Identification of a tachykinin receptor and its implication in carbohydrate and lipid homeostasis in Rhodnius prolixus, a chagas disease vector.

Neuropeptides and their receptors are fundamentally important in regulating many physiological and behavioural processes in insects. In this work, we have identified, cloned, and sequenced the tachykinin receptor (Rhopr-TKR) from Rhodnius prolixus, a vector of Chagas disease. The receptor is a G protein-coupled receptor belonging to the Rhodopsin Family A. The total length of the open reading frame of the Rhopr-TKR transcript is 1110 bp, which translates into a receptor of 338 amino acids. Fluorescent in-situ RNA-hybridization (FISH) for the Rhopr-TKR transcript shows a signal in a group of six bilaterally paired neurons in the protocerebrum of the brain, localized in a similar region as the insulin producing cells. To examine the role of tachykinin signaling in lipid and carbohydrate homeostasis we used RNA interference. Downregulation of the Rhopr-TKR transcript led to a decrease in the size of blood meal consumed and a significant increase in circulating carbohydrate and lipid levels. Further investigation revealed a close relationship between tachykinin and insulin signaling since the downregulation of the Rhopr-TKR transcript negatively affected the transcript expression for insulin-like peptide 1 (Rhopr-ILP1), insulin-like growth factor (Rhopr-IGF) and insulin receptor 1 (Rhopr-InR1) in both the central nervous system and fat body. Taken together, these findings suggest that tachykinin signaling regulates lipid and carbohydrate homeostasis via the insulin signaling pathway.

Expression and functional characterization of tachykinin-related peptides in the blood-feeding bug, Rhodnius prolixus.

Tachykinins (tachykinin-related peptides, TRPs) are multifunctional neuropeptides that have widespread distribution in the central nervous system (CNS) and in the gastrointestinal tract of many insects, and most have been shown to stimulate contractions of visceral muscles. Invertebrate TRPs carry a characteristic conserved C-terminal pentapeptide (FXGXR-amide) and most of them share some amino acid sequence similarities (approx. 45%) with the vertebrate and mammalian tachykinin family. We have functionally characterized the tachykinins in R. prolixus (Rhopr-TKs) and partially cloned the transcript that encodes for the peptide precursor. The transcript encodes 8 Rhopr-TKs, 7 of which are unique with Rhopr-TK 5 having 2 copies. The spatial distribution analysis of the Rhopr-TK transcript indicates that the highest expression levels are in the CNS, but transcript expression is also associated with salivary glands, fat body, dorsal vessel, and the various gut compartments. Rhopr-TK 1, 2 and 5 significantly increase the frequency and amplitude of peristaltic contractions of the salivary glands. Hindgut muscle also displayed a dose-dependent increase in basal tonus in response to Rhopr-TK1, 2 and 5. TK-like immunoreactivity was seen in a small group of processes that are situated on the lateral margins of the hindgut. Interestingly, kinin-like immunoreactivity is seen in immunoreactive processes on the lateral margin of the hindgut as well as fine processes covering the entire hindgut. Co-localization studies show that TK-like staining is always co-localized with kinin-like immunoreactivity, whereas kinin-like staining is seen in the fine processes that are devoid of TK-like immunoreactivity indicating that TKs are most likely released together with kinins to act on the hindgut. Rhopr-Kinin 2 is a potent stimulator of hindgut muscle contraction in R. prolixus. Addition of Rhopr-Kinin 2 and Rhopr-TK 2 to the hindgut leads to a contraction that was additive of the effects of Rhopr-Kinin 2 and Rhopr-TK 2 alone.

Adipokinetic hormone signalling system in the Chagas disease vector, Rhodnius prolixus.

Neuropeptides and their G protein-coupled receptors are widespread throughout Metazoa and in several cases, clear orthologues can be identified in both protostomes and deuterostomes. One such neuropeptide is the insect adipokinetic hormone (AKH), which is related to the mammalian gonadotropin-releasing hormone. AKH has been studied extensively and is known to mobilize lipid, carbohydrates and proline for energy-consuming activities such as flight. In order to determine the possible roles for this signalling system in Rhodnius prolixus, we isolated the cDNA sequences encoding R. prolixus AKH (Rhopr-AKH) and its receptor (Rhopr-AKHR). We also examined their spatial expression pattern using quantitative PCR. Our expression analysis indicates that Rhopr-AKH is only expressed in the corpus cardiacum of fifth-instars and adults. Rhopr-AKHR, by contrast, is expressed in several peripheral tissues including the fat body. The expression of the receptor in the fat body suggests that AKH is involved in lipid mobilization, which was confirmed by knockdown of Rhopr-AKHR via RNA interference. Adult males that had been injected with double-stranded RNA (dsRNA) for Rhopr-AKHR exhibited increased lipid content in the fat body and decreased lipid levels in the haemolymph. Moreover, injection of Rhopr-AKH in Rhopr-AKHR dsRNA-treated males failed to elevate haemolymph lipid levels, confirming that this is indeed the receptor for Rhopr-AKH.

