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SUMMARY The mechanism of primary urine production and the activity of a diuretic kinin, Achdo-KII, were investigated in Malpighian tubules of Acheta domesticus by measuring intracellular Na+, K+ and Cl− activities, basolateral membrane voltage (Vb), fluid secretion and transepithelial ion transport. Calculated electrochemical gradients for K+ and Cl− across the basolateral membrane show they are actively transported into principal cells, and basolateral Ba2+-sensitive K+ channels do not contribute to net transepithelial K+ transport and fluid secretion. A basolateral Cl− conductance was revealed after the blockade of K+ channels with Ba2+, and a current carried by the passive outward movement of Cl− accounts for the hyperpolarization of Vb in response to Ba2+. Ion uptake via Na+/K+/2Cl− cotransport, driven by the inwardly directed Na+ electrochemical gradient, is thermodynamically feasible, and is consistent with the actions of bumetanide, which reduces fluid secretion and both Na+ and K+ transport. The Na+ gradient is maintained by its extrusion across the apical membrane and by a basolateral ouabain-sensitive Na+/K+-ATPase. Achdo-KII has no significant effect on the intracellular ion activities or Vb. Electrochemical gradients across the apical membrane were estimated from previously published values for the levels of Na+, K+ and Cl− in the secreted fluid. The electrochemical gradient for Cl− favours passive movement into the lumen, but falls towards zero after stimulation by Achdo-KII. This coincides with a twofold increase in Cl− transport, which is attributed to the opening of an apical Cl− conductance, which depolarises the apical membrane voltage.

Serotonin stimulates secretion by Malpighian tubules (MT) of a number of insects, and functions as a diuretic hormone in Rhodnius prolixus and in larval Aedes aegypti. Serotonin is here shown to be a potent stimulant of secretion by MT of the house cricket, Acheta domesticus, with an apparent EC(50) of 9.4 nmol L(-1), although its diuretic activity is just 25% of the maximum achievable with either the native CRF-related peptide, Achdo-DH, or a crude extract of the corpora cardiaca. In this respect, the diuretic activity of serotonin is similar to that of the cricket kinin Achdo-KI, and when tested together their actions are not additive, which suggests they target the same transport process. Consistent with this suggestion, the activity of serotonin is chloride-dependent and is associated with a non-selective stimulation of NaCl and KCl transport. In common with Achdo-KI, serotonin has no effect on cAMP production by isolated MT, and both act synergistically with exogenous 8bromo-cAMP in stimulating fluid secretion, most likely by promoting the release of Ca(2+) from intracellular stores. A number of serotonin agonists and antagonists were tested to determine the pharmacological profile of receptors on cricket MT. The results are consistent with the diuretic activity of serotonin being mediated through a 5-HT(2)-like receptor.

Spined soldier bugs, Podisus maculiventris, are heteropteran insects that feed voraciously on other insects, particular the soft bodied larval forms of Lepidoptera and Coleoptera. The response of P. maculiventris Malpighian tubules (MTs) to serotonin and known diuretic and antidiuretic peptides has been investigated, and is compared with that of MT from the hematophagous and phytophagous heteropteran bugs Rhodnius prolixus and Acrosternum hilare, respectively. A CRF-related peptide diuretic hormone (DH) from the termite Zootermopsis nevadensis (Zoone-DH) stimulated MT secretion, which was reversed by a member of the CAP(2b) family of peptides from A. hilare (Acrhi-CAP(2b)-2), an antidiuretic effect. Serotonin had no effect on secretion, neither did a representative calcitonin-like DH, kinin, tachykinin-related peptide, and an antidiuretic factor from the mealworm Tenebrio molitor (Tenmo-ADFb) in both P. maculiventris or A. hilare. Serotonin is a DH in R. prolixus, and its lack of effect on MT from P. maculiventris and A. hilare suggests this is an adaptation to hematophagy. On the other hand, the antidiuretic activity of members of the CAP(2b) family in all three bugs is consistent with this being a heteropteran feature rather than a specialism for hematophagy.

Plant sucking heteropteran bugs feed regularly on small amounts of K(+)-rich plant material, in contrast to their hematophagous relatives which imbibe large volumes of Na(+)-rich blood. It was anticipated that this would be reflected in the endocrine control of Malpighian tubule (MT) secretion. To explore this, neuroendocrine factors known to influence MT secretion were tested on MT of the pentatomid plant sucking stink bugs, Acrosternum hilare and Nezara viridula, and the results compared with previously published data from Rhodnius prolixus. Serotonin had no effect on N. viridula MT, although it stimulates secretion by R. prolixus MT1000-fold, and initiates a rapid diuresis to remove excess salt and water from the blood meal. Kinins had no effect on stink bug MT, but secretion was increased by Zoone-DH, a CRF-like peptide, although the response was a modest 2-3-fold acceleration compared with 1000-fold in R. prolixus. Native CAPA peptides, which have diuretic activity in dipteran flies, had antidiuretic activity in MT of the stink bug (Acrhi/Nezvi-CAPA-1 and -2), as previously shown with Rhopr-CAPA-2 in R. prolixus. The antidiuretic activity of Rhopr-CAPA-2 has been linked with terminating the rapid diuresis, but results with stink bugs suggest it is a general feature of heteropteran MT.

