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When injected into adult or nymphal Locusta that have been deprived of food for 2 h, immunogens such as laminarin and bacterial LPS can induce an almost immediate dose-dependent state of anorexia for at least 1 h. Such anorexia is a component of a medley of physiological and behavioural changes called collectively ‘sickness behaviour’ that occurs in a wide range of animals in response to infection or immune challenge. Sub-optimal amounts of injected laminarin allow some locusts to feed, but with a longer latency than in controls, although the length of the first meal is unaffected. The feeding behaviour of fifth instar nymphs is more sensitive to laminarin than that of adults, but both stages respond to amounts of immunogen that are lower than those required to activate the phenoloxidase cascade. Injection of adipokinetic hormone (AKH) before the period of food deprivation prevents the anorexigenic action of the laminarin in adults but not in nymphs. It is argued that the effect of the AKH may be indirect, through its lipid-mobilising action. The insecticide pymetrozine increases the latency to feed but also reduces the length of the first meal, and its anorexigenic activity is not affected by injection of AKH. The present data support the concept that laminarin-induced anorexia involves a central lack of motivation to eat, rather than a ‘stop eating’ signal. Others have shown that the mechanism of action of pymetrozine involves the serotonergic system and can be blocked by mianserin, so it is intriguing that in the present study injection of mianserin prior to that of laminarin modulates the anorexigenic effect of the immunogen. This suggests that biogenic amines are involved in the control of appetitive behaviour in locusts, as they are in vertebrates. The possible usefulness of the locust model in studying sickness-induced anorexia is discussed briefly.

A key step in Escherichia coli K1 meningitis is the crossing of the blood-brain barrier by the bacteria in order to gain entry into the central nervous system (CNS). In this study, a novel ex vivo model to study E. coli K1 invasion of the CNS is described that uses the African migratory locust, Locusta migratoria. By injecting bacteria into isolated locust head capsules, it was demonstrated that E. coli K1 invade the locust brain within 2 h in numbers depending on the concentration of bacteria injected. Using several mutants derived from K1, it was shown that outer-membrane protein A is a critical bacterial determinant required for the E. coli K1 invasion. The isogenic gene-deletion mutants, DeltafimH, Deltacnf1, DeltaneuDB and a rough LPS mutant showed significantly reduced invasion of locust brain. This novel model for the study of E. coli K1 pathogenesis offers several advantages over existing mammalian models in relation to its relative ease of use, cost-effectiveness and ethical acceptability.

It is shown here for the first time that locusts can be used as a model to study Escherichia coli K1 pathogenesis. E. coli K-12 strain HB101 has very low pathogenicity to locusts and does not invade the locust brain, whereas the injection of 2 × 10 6 E. coli K1 strain RS218 (O18:K1:H7) kills almost 100% of locusts within 72 h and invades the brain within 24 h of injection. Both mortality and invasion of the brain in locusts after injection of E. coli K1 require at least two of the known virulence determinants shown for mammals. Thus, deletion mutants that lack outer membrane protein A or cytotoxic necrotizing factor 1 have reduced abilities to kill locusts and to invade the locust brain compared to the parent E. coli K1. Interestingly, deletion mutants lacking FimH or the NeuDB gene cluster are still able to cause high mortality. It is argued that the likely existence of additional virulence determinants can be investigated in vivo by using this insect system.

Injections of Bacillus, or of blastospores from the entomopathogenic fungus, Metarhizium anisopliae, activate the prophenoloxidase (PPO) cascade, and coinjection of adipokinetic hormone-I (AKH) enhances and prolongs these responses. When injected concurrently with an immunizing dose of live bacteria, AKH suppresses the appearance of antimicrobial activity and, after a short delay, increases the growth of bacteria within the hemocoel. Injections of live Escherichia coli or Pseudomonas aeruginosa into locusts fail to activate PPO in the hemolymph, even when coinjected with AKH. The coinjection of bacteria and hormone is rarely lethal to the locust. However, if locusts are injected with AKH when they are infected with Metarhizium, they die more rapidly than if no AKH is administered.

Our knowledge on primary structure, synthesis, release, receptor binding, structure-activity relationships, mode of action and degradation of, mainly, neuropeptides from insects has increased dramatically during the last 10 years or so. Here, five case studies are presented, which deal selectively with effects on: reproduction (trypsin modulating oostatic factor in mosquito); energy metabolism, locomotion and the immune system (adipokinetic hormones); water and ion balance, and feeding behaviour (diuretic hormones, kinins, sulfakinins); sex attraction (pheromone biosynthesis activating neuropeptide); and growth and development, and muscle activity (allatostatins). The literature is reviewed in the context of how the knowledge on neuropeptides has been and can be used for the design of novel, safe and selective compounds to control pest insects in the foreseeable future.

The potencies of natural adipokinetic hormones and synthetic variants have been determined in Locusta migratoria using the lipid mobilisation assay in vivo, and/or the acetate uptake assay in vitro. These data are combinations of previously published and unpublished data (a total of sixty-nine analogues), and form data sets for the construction of mathematical models of the hormone potencies. The sequence variations of amino acids in both natural and artificial adipokinetic hormone analogues were described using continuous descriptor scales z1′, z2′, and z3′, each previously published scale being derived from various properties of the amino acids. By means of these z′-scales and partial least squares regression we attempted to model the potencies in Locusta migratoria of adipokinetic hormones in the two assays. Correlations (r2 values) between predicted and actual potencies of the different peptides were up to 0.73. We discuss the potential of the partial least squares method for formulating quantitative relationships between different hormone structures and their potencies, and describe how the procedure might be used in structure–activity prediction with the construction of an optimised peptide data set.