Identification, characterization and expression of a receptor for the unusual myosuppressin in the blood-feeding bug, Rhodnius prolixus.

Myosuppressins are a family of the FMRFamide-like peptides. They have been characterized in many insects and shown to inhibit visceral muscle contraction. Rhodnius prolixus possesses an unusual myosuppressin in that the typical FLRFamide C-terminal motif is unique and ends with FMRFamide. In the present study, we isolated the cDNA sequence for the R. prolixus receptor for this unusual myosuppressin (RhoprMSR). Quantitative PCR indicates high relative transcript expression of RhoprMSR in the central nervous system and also supports the previously described physiological effects of RhoprMS on the digestive system, with expression of the RhoprMSR transcript in the midgut and hindgut. Expression of the RhoprMSR transcript was also found in the female and male reproductive system of 5th instar nymphs, with transcript expression greater in the female reproductive tissues. No expression was found in the salivary glands or Malpighian tubules. A functional receptor expression assay confirmed that the cloned RhoprMSR is indeed activated by RhoprMS (half maximum effective concentration = 42.7 nM). Structure-activity studies based upon both functional receptor assays and physiological assays showed the importance of the HVFMRFamide moiety, as further N-terminal truncation removed all activity.

Reprint of "The distribution and physiological effects of three evolutionarily and sequence-related neuropeptides in Rhodnius prolixus: Adipokinetic hormone, corazonin and adipokinetic hormone/corazonin-related peptide".

We have examined the distribution and physiological effects of three evolutionarily and sequence-related neuropeptides in Rhodnius prolixus. These neuropeptides, adipokinetic hormone (RhoprAKH), corazonin (CRZ) and adipokinetic hormone/corazonin-related peptide (RhoprACP) are present in distinct, non-overlapping neuronal subsets in the central nervous system (CNS), as determined by immunohistochemistry. Corazonin-like immunoreactive cell bodies are present in the brain and ventral nerve cord, whereas ACP-like immunoreactive cell bodies are only present in the brain, and AKH-like immunoreactive cell bodies only present in the corpus cardiacum (CC). The immunoreactivity to ACP, CRZ and AKH in R. prolixus suggests that ACP and CRZ are released within the CNS, and that CRZ and AKH are released as neurohormones from the CC. Injection of RhoprAKH into adult males elevated haemolymph lipid levels, but injection of CRZ or RhoprACP failed to have any effect on haemolymph lipid levels. Corazonin stimulated an increase in heart-beat frequency in vitro, but RhoprAKH and RhoprACP failed to do so. Thus, although all three neuropeptides share sequence similarity, the AKH and CRZ receptors only respond to their own ligand.

The distribution and physiological effects of three evolutionarily and sequence-related neuropeptides in Rhodnius prolixus: Adipokinetic hormone, corazonin and adipokinetic hormone/corazonin-related peptide.

We have examined the distribution and physiological effects of three evolutionarily and sequence-related neuropeptides in Rhodnius prolixus. These neuropeptides, adipokinetic hormone (RhoprAKH), corazonin (CRZ) and adipokinetic hormone/corazonin-related peptide (RhoprACP) are present in distinct, non-overlapping neuronal subsets in the central nervous system (CNS), as determined by immunohistochemistry. Corazonin-like immunoreactive cell bodies are present in the brain and ventral nerve cord, whereas ACP-like immunoreactive cell bodies are only present in the brain, and AKH-like immunoreactive cell bodies only present in the corpus cardiacum (CC). The immunoreactivity to ACP, CRZ and AKH in R. prolixus suggests that ACP and CRZ are released within the CNS, and that CRZ and AKH are released as neurohormones from the CC. Injection of RhoprAKH into adult males elevated haemolymph lipid levels, but injection of CRZ or RhoprACP failed to have any effect on haemolymph lipid levels. Corazonin stimulated an increase in heart-beat frequency in vitro, but RhoprAKH and RhoprACP failed to do so. Thus, although all three neuropeptides share sequence similarity, the AKH and CRZ receptors only respond to their own ligand.