The multifunctional arthropod 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide motif Phe-X(1)-X(2)-Trp-Gly-NH(2), where X(1)=His, Asn, Ser, or Tyr and X(2)=Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects. Compounds with similar biological activity could be exploited for the control of arthropod pest populations such as the mosquito Aedes aegypti (L.) and the southern cattle tick Rhipicephalus (Boophilus) microplus (Canestrini), vectors of human and animal pathogens, respectively. Insect kinins, however, are susceptible to fast enzymatic degradation by endogenous peptidases that severely limit their use as tools for pest control or for endocrinological studies. To enhance resistance to peptidases, analogs of the insect kinins incorporating bulky alpha,alpha-disubstituted amino acids in positions adjacent to both primary and secondary peptidase hydrolysis sites were synthesized. In comparison with a control insect kinin, several of these analogs are highly stable to hydrolysis by degradative enzymes ANCE, neprilysin and Leucine aminopeptidase. Six analogs were evaluated by calcium bioluminescence assay on recombinant receptors from mosquito and tick. Four of these analogs either matched or exceeded the potency of the control kinin peptide agonist. One of these was about 5-fold more potent than the control agonist on the tick receptor. This analog was 8-fold more potent than the control agonist on the mosquito receptor, and twice more potent than the endogenous Aedes kinin-II. The analog also demonstrated potent activity in an in vitro Aedes Malpighian tubule fluid secretion assay. Similar comparisons of analog potency cannot be made to tick kinins because no endogenous kinin has yet been identified. These potent, biostable analogs represent ideal new tools for endocrinologists studying arthropod kinin-regulated processes in vivo, particularly for ticks in which their role remains to be established.

Insect kinin neuropeptides share a common C-terminal pentapeptide sequence Phe(1)-Xaa(1)(2)-Xaa(2)(3)-Trp(4)-Gly(5)-NH(2) (Xaa(1)(2)= His, Asn, Phe, Ser or Tyr; Xaa(2)(3)= Pro, Ser or Ala) and have been isolated from a number of insects. They have been associated with the regulation of such diverse processes as hindgut contraction, diuresis, and the release of digestive enzymes. In this review, the chemical, conformational, and stereochemical aspects of the activity of the insect kinins with expressed receptors and/or biological assays are reviewed. With this information, both nonselective and selective biostable analogues have been designed that protect peptidase-susceptible sites in the insect kinin sequence and demonstrate significant retention of activity in both receptor and biological assays. C-terminal aldehyde insect kinin analogues modify the activity of the insect kinins, leading to inhibition of weight gain and mortality in corn earworm larvae and selective inhibition of diuresis in the housefly. Promising mimetic analogue leads in the development of selective agents capable of disrupting insect kinin-regulated processes have been identified that may provide interesting tools for arthropod endocrinologists and new pest insect management strategies in the future.

SUMMARYThe pioneering work of Simon Maddrell established that the rapid postprandial diuresis of the haematophagous insect Rhodnius prolixusis controlled by a diuretic hormone and demonstrated the role of the Malpighian tubules in meeting the volumic, osmotic and ionic challenges posed by an enormous blood meal. A number of diuretic and antidiuretic hormones that control secretion of primary urine by Malpighian tubules have now been identified, but little is known of the interplay between these hormones and those that regulate transport processes in the hindgut. This review therefore focuses on the control of ionic homeostasis in Rhodnius and mosquitoes, because primary urine is voided virtually unchanged during the rapid diuresis that follows a blood meal. At such times, the hindgut has a negligible impact on the volume and composition of the final urine, and neurohormones acting on the Malpighian tubules have a dominant role in the control of ionic homeostasis.

SUMMARY Acheta domesticus is reported to have an antidiuretic hormone that reduces Malpighian tubule secretion. Identified peptides known to work in this way (Tenmo-ADFa and ADFb, and Manse-CAP2b) were tested as candidates for the unidentified hormone, along with their second messenger,cyclic GMP. Only Tenmo-ADFb was active, but was diuretic, as was 8-bromo cyclic GMP. The activity of Tenmo-ADFb is comparable to that of the cricket kinin neuropeptide, Achdo-KII, but it is much less potent. Its activity was unaffected by deleting either the six N-terminal residues or the C-terminal phenylalanine. At high concentrations, tubule secretion is doubled by Tenmo-ADFb and Achdo-KII, but their actions are non-additive, suggesting they have a similar mode of action. Both stimulate a non-selective KCl and NaCl diuresis, which is consistent with the opening of a transepithelial Cl–conductance. In support of this, the diuretic response to Tenmo-ADFb and Achdo-KII is prevented by a ten-fold reduction in bathing fluid chloride concentration, and both peptides cause the lumen-positive transepithelial voltage to collapse. The Cl– conductance pathway appears likely to be transcellular, because the Cl– channel blocker DPC reduces both basal and peptide-stimulated rates of secretion. The effects of 8-bromo cyclic GMP on transepithelial voltage and composition of the secreted fluid are markedly different from those of Tenmo-ADFb. This is the first report of the antidiuretic factor Tenmo-ADFb stimulating tubule secretion. Although the actions of Tenmo-ADFb are indistinguishable from those of Achdo-KII, it is unlikely to act at a kinin receptor, because the core sequence (residues 7–12) lacks the Phe and Trp residues that are critical for kinin activity.

The insect kinins are present in a wide variety of insects and function as potent diuretic peptides in flies. A C-terminal aldehyde insect kinin analog, Fmoc-RFFPWG-H (R-LK-CHO), demonstrates stimulation of Malpighian tubule fluid secretion in crickets, but shows inhibition of both in vitro and in vivo diuresis in the housefly. R-LK-CHO reduced the total amount of urine voided over 3 h from flies injected with 1 microL of distilled water by almost 50%. The analog not only inhibits stimulation of housefly fluid secretion by the native kinin Musdo-K, but also by thapsigargin, a SERCA inhibitor, and by ionomycin, a calcium ionophore. The activity of R-LK-CHO is selective, however, as related C-terminal aldehyde analogs do not demonstrate an inhibitory response on housefly fluid secretion. The selective inhibitory activity of R-LK-CHO on housefly tubules represents an important lead in the development of environmentally friendly insect management agents based on the insect kinins.

