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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.

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.

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

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.

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.

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.

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.

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.

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.

A series of truncated and Ala-replacement analogs of the peptide Manse-CAP2b (pELYAFPRV-NH(2)) were assayed for diuretic activity on Malpighian tubules of the housefly Musca domestica (M. domestica). The C-terminal hexapeptide proved to be the active core, the minimum sequence required to retain significant diuretic activity. However, full activity required the C-terminal heptapeptide, which was equipotent with the most active of the native housefly CAP2b peptides. Replacement of Arg(7) and Val(8) with Ala led to inactivity and a large 70-fold drop in potency, respectively, indicating that these were critical residues. The Leu(2) was semicritical, where a six-fold loss in potency was observed. Conversely, the replacement of all other residues with Ala led to much smaller effects on potency and these positions were considered to be noncritical. This structure-activity relationship data can aid in the design of mimetic agonist/antagonist analogs of this diuretic peptide family with enhanced biostability and bioavailability, as tools for arthropod endocrinologists and as potential pest management agents capable of disrupting the water balance in pest flies.

MALDI-TOF mass spectrometric analysis of single lateral abdominal nerves (LANs) demonstrate the presence of the insect kinin Musdo-K in the housefly Musca domestica, and identify heretofore unknown insect kinins in two other Dipteran species as Musdo-K in the stable fly Stomoxys calcitrans and horn fly Haematobia irritans. The insect kinin native to the flesh fly Neobellieria bullata is identified as Drome-K. Musdo-K and Drome-K are identical save for the conservative substitution of Ser for Thr in position 2. The sequences of the insect kinins are, therefore, remarkably conserved throughout Dipterans. The in vitro Malpighian tubule fluid secretion activity of Musdo-K in the stable fly is similar to that in the housefly, whereas that of Drome-K is 30-fold more potent in the flesh fly than in the fruit fly. Given the structural identities of the kinins and CRF-like diuretic hormones of these Dipteran species, the housefly can serve as a model insect for the study of diuretic peptides and their functions in the stable fly and horn fly, both livestock pests.

The diuretic/myotropic insect kinin neuropeptides, which share the common C-terminal pentapeptide core FX(1)X(2)WG-NH(2), reveal primary (X(2)-W) and secondary (N-terminal to F) sites of susceptibility to peptidases bound to corn earworm (H. zea) Malpighian tubule tissue. Analogs designed to enhance resistance to tissue-bound peptidases, and pure insect neprilysin and ACE, demonstrate markedly enhanced in vivo activity in a weight gain inhibition assay in H. zea, and strong in vivo diuretic activity in the housefly (M. domestica). The peptidase-resistant insect kinin analog pQK(pQ)FF[Aib]WG-NH(2) demonstrates a longer internal residence time in the housefly than the native muscakinin (MK), and despite a difference of over 4 orders of magnitude in an in vitro Malpighian tubule fluid secretion assay, is equipotent with MK in an in vivo housefly diuretic assay. Aminohexanoic acid (Ahx) is shown to function as a surrogate for N-terminal Lys, while at the same time providing enhanced resistance to aminopeptidase attack. Peptidaese-resistant insect kinin analogs demonstrate enhanced inhibition of weight gain in larvae of the agriculturally destructive corn earworm moth. Potent peptidase resistant analogs of the insect kinins, coupled with an increased understanding of related regulatory factors, offer promise in the development of new, environmentally friendly pest insect control measures.

Musca kinin (Musdo-K; NTVVLGKKQRFHSWG-NH 2 ) and N-terminal truncated analogs of 4–14 residues in length were assayed for diuretic and myotropic activity on housefly Malpighian tubules and hindgut, respectively. The pentapeptide was the minimum sequence required for biological activity, but it was > 5 orders of magnitude less potent than the intact peptide. The pharmacological profiles of the different analogs in the two assays were very similar, suggesting the same receptor is present on both tissues. Potency was little affected by the deletion of Asn 1 , but was reduced > 10-fold after the removal of Thr 2 . Deletion of the next 5 residues had relatively little effect, but after the second lysyl residue (Lys 8 ) was removed potency fell by one to two orders of magnitude. There was a similar drop in potency after the removal of Arg 10 , and at 100 μM the pentapeptide had only 20% of the diuretic activity of the intact peptide. The importance of Arg 10 was confirmed by comparing dose-response curves for Musdo-K [6–15] and Acheta kinin-V (AFSHWG-NH 2 ) in the diuretic assay; the substitution of arginine by alanine produced a significant reduction in potency and some loss of activity.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic hormone was identified in a BLAST search of the Drosophila genome database. The predicted 31-residue amidated peptide (D. melanogaster DH31; Drome-DH31) was synthesised and tested for activity on fruit fly Malpighian tubules. It increases tubule secretion by approximately 35 % of the response obtained with a myokinin from the housefly Musca domestica (muscakinin; Musdo-K) and has an EC50 of 4.3 nmol l−1. The diuretic activities of Drome-DH31 and Musdo-K were additive when tested at threshold and supra-maximal concentrations, which suggests that they target different transport processes. In support of this, Drome-DH31 increased the rate of secretion by tubules held in bathing fluid with a reduced Cl− concentration, whereas Musdo-K did so only in the presence of Drome-DH31. Stimulation with Drome-DH31 increased the lumen-positive transepithelial potential in the main secretory segment of the tubule. This was attributed to activation of an apical electrogenic proton-translocating V-ATPase in principal cells, since it was associated with hyperpolarisation of the apical membrane potential and acidification of secreted urine by 0.25 pH units. Exogenous 8-bromo-cyclic AMP and cyclic GMP increased tubule secretion to the same extent as Drome-DH31 and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH31 resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol l−1 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH31 activating an apical membrane V-ATPase via cyclic AMP. Since the K+ concentration of the secreted urine was unchanged, it is likely that Drome-DH31 has an equal effect on K+ and Na+ entry across the basolateral membrane.

