Myostimulatory neuropeptides in cockroaches: structures, distribution, pharmacological activities, and mimetic analogs

Journal of Insect Physiology 47 (2001) 311–324 www.elsevier.com/locate/jinsphys Mini-Review Myostimulatory neuropeptides in cockroaches: structures, distribution, pharmacological activities, and mimetic analogs Reinhard Predel a, b , Ronald J. Nachman c, Gerd Gäde b, d,* a c Institut für Allgemeine Zoologie und Tierphysiologie, Friedrich-Schiller-Universität, Erbertstr. 1, 07743 Jena, Germany b Zoology Department, University of Cape Town, Rondebosch 7701, South Africa Southern Plains Agricultural Research Center, Agricultural Research Service, US Department of Agriculture, 2881 F & B Road, College Station, TX 77845, USA d 14 Landseer Road, Mowbray 7700, South Africa Received 2 May 2000; accepted 30 August 2000 Abstract In this brief overview we give the historical background on the discovery of myostimulatory neuropeptides in cockroaches. Related peptides were later found in other insect groups as well. We summarize the current knowledge on primary structures, localization, physiological and pharmacological effects of the different cockroach neuropeptides, including kinins, sulfakinins, pyrokinins, tachykinin-related peptides, periviscerokinins, corazonin, and proctolin. In addition, we briefly comment on the development of mimetic pseudopeptide analogs in the context of their possible use in insect pest management.  2001 Elsevier Science Ltd. All rights reserved. Keywords: Insect neuropeptides; Cockroaches; Myotropin; Mimetic analogs; Pest management 1. Introduction To date, the majority of neuropeptides that have been completely chemically identified in cockroaches exhibit myotropic activities, i.e. they either stimulate or inhibit the activity of muscles. A number of these peptides are putative hormones. Indeed, we know of not less than 20 myostimulatory neuropeptides from neurohemal organs of the American cockroach—more than from any other insect. The detection of myoactive substances in insects dates back to the 1950s when a very simple bioassay was established with cockroach hearts (Cameron, 1953; Unger, 1957). By this time, some scientists had already focused their research interests on the corpora cardiaca (CC), which were correctly believed to be a source of myotropic neurohormones (see, for example, Brown, 1965; Kater, 1968; Hertel, 1971; Gersch, 1972; Holman and Marks, 1974). With the robust heart bioassay in hand, myotropins were shown not to be restricted to the * Corresponding author. Tel.: +27-21-650 3615; fax: +27-21-650 3301. E-mail address: ggade@botzoo.uct.ac.za (G. Gäde). retrocerebral complex but to be distributed throughout the central nervous system (Ralph, 1962; Smith and Ralph, 1967; Rounds and Gardner, 1968; Gersch, 1974). Such a pattern of distribution was later corroborated using visceral muscles such as hindgut, foregut and oviduct to test for myoactive substances. As early as the mid-1960s, peptides were assumed to be the major candidates responsible for myotropic activity (Brown, 1965; Kater, 1968). It was, thus, an important stimulus for the young discipline of invertebrate neuropeptide research, when the first neuropeptide was identified from whole-body extracts of an insect, the American cockroach, Periplaneta americana (Brown and Starratt, 1975). The peptide was code-named proctolin, and it turned out to be a typical neuromodulator in insects, released in the vicinity of visceral and skeletal muscles (see Orchard et al., 1989). The first peptidergic neurohormones that were chemically fully elucidated from cockroach CC, were members of the adipokinetic hormone (AKH) family (Baumann and Penzlin, 1984; Scarborough et al., 1984; Witten et al., 1984). Despite the fact that AKHs show a certain degree of myoactivity in various muscle assays in cockroaches (Baumann et al., 1990; Keeley et al., 1991), their role in the control of 0022-1910/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 0 ) 0 0 1 2 9 - 3 312 R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 muscle activity is not well established. These metabolic peptides are substances which are synthesized in the glandular lobe of the CC and, therefore, are highly concentrated in these organs. This may explain why AKHs were the first ‘myotropins’ identified from the retrocerebral complex of cockroaches. Structures, functions and modes of action of cockroach AKHs have been reviewed by Gäde (1997). Since 1986, 12 myotropic neuropeptides have been isolated from head extracts of the Madeira cockroach, Leucophaea maderae. Structures, isolation procedures and the hindgut bioassay which was used for the detection of L. maderae myotropins are discussed elsewhere (Cook and Wagner, 1990; Holman et al., 1991a). These peptides included kinins, pyrokinins, sulfakinins and leucomyosuppressin. All the identified peptides were members of novel neuropeptide families which were later also found in other insect orders (see Gäde, 1997). Further neuropeptides of hitherto unknown peptide families were later directly isolated from neurohemal organs of P. americana, namely corazonin and the periviscerokinins (Veenstra, 1989a; Predel et al., 1995). In this overview, we summarize the current knowledge on structures, localization and pharmacology of myostimulatory neuropeptides in cockroaches, which have been favourite experimental objects in neurophysiology/endocrinology for a long time (see Huber et al., 1990). Additionally, we summarize the development of synthetic analogs of cockroach myotropins which are resistant to degradation. Such analogs are developed with the aim to specifically control pest insects in the future. The review deliberately omits work on myoinhibitory peptides which would have necessitated the inclusion of the large group of allatostatins, as well as leucomyosuppressin. These have been reviewed elsewhere (see, for example, Bendena et al., 1997; Gäde, 1997; Weaver et al., 1998). 2. Primary structures and characterization of myostimulatory peptides in cockroaches Seven neuropeptide families with myostimulatory properties are known from cockroaches: proctolin, corazonin, kinins, sulfakinins, pyrokinins, tachykinin-related peptides, and periviscerokinins (Table 1). Cockroach myotropins were mainly isolated from two species, L. maderae and P. americana, which belong to different suborders of the Blattariae, namely the Blaberoidea and Blattoidea. Proctolin and corazonin were first isolated from P. americana, but are widely distributed in other insects also (Orchard et al., 1989; Veenstra, 1991). The half life of corazonin in the hemolymph has not been studied yet, but it is assumed that this undecapeptide is not easily enzymatically cleaved, because it has a blocked N-ter- minus (pyroglutamate) and a C-terminal amidation. To date, it is not clear whether there is a core sequence of corazonin necessary for biological activity. The pentapeptide, proctolin, is the only cockroach myotropin with a non-amidated C-terminus, which results in a fast degradation in the hemolymph (Starratt and Steele, 1984; Quistad et al., 1984). Structure–activity studies on proctolin established that the complete sequence of this peptide is required to exhibit full potency (Starratt and Brown, 1979). To date, a large number of analogs of proctolin have been used to investigate the structural requirements to bind to the proctolin receptor(s). It was shown that the size of the aromatic ring of tyrosine (Sullivan and Newcomb, 1982) but not a free hydroxyl group on this amino acid (Starratt and Brown, 1979) is necessary for full efficacy. In various studies, Konopinska and Rosinski (1999) confirmed the importance of the following for full biological activity of proctolin: (1) the pyrrolidine ring in proline, (2) intact arginine residues and (3) a polar group in the position of the hydroxyl group of tyrosine. Using a proctolin antagonist (cycloproctolin), the myotropic effect of proctolin was reduced by 40%, suggesting the presence of at least two subtypes of proctolin receptors (Gray et al., 1994; Baines et al., 1996). Studies on tissue extracts from locust foregut revealed a stimulation of the inositol phosphate metabolism (Hinton and Osborne, 1995) but, despite the above mentioned results, the nature of the proctolin receptor(s) is still largely unknown. Recently, a proctolin-binding protein was purified from the foregut of the cockroach Blaberus craniifer (Mazzocco and Puiroux, 2000). Sulfakinins occur as two isoforms in all insects studied so far (see Gäde, 1997). They are characterized by the C-terminal hexapeptide Y(SO3H)GHMRFamide which is essential for biological activity (Nachman et al., 1988). In addition, they always have acidic residues prior to the tyrosine residue, which are necessary as