MALDI-TOF/TOF tandem mass spectrometry has been applied to determine the complete sequences of the PVK/CAP2b neuropeptides in the stable fly Stomoxys calcitrans and horn fly Haematobia irritans, insect pests of livestock. This peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The sequences are as follows: Stoca-PVK/CAP2b-1, AGGASGLYAFPRVa; Stoca-PVK/CAP2b-2, NAKLYPVPRVa; and Haeir-PVK/CAP2b-1, AGGASGLYAFPRVa; Haeir-PVK/CAP2b-1, NAKLYPMPRVa. Both Stoca-PVK/CAP2b-1 and -2 stimulate Malpighian tubule fluid secretion in the stable fly, with EC50 values between 3 and 11 nM. The identification of these novel neuropeptides adds to our knowledge of the peptidomes of flies, and can aid in the development of neuropeptide-based control strategies of these insect pests.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF–TOF) tandem mass spectrometry has been applied for the first time on an insect/arthropod target, focusing on PVK/CAP2b neuropeptides in the housefly Musca domestica and flesh fly Neobellieria bullata. The peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The confirmation of side-chain fragments which allows assignment of Leu/Ile even from samples as small as neurohemal organs will greatly accelerate the identification of novel neuropeptides that are implicated in the regulation of critical physiological processes in insects. The unnatural Ile analog is 4.5 times more active than the native Leu sequence in a housefly Malpighian tubule fluid secretion assay, which reinforces the caveat that potency values in a biological assay cannot be relied upon to predict the native sequence. © 2005 Published by Elsevier Inc.

Based on a receptor interaction model, simplified analogues of insect kinins were prepared. The compounds were templated on a conformationally restricted amino piperidinone carboxylate scaffold. The conformation of the analogues was studied by NMR and the biological activity of the compounds was tested in a bio-assay.

SUMMARYMosquito natriuretic peptide (MNP), an uncharacterised peptide from the yellow fever mosquito, Aedes aegypti, acts via cyclic AMP to stimulate secretion of Na+-rich urine by opening a Na+ conductance in the basolateral membrane of Malpighian tubule principal cells. Corticotropin releasing factor (CRF)-related peptides and calcitonin (CT)-like diuretic peptides use cyclic AMP as a second messenger and were therefore considered likely candidates for MNP. BLAST searches of the genome of the malaria mosquito Anopheles gambiae, gave sequences for the CRF-related peptide Anoga-DH44 and the CT-like peptide Anoga-DH31, which were synthesised and tested for effects on Malpighian tubules from An. gambiae and Ae. aegypti,together with 8-bromo-cyclic AMP.The cyclic AMP analogue stimulated secretion of Na+-rich urine by An. gambiae Malpighian tubules, reproducing the response to MNP in Ae. aegypti. It also depolarised the principal cell basolateral membrane voltage (Vb) while hyperpolarising the transepithelial voltage (Vt) to a similar extent.Anoga-DH44 and Anoga-DH31 stimulated production of cyclic AMP, but not cyclic GMP, by Malpighian tubules of An. gambiae. Both peptides had diuretic activity, but only Anoga-DH31 had natriuretic activity and stimulated fluid secretion to the same extent as 8-bromo-cyclic AMP. Likewise, Anoga-DH31 reproduced the effects of cyclic AMP on tubule electrophysiology, whereas Anoga-DH44initially hyperpolarised Vb and depolarised Vt, which is the opposite of the effect of Anoga-DH31.Anoga-DH44 and Anoga-DH31 were also tested for effects on fluid secretion and ion transport by Ae. aegypti tubules. As in An. gambiae, the CRF-related peptide Anoga-DH44 had a non-specific effect on the transport of Na+ and K+,whereas the CT-like peptide Anoga-DH31 specifically stimulated transepithelial Na+ transport.We conclude that the CT-like peptide Anoga-DH31 is the previously uncharacterised mosquito natriuretic peptide.

This minireview considers various aspects of the control of hydromineral balance in insects with particular reference to the control of diuresis and natriuresis in mosquitoes, with new information on the diuretic peptides of Anopheles gambiae.

Water loss from adult male houseflies was continuously recorded using a flow-through humidity meter, which enabled losses to be apportioned between the sum of cuticular and respiratory transpiration, salivation and excretion. Transpiration accounted for >95% of water lost from sham-injected flies, compared with excretion (3.0%) and salivation (2.4%). In contrast, excretion accounted for 40% of water lost from flies injected with > or =3 microl of saline, whereas salivary losses were unchanged. Saline injections (1-5 microl) expanded the abdomen in the dorsal-ventral plane, and this expansion was positively correlated with the magnitude of the ensuing diuresis, suggesting the signal for diuretic hormone release originates from stretch receptors in abdominal tergal-sternal muscles. The effects of decapitation, severing the ventral nerve cord within the neck or ligaturing the neck, showed the head was needed to initiate and maintain diuresis, but was neither the source of diuretic hormone nor did it control the discharge of urine from the anus. These findings indicate the head is part of the neural-endocrine pathway between abdominal stretch receptors and sites for diuretic hormone release from the thoracic-abdominal ganglion mass. Evidence is presented for Musdo-K having a hormonal role in the control of diuresis, although other neuropeptides may also be implicated.

