Characterization and deorphanization of RYamide signaling in Aedes aegypti : a potential regulator of hindgut-associated physiology

Characterization and deorphanization of RYamide signaling in Aedes aegypti: a potential regulator of hindgut-associated physiology | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Characterization and deorphanization of RYamide signaling in Aedes aegypti : a potential regulator of hindgut-associated physiology View ORCID Profile J Tan , T Luong , View ORCID Profile JP Paluzzi doi: https://doi.org/10.1101/2025.10.17.683104 J Tan 1 Department of Biology, York University , Toronto, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for J Tan T Luong 1 Department of Biology, York University , Toronto, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site JP Paluzzi 1 Department of Biology, York University , Toronto, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for JP Paluzzi For correspondence: paluzzi{at}yorku.ca Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Neuropeptide Y-related signaling, consisting of various neuropeptides and their receptors including, for example, the mammalian neuropeptide Y (NPY), pancreatic polypeptide (PP), and peptide YY (PYY) families, along with neuropeptide Y-like peptides in insects, is evolutionarily conserved across vertebrates and invertebrate organisms. Vertebrate NPY is known as an important regulator of energy homeostasis and feeding behaviour, while studies on one NPY-associated signaling system in arthropods, known as RYamide, have mainly focused on functions regulating feeding-related behaviours. The current study aimed to begin exploring an additional functional role of RYamide signaling related to excretory physiology in Aedes aegypti given that a candidate RYamide receptor, RYaR2, is enriched in the hindgut of adult mosquitoes based on publicly available RNA-seq databases. Herein, we report that the RYamide transcript is expressed in all post-embryonic stages with greatest abundance in adult male mosquitoes. Specifically, the central nervous system and the ventral nerve cord were demonstrated as the major sources of RYamide, as supported by RT-qPCR and intense RYamide immunoreactivity observed in the protocerebral posterior dorsomedial neurons and protocerebral anterolateral neurons in the mushroom body region, along with an intensively stained pair of neurons in the terminal abdominal ganglion (TAG). The pair of immunoreactive neurons in the TAG contain elaborate processes extending onto the hindgut, with a branch extending posteriorly over the rectum where fine processes are in close proximity to the rectal papillae (pads) while an anterior branch extends over the ileum terminating at the midgut-hindgut junction forming a fine circular plexus over the pyloric valve. Importantly, two orphan GPCRs, previously referred to as NPYLRs, were functionally deorphanized as bona fide RYamide receptors. Their responses to diverse peptidergic ligands were assessed, revealing that RYamides elicited the strongest receptor activation, with half maximal effective concentrations in the pico- to nanomolar range. Notably, our phylogenetic analysis revealed that while only a single RYaR is commonly found across most arthropods, culicine mosquito species including A. aegypti possess two RYaRs. The characterization of RYamide signaling with respect to excretory physiology involving the hindgut, particularly in species having two receptor isoforms such as culicine mosquitos, may provide valuable insights for development of novel, species-specific pest control strategies. 1. Introduction The yellow fever mosquito, Aedes aegypti , an African originated mosquito species that is widely distributed in tropical and sub-tropical regions ( Carvalho and Moreira, 2017 ; Kraemer et al., 2015 ), is recognized as a principal vector capable of transmitting dengue, yellow fever, chikungunya and other disease agents through its blood feeding behaviour ( Kotsakiozi et al., 2017 ; Morrison et al., 2008 ). Expanded habitats have led to an increased risk of mosquito-borne diseases among human populations ( Juliano and Philip Lounibos, 2005 ). Global climate change and increased urbanization are key factors for expanding geographic locations that are suitable habitats of Ae. aegypti ( Caminade et al., 2019 ; Jansen and Beebe, 2010 ; Reinhold et al., 2018 ; Sprenger and Reiter, 1987 ). The anthropophilic characteristic of Ae. aegypti and their exceptional ability to adapt to the urban environment elevate health risks in human communities. In addition to current mosquito control strategies, such as Wolbachia infection ( Mains et al., 2019 ), male sterilization ( Tussey et al., 2023 ), and traditional insecticides ( McGregor and Connelly, 2021 ), there is a growing need to develop novel, more targeted approaches for effective mosquito control. Recently, neuropeptide Y-like receptor 7 (NPYLR7), but not a closely related NPY-like receptor 5 (NPYLR5), has been shown to act as a key regulator of host-seeking behaviour in female mosquitoes, with its activation by a small-molecule agonist resulting in suppression of host-seeking and blood feeding ( Duvall et al., 2019 ; Zeledon et al., 2024 ), making it a potential target for pest management. Nonetheless, despite NPYLR7 being activated by a human neuropeptide Y (NPY) Y2 receptor agonist and several synthetic small-molecule agonists ( Duvall et al., 2019 ), it remained an orphan receptor in mosquitoes as no endogenous ligands were identified. While the endogenous peptidergic ligands for NPYLR5 and NPYLR7 remain unknown in mosquitoes, earlier phylogenetic analysis examining luqin-type neuropeptide signaling provided evidence that NPYLR5 belongs to an orthologous group that includes receptors for arthropod RYamide receptors ( Jékely, 2013 ; Mirabeau and Joly, 2013 ; Yañez-Guerra et al., 2018 ). The RYamide prepropeptide, encoded by a single gene, is widely conserved across arthropods ( Hauser et al., 2010 ), such as Drosophila melanogaster ( Ida et al., 2011 ; Veenstra and Khammassi, 2017 ), Ae. aegypti , Bombyx mori ( Roller et al., 2016 ). However, unlike the parasitic wasp Nasonia vitripennis , where seven peptide isoforms are produced, the prepropeptide in most insects, including Ae. aegypti , is typically cleaved forming only two peptide isoforms of RYamide and/or RFamide, which share a conserved FFXXXRY/Famide motif ( Hauser et al., 2010 ). RYamide shares a similar C-terminal sequence (GxRYamide, where X is S in most examined species) ( Hauser et al., 2010 ) with insect neuropeptide F (NPF) peptides that often contain RxRFamide, where X is often P, A or V and terminal F sometimes substituted with a Y residue ( Nässel and Wegener, 2011 ), which are related to the vertebrate NPY family (NPY, pancreatic polypeptide (PP), and peptide YY (PYY)) known to regulate feeding behaviour, energy expenditure and energy homeostasis ( Inui, 1999 ; Loh et al., 2015 ; Qi et al., 2022 ). Additionally, other neuropeptides containing an RF or RYamide motif at the C-terminus, such as short neuropeptide F (sNPF) ( Christ et al., 2018 , 2017 ), have also been identified in mosquitoes. However, there is no evidence suggesting that the genes encoding RYamide, sNPF, and NPF share a common evolutionary origin ( Hauser et al., 2010 ; Nässel and Wegener, 2011 ; Strand et al., 2016 ). Subsequent studies have deorphanized a GPCR encoded by the D. melanogaster gene CG5811 and its orthologs in the red flour beetle Tribolium castaneum and the silkworm Bombyx mori as authentic receptors for RYamides (RYaRs) ( Collin et al., 2011 ; Guo et al., 2021 ; Ida et al., 2011 ), with evidence indicating that the RYamide signaling pathway in silkworms involves Gαq and membrane-bound phospholipase C (PLC)-IP3 ( Guo et al., 2021 ). The localization of RYamide demonstrated that the brain is an important source of RYamide production in insects. For example, eight RYamide-producing neurons are localized in the protocerebrum and optic lobes of adult silkworms ( Roller et al., 2016 ). Additionally, RNA-seq data have confirmed a high RYamide transcript (AAEL011702) abundance in the adult mosquito brain ( Hixson et al., 2022 ; Matthews et al., 2016 ). The enrichment of a candidate GPCR (AAEL017005; NPYLR5) detected in the head ( Hixson et al., 2022 ), which was initially demonstrated to have a weak response to NPF ( Liesch et al., 2013 ), further suggests that the brain may also function as a target in the RYamide signaling system. In the bean pod borer Maruca vitrata , RYamide receptor transcript expression in the brain appears to undergo circadian rhythm with greatest abundance during mid photophase ( Lee, 2022 ). In addition to its distribution within the brain, two RYamide producing neurons have been immunolocalized in the abdominal ganglia of adult D. melanogaster , with projections extending towards the hindgut rectal pads ( Veenstra and Khammassi, 2017 ). The insect hindgut is an organ responsible for modifying primary urine produced by Malpighian tubules through selective reabsorption of nutrients, ions and water into the haemolymph depending on demands of the body ( Donini and O’Donnell, 2005 ; Drake et al., 2015 ; Marusalin et al., 2012 ; Phillips et al., 1987 ; Picinic et al., 2024 ; Ramsay, 1950 ). In insects, innervation of the hindgut from the terminal abdominal ganglion has been reported ( Cook et al., 1991 ; Van Handel and Klowden, 1996 ), suggesting that RYamide may play a role in regulating excretory physiology and hydromineral homeostasis. Corroborating this hypothesis, RYaR (CG5811) transcript is highly enriched in the hindgut in adult Drosophila , including in the rectal pads ( Leader et al., 2018 ), which are known for intensive ion transport activities within the hindgut ( Bradley, 1987 ; Hanrahan and Phillips, 1983 ; Hopkins, 1967 ; Patrick et al., 2006 , 2002 ). A similar expression profile of RYaR transcript is reported in the hindgut of B. mori larvae ( Guo et al., 2021 ). It is worth noting that the second RYamide receptor (AAEL019786; NPYLR7) candidate examined in the current study, which was previously reported to respond to sNPF and human PYY at relatively high concentrations ( Liesch et al., 2013 ), is highly abundant in the adult mosquito hindgut ( Hixson et al., 2022 ). However, previous studies in insects have predominantly focused on the role of RYamides in regulating feeding-related behaviours. RYa1 has been shown to reduce feeding motivation in the blowfly Phormia regina , as evidenced by a ten-fold higher sucrose concentration required to induce a proboscis extension reflex in flies injected with this neuropeptide ( Ida et al., 2011 ). Additionally, electrophysiological recordings revealed a significant reduction in sugar receptor neuron activity in flies following dRYa1 injection, further supporting its role in feeding suppression ( Maeda et al., 2015 ). Similar inhibitory effects on feeding behaviour have also been observed with dRYa2. In mosquitoes, the aforementioned NPYLR7-associated signaling has been reported to play a suppressive role in host seeking and blood-feeding behaviour of female mosquitoes ( Duvall et al., 2019 ; Zeledon et al., 2024 ). Aside from feeding behaviour-related actions, RYamides appear to be involved in regulating pharynx and ileum muscle contractions that potentially relate to feeding and digestion in fed silkworm larvae ( Matsumoto et al., 2019 ). To explore potential osmoregulatory role of RYamide signaling and to gain a better understanding of excretory physiology in mosquitoes, we investigated transcript expression of RYamide over the course of mosquito development and in various tissues of adult males and females including the nervous system and peripheral organs. Using a custom antibody generated against Ae. aegypti RYamides, we mapped immunoreactivity within neurons in the nervous system including the brain and terminal abdominal ganglia as well as axonal processes associated with excretory organs including the hindgut. Importantly, our study set out to deorphanize two Ae. aegypti RYamide receptor candidates by assessing the specificity and ligand activity of endogenous RYamides as well as several structurally related neuropeptides. This is particularly important for the RYamides since they share structural similarity with a number of distinct insect neuropeptide families, including FMRFamides (FMRFa) ( Brown et al., 1986 ; Godoy et al., 2021 ), neuropeptide F (NPF) ( Brown et al., 1999 ; Stanek et al., 2002 ), short neuropeptide F (sNPF) ( Christ et al., 2017 ; Nässel and Wegener, 2011 ), sulfakinins ( Guo et al., 2020 ; Wei et al., 2000 ), and myosuppressins ( Dickerson et al., 2012 ; McCormick and Nichols, 1993 ). Lastly, the current study aims to understand the phylogeny and evolutionary history of RYamide receptors across arthropods (and more broadly in other animals), which was prompted by the presence of two RYamide receptors in Ae. aegypti , whereas all other reported insect species to date, including D. melanogaster , T. castaneum , and B. mori , possess only a single receptor ( Collin et al., 2011 ; Guo et al., 2021 ). 