Thirst-driven hygrosensory suppression promotes water seeking in Drosophila.

Survival in animals relies on navigating environments aligned with physiological needs. In Drosophila melanogaster, antennal ionotropic receptors (IRs) sensing humidity changes govern hygrotaxis behavior. This study sheds light on the crucial role of IR8a neurons in the transition from high humidity avoidance to water-seeking behavior when the flies become thirsty. These neurons demonstrate a heightened calcium response toward high humidity stimuli in satiated flies and a reduced response in thirsty flies, modulated by fluctuating levels of the neuropeptide leucokinin, which monitors the internal water balance. Optogenetic activation of IR8a neurons in thirsty flies triggers an avoidance response similar to the moisture aversion in adequately hydrated flies. Furthermore, our study identifies IR40a neurons as associated with dry avoidance, while IR68a neurons are linked to moist attraction. The dynamic interplay among these neurons, each with opposing valences, establishes a preference for approximately 30% relative humidity in well-hydrated flies and facilitates water-seeking behavior in thirsty individuals. This research unveils the intricate interplay between sensory perception, neuronal plasticity, and internal states, providing valuable insights into the adaptive mechanisms governing hygrotaxis in Drosophila.

AI protein structure prediction-based modeling and mutagenesis of a protostome receptor and peptide ligands reveal key residues for their interaction.

The protostome leucokinin (LK) signaling system, including LK peptides and their G protein-coupled receptors, has been characterized in several species. Despite the progress, molecular mechanisms governing LK peptide-receptor interactions remain to be elucidated. Previously, we identified a precursor protein for Aplysia leucokinin-like peptides (ALKs) that contains the greatest number of amidated peptides among LK precursors in all species identified so far. Here, we identified the first ALK receptor from Aplysia, ALKR. We used cell-based IP1 activation assays to demonstrate that two ALK peptides with the most copies, ALK1 and ALK2, activated ALKR with high potencies. Other endogenous ALK-derived peptides bearing the FXXWX-amide motif also activated ALKR to various degrees. Our examination of cross-species activity of ALKs with the Anopheles LK receptor was consistent with a critical role for the FXXWX-amide motif in receptor activity. Furthermore, we showed, through alanine substitution of ALK1, the highly conserved phenylalanine (F), tryptophan (W), and C-terminal amidation were each essential for receptor activation. Finally, we used an artificial intelligence-based protein structure prediction server (Robetta) and Autodock Vina to predict the ligand-bound conformation of ALKR. Our model predicted several interactions (i.e., hydrophobic interactions, hydrogen bonds, and amide-pi stacking) between ALK peptides and ALKR, and several of our substitution and mutagenesis experiments were consistent with the predicted model. In conclusion, our results provide important information defining possible interactions between ALK peptides and their receptors. The workflow utilized here may be useful for studying other ligand-receptor interactions for a neuropeptide signaling system, particularly in protostomes.

Two Lys-vasopressin-like peptides, EFLamide, and other phasmid neuropeptides.

Phasmid neuropeptide genes were identified in the genomes of two phasmids, Timema cristinae and Clitarchus hookeri. The two species belong to two sisters groups, the Timematodea and Euphasmatodea respectively. Neuropeptide genes were identified using the BLAST+ program on the genome assemblies and the absence of some neuropeptides was confirmed by the concomitant absence of their G-protein coupled receptors. Both genomes were assembled using short reads and the average coverage of the genome is more than 166 times for both species. This makes it virtually impossible that there would not be a single short read for at least one of the conserved transmembrane regions of a GPCR coded by such a genome. Hence, when not a single read can be found for a specific GPCR, it can be concluded that the particular gene is absent from that species. Most previously identified insect neuropeptides are used by these two species. Of the three arthropod allatostatin C related peptides, only allatostatins CC and CCC are present. Both species lack leucokinin, while sulfakinin and dilp8 signaling is absent from Clitarchus, but present in Timema. Interestingly, whereas Timema has lost a vasopressin-related peptide, the gene coding such a peptide is amplified in the Clitarchus genome. Furthermore, while Clitarchus has a specific tryptopyrokinin gene, Timema does not and in this species tryptopyrokinin is coded only by the pyrokinin and periviscerokinin genes. Finally, both species have genes coding EFLamide and its GPCR; in phasmids these genes codes for one (Clitarchus) or two (Timema) EFLamide paracopies.

Evaluation of Aib and PEG-polymer insect kinin analogs on mosquito and tick GPCRs identifies potent new pest management tools with potentially enhanced biostability and bioavailability.

Insect kinins modulate aspects of diuresis, digestion, development, and sugar taste perception in tarsi and labellar sensilla in mosquitoes. They are, however, subject to rapid biological degradation by endogenous invertebrate peptidases. A series of α-aminoisobutyric (Aib) acid-containing insect kinin analogs incorporating sequences native to the Aedes aegypti mosquito aedeskinins were evaluated on two recombinant kinin invertebrate receptors stably expressed in cell lines, discovering a number of highly potent and biostable insect kinin mimics. On the Ae. aegypti mosquito kinin receptor, three highly potent, biostable Aib analogs matched the activity of the Aib-containing biostable insect kinin analog 1728, which previously showed disruptive and/or aversive activity in aphid, mosquito and kissing bug. These three analogs are IK-Aib-19 ([Aib]FY[Aib]WGa, EC50 = 18 nM), IK-Aib-12 (pQKFY[Aib]WGa, EC50 = 23 nM) and IK-Aib-20 ([Aib]FH[Aib]WGa, EC50 = 28 nM). On the Rhipicephalus (Boophilus) microplus tick receptor, IK-Aib-20 ([Aib]FH[Aib]WGa, EC50 = 2 nM) is more potent than 1728 by a factor of 3. Seven other potentially biostable analogs exhibited an EC50 range of 5-10 nM, all of which match the potency of 1728. Among the multi-Aib hexapeptide kinin analogs tested the tick receptor has a preference for the positively-charged, aromatic H over the aromatic residues Y and F in the X1 variable position ([Aib]FX1[Aib]WGa), whereas the mosquito receptor does not distinguish between them. In contrast, in a mono-Aib pentapeptide analog framework (FX1[Aib]WGa), both receptors exhibit a preference for Y over H in the variable position. Among analogs incorporating polyethylene glycol (PEG) polymer attachments at the N-terminus that can confer enhanced bioavailability and biostability, three matched or surpassed the potency of a positive control peptide. On the tick receptor IK-PEG-9 (P8-R[Aib]FF[Aib]WGa) was the most potent. Two others, IK-PEG-8 (P8-RFFPWGa) and IK-PEG-6 (P4-RFFPWGa), were most potent on the mosquito receptor, with the first surpassing the activity of the positive control peptide. These analogs and others in the IK-Aib series expand the toolbox of potent analogs accessible to invertebrate endocrinologists studying the structural requirements for bioactivity and the as yet unknown role of the insect kinins in ticks. They may contribute to the development of selective, environmentally friendly pest arthropod control agents.

Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila.

Multiple neuropeptides are known to regulate water and ion balance in Drosophila melanogaster. Several of these peptides also have other functions in physiology and behavior. Examples are corticotropin-releasing factor-like diuretic hormone (diuretic hormone 44; DH44) and leucokinin (LK), both of which induce fluid secretion by Malpighian tubules (MTs), but also regulate stress responses, feeding, circadian activity and other behaviors. Here, we investigated the functional relations between the LK and DH44 signaling systems. DH44 and LK peptides are only colocalized in a set of abdominal neurosecretory cells (ABLKs). Targeted knockdown of each of these peptides in ABLKs leads to increased resistance to desiccation, starvation and ionic stress. Food ingestion is diminished by knockdown of DH44, but not LK, and water retention is increased by LK knockdown only. Thus, the two colocalized peptides display similar systemic actions, but differ with respect to regulation of feeding and body water retention. We also demonstrated that DH44 and LK have additive effects on fluid secretion by MTs. It is likely that the colocalized peptides are coreleased from ABLKs into the circulation and act on the tubules where they target different cell types and signaling systems to regulate diuresis and stress tolerance. Additional targets seem to be specific for each of the two peptides and subserve regulation of feeding and water retention. Our data suggest that the ABLKs and hormonal actions are sufficient for many of the known DH44 and LK functions, and that the remaining neurons in the CNS play other functional roles.

Discovery of leucokinin-like neuropeptides that modulate a specific parameter of feeding motor programs in the molluscan model, Aplysia.

A better understanding of neuromodulation in a behavioral system requires identification of active modulatory transmitters. Here, we used identifiable neurons in a neurobiological model system, the mollusc Aplysia, to study neuropeptides, a diverse class of neuromodulators. We took advantage of two types of feeding neurons, B48 and B1/B2, in the Aplysia buccal ganglion that might contain different neuropeptides. We performed a representational difference analysis (RDA) by subtraction of mRNAs in B48 versus mRNAs in B1/B2. The RDA identified an unusually long (2025 amino acids) peptide precursor encoding Aplysia leucokinin-like peptides (ALKs; e.g. ALK-1 and ALK-2). Northern blot analysis revealed that, compared with other ganglia (e.g. the pedal-pleural ganglion), ALK mRNA is predominantly present in the buccal ganglion, which controls feeding behavior. We then used in situ hybridization and immunohistochemistry to localize ALKs to specific neurons, including B48. MALDI-TOF MS on single buccal neurons revealed expression of 40 ALK precursor-derived peptides. Among these, ALK-1 and ALK-2 are active in the feeding network; they shortened the radula protraction phase of feeding motor programs triggered by a command-like neuron. We also found that this effect may be mediated by the ALK-stimulated enhancement of activity of an interneuron, which has previously been shown to terminate protraction. We conclude that our multipronged approach is effective for determining the structure and defining the diverse functions of leucokinin-like peptides. Notably, the ALK precursor is the first verified nonarthropod precursor for leucokinin-like peptides with a novel, marked modulatory effect on a specific parameter (protraction duration) of feeding motor programs.

Leucokinin signaling regulates hunger-driven reduction of behavioral responses to noxious heat in Drosophila.

In the fruitfly Drosophila melanogaster, hunger has a significant impact on its sensory systems and brain functions, and consequently modifies related behaviors. However, it remains unclarified whether hunger affects nociceptive behavioral responses to heat stimuli. In this study, we show that food deprivation reduces responses to noxious heat in wild-type flies. We further identified that the neuropeptide Leucokinin (Lk) and its receptor (Lkr) are essential for the reduction of responses to noxious heat. Temporal silencing of Lk-expressing neurons and a knockout mutation of Lkr generated using the CRISPR/Cas9 system inhibited the reduction of responses to noxious heat. Thus, our results reveal that hunger induces reduction of responses to noxious heat through the Lk/Lkr signaling pathway in Drosophila.

Origin and specification of the brain leucokinergic neurons of Drosophila: similarities to and differences from abdominal leucokinergic neurons.

BACKGROUND: The Drosophila central nervous system contains many types of neurons that are derived from a limited number of progenitors as evidenced in the ventral ganglion. The situation is much more complex in the developing brain. The main neuronal structures in the adult brain are generated in the larval neurogenesis, although the basic neuropil structures are already laid down during embryogenesis. The embryonic factors involved in adult neuron origin are largely unknown. To shed light on how brain cell diversity is achieved, we studied the early temporal and spatial cues involved in the specification of lateral horn leucokinin peptidergic neurons (LHLKs). RESULTS: Our analysis revealed that these neurons have an embryonic origin. We identified their progenitor neuroblast as Pcd6 in the Technau and Urbach terminology. Evidence was obtained that a temporal series involving the transcription factors Kr, Pdm, and Cas participates in the genesis of the LHLK lineage, the Castor window being the one in which the LHLKs neurons are generated. It was also shown that Notch signalling and Dimmed are involved in the specification of the LHLKs. CONCLUSIONS: Serial homologies with the origin and factors involved in specification of the abdominal leucokinergic neurons (ABLKs) have been detected.

Functional Identification and Characterization of Leucokinin and Its Receptor in the Fall Webworm, Hyphantria cunea.

Neuropeptides function as central neuromodulators and circulating hormones that modulate insect behavior and physiology. Leucokinin (LK) is an intercellular signaling molecule that mediates many physiological and behavioral processes. However, the functions of LK associated with environmental stress and feeding behavior in the fall webworm, Hyphantria cunea, is little known. Our primary objective is to understand the function of LK and LK receptor (LKR) neuroendocrine system in H. cunea. In the present study, the results showed that LK/LKR are expressed at different developmental stages and in various tissues of H. cunea. A candidate receptor-ligand pairing for LK was identified in the larval transcriptome of H. cunea. In a heterologous expression system, the calcium assay was used to demonstrate that LKR is activated by HcLKs in a dose-dependent manner, with 50% effective concentration (EC50) values of 8.44-90.44nM. Knockdown of HcLK and HcLKR by microinjecting target-specific dsRNA leads to several effects in H. cunea, including feeding promotion, increase in resistance to desiccation and starvation stress, and regulation of water homeostasis. The transcript levels of HILP2 (except in the LK knockdown group), HILP5, and HILP8 increased, whereas those of HILP3, HILP4, and HILP6 decreased; HILP1, HILP2 (in the LK knockdown group), and HILP7 gene expression was not influenced after LK and LKR knockdown. Variations in mRNA expression levels in insulin-like peptide genes in the knockdown larvae suggest an essential role of these genes in survival in H. cunea. To our knowledge, the present study is the first comprehensive study of LK and LKR - from gene to behavior - in H. cunea.

Activity of native tick kinins and peptidomimetics on the cognate target G protein-coupled receptor from the cattle fever tick, Rhipicephalus microplus (Acari: Ixodidae).

BACKGROUND: Kinins are multifunctional neuropeptides that regulate key insect physiological processes such as diuresis, feeding, and ecdysis. However, the physiological roles of kinins in ticks are unclear. Furthermore, ticks have an expanded number of kinin paracopies in the kinin gene. Silencing the kinin receptor (KR) in females of Rhipicephalus microplus reduces reproductive fitness. Thus, it appears the kinin signaling system is important for tick physiology and its disruption may have potential for tick control. RESULTS: We determined the activities of endogenous kinins on the KR, a G protein-coupled receptor, and identified potent peptidomimetics. Fourteen predicted R. microplus kinins (Rhimi-K), and 11 kinin analogs containing aminoisobutyric acid (Aib) were tested. The latter incorporated tick kinin sequences and/or were modified for enhanced resistance to arthropod peptidases. A high-throughput screen using a calcium fluorescence assay in 384-well plates was performed. All tested kinins and Aib analogs were full agonists. The most potent kinin and two kinin analogs were equipotent. Analogs 2414 ([Aib]FS[Aib]WGa) and 2412 ([Aib]FG[Aib]WGa) were the most active with EC50 values of 0.9 and 1.1 nM, respectively, matching the EC50 of the most potent tick kinin, Rhimi-K-14 (QDSFNPWGa) (EC50  = 1 nM). The potent analog 2415 ([Aib]FR[Aib]WGa, EC50  = 6.8 nM) includes both Aib molecules for resistance to peptidases and a positively charged residue, R, for enhanced water solubility and amphiphilic character. CONCLUSION: These tick kinins and pseudopeptides expand the repertoire of reagents for tick physiology and toxicology towards finding novel targets for tick management. © 2019 Society of Chemical Industry.

The Cattle Fever Tick, Rhipicephalus microplus, as a Model for Forward Pharmacology to Elucidate Kinin GPCR Function in the Acari.

The success of the acaricide amitraz, a ligand of the tick tyramine/octopamine receptor (a G protein-coupled receptor; GPCR), stimulated interest on arthropod-specific GPCRs as targets to control tick populations. This search advances tick physiology because little is known about the pharmacology of tick GPCRs, their endogenous ligands or their physiological functions. Here we explored the tick kinin receptor, a neuropeptide GPCR, and its ligands. Kinins are pleiotropic insect neuropeptides but their function in ticks is unknown. The endogenous tick kinins are unknown and their cDNAs have not been cloned in any species. In contrast, more than 271 insect kinin sequences are available in the DINeR database. To fill this gap, we cloned the kinin cDNA from the cattle fever tick, Rhipicephalus microplus, which encodes 17 predicted kinins, and verified the kinin gene structure. We predicted the kinin precursor sequences from additional seven tick species, including Ixodes scapularis. All species showed an expansion of kinin paracopies. The "kinin core" (minimal active sequence) of tick kinins FX1X2WGamide is similar to those in insects. Pro was predominant at the X2 position in tick kinins. Toward accelerating the discovery of kinin function in ticks we searched for novel synthetic receptor ligands. We developed a dual-addition assay for functional screens of small molecules and/or peptidomimetics that uses a fluorescent calcium reporter. A commercial library of fourteen small molecules antagonists of mammalian neurokinin (NK) receptors was screened using this endpoint assay. One acted as full antagonist (TKSM02) with inhibitory concentration fifty (IC50) of ∼45 µM, and three were partial antagonists. A subsequent calcium bioluminescence assay tested these four antagonists through kinetic curves and confirmed TKSM02 as full antagonist and one as partial antagonist (TKSM14). Antagonists of NK receptors displayed selectivity (>10,000-fold) on the tick kinin receptor. Three peptidomimetic ligands of the mammalian NK receptors (hemokinin 1, antagonist G, and spantide I) were tested in the bioluminescence assay but none were active. Forward approaches may accelerate discovery of kinin ligands, either as reagents for tick physiological research or as lead molecules for acaricide development, and they demonstrate that selectivity is achievable between mammalian and tick neuropeptide systems.

Variability in the number of abdominal leucokinergic neurons in adult Drosophila melanogaster.

Developmental plasticity allows individuals with the same genotype to show different phenotypes in response to environmental changes. An example of this is how neuronal diversity is protected at the expense of neuronal number under sustained undernourishment during the development of the Drosophila optic lobe. In the development of the Drosophila central nervous system, neuroblasts go through two phases of neurogenesis separated by a period of mitotic quiescence. Although during embryonic development much evidence indicates that both cell number and the cell fates generated by each neuroblast are very precisely controlled in a cell autonomous manner, after quiescence extrinsic factors control the reactivation of neuroblast proliferation in a fashion that has not yet been elucidated. Moreover, there is very little information about whether environmental changes affect lineage progression during postembryonic neurogenesis. Using as a model system the pattern of abdominal leucokinergic neurons (ABLKs), we have analyzed how changes in a set of environmental factors affect the number of ABLKs generated during postembryonic neurogenesis. We describe the variability in ABLK number between individuals and between hemiganglia of the same individual and, by genetic analysis, we identify the bithorax-complex genes and the ecdysone hormone as critical factors in these differences. We also explore the possible adaptive roles involved in this process. J. Comp. Neurol. 525:639-660, 2017. © 2016 Wiley Periodicals, Inc.