In vertebrates, the peptides cholecystokinin (CCK), neuropeptide Y, galanin, and bombesin are known to be involved in the control of food intake. We report here that insect sulfakinins, peptides which display substantial sequence similarities with the vertebrate gastrin/CCK peptide family, significantly inhibit food uptake in fifth instar nymphs of the locust, Schistocerca gregaria. Upon injection of Lom-sulfakinin, a neuropeptide present in the corpus cardiacum of locusts, food intake was significantly reduced in a dose-dependent manner within a fixed 20 min time period. The induced effect ranged from 13% inhibition (10 pmol of injected peptide) to over 50% inhibition at 1 nmol. Other naturally occurring sulfakinins from different insect species also elicited this satiety effect. Analogous to the satiety effect of CCK in vertebrates, the sulfate group is required for activity. No effect on the palptip resistance was found after injection with sulfakinin. Therefore it seems unlikly that sulfakinins reduce food intake by decreasing the sensitivity of the taste receptors.

Several structural characteristics in the molecule of the locust adipokinetic hormone, AKH-I, have been investigated in terms of their importance in determining biologic activity. All modifications tested in this study resulted in analogues with decreased potency in comparison with the parent molecule. However, all analogues that were found to be active gave a full response, although often only at very high doses of peptide. This study has highlighted for the locust receptor(s) the vital role of the side chain of Thr(5), and the importance of positions 4 and 8. For example, when Trp(8) and Phe(4) were exchanged, the resulting analogue (Trp(4),Phe(8)-AKH-I) was one of the least active analogues tested in this study. Although Trp is tolerated quite well as a substitute for Phe(4), with only a 10-fold loss of potency, Phe is not favored as a substitute for Trp(8) (>300 times decrease in potency). On the other hand, 3-[2-napthyl] alanine (Nal) is a better substitute for Trp(8) (only a 100-fold loss in potency). We conclude that position 4 requires a phenyl ring in the side chain, and position 8 an indole ring.

Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) stimulates lipid synthesis in locust fat body in vitro, and is able to reverse the inhibitory effects of AKH-I on lipid synthesis. Effective stimulatory concentrations of TLCK were in the range of 0.2–1.0 mM. Similar stimulatory effects were also achieved with phenylalanine chloromethyl ketone (PheCK) and leucine chloromethyl ketone (LeuCK), but not with tosyl-phenylalanine chloromethyl ketone (TPCK), dansyl-glu-gly-arg-CK, chloroacetone, chloroacetic acid, chloroacetamide, chloroacetaldehyde, chloroacetyl-L-leucine or acetylated or fluorescamine-labelled TLCK, PheCK, and LeuCK. The level of stimulation caused by TLCK was dependent on incubation time, so that after a 5-h preincubation of fat body tissue with TLCK the stimulated rate was severalfold higher than the control. TLCK also increased the rate of uptake of trehalose and uridine, but not glucose, deoxyglucose or glycine. Increasing concentrations of bovine serum albumin (BSA) in the incubation medium caused a reduction in the rate of TLCK-stimulated acetate uptake, such that levels of uptake were no higher with 1% BSA than in the controls. A range of more specific protease and kinase inhibitors was tested, but none caused stimulation; thus the mode of action of TLCK on the stimulation of acetate uptake has yet to be identified. Elucidation of the mode of action of TLCK may facilitate the development of novel compounds for insect pest control. Arch. Insect Biochem. Physiol. 39:9–17, 1998. © 1998 Wiley-Liss, Inc.

The secondary structure of six members of the AKH/RPCH family of arthropod neuropeptides has been studied by circular dichroism spectroscopy. None of the peptides examined shows a clear ordered conformation in aqueous solution, pH 7.5 at room temperature. At low temperatures in ethanediol/aqueous buffer (2:1, pH 7.5), however, a P II extended conformation becomes apparent in all of the peptides tested. For the peptides that are most active in the lipid mobilization assay, interaction with SDS micelles induces the formation of a β -structure.

To investigate the receptor tolerances to N-terminal variation, novel analogues to Locusta AKH-I (adipokinetic hormone) have been synthesized with modifications at the N-terminus. Analogues were made where the N-terminal pyroglutamyl residue was spaced further from the remainder of the molecule by the insertion of glycine residues between either pGlu 1 and Leu 2 (Gly 1a –AKH-I) or Leu 2 and Asn 3 (Gly 2a –AKH-I and Gly 2ab –AKH-I). Other modified hormones with N-terminal extensions were: (Ahx) n –AKH-I (Ahx, aminohexanoic acid); HPP(Ahx) n –AKH-I (HPP, hydroxyphenyl propionate) and Ac(Ahx) n –AKH-I (where n =0–3). Finally, acetylated and non-acetylated amino acids were substituted for pGlu 1 : Glu, Pro, Ala and Tyr. The effects of these modifications on biological potency were tested in the lipid mobilization assay in vivo and acetate uptake assay in vitro . The potency of AKH-I was reduced much more by insertion of glycine between pGlu 1 and Leu 2 , than between Leu 2 and Asn 3 , perhaps suggesting that a hydrophobic residue is required adjacent to the pGlu for biological activity. In addition, a residue N-terminal to Leu 2 is necessary for activity (i.e., [despGlu]–AKH-I is inactive) unless the free N-terminus is acetylated: Ac[despGlu]–AKH-I is active, but has low potency. The potencies of HPP(Ahx) 0–3 –AKH-I, Ac(Ahx) 1–3 –AKH-I and glycine-inserted analogues decreased consistently with increasing extension of the N-terminus away from the remainder of the molecule. However, potencies of the unblocked (Ahx) n –AKH-I analogues did not, and potency in either assay did not appear related to the number of aminohexanoic residues. Similarly, while hormonal activity was retained by substitution of pGlu 1 by Tyr, Pro, Ala or Glu in both assays, acetylation of the resulting analogues did not provide a consistent increase in potency, but actually decreased for AcGlu 1 –AKH-I compared with its unblocked analogue. HPP 1 –AKH-I was the most potent of the modified peptides tested, with almost the same potency in the assay in vitro as the natural peptide.

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.

A series of Locusta adipokinetic hormone I (AKH-I),QLNFTPNWGTa, analogues, were synthesized with modifications at the C-terminal threonine residue using a combination of solid- and liquid-phase methodology and evaluated in Locusta migratoria, in a lipid mobilization assay in vivo and an acetate uptake assay in vitro. Modifications at Thr10 of AKH-I involved replacement of its C-terminal amide by the groups -OH, -OCH3, -NHCH3, -N(CH3)2, and -NHC6H5; the last three groups were also applied to the amide of AKH-I-[Thr(Bzl)10]. The methyl ester, monomethyl, and dimethyl analogues were all of lower activity than the parent in the lipid mobilization assay, but lost less than two orders of potency. In the acetate uptake assay, again the methyl ester analogue showed the greatest retention of biological activity of all modified peptides. A cyclic analogue, cyclo (PLNFTPNWGT), was active in both assays, but only at very high concentrations. Almost all analogues were more active in the acetate uptake assay than in the lipid assay, but unusually, AKH-I-NHCH, and AKH-I-N(CH3)2, together with cyclo(PLNFTPNWGT), were more active in the lipid mobilization assay. In addition, the acid AKH-I analogue did not suffer as large a loss in potency in the lipid mobilization assay as in the acetate uptake assay, although it was less potent in the former. The relative potencies of these two methyl analogues contrast with those for AKH-I[Thr(Bzl)10]-NHCH3 and AKH-I-[Thr(Bzl)10]-N(CH3)2, which, together with both phenyl analogues, were significantly more active in the acetate uptake assay. We conclude that the acetate uptake assay has a greater preference for a hydrophobic C-terminus, compared with the lipid mobilization assay.

Extracts of brains and corpora cardiaca from adult Locusta migratoria inhibit total RNA synthesis in vitro in the fat body of Locusta migratoria. Adipokinetic hormones (AKHs) appear to be responsible for the inhibitory activity in extracts of corpora cardiaca, but these peptides are not the active factor(s) in the brain. Locusta adipokinetic hormones-I, -II and -III (AKH-I, AKH-II and AKH-III) inhibit RNA synthesis in vitro in both females and in males. In males, the responses are dose-dependent with their potencies decreasing in the order: AKH-III > AKH-II > AKH-I. All three AKHs are equally efficacious, suppressing RNA synthesis to levels similar to those measured when actinomycin D is added to the incubations of fat body tissue. Mature adults (25 days old of either sex) are the most sensitive, while younger insects show practically no inhibition of RNA synthesis in response to injected adipokinetic peptides. At supramaximal doses, however, the peptides cease to be effective in females, while they retain their efficacy in males. The chemical nature of the active material in the extracts of brain remains to be determined, but its mechanism of action in inhibiting RNA synthesis differs from that of the AKHs: the latter require extracellular Ca2+ for their action, while extracts of brain do not. Neither mechanism appears to involve cAMP.

A method is described for the rapid determination of the activities of adipokinetic peptides, based on their inhibition in fat body of the synthesis of lipid from [1-14C] acetate. The main purpose of this study was to develop a method for measuring adipokinetic hormone (AKH) activity in vitro. The assay employs microfiltration plates which allow dose response curves to three different test peptides to be obtained in one day. The optimum conditions for the assay were Schneider's: Minimum Essential medium Eagle (1:1) at pH 7.2, adjusted with sucrose to 450 mOsm. A comparison between the three naturally occurring AKHs of Locusta migratoria, reveals AKH-III to be an outstandingly potent inhibitor of acetate uptake; EC50 values are approx. 0.2 pmol 300 μ l (6.67 × 10−10 M) for both AKH-I and II, but only about 40 fmol 300 μ l (1.33 × 10−10M) for AKH-III. The sensitivity of this uptake assay to other substances was tested (unrelated hormones, second messengers and their analogues and inhibitors of prospective second messenger pathways) and the specificity of this assay for a variety of AKH/RPCH-like hormones was demonstrated.

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.