Insect kinin analogues of the sequence Phe-Phe-psi[CN(4)]-Ala-Trp-Gly-NH(2) containing (L-Phe(2),L-Ala(3)) and (L-Phe(2),D-Ala(3)) stereochemical variants of the tetrazole moiety, a mimic of the type VI beta-turn, demonstrate significant agonist and partial antagonist activity, respectively, in a cricket diuretic bioassay. A comparison of the solution conformations of these two stereochemical variants indicates a structural basis for their divergent bioactivities. The (D-Phe(2),D-Ala(3)) stereochemical variant was synthesized and found to demonstrate significant agonist activity. The results further define stereochemical requirements for the diuretic activity of insect kinins in crickets and provide valuable information for the design of biostable analogues capable of disrupting digestive and diuretic processes in pest insects.

The insect kinins are potent diuretic peptides that preferentially form a cis-Pro, type VI -turn. An insect kinin analog containing (2S,4S)-4-aminopyroglutamate, a novel cis-peptide bond, type VI -turn motif, demonstrates significant activity in the physiological range in a cricket diuretic assay. This is the first instance of a 4-aminopyroglutamate analog of a peptide with a preference for a type VI turn that demonstrates significant bioactivity. The results provide further confirmatory evidence for the active conformation of the insect kinins, and a new scaffold with which to design biostable, peptidomimetic analogs capable of disrupting critical insect kinin-regulated processes in insects. © 2004 Wiley Periodicals, Inc.* Biopolymers 75: 412- 419, 2004

The first reported examples of C-terminal aldehyde analogs of an insect neuropeptide are described. They are hexapeptide insect kinin analogs Boc-VFFPWG-H and Fmoc-RFFPWG-H. Activity observed for these modified analogs in an in vitro insect diuretic assay confirms that the C-terminal aldehyde group is tolerated by an insect kinin receptor. The two analogs demonstrate greatly enhanced activity over standard C-terminal amide insect kinins in a larval weight gain inhibition assay in the corn earworm Helicoverpa zea. Treatment with Boc-VFFPWG-H led to significant increases in larval mortality at doses of 500 pm (45%) and 5 nm (67%). Boc-VFFPWG-H represents a lead analog in the development of novel, environmentally friendly pest insect management agents based on the insect kinin class of neuropeptides.

The insect kinin neuropeptides have been implicated in the regulation of water balance, digestive organ contraction, and energy mobilization in a number of insect species. A previous solution conformation study of an active, restricted-conformation cyclic analog, identified two possible turn conformations as the likely active conformation adopted by the insect kinins at the receptor site. These were a cisPro type VI beta-turn over C-terminal pentapeptide core residues 1-4 and a transPro type I-like beta-turn over core residues 2-5, present in a ratio of 60:40. Synthesis and evaluation of the diuretic activity of insect kinin analogs incorporating a tetrazole moiety, which mimics a cis peptide bond, identifies the active conformation as the former. The discovery of a receptor interaction model can lead to the development of potent agonist and antagonist analogs of the insect kinins. Indeed, in this study a tetrazole analog with D stereochemistry has been shown to demonstrate partial antagonism of the diuretic activity of natural insect kinins, providing a lead for more potent and effective antagonists of this critical neuropeptide family. The future development of mimetic agonists and antagonists of insect kinin neuropeptides will provide important tools to neuroendocrinologists studying the mechanisms by which they operate and to researchers developing new, environmentally friendly pest insect control strategies.

Diuresis was studied in vivo by measuring the loss of tritiated water. The basal rate of water loss (5 nl/min) represents respiratory and cuticular losses, whereas higher rates reflect urine output, which reaches 20 nl/min after injection of 1 microl distilled water. This response to hypervolemia involves release of a diuretic hormone(s) into the hemolymph. However, housefly diuretic peptides increased urine output to a maximum of only 7 nl/min, and higher rates may require fluid reabsorption from the hindgut to be reduced. Diuresis is partially blocked by injected anti-muscakinin antibodies, providing evidence of a hormonal function for this insect myokinin.

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.

Insect diuretic hormones are crucial for control of water balance. We isolated from the cockroach Diploptera punctata two diuretic hormones (DH), Dippu-DH 31 and Dippu-DH 46 , which increase cAMP production and fluid secretion in Malpighian tubules of several insect species. Dippu-DH 31 and -DH 46 contain 31 and 46 amino acids, respectively. Dippu-DH 46 belongs to the corticotropin-releasing factor (CRF)-like insect DH family, whereas Dippu-DH 31 has little sequence similarity to the CRF-like DH, but is similar to the calcitonin family. Dippu-DH 46 and -DH 31 have synergistic effects in D. punctata but have only additive effects in Locusta migratoria . Dippu-DH 31 represents a distinct type of insect DH with actions that differ from those of previously identified insect peptides with diuretic activity.

Three isoforms of trypsin were identified in midgut preparations from Locusta migratoria. Ammonium-sulphate-fractionated luminal contents of midguts were subjected to benzamidine affinity chromatography; proteins eluted by benzamidine were then separated by anion-exchange chromatography. Cationic (TRY 1) and anionic (TRY 2) trypsin activities were eluted from the DEAE column. TRY 1 was homogeneous, producing a single band of Mr 23,000 on SDS-PAGE. TRY 2 comprised two trypsins, TRY 2A (Mr 27,000) and TRY 2B (Mr 29,000). Following a subsequent chromatography step using a Bio-Rad UNO Q column, TRY 2A and TRY 2B were resolved to homogeneity. When homogenates of midgut caecae were the starting material for chromatography, SDS-PAGE of benzamidine-eluted proteins revealed an additional putative trypsin of Mr 17,000 (termed SERP 17) which had been absent from luminal enzyme preparations. Determination of the N-terminal 11 amino acid residues of each protein revealed unique, but similar sequences. The four sequences all began with IVGG, a motif which signifies all four proteins are serine proteases. TRY 1, TRY 2A and TRY 2B were shown to contain only trypsin activity and the preparations were sensitive to inhibition by AEBSF, PMSF, TLCK, benzamidine, leupeptin, SBTI, BPTI and E64.