Isolation, cloning and expression of the Crustacean Cardioactive Peptide gene in the Chagas' disease vector, Rhodnius prolixus.

The blood-gorging bug, Rhodnius prolixus, is a major vector of Chagas' disease in Central and South America. We have cloned and characterized the crustacean cardioactive peptide (CCAP) gene in R. prolixus. The RhoprCCAP gene contains five exons and four introns, and encodes a 129 amino acid prepropeptide, which following post-translation processing, produces CCAP. The predicted RhoprCCAP amino acid sequence is identical to CCAP of crustaceans and other insects, i.e. it is highly conserved. RhoprCCAP mRNA is observed in the central nervous system (CNS) using reverse transcriptase (RT) PCR, but not in the gut and salivary glands. In situ hybridization reveals that the expression of CCAP mRNA is localized to a small number of dorsally situated bilaterally paired neurons within the CNS.

The roles of Dippu-allatostatin in the modulation of hormone release in Locusta migratoria.

Dippu-allatostatins (ASTs) have pleiotropic effects in Locusta migratoria. Dippu-ASTs act as releasing factors for adipokinetic hormone I (AKH I) from the corpus cardiacum (CC) and also alter juvenile hormone (JH) biosynthesis and release from the corpus allatum (CA). Dippu-AST-like immunoreactivity is found within lateral neurosecretory cells (LNCs) of the brain and axons within the paired nervi corporis cardiaci II (NCC II) to the CC and the CA, where there are extensive processes and nerve endings over both of these neuroendocrine organs. There was co-localization of Dippu-AST-like and proctolin-like immunoreactivity within these regions. Dippu-ASTs increase the release of AKH I in a dose-dependent manner, with thresholds below 10(-11)M (Dippu-AST 7) and between 10(-13) and 10(-12)M (Dippu-AST 2). Both proctolin and Dippu-AST 2 caused an increase in the cAMP content of the glandular lobe of the CC. Dippu-AST 2 also altered the release of JH from the locust CA, but this effect depended on the concentration of peptide and the basal release rates of the CA. These physiological effects for Dippu-ASTs in Locusta have not been shown previously.

Octopamine modulates spermathecal muscle contractions in Locusta migratoria.

Octopamine was identified in the spermathecal tissue of Locusta migratoria using HPLC and immunohistochemical techniques. Octopamine-like immunoreactive unpaired median neurons were identified in the VIIth and VIIIth (terminal) abdominal ganglia and octopamine-like immunoreactive axons were present in the ventral ovipositor nerve (branches from this nerve innervate the spermatheca). Stimulatory actions of octopamine on myogenic and neurogenic contractions were observed. Dose-dependent increases in the frequency of myogenic contractions and the amplitude of neurogenic contractions were elicited by the application of octopamine to the spermathecal muscle. Non-sustained basal tension increases were noted in some preparations, although these were not found to be dose-dependent. SchistoFLRFamide (PDVDHVFLRFamide) inhibited octopamine-induced contractions by a maximum of about 30%. In the presence of 3-isobutyl-1 -methylxanthine, octopamine increased cAMP levels in all regions of the spermathecal. The largest increase in cAMP content was found in the spermathecal sac, followed by the straight duct and coil duct. Phentolamine blocked octopamine-induced increases in cAMP levels and abolished the actions of octopamine on myogenic contractions.

Evidence of a neural loop involved in controlling spermathecal contractions in Locusta migratoria.

The control of spermathecal contractions in Locusta migratoria via a neural loop was demonstrated using mechanical stimulation and electrophysiological recordings. Extracellular electrophysiological recordings from the receptaculum seminis nerve (N2B2), which innervates the spermathecal sac, were conducted during mechanical stimulation of the genital chamber sensory cells. Activation of the genital chamber sensory cells, using a glass probe approximating the shape and size of an egg, was found to increase the action potential frequency and initiate bursts of action potentials if a tonic frequency of action potentials was present prior to stimulation. If the motor pattern initially consisted of bursts of action potentials, then mechanical stimulation of the genital chamber sensory cells resulted in an increase in firing frequency, in most preparations, with the bursting remaining. Removal of the probe from the genital chamber always returned the motor activity to that noted prior to sensory cell stimulation. Simultaneous electrophysiological recordings from both the left and right receptaculum seminis nerves (N2B2) revealed that the bursts of action potentials were coordinated, although individual action potentials were not coupled one to one. Activation of the genital chamber sensory cells also resulted in increases in spermathecal contraction frequency, an effect which was coordinated with the changes in motor activity. It is proposed that an egg in the genital chamber activates the sensory cells resulting in increases in spermathecal contraction frequency and the subsequent release of spermatozoa onto the micropyle of the egg for fertilization.