2. Materials and methods 2.1 Animal rearing A colony of Ae. aegypti (Liverpool strain) was maintained by regularly providing female mosquitoes with sheep’s blood in Alsever’s solution (Cedarlane Laboratories Ltd., Burlington, ON), which allows them to produce and later deposit eggs on dampened filter papers (Fisher Scientific) placed within BugDorm insect cages (MegaView Science Co., Ltd). Eggs were collected and hatched in plastic containers with dechlorinated water, and reared in an incubator at 25°C on a 12:12hr light: dark cycle as previously described ( Wahedi and Paluzzi, 2018 ). Larval food was prepared containing 2% (w/v) brewer’s yeast (Bulk Barn, Milton, ON) and 2% (w/v) beef liver powder (NOW Foods, Bloomingdale, Illinois) suspended in distilled water with ∼2mL of this suspension provided daily to each litre of larval bath. Adult mosquitoes were provided 10% sucrose solution in a small petri-dish covered with parafilm and fitted with a cotton ball allowing them to feed ad libitum . 2.2 Design of primers for qPCR, receptor expression constructs and dsRNA synthesis Primers for reverse transcriptase quantitative PCR (RT-qPCR), synthesizing double-strand RNA (dsRNA) that targets the RYamide transcript, and mammalian expression constructs containing the two candidate RYamide receptors, specifically AAEL017005, NPYLR5 and AAEL019786, NPYLR7 (see Supplementary Table S1) were designed using the Primer3 tool in Geneious Pro Bioinformatics Software (Biomatters Ltd). The amplification efficiency of RT-qPCR primer pairs was determined by a standard curve analysis using head cDNA from adult female mosquitoes. Specificity of amplification was confirmed by observing no detection in negative controls including a no reverse transcriptase control group that accounts for false positive amplification from potential genomic DNA contamination and a no template control that eliminates the possibility of any non-specific amplification including primer-dimers. 2.3 Profiling developmental and spatial expression of RYamides via RT-quantitative PCR Whole body of Ae. aegypti of 4 th larva, early and late pupa, one- and four-day-old adults (10 individuals per biological replicate), and four-day old dsRNA-injected adults (72h post-injection) were collected and stored in 1X RNA protection buffer for subsequent RNA extraction (Monarch Total RNA Miniprep kit, New England Biolabs, Whitby, ON). Tissues/organs (head, thorax, midgut, Malpighian tubules (MTs), reproductive system including primary and accessory reproductive organs, and remaining carcass including the cuticle with underlying epidermis, skeletal muscle, fat body and abdominal ganglia of anaesthetized one-day-old adult mosquitoes (40 individuals per biological replicate) were dissected in Dulbecco’s phosphate buffered saline (DPBS; Wisent Corporation, St. Bruno, QC, Canada). Total RNA of whole animal body and dissected tissues/organs was extracted using a Monarch® Total RNA Miniprep Kit (New England Biolabs, Whitby, ON, Canada) following the manufacture’s protocol. The RNA concentration was quantified using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) and cDNA was synthesized from purified total RNA using 500ng for whole insect samples and 15-50ng for tissue/organ samples, using iScript TM Reverse Transcription Supermix (Bio-Rad, Mississauga, ON). Synthesized cDNA was then diluted at least 10-fold with nuclease-free water prior to RT-qPCR analysis. Transcript abundance of RYamide (AAEL011702) was assessed on a StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, United States) using PowerUP TM SYBR® Green Master Mix (Applied Biosystems, Carlsbad, CA, United States). RT-qPCR cycling condition were as follow: 95°C for 20 seconds, and then 40 cycles of (i) 95°C for 3 seconds and (ii) 62°C for 30 seconds. Through ΔΔCT method the expression of RYamide was normalized using two reference genes, ribosomal protein 49 (rp49) and rpS18, as optimal endogenous reference genes ( Paluzzi et al., 2014 ). To confirm reproducibility of the results, 9 biological replicates were collected for determining developmental transcript abundance of RYamides, 4-5 biological replicates for spatial expression profiles, and 5 biological replicates for validating RYamide knockdown efficiency. 2.4 Whole mount immunohistochemistry To examine cellular distribution of RYamide in adult Ae. aegypti , the brain, gut, and the abdominal ganglia were dissected in nuclease-free Dulbecco’s phosphate-buffered saline (DPBS; Wisent Corporation, St. Bruno, QC, Canada) on a silicone-lined petri dish and then fixed in 4% paraformaldehyde (PFA) overnight at 4°C. Following overnight fixation, tissues were washed three times for 10 minutes each with DPBS followed by a 1h incubation with 4% Triton X-100, 2% bovine serum albumin (BSA) and 10% normal sheep serum (NSS) on a rocking platform at room temperature. Permeabilized tissues were incubated with primary antibody anti- Aedae RYa1 for 48h at 4°C on a rocking platform with gentle agitation. The custom rabbit polyclonal primary antibody, anti- Aedae RYa1 (Biomatik Corp., Kitchener, ON) (5µg/ml), was prepared 12h prior to use in a solution containing 0.4% Triton X-100, 2% bovine serum albumin (BSA), and 10% normal sheep serum (NSS) with 10µM synthetic Aedes neuropeptides, including NPF, sNPF, FMRFa3, CAPA and pyrokinin1, to reduce the possibility of cross-reactivity in immunolocalization (see Supplementary Table S2). Following primary antibody incubation, DPBS washed tissues (three times) were treated with a goat anti-rabbit Alexa Fluor® 568 IgG (H+L) conjugated secondary antibody (Molecular Probes, Life Technologies, Eugene, OR, USA) diluted 1:200 in 10% NSS and DPBS, incubating for another 48h at 4°C on a rocker. After three DPBS rinses (10 minutes each), tissues were mounted onto microscope slides using a mounting buffer containing glycerol and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Molecular Probes, Eugene, OR, USA) to stain cell nuclei. A Lumen Dynamics XCite™ 120Q Nikon fluorescence microscope (Nikon, Mississauga, ON, Canada) and a Zeiss Laser Scanning LSM 700 confocal microscope (Carl Zeiss Canada Ltd., Toronto, ON, Canada) were used to examine samples and acquire images. 