A comparison of solution conformations of active, restricted-conformation analogues of two sequence-similar insect/vertebrate neuropeptide family pairs shed light on the potential existence of molecular evolutionary relationships. Analogues of the locustatachykinins and the mammalian tachykinin substance P, containing a sterically hindered Aib-NMePhe/Tyr residue block, share similar low-energy turn conformations incorporating a cis peptide bond. Conversely, restricted conformation analogues of the insect kinins and the mammalian opiate peptide Tyr-W-MIF-1, with near identical C-terminal tetrapeptide sequences, adopt different conformations. The insect kinins adopt a cisPro 1-4 beta-turn, in which the Phe1 is critical for bioactivity. Tyr-W-MIF-1 prefers a transPro 2-5 turn, and an additional N-terminal Phe severely inhibits mu-opiate receptor binding. Comparisons of the chemical/conformational requirements for receptor interaction are consistent with a distant evolutionary relationship between the insectatachykinins and tachykinins, but not between the insect kinins and Tyr-W-MIF-1. Therefore, analogues of the insect kinins with pest control potential can be readily designed to avoid mammalian interactions.

Two chymotrypsin isozymes (CTR 1 and CTR 2) from the midgut lumen of Locusta migratoria have been identified and purified. MALDI-TOF mass spectrometry revealed an M r of 22 679 (±30) for CTR 1 and 22 592 (±30) for CTR 2. Both chymotrypsins hydrolysed S-(Ala) 2 ProPhe-pNA (CTR 1: K m =0.29±0.01 mM, V max =83.0±1.4 U/mg; CTR 2: K m =0.42±0.01 mM, V max =48.9±1.1 U/mg) and S-(Ala) 2 ProLeu-pNA (CTR 1: K m =0.50±0.04 mM, V max =1.7±0.1 U/mg; CTR 2: K m =1.12±0.08 mM, V max =11.4±0.6 U/mg), but neither enzyme hydrolysed BTpNA, S-Phe-pNA, Ac-Leu-pNA or S-(Ala) 3 -pNA. CTR 1 and CTR 2 activities were effectively inhibited by AEBSF, PMSF, TPCK, chymostatin, SBTI and BPTI. Using S-(Ala) 2 ProPhe-pNA as the substrate, CTR 1 gave optimal activity between pH 8.0 and 10.0, while CTR 2 was optimally active over the range pH 8.0–11.0. The N-terminal 15 amino acids of the purified chymotrypsins were determined, revealing their unique sequences which are also different from another, previously characterised Locusta chymotrypsin.

A competitive ELISA employing a polyclonal antiserum raised against leucokinin-I was used to isolate and purify a myokinin (muscakinin) from 1.05 kg of adult houseflies ( Musca domestica ). Following solid-phase purification, seven HPLC column steps were used to purify 4.8 nmol of leucokinin-immunoreactive material. Sequence analysis and mass spectrometry were consistent with the structure Asn-Thr-Val-Val-Leu-Gly Lys-Lys-Gln-Arg-Phe-His-Ser-Trp-Gly NH 2 . This peptide was synthesized and co-eluted with the natural peptide on three different HPLC columns. The activities of natural and synthetic muscakinin were identical, with both producing a 4–5 fold increase in fluid secretion by housefly Malpighian tubules at nanomolar concentrations. The presence of a pair of basic residues (Lys-Lys) suggested muscakinin might be processed further, with the peptide pGlu-Arg-Phe-His-Ser-Trp-Gly NH 2 being produced by conversion of an N-terminal glutamine to pyroglutamic acid. However, this analog was 1000-fold less active than the intact peptide, comparable to the activity of AK-V which shares the same C-terminal pentapeptide sequence. The diuretic activity of muscakinin is more than double that of a previously identified CRF-related diuretic peptide ( Musca -DP) from the housefly, and the two peptides act synergistically in stimulating fluid secretion. Muscakinin also increased the frequency and amplitude of contractions by housefly hindgut which might further contribute to the excretory process.

This paper reports the purification of three myotropic neuropeptides from the white shrimp Penaeus vannamei. The central nervous systems of 3500 shrimps were extracted in an acidified solvent, after which four to five HPLC column systems were used to obtain pure peptides. A cockroach hindgut muscle contraction bioassay was used to monitor all collected fractions. The pure peptides were submitted to Edman degradation based automated microsequencing. Mass spectrometry and chemical synthesis confirmed the sequences. Ala-Pro-Ser-Gly-Phe-Leu-Gly-Met-Arg-NH2 (Pev-tachykinin, 934.1 Da) belongs to the tachykinin family with identified members in all vertebrate classes and some invertebrate classes: arthropods, annelids and molluscs. A very specific Pev-tachykinin antiserum was developed, which labels 4 neurosecretory cells in the brain. Ala-Ser-Phe-Ser-Pro-Trp-Gly-NH2 (Pev-kinin 1, 749.8 Da) and Asp-Phe-Ser-Ala-Trp-Ala-NH2 (Pev-kinin 2, 694.7 Da) are the first crustacean kinins. Pev-kinin 2 is the first kinin with a Trp-Ala-NH2 instead of a kinin-typical Trp-Gly-NH2 carboxyterminus. ispartof: Biochemical and Biophysical Research Communications vol:248 issue:2 pages:406-411 ispartof: location:United States status: published

SYNOPSIS. Diuretic peptides stimulate primary urine production by insect Malpighian tubules and, along with antidiuretic hormones, regulate water loss from the excretory system. There are two structurally distinct families of diuretic peptides. CRF-related diuretic peptides belong to the vertebrate CRF/sauvagine/urotensin I/urocortin superfamily. They increase urine production via a cyclic AMP-dependent mechanism, and there is evidence for one of them (Lom-DH) having a hormonal function in the control of post-feeding diuresis in locusts. Related peptides appear to trigger post-eclosion diuresis in moths. The insect kinins act via a Ca2+-dependent mechanism to stimulate urine production. They are found in hemolymph at levels sufficient to stimulate tubule secretion, but their role in the control of diuresis in vivo has still to be defined. In locusts, these two classes of diuretic peptide act synergistically to accelerate tubule secretion, which may be of physiological relevance. Indeed, synergism may be the norm in controlling tubule secretion, and can involve both neuropeptides and biogenic amines (notably serotonin). Although other diuretic peptides have been identified, their role as circulating hormones has yet to be established.

Coast, G. M. The influence of neuropeptides on Malpighian tubule writhing and its significance for excretion. Peptides 19 (3) 469–480, 1998.—Diuretic peptides (locustakinin and Locusta -DH) increase the spontaneous contractile activity of visceral muscle fibers associated with Malpighian tubules from the migratory locust ( Locusta migratoria ) at concentrations that increase urine production. Muscle activity is shown to assist the flow of material in the tubule lumen, but is not essential for diuresis. Tubule writhing also serves to reduce unstirred layers (USLs) at the basolateral surface of the epithelium and thereby facilitates the excretion of solutes entering the lumen by passive diffusion.

The myotropic activity and mode of action of an endogenous corticotropin-releasing factor (CRF)-related diuretic peptide ( Acheta -DP) was investigated using isolated denervated foreguts from the house cricket, Acheta domesticus . Acheta -DP increased the frequency and amplitude of contractions of the foregut at a threshold concentration of 3.74 ± 0.002 n m . The response to the peptide was unaffected by tetrodotoxin (TTX), and is likely to be a direct action on the muscle cells. Acheta -DP caused a 10-fold increase in cyclic AMP production by isolated foreguts. The ability of IBMX to potentiate the myotropic activity of Acheta -DP suggests the peptide acts via a cyclic AMP-dependent mechanism. In support of this, forskolin produced a peptide-like response, and enhanced the effect of Acheta -DP. Myotropic activity was dependent on extracellular calcium, and was inhibited by the L-type calcium channel antagonist, nifedipine. Bay K 8644 enhanced the response of the foregut to both Acheta -DP and to dibutyryl cyclic AMP, suggesting that the physiological response arose from calcium influx through L-type calcium channels. It is proposed that Acheta -DP binds to a receptor on muscle fibre membranes activating adenylyl cyclase. The resultant increase in intracellular cyclic AMP is thought to open L-type calcium channels and thereby stimulate gut contractions.

This study provides clear evidence for synergism between diuretic peptides from Locusta migratoria, Locusta-DP (a CRF-related peptide) and locustakinin (an insect myokinin). At low concentrations, the peptides act co-operatively to stimulate Malpighian tubule fluid secretion by more than the sum of their separate responses. They work via different second messengers, and their synergistic actions can be mimicked with exogenous 8-bromo-cyclic AMP replacing Locusta-DP, and/or with thapsigargin substituting for locustakinin. Additionally, 5-HT, which had been shown previously to stimulate tubule secretion via a cyclic AMP-independent mechanism, potentiated the response to Locusta-DP. Potassium is invariably the dominant cation secreted by locust tubules, but Locusta-DP, whether in the absence or presence of locustakinin, increases JNa at the expense of JK. In contrast, JNa and JK increase in parallel with JV after stimulation by locustakinin. These findings are consistent with the known actions of cyclic AMP and leucokinin-VIII on mosquito tubules. The ability to vary the relative amounts of Na+ and K+ delivered to the hindgut may be important for the maintenance of haemolymph volume and composition in recently fed locusts.

A series of insect kinin analogues, AFFPWG-X, modified at the C -terminal group, were evaluated in a cricket Malpighian tubule secretion bioassay. The results were compared with activity profiles observed in a cockroach hindgut myotropic bioassay for these analogues. Although the replacement of the C -terminal amide group with a negatively charged acid leads to a precipitous drop in diuretic activity, it can be partially restored with the introduction of ester groups such as methyl or benzyl. The presence of branched chain character in the C -terminal group or a C -terminal α-carbon-amide distance spanning five methylene group spacers is incompatible with the receptor interaction required for biological activity. Significant diuretic activity is retained with four or fewer methylene groups in this region. C -terminal group analogues containing -SCH 3 , -NHCH 2 CH 2 OCH 3 , or -OCH 2 (C 6 H 5 ) offered the greatest retention of diuretic activity while providing increased hydrophobicity and/or steric bulk. The data are of potential value in the development of mimetic analogues of this insect neuropeptide family. Mimetic analogues are potentially valuable tools to insect neuroendocrinologists studying diuresis and/or engaged in the development of future pest management strategies.