The effects of analogs of the diuretic peptides Locmi-DH, Dippu-DH(46) and Dippu-DH(31) on two aspects of appetitive behavior are investigated in previously food-deprived nymphs of Locusta migratoria. The analogs tested are the C-terminal 15-mer and nonapeptides and their corresponding cyclic analogs. At a nominal dose of 1pmol injected per nymph, the linear fragments and their cyclic analogs of Dippu-DH(46) display no significant effects on the latency to feed or on the length of the first meal in nymphs. However, at the same dose, the linear fragments of Dippu-DH(31) and their cyclic analogs, and analogs of Locmi-DH modulate appetitive behavior: they are anorexigenic in reducing the duration of the first meal, and generally increasing the latency to feed. The cyclic analogs of Dippu-DH(31) are at least as effective as their linear counterparts in influencing these aspects of appetitive behavior in locust nymphs.

Background Many aspects of Acanthamoeba granulomatous encephalitis remain poorly understood, including host susceptibility and chronic colonization which represent important features of the spectrum of host-pathogen interactions. Previous studies have suggested locusts as a tractable model in which to study Acanthamoeba pathogenesis. Here we determined the mode of parasite invasion of the central nervous system (CNS). Results Using Acanthamoeba isolates belonging to the T1 and T4 genotypes, the findings revealed that amoebae induced sickness behaviour in locusts, as evidenced by reduced faecal output and weight loss and, eventually, leading to 100% mortality. Significant degenerative changes of various tissues were observed by histological sectioning. Both isolates produced disseminated infection, with viable amoebae being recovered from various tissues. Histological examination of the CNS showed that Acanthamoeba invaded the locust CNS, and this is associated with disruption of the perineurium cell/glial cell complex, which constitutes the locust blood-brain barrier. Conclusions This is the first study to demonstrate that Acanthamoeba invades locust brain by modulating the integrity of the insect's blood-brain barrier, a finding that is consistent with the human infection. These observations support the idea that locusts provide a tractable model to study Acanthamoeba encephalitis in vivo. In this way the locust model may generate potentially useful leads that can be tested subsequently in mammalian systems, thus replacing the use of vertebrates at an early stage, and reducing the numbers of mammals required overall.

A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides.

Exogenous cAMP, cAMP analogues and forskolin all stimulated fluid secretion by the isolated Malpighian tubules of Acheta domesticus . IBMX potentiated the effects of 8-bromo cAMP and forskolin, and the diuretic activity of extracts of corpora cardiaca. The effects of forskolin and corpora cardiaca on cAMP production were determined on isolated tubules both in the presence and absence of IBMX. Such treatments increased intracellular levels of cAMP, with this response paralleling or preceding the increase in fluid secretion. Fluid secretion increased with intracellular cAMP concentrations up to about 8 μM, but further increases in cAMP were not associated with any further increases in fluid secretion. The adenylyl cyclase activity of Malpighian tubule plasma membrane preparations was investigated and shown to be stimulated in a dose-dependent manner by extracts of whole heads. Results are discussed in relation to the role of cAMP as a second messenger in the Malpighian tubules of Acheta , and consideration is given to the establishment of a convenient assay for diuretic peptides which stimulates adenylyl cyclase activity.

An octapeptide which mobilizes lipid and inhibits the incorporation of [ 3 H]leucine into haemolymph proteins in both Acheta domesticus and Locusta migratoria has been purified from the corpora cardiaca of Acheta . The primary sequence of this Acheta adipokinetic hormone, determined by pulsed liquid-phase Edman sequencing after enzymatic removal of the N -terminal pyroglutamate is: (pGlu)-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH 2 . This sequence is identical to that of an octapeptide isolated previously from Gryllus bimaculatus and Romalea microptera . Synthetic material with the assigned structure is biologically active and is chromatographically identical to the natural peptide. In both crickets and locusts, the sensitivity of [ 3 H]leucine incorporation to inhibition by Acheta adipokinetic hormone (EC 50 = 5 × 10 −11 M) is two orders of magnitude greater than that of the adipokinetic response (EC 50 = 3 × 10 −9 M). Acheta adipokinetic hormone elicits a full adipokinetic response in both Locusta and in Acheta .

Sir, In recent years, the burden of infectious diseases has exacerbated with the emergence of antimicrobial resistance and lessening efficacy of the available antimicrobial compounds. 1 Vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus (MRSA) have recently emerged as major threats to public health. In fact, the drug of choice to treat MRSA is vancomycin, but it has emerged recently that vancomycinintermediate S. aureus is exhibiting some levels of resistance against vancomycin. In addition, there are reports from the USA of MRSA showing high-level resistance to glycopeptides due to acquisition of the vanA gene complex. 2 Thus, new antimicrobial agents are required to meet the challenge posed by the emergence of multidrug-resistant microorganisms. The search for new antibiotic compounds originating from natural resources is an important research area. Insects are the largest (80% of all fauna) and most widespread group within the Animal Kingdom, and synthesize a variety of antibacterial compounds, including the defensins and cecropins, as part of their innate immune response to infection. 3‐5 Up to 50% of the

Background Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. Results Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF) suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC). Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like) suggesting a role in blood-brain barrier perturbations. Conclusion Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s) in invasion of the brain tissue by amoebae.

Two isolates of Metarhizium anisopliae var acridum were tested for their effects on the locust immune system and for comparison with the effects of challenge by injection with laminarin. Isolate IMI 330189 (referred to hereafter as Met 189) is highly pathogenic whether applied topically as conidia or injected as blastospores. However, isolate ARSEF 728 (referred to hereafter as Met 728) is pathogenic only when injected as blastospores, suggesting that the lack of pathogenicity of topically applied conidia from this isolate is due to a failure to penetrate the insect cuticle and gain access to the haemocoel. After topical application of conidia from Met 189, no activation of prophenoloxidase is detected, but injection of blastospores from Met 189 brings about a transient increase in phenoloxidase activity in the haemolymph in both adult locusts and 5th instar nymphs, although this does not prevent fungal-induced mortality. Co-injection of adipokinetic hormone-I (AKH-I) with blastospores prolongs the activation of prophenoloxidase in the haemolymph of adult locusts, and enhances it in nymphs. It is argued that the lack of activation of prophenoloxidase in nymphs shown previously (Mullen, L., Goldsworthy, G., 2003. Changes in lipophorins are related to the activation of phenoloxidase in the haemolymph of Locusta migratoria in response to injection of immunogens. Insect Biochemistry and Molecular Biology 33, 661-670), reflects differences in the sensitivity of the immune system between adults and nymphs rather than distinct qualitative differences, and this is confirmed in this study by the demonstration that doses of laminarin higher than those used previously (>or=100 microg) do activate the prophenoloxidase cascade in 5th instar nymphs. Nodules are formed in locusts of all ages in response to fungal infection or injection of laminarin, although there is wide variation in the number, size and distribution of nodules formed. During the examination of 5th instar nymphs for nodule formation, a previously unknown phenomenon was observed in which the salivary glands melanise in response to injections of blastospores or high doses of laminarin. In c. 85% of such nymphs, this reaction is so strong that the whole salivary gland is intensely black. Such a response is not observed in the salivary glands of mature adult locusts.

Injections of immunogens, such as beta-1,3-glucan or lipopolysaccharide (LPS), bring about a marked hyperlipaemia with associated changes in lipophorins and apolipophorin-III in the haemolymph of Locusta migratoria. These changes are similar to those observed after injection of adipokinetic hormone (AKH). The possibility that endogenous AKH is released as part of the response to these immunogens is investigated using passive immunisation against AKH-I, and measurement of AKH-I titre in the haemolymph after injection of immunogens. The data presented show that, despite the similarity of the changes brought about by the presence of immunogens in the haemolymph to those brought about by AKH, there is no release of endogenous AKH after injection of laminarin or LPS. A direct effect of the immunogens on release of neutral lipids by the fat body cannot be demonstrated in vitro, and the mechanism by which hyperlipaemia is induced during immune challenge remains uncertain.

The synthesis is described of an analogue of the locust CRF-like diuretic peptide in which methionine in positions 1,3, and 13 is replaced by isosteric methyl-homoserine residues. This analogue has been tested for biological activity on Malpighian tubules in vitro, and feeding behavior in vivo. It is highly active in stimulating fluid secretion and accumulation of cAMP in tubules, and on increasing the latency to feed and reducing meal duration. A 15 residue fragment from the C-terminus of the CRF-like peptide, Locmi-DP(32-46), is fully active in the feeding assay, but has only weak ability to stimulate the accumulation of cAMP in tubules. Two smaller fragments, Locmi-DP(32-37) and Locmi-DP(41-46), were tested but neither had consistent biological activity in any of the assays used here. None of the peptides tested have any substantive activity in increasing cGMP in tubules.

Our knowledge on primary structure, synthesis, release, receptor binding, structure-activity relationships, mode of action and degradation of, mainly, neuropeptides from insects has increased dramatically during the last 10 years or so. Here, five case studies are presented, which deal selectively with effects on: reproduction (trypsin modulating oostatic factor in mosquito); energy metabolism, locomotion and the immune system (adipokinetic hormones); water and ion balance, and feeding behaviour (diuretic hormones, kinins, sulfakinins); sex attraction (pheromone biosynthesis activating neuropeptide); and growth and development, and muscle activity (allatostatins). The literature is reviewed in the context of how the knowledge on neuropeptides has been and can be used for the design of novel, safe and selective compounds to control pest insects in the foreseeable future.

Interactions between the locust endocrine and immune systems have been studied in vivo in relation to nodule formation and activation of the prophenoloxidase cascade in the haemolymph. Injection of bacterial lipopolysaccharide (LPS) extracted from Escherichia coli induces nodule formation in larval and adult locusts but does not increase phenoloxidase activity in the haemolymph. Nodule formation starts rapidly after injection of LPS and is virtually complete within 8 h, nodules occurring mainly associated with the dorsal diaphragm on either side of the heart, but sometimes with smaller numbers associated with the ventral diaphragm on either side of the nerve cord. Co-injection of adipokinetic hormone-I (Lom-AKH-I) with LPS stimulates greater numbers of nodules to be formed in larval and adult locusts, and activates phenoloxidase in the haemolymph of mature adults but not of nymphs. The effect of co-injection of Lom-AKH-I with LPS on nodule formation is seen at low doses of hormone; only 0.4 pmol of Lom-AKH-I per adult locust is needed to produce a 50% increase in the number of nodules formed. When different components of LPS from the E. coli Rd mutant are tested, the mono- and the diphosphoryl Lipid A components have similar effects to the intact LPS. Remarkably, detoxified LPS activates phenoloxidase in the absence of Lom-AKH-I, although co-injection with hormone does enhance this response. Both diphosphoryl Lipid A and detoxified LPS induce a level of nodule formation that is enhanced by co-injection of Lom-AKH-I, but monophosphoryl Lipid A does not initiate nodule formation even when injected with hormone. Co-injection of a water-soluble inhibitor of eicosanoid synthesis, diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid), reduces nodule formation in response to injections of LPS (both in the absence and presence of hormone) in a dose-dependent manner, but does not prevent activation of phenoloxidase in adult locusts. It is shown that nodule formation and activation of the prophenoloxidase in locust haemolymph can both be enhanced by Lom-AKH-I, but it is argued that these processes involve distinct mechanisms in which eicosanoid synthesis is important for nodule formation, but not for the increased phenoloxidase activity.

In Locusta migratoria, activation of phenoloxidase in the haemolymph in response to injection of laminarin is age-dependent: being absent in fifth instar nymphs and newly emerged adults, and only becoming evident four days after the final moult. This pattern of change in phenoloxidase activation correlates with the pattern of change in the concentration of apolipophorin-III (apoLp-III) in the haemolymph. Injection of a conspecific adipokinetic hormone (Lom-AKH-I) has no effect on the phenoloxidase response in nymphs or newly emerged adults but, in adults older than four days, co-injection of the hormone with laminarin prolongs the activation of phenoloxidase in the haemolymph: a similar enhancement of the response to laminarin is observed in locusts that have been starved for 48 h but not injected with AKH-I. During most of the fifth stadium, injection of laminarin results in a decrease in the level of prophenoloxidase in the haemolymph; an effect that is not observed in adults of any age. Marked changes in the concentration of apoLp-III, and the formation of LDLp in the haemolymph, are observed after injection of laminarin (or LPS) and these are remarkably similar, at least qualitatively, to those that occur after injection of AKH-I. The involvement of lipophorins in the activation of locust prophenoloxidase in response to immunogens is discussed.

The development of an enzyme-linked immunoassay (ELISA) for the adipokinetic neuropeptide hormone, Pya-AKH, from the firebug Pyrrhocoris apterus L. is described. The ELISA measures as little as 20 fmol of Pya-AKH. Tested against a range of synthetic peptides, the assay has a high sensitivity for peptides containing the C-terminal motif FTPNWamide. The amounts of Pya-AKH in the brain, corpora cardiaca, suboesophageal ganglia, and fused thoracic and abdominal ganglionic mass are very small, with only the corpora cardiaca containing appreciable levels of the hormone (ca. 4 pmol per bug). Preliminary estimates of the persistence of the hormone in the haemolymph are consistent with values determined for AKHs in other insects, and suggest that Pya-AKH has a rapid turnover with a half-life of ca. 18 min. Measurements of circulating titres of AKH in Pyrrhocoris are only possible in the ELISA described here by using pooled samples of haemolymph, and after preliminary clean-up of the haemolymph samples. The titre of Pya-AKH in resting reproductive female Pyrrhocoris is ca. 1 fmol/mgr;l.

Lom-AKH-I enhances the activation in vivo of prophenoloxidase in the haemolymph of the African migratory locust, Locusta migratoria, in response to challenge with laminarin. AKH does not influence the speed or initial magnitude of the phenoloxidase response to laminarin, but prolongs the period of activation of the enzyme in a dose-dependent manner. Injections of preparations of bacterial lipopolysaccharide (LPS) do not activate prophenoloxidase in vivo, but co-injection of Lom-AKH-I with commercial preparations of LPS from Klebsiella pneumoniae, Escherichia coli, or Shigella flexneri (but not one from Pseudomonas aeroginosa) results in dose-dependent increases in the levels of phenoloxidase that persist in the haemolymph for several hours. It is argued that the effects of AKH on phenoloxidase activation in locusts described here are, at least in part, related directly to changes in lipid metabolism brought about by the hormone.

The prophenoloxidase cascade in the haemolymph of mature adult Locusta migratoria migratorioides (R & F) is activated in response to injection of laminarin, a -1,3 glucan. Co-injection of adipokinetic hormone-I (Lom-AKH-I) and laminarin prolongs the activation of the enzyme in a dose-dependent manner. However, injections of bacterial lipopolysaccharide (LPS) do not activate prophenoloxidase unless AKH is co-injected, when there is a dose-dependent increase in the level of phenoloxidase that persists in the haemolymph for several hours. Even when AKH is co-injected, the highest levels of phenoloxidase activity are always greater after injection of laminarin than after LPS, and these two immunogens must activate the prophenoloxidase cascade by quite distinct pathways. In the present study, interactions between the endocrine and immune systems were examined with respect to activation of prophenoloxidase and the formation of nodules: injection of LPS induces nodule formation in adult locusts. With LPS from Pseudomonas aeruginosa, nodules form exclusively in dense accumulations in the anterior portion of the abdomen on either side of the dorsal blood vessel associated with the dorsal diaphragm. However, with LPS from Escherichia coli, fewer nodules are formed but with a similar distribution, except that occasionally some nodules are aligned additionally on either side of the ventral nerve cord. Co-injection of Lom-AKH-I with LPS from either bacteria stimulates greater numbers of nodules to be formed. This effect of coinjection of AKH on nodule formation is seen at low doses of hormone with only 0.3 or 0.4 pmol of Lom-AKH-1, respectively, increasing the number of nodules by 50%. Injections of octopamine or 5-hydroxytryptamine do not mimic either of the actions of Lom-AKH-I described here. Co-injection of an angiotensin-converting enzyme inhibitor, captopril, reduces nodule formation in response to injections of LPS but has no effect on the activation of phenoloxidase. Co-injection of an inhibitor of eicosanoid synthesis, dexamethasone, with LPS influences nodule formation (with or without AKH) in different ways according to the dose of dexamethasone used, but does not affect activation of prophenoloxidase. Eicosanoid synthesis is important for nodule formation, but not for the activation of the prophenoloxidase cascade in locust haemolymph.

This review is concerned mainly with the adipokinetic hormones (AKHs) of locusts: their molecular conformations, actions and functions and the development of microfiltration assays in vitro. The physiological significance of having multiple hormones with overlapping actions whose efficacy changes during development is discussed in relation to the possibility that these reflect variations in populations of receptors and/or the pharmacokinetics of the peptides. The involvement of second messengers in the transduction mechanism of AKHs is reviewed, and we describe hormone-induced changes of intracellular calcium in single dispersed fat body cells. The structure activity relationships of the three locust AKHs and a number of analogues with variations at the N- and C-termini are discussed. A number of areas are identified where there are gaps in our understanding of these hormones, and some of these will be the focus of our future research.

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.

A biologically active 125 I-labeled analogue of AK-II (3′-hydroxyphenyl propionic-Gly-Gly-Gly-Phe-Ser-Pro-Trp-Gly-NH 2 ) was used to investigate the properties of achetakinin binding sites on plasma membranes from Malpighian tubules of Acheta domesticus . With optimized conditions, binding was rapid, reversible, and specific, and saturation studies revealed a single class of binding sites with K d 0.55 n M and B max 39.9 fmol/mg membrane protein. The affinities of achetakinins for binding sites on tubule membranes ranked AK-V > AK III > AK-II > AK-I ≥ AK-IV, in general agreement with their potencies in functional assays. However, IC 50 values were several orders of magnitude higher than corresponding values for EC 50 , which suggests a considerable receptor reserve.

A convenient method of synthesis, using a combination of solid and liquid phase methodology, for locust Adipokinetic Hormone-I (AKH-I) and its analogues with modifications at the threonine residues are reported. The N-terminal nonapeptide acid of AKH-I is synthesized in the solid phase using the 2-chlorotrityl chloride resin and the Fmoc/t-Bu strategy. Quantitative cleavage of the nonapeptide acid from the resin, with the tert-butyl type side-chain protection intact, is achieved with a mixture of acetic acid/trifluoroethanol/ dichloromethane. The nonapeptide acid is then coupled in solution to the threonine derivatives, H-Thr-NH2 or H-Thr(Bz1)-NH2, with the DCC/HOBt method. The efficiency of this approach in the synthesis of AKH-I is demonstrated by the high yields and purity of the synthesized peptides. All the synthesized peptides were tested in two ways: first, in a lipid mobilization assay in locusts in vivo; and second, in a novel assay in vitro concerned with the uptake of radiolabelled acetate into locust tissue. Replacement of the hydroxyl hydrogen in Thr5 of locust AKH-I by the bulky and highly lipophilic tert-butyl group reduced the potency markedly, whereas efficacy is unaffected, but when the hydroxyl hydrogen of Thr10 in AKH-I is replaced by a benzyl group, the activity of the resulting analogue is identical to that of the natural peptide. Structure-activity relationships are discussed. © Munksgaard 1994.

Insect diuretic peptides differ chemically and, even within a single species, several forms of diuretic peptide may be present (Wheeler and Coast, 1989). Thus, in Locusta migratoria, diuretic peptides have been described as AVP-like, ACTH-like, and as novel peptides. Only two insect diuretic peptides have been fully characterised, one from Locusta (Proux et al., 1987), and the other from Manduca sexta (Kataoka et al., 1989). They have no structural analogy to each other, but each has similarities with some vertebrate peptides. Fractionation of aqueous extracts of the corpora cardiaca of Acheta domesticus yields a number of peptides which stimulate fluid secretion by isolated Malpighian tubules (Coast and Wheeler, 1989). One peptide, Acheta peptide-I (AP-I), is associated with a major UV absorbing peak, and was selected for sequence analysis.

In locusts, adipokinetic hormones have well defined actions: they mobilise diacylglycerols from the fat body and stimulate oxidation of lipids by the flight muscles (Goldsworthy and Wheeler, 1989). Their actions presumably depend on interactions with specific receptors in the tissues, but there have been no direct studies of AKH receptors. Some information about receptors can, however, be inferred from biological assay data. For most actions, AKH-I and to a lesser extent AKH-II are both effective agonists, but AKH-II stimulates cAMP accumulation in the fat body more than AKH-I (Goldsworthy et al., 1986), and AKH-II releases carbohydrate from the fat body, whereas AKH-I does not (Loughton and Orchard, 1981). These differences in actions suggest that AKH-I and -II may act via different receptors at the fat body, although their high degree of cross reactivity in other assays indicates that they may also stimulate the same receptors. The availability of synthetic naturally occurring analogues of adipokinetic peptides has prompted an examination of their structure/activity relationships.

During trivial flight in locusts, carbohydrates are the main energy source for the flight muscles, but during long-term (migratory) flight, lipids mobilised from the fat body are utilised. The supply of fuels to the flight muscles, and the balance between fuels, is determined by peptidic adipokinetic hormones, AKH-I and AKH-II, released from the corpora cardiaca. These peptides also indirectly effect changes in lipid-transporting mechanisms in the haemolymph, and regulate a lipoprotein lipase necessary for lipid uptake by the flight muscles. The sequences of the AKHs of locusts are known and they are members of a family of invertebrate neuropeptides. Extensive structure/activity studies for the action of AKHs on lipid mobilisation have been carried out and, together with studies on secondary structure, the structural features necessary for biological activity are being elucidated. Such information will be of great importance in receptor studies for these hormones, and in the development of powerful agonists and antagonists.

The structures of 3 locust adipokinetic hormones are known: the decapeptide adipokinetic hormone I is common to all species of locust investigated so far, but the octapeptide AKH-II’s of Locusta (AKH-IIL) and Schistocerca (AKH-IIS) differ by a single amino acid (see Table 1). In addition to these locust peptides, structurally related but often functionally different peptides from other insect species have been sequenced. These include the adipokinetic peptide from Heliothis zea (Jaffe et al., 1986), which is identical to that in Manduca sexta (Ziegler et al., 1985); hypertrehalosaemic peptides I and II from Periplaneta americana (sequenced independently by various groups; see Goldsworthy et al., 1986a); and hypertrehalosaemic factor II from Carausius morosus (Gade and Rinehart, 1986). Together with a crustacean peptide, Red Pigment Concentrating Hormone, these peptides apparently represent a group of closely related molecules, the AKH/RPCH family: they possess striking sequence and structural similarities, including blocked amino and carboxy terminals. We have compared the activities of these peptides in the locust hyperlipaemic assay to give an insight into the structural features necessary for the fit of the AKH molecule to its receptor on the fat body.

Adipokinetic hormones (AKH) I and II are released into the haemolymph during long-term flight in locusts and have several well defined sites of action: they increase the rate of release of diacylglycerols from the fat body and stimulate the uptake and utilisation of these lipids in the flight muscles (see Goldsworthy, 1983)• Adipokinetic hormones also promote indirect effects such as haemolymph lipoprotein transformations (Goldsworthy et al., 1985). Lipids released from the fat body are transported as part of lipoproteins; in resting locusts the majority of haemolymph diacylglycerol is carried as part of lipoprotein Ayellow, but after hormone injection Ayellow becomes lipid-loaded and associates with non-lipid carrying apoproteins to become lipoprotein A+, which carries 10 to 15X as much lipid as Ayellow (Mwangi and Goldsworthy, 1977; Van Der Horst et al., 1981a,b; Wheeler and Goldsworthy, 1983a,b).

List of contributors Preface 1. What is special about peptides as neuronal messengers? J. Joosse Part I. Immunocytochemistry and Ultrastructure: 2. The new neurobiology - ultrastructural aspects of peptide release as revealed by studies of invertebrate nervous systems D. W. Golding and D. V. Pow 3. Immunocytochemistry of hormonal peptides in molluscs: optical and electron microscopy and the use of monoclonal antibodies H. H. Boer and J. van Minnen 4. Immunocytology of insect peptides and amines C. Remy and J. Vieillemaringe 5. Immunocytochemistry and ultrastructure of crustacean endocrine cells G. Martin Part II. Arthropod Neurohormones: 6. Characterisation of insect neuropeptides W. Mordue and K. J. Siegert 7. The isolation and characterisation of vertebrate-type peptides in insects H. Duve and A. Thorpe 8. Humoral functions of insect neuropeptides C. H. Wheeler, G. Gade and G. J. Goldsworthy 9. Functions of aminergic and peptidergic skeletal motoneurones in insects M. O'Shea, S. Hekimi, J. Witten and M. K. Worden 10. Physiology and biochemistry of crustacean neurohormonal peptides S. G. Webster and R. Keller Part III. Neurohormones in Coelenterates, Annelids and Protochordates: 11. Structure, location and possible actions of Arg-Phe-amide peptides in coelenterates C. J. P. Grimmelikhuijzen, D. Graff and A. N. Spencer 12. Neuropeptides and monoamines in annelids M. Porchet and N. Dhainaut-Courtois 13. Functional aspects of peptide neurohormones in protochordates M. C. Thorndike and D. Georges Part IV: Neurohormones in Molluscs: 14. Bioactive peptides in molluscs W. P. M. Geraerts, E. Vreugdenhil and R. H. M. Ebberink 15. Actions and roles of the FMRFamide peptides in Helix G. A. Cottrell, N. W. Davies, J. Turner and A. Oates 16. Evolution of peptide hormones: an Aplysia CRF-like peptide R. Taussig, J. R. Nambu and R. H. Scheller Index.