Rhodnius prolixus, a blood-feeding hemipteran insect, ingests large meals which are followed by rapid diuresis to eliminate excess water and salt. In Rhodnius, serotonin and an unidentified peptide(s) [33,34] have been shown to act as neurohormonal diuretic factors. In other insects, two families of diuretic peptides, the corticotropin releasing factor (CRF)-like, and kinin peptides [9], have been identified and sequenced. Recently, we demonstrated the presence of a CRF-like diuretic peptide in the CNS and digestive system of Rhodnius [47] using immunohistochemistry and bioassay. In this study, combining immunohistochemistry and radioimmunoassay (RIA) techniques, we show the presence of leucokinin-like peptide(s) in the CNS and digestive system of Rhodnius 5th instar. Additionally, double-label immunohistochemistry demonstrates that the leucokinin-like and CRF-like peptides are co-localized in the posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM) and in neurohaemal areas on abdominal nerves one and two, suggesting the possibility of co-release of the peptides into the hemolymph.Partially purified extracts of the CNS and neurohaemal tissue were tested in vitro on Malpighian tubule secretion and cAMP assays. The factors eluting with increasing acetonitrile percentages from Sep-Pak cartridges were assayed in the presence or absence of ketanserin, a serotonin antagonist which blocks the effects of serotonin on Malpighian tubules. The results indicate activity of serotonin and a CRF-like diuretic peptide on Rhodnius Malpighian tubules, but fail to demonstrate activity of the leucokinin-like peptide(s). The rapid diuresis following feeding is a highly coordinated event, requiring the movement of water and salt across the epithelial cells of the crop into the hemolymph, and from the hemolymph across the cells of the Malpighian tubules. The urine then travels along the Malpighian tubules into the hindgut in order to be expelled. The presence of a leucokinin-like peptide(s) in the CNS and digestive system, which co-localizes with a CRF-like peptide(s), suggests that kinins may play a role in the rapid diuresis, although possibly not directly on the Malpighian tubules.

In the migratory locust, the CRF-related diuretic hormone that stimulates fluid secretion by the Malpighian tubules, and the ovary maturing parsin, a neurohormone able to stimulate oogenesis, are produced by the same neuroendocrine cells of the pars intercerebralis in the brain.

Nachman, R. J., R. E. Isaac, G. M. Coast and G. M. Holman. Aib-containing analogues of the insect kinin neuropeptide family demonstrate resistance to an insect angiotensin-converting enzyme and potent diuretic activity. Peptides 18(1) 53–57, 1997.—Analogues of the insect kinin family in which the Xaa 2 residue of the C -terminal pentapeptide core sequence Phe-Xaa 1 -Xaa 2 -Trp-Gly-NH 2 (Xaa 1 = Asn, His, Phe, Ser, or Tyr; Xaa 2 = Ala, Ser, or Pro) is replaced with sterically hindered aminoisobutyric acid (Aib) prove to be resistant to hydrolysis by housefly ( Musca domestica ) angiotensin-converting enzyme (ACE), an endopeptidase capable of hydrolysis and inactivation of the naturally occurring insect kinin peptides. The Aib residue is compatible with formation of a turn in the active core region that is important for the biological activity of the insect kinins. One of the Aib-containing analogues, pGlu-Lys-Phe-Phe-Aib-Trp-Gly-NH 2 , is five- and eightfold more active than the most active endogenous insect kinins in cockroach ( Leucophaea maderae ) hindgut myotropic and cricket ( Acheta domesticus ) Malpighian tubule fluid secretion assays, respectively. As the analogue is blocked at both the amino- and the carboxyl-terminus and resistant to an endopeptidase present in insects, it is better adapted than the endogenous peptides to survive for long periods in the hemolymph. Enzyme-resistant insect kinin analogues can provide useful tools to insect researchers studying the neuroendocrine control of water and ion balance and the physiological consequences of challenging insects with diuretic factors that demonstrate enhanced resistance to peptidase attack. If these analogues, whether in isolation or in combination with other factors, can disrupt the water and/or ion balance, they hold potential utility for the control of pest insect populations in the future.

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.

An antiserum against an achetakinin analog selectively localized leucokinin VIII (LKVIII) in the CNS of Leucophaea maderae. Preabsorption studies of the achetakinin antiserum with either preimmune serum or LKVIII prevented a positive reaction in both ELISA and immunocytochemical procedures. LKVIII immunoreactive neurons were found in the brain, frontal, and subesophageal ganglion, all 3 thoracic ganglia and the terminal ganglion. Nerves originating from the thoracic and terminal abdominal ganglia contain LKVIII material. Lateral and medial neurosecretory cells synthesizing LKVIII-like products contribute axons to the nervi corporis cardiaci that terminate in neurohemal sites in the corpora cardiaca and nervi corporis allati. Thus, leucokinin VIII, like leucokinin I (LKI) and leucomyosuppressin (LMS), appears to have both a neurohemal and neurotransmitter mode of regulating target cells in L. maderae.

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.

A diuretic peptide Locusta-DP, identified by its ability to increase cyclic AMP production in locust Malpighian tubules in vitro, has been isolated and characterized from whole heads of Locusta migratoria. The purified peptide stimulates fluid secretion by Malpighian tubules maximally in vitro. The primary structure of Locusta-DP was established as a 46 residue amidated peptide: MGMGPSLSIVNPMDVLRQRLLLEIARRRLRDAEEQIKANKDFLQQI-NH2. Locusta-DP has 48% sequence identity with Acheta-DP and 49% identity with Manduca-DH, and provides further evidence for the presence of a family of diuretic peptides in insects.