Feeding state influences the content of FMRFamide- and tachykinin-related peptides in endocrine-like cells of the midgut of Locusta migratoria.

The midgut of 5th instar male African migratory locust, Locusta migratoria, was found to contain endocrine-like cells that stained positively for FMRFamide-like immunoreactivity. These cells have cell bodies which are tear-drop in shape with processes extending from the cell body. FMRFamide-like immunoreactivity has been described in similar cells in adult midgut tissue [16]. The midgut tissue content of FMRFamide-like immunoreactivity is differentially distributed throughout various regions of the midgut (gastric cecae, anterior and posterior midgut) in 5th instar and varied ages of adult. FMRFamide-like immunoreactivity in midgut tissues decreases significantly by 24 h of starvation, whereas locustatachykinin I-like immunoreactivity does not decrease until 48 h of starvation indicating that there are differential timing effects of these two peptide families on midgut content. HPLC analysis, combined with RIA, of different regions of the midgut tissue from both fed and starved locusts revealed that the relative proportions of the members of the two peptide families vary depending upon the feeding state. These results indicate that the contents of these endocrine-like cells appears to be differentially influenced by the feeding state of the locust.

N-terminal modified analogs of HVFLRFamide with inhibitory activity on the locust oviduct.

New N-terminal analogs of the peptide HVFLRFamide, the minimum sequence of the insect myosuppressins capable of inhibiting spontaneous and induced contractions of the locust oviduct, were synthesized and tested for biologic activity on locust oviduct. Most active, as judged by the ability to inhibit proctolin-induced contractions of locust oviduct, was (N(alpha)-acetyl)-HVFLRFamide. D-Pro-HVFLRFamide was also highly inhibitory. Interestingly, low doses of the pentapeptide analog (N(alpha)-imidazoleacrylyl)-VFLRFamide inhibited oviduct contractions. This is the first pentapeptide analog shown to inhibit contractions of locust oviduct, and this result indicates that the alpha-amino group of His is not absolutely required for inhibitory activity. In all cases when His was replaced by a D-amino acid, the analogs were stimulatory, resulting in an increase in basal tonus of the locust oviduct. The results provide further insight into the structural features of the HVFLRFamide molecule that are required for inhibitory activity on locust oviduct muscle.

The neural control of spermathecal contractions in the locust, Locusta migratoria.

The innervation of the spermatheca and demonstration of neural control of spermathecal contractions in Locusta migratoria was illustrated using anterograde and retrograde fills, combined with electrophysiological stimulation and recording. The anterior portion of the spermatheca receives innervation via the receptaculum seminis nerve (N2B2) from two large ventral neurons and one dorsal neuron. All were bilaterally paired and situated in the VIIIth abdominal ganglion. Three ventral bilaterally paired neurons situated in the VIIIth abdominal ganglion also provide innervation to the posterior portion of the spermatheca via the ductus seminalis aperture nerve (N2B3). Six DUM neurons, located in the VIIIth abdominal ganglion, in addition to two centroposteriorly situated DUM neurons in the VIIth abdominal ganglion, are also associated with these two nerves. N2B4 also provides innervation to the posterior portion of the spermatheca. N2B6b is associated with sensory cells identified in the anterior lateral regions of the genital chamber. The spermatheca contracts spontaneously, with peristaltic contractions beginning at the spermathecal sac and continuing along the length of the spermathecal duct. However electrical stimulation of the ventral ovipositor nerve (VON or N2B), receptaculum seminis nerve (N2B2) and the ductus seminalis aperture nerve (N2B3) indicates that contractions are also under neural control. In particular contractions of the spermathecal sac, coil duct and anterior straight duct are initiated via motor projections from the receptaculum seminis nerve (N2B2) and posterior straight duct contractions are controlled by motor input from the ductus seminalis aperture nerve (N2B3). The results suggest that spermathecal contractions of the anterior and posterior portions of the spermatheca are under separate neural control.

The distribution and myotropic activity of locustatachykinin-like peptides in locust midgut.

