Is the Regulation of Corpora Allata Activity Their Primary Function?*

REVIEW Eur. J. Entomol. 96: 255-266, 1999 ISSN 1210-5759

Allatostatins and allatotropins: Is the regulation of corpora allata activity their primary function?*

K laus H. HOFFMANN, M artina MEYERING-VOS and M atthias W. LORENZ

Tierökologie 1, Universität Bayreuth, D-95440 Bayreuth, Germany; e-mail: [email protected]

Key words. Allatostatin, allatotropin, JH-biosynthesis, myotropic activity, immunocytochemistry, cDNA, prohormone gene sequences, second messenger, protostomes, Insecta, Crustacea

Abstract. More than 60 neuropeptides that inhibit juvenile hormone synthesis by the corpora allata have been isolated from the brains of various insect species. Most of them are characterized by a common C-terminal pentapeptide sequence Y/FXFGL/l/V (alla tostatin A family, allatostatin superfamily). Besides the allatostatin A family, allatostatic neuropeptides belonging to other two pep tide families (W2W9-allatostatins or allatostatin B family; lepidopteran allatostatin) were reported. So far, only one allatotropin has been identified. Here we discuss latest literature on the multiplicity and multifunctionality of the allatoregulating neuropeptides, their physiological significance as well as their evolutionary conservation in structure and function.

INTRODUCTION Since 1989, more than 60 neuropeptides that inhibit JH Development and reproduction of insects are regulated production by the CA in homologous or heterologous bio to a large extent by juvenile hormones (JH) and ecdyster- assays in vitro have been isolated from the brains of a few oids. During the larval stages, these hormones control insect species. Most of them are characterized by a com moulting and metamorphosis whereas in adult insects, mon C-terminal pentapeptide sequence Y/FXFGL/I-NH2 they are involved in the regulation of vitellogenesis in fe (Duve et al., 1997a; Gade et al., 1997; Veenstra et al., males and spermatogenesis and growth of the accessory 1997; Veelaert et al., 1998). The sequences of many more reproductive glands in males (Koeppe et al., 1985; allatostatins of the Y/FXFGL/I-NH 2 family were deduced Nijhout, 1994; Riddiford, 1994; Gade et al., 1997). Juve from prohormone gene sequences (Donly et al., 1993; nile hormones are synthesized and released from the cor Ding et al., 1995; East et al., 1996; Vanden Broeck et al., pora allata (CA) which are present in all insect stages. 1996; Veenstra et al., 1997; Belles et al., 1999). Besides Juvenile hormone III (C16-JH) is the predominant insect the Y/FXFGL/I-NH2 allatostatins (allatostatin A family, JH; the higher homologues (JH 0 ,1, II) are only produced allatostatin superfamily), allatostatic neuropeptides be in Lepidoptera. A new type of JH has been identified in longing to another two peptide families were reported higher dipterans, the JH III bisepoxide (JHB3) (Richard et (Kramer et al., 1991; Lorenz et al., 1995a). So far, only al., 1989). one allatotropin has been identified (Kataoka et al., Haemolymph JH levels may change dramatically over 1989). relatively short periods of time. The finite haemolymph or Allatostatins have been the subject of several reviews in tissue levels of JH are a balance between the rate at which which more extensive bibliographies can be found (Stay hormones are biosynthesized by the CA and the rate at et al., 1994a; Tobe et al., 1995; Bendena et al., 1997; which these molecules are metabolized or excreted. Al Weaver et al., 1998). In the present review we discuss lat though JH-degrading enzymes are known to play a role in est literature on the multiplicity and multifunctionality of determining physiological levels of JH, it is likely that the the allatostatins (and also of the one known allatotropin), overall control of tissue and haemolymph levels is more the physiological significance of such a variety of related intimately linked with changes in the rates of JH biosyn peptides, as well as their evolutionary conservation in thesis (Weaver et al., 1998). structure and function. During the last decade, interest has focused on factors NEUROPEPTIDES OF THE ALLATOSTATIN that regulate JH biosynthesis by the CA (Stay et al., SUPERFAMILY 1994a). Juvenile hormone biosynthesis is regulated by The name allatostatin was originally introduced to de both peptidergic and aminergic inputs (Rachinsky & fine unknown regulatory factors that inhibited JH biosyn Tobe, 1996). Depending upon the species and develop thesis in vitro by the CA of insects (Tobe, 1980). When ment stage, their signals may be either stimulatory or in these structures were isolated at first from brain extracts hibitory (allatotropins and allatostatins), and the signals of the cockroach Diploptera punctata (Woodhead et al., may reach the glands via the haemolymph or via nervous 1989; Pratt et al., 1989) and identified as neuropeptides, connections (Stay et al., 1994a). the term allatostatin was retained. Meanwhile, further

* This paper is based on a lecture presented at the 6th European Congress of Entomology held in České Budějovice, Czech Repub lic, August 1998.

255 members of the allatostatin superfamily have been iso THE ALLATOSTATIN SUPERFAMILY GENES lated and identified from the cockroaches Periplaneta The amino acid sequence of the first allatostatin precur americana (Weaver et al., 1994) andBlattella germanica sor was deduced from a cDNA sequence derived from (Belles et ah, 1994), the cricket Gryllus bimaculatus (Lo mRNA isolated from the brain of virgin female D. punc renz et ah, 1995b), the locust Schistocerca gregaria (Vee- tata (Donly et ah, 1993). Specific primers designed laert et ah, 1996a), the stick insect Carausius morosus against sequences within the coding region of the D. (Lorenz et ah, 1998a), the blowfly Calliphora vomitoria punctata cDNA were subsequently used to amplify allato (Duve et ah, 1993), the mosquito Aedes aegypti (Veenstra statin specific DNA fragments by PCR from both P. et ah, 1997), the lepidopteran species Cydia pomonella, americana genomic DNA and brain-derived cDNA (Ding Helicoverpa armigera (Duve et ah, 1997a) and Manduca et ah, 1995). The pre-proallatostatins of D. punctata and sexta (Davis et ah, 1997), and the honeybee, Apis mellif- P. americana, two distantly related cockroach species, are era (Unnithan et ah, 1995; H. Kaatz, pers. commun.). In similar in sequence, in size and in organization. Each pre S. gregaria, one peptide (Scg-AST 1) ends with Val at the cursor is organized into domains beginning with a hydro- C-terminus and, therefore, the allatostatin superfamily phobic signal sequence domain followed by the allatosta should be named Y/FXFGL/I/V-NH 2 family. In flies, an tin peptides with associated sequence Gly-Lys/Arg-Arg other sub-group of the same peptide family ending C- required for a-amidation and endoproteolytic cleavage, terminally with Met-NLL instead of Leu/Ile/Val-NH2 was and four acidic spacer regions which interrupt the ar found (Duve et ah, 1993, 1994; 1995a). Three of the four rangement of peptides and probably serve to compensate known Met-callatostatins originate through post- the basic charge contribution of the cleavage sites. In ad translational modifications. [Hyp3] Met-callatostatin dition to the already purified peptide sequences, a number (Cav-AST 6) and [Hyp2] Met-callatostatin (Cav-AST 7) of other putative allatostatin-like peptides were discov are hydroxylated analogs of Cav-AST 5; the third one ered by analysing their precursors. The P. americana pre represents a truncated hexapeptide (Cav-AST 8), des Gly- cursor is cleaved into 14 different allatostatins, compared Pro Cav-AST 5. The post-translational modification of to 13 peptides that derived from the D. punctata prolyl hydroxylation may provide the peptides with a precursor. The difference in peptide number is the result higher resistance to enzymatic degradation. When using of a carboxyl-terminal substitution/deletion in the D. the allatostatins in the rapid partition assay for determina punctata counterpart to Pea-AST 12 which has destroyed tion of in vitro JH-release (Feyereisen & Tobe, 1981) in a the amidation signal that is required for biological activity homologous way, only in cockroaches and crickets a (Weaver et al., 1998). dose-dependent, rapid and reversible inhibition of JH- Further gene sequences of allastostatins have been re release was observed (Woodhead et ah, 1989; Pratt et ah, ported from the cockroaches Blattella germanica, Blatta 1991a; Belles et ah, 1994; Weaver et ah, 1994; Wood- orientalis, Supella longipalpa, and Blaberus craniifer head et ah, 1994; Lorenz et ah, 1995b). Structure activity (Bellés et al., 1999), the orthopteran Schistocerca gre studies of the cockroach and cricket allatostatins have garia (Vanden Broeck et al., 1996), the dipterans Calli shown that these peptides are capable of inhibiting JH III- phora vomitoria, Lucilia cuprina (East et al., 1996) and biosynthesis in CA, but with varying degrees of efficacy Aedes aegypti (Veenstra et al., 1997), and the lepidop (Lange et ah, 1995; Weaver et ah, 1998). In the two cock teran Helicoverpa armigera (in Duve et al., 1997a). The roach species, D. punctata and P. americana, there are precursors of all the cockroach species are similar in size, major differences in the sensitivity of the CA to the alla and the organisation of the allatostatins that they contain tostatins at different stages of development and particu (13 or 14, depending on the species) has been conserved. larly during the female reproductive cycle (Pratt et ah, The four acidic domains separate the individual func 1990, 1991b; Stay et ah, 1991; Weaver 1991; Fairbaim & tional peptides into five groups: peptides 1^1; 5-10; 11; Stay, 1995; Weaver et ah, 1995). Unnithan & Feyereisen 12 in the case of P. americana and B. orientalis', 13-14. (1995) demonstrated that the sensitivity to allatostatins The schistostatin precursor differs from that of cockroach can be experimentally manipulated (CA transplantation, pre-proallatostatins in size, in sequence, and in organisa CA denervation, ovariectomy) and suggest the existence tion. It contains 10 peptides that are interrupted only once of humoral factor(s) (from the ovary?) responsible to the by an acidic spacer (schistostatins 1—4 and 5-10). changes of CA sensitivity. Such a difference in sensitivity The octadecapeptide 2 has a rather atypical hydropho of the CA was not observed in G. bimaculatus (M.W. Lo bic nature, a high tyrosine content and a dibasic cleavage renz, unpubh). site Lys9-Arg10 within the internal N-terminal sequence. Neuropeptides of the allatostatin superfamily isolated To date, the cleavage product (carboxyl portion amino from stick insects, locusts, flies, moths and bees inhibit acid residues 11-18) has not been identified in cock JH-biosynthesis by CA of cockroaches or crickets in the roaches but has been found in brain extracts of the locust, heterologous bioassay, but are not at all active when us S. gregaria (Veelaert et al., 1996b). Scg-AST 2 " '18 did ing CA of the donor species. Because of their sequence not inhibit JH biosynthesis by the CA of the cockroach D. homology at the C-terminus they are recognized as prob punctata. able homologues of an allatostatin superfamily and were The prohormones of flies and moths have the same ba referred to as carausiustatins, schistostatins, callatostatins, sic organisation, with the functional peptides arranged in cydiastatins and helicostatins, respectively. tandem blocks separated by acidic spacer regions, but

