pacifastin-related precursors 1J4 (SGPP-1J4) were measured in different tissues, that is, brain (Br), fat body (Fb), foregut (Fg), midgut (Mg), hindgut (Hg), gonads (G) and male accessory glands (Acg) from isolated-reared (S) and crowd-reared (G) desert locusts. No data were recorded in female accessory glands (N). Then, per SGPP-transcript, the normalized transcript levels, corresponding to all the different experimental conditions, were expressed as a percentage of the maximal expression level (%MAX). In the schematic color presentation, the grey-black color intensity (> 0% and = 100%) corresponds to the %MAX-value: %MAX-values < 0.5% are indicated asJ, 0.5% %MAX-values 5% are indicated by a grey tone of 5% and so on. Thus, the highest SGPP-4-transcript level, which is recorded in fat body from gregarious males, corresponds to %MAX = 100% and is represented in black. Accordingly, the relative SGPP-4-mRNA content in fat body from solitary males, corresponding to 10%  %MAX-values  15%, is represented by a 15%-grey tone.

ganglia) will be generated and labeled separately. All sequence information will be implemented in a single EST database. The new data will be complementary with the EST data of Locusta as we mainly focused on the CNS (brain, sub-esophageal ganglion, thoracic and abdominal ganglia), which plays undoubtedly a major role in phase transition.

Discussion Locusts continue to be a major insect plague in numerous countries worldwide. The intermittent character of the outbreaks hampers sustained research. After a few years of severe damage, the harmless solitary phase becomes predominant and, for lack of alarming pictures in the news media, governments and granting agencies tend to lower the budget for locust research. It is thanks to a limited number of laboratories specializing in locust physiology Insect Science 13, 3J12

and ecology, that there is some continuity. The progress in better understanding the molecular basis of phase transition realized in recent years is substantial. When going through the data mentioned before, it is clear that we are still missing the major physiological events, in particular the primordial triggering ones. The discovery that corazonin is the hormone that is responsible for bringing about the dark colororation of the cuticle, a typical feature of the gregarious phase state, and this in both Locusta and Schistocerca (Tawfik et al., 1999), incited further investigations to investigate whether this neuropeptide might also induce all other major gregarious phase state characteristics. Indeed it influences the morphometry (Hoste et al., 2002a) but it turned out not be causal to the changes in behavior that typically accompany phase transition. Because of major technical advances in the speed and cost of sequencing, EST libraries of locusts have come within reach. The EST library of Locusta has already been

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published (Kang et al., 2004) and also that of the nervous system of Schistocerca is under construction. It can be expected that this approach, not yet perfect, will nevertheless stimulate further analysis of the molecular differences between the phase states. In a few years from now, the search forthe needle in a haystack approachthat we had to adopt willy-nilly until now, will be replaced, to a large extent, by much more straightforward techniques in gene expression analysis.

Acknowledgments The authors gratefully acknowledge the K.U. Leuven Research Foundation (GOA/2000/04) and the Fund for Scientific Research, Flanders (Belgium) (F.W.O.Vlaanderen) for financial support. P. Verleyen and J. Huybrechts are postdoctoral researchers of the Fund for Scientific Research, Flanders, G. Simonet and I. Claeys obtained a postdoctoral fellowship from the K.U. Leuven Research Foundation, F. Sas and T. Vandersmissen obtained a PhD fellowship from the K.U. Leuven Research Foundation (GOA/2000/04). We also thank J. Puttemans for preparing the fine figures.

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Part B, 132, 107J115. Colgan, D.J. (1987) Developmental changes of isoenzymes catalysing glycolitic and associated reactions in Locusta migratoria in relation to the rearing density of hatchlings. Insect Biochemistry, 17, 303J308. Fournier, B. and Girardie, J. (1988) A new function for the locust neuroparsins: stimulation of water reabsorption. Journal of Insect Physiology, 34, 309J313. Girardie, J., Boureme, D., Couillaud, F., Tamarelle, M. and Girardie, A. (1987) Anti-juvenile effect of neuroparsin A, a neuroprotein isolated from locust corpora cardiaca. Insect Biochemistry, 17, 977J983. Girardie, J., Girardie, A., Huet, J.C. and Pernollet, J.C. (1989) Amino acid sequence of locust neuroparsins. FEBS Letters, 245, 4J8. Graf, R., Neuenschwander, S., Brown, M.R. and Ackermann, U. (1997) Insulin-mediated secretion of ecdysteroids from mosquito ovaries and molecular cloning of the insulin receptor homolog from ovaries of bloodfed Aedes aegypti. Insect Molecular Biology, 6, 151J163. Hartfelder, K. and Emlen, D.J. (2005) Endocrine control of insect polyphenism. Comprehensive Molecular Insect Science, 3, 651J703. Hietter, H., Van Dorselaer, A. and Luu, B. (1991) Characterization of three structurally related 8J9 kDa monomeric peptides present in the corpora cardiaca of Locusta: a revised structure for the neuroparsins. Insect Biochemistry, 21, 219J224. Hoste, B., Simpson, S.J., Tanaka, S., Zhu, D.-H., De Loof, A. and Breuer, M. (2002a) Effects of [His7]-corazonin on the phase state of isolated-reared (solitarious) desert locusts, Schistocerca gregaria. Journal of Insect Physiology, 48, 981J990. Hoste, B., Simpson, S.J., Tanaka, S., De Loof, A. and Breuer, M. (2002b) A comparison of phase-related shifts in behavior and morphometrics of an albino strain, deficient in [His7]-corazonin, and a normally colored Locusta migratoria strain. Journal of Insect Physiology, 48, 791J801. Hoste, B., Simpson, S.J., De Loof, A. and Breuer, M. (2003) Behavioural differences in Locusta migratoria associated with albinism and their relation to [His7]-corazonin. Physiological Entomology, 28, 32J38. Janssen, T., Claeys, I., Simonet, G., De Loof, A. and Vanden Broeck, J. (2001) cDNA cloning and transcript distribution of two different neuroparsin precursors in the locust, Schistocerca gregaria. Insect Molecular Biology, 10, 183J189. Kang, L., Chen, X., Zhou, Y., Liu, B., Zheng, W., Li, R., Wang, J. and Yu, J. (2004) The analysis of large-scale gene expression correlated to the phase changes of the migratory locust. Proceedings of the National Academy of Sciences USA, 101, 17611J17615. Kim, Y.J., Spalovska-Valachova, I., Cho, K.H., Zitnanova, I., Park, Y., Adams, M.E. and Zitnan, D. (2004) Corazonin receptor signaling in ecdysis initiation. Proceedings of the

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Accepted November 10, 2005

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