2.5 Preparation of RYamide receptor mammalian expression constructs The full open reading frame of two putative Ae. aegypti RYamide receptors (AAEL017005; NPYLR5 and AAEL019786; NPYLR7) were amplified using high-fidelity Q5 polymerase (New England Biolabs, Whitby, ON) as described previously ( Tan et al., 2024 ) and primers incorporating a Kozak translation initiation sequence ( Kozak, 1986 ), stop codon and restriction sites for directional subcloning (see Supplementary Table S1). The digested amplicons were then subcloned into mammalian expression vector pcDNA3.1+, followed by bacterial transformation and plasmid DNA extraction to obtain a large quantity of receptor constructs using ZymoPURE II Plasmid Midiprep Kit (Zymo Research, Tustin, CA, USA). Prepared constructs were sequenced to verify base accuracy by whole plasmid sequencing performed by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation. 2.6 Heterologous functional activity bioluminescence assay At 48h prior to the bioluminescence assay, a previously established Chinese hamster ovary cell line K1 (CHO-K1) stably expressing the calcium bioluminescent reporter protein aequorin ( Paluzzi et al., 2012 ) was seeded into T75 culture flasks at 90% confluency, and followed by a 6h incubation at 37°C. Following the manufacture’s recommendation, mammalian expression constructs were then prepared for transfection using PolyJet TM DNA in vitro transfection reagent (FroggaBio Inc., Concord, ON). Cells were prepared for the functional activity bioluminescence assay 42h post-transfection by incubating in assay media (BSA media) containing DMEM: F12 basal media, 10% Bovine Serum Albumin (BSA) and 1X antibiotic-antimycotic, with 5µM coelenterazine-h, in the dark at room temperature for 3 hours. After a 10-fold dilution in BSA media, cells were incubated an additional 45 minutes in the dark. A library of mosquito neuropeptides (Supplementary Table S2) were commercially synthesized (Shanghai Royobiotech Co., Ltd, Shanghai, China) at purity >95% and used initially at a final titre of 10 -6 M to examine the ligand selectivity and responsiveness of the two putative RYaRs. Dose-dependent responses of neuropeptides were then investigated by measuring bioluminescent responses of serial dilutions ranging from 10 -5 M to 10 -16 M. As a positive control, 100µM ATP was used to normalize the bioluminescent response in individual wells triggered by endogenous purinoceptors expressed in CHO-K1 cells ( Iredale and Hill, 1993 ). Measurements of Ca 2+ kinetics was acquired with a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). 2.7 RYamide receptor phylogenetic analysis RYamide receptor sequences and tachykinin receptor sequences from various species across arthropods were collected on NCBI website ( https://www.ncbi.nlm.nih.gov/ ) by blast search of deduced Ae. aegypti RYaR1 (AAEL017005) and RYaR2 (AAEL019786) protein sequences and D. melanogaster tachykinin-like receptor (AAF56979.2) within the database. Following the protocol described previously ( Wahedi and Paluzzi, 2018 ), protein sequences were aligned using ClustalX2 ( Larkin et al., 2007 ). Evolutionary relationships between receptor sequences among examined species were explored by maximum-likelihood phylogenetic analysis methods supported by 1000 bootstraps using MEGA-X software ( Jones et al., 1992 ; Kumar et al., 2018 ). 2.8 Statistical analysis One-way ANOVA with Tukey’s multiple comparisons test or two-way ANOVA with Šídák’s multiple comparisons test was used to analyze the log-transformed developmental/tissue-specific transcript expression data, and luminescent responses from the functional activity bioluminescence assay, respectively. A two-tailed t-test was used to assess significance of RYamide knockdown as determined by RT-qPCR analysis. Dose-response curves and EC 50 values were determined using the sigmoidal dose-response model as previously described ( Tan et al., 2024 ). Statistical analysis was conducted using GraphPad Prism 9 adopting a 0.05 significance threshold. 3. Results 3.1 Expression profiles of Ae. aegypti RYamide over developmental stages and in adult organs The RYamide transcript was expressed throughout the post-embryonic development of Ae. aegypti , with a relatively consistent transcript abundance. However, adult males at both early (1-day old) and later (4-day old) adult stages showed significantly higher transcript abundance compared to the 4 th instar larvae ( Fig. 1 ). In addition, the RYamide transcript showed differential expression between adult males and females, with a significant difference between sexes in 4-day old adults. Download figure Open in new tab Figure 1. Expression profile of Ae. aegypti RYamide (RYa) transcript in different post-embryonic developmental stages determined by RT-qPCR. Transcript levels of various developmental stages relative to the fourth instar larva. Abbreviations: 1do M = 1-day-old male adult, 1do F = 1-day-old female adult, 4do M = 4-day-old male adult, 4do F = 4-day-old female adult. Statistical differences are denoted with different letters, as determined by a one-way ANOVA with Tukey’s multiple comparison on log-transformed data (p<0.05). Data represents the mean ± SEM (n=9). Organ-specific transcript expression profiles of RYamide in one-day old mosquitoes was examined, revealing consistent abundance profile between both sexes. The nervous system appeared to be a primary source of RYamide transcript production, as the head (brain) showed high enrichment of the transcript ( Fig. 2 ). Additionally, moderate RYamide transcript abundance was detected in the thorax and carcass, that contain the thoracic and abdominal ganglia (respectively) as well as other tissues including (but not limited to) fat body and epidermis. In comparison with the RYamide transcript enrichment in the head, a significantly lower abundance was detected in the hindgut and reproductive tissues of male mosquitoes ( Fig. 2A ). A similar trend was noted in female mosquitoes, although without any statistical significance ( Fig. 2B ). Download figure Open in new tab Figure 2. Organ-specific expression profile of RYamide (RYa) transcript in male (A) and female (B) 1-day-old adult Ae. aegypti determined by RT-qPCR. Transcript levels of various tissues/organs relative to the head. Tissue/organ abbreviations: head, thorax, midgut (MG), Malpighian tubules (MTs), hindgut (HG), reproductive system and carcass. Statistical differences are denoted with different letters, as determined by a one-way ANOVA with Tukey’s multiple comparison on log-transformed data (p<0.05). Data represents the mean ± SEM (n=4-5). Across all tested tissues and in both sexes, MTs had the lowest RYamide transcript abundance detected with significantly lower levels compared to the brain. This expression profile aligns with publicly available RNA-seq data ( Hixson et al., 2022 ), which indicates enrichment in the head of female Ae. aegypti (Supplementary Fig. S1). 