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The synthesis and biological activity of a bifunctional, heterodimeric insect neuropeptide analog are described. The heterodimer is composed of the C-terminal pentapeptide active core regions of the leucokinin/achetakinin and pyrokinin neuropeptide families linked via their N-terminal amino groups with a succinyl diacid moiety. Members of the leucokinin/achetakinin family can induce fluid secretion in malpighian tubules of the house cricket, Acheta domesticus, whereas the pyrokinins demonstrate activity in a cricket oviduct myotropic bioassay. No cross-activity is observed for the two neuropeptide families in these bioassays. However, the heterodimer elicits responses in both Acheta bioassays. Such a bifunctional analog may in future serve as a template for the design of stable, bifunctional pest insect control agents of greater efficiency.

This is an investigation of an endocrine cell type in the midgut of the migratory locust Locusta migratoria. This cell type is found in the posterior region of the midgut and is especially common in the ampullae through which Malpighian tubules drain into the gut at the midgut-hindgut junction. Strong Locusta diuretic hormone-like immunoreactivity in these cells was colocalized with FMRFamide- and substance P-like immunoreactivities. At the ultrastructural level,immunoreactivity for Locusta diuretic hormone was found in spherical granules (mean diameter of 450 nm), the contents of which showed variable electron density. Fractionation of a methanolic extract of the ampullae by reversed-phase high performance liquid chromatography revealed the presence of two peaks of Locusta diuretic hormone-like immunoreactive material, both of which stimulate cyclic AMP production by isolated Malpighian tubules. The more hydrophobic material is most likely Locusta diuretic hormone, which has the same retention time when chromatographed under identical conditions.

A radioimmunoassay was developed using 125I-labeled-[TyrO]Locusta-DH and polyclonal antibodies raised against Locusta-DH (29-46). The assay had a detection limit of 50 pM, and displayed limited cross-reactivity for other CRF-related peptides, but not for unrelated peptides. About 60% of the total immunoreactive material in locust hemolymph was attributable to Locusta-DH. The circulating level of diuretic hormone increases fivefold in fed insects, sufficient to stimulate primary urine production, and is correlated with the duration of the meal. This is consistent with the role of Locusta-DH in the control of postfeeding diuresis.

Locusta-DP is a corticotropin-releasing factor (CRF)-related diuretic peptide isolated from the migratory locust Locusta migratoria. At nanomolar concentrations, synthetic Locusta-DP stimulated fluid secretion and cyclic AMP production by Malpighian tubules isolated in vitro and increased the rate of amaranth clearance in starved locusts to levels comparable with those observed during post-feeding diuresis. The peptide also caused a marked (approximately 10 %), but short-lived, reduction in the haemolymph volume of starved locusts. A polyclonal antiserum raised against Locusta-DP(29-46) was shown to block peptidergic signal transfer in vitro and in vivo. Pre-treatment of Locusta-DP (5 nmol l-1) with antiserum diluted 1:100 resulted in a rapid reduction in the free peptide concentration to less than 1 nmol l-1, the threshold for a measurable effect on cyclic AMP production by isolated tubules. In intact insects, passive immunization with Locusta-DP antiserum blocked increases in the rate of amaranth clearance in response to exogenous diuretic peptide or in response to feeding. The latter was due specifically to the binding of Locusta-DP, because when the relevant antibodies were preadsorbed with Locusta-DP(29-46), the antiserum had no effect on amaranth clearance by recently fed insects. This provides unequivocal evidence of a hormonal function for Locusta-DP in the control of primary urine production.

Achetakinin-like immunoreactive material in tissues and haemolymph of adult male crickets was quantified by radioimmunoassay. Achetakinin-like material was found in the brain, suboesophageal ganglia and the thoracic and abdominal ganglia, but the largest amount was within the retrocerebral complex. A Ca2+-dependent release of achetakinin-like immunoreactive material was demonstrated from retrocerebral complexes incubated in vitro in saline containing a high concentration of K+. The concentration of achetakinin-like material in haemolymph from fed crickets was estimated to be 2.8 nmol l−1 and increased more than 10-fold in insects starved for 48 h without access to water. The presence of achetakinin-like material in haemolymph suggests that these peptides are released in vivo and function as circulating neurohormones.