The CAP2b neuropeptide family plays an important role in the regulation of the processes of diuresis and/or antidiuresis in a variety of insects. While Manse-CAP2b (pELYAFPRV-NH2) and native CAP2bs elicit diuretic activity in a number of species of flies, native CAP2b sequences have been shown to elicit antidiuretic activity in the kissing bug Rhodnius prolixus and the green stink bug Acrosternum hilare, the latter being an important pest of cotton and soybean in the southern United States. Analogs of CAP2b containing either a (Z)-alkene, cis-Pro or an (E)-alkene, trans-Pro isosteric component were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretion by Malpighian tubules isolated from A. hilare. The conformationally constrained trans-Pro analog demonstrated statistically significant antidiuretic activity, whereas the cis-Pro analog failed to elicit activity. The results are consistent with the adoption of a trans orientation for the Pro in CAP2b neuropeptides during interaction with receptors associated with the antidiuretic process in the stink bug. In addition, the results are further consistent with a theory of ligand-receptor coevolution between the CAP2b and pyrokinin/PBAN neuropeptide classes, both members of the '-PRXamide' superfamily. This work further identifies a scaffold with which to design mimetic CAP2b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting CAP2b-regulated diuretic/antidiuretic functions.

Book synopsis: The publication of the extensive seven-volume work Comprehensive Molecular Insect Science provided a complete reference encompassing important developments and achievements in modern insect science. One of the most swiftly moving areas in entomological and comparative research is endocrinology, and this volume, Insect Endocrinology, is designed for those who desire a comprehensive yet concise work on important aspects of this topic. Because this area has moved quickly since the original publication, articles in this new volume are revised, highlighting developments in the related area since its original publication. Insect Endocrinology covers the mechanism of action of insect hormones during growth and metamorphosis as well as the role of insect hormones in reproduction, diapause and the regulation of metabolism. Contents include articles on the juvenile hormones, circadian organization of the endocrine system, ecdysteroid chemistry and biochemistry, as well as new chapters on insulin-like peptides and the peptide hormone Bursicon. This volume will be of great value to senior investigators, graduate students, post-doctoral fellows and advanced undergraduate research students. It can also be used as a reference for graduate courses and seminars on the topic. Chapters will also be valuable to the applied biologist or entomologist, providing the requisite understanding necessary for probing the more applied research areas.

An identical CRF-related diuretic peptide (Musca-DP) was isolated and characterized from whole-body extracts of the house fly, Musca domestica, and stable fly, Stomoxys calcitrans. The peptide stimulates cyclic AMP production in Manduca sexta Malpighian tubules and increases the rate of fluid secretion by isolated Musca domestica tubules. The 44-residue peptide, with a mol.wt. of 5180, is amidated, and has the primary structure: NKPSLSIVNPLDVLRQRLLLEIARRQMKENTRQVELNRAILKNV-NH2. Musca-DP has a high percentage of sequence identity with other characterized CRF-related insect diuretic peptides.

The multifunctional 'insect kinins' of arthropods share the evolutionarily conserved C-terminal pentapeptide core sequence Phe-X(1)-X(2)-Trp-Gly-NH(2), where X(1)=His, Asn, Ser, or Tyr and X(2)=Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects, including the house cricket, Acheta domesticus. Insect kinins, however, are susceptible to fast enzymatic degradation by endogenous peptidases that severely limit their potential use as tools for pest control or for endocrinological studies. To enhance resistance to peptidases, the core insect kinin sequence was structurally modified in this study to replace native peptide bonds susceptible to proteolytic degradation. These modifications include incorporation of two stereochemical variants of the β-turn mimetic motif 4-aminogutamate in place of the X(1)-X(2) residues, insertion of a reduced peptide bond between residues Trp-Gly, and replacement of the Phe residue with a hydrocinnamyl group. The resulting biostable, peptidomimetic analogs contain no native peptide bonds and yet retain significant diuretic activity in an in vitro cricket Malpighian tubule fluid secretion assay, matching the efficacy of a native A. domesticus kinin (Achdo-KI). These novel analogs represent ideal new tools for endocrinologists studying arthropod kinin regulated processes in vivo, and provide leads in the development of novel, environmentally friendly pest insect management agents capable of disruption of the critical processes that kinins regulate.

ELISA experiments revealed that an antiserum raised against an achetakinin-analog could specifically detect the recently isolated Culekinin Depolarizing Peptide (CDP)-II from the mosquito, Culex salinarius. The characterization indicated that two different epitopes in the C-terminal region of achetakinin I and CDP-II are recognized. One epitope is the -F-Y-region, the other is the -P-W-region. Among the peptides isolated from C. salinarius, the antiserum reacts only with CDP-II. Pre-absorption tests of the antiserum with CDP-II in immunohistological stainings abolished the reaction, while tests with pre-immune sera did not cause any immunopositive reactions. In the mosquito head ganglia, immunoreactive neurons were detected in the pars lateralis, the optic lobe and the suboesophageal ganglion. Although some immunopositive axons extended into the nervi corporis cardiacii II, no immunoreactivity was observed in the retrocerebral complex. In the thoracic ganglia, immunoreactive neurons were found in the pro-, meso- and metathoracic neuromeres. No immunoreactivity was found elsewhere. With this study we demonstrate that CDP-II, isolated from a whole body extract, is truly a neuropeptide, and the data suggest that its function is neuromodulating or neurotransmitting rather than neurohormonal.

The nomenclature currently in use for insect neuropeptide and peptide hormone families is reviewed and suggestions are made as to how it can be rationalized. Based upon this review, a number of conventions are advanced as a guide to a more rationale nomenclature. The scheme that is put forward builds upon the binomial nomenclature scheme proposed by Raina and Gade in 1988 [100], when just over 20 insect neuropeptides had been identified. Known neuropeptides and peptide hormones are assigned to 32 structurally distinct families, frequently with overlapping functions. The names given to these families are those that are currently in use, and describe a biological function, homology to known invertebrate/vertebrate peptides, or a conserved structural motif. Interspecific isoforms are identified using a five-letter code to indicate genus and species names, and intraspecific isoforms are identified by Roman or Arabic numerals, with the latter used to signify the order in which sequences are encoded on a prepropeptide. The proposed scheme is sufficiently flexible to allow the incorporation of novel peptides, and could be extended to other arthropods and non-arthropod invertebrates.