The midgut of the African migratory locust, Locusta migratoria, was found to contain endocrine-like cells that stained positively for locustatachykinin I (Lom TK I)-like immunoreactivity. These cells were distributed in an unequal manner throughout the midgut of the locust, with a greater density of Lom TK I-like immunoreactive endocrine-like cells occurring in the posterior region of the midgut. These singly occurring cells appear elongate with an apical extension projecting toward the midgut lumen and a smaller projection extending towards the midgut basal lamina. No immunoreactive neuronal processes were detected along the midgut wall. Radioimmunoassays revealed that the female midgut contained two to three times more Lom TK I-like material than the male midgut, and radioimmunoassay coupled to high-performance liquid chromatography analysis revealed that at least five locustatachykinin isoforms appear to be present in the midgut. This distribution of Lom TK I-like material suggests possible functional differences in the various regions of the midgut. The role that these cells may play in locust midgut secretory activity and motility remains unknown. However, the addition of synthetic Lom TK I through IV to a ring type midgut muscle preparation stimulated contraction of midgut circular muscles, suggesting a possible physiological role for these peptides. Dose-response curves constructed for Lom TK I-IV revealed that the peptide-induced contractions increased in a dose-dependent manner.

Locustatachykinin isoforms in the locust: distribution and quantification in the central nervous system and action on the oviduct muscle.

The presence of locustatachykinin (LomTK)-like immunoreactivity is demonstrated in the central nervous system (CNS) of Locusta migratoria with the use of a polyclonal antiserum raised against LomTK1. By developing a radioimmunoassay with the same antiserum, we have demonstrated picomolar amounts of LomTK-like material in the tissues of the central nervous system. In contrast, only femptomolar amounts of LomTK-like material are associated with the oviduct tissue. The relative amounts of the different LomTK isoforms in the brain and the abdominal ganglionic chain were examined by separating the native peptides on high-performance liquid chromatography and comparing their retention times to synthetic LomTK standards. The amounts of the different isoforms of LomTK differed between and within the two regions of the central nervous system. However, the ratios of the different isoform amounts were similar between the two regions. The myostimulatory activities of LomTKs 1 to 4 were characterized by using the locust oviduct bioassay. LomTKs 1, 2, and 3 appeared to be more efficacious than LomTK4.

The modulation of skeletal muscle contraction by FMRFamide-related peptides of the locust.

The external ventral protractor muscle of the VIIth abdominal segment, M234, is a skeletal muscle that possesses receptors that recognize a range of FMRFamide-related peptides and application of these peptides results in an increase in the amplitude of neurally evoked contractions with little or no effect on basal tonus. FLRFamide itself has the same biologic activity as the extended peptides, whereas truncation to LRFamide or RFamide results in a peptide with no biologic activity. The receptors recognizing these extended FLRFamides, which include SchistoFLRFamide, seem to be different from the SchistoFLRFamide receptors found on locust oviduct visceral muscle. SchistoFLRFamide and the non-peptide mimetic, benzethonium chloride (Bztc), increase the frequency and amplitude of miniature endplate potentials, increase the amplitude of neurally evoked excitatory junction potentials, and result in a hyperpolarisation of resting membrane potential. Bztc, however, also abolishes the active membrane response that may explain its ability to decrease neurally evoked contractions.

The effects of SchistoFLRFamide on contractions of locust midgut.

The midgut of insects has recently been shown to contain numerous endocrine-like cells and the midgut is now considered one of the largest endocrine organs in the insect. Using immunohistochemistry, radioimmunoassay, and muscle bioassay techniques, the midgut of the adult locust, Locusta migratoria, has been investigated for the distribution and possible function of FMRFamide-related peptides contained within these endocrine-like cells and innervation. Endocrine-like cells containing RFamide-like immunoreactivity were observed to be unequally distributed throughout the midgut. RFamide-like immunoreactivity was also seen in the ingluvial ganglion and in the nerves projecting posteriorly to the midgut. These axonal tracts resulted in extensive arborizations over the posterior midgut which were RFamide-like immunoreactive. Radioimmunoassay indicated larger amounts of FMRFamide equivalents in female locust midgut as compared to males with an unequal distribution of FMRFamide-like immunoreactivity in the gastric caeca and in the anterior and posterior parts of the midgut. Circular muscle contraction of the midgut was monitored using a ring-type preparation. Structure/activity studies have shown that the only FMRFamide-related peptides tested that alter circular muscle contraction of the midgut are those that belong to the subfamily referred to as myosuppressins. SchistoFLRFamide, leucomyosuppressin, and ManducaFLRFamide were each capable of lowering basal tonus and inhibiting spontaneous and proctolin-induced contractions of midgut muscle. Further structure/activity studies indicated that HVFLRFamide is the minimum sequence required to achieve inhibition comparable to the parent compound. This work suggests that a possible function for the FMRFamide-related peptides contained within the endocrine cells and innervation of the midgut of the locust may be in modulating midgut contraction and thereby playing a role in digestion.