256 there are substantial changes in peptide number and in that the cricket prohormone contains one peptide in two primary structure between the orders. In the two blowfly copies, AGGRQYGFGL-NH2 at positions 3 and 4. The species C. vomitoria and L. cuprina, the prohormones are ARPYSFGL-NH2 peptide is not present in G. bimacula essentially identical and the peptides are present in two tus whereas the cockroach octadecapeptide 2 is small blocks separated by a short spacer region (Duve et conserved. Again, one peptide ends with Val at the C- al., 1998). If all the potential endoproteolytic processing terminus. sites of the prohormone were cleaved, five different octa- IMMUNOCYTOCHEMICAL DISTRIBUTION OF peptides would be produced, three in the first block and PEPTIDES OF THE ALLATOSTATIN SUPERFAMILY two in the second block. Examination of the precursor se A monoclonal antibody against D. punctata allatostatin quence revealed that two additional peptides, a heneicosa- 7 (Dip-AST 7) has been used to localize allatostatin im- peptide and a hexadecapeptide could be produced if munoreactive cells in nerve fibers in the brain/retrocere- processing at two of the dibasic sites did not proceed to bral complex of the cockroaches D. punctata (Stay et al., completion. The hexadecapeptide has already been identi 1992) andP. americana (Agricola et al., 1992), and the fied in tissue extracts of C. vomitoria (Duve et al., 1993); crickets G. bimaculatus and A. domesticus (Neuháuser et the other is structurally similar to the octadecapeptide 2 in al., 1994; Witek et al., 1999). The results demonstrated cockroaches and locusts. In the prohormone of A. aegypti that allatostatins produced in lateral neurosecretory cells also five peptides are present in two blocks separated by a are delivered to the CA cells through the nervi corporis spacer region, but two in the first block and three in the cardiaci (NCC) II. Four large anterior medial cells in the second block. The differences between the peptides from pars intercerebralis showed strong immunoreactivity but the blowflies and the mosquito are much more pro rather surprisingly, the NCC I from the medium neurose nounced than between the different cockroach species. cretory cells that innervate the corpora cardiaca (CC) From the Aedes pre-proallatostatin cDNA structure, the were lacking immunoreactivity. The distribution of im existence of an octadecapeptide such as isolated from munoreactivity in the CC suggests this neurohaemal or cockroaches and locusts (and which was also found in the gan as a site of release of allatostatins into the honeybee, where Ser is replaced by Thr; in Veenstra et haemolymph. The same antibody has revealed many cells al., 1997) cannot be predicted. Interestingly, however, the in the suboesophageal and abdominal ganglia of the cock Aedes peptide 2 has some structural resemblance to the roaches that are allatostatin immunoreactive. Tissues and cockroach allatostatin 2, amino acid residues 11-18. organs that are innervated by allatostatin-immunoreactive Within the helicostatin gene ( H. armígera) nine peptides neurones include the midgut and hindgut and midgut neu are arranged in two groups of 3 and 6, respectively. The rosecretory cells, the antennal heart, the prothoracic, sali peptide at position 2 represents an octadecapeptide identi vary and male accessory glands and the abdominal cal to that in cockroaches. In extracts of H. armígera perisympathetic organs (see Weaver et al., 1998). InD. there appeared to be almost equal amounts of the long punctata, as in P. americana, the nerves of the antennal (octadecapeptide) and short (amino acid residues 11-18; pulsatile organ and hindgut muscle were immunopositive octapeptide) form of this peptide, whereas in C. pomo- (Woodhead et al., 1992; Lange et al., 1993). Immunocy- nella the octapeptide predominated over the octadecapep tochemistry, and also in situ hybridization, have demon tide in the ratio of 10 : 1 (Duve et al., 1997a). One strated the presence of numerous allatostatin positive peptide, ARPYSFGL-NH2 has been identified in every endocrine cells in the midgut of D. punctata (Reichwald species where the prohormone has been characterized, ex et al., 1994; Yu et al., 1995a). Ude & Agricola (1995) cept in A. aegypti. demonstrated the coexistence of FMRF-amide-like and The high number of homologous peptides within one allatostatin-like immunoreactivity in the lateral heart prohormone and the high variability both in number and nerve of P. americana. Recently, Maestro et al. (1998) structure when comparing species from different orders described the anatomical distribution of allatostatins in suggest that the allatostatin sequences were generated the cockroach B. germánica by using an antiserum raised through a process of internal gene duplication which oc against callatostatin 3 from the blowfly C. vomitoria. Be curred before these species diverged from each other in sides intensive cell and nerve staining in the protocere evolutionary time (Belles et al., 1999), and that the proc brum and tritocerebrum of the brain, stained nerve esses of gene duplication and mutation may have con terminals were shown in the CA. These terminals come ferred functional advantages that were selected for during from perikarya situated in the lateral neurosecretory cells evolution (Duve et al., 1997b). Very recently, we charac in the pars lateralis and in the suboesophageal ganglion. terized the precursor cDNA for allatostatins of the allato Immunoreactive axons from median neurosecretory cells statin superfamily from the cricket G. bimaculatus enter the stomatogastric nerve system and innervate the (Meyering-Vos et al., 1998 and unpublished results). The foregut and midgut. The hindgut is innervated by neu size of the cricket precursor cDNA and arrangement of rones, originating from perikarya that are located in the peptides are very similar to those in cockroaches with a last abdominal ganglion. Endocrine cells of the midgut total number of 14 allatostatins. A single mutation/dele- epithelium were also stained. In the locust species S. gre tion in the coding region between the third and fourth garia and Locusta migratoria strongly immunoreactive acidic spacer seems to have reduced the number of pep cells were stained with a polyclonal antibody against Dip- tides from fifteen to fourteen. Another interesting point is AST 2 in the pars lateralis of the brain with the axons

257 (NCC II and NCA I) extending to and arborizing in the and fiber tracts in the neuroendocrine system of the ear CC and the CA (although neither schistostatins nor cock wig Euborellia annulipes. Earwigs are more closely re roach allatostatins inhibited CA activity, in vitro), lated to orthopterans than to dipterans or lepidopterans. It whereas NCC I and fibers in the NCA II connecting the was surprising, therefore, that in E. annulipes the CC CA to the suboesophageal ganglion remained unstained. cells were not immunoreactive, nor were the neurosecre Also no immunoreactivity was present in the glandular tory endings of fiber tracts from the brain to the CC. Also part of the CC (Veelaert et ah, 1995a). In the brain of S. no immunoreactivity was found in the CA, which agreed gregaria, more than 2,000 neurones exhibit Dip-AST 7- with the fact that the earwig CA could not be inhibited by like immunoreactivity (Vitzthum et ah, 1996). cockroach allatostatins. Strong immunoreactivitiy was Immunocytochemical studies with a range of Leu- found in some cells of the brain as well as in the recurrent callatostatin antisera (Duve & Thorpe, 1994) revealed a and oesophageal nerves. The last abdominal ganglion high number of immunopositive neurones throughout the contained immunoreactive somata and immunoreactive central nervous system, the peripheral nervous system axons of the proctodeal nerve innervated the rectum, ante and in gut endocrine cells of the blowfly C. vomitoria. rior intestine and posterior midgut. No reactive endocrine Leu-callatostatin immunoreactivity was also demon cells in the midgut were detected. Veiy recently, the same strated in varicosities and axons within the cardiac- authors (Rankin et al., 1998b) found at least two peaks of recurrent nerve bundle in the CC (Duve et ah, 1998). In allatostatin-immunoreactivity in brain extracts from adult contrast to the CC, the CA showed no immunoreactivity, female earwigs which had been separated by high per consistent with the lack of effect of these peptides as true formance liquid chromatography (HPLC). The HPLC- allatostatins in flies. Throughout the hindgut, immunore- separated fractions inhibited cockroach, D. punctata, CA active nerve fibers were found in close association with and suppressed earwig hindgut contractions. Although both longitudinal and circular muscle fibers, whereas the synthetic Dip-AST 7 did not inhibit JH synthesis by ear foregut and almost the entire length of the midgut of C. wig CA, a crude brain extract of earwigs suppressed ear vomitoria contained no immunoreactive nerve fibers. wig JH biosynthesis in vitro in glands of low, but not in Positive neurosecretory cells of the peripheral nervous glands of high activity. In another earwig, Labidura ripa system were observed along the length of certain nerve ria, a polyclonal antibody raised against Blg-AST 4 and muscle fibers, notably on the surface of the major (BLAST 3) has been used to immunolocalize allatostatin- nerve trunks to the legs and in association with integu- like peptides in the brain/retrocerebral complex of adult mental muscle. In situ hybridization with a Leu- females (Sayah et al., 1998). Besides strongly stained im callatostatin gene probe (East et al., 1995) has mainly munoreactive cells in the pars intercerebralis and in the confirmed the results of the immunocytochemical studies. pars lateralis, fibres leading to the CA were also stained. Antisera raised against Leu-callatostatins have also The results indicate that in this species allatostatin-like been used to study the tissue distribution of peptides of material is produced periodically in correlation with low the allatostatin superfamily in fourth and fifth instar lar levels of JH and the absence of vitellogenesis. vae of the codling moth C. pomonella (Duve et ah, Allatostatins or allatostatin-like molecules may occur 1997a). Midgut endocrine cells showed immuno also in many non-arthropods which do not have JH or reactivity, as did several paired neurones in the brain and even do not possess a discrete endocrine system. Cock ventral nerve cord. Within the visceral nervous system, roach allatostatin-like immunoreactivity has been demon the frontal ganglion contained positive neurones. Axons strated in ganglia of the freshwater snails Bulinus globo- from these cells project within the nervi cardiostomato- sus and Stagnicola elodes (Rudolph & Stay, 1997), and in gastrici to innervate the foregut. All these observations the nervous systems of Hydra oligactis (Hydrozoa), Mo- led to the term “brain/gut peptides” for members of the al niezia expansa (Cestoda), Schistosoma mansoni (Trema- latostatin superfamily. toda), Artioposthia triangúlala (Turbellaria), Ascaris Davis et al. (1997) used an antiserum against cockroach suum (Nematoda), Lumbricus terrestris (Oligochaeta), Li- allatostatins to map immunoreactive neurones in larvae of max pseudoflavus (Gastropoda), and Elodene cirrhosa M. sexta. In the larval CNS, a high number of immu- (Cephalopoda), but not in the chordate Ciona intestinalis nostained intemeurones was found to form an extensive (Ascidiacea) (Smart et al., 1994). In a range of helminths, neuropil. Immunoreactive neuroendocrine cells in the immunostaining was observed in the nerve cords and an brain, abdominal ganglia, and their respective neurohae- terior ganglia, but also in the subtegumental nerves and mal organs appear to release peptides as neurohormones. around the reproductive apparatus or in neurones in the Interestingly, in these neuroendocrine cells immunoreac pharynx. Immunostaining was also found in fibers inner tivity was found to be co-localized with that for M. sexta vating the anterior sense organs in two nematode species diuretic hormone. Innervation of the CA by allatostatin (Smart et al., 1995). superfamily immunoreactive processes was very sparse. THE ALLATOSTATIN B FAMILY Many thoracic motor neurones were also immuno (W2W9-ALLATOSTATINS) reactive, but this immunoreactivity disappeared early in metamorphosis and did not reappear in the adult. Inhibition of JH biosynthesis by extracts of the brain of Rankin and co-workers (Rankin et al., 1998a) demon the cricket G. bimaculatus has led to the isolation of five strated the presence of allatostatin-immunoreactive cells peptides so far (Lorenz et al., 1995a; Hoffmann &

258 Lorenz, 1997; Lorenz & Hoffmann, 1998), all nonapep- Malpighian tubules (Audsley et al., 1998). Mas-AST tides, which show high sequence similarity to the myoin- failed to inhibit in vitro JH synthesis by CA of P. hibiting peptides isolated from L. migratoria (Lom-MIP) unipuncta sixth-instar larvae or newly emerged adults, (Schoofs et al., 1991) and M. sexta (Blackburn et al., but inhibited JH biosynthesis in 5-day-old adult females. 1995) . These peptides have been designated B- A similar precursor organization and peptide sequence as allatostatins or W2W9-allatostatins in accordance with in the moths has been deduced from a gene sequence iso their structure and biological activity. In general, the B- lated from the dipteran D. melanogaster (Bendena et al., allatostatins caused 50% inhibition of JH synthesis at 1995; Tobeetal., 1995). doses that were up to one order of magnitude higher com MULTIFUNCTIONAL ROLE OF ALLATOSTATINS pared to the A-allatostatins. The two members Grb-AST A5 and B5, however, showed the same high potency in Allatostatins of the Y/FXFGL/I/V-amide type from inhibiting JH biosynthesis in vitro (Lorenz et al., 1999). cockroaches and crickets as well as the Mas-AST were Even after long exposure of the CA to relatively high first discovered because of their inhibiting action on in doses (10 6M) of B-allatostatins the glands recovered sect JH biosynthesis. However, callatostatins or schisto- completely from inhibited rates after the peptide had been statins, for example, do not act upon JH biosynthesis in withdrawn from the incubation medium. No synergistic the blowfly or locust, respectively, but are potent inhibi effect of the allatostatins from the two different peptide tors of either peristaltic movements of the ileum (Duve et families could be observed. The B-allatostatins proved to al., 1998) or of the spontaneous contraction of the lateral be neither sex/stage nor species specific. Although ex oviducts (Schoofs et al., 1997). Duve et al. (1998) dem tracted from brains of adult females, they inhibited CA onstrated that different Leu-callatostatins have potent, im activity in adult males and last instar larvae as well. An mediate and reversible myoinhibiting effects at different allatostatic activity of the B-allatostatins was also shown regions of the hindgut of C. vomitoria. In that bioassay, in the house cricket, Acheta domesticus (Lorenz et al., the two post-translational hydroxylated Met-callatostatins 1995a). Immunohistochemical data (Witek et al., 1999) had an even greater potency than the Met-callatostatin show immunoreactive material in the CC as well as in the Cav-AST 5, and both showed a biphasic response com arborizing nerve fibers within the CA of adult female pared with the monophasic response of the Met- crickets. When Schoofs and co-workers (Schoofs et al., callatostatin (Duve et al., 1995a). Similarly, cockroach al 1996) used an antiserum raised against Lom-MIP to study latostatins have been shown to elicit antimyotropic activ the distribution of immunoreactivity in the nervous sys ity in gut tissues in D. punctata (Lange et al., 1995). tem of adult locusts, they found immunoreactive fibers in When applied simultaneously with proctolin, Dip- the NCC II to the CC, within the glandular part of the CC, allatostatins antagonized the stimulatory effect of procto and also in the NCA I connecting the CC to the CA and lin in a dose-dependent manner. In contrast to the modu arborizing there. The intrinsic cells of the CA, however, lation by allatostatins of spontaneous contraction of hind- were unstained. Chromatographic purification of brain gut, antennal heart muscle did not show any response to extracts of the stick insect, C. morosus, led to the isola application of allatostatin alone in D. punctata or in P. tion of several peptides of the W2W9-allatostatin family americana (Hertel & Penzlin, 1992), although allato which inhibit CA activity in crickets but exert no activity statins may also act on the antennal heart muscle to on Carausius CA (Lorenz et al., 1998a). modulate the action of a myostimulating peptide - most likely proctolin. In the cockroach Leucophaea maderae, THE LEPIDOPTERAN (M SEXTA) ALLATOSTATIN spontaneous contractile activities of the foregut, but not A single non-amidated peptide, pEVRFRQCYFNPISCF the hindgut, are inhibited by callatostatins (Duve et al., -OH (Mas-AST) purified from brains of M. sexta strongly 1995b). Cydiastatins inhibit reversibly all measurable inhibits JH biosynthesis in vitro by CA of fifth instar lar forms of spontaneous contractile activity of the foregut of vae and adult females of the moth (Kramer et al., 1991). the moth, including closure of the oesophageal valve. It also inhibits the CA of adult females of another lepi- Complete myoinhibition was observed at very low pep dopteran species, Heliothis (Helicoverpa) zea, but has no tide concentrations from 10_1°to 10“I6M. effect on the activity of CA from adult females of the bee Experiments with all the 13 or 14 allatostatins of D. tle Tenebrio molitor, the grasshopper Melanoplus san- punctata and P. americana, respectively, indicated that guinipes, or the cockroach P. americana. A search for a their rank order for biological activities, and presumably similar peptide using recombinant DNA methods was the interaction with receptors, depends upon the particular conducted in the true armyworm, Pseudaletia unipuncta function under study. Dip-AST 2, for example, is the (Jansons et al., 1996). The precursor contains a single most potent in inhibiting cockroach CA activity, but is peptide with a sequence identical to Mas-AST. Similar re hardly effective in modulation of myotropic activity sults, using the same probe, have recently been obtained (Lange et al., 1995; Bendena et al., 1997; Weaver et al., in another noctuid, Lacanobia oleracea (Weaver et al., 1998). 1998). The tomato moth,L. oleracea, contains Mas-AST The immunocytochemical data implicate members of in brain extracts and in the haemolymph. Mas-AST-like the allatostatin superfamily in functions as neurotransmit immunoreactivity was also distributed throughout the ters, or neuromodulators within the CNS, and the pres central nervous system and associated with midgut and ence of immunoreactive material as well as their

259 expression in midgut endocrine cells and neurohaemal or treatment. The authors suggest that a factor from the gans suggest a humoral pathway for the action of these thorax/abdomen, which reaches the head mainly through peptides in addition to neural pathways. In last instar lar a nervous pathway, is released during starvation and in vae of D. punctata, the haemolymph concentration of al- hibits CA development. This factor may stimulate allato latostatins was low in the first half of the stadium but statin production or release, or may well be an allatostatin increased dramatically in the latter half, when JH hor itself. mone production becomes undetectable (Yu et al., 1993). Members of the cricket allatostatin B family, in addi In haemolymph of mated females, allatostatin immunore- tion to their potent inhibitory action on JH biosynthesis, active material varied with the reproductive cycle. Low exerted myoinhibitory activity in the L. maderae hindgut during vitellogenesis, the titer sharply increased shortly assay (Hoffmann et al., 1998), and effectively inhibited after ovulation and reached a maximum early in preg ovarian ecdysteroid biosynthesis in G. bimaculatus, thus nancy. To demonstrate that the quantities of allatostatins acting as an ecdysiostatin (Lorenz et al., 1997a). When found in haemolymph may be sufficient to inhibit the CA injected into adult female crickets, effects on ovarian de in vivo, peptides were injected into mated females of D. velopment and haemolymph vitellogenin titer were simi punctata at various intervals after ecdysis. These treat lar to those after injection of Grb-AST AL Haemolymph ments retarded basal oocyte growth and reduced JH bio ecdysteroid titers were also reduced after allatostatin B synthesis (Woodhead et ah, 1993). Injection of Grb-AST treatment. Since cricket B allatostatins belong to the same Al into adult female G. bimaculatus did not affect the ca peptide family as the Lom-MIP, it may be of interest to pacity of the CA to produce JH ex vivo, but lowered body note that Lom-MIP displays an allatostatic activity on CA weight, ovary weight, number of eggs per ovary, and of crickets which is lower than that of the Gryllus B- haemolymph titers of ecdysteroids (Lorenz et ah, 1998b). allatostatins (Lorenz et al., 1995a). Lom-MIP suppresses The haemolymph titers of vitellogenins were almost twice spontaneous contractions of both the hindgut and the ovi as high in the allatostatin-injected females as in the con duct of L. migratoria. trol animals. Injection of a cocktail of schistostatins into For Mas-AST, no other function than the allatostatic the haemolymph of S. gregaria induced the presence of one has been so far presented. However, the widespread an 88 kDa haemolymph protein of yet unknown function distribution of Mas-AST immunoreactivity in L. olerácea (Schoofs et ah, 1997). (Audsley et al., 1998) suggests alternative roles in addi Using a monoclonal antibody against Dip-AST 7 and in tion to the control of JH biosynthesis for this peptide. In situ hybridization with a cRNA probe, it was demon P. unipunctata, the pattern of Mas-AST prohormone gene strated that this allatostatin type is present in and synthe expression does not correlate well with the profile of JH sized by granular haemocytes of D. punctata (Skinner et biosynthesis, suggesting additional or alternative func ah, 1997). Allatostatins produced in haemocytes may tions for this peptide, possibly associated with migratory contribute to one or more of the proposed functions of flight of that species (Jansons et al., 1996). these peptides in cockroaches, i.e. modulation of activity BIOCHEMICAL AND MOLECULAR ACTION OF of CA, muscle and nerves. However, because allatostatins ALLATOSTATINS are rapidly degraded in the haemolymph (Garside et ah, 1997) , it is more likely that allatostatin-containing haemo The inhibition of JH synthesis by peptides of the allato cytes are acting locally, e.g. to regulate specific functions statin superfamily in cockroaches and crickets is their of other haemocytes. only function that has been biochemically characterized In the search for possible new functions of allatostatins, so far. Two approaches can be taken to study the mode of Martin et ah (1996) focused their attention on the fat action of allatostatin on the CA. The binding of peptides body of the German cockroach, B. germanica. The ex to putative receptors and elicitation of second messenger periments showed that Blg-AST 5 (BLAST-2) impaired responses can be followed to the ultimate biochemical tar vitellogenin release in fat bodies incubated in vitro, and gets.) Alternatively, the biochemical steps involved in that this effect appears to be mediated by the inhibition of the de novo synthesis of JH can be analyzed to determine vitellogenin glycosylation. Whereas allatostatin deamida which one(s) are affected by incubation of the CA with tion at the C-terminus abolishes the inhibition of JH pro the allatostatins (Sutherland & Feyereisen, 1996). Earlier duction, non-amidated analogs of the Y/FXFGL/I/V- studies showed that the target for allatostatins occurs -amide type inhibited vitellogenin release in Blattella early in the JH biosynthetic pathway, at least before the (Martin et ah, 1998). The same group (Osorio et ah, conversion of famesoic acid to methyl famesoate (Fey 1998) usedB. germanica as a model to study the effects ereisen et al., 1981). One study by Wang and co-workers of feeding upon CA development and function. During (Wang et al., 1994) claimed to demonstrate that allato each gonadotropic cycle, an increase in food consumption statins inhibit JH biosynthesis as a result of inhibition of is followed by an increase in CA volume, which in turn terminal enzymes (methyltransferase, epoxidase). The precedes JH production. Starved females do not develop same study, however, reported that famesoic acid, a JH the CA and produce very low amounts of JH. However, III precursor, stimulated JH III synthesis both in the pres CA volume in isolated heads from starved and decapi ence and absence of allatostatin. In adult males of G. bi tated females was able to increase to levels similar to fed maculatus, Grb-AST A5 and B5, respectively, decrease controls, but this increase was abolished by allatostatin methyl famesoate accumulation in the CA which have

260 been incubated in medium containing 200 jiM famesol M. sexta (Kataoka et al., 1989). It increases JH synthesis (Lorenz et al., 1999). Sutherland & Feyereisen (1996) in adult animals but did not affect CA activity of larvae or proposed from their experiments on the cockroach D. pupae. Furthermore, CA from the beetle T. molitor, the punctata that inhibition of JH III biosynthesis by Dip- locust S. gregaria, and the cockroach P. americana were AST 7 occurs at the first committed step(s) of JH synthe not activated by the synthetic allatotropin, whereas the sis, i.e. the transfer of C 2-units from mitochondria to the CA of the noctuid moths, H. virescens (Kataoka et al., cytoplasm by the tricarboxylate carrier and/or the ATP- 1989),L. oleracea (Audsley et al., 1999) andSpodoptera citrate lyase. frugiperda (U. Oeh and K. H. Hoffmann, unpubl.) were Receptors for allatostatins have been identified and par stimulated, suggesting order specificity. Studies on NH2- tially characterized from both brain/retrocerebral com terminal truncated sequences of the synthetic peptide sug plexes (Yu et al., 1995b) and gut membrane preparations gested that the amino acids 6-13 are the biologically (Tobe et al., 1998). The presence of multiple allatostatin active core. In addition to its effect in stimulating JH pro species might suggest a priori the existence of individual duction, Mas-AT is a potent cardioaccelerating peptide in receptors for each substance. It remains to be determined, pharate adult M. sexta (Veenstra et al., 1994). however, whether each allatostatin species is associated The effect of Mas-AT upon JH biosynthesis in the adult with a different receptor/receptor subtype. Pratt et al. moth is presumably exerted via second messengers such (1997) proposed the existence of at least two receptor as inositol phosphate (IP 3) and Ca2+ (Reagan et al., 1992). types in the CA of the cockroach D. punctata and called An enzyme immunoassay for Mas-AT was used to them a and p receptor types. The a-type receptor is char study the distribution of allatotropin within the neurones acterized by a high affinity interaction with “short” allas- and endocrine system of M. sexta (Veenstra & Hagedom, tostatins, whereas the P-type is selective for peptides such 1993). The peptide is present in the retrocerebral as the Dip-AST 2 (18mer), with a lower affinity, and rec complex, the brain, and the ventral nerve cord of the ognizes the larger N-terminal address sequence. Penta- moths. A Mas-AT-immunoreactive peptide was also peptides of the YXFGL-amide structure were the shortest found in the nervous system of P. americana suggesting fully active allatostatins. Two such peptides (YAFGL- that related peptides are most generally present in insects. NH2, YSFGL-NH2) were recently identified in the shore The function of the peptide in P. americana remains to be crab Carcinus maenas (Duve et al., 1997b). Further struc elucidated. From the locust L. migratoria, the [Ala6, Leu7, ture activity studies have shown that unamidated peptides Ser8]-homologue (Lom-AG-MT 1) of Mas-AT was iso are inactive, and that the Phe at the C-terminus, that lated from male accessory glands and showed a myo- seems to be conserved in all members of the allatostatin tropic effect on the oviduct of the same species (Paemen superfamily, is essential for receptor binding. The C- et al., 1991). terminal structural elements may be oriented in three- Lee et al. (1998) have shown that Mas-AT may exert a dimensional space by a conformation on the receptor like biological role in larval insects that differs from its role in a type II p-tum centered around the Phe-Gly position the adult. The peptide rapidly inhibits in vitro active ion (Hayes et al., 1994; Nachman et al., 1998). transport across the midgut epithelium of day 2 (feeding) Piulachs et al. (1997) have replaced the Leu3-Tyr4 pep fifth-instar tobacco homworms. The inhibition was elimi tide bond of Blg-AST 2 with the methyleneamino nated by removing the peptide. Midguts dissected from HCH2NH] and ketomethylene v|/[COCH2] surrogates, re pharate fifth instars or wandering fifth instars were not af spectively, with the aim of increasing the resistance to en fected by the peptide. Recent studies by Bhatt & Horo- zymatic degradation. These analogs were less active than dyski (1999) on the expression of the M. sexta allato the prototype peptide when tested for inhibition of JH tropin gene in cells of the central and enteric nervous sys synthesis in vitro by CA from B. germanica, but more ac tems substantiate and strengthen the hypothesis that tive when tested in vivo as an inhibitor of JH synthesis Mas-AT exhibits multiple diverse functions, some of and vitellogenin production. which may be specific to a particular life stage. The array of allatostatins and the likely occurrence of A stimulatory mode of control of JH synthesis has also multiple receptor subtypes raises the possibility of multi been observed, for example, in larvae of the wax moth ple signal transduction mechanisms for these peptides. Galleria mellonella (Bogus & Scheller, 1996), in adult lo Signal transduction within the CA occurs by way of custs (L. migratoria', Lehmberg et al., 1992 and S. gre known second messengers, including cyclic nucleotides, garia', Veelaert et al., 1995b) and crickets (G. bimacula- Ca2+, and the phosphoinositides (Rachinsky & Tobe, tus\ Lorenz & Hoffmann, 1995), and in adult females of 1996). Differences in signal transduction between species the linden bug Pyrrhocoris apterus (Hodkova et al., (locust, cockroach, moth) may reflect differences in the 1996). So far, however, none of these allatotropic factors peptide signals that regulate JH biosynthesis and in the in has been identified. trinsic state of the CA (Rachinsky & Tobe, 1996). CONCLUSIONS ALLATOTROPINS More than 60 so-called “allatostatins” belonging to To date, only one allatotropin (Mas-AT; GFKNVQMM three peptide families have been isolated and character TARGF-NH2) has been isolated and characterized. This ized in the last decade from a variety of insect species: the allatotropin was from heads of pharate adults of the moth Y/FXFGL/I/V-NH2 allatostatin superfamily, the W2W9-

261 allatostatin (B) family, and the M. sexta allatostatin. cockroach CA in vitro. In last instar larvae of the blowfly Members of the allatostatin superfamily inhibited JH bio L. cuprina, synthesis of JHB 3 also seems to be controlled synthesis in CA from cockroaches and crickets. The alla- by at least three distinct mechanisms (Sutherland & East, tostatic activity of the B-allatostatins seems to be 1997). In the second step, a putative brain allatostatin of restricted to crickets (Lorenz et al., 1997b). Within the yet unknown structure may act directly on the CA. Lepidoptera, questions remain about the universality of All these results demonstrate that many more in vivo allatostatic function of Mas-AST throughout this order and in situ studies will be necessary to unequivocally (Weaver et ah, 1998). The occurrence of members of the clarify roles and functions of the known “allatostatins” three allatostatin families in insect species other than and “allatotropins” in a wide range of invertebrates. Be those where they are effective upon JH biosynthesis, in yond that, there is no doubt that novel (primary?) allato conjunction with the reports on their neuro- and myo- statins and allatotropins will be detected in JH producing modulatory activities, strongly suggest that inhibition of arthropods. JH biosynthesis by CA may represent an altered or secon ACKNOWLEDGEMENTS. The authors are very grateful to the dary role for these neuropeptides which is present in only Deutsche Forschungsgemeinschaft (Ho 631/15-2) and the a few insect species. DLR/BMBF (TSR-072-97) for financial support of their re This suggestion is supported by the isolation of 20 neu search projects. 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