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.

Previously, a corticotropin releasing factor (CRF)-like diuretic peptide (Man-duca-DH) has been isolated from Manduca sexta and shown to stimulate fluid excretion in vivo in post-eclosion Pieris rapae adults and in pre-wandering postfeeding Manduca sexta larvae. However, Manduca-DH was reported to have no effect on Malpighian tubules in vitro. Manduca-DH and [Nle2,11]-Manduca-DH were synthesized in Texas and assayed in London on isolated Malpighian tubules of Acheta domesticus. Man-duca-DH stimulated fluid secretion by about 60% of the maximum response achievable with extracts of corpora cardiaca and increased the production of cyclic AMP. In combination with 10−4moll−1 3-isobutyl-l-methyl xanthine (IBMX), Manduca-DH stimulated maximal secretion. A number of CRF-related peptides also stimulated fluid secretion and cyclic AMP production in cricket tubules, and the CRF antagonist α-helical-CRF[9–41] blocked the stimulation of fluid secretion by Manduca-DH. [Nle2,11]-Manduca-DH was more active than Manduca-DH in both assays, suggesting that methionine residues in the natural peptide may become oxidized. Taken in conjunction with previous in vivo studies, the present findings suggest that a Manduca-DH-like diuretic peptide is the hormone controlling post-eclosion diuresis in butterflies, and Manduca-DH was shown to stimulate both fluid secretion and cyclic AMP production in Malpighian tubules from 1–12 h posteclosion Pieris rapae adults. The function of the peptide in Manduca sexta is discussed.

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.

Direct and indirect assays of diuresis in insects are reviewed and their advantages and disadvantages discussed in relation to their use during the isolation and characterisation of diuretic factors. The nature of known and putative diuretic peptides is contrasted and points of controversy over the identity of these peptides highlighted. The isolation and characterisation of a putative diuretic peptide from Acheta domesticus is discussed as a case study in the purification and characterisation of such peptides.

At 10−9 M achetakinins double the rate of fluid secretion by isolated Malpighian tubules of the house cricket, Acheta domesticus. They appear to act independently of cAMP, and the diuretic responses to a cAMP analogue (8-bromo-cAMP) and achetakinin-I are additive. At 10−9 M achetakinin-I potentiates the diuretic response to exogenous 8-bromo-cAMP. In the presence of 10−5 M 8-bromo-cAMP, achetakinins, I, II, III, IV and V stimulate fluid secretion maximally, and have ED50 values ranging from 1.8 × 10−11 M to 3.2× 10−10 M. The pentapeptide C-terminal sequence FX1X2WG. NH2 (where X1 = H, N, S, Y, and X2 = S or P), common to achetakinins I–V, is all that is required for full biological activity. A possible role for achetakinins as diuretic hormones in vivo is discussed.

Insect neuropeptides of the insect kinin class share a common C-terminal pentapeptide sequence F(1)X(1)(2)X(2)(3)W(4)G(5)-NH(2) (X(2)(3) = P, S, A) and regulate such critical physiological processes as water balance and digestive enzyme release. Analogs of the insect kinin class, in which the critical residues of F(1), P(3), and W(4) were replaced with beta(3)-amino acid or their beta(2)-homo-amino acid variants, have been synthesized by the solid phase peptide strategy. The resulting single- and double-replacement analogs were evaluated in an insect diuretic assay and enzyme digestion trials. Analogs modified in the core P(3) position produce a potent and efficacious diuretic response that is not significantly different from that obtained with the endogenous achetakinin peptides. The analogs also demonstrate enhanced resistance to hydrolysis by ACE and NEP, endopeptidases that inactivate the natural insect neuropeptides. This paper describes the first instance of beta-amino acids analogs of an arthropod peptide that demonstrate significant bioactivity and resistance to peptidase degradation.

An overview is given of the role of Malpighian (renal) tubules and the hindgut (ileum and rectum) in the excretory process of insects. The review focuses on the mechanism of primary urine production by Malpighian tubules and its control by neurohormones, which includes serotonin and neuropeptides resembling mammalian corticotropin-releasing factor (CRF) and calcitonin. Particular emphasis is given to in vitro studies of the effect of neurohormones on Malpighian tubule ion transport and a consideration of their likely role in the regulation of salt balance in vivo.

The insect kinins are present in a wide variety of insects and function as potent diuretic peptides, though they are subject to rapid degradation by internal peptidases. Insect kinin analogs incorporating stereochemical variants of (2S,4S)-4-aminopyroglutamate (APy), a cis-peptide bond motif, demonstrate significant activity in a cricket diuretic assay. Insect kinin analogs containing (2R,4R)-APy, (2S,4R)-APy and (2S,4S)-APy are essentially equipotent on an insect diuretic assay, with EC50 values of about 10−7M, whereas the (2R,4S)-APy analog is at least 10-fold more potent (EC50 = 7 × 10−9M). Conformational studies in aqueous solution indicate that the (2R,4S)-APy analog is considerably more flexible than the other three variants, which may explain its greater potency. The work identifies the optimal stereochemistry for the APy scaffold with which to design biostable, peptidomimetic analogs with the potential to disrupt critical insect kinin-regulated processes in insects. © 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88:1–7, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.