3.2 Deorphanization of RYamide receptors The prepared mammalian expression constructs were validated by comparing the responsiveness of cells transfected with the control construct, pBud-EGFP, compared to cells transfected with each of the RYamide receptor constructs. The RYaR1 and RYaR2 transfected cells exhibited strong luminescent response following application of RYamides while no luminescent response to RYamides was observed in cells transfected with the control construct, pBud-EGFP (Supplementary Fig. S2). A variety of mosquito neuropeptides that share the RF/RYamide sequence at the C-terminus (Supplementary Table S2) were tested to examine the sensitivity and specificity of the two putative RYaRs. At a high dosage (10 -6 M), which is considered a saturating concentration ( Collin et al., 2011 ; Ida et al., 2011 ; Lajevardi and Paluzzi, 2020 ), Aedae RYa1 and Aedae RYa2 elicited strong luminescent responses in RYaR1, which were significantly stronger than the BSA assay media alone, as well as other tested ligands ( Fig. 3A ). Moreover, certain structurally related peptidergic ligands, including full length sNPF1, sNPF2, FMRFa3, FMRFa9, and NPF, were able to induce low luminescent responses by the RYaR1 that were statistically significant from BSA assay media alone, while RYaR1 was unresponsive to other tested neuropeptides which yielded only background level of luminescence ( Fig. 3A ). In contrast, RYaR2 was less selective at the high saturating concentration (10 -6 M) since all tested neuropeptides were found to activate the receptor with levels of luminescence detected that were all significantly higher compared to the negative control with BSA assay media alone ( Fig. 3B ). It is worth noting that Aedae RYa1 still exhibited the strongest response in activating RYaR2 among all tested ligands. Download figure Open in new tab Figure 3. Luminescent response of CHO-K1 cells expressing Ae. aegypti RYaR1 and RYaR2. RYa1 and RYa2 elicited strong luminescent response activating RYaR1 (A) and RYaR2 (B) at concentration of 10 -6 M. Statistical differences are denoted by asterisks (*), as determined by a one-way ANOVA with Dunnett’s multiple comparisons test when compared to the BSA (vehicle control) group (p < 0.05) (n=3). Curves were fitted following a sigmoidal dose-response model where RYa1 and RYa2 were found to activate RYaR1 (C) and RYaR2 (D), but with different binding affinities. RYaR1 showed a lower sensitivity compared to RYaR2 with respect to receptor activation by over two orders of magnitude. EC 50 values shown determined based on averages of all biological replicates (n=3-6). A dose-response analysis of RYaR1 revealed that Aedae RYa1 has a slightly higher (∼3.5-fold greater) activity in terms of receptor activation with a half maximal effective concentration (EC 50 ) of 0.47nM compared to Aedae RYa2 with a EC 50 of 1.64nM ( Fig. 3C ). Conversely, RYaR2 shows no capability to distinguish between the two RYamides ( Aedae RYa1 EC 50 =14.08pM; Aedae RYa2 EC 50 =12.95pM) ( Fig. 3D ). Moreover, the results suggest that RYaR2 is more sensitive to both RYamides in comparison to RYaR1, since pico-molar concentrations of RYamides are able to activate RYaR2. RYamide induced luminescent response of RYaR2 reached the plateau at a concentration ∼10 -10 M, while a much higher saturation concentration was measured in RYaR1 (∼10 -7 M) ( Fig. 3C-D ). On the other hand, RYaR1 was more selective in terms of differentiating between RYamides and other structurally related neuropeptides. Only low luminescent responses were detected when NPF, sNPFs, and FMRFas were applied against RYaR1 at high doses, with EC 50 values approximately in the millimolar to micromolar range ( Fig. 3C ). Comparatively, RYaR2 is activated by NPF, sNPF, and FMRFa more readily, with responses detectable at ∼10 -8 M and beyond. Although RYaR2 responded to all tested peptides in the micromolar range, the EC 50 of RYamides to activate RYaR2 was several orders of magnitude lower compared to these other peptidergic ligands ( Fig. 3D ). Notably, all tested neuropeptides other than RYamides failed to reach the plateau of the dose response curves (representing maximal receptor activation) at the highest concentration tested (10 -5 M), indicating that RYamides are indeed the natural ligands of these two previously orphan receptors ( Fig. 3C-D ). 3.3 Distribution of RYamide immunoreactivity To localize RYamide within the central and peripheral nervous system, whole-mount immunohistochemistry was conducted on several tissues of 1-day-old and 4-day-old adult mosquitoes of both sexes using a customized anti- Aedae RYa1 antibody (see Methods section). While no immunoreactivity was found in cells localized to the midgut, a ring of prominent RYamide immunoreactivity was observed encircling the pyloric valve, which is localized at the junction between the midgut and hindgut ( Fig. 4A, 4G, 4H ). Strong RYamide immunoreactive staining was observed over the rectum in processes extending into, and in close proximity to the surface of, each of the rectal papillae (pads), with six in females and four in males ( Fig. 4A, 4D, 4E ). RYamide immunoreactivity was also observed in the CNS, including the ventral nerve cord and the brain. Specifically, a pair of immunoreactive neurons located at the middle of the anterior region of the terminal abdominal ganglion (TAG) were detected. Additionally, RYamide immunoreactive axonal projections emanating from this pair of neurons extended over the rectum and, as mentioned above, associated with each of the rectal papillae ( Fig. 4D , 4E). Download figure Open in new tab Figure 4. RYamide immunoreactive cells were localized in the terminal