Manduca sexta diuretic hormone (Mas-DH) stimulates fluid secretion by adult Malpighian tubules of M. sexta, demonstrating its site of diuretic action in M. sexta for the first time. It was not possible to develop a suitable bioassay to measure fluid secretion in larval proximal tubules. Mas-DH has an antidiuretic action on the cryptonephric complex of larval M. sexta because it increases fluid absorption from the rectum. It appears that in this complex Mas-DH is acting on a Na+/K+/2Cl− co-transporter, presumably on the basal membrane of the cryptonephric Malpighian tubules, because Mas-DH-stimulated fluid absorption by the cryptonephric complex is inhibited by bumetanide or the removal of Cl-, Na+ or K+ from the haemolymph side of the tissue. This is the first demonstration of hormonal control of fluid absorption by the cryptonephric complex. Concomitant with the stimulation of fluid transport, Mas-DH increases the amount of cyclic AMP secreted by adult Malpighian tubules and the cryptonephric complex. In addition, Mas-DH promotes cyclic AMP production by the larval proximal tubules.

Dose-response curves are presented for the diuretic activity in aqueous extracts of brain, retrocerebral complex, and ventral nerve cord ganglia from Acheta domesticus. Diuretic activity is highest in extracts of brain and corpora cardiaca. In comparison with such extracts, those of the suboesophageal ganglia and thoracic ganglia I-III produce truncated responses, whilst abdominal ganglia 1–4 show evidence of an inhibition of the diuretic response at high doses. ED50 values, obtained from Hill plots, are similar for extracts of brain, corpora cardiaca, corpora allata, and abdominal ganglia, but are 3–4 times higher for extracts of suboesophageal and thoracic ganglia. Separation of aqueous extracts of corpora cardiaca by reversed-phase HPLC yields a number of fractions which stimulate fluid secretion in isolated tubules. Diuretic activity in these fractions is destroyed by treatment with Pronase E, and on this basis is identified as peptidic. In general, diuretic activity is found in the same RP-HPLC fractions prepared from aqueous extracts of brain, suboesophageal ganglia, thoracic ganglia I-III, and abdominal ganglia 1–4.

In insects primary urine is produced by the Malpighian tubules under hormonal control. Here we have analysed the effects of the peptide locustatachykinin I (Lom-TK-I) on secretion in isolated Malphigian tubules. We also mapped the distribution of Lom-TK immunoreactivity in the gut in comparison with Locusta diuretic hormone (Lom-DH) and serotonin, two other factors that are active on locust tubules. Lom-TK-I produces an immediate, potent and long-lasting stimulation of fluid secretion. Furthermore, we show that Lom-TK-I acts synergistically with Lom-DH on fluid secretion and demonstrate that Lom-TKs are co-localised with Lom-DH in endocrine cells of the midgut ampullae. Thus, the two peptides might be released together to act synergistically on fluid secretion. Also serotonin and Lom-DH act synergistically and we can demonstrate a plexus of serotonin-containing axon processes over the midgut.

The distribution of a CRF-related diuretic peptide (Musca-DP) and the diuretic/myotropic insect myokinins in the central nervous system of larval and adult houseflies was investigated using antisera raised against Locusta-DH and leucokinin-I, respectively. Two separate, small populations of immunoreactive neurons are present in the brain and fused thoracic-abdominal ganglion mass. There is no evidence for these immunoreactivities being colocalised either within single neurons or at neurohaemal release sites. Crude extracts of tissues containing immunoreactive material increase fluid secretion by isolated Malpighian tubules from adult flies. Diuretic activity is highest in tissues containing myokinin-immunoreactive material. Consistent with this observation, myokinin analogues produce a four- to five-fold increase in fluid secretion, which is more than twice the response to Musca-DP. These effects are mimicked by treatments that increase intracellular calcium and cyclic AMP, respectively. When tested at threshold concentrations, the two classes of diuretic peptide act synergistically to accelerate tubule secretion, and their separate localisation may be important for the precise control of diuresis.

Locusta -DH is known to have a hormonal function in the control of post-feeding diuresis in the migratory locust. This study has quantified Locusta- DH in tissues from V th instar nymphs and adults, and investigated the K + -induced release of the peptide from corpora cardiaca. Locusta -DH is present in thoracic and abdominal ganglia, but the amounts are small (25–200 fmol) compared with brain (∼1 pmol) and corpora cardiaca (>5 pmol) from 14-day old locusts. About 50% of the immunoreactive material in corpora cardiaca coelutes with Locusta- DH on reversed-phase HPLC. An earlier eluting fraction is also biologically active, suggesting locusts have a second, previously undetected, CRF-related peptide. The amount of peptide stored in corpora cardiaca varies with age and physiological status. Reductions on day 1 of the adult instar and immediately after feeding suggest Locusta- DH controls post-eclosion as well as post-feeding diureses. Locusta -DH is released by a Ca 2+ -dependent mechanism from corpora cardiaca held in salines containing ≥40 mM K + . This is blocked by verapamil, implicating L-type Ca 2+ channels. Release is most rapid shortly after transfer to a high K + saline, and more peptide is released from glands allowed to recover in normal saline between successive K + depolarisations.

Locust abdominal ganglia are shown to colocalize Locusta-diuretic peptide-, leucokinin I-, and lysine vasopressin-like immunoreactivity in posterior lateral neurosecretory cells. Extracts of abdominal ganglia were partially purified by RP-HPLC then dot immunoassay screened with the same antisera used for immunocytochemistry. Locusta-diuretic peptide-like immunoreactive material coeluted with synthetic Locusta-diuretic peptide, and leucokinin-like immunoreactive material coeluted with locustakinin. Lysine vasopressin-like material eluted in fractions that also showed Locusta-diuretic peptide and leucokinin I immunoreactivity. The diuretic activity of synthetic Locusta-diuretic peptide and locustakinin is demonstrated, and they are shown to act at least additively to promote Malpighian tubule fluid secretion. The immunoreactive neurosecretory cells are assumed to express at least these two peptides, and a model for promoting fluid secretion is proposed.

Part I. Moulting, Metamorphosis and Reproduction: 1. Structures, functions and occurence of insect allatostatic peptides R. J. Weaver, J. P. Edwards, W. G. Bendena, and S. S. Tobe 2. Neuropeptides inhibiting growth and reproduction of crustaceans S. G. Webster 3. Molecular, cytological and physiological aspects of the crustacean hyperglycemic hormone family F. Van Herp 4. Endocrine effectors in insect vitellogenesis X. Belles 5. Endocrine regulation of development and reproduction in Acarines L. O. Lomas, and H. H. Rees 6. Ecdysteroid synthesis in the crustacean Y-organ: role of cyclic nucleotides and Ca 2+ D. Sedlmeier, and A. Seinsche 7. Regulation of steroidogenesis: role of transaldolase in crab moulting glands F. Lachaise, and G. Somme Part II. Control of Intermediary Metabolism, Ion and Water Balance: 8. New perspectives on the structures, assays and actions of locust adipokinetic hormones M. J. Lee, and G. J. Goldsworthy 9. Signal transduction of adipokinetic hormone W. J. A. Van Marrewijk, and D. J. Van der Horst 10. The regulation of primary urine production in insects G. M. Coast 11. Locust ion transport peptide (ITP): function, structure, DNA and expression J. E. Phillips, J. Meredith, N. Audsley, M. Ring, A. Macins, H. Brock, D. Theilmann, and D. Littleford Part III. Myotropic and Myoininhibitory Arthropod Neuropeptides: Structures and Functions?: 12. The dipteran Leu-callatostatins: structural and functional diversity in an insect neuroendocrine peptide family H. Duve, A. Thorpe, A. H. Johnsen, J-L. Maestro, A. G. Scott, and P. D. East 13. An insect peptide family in search of functions the tachykinin-related peptides D. R. Nassel, C. T. Lundquist, J. E. Muren, and A. S. A. Winther 14. The distribution, biological activity, and pharmacology of SchistoFLRFamide and related peptides in insects I. Orchard, and A. B. Lange 15. Ontogenetic, phylogenetic and physiological aspects of the conserved crustacean cardioactive peptide (CCAP) neural networks in arthropods H. Dirksen 16. Control of the insect oviduct: the role of the neuropeptide CCAP in the tobacco hornworm, Manduca sexta A. K. Marshall, and S. E. Reynolds Part IV. Peptidases, Peptide and Pseudopeptide Mimetics: Toward New Strategies of Insect Pest Control?: 17. Insect angiotensim-converting enzyme: comparative biochemistry and evolution R. E. Isaaac, D. Coates, T. A. Williams, and L. Schoofs 18. Mimetic analogues of the myotropic diuretic insect kinin neuropeptide family R. J. Nachman, G. M. Holman, and G. M. Coast.

A simple modification of the Ramsay method is described for the isolation in vitro of Malpighian tubules from Acheta domesticus. Isolated tubules continue to secrete for many hours in a simple Ringer solution. The rate of secretion is linearly related to the length of tubule within the bathing fluid. Secretion rates during the immediate post-equilibration period relate to the physiological state of the donor insect. Highest rates are recorded in tubules removed from animals during the scotophase when the crickets are most actively feeding. Starvation reduces the rate of secretion and when previously starved insects are given access to food the rate increases. These results are consistent with variations in the haemolymph titre of DH that are induced by feeding. Tubule fluid secretion is stimulated by either CC extract (1 gland pair/50 μ1) or dibutyryl cAMP (10-3M). CC extracts increase the rate of fluid secretion by c. 200 pl/mm/min for up to 6 h after the tubules have been isolated. The response varies little with the age or physiological state of the donor insect. However, tubules from newly moulted insects secrete at very low rates and respond poorly to CC extract, although they are stimulated by cAMP. The significance of these results is discussed with particular reference to the use of Acheta tubules as a bioassay preparation for DH.

Fluid secretion by single isolated Malpighian tubules of Acheta domesticus(L.) is stimulated by aqueous extracts of nervous and neuroendocrine tissues from the cricket. Diuretic activity, expressed as the increase in rate of secretion per microgram tissue protein (pl/mm/min/ug protein), is highest in the CA and CC. The response to aqueous CC extracts is dose-dependent. The maximum increase in secretion rate is 300–350 pl/mm/min and the EDmax and ED50 are 0.1–0.2 and 0.02–0.03 gland pairs respectively. The diuretic activity in the CC is retained after 5min at 95C, and is freely soluble in 80% methanol. Diuretic activity is, however, greatly reduced after prolonged heating or after treatment with either pronase or chymotrypsin. It is conluded that the diuretic factor(s) is a low molecular weight peptide. A loss of activity after incubation with pyroglutamate amino peptidase suggests that some active peptides present are N-terminally blocked. The diuretic activity of crude aqueous CC extracts is rapidly lost on standing at room temperature. This is partly prevented by precipitation of protein enzymes by either heat treatment or extraction in methanol. Synthetic vertebrate and insect neuropeptides are generally low in activity increasing fluid secretion by no more than 60pl/mm/min. However, extracts of neuroendocrine tissues from a wide range of insect species are potent stimulants of tubule secretion. Various biogenic amines were tested and have little effect on fluid secretion; thus they cannot contribute greatly to the response of cricket tubules to aqueous tissue extracts.