Proctolin's role in neurally evoked contractions of the locust oviducts.

The effects of proctolin (RYLPT) on neurally evoked contractions of locust oviduct muscle were studied to examine the role of proctolin as a cotransmitter. Increasing the number of stimuli in a burst (from one to 30 stimuli) resulted in an increase in amplitude of contraction of locust oviduct muscle. Proctolin was capable of increasing the amplitude of neurally evoked contractions at lower-stimulus regimes (one- and two-stimulus bursts) but did not do so at higher-stimulus regimes (five- and 10-stimulus bursts). The effects of proctolin were dose dependent within the one- and two-stimulus regimes, with thresholds at 10(-9) M and maxima at 2.5 x 10(-8) M. Addition of proctolin increased the basal tonus and size of a postcontraction relaxation of the oviduct muscle in a dose-dependent manner during all stimulus regimes. However, the effect of proctolin on basal tonus and the postcontraction relaxation was much less at the higher stimulus regimes. Previously, several proctolin analogues have been tested for their ability to antagonize proctolin-induced contractions of the oviduct muscle. Since proctolin is proposed to be a cotransmitter at this neuromuscular junction, one of these analogues, cycloproctolin, was used to antagonize proctolin's effects on neurally evoked contractions. In the presence of the antagonist, the maximum amplitude induced by application of proctolin was decreased by 22.7%, while the proctolin-induced increase in basal tonus was decreased by 45.8%. Finally, the maximum increase in the size of the postcontraction relaxation caused by proctolin was lowered by 32.0%. The results of the present study show that exogenously applied proctolin is an excitant of the oviduct muscle at lower, rather than higher, stimulus regimes, and this latter inaction may be due to the corelease of endogenous proctolin during increased neural stimulation.

Localization and neurohemal release of FMRFamide-related peptides in the stick insect Carausius morosus.

FMRFamide-like immunoreactivity was localized immunohistochemically in the central stomatogastric nervous systems, visceral tissues, and the neurohemal corpora cardiaca, transverse, and segmental nerves. Each of these neurohemal areas contains one morphologically distinct type of immunoreactive neurosecretory granule. The hemolymph level of FMRFamide-like peptides, quantified by RIA, is higher in animals sampled 2 h into the dark cycle, relative to those sampled at mid-light cycle or 9 h into the dark cycle. High potassium depolarization evokes the calcium-dependent release of FMRFamide-like peptides from neurohemal areas in vitro and HPLC fractionation of hemolymph, corpora cardiaca, and their bathing medium suggests that these organs contribute a single peptide to the FMRFamide-related peptides circulating in the hemolymph of active animals.

Proctolin analogues and a proctolin metabolite as antagonist of the peptide proctolin.

The locust oviduct bioassay was used to assess a variety of proctolin analogues as possible agonists and antagonists of the peptide proctolin. Both [alpha-methyl-L-tyrosine2]proctolin and [N-methyl-L-tyrosine2] proctolin were antagonists of proctolin with thresholds of 5 x 10(-9) M. Interestingly, at these threshold doses the analogues were antagonists when applied along with proctolin, being capable of shifting the dose-response curve for proctolin an order of magnitude to the right. Of the three tripeptides tested Tyr-Arg-Thr and Arg-Tyr-Thr showed no agonistic effects and were incapable of antagonizing proctolin-induced contractions. The third tripeptide, Leu-Pro-Thr, showed minimal agonistic effects and when applied with proctolin, significantly decreased the maximum response and increased the ED50 values of the parent compound. Interestingly, this tripeptide is a degradation product of proctolin. Cycloproctolin possessed no agonistic activity up to 10(-5) M but did antagonize proctolin's response in a dose-dependent manner with 2 x 10(-5) M cycloproctolin shifting the proctolin curve nearly two orders of magnitude to the right. Simultaneous application of 10(-9) M [alpha-methyl-L-tyrosine2]proctolin and 10(-5) M cycloproctolin showed some synergistic effect as the maximum response to the peptide was decreased by 21.6% and the dose-response curve shifted further to the right. These proctolin antagonist will be useful tools for examining the physiological importance of proctolin in insects as well as helping to identify receptor subtypes.