abdominal ganglion with processes extending to the hindgut of 4-day-old adult Ae. aegypti reared under standard laboratory conditions via a custom anti-RYa1 antibody. (A) An overview of RYamide immunoreactivity throughout the gut and abdominal ganglia. (B, C) Male and female mosquitoes were injected with dsRNAs targeting either EGFP (dsEGFP) or the Ae. aegypti RYamide gene (dsRYa). Knockdown efficiency was assessed by RT-qPCR analysis on 4-day-old mosquitoes (3 days post dsRNA injection). Statistical differences are denoted by asterisks (*), as determined by a two-tailed t-test on log-transformed data (p < 0.05) with data represent the mean ± SEM (n = 5). Terminal abdominal ganglion (TAG) and the hindgut of (D) non-injected (NI), (E) dsEGFP-, and (F) dsRYa-injected mosquitoes. Two RYamide producing neurons (encircled by dashed line) were localized in the terminal abdominal ganglion of non-injected and dsEGFP-injected mosquitoes but were not observable in dsRYa-injected mosquitoes. RYamide processes (white arrows) projecting from RYamide-immunoreactive anteromedial neurons of the TAG into the rectal papillae and extending anteriorly over the ileum towards the pyloric valve were observed in non-injected and dsEGFP mosquitoes, while much weaker immunoreactivity was detected in knockdown mosquitoes. (G-I) A ring-shaped RYamide immunoreactive plexus was observed encircling the pyloric valve in non-injected and dsEGFP-injected mosquitoes, while reduced immunostaining intensity was observed in dsRYa-injected mosquitoes. The same pattern of immunolocalization was observed in both males and female adult mosquitoes, with no differences observed between sexes. Scale bars: (A) 200μm; (D-F) 50μm; (G-I) 20μm. Furthermore, this same pair of RYamide immunoreactive processes projecting from the terminal abdominal ganglion to the rectum also continued anteriorly over the ileum towards the pyloric valve ( Fig. 4D, 4E, 4G, 4H ). A few scattered neurons on either side of the terminal ganglion with much weaker immunoreactive staining were also detected, which as described below, may reflect non-specific staining with the antibody ( Fig. 4D ). Multiple immunoreactive neurons detected using the anti-RYamide antibody were bilaterally localized in the lateral protocerebrum and mushroom bodies of the brain ( Fig. 5 ). Moreover, RYamide immunoreactivity was found across the bilaterally paired optic lobes, and specifically clustered in the lamina regions of the brain. Download figure Open in new tab Figure 5. RYamide immunoreactivity in the brain of 4-day-old adult Ae. aegypti reared under standard laboratory conditions via custom anti- Aedae RYa1 antibody. RYamide-like immunoreactivity was localized in several pairs of neurons within the anterior lateral protocerebrum, the pars lateralis (PL), while immunoreactive processes were observed in the medulla of the optic lobe. PI: pars intercerebralis . (A) Non-injected (control) mosquitoes. (B) dsEGFP mosquitoes. (C) dsRYa mosquitoes. In general, the same pattern and distribution of RYamide-immunoreactivity was observed in the brain of male and female mosquitoes. Scale bars: 200μm. To clarify possible cross-reactivity between the anti-RYamide antibody with neuropeptides that share similar structural features to Ae. aegypti RYamides, as well as to confirm the localization of RYamide, whole-mount immunohistochemistry was also conducted following RYamide knockdown. The efficiency of RYamide knockdown was determined by RT-qPCR indicating approximately 60% and 70% reduction in RYamide transcript abundance in male and female mosquitoes, respectively ( Fig. 4B , 4C). In comparison to non-injected and dsEGFP-injected control mosquitoes, intensive RYamide immunoreactivity within each of the rectal papillae was no longer observable in RYamide knockdown mosquitoes, whereas only faintly stained RYamide immunoreactive processes were visible over the ileum and rectum ( Fig. 4D-F ). Similarly, RYamide immunoreactive staining in processes over the pyloric valve was greatly reduced in dsRYa-injected mosquitoes ( Fig. 4G-I ). Furthermore, while the strongly staining pair of RYamide immunoreactive neurons localized in the middle of the anterior region of the terminal abdominal ganglion were no longer detectable, other immunoreactive neurons localized on either side of the terminal abdominal ganglion were still observable in RYamide knockdown mosquitoes ( Fig. 4D-F ). This supports the notion that the strongly stained RYamide immunoreactive pair of neurons in the anterior of the terminal abdominal ganglion is the source of RYamide, but other immunoreactive cells detected in this ganglion are likely not RYamide producing neurons. In the brain, all RYamide immunoreactive neurons localized in the protocerebrum and mushroom body as well as most interneuron staining across the optic lobes and lamina regions ( Fig. 5A-B ) were no longer visible following RYamide knockdown ( Fig. 5C ). 3.4 Phylogenetic analysis of RYamide receptors among insects and other species While earlier studies on D. melanogaster and T. castaneum have only characterized and functionally deorphanized a single RYamide receptor ( Collin et al., 2011 ; Ida et al., 2011 ), the fact that Ae. aegypti possesses two bona fide RYamide receptors encoded by two separate genes is fascinating. This observation raises the question of whether the presence of two RYamide receptors is unique to Ae. aegypti mosquitoes or a more common phenomenon among other arthropods. Phylogenetic analysis using maximum-likelihood methods was performed to investigate the evolutionary relationships of RYamide receptors across arthropods and to gain insights into their evolutionary history. The concise phylogenetic tree ( Fig. 6 ) and a more comprehensive tree (Supplementary Fig. S4) including RYamide/luqin-type receptors from more diverse and a greater number of taxa revealed that Ae. aegypti RYaR1 (AAEL017005) and RYaR2 (AAEL019786) grouped into two separate clades, positioned as sister groups to each other. This sequence relationship supports the presence of two distinct RYamide receptors in Ae. aegypti and corroborates the functional deorphanization findings that revealed these two receptors are highly selective for endogenous RYamide peptides. Even more intriguing is that only the Culicidae family possesses two types of RYamide receptors. Specifically, species in the Culicinae