recognition site for the tyrosylprotein sulfotransferase (Hortin et al., 1986). These requirements obviously restrict the structural diversity of the sulfakinin family. A number of post-translational modifications such as pyroglutamate formation, sulfation or O-methylation of glutamic acid at the N-terminus have been found for sulfakinins in P. americana (Predel et al., 1999b). Different physiological properties and degradation rates may exist for these forms. It is interesting to note that sulfakinins show a high degree of sequence similarity with the vertebrate gastrins/cholecystokinins. Eight peptides of the kinin family were isolated from L. maderae, as well as from P. americana. Only two of them, Lem-K-7 and Lem-K-8, were found in both species. All cockroach kinins share the same C-terminal pentapeptide sequence, FXSWGamide (X=H,N,S,Y), which was shown to be essential for biological activity, using the cockroach hindgut as an in vitro bioassay system R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 313 Table 1 Primary structures of myostimulatory peptides isolated from cockroaches. Pea: P. americana; Lem: L. maderae; Pef: P. fuliginosa. Peptides marked with an asterisk were also found in P. americana (Predel et al., 1997b, 1999b) Peptide name Sequence Reference Proctolin Corazonin RYLPT–OH pQTFQYSRGWTN–NH2 Brown and Starratt, 1975 Veenstra, 1989a Sulfakinins Lem-SK-1 Lem-SK-2* Pea-SK EQFEDY(SO3)GHMRF–NH2 pQSDDY(SO3)GHMRF–NH2 EQFDDY(SO3)GHMRF–NH2 Nachman et al., 1986a Nachman et al., 1986b Veenstra, 1989b Kinins Lem-K-1 Lem-K-2 Lem-K-3 Lem-K-4 Lem-K-5 Lem-K-6 Lem-K-7* Lem-K-8* Pea-K-1 Pea-K-2 Pea-K-3 Pea-K-4 Pea-K-5 Lom-K* DPAFNSWG–NH2 DPGFSSWG–NH2 DQGFNSWG–NH2 DASFHSWG–NH2 GSGFSSWG–NH2 pQSSFHSWG–NH2 DPAFSSWG–NH2 GADFYSWG–NH2 RPSFNSWG–NH2 DASFSSWG–NH2 DPSFNSWG–NH2 GAQFSSWG–NH2 SPAFNSWG–NH2 AFSSWG–NH2 Holman et al., 1986a Holman et al., 1986a Holman et al., 1986b Holman et al., 1986b Holman et al., 1987a Holman et al., 1987a Holman et al., 1987b Holman et al., 1987b Predel et al., 1997b Predel et al., 1997b Predel et al., 1997b Predel et al., 1997b Predel et al., 1997b Schoofs et al., 1992 Pyrokinins Lem-PK Pea-PK-1 Pea-PK-2 Pea-PK-3 Pea-PK-4 Pef-PK-4 Pea-PK-5 Pea-PK-6 pQTSFTPRL–NH2 HTAGFIPRL–NH2 SPPFAPRL–NH2 LVPFRPRL–NH2 DHLPHDVYSPRL–NH2 DHLSHDVYSPRL–NH2 GGGGSGETSGMWFGPRL–NH2 SESEVPGMWFGPRL–NH2 Holman et al., 1986c Predel et al., 1997a Predel et al., 1997a Predel et al., 1999a Predel et al., 1999a Predel and Eckert, 2000 Predel et al., 1999a Predel and Eckert, 2000 Tachykinins Lem-TRP-1 Lem-TRP-2 Lem-TRP-3 Lem-TRP-4 Lem-TRP-5 Lem-TRP-6 Lem-TRP-7 Lem-TRP-8 Lem-TRP-9 APSGFLGVR–NH2 APEESPKRAPSGFLGVR–NH2 NGERAPGSKKAAPSGFLGTR–NH2 APSGFMGMR–NH2 APAMGFQGVR–NH2 APAAGFFGMR–NH2 VPASGFFGMR–NH2 GPSMGFHGMR–NH2 APSMGFQGMR–NH2 Muren Muren Muren Muren Muren Muren Muren Muren Muren and and and and and and and and and Periviscerokinins Pea-PVK-1 Pea-PVK-2 Lem-PVK-1 Lem-PVK-2 Lem-PVK-3 GASGLIPVMRN–NH2 GSSSGLISMPRV–NH2 GSSGLIPFGRT–NH2 GSSGLISMPRV–NH2 GSSGMIPFPRV–NH2 Predel Predel Predel Predel Predel et et et et et (Nachman and Holman, 1991). A single isoform, LemK-6, contains a blocked N-terminus (pyroglutamate). Six members of the pyrokinin family were reported from the American cockroach. These peptides are named pyrokinins because of the N-terminal pyroglutamate of leucopyrokinin (Lem-PK) which was the first identified member of this peptide family. All other known cockroach pyrokinins, however, do not have blocked N-ter- Nässel, Nässel, Nässel, Nässel, Nässel, Nässel, Nässel, Nässel, Nässel, al., al., al., al., al., 1996a 1996a 1996a 1996a 1996a 1997 1997 1997 1997 1995 1998 2000 2000 2000 mini. Different species of the family Blattidae (P. australasiae, P. fuliginosa, Blatta orientalis, Neostylopyga rhombifolia) express Pea-PK-1 to 5 (with one exception: Pef-PK-4 in P. fuliginosa, see Table 1), whereas a sixth isoform varies more considerably in primary structure between these species (R. Predel, unpublished data). Pyrokinins share a C-terminal pentapeptide (F/YXPRLamide) which is required for myotropic 314 R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 activity (Nachman et al., 1986c). The N-terminus, as well as the amino acid at the X-position, differ remarkably, and this results in very different bioactivities (see later). Tachykinin-related peptides (TRPs) were first isolated in insects from a locust (Schoofs et al., 1990) but later were also found in the brain and midgut of L. maderae. These peptides show some sequence similarity to vertebrate tachykinins. The presumed similarity with mammalian tachykinin receptors was successfully exploited to clone putative TRP receptors from Drosophila (Li et al., 1992; Monnier et al., 1992) and Stomoxys calcitrans (Guerrero, 1997); the endogenous ligand, however, has not been identified yet. Recently, a clone encoding a part of a putative Lem-TRP receptor was isolated from a cDNA library of L. maderae (cited in Nässel, 1999). Antisera raised against a partial sequence of this receptor stained neuropils in the cockroach brain which also showed TRP-like immunoreactivity. The immunostainings, however, did not match completely. This suggests that several types of TRP receptors may exist in L. maderae (Nässel, 1999). The nine TRPs of L. maderae all share the C-terminal sequence GFXGXRamide. Studies with fragments of the related Locusta TK-1 revealed that the C-terminal heptapeptide of this substance is required for full biological activity on the cockroach hindgut (Winther et al., 1998). The periviscerokinin family is presently known only from cockroaches. It is characterized by GXSGLI (with X being A, S or SS) at the N-terminus, whereas the Cterminus is remarkably different; the exception is an arginine residue as the penultimate amino acid residue. This structural feature is unique among families of insect myostimulatory neuropeptides which usually share a common C-terminus. 3. Distribution of myotropins with special emphasis on neurohemal release sites Initially, cockroach myotropins were isolated from whole-body extracts (proctolin) or head extracts (leucokinins, leucopyrokinin, leucosulfakinins). Thereafter, most myotropins from other insects were identified from different parts of the central nervous system such as brain/retrocerebral complex and ventral nerve cord (see Schoofs et al., 1997; Gäde, 1997). It was, therefore, not clear if these peptides were candidates for hormonal regulation of visceral muscle activity. It was only in the American cockroach that neurohemal organs were directly investigated for their inventory of putative myotropic neurohormones. These studies revealed that all myotropins, with the exception of proctolin and the TRPs, are largely concentrated in neurohemal organs of the central nervous system and, thus, most likely play an important role in the hormonal regulation of physiological processes. In this paragraph we summarize the current knowledge about the distribution of myostimulatory neuropeptides in major neurohemal organs, and the cellular origin of these putative hormones (see Fig. 1). The best-known neurohemal release site of insects is the retrocerebral complex which consists of glandular parts (corpora allata, glandular lobes of the corpora cardiaca), as well as neurohemal tissues (storage lobes of the corpora cardiaca). The corpora allata and nerves in the vicinity of the retrocerebral complex are also thought to release, to a certain degree, neurohormones which are produced in the CNS. Kinins, sulfakinins, five of six pyrokinins and corazonin are all concentrated in the corpora cardiaca/allata of P. americana. Immunocytochemical studies revealed the putative sites of synthesis of these substances in the CNS of cockroaches. Kinin-like peptides are produced in neurosecretory cells of the pars intercerebralis and pars lateralis of the protocerebrum (L. maderae) and transported via the nervi corporis cardiaci1 (NCC-1) and NCC-2 to the storage lobes of the corpora cardiaca (Nässel et al., 1992; Meola et al., 1994). No immunoreactivity was detected in the corpora allata. Obviously, all kinins are stored together in the corpora cardiaca, as revealed by a combination of radioimmunoassay and HPLC-analysis (Winther et al., 1996). Sulfakinin-like peptides were found in neurosecretory cells of the pars intercerebralis and transported via the NCC1 to the storage lobe of the CC (Agricola and Bräunig, 1995; East et al., 1997). Antisera raised against corazonin stained neurosecretory cells in the pars lateralis (P. americana) from where immunopositive material can be traced along the NCC-2 to the storage lobes of the corpora cardiaca (Veenstra and Davis, 1993; Predel et al., 1994). Immunostaining was also detectable in the corpora allata. A similar localization of corazonin-like immunoreactive material was found in the brain-retrocerebral complex of L. maderae (Predel et al., 1994). The distribution of pyrokinins in the CNS of P. americana was investigated using a combination of immunocytochemistry, isolation procedures and mass spectrometric analysis of single organs and nerves (Predel and Eckert, 2000). Pea-PK-1 to 4 and Pea-PK-6 are stored in the retrocerebral complex and are produced in cells located in both the suboesophageal ganglion and the tritocerebrum. These peptides reach the corpora cardiaca/allata via the NCC-1, NCC-3, and nervi corporis allati-2. These peptides were also concentrated in the storage lobes of the corpora cardiaca and accumulated particularly in and around the corpora allata. No other known peptides were detectable in such high concentrations in the corpora allata of adult American cockroaches. The abdominal perisympathetic organs (PSOs) of the American cockroach contain a totally different inventory of myotropic neuropeptides compared with that of the retrocerebral complex (see Predel et al., 1999c). Alto- R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 315 Fig. 1. Current knowledge about distribution, and cellular origin of myostimulatory neuropeptides in major neurohemal release sites of cockroaches (for references, see text). gether, only three myostimulatory peptides have been identified from these organs: periviscerokinin-1 and 2, and pyrokinin-5 (Predel et al. 1995, 1998; Predel et al., 1999a). Immunochemical studies confirmed that these myotropic peptides are indeed typical of the neurohemal system in the abdomen: both periviscerokinins and pyrokinin-5 were detected in abdominal PSOs, whereas immunoreactive material was not localized either in the retrocerebral complex or in the thoracic PSOs (Eckert et al., 1999; Wegener et al., 1999). PK-5-like immunoreactive material was co-localized with periviscerokinins in three cell clusters in the midline of each unfused abdominal ganglion (Predel et al., 1999c). The neurons of these cell groups project via the anterior median/transverse nerves into the abdominal PSOs. PeaPVK-1-like immunoreactive material was similarly distributed in six cockroach species belonging to different families (Wegener and Eckert, 1998). Recently, a novel member of the pyrokinin peptide family (Pea-PK-6) was isolated and identified from abdominal PSOs of the American cockroach (Predel and Eckert, 2000). This peptide was also found in the retrocerebral complex where it is stored in high concentrations. Thus, Pea-PK-6 is the first known peptide present in neurohemal organs of the brain, as well as those of the ventral nerve cord. To date, no biological assays have been conducted using Pea-PK-6 and we do not know whether it is indeed myostimulatory. Interestingly, a study investigating HPLC-generated fractions of an extract of 20 thoracic PSOs of P. americana by MALDI-TOF MS, identified masses of 14 substances, none of which was identical to that of a known myostimulatory peptide (Predel, 1999). This suggests that thoracic PSOs contain unique neuropeptides and substantiates the hypothesis of a spatially unique distribution of myotropic neuropeptides in the various neu- 316 R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 rohemal organs of the three body regions (tagmata) of the American cockroach. Immunocytochemical studies with antisera raised against Pea-PVK-1 and Pea-PK-5 confirm this hypothesis (Eckert et al., 1999; Predel and Eckert, 2000). Immunocytochemical studies using antisera against myotropins from the retrocerebral complex always revealed, in addition to the sites of synthesis of putative hormones, a number of immunopositive interneurons, as well as extensive arborizations in the ganglia of the CNS. This is typical of the distribution of neuropeptides in insects (see Homberg, 1994; Nässel 1993, 1996). Their role in the CNS, however, remains unclear. It is also difficult to confirm the chemical nature of immunopositive material in these neurons. In contrast to the pattern in immunostaining described above, antisera raised against myotropins from abdominal PSOs stained neurosecretory cells in the abdominal ganglia which have no interganglionic connections and no extensive arborizations in the abdominal ganglia itself (Eckert et al., 1999; Predel and Eckert, 2000). TRPs were not found to be concentrated in neurohemal organs of the central nervous system, although they are present. TRP-like immunoreactivity (IR) was measured in a ratio 1:25 in the retrocerebral complex and brain of L. maderae, respectively (Muren and Nässel, 1996a). This ratio, in comparison with that estimated for kinin-like IR (ratio of 3:1; Muren et al., 1993), suggests a role in the retrocerebral complex itself rather than as a released hormone of physiological importance. Immunostaining in the corpora cardiaca of L. maderae revealed a few TRP-like immunoreactive processes in the glandular lobes but not in the storage lobes (Muren et al., 1995). In locusts, TRPs were found to be candidates for regulating the release of adipokinetic hormones (Nässel et al., 1999). TRPs are highly abundant in visceral organs, such as the intestine, especially in the midgut (Muren and Nässel, 1996a,b). Some TRPs found in the midgut of L. maderae are different from those isolated from the brain of the same species (Muren and Nässel, 1997). It appears that TRPs are not only expressed in a tissue-specific manner (Muren and Nässel, 1997), but are even differentially distributed in cells of the midgut itself (Winther et al., 1999). As is the case for TRPs, proctolin also does not seem to be highly concentrated in neurohemal organs of the central nervous system of cockroaches. As little as 60 fmol/CC was found for the American cockroach, calculated after a three-step HPLC purification (R. Predel, R. Kellner, G. Gäde, unpublished results). Proctolin is widely distributed in neurons of the central nervous system, in motor neurons, and proctolin-containing fibers extensively innervate visceral and skeletal muscles. The literature on the occurrence of proctolin is reviewed by Orchard et al. (1989) and Gäde (1997). Recently, procto- lin was also identified in antennal pulsatile organs of the American cockroach (Hertel et al., 1997). 4. Muscle-specific efficacy Prior to 1989, all myostimulatory neuropeptides of cockroaches, including kinins, sulfakinins, pyrokinins and proctolin, were identified by monitoring their effects on the frequency and amplitude of the hindgut of L. maderae in vitro (Holman et al., 1991b). Later, the TRPs of this cockroach were also identified using this popular bioassay (Muren and Nässel 1996b, 1997). A superficial screening of data on insect neuropeptides available at that time could easily be intrepreted by suggesting that (1) most insect neuropeptides have myotropic potency and (2) all cockroach myotropins act on the same muscle tissue. This view did not change much during the following couple of years, although a few myostimulatory peptides were discovered which are not active on the hindgut, such as corazonin and the periviscerokinins. To date, there is quite a substantial body of information available suggesting different functions of myotropins. Insect kinins stimulate fluid secretion of isolated Malpighian tubules of different insects, including crickets, mosquitos, locusts and flies (Hayes et al., 1989; Coast et al., 1990; Thompson et al., 1995; O’Donnell et al., 1996). Sulfakinins stimulate the release of the digestive enzyme α-amylase in a beetle (Nachman et al., 1997). TRPs may be necessary to initiate AKH-release from the corpora cardiaca of locusts (see above). Members of FXPRLamides (which include pyrokinins) have been found to regulate diverse processes such as activation of pheromone biosynthesis (Raina, 1993), induction of egg diapause (Imai et al., 1991) and melanization (Matsumotu et al., 1992) in Lepidoptera. Recently, it was reported that [His7]-corazonin is responsible for dark coloration in gregarious locusts (Tawfik et al., 1999). Surprisingly, it is not yet clear whether the above-mentioned functions occur in cockroaches. The field is, therefore, wide open for physiologists. Comparative studies investigating the effects of the myostimulatory peptides on different visceral muscles are rare (Cook et al., 1989; Wagner and Cook, 1993; Predel et al., 1994). To fill this gap, the efficacy of myotropins in assays using visceral muscles of the American cockroach has been investigated, and the data are summarized in Table 2. To date, no TRPs have been identified from P. americana but Lem-TRPs were shown to be active on the isolated foregut of this species (Nässel et al., 1998). In addition, a mammalian tachykinin, substance P, was highly effective in stimulating the activity of the oviduct, but this peptide was not effective on the hyperneural muscle and antennal heart preparations (Penzlin et al., 1989). From these few data it already seems clear that members of the various peptide R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 317 Table 2 Efficacy of myostimulatory peptides on isolated visceral muscle preparations of P. americana (previously unpublished data). Threshold concentrations were calculated from testing five muscle preparations. +++, threshold #1029 M; +, threshold #1027 M; (+), some but not all of the five preparations showed a reaction, at .1027 M; –, no reaction at #1027 M Foregut Hindgut Heart Antennal heart HNM Oviduct Pyrokinins (Pea-PK-1) + +++ +++ +++ +++ +++ Kinins (Lem-K-7) – +++ – – – – Corazonin (+) – +++ +++ +++ – Proctolina +++ +++ + +++ +++ +++ Sulfakinins (Pea-SK) (+) +++ +++ + – – Periviscerokinins (Pea-PVK-2) +++ – +++ +++ +++ – a For further previously published data, see Konopinska and Rosinski (1999). families act in a remarkably muscle-specific manner, indicating a functional diversity even among the visceral muscles. Some of the myotropic properties of neuropeptides shown in Table 2 for P. americana are also known for L. maderae: stimulation of hindgut activity by proctolin, kinins, and sulfakinins, and the effects of leucopyrokinin on a variety of visceral muscles (see Cook and Wagner, 1991). Other peptides, however, act in a rather groupor even species-specific manner. This is true for the potency of TRPs on the cockroach foregut (Nässel et al., 1998) and for corazonin (Predel et al., 1994). Both peptides are very effective in stimulating certain visceral muscles in the American cockroach but failed to activate the homologous muscles in L. maderae. The TRP sensitivity in the foregut of the American cockroach seems to be correlated with positive immunostainings in nerve fibers supplying muscles of the foregut in this species, whereas the foregut of L. maderae is not innervated by TRP-immunoreactive nerve fibers (Nässel et al., 1998). There is no general rule, however, concerning the correlation of positive immunostainings and myotropic potency in visceral muscles of cockroaches, although this phenomenon has not yet been investigated thoroughly. Visceral muscles, which are sensitive to certain peptides, may contain immunopositive nerve fibers and/or endocrine cells indicating the presence of these peptides, as was shown for periviscerokinin-1 (Eckert et al., 1999) and sulfakinins (Agricola and Bräunig, 1995). This is, however, not the case for corazonin (Predel et al., 1994), kinins (Nässel et al., 1992), and Pea-PK-1 to 4 (Predel and Eckert, 2000). Proctolin-immunoreactive fibers always seem to innervate visceral muscles (see Orchard et al., 1989; Hertel et al., 1997). This is consistent with the proposed role of proctolin as a neurotransmitter/neuromodulator rather than a neurohor- mone. Target-specificity of proctolin is likely to be achieved by local release. This peptide was shown to be involved in the antagonistic neuronal control of the hyperneural muscle of P. americana (Penzlin, 1994). A local release of proctolin from transverse nerves 2 or 7 results in a contraction of this muscle, whereas the stimulation of transverse nerves 3–6 induces muscle relaxation, probably via the release of a biogenic amine (octopamine). The occurrence of neuropeptide families with multiple forms, which is typical of insect myotropic peptides, raises the question of different degradation rates or a possible functional diversification by such forms. To date, no convincing data have been presented that different isoforms of any neuropeptide family in cockroaches fulfil different functions. In contrast, multiple members of insect kinins, tachykinins and sulfakinins are very similar in their myotropic potency (Cook et al., 1989; Predel et al., 1997b, 1999b; Muren and Nässel, 1997; Nässel et al., 1998). Pyrokinins of the American cockroach, however, exert dramatically different potencies with regard to various visceral muscles, with threshold concentrations differing by more than three orders of magnitude (Predel and Nachman, 2001). We propose that these peptides have clearly different functions, which is supported by their differential distribution in the CNS and neurohemal organs (Predel and Eckert, 2000). 5. Active conformation and pseudopeptide mimetic analogs While the myostimulatory neuropeptides modulate a number of physiological systems important for normal insect behavior and survival (Gäde, 1997), they are, by design, unsuitable for optimal application as agents for 318 R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 insect management and/or as tools for endocrinologists studying neuropeptide-mediated functions (Nachman et al., 1993a). As molecular messengers, neuropeptides are necessarily subject to degradation by peptidases in the hemolymph and tissues so that the message conveyed can be terminated when it is no longer required. Furthermore, neuropeptides fail to cross the hydrophobic cuticle and cannot, therefore, act efficiently when applied in a topical fashion. The replacement of part of the peptide nature with, and/or addition of, non-peptide components in neuropeptides, as well as incorporation of unnatural residues can alleviate these innate liabilities (Nachman et al., 1993a). In this section, information on the active conformations adopted by myostimulatory neuropeptides at the receptor site is reviewed, and the development of pseudopeptide mimetic analogs with enhanced cuticle penetrating ability and/or resistance to peptidase attacks are described. 5.1. Pyrokinins Analysis of a rigid, constrained analog, as opposed to highly flexible linear forms, was a requirement for a valid assessment of the conformation adopted by the pyrokinins at various receptor sites. A rigid, unnatural cyclic analog of the pyrokinins (cyclo[Asn–Thr–Ser– Phe–Thr–Pro–Arg–Leu]) was synthesized (Nachman et al., 1991), and demonstrated significant activity on the isolated cockroach hindgut and oviduct (Nachman et al., 1997). Through a combination of spectroscopic and computer molecular dynamics techniques, the aqueous solution structure was found to consist of a rigid and prominent transPro type I β-turn encompassing residues Thr–Pro–Arg–Leu within the active core region (Nachman et al., 1991). Knowledge of this conformational preference was used to design a pseudopeptide mimetic analog by incorporating non-peptide replacements for three of the four amino acids. In order to promote a conformational preference for a β-turn-like structure, carbocyclic Pro-mimetic moieties were incorporated into the pseudopeptide analogs. A five membered carbocyclic ring containing two adjacent carboxyl groups was used as a replacement for the Pro and appended onto the N-terminus of the critical C-terminal dipeptide Arg–Leu–NH2. To the second carboxyl group a 4-phenylbutylamino component was appended to introduce the phenyl ring normally present in the first position of the pyrokinin active core. The resulting analog lacks the ring nitrogen of Pro and features a reverse-amide bond, which is not susceptible to peptidase attack. Molecular modelling studies demonstrate that the analog can readily mimic the β-turn structure of the pyrokinin active core region. The mimetic analog retains significant biological activity on the isolated cockroach hindgut (Nachman et al. 1995, 1997). An important step towards the implementation of pseudopeptide mimetic analogs in pest management strategies was the development of analogs of the pyrokinin peptide family capable of transmigration through the insect cuticle. The problem was addressed by development of pseudopeptide analogs with amphiphilic character, i.e. peptides containing both a highly charged, polar species and a highly hydrophobic component. It was reasoned that amphiphilic analogs with surfactant properties would not only penetrate the hydrophobic cuticular layer but maintain the water solubility necessary for them to re-emerge in the insect circulatory system and reach the target receptor site. The pyrokinin C-terminal pentapeptide core already possesses one of the components necessary for amphiphilic character, viz. the highly charged, basic Arg residue. A highly hydrophobic component was introduced via replacement of the phenyl ring of the core N-terminal Phe residue with the cage-like, ball-shaped o-carborane moiety (Nachman et al., 1996), or by addition of organic acids containing aromatic groups to the N-terminus of the pentapeptide core sequence (Abernathy et al., 1996; Teal and Nachman, 1997). The extent of the cuticleretention properties of the amphiphilic analogs was found to be dependent on the size and polarity of the hydrophobic component (Teal and Nachman, 1997; Teal et al., 1999). The amphiphilic pyrokinin analogs were primarily studied with the pheromonotropic assay in Heliothis virescens, where it was demonstrated that a single topical application could induce unnatural production of high pheromone titres for over 20 h (Nachman et al., 1996; Abernathy et al., 1996; Teal and Nachman, 1997). However, the amphiphilic pyrokinin analogs were also capable of penetrating the cuticle of cockroaches (Teal et al., 1999). Experiments with isolated cockroach cuticle, a more dense and sclerotized structure than moth cuticle, demonstrated that these analogs generally show increased cuticle-retention properties in the cockroach. From these experiments it is clear that amphiphilic analogs of insect neuropeptides can be designed to demonstrate topical activity preferentially for one class of insects over another, based on the difference in cuticle structure and chemical make-up. 5.2. Kinins, TRPs, and sulfakinins Kinins adopt a cisPro type VI β-turn encompassing residues Phe–Phe–Pro–Trp of the active core (Nachman et al., 1999). Accordingly, pseudopeptide mimetic analogs with enhanced resistance to peptidases were designed and synthesized (Nachman et al., 1993b; Nachman et al., 1998a). Successful interaction with the insect kinin receptor requires only the C-terminal Trp–Gly– NH2 with an N-terminal acyl extension that incorporates a phenyl ring to represent the Phe core residue. The biological potency can be enhanced markedly by inducing the correct conformational relationship between the Trp– R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 Gly–NH2 and the phenyl ring, such as what occurs when a Pro-mimetic carbocyclic ring is incorporated into this kinin pseudodipeptide framework (Nachman et al., 1993b). The insect kinins are susceptible to degradation by insect angiotensin-converting enzyme (ACE), which targets the active core region via removal of the C-terminal dipeptide fragment. Replacement of the Pro (or Ser) within the active core pentapeptide with a bulky aminoisobutyric acid (Aib) group leads to an analog with complete resistance to housefly ACE and full myotropic potency on the cockroach hindgut (Nachman et al., 1998a). With respect to the TRPs, an ACE-resistant analog of Lem-TRP-1 has been synthesized by replacing the Gly residue, adjacent to the ACE-susceptible peptide bond between Gly and Val, with the Aib residue (pGlu–Ala– Pro–Ser–Gly–Phe–Leu–[Aib]–Val–Arg–NH2) (Nachman et al., 1998b). This analog was not only completely resistant to recombinant fruitfly ACE but also had the same potency as Lem-TRP-1 in inducing contractions of the isolated hindgut of L. maderae (Nachman et al., 1998b). A distinguishing feature of the sulfakinins is the sulfated Tyr residue, a highly unstable component that is readily hydrolyzed under acidic conditions (Nachman et al., 1993b). For the development of stable mimetic sulfakinin analogs with potential pest insect control applications, the acidic, negatively-charged sulfate group was replaced by the acidic, negatively-charged, but more stable, carboxyl group. This was effected in one case by the replacement of the two core residues Tyr(SO3H)– Gly with the aliphatic diacid dodecanedioc acid in the pseudopeptide analog HO2C(CH2)10C(O)–His–Nle– Arg–Phe–NH2 (Nle replaces oxidation-susceptible Met). An even more effective mimic of the Tyr(SO3H) group is the novel residue [Phe(CH2CO2H)], which, when incorporated in the sulfakinin sequence, led to an analog that was active within the physiological range on the isolated cockroach hindgut (Nachman et al., 1993b). The carbon–carbon bonds linking the negatively-charged carboxyl group to the rest of the sequence in these two sulfakinin analogs are not susceptible to hydrolysis. 319 in large amounts in the intestine. Interestingly, the former groups of neuropeptides are stored in similar quantities in the respective neurohemal organs. This has led to the assumption that, in order to regulate the activity of visceral muscles, a finite amount of the various forms of each myostimulatory neuropeptide family has to be released and that this threshold amount has a similar magnitude for the different families. Unfortunately, however, we still do not have sufficiently conclusive information about a true hormonal role for most of these neuropeptides. For example, the following questions are, as yet, unanswered: are the myostimulatory peptides released from the neurohemal areas of cockroaches in vivo and what are the concentrations of these putative hormones in the hemolymph? Data on these topics are only available for kinin-like IR substances in L. maderae (Muren et al., 1993) and FMRF-like IR substances in P. americana (Elia et al., 1995). Another promising area of future research will be investigations into the potency of the various myostimulatory neuropeptides in vivo. This will be no easy task, since difficulties have already been experienced performing pharmacological studies with these peptides on isolated visceral muscles. To date, a number of research groups used different experimental conditions for such assays, that is, non-standard procedures. As depicted in Fig. 2 as an example, an isolated muscle is still quite a 6. Final remarks In the past 15 years, our knowledge on myotropic neuropeptides has increased tremendously, especially with respect to their primary sequences. These various neuropeptides can be grouped into distinct families. Most of the original members of such families were initially isolated from cockroaches and were later also found in other insects. The majority of myostimulatory peptides belonging to the families of corazonin, kinins, sulfakinins, periviscerokinins and pyrokinins occur in neurohemal organs, whereas proctolin and TRPs are found Fig. 2. Efficacy of synthetic proctolin (1028 M) in the antennal heart preparation of P. americana using different flow rates in the perfusion chamber. The same muscle preparation responded with a clear increase in frequency when higher flow rates were tested. The resulting dose– response curve drastically illustrates the influence of operating conditions in a visceral muscle assay. Each point represents the mean±SEM of five preparations. 320 R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 complex system which often reacts with a different response to the same substance when the operating conditions (here: flow rate of perfusion) are changed. Hence, it is (1) necessary to specify the experimental conditions in great detail and (2) it is desirable, for comparative interpretations, to first challenge and monitor the muscle under investigation with a standard dose of proctolin before experiments with other peptides are carried out. Knowledge on the mode of action of myostimulatory peptides of cockroaches are scarce. Wegener and Nässel (2000) investigated the influence of periviscerokinin-2 and proctolin on Ca2+ movements in a tonic cockroach muscle (hyperneural muscle) and found that both peptides induce Ca2+ influx by an activation or modulation of dihydropyridine-sensitive and voltage-independent sarcolemmal Ca2+ channels. Ca2+-induced Ca2+ release appears to be the main mechanism by which both peptides induce contractions of the hyperneural muscle. A major gap in current research on cockroach myostimulatory neuropeptides is definitely the lack of molecular biological data. Intensification of such studies should result in much-needed information regarding the receptors for the different peptide families. In Drosophila, several putative neuropeptide receptors have been cloned after designing oligonucleotide primers based on DNA sequences encoding conserved regions of known mammalian receptors (Li et al., 1992; Monnier et al., 1992; Hauser et al., 1998; Birgül et al., 1999). Data obtained from receptor studies on Drosophila should be used to characterize related receptors in other insects, including cockroaches. Subsequently, it will be possible to identify putative target structures for the different peptide families and to clarify the mode of action of these peptides. A possible detection and pharmacological investigation of multiple receptor subtypes could contribute to a better understanding of the function of multiple peptide forms. Large and well-studied insects, such as cockroaches, locusts and moths, will also be used to study the effects of peptide deficiencies in order to complement Drosophila experiments. In many respects, physiological and behavioral data will be much easier to obtain from an American cockroach or a locust than a fruitfly. At the beginning of the new millennium, most families of myostimulatory neurohormones of cockroaches are structurally known owing to the development of sensitive HPLC and peptide sequencing techniques. The challenge for the coming years will be to establish the neuropeptide pattern/inventory in single neurohemal organs or even neurons during different developmental stages and/or different sexes and/or during different physiological conditions such as movement and reproduction. Recently introduced, sensitive mass spectrometric methods (see, for instance, Worster et al., 1998) allow a fast screening of abundant neuropeptides in single neurohemal organs, nerves and neurons. If such methods are combined with the latest available micro- or nanobore HPLC techniques to separate and quantify myostimulatory neuropeptides, it should be feasible to monitor fluctuations in the rate of their synthesis, storage and/or release. Insect physiologists yearn for such data to gain insights into a coordinated hormonal action of the myostimulatory neuropeptides. Acknowledgements The authors gratefully acknowledge the financial support by National Science Foundation (Pretoria) and the Rotary Club (Rondebosch, Cape Town; GG, RP) and by Deutsche Forschungsgemeinschaft (Predel 595/1-1,2,6), and thank Heather G. Marco for critically correcting the English text. References Abernathy, R.L., Teal, P.E.A., Meredith, J.A., Nachman, R.J., 1996. Induction of moth sex pheromone production by topical application of an amphiphilic pseudopeptide mimic of pheromonotropic neuropeptides. Proceedings of the National Academy of Sciences of the USA 93, 2621–2625. Agricola, H., Bräunig, P., 1995. Comparative aspects of peptidergic signalling pathways in the nervous systems of arthropods. In: Breidbach, O., Kutsch, W. (Eds.), The Nervous System of Invertebrates: An Evolutionary and Comparative Approach. Birkhäuser, Basel, pp. 303–327. Baines, R.A., Walther, C., Hinton, J.M., Osborne, R.H., Konopinska, D., 1996. Selective activity of an proctolin analogue reveals the existence of two receptor subtypes. Journal of Neurophysiology 75, 2647–2650. Baumann, E., Penzlin, H., 1984. Sequence analysis of neurohormone D, a neuropeptide of an insect, Periplaneta americana. Biomedica Biochimica Acta 43, K13–K16. Baumann, E., Gäde, G., Penzlin, H., 1990. Structure–function studies on neurohormone D: activity of naturally-occurring hormone analogues. Journal of Comparative Physiology B 160, 423–429. Bendena, W.G., Donly, B.C., Fuse, M., Lee, E., Lange, A.B., Orchard, I., Tobe, S., 1997. Molecular characterization of the inhibitory myotropic peptide leucomyosuppressin. Peptides 18, 157–163. Birgül, N., Weise, C., Kreienkamp, H.J., Richter, D., 1999. Reverse physiology in Drosophila: identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family. The EMBO Journal 18, 5892–5900. Brown, B.E., 1965. Pharmacologically active constituents of the cockroach corpus cardiacum: resolution of some characteristics. General and Comparative Endocrinology 5, 387–401. Brown, B.E., Starratt, A.N., 1975. Isolation of proctolin, a myotropic peptide, from Periplaneta americana. Journal of Insect Physiology 23, 1879–1881. Cameron, M.L., 1953. Some pharmacologically active substances found in insects. D.Sc. thesis, Cambridge University, Cambridge. Coast, G.M., Holman, G.M., Nachman, R.J., 1990. The diuretic activity of a series of cephalomyotropic neuropeptides, the achetakinins, on isolated Malpighian tubules of the house cricket, Acheta domesticus. Journal of Insect Physiology 36, 481–488. Cook, B.J., Holman, G.M., Wagner, R.M., Nachman, R.J., 1989. Pharmacological actions of a new class of neuropeptides, the leucokin- R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 ins I–IV, on the visceral muscles of Leucophaea maderae. Comparative Biochemistry and Physiology C 93, 257–262. Cook, B.J., Wagner, R.M., 1990. Isolation and characterization of cockroach neuropeptides: the myotropic and hyperglycemic peptides. In: Huber, I., Masler, E.P., Rao, B.R. (Eds.), Cockroaches as Models for Neurobiology: Applications in Biomedical Research, Vol. 2. CRC Press, Boca Raton, FL, pp. 53–84. Cook, B.J., Wagner, R.M., 1991. Myotropic neuropeptides: physiological and pharmacological actions. In: Menn, J.J., Kelly, T.J., Masler, E.P. (Eds.), Insect Neuropeptides. Chemistry, Biology and Action. ACS Symposium Series 453. American Chemical Society, Washington, DC, pp. 51–64. East, P.D., Hales, D.F., Cooper, P.D., 1997. Distribution of sulfakininlike peptides in the central and sympathetic nervous system of the American cockroach, Periplaneta americana (L.) and the field cricket, Teleogryllus commodus (Walker). Tissue and Cell 29, 347–354. Eckert, M., Predel, R., Gundel, M., 1999. Periviscerokinin-like immunoreactivity in the nervous system of the American cockroach. Cell and Tissue Research 295, 159–170. Elia, A.J., Money, T.G.A., Orchard, I., 1995. Flight and running induce elevated levels of the FMRFamide-related peptides in the haemolymph of the cockroach, Periplaneta americana (L.). Journal of Insect Physiology 41, 565–570. Gäde, G., 1997. The explosion of structural information on insect neuropeptides. In: Herz, W., Kirby, G.W., Moore, R.E., Steglich, W., Tamm, C. (Eds.), Progress in the Chemistry of Organic Natural Products, Vol. 71. Springer, Wien, pp. 1–128. Gersch, M., 1972. Experimentelle Untersuchungen zum Freisetzungsmechanismus von Neurohormonen nach elektrischer Reizung der corpora cardiaca von Periplaneta americana in vitro. Journal of Insect Physiology 18, 2425–2439. Gersch, M., 1974. Experimentelle Untersuchungen zur Auschüttung von Neurohormonen aus Ganglien des Bauchmarks von Periplaneta americana nach elektrischer Reizung in vitro. Zoologische Jahrbücher Physiologie 78, 138–149. Gray, A.S., Osborne, R.H., Jewess, P.J., 1994. Pharmacology of proctolin receptors in the isolated foregut of the locust Schistocerca gregaria—identification of [α-methyl-l-tyrosine2]-proctolin as a potent receptor antagonist. Journal of Insect Physiology 40, 595– 600. Guerrero, F.D., 1997. Transcriptional expression of a putative tachykinin-like peptide receptor gene from the stable fly. Peptides 18, 1–5. Hauser, F., Sondergaard, L., Grimmelijkhuijzen, C.J.P., 1998. Molecular cloning, genomic organization and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to gonadotropin-releasing hormone receptors from vertebrates. Biochemical and Biophysical Research Communications 249, 822– 828. Hayes, T.K., Pannabecker, T.L., Hinckley, D.J., Holman, G.M., Nachman, R.J., Petzel, D.H., Beyenbach, K.W., 1989. Leucokinins, a new family of ion transport stimulators and inhibitors in insect Malpighian tubules. Life Science 44, 1259–1266. Hertel, W., 1971. Untersuchungen zur neurohormonalen Steuerung des Herzens der Amerikanischen Schabe Periplaneta americana (L.). Zoologische Jahrbücher Physiologie 76, 152–184. Hertel, W., Rapus, J., Richter, M., Eckert, M., Vettermann, S., Penzlin, H., 1997. The proctolinergic control of the antenna-heart in Periplaneta americana (L.). Zoology (ZACS) 100, 70–79. Hinton, J.H., Osborne, R.H., 1995. Proctolin receptor in the foregut of the locust Schistocerca gregaria is linked to an inositol phosphate second messenger system. Journal of Insect Physiology 41, 1027–1033. Holman, G.M., Marks, E.P., 1974. Synthesis, transport, and release of a neurohormone by cultured neuroendocrine glands from the cockroach, Leucophaea maderae. Journal of Insect Physiology 20, 479–484. 321 Holman, G.M., Cook, B.J., Nachman, R.J., 1986a. Isolation, primary structure and synthesis of two neuropeptides from Leucophaea maderae: members of a new family of cephalomyotropins. Comparative Biochemistry and Physiology C 84, 205–211. Holman, G.M., Cook, B.J., Nachman, R.J., 1986b. Isolation, primary structure and synthesis of two additional neuropeptides from Leucophaea maderae: members of a new family of cephalomyotropins. Comparative Biochemistry and Physiology C 84, 271–276. Holman, G.M., Cook, B.J., Nachman, R.J., 1986c. Primary structure and synthesis of a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea maderae. Comparative Biochemistry and Physiology C 85, 219–224. Holman, G.M., Cook, B.J., Nachman, R.J., 1987a. Isolation, primary structure and synthesis of leucokinins V and VI: myotropic peptides of Leucophaea maderae. Comparative Biochemistry and Physiology C 88, 27–30. Holman, G.M., Cook, B.J., Nachman, R.J., 1987b. Isolation, primary structure and synthesis of leucokinins VII and VIII: the final members of cephalomyotropic peptides isolated from head extracts of Leucophaea maderae. Comparative Biochemistry and Physiology C 88, 31–34. Holman, G.M., Nachman, R.J., Wright, M.S., Schoofs, L., Hayes, T.K., DeLoof, A., 1991a. Insect myotropic peptides: isolation, structural characterization, and biological activities. In: Menn, J.J., Kelly, T.J., Masler, E.P. (Eds.), Insect Neuropeptides. Chemistry, Biology and Action. ACS Symposium Series 453. American Chemistry Society, Washington, DC, pp. 40–50. Holman, G.M., Nachman, R.J., Schoofs, L., Hayes, T.K., Wright, M.S., DeLoof, A., 1991b. The Leucophaea maderae hindgut preparation—a rapid and sensitive bioassay tool for the isolation of insect myotropins of other insect species. Insect Biochemistry 21, 107–112. Homberg, U., 1994. Distribution of neurotransmitters in the insect brain. In: Rathmayer, W. (Ed.), Progress in Zoology, Vol. 40. Gustav Fischer Verlag, Stuttgart/Jena/New York, pp. 1–88. Hortin, G., Folz, R., Gordon, J.I., Strauss, A.W., 1986. Characterization of sites of tyrosin sulfation in proteins and criteria for predicting their occurrence. Biochemical and Biophysical Research Communications 141, 326–333. Huber, J., Masler, E.B., Rao, B.R. (Eds.), 1990. Cockroaches as models for neurobiology: applications in biomedical research, Vol. I/II. CRC Press, Boca Raton, FL. Imai, K., Konno, T., Nakazawa, Y., Komiya, T., Isobe, M., Koga, K., Goto, T., Yaginuma, T., Sakakibara, K., Hasegawa, K., Yamashita, O., 1991. Isolation and structure of diapause hormone of the silkworm, Bombyx mori. Proceedings of the Japanese Academy 67B, 98–101. Kater, S.B., 1968. Cardioaccelerator release in Periplaneta americana (L.). Science 160, 765–766. Keeley, L.L., Hayes, T.K., Bradfield, J.Y., Sowa, S.M., 1991. Physiological actions by hypertrehalosemic hormone and adipokinetic peptides in adult Blaberus discoidalis cockroaches. Insect Biochemistry 21, 121–129. Konopinska, D., Rosinski, G., 1999. Proctolin, an insect neuropeptide. Journal of Peptide Science 5, 533–546. Li, X.J., Wolfgang, W., Wu, Y.N., North, R.A., Forte, M., 1992. Cloning, heterologous expression and developmental regulation of a Drosophila receptor for tachykinin-like peptides. The EMBO Journal 10, 3221–3229. Matsumotu, S., Yamashita, O., Fonagy, A., Kurihara, M., Uchiumi, K., Nagamine, T., Mitsui, T., 1992. Functional diversity of a pheromonotropic neuropeptide: induction of cuticular melanization and embryonic diapause in lepidopteran insects by Pseudaletia pheromonotropin. Journal of Insect Physiology 38, 847–851. Mazzocco, C., Puiroux, J., 2000. Purification of proctolin-binding proteins from the foregut of the insect Blaberus craniifer. European Journal of Biochemistry 267, 2252–2259. 322 R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 Meola, S.M., Clottens, F.L., Coast, G.M., Holman, G.M., 1994. Localization of leucokinin VIII in the cockroach Leucophaea maderae, using an antiserum directed against an achetakinin-I analog. Neurochemical Research 19, 805–814. Monnier, D., Colas, J.F., Rosay, P., Hen, R., Borelli, E., Maroteaux, L., 1992. NKD, a developmentally regulated tachykinin receptor in Drosophila. Journal of Biological Chemistry 267, 1298–1302. Muren, J.E., Lundquist, C.T., Nässel, D.R., 1993. Quantitative determination of myotropic neuropeptide in the nervous system of the cockroach Leucophaea maderae: distribution and release of leucokinins. Journal of Experimental Biology 179, 289–300. Muren, J.E., Lundquist, C.T., Nässel, D.R., 1995. Abundant distribution of locustatachykinin-like peptide in the nervous system and intestine of the cockroach Leucophaea maderae. Philosophical Transactions Royal Society London B 348, 423–444. Muren, J.E., Nässel, D.R., 1996a. Radioimmunoassay determination of tachykinin-related peptide in different portions of the central nervous system and intestine of the cockroach Leucophaea maderae. Brain Research 739, 314–321. Muren, J.E., Nässel, D.R., 1996b. Isolation of five tachykinin-related peptides from the midgut of the cockroach Leucophaea maderae: existence of N-terminally extended isoforms. Regulatory Peptides 65, 185–196. Muren, J.E., Nässel, D.R., 1997. Seven tachykinin-related peptides isolated from the brain of the Madeira cockroach: evidence for tissue specific expression of isoforms. Peptides 18, 7–15. Nachman, R.J., Holman, G.M., Haddon, W.F., Ling, N., 1986a. Leucosulfakinin, a sulfated insect neuropeptide with homology to gastrin and cholecystokinin. Science 234, 71–73. Nachman, R.J., Holman, G.M., Cook, B.J., Haddon, W.F., Ling, N., 1986b. Leucosulfakinin-II, a blocked sulfated insect neuropeptide with homology to cholecystokinin and gastrin. Biochemical and Biophysical Research Communications 140, 357–364. Nachman, R.J., Holman, G.M., Cook, B.J., 1986c. Active fragments and analogs of the insect neuropeptide leucopyrokinin: structurefunction studies. Biochemical and Biophysical Research Communications 137, 936–942. Nachman, R.J., Holman, G.M., Haddon, W.F., 1988. Structural aspects of gastrin/CCK-like insect leucosulfakinins and FMRF-amide. Peptides 9 (suppl. 1), 137–143. Nachman, R.J., Roberts, V.A., Dyson, H.J., Holman, G.M., Tainer, J.A., 1991. Active conformation of an insect neuropeptide family. Proceedings of the National Academy of Sciences USA 88, 4518–4522. Nachman, R.J., Holman, G.M., Haddon, W.F., 1993a. Leads for insect neuropeptide mimetic development. Archives of Insect Biochemistry and Physiology 22, 181–197. Nachman, R.J., Holman, G.M., Hayes, T.K., Beier, R.C., 1993b. Acyl, pseudotetra-, tri-, and dipeptide active-core analogs of insect neuropeptides. International Journal of Peptide and Protein Research 42, 372–377. Nachman, R.J., Roberts, V.A., Holman, G.M., Beier, R.C., 1995. Pseudodipeptide analogs of the pyrokinin/PBAN (FXPRLa) family containing carbocyclic Pro-mimetic conformational components. Regulatory Peptides 57, 359–370. Nachman, R.J., Teal, P.E.A., Radel, P., Holman, G.M., Abernathy, R.L., 1996. Potent pheromonotropic/myotropic activity of a carboranyl pseudotetrapeptide analog of the insect pyrokinin/PBAN neuropeptide family administered via injection and topical application. Peptides 17, 747–752. Nachman, R.J., Giard, W., Favrel, P., Suresh, T., Sreekumar, S., Holman, G.M., 1997. Insect myosuppressins stimulate release of the digestive enzyme α-amylase in two invertebrates: the scallop Pecten maximus and insect Rhynchophorus ferrugineus. In: Strand, F., Beckwith, W., Sandman, C. (Eds.), Neuropeptides in Developing and Aging. Annals of the New York Academy of Sciences 814, 335–338. Nachman, R.J., Holman, G.M., Coast, G.M., 1998a. Mimetic analogues of the myotropic/diuretic insect kinin neuropeptide family. In: Coast, G.M., Webster, S.G. (Eds.), Recent Advances in Arthropod Endocrinology, SEB Seminar Series 65. Cambridge University Press, Cambridge, pp. 379–391. Nachman, R.J., Muren, J.E., Isaac, R.E., Lundquist, C.T., Karlson, A., Nässel, D., 1998b. An isobutyric acid-containing analogue of the cockroach tachykinin-related peptide, LemTRP-1, with potent bioactivity and resistance to an insect angiotensin-converting enzyme. Regulatory Peptides 74, 61–66. Nachman, R.J., Moyna, G., Williams, H.J., Zabrocki, J., Zadina, J.E., Coast, G.M., Vanden Broeck, J., 1999. Comparison of active conformations of the insectatachykinin/tachykinin and insect kinin/Tyr-W-MIF-1 neuropeptide pairs. In: Sandman, C., Strand, F., Beckwith, B., Chronwall, B.M., Flynn, F.W., Nachman, R.J. (Eds.), Annals of the New York Academy of Sciences 897, 388– 400. Nässel, D.R., Cantera, R., Karlsson, A., 1992. Neurons in the cockroach nervous system reacting with antisera to the neuropeptide leucokinin I. Journal of Comparative Neurology 322, 45–67. Nässel, D.R., 1993. Neuropeptides in the insect brain: a review. Cell Tissue Research 273, 1–29. Nässel, D.R., 1996. Neuropeptides, amines and amino acids in an elementary insect ganglion; functional and chemical anatomy of the unfused abdominal ganglion. Progress in Neurobiology 48, 325–420. Nässel, D.R., Vullings, H.G.B., Passier, P.C.C.M., Lundquist, C.T., Schoofs, L., Diederen, J.H.B., Van der Horst, D.J., 1999. Several isoforms of locustatachykinins may be involved in cyclic AMPmediated release of adipokinetic hormones from locust corpora cardiaca. General and Comparative Endocrinology 113, 401–412. Nässel, D.R., Eckert, M., Muren, J.E., Penzlin, H., 1998. Species-specific action and distribution of tachykinin-related peptides in the foregut of the cockroaches Leucophaea maderae and Periplaneta americana. Journal of Experimental Biology 201, 1615–1626. Nässel, D., 1999. Tachykinin-related peptides in invertebrates: a review. Peptides 20, 141–158. O’Donnell, M.J., Dow, J.A.T., Huesman, G.R., Tublitz, N.J., Maddrell, S.H.P., 1996. Separate control of anion and cation transport in Malpighian tubules of Drosophila melanogaster. Journal of Experimental Biology 199, 1163–1175. Orchard, I., Belanger, J.H., Lange, A.B., 1989. Proctolin: a review with emphasis on insects. Journal of Neurobiology 20, 470–496. Penzlin, H., Wieduwilt, I., Hertel, W., 1989. Evidence for a myotropic effect of substance P in Periplaneta americana (L.). General and Comparative Endocrinology 75, 88–95. Penzlin, H., 1994. Antagonistic control of the hyperneural muscle in Periplaneta americana (L.) (Insecta, Blattaria). Journal of Insect Physiology 40, 39–51. Predel, R., Agricola, H., Linde, D., Wollweber, L., Veenstra, J.A., Penzlin, H., 1994. The insect neuropeptide corazonin: physiological and immunocytochemical studies in Blattariae. Zoology (ZACS) 98, 35–49. Predel, R., Linde, D., Rapus, J., Vettermann, S., Penzlin, H., 1995. Periviscerokinin: a novel myotropic neuropeptide from the perisympathetic organs of the American cockroach. Peptides 16, 61–66. Predel, R., Kellner, R., Kaufmann, R., Penzlin, H., Gäde, G., 1997a. Isolation and structural elucidation of two pyrokinins from the retrocerebral complex of the American cockroach. Peptides 18, 473–478. Predel, R., Kellner, R., Rapus, J., Penzlin, H., Gäde, G., 1997b. Isolation and structural elucidation of eight kinins from the retrocerebral complex of the American cockroach, Periplaneta americana. Regulatory Peptides 71, 199–205. Predel, R., Rapus, J., Eckert, M., Holman, G.M., Nachman, R.J., Wang, Y., Penzlin, H., 1998. Isolation of periviscerokinin-2 from R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 the abdominal perisympathetic organs of the American cockroach, Periplaneta americana. Peptides 19, 801–809. Predel, R., 1999. Tagmata-specific expression of neuropeptides in insects. In: Roubos, E.W., Wendelaar Bonga, S.E., Vaudry, H., DeLoof, A. (Eds.), Recent Developments in Comparative Endocrinology and Neurobiology. Shaker Publishers, Maastricht, pp. 283–284. Predel, R., Kellner, R., Nachman, R.J., Holman, G.M., Rapus, J., Gäde, G., 1999a. Differential distribution of pyrokinin-isoforms in cerebral and abdominal neurohemal organs of the American cockroach. Insect Biochemistry and Molecular Biology 29, 139–144. Predel, R., Brandt, W., Kellner, R., Rapus, J., Nachman, R.J., Gäde, G., 1999b. Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid. European Journal of Biochemistry 263, 552–560. Predel, R., Eckert, M., Holman, G.M., 1999c. The unique neuropeptide pattern in abdominal perisympathetic organs of insects. In: Sandman, C., Strand, F., Beckwith, B., Chronwall, B.M., Flynn, F.W., Nachman, R.J. (Eds.), Annals of the New York Academy of Sciences, 897, 282–290. Predel, R., Eckert, M., 2000. Tagma-specific distribution of FXPRLamides in the nervous system of the American cockroach. Journal of Comparative Neurology 419, 352–363. Predel, R., Kellner, R., Baggerman, G., Steinmetzer, T., Schoofs, L., 2000. Identification of novel periviscerokinins from single neurohaemal release sites in insects—MS/MS fragmentation complemented by Edman degradation. European Journal of Biochemistry 267, 3869–3874. Predel, R., Nachman, R.J., 2001. Efficacy of native FXPRLamides (pyrokinins) and synthetic analogs on visceral muscles of the American cockroach. Journal of Insect Physiology, in press. Quistad, G.B., Adams, M.E., Scarborough, R.M., Carney, R.L., Schooley, D.E., 1984. Metabolism of proctolin, a pentapeptide neurotransmitter in insects. Life Sciences 34, 569–576. Raina, A.K., 1993. Neuroendocrine control of sex pheromone biosynthesis in Lepidoptera. Annual Review of Entomology 38, 329–349. Ralph, C.L., 1962. Heart accelerators and decelerators in the nervous system of Periplaneta americana (L.). Journal of Insect Physiology 8, 431–439. Rounds, H.D., Gardner, E.F., 1968. A quantitative comparison of the activity of cardioaccelerator extracts from various portions of the cockroach nerve cord. Journal of Insect Physiology 14, 495–497. Scarborough, R.M., Jamieson, G.C., Kalish, F., Kramer, S.J., McEnroe, G.A., Miller, C.A., Schooley, D.A., 1984. Isolation and primary structure of two peptides with cardioacceleratory and hyperglycemic activity from the corpora cardiaca of Periplaneta americana. Proceedings of the National Academy of Sciences USA 81, 5575–5579. Schoofs, L., Holman, G.M., Hayes, T.K., De Loof, A., 1990. Locustatachykinin I and II, two novel insect neuropeptides with homology to peptides with the vertebrate tachykinin family. FEBS Letters 261, 397–401. Schoofs, L., Veelaert, D., Vanden Broeck, J., De Loof, A., 1997. Peptides in the locusts, Locusta migratoria and Schistocerca gregaria. Peptides 18, 145–156. Smith, N.A., Ralph, C.L., 1967. Some characteristics of heart accelerating substance from the cockroach ventral nerve cord. American Zoology 7, 199. Starratt, A.N., Brown, B.E., 1979. Analogs of the insect myotropic peptide proctolin: synthesis and structure–activity studies. Biochemical and Biophysical Research Communications 90, 461–466. Starratt, A.N., Steele, R.W., 1984. In vivo inactivation of the insect neuropeptide proctolin in Periplaneta americana. Insect Biochemistry 14, 97–102. Sullivan, R.E., Newcomb, R.W., 1982. Structure and function analysis 323 of an arthropod peptide hormone: proctolin and synthetic analogues compared on the cockroach hindgut receptor. Peptides 3, 337–344. 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. Proceedings of the National Academy of Sciences USA 96, 7083–7087. Teal, P.E.A., Nachman, R.J., 1997. Prolonged pheromonotropic activity of pseudopeptide mimics of insect pyrokinin neuropeptides after topical application or injection into a moth. Regulatory Peptides 72, 161–167. Teal, P.E.A., Meredith, J.A., Nachman, R.J., 1999. Comparison of rates of penetration through insect cuticle of amphiphilic analogs of insect pyrokinin neuropeptides. Peptides 20, 63–70. Thompson, K.S.J., Rayne, R.C., Gibbon, C.R., May, S.T., Patel, M., Coast, G.M., Bacon, J.P., 1995. Cellular co-localization of diuretic peptides in locusts: a potent control mechanism. Peptides 16, 95– 104. Unger, H., 1957. Untersuchungen zur neurohormonalen Steuerung der Herztätigkeit bei Schaben. Biologisches Zentralblatt 76, 204–225. Veenstra, J.A., 1989a. Isolation and structure of corazonin, a cardioactive peptide from the American cockroach. FEBS Letters 250, 231–234. Veenstra, J.A., 1989b. Isolation and structure of two gastrin/CCK-like neuropeptides from the American cockroach homologous to the leucosulfakinins. Neuropeptides 14, 145–149. Veenstra, J.A., 1991. Presence of corazonin in three insect species, and isolation and identification of [His7] corazonin from Schistocerca americana. Peptides 12, 1285–1289. Veenstra, J.A., Davis, N.T., 1993. Localization of corazonin in the nervous system of the cockroach Periplaneta americana. Cell and Tissue Research 274, 57–64. Wagner, R.M., Cook, B.J., 1993. Comparative actions of the neuropeptides leucopyrokinin and periplanetin CCI on the visceral muscle systems of the cockroaches Leucophaea maderae and Periplaneta americana. Comparative Biochemistry and Physiology C 106, 679–687. Weaver, R.J., Edwards, J.P., Bendena, W.G., Tobe, S.S., 1998. Structures, functions and occurrences of insect allatostatic peptides. In: Coast, G.M., Webster, S.M. (Eds.), Recent Advances in Arthropod Endocrinology, SEB Seminar Series 65. Cambridge University Press, Cambridge, pp. 3–32. Wegener, C., Eckert, M., 1998. Periviscerokinin-like immunoreactivity in the thoracic and abdominal ganglia of cockroaches (Blattodea). In: Konopinska, D. (Ed.), Insects: Chemical, Physiological and Environmental Aspects. Wroclaw University Press, Wroclaw, pp. 88–92. Wegener, C., Predel, R., Eckert, M., 1999. Quantification of periviscerokinin-1 in the nervous system of the American cockroach, Periplaneta americana: an insect neuropeptide with unusual distribution. Archives of Insect Biochemistry and Physiology 40, 203– 211. Wegener, C., Nässel, D.R., 2000. Peptide-induced Ca2+ movements in a tonic insect muscle: effects of proctolin and periviscerokinin-2. Journal of Neurophysiology 84, 3056–3068. Winther, A.M.E., Lundquist, C.T., Nässel, D.R., 1996. Multiple members of the leucokinin neuropeptide family are present in cerebral and abdominal neurohemal organs in the cockroach Leucophaea maderae. Journal of Neuroendocrinology 8, 785–792. Winther, A.M.E., Muren, J.E., Lundquist, C.T., Osborne, R.H., Nässel, D.R., 1998. Characterization of actions of Leucophaea tachykininrelated peptides (LemTRPs) and proctolin on cockroach hindgut contractions. Peptides 19, 445–458. Winther, A.M.E., Muren, J.E., Ahlborg, N., Nässel, D.R., 1999. Differential distribution of isoforms of Leucophaea tachykinin-related peptides (LemTRPs) in endocrine cells and neuronal processes of 324 R. Predel et al. / Journal of Insect Physiology 47 (2001) 311–324 the cockroach hindgut. Journal of Comparative Neurology 406, 15–28. Witten, J.L., Schaffer, M.H., O’Shea, M., Cook, J.C., Hemling, M.E., Rinehart, K.L., 1984. Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry. Biochemical and Biophysical Research Communications 124, 350–358. Worster, B.M., Yeoman, M.S., Benjamin, P.R., 1998. Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the pattern of peptide expression in single neurons resulting from alternative mRNA splicing of the FMRFamide gene. European Journal of Neuroscience 10, 3498–3507.