subfamily, such as Toxorhynchites rutilus septentrionalis , Malaya genurostris , Wyeomyia smithii , Culex pipiens pallens , Aedes albopictus , and Aedes aegypti , have both RYaR1 and RYaR2 receptors, while members of the Anophelinae subfamily (e.g. Anopheles gambiae and Anopheles coluzzii ) possess only a single receptor, namely RYaR1. Other species within the order Diptera also possess only RYaR1 and are positioned as a sister group to the mosquito RYaR1 clade. Aside from dipterans, representative species from the orders Coleoptera, Hemiptera, Blattodea and Hymenoptera also have receptors that share phylogenetic relationship with dipteran RYaR1. On the other hand, receptors from representative species of Lepidoptera (butterflies and moths) were positioned within a sister group to the mosquito Culicinae subfamily RYaR2 monophyletic clade ( Fig. 6 ). Additionally, evidence of a potential RYaR2 gene duplication was observed in moths, such as Bombyx , Cydia and Ostrinia , but no evidence supporting a RYaR2 gene duplication was observed in butterflies. Notably, an RYamide receptor ortholog of either subtype was not found in available orthopteran (grasshoppers, crickets and locusts) online datasets or in the long-established model for insect physiology, Rhodnius prolixus , suggesting selective loss of this system broadly in specific orders (e.g. Orthoptera) and more specifically in a subset of species in other orders (e.g. Hemiptera). Beyond the class Insecta, phylogenetic analysis revealed RYamide-related receptors in other arthropods, including in decapod (crabs, lobsters, crayfish, shrimp, and prawns), thecostracan (barnacles) and branchiopod (water fleas) crustaceans with evidence of expansion in decapods ( Fig. 6 ). In another ecdysozoan phylum, Nematoda, the analysis supported the occurrence of RYamide receptor orthologs in several species (Supplementary Fig. S4). Furthermore, RYamide-like receptors were also detected in a variety of arachnids including representatives from the superorder Parasitiformes (ticks and mites), and the orders Araneae (spiders) and Scorpiones (scorpions), which clustered as separate groups basal to the monophyletic group consisting of crustacean and insect RYamide receptors. Our expanded analysis provides evidence supporting the occurrence of RYamide/luqin-related receptors in diverse molluscan, annelid and priapulida representative taxa (Supplementary Fig. S4). Collectively, RYamide receptors are broadly distributed in arthropods and are orthologs of luqin-type receptors functionally characterized in deuterostomes ( Yañez-Guerra et al., 2018 ) supporting earlier findings that RYamide/luqin-type receptors and their ligands are evolutionarily ancient signaling systems found across bilaterian organisms ( Mirabeau and Joly, 2013 ), despite being absent in chordates ( Yañez-Guerra and Elphick, 2020 ). Download figure Open in new tab Figure 6. Phylogenetic analysis showing the relationship of RYamide-related receptors across arthropods and more widely in more diverse organisms. The tree was constructed using maximum-likelihood phylogenetic analysis methods (with 1000 bootstrap replicates). The numbers adjacent to nodes indicate the statistical support for the clustering of related sequences within the respective clade. Various tachykinin, NPF and sNPF receptors, as well as NPY receptors of Homo sapiens , were included to demonstrate the evolutionary relationship between vertebrate NPY receptors and invertebrate RYamide, NPF, and sNPF receptors. Homo sapiens NPYR receptors was included in the analysis and imposed as the outgroup. 4. Discussion The RYamide neuropeptides and their associated GPCRs were identified across arthropods over a decade ago ( Collin et al., 2011 ; Hauser et al., 2010 ; Ida et al., 2011 ). However, research on RYamide distribution, their associated receptors and their phylogeny, and the physiological roles of RYamide signaling in many other insects, including Ae. aegypti , is largely unknown. Ae. aegypti is a notorious human disease vector, capable of transmitting various disease agents such as dengue, Zika and yellow fever disease. Factors such as climate change and urbanization have expanded mosquito habitats, particularly for anthropophilic species including as Ae. aegypti , further increasing the risk to public health ( Jansen and Beebe, 2010 ; Reinhold et al., 2018 ). Studying RYamides and their receptors advances our understanding of neuropeptide signaling, which can be conserved or divergent across species and may provide insight for novel pest control strategies if such pathways regulate critical biological functions and could be disrupted using environmentally benign agents. 4.1 Deorphanization and evolutionary insights of RYamide receptors Our findings represent a breakthrough in the deorphanization of GPCRs encoded by the genes AAEL017005 and AAEL019786, validating them as authentic RYamide receptors in Ae. aegypti . No study had previously linked the orphan receptors NPYLR5 and NPYLR7 with their RYamide peptide ligands ( Duvall et al., 2019 ; Liesch et al., 2013 ). Herein, we provide evidence demonstrating that Ae. aegypti NPYLR5 and GPCR-AAEL017005 as well as NPYLR7 and GPCR-AAEL019786, are identical with regards to their deduced amino acid sequences and, as a result of identifying their endogenous and highly active RYamide ligands, accordingly we renamed them as Aedae RYaR1 and Aedae RYaR2, respectively. Aedae RYaR1 exhibited a nearly equal response to its two endogenous RYamides peptide ligands, which aligns with relative ligand potencies reported earlier for RYaR in D. melanogaster ( Collin et al., 2011 ; Ida et al., 2011 ). However, the binding and activation of RYaR1 by RYamides in Ae. aegypti is more sensitive in terms of the half-maximal response (EC 50 ), which is lower compared to receptor orthologs in T. castaneum ( Collin et al., 2011 ), Caenorhabditis elegans ( Ohno et al., 2017 ), and B. mori ( Guo et al., 2021 ). Furthermore, Ae. aegypti RYaR2 demonstrated a more potent binding and activation sensitivity to RYamides with a pico-molar range EC 50 , which is orders of magnitude more sensitive compared to Ae. aegypti RYaR1 or any previously described RYamide-related receptor ( Collin et al., 2011 ; Guo et al., 2021 ; Ida et al., 2011 ; Ohno et al., 2017 ). However, while it is unlikely to hold any physiological relevance, the specificity and selectivity of Ae. aegypti RYaR1 and RYaR2 was compromised at higher concentrations (particularly for the latter) as there was some promiscuous activation evident by neuropeptides sharing some structural similarities to RYamides including members of the RFamide peptide family. This cross activation of Ae. aegypti RYamide receptors by other structurally related ligands was not totally unexpected, as it has been observed in functionally deorphanized orthologous receptors in other arthropods ( Collin et al., 2011 ; Guo et al., 2021 ; Ida et al., 2011 ; Ohno et al., 2017 ). To determine whether the presence of a second RYamide receptor is unique to Ae. aegypti and other mosquitoes, or if it is more widely found across arthropods, a phylogenetic analysis was conducted to examine the evolutionary relationship between two RYamide receptor sequences across various arthropod orders. The phylogenetic analysis revealed that only one of the two currently recognized mosquito subfamilies ( Harbach, 2007 ; Lorenz et al., 2021 ; Reidenbach et al., 2009 ), specifically members of the Culicinae, possess two RYamide receptor subtypes. Astonishingly, members of the Anophelinae subfamily including Anopheles mosquitoes, along with other dipteran species such as Drosophila and Musca domestica , have only a single RYamide receptor, RYaR1. Relatedly, our analysis also revealed that diverse members of the orders Hymenoptera, Blattodea, Coleoptera Siphonaptera and Hemiptera orders also have a receptor isoform sharing closest similarity to the RYaR1 subtype. Comparatively, Lepidoptera possess only RYaR2 with evidence of duplication of this receptor subtype within this order. Interestingly, we did not find any RYamide-related receptors in genomic data from members of the orders Orthoptera and Trichoptera, while RYamide receptor orthologs were identified in available genomes from species in the orders Odonata, Thysanoptera and Psocodea. Beyond the class Insecta, the phylogenetic analysis indicates that other arthropods possess RYamide-related receptors including members of the subphylum Crustacea such as barnacles and decapod crustaceans, with evidence of expansion in the latter. In Chelicerta, RYamide-related receptors are present in scorpions, spiders and ticks, with evidence of expansion in all but the latter group. Notably, we did not find any evidence of RYamide-related receptors in available genome data from members of the subphylum Myriapoda. In other protostomes, RYamide-related receptors were identified in Mollusca with presence in cephalopods (octopus), bivalves (mussel, oyster) and gastropods (limpet) where evidence supports expansion in the latter two molluscan classes (bivalves and gastropods). Our analysis also detected RYamide-related receptors within members of the Nematoda, Nemertea, Tardigrada, Brachiopoda, Annelida and Priapulida phyla. The current data supports that an ancestral RYamide receptor in arthropods along with other protostomes and the Luqin-type receptors in Ambulacraria ( Yañez-Guerra and Elphick, 2020 ), such as luqin receptor 1 and 2 characterized in the starfish Asterias rubens ( Yañez-Guerra et al., 2018 ), share a common ancestor. Indeed, luqin-type and, relatedly, RYamide-related signaling originated in a common ancestor of bilaterian organisms and is paralog of tachykinin-like signaling systems, while these two clades are evolutionarily related to NPY/NPF related signaling ( Yañez-Guerra et al., 2018 ; Yañez-Guerra and Elphick, 2020 ). Based on the data in insects, the results suggest a gene duplication of RYa1R occurred in a Mecopterida ancestor prior to the divergence of Antliophora and Amphiesmenoptera superorders within the Holometabola ( Peters et al., 2014 ; Song et al., 2016 ), resulting in a second receptor subtype (RYaR2) retained in Lepidoptera and both receptor subtypes occurring in select dipterans, specifically in the Culicinae subfamily of mosquitoes. 4.2 The nervous system as source and the hindgut as target The expression profile of RYamide transcript was examined during developmental and in individual organs/tissues of early and later stage adult Ae. aegypti . The developmental expression profile revealed enrichment of RYamide transcripts following the transition from pupal to adult stages. Specifically, the enrichment of RYamide was detected in both early and later stage male adults relative to fourth instar larvae, and this could be indicative of a sex-specific post-eclosion function for RYamide signaling. In contrast to these findings, the number of RYamide expressing neurons in the nervous system including the brain and the abdominal ganglia, as well as in enteroendocrine cells in the midgut of B. mori , reduced through the development, as the highest number of RYamide expressing cells was visualized in larva of B. mori ( Roller et al., 2016 ). Spatial transcript expression profiling and immunolocalization of RYamide in adult Ae. aegypti aimed to unveil potential physiological functions for RYamide and its corresponding receptors by determining RYamide producing cells and target tissues/organs. RYamide transcript was found to be highly enriched in the brain of both sexes, corroborating RYamide producing neurons immunolocalized in the brain and confirmed through RNAi-mediated knockdown. In particular, clusters of RYamide immunoreactive neurons were localized in the anterior lateral protocerebrum region of the brain along with the area above the calyx region covered by intrinsic neurons, Kenyon cell bodies ( Heuer et al., 2012 ) within the mushroom body. Insect mushroom bodies are complex in their neuronal connections and, unsurprisingly, are known to be involved in diverse functions such as learning and memory ( Cassenaer and Laurent, 2012 ; Keene and Waddell, 2007 ; Mizunami et al., 1998 ; Zars, 2000 ), sensory information processing, including olfactory ( Galizia and Rössler, 2010 ), acoustic ( Li and Strausfeld, 1999 ; Schildberger, 1984 ), and gustatory information ( Farris, 2008 ; Masek and Scott, 2010 ), and sleep regulation ( Guo et al., 2011 ). Interestingly, extrinsic neurons, such as octopaminergic and dopaminergic neurons, which form synapses on mushroom body Kenyon cell axonal projections, have been suggested to be associated with feeding-related behavioural responses ( Schroll et al., 2006 ; Schwaerzel et al., 2003 ). The previously reported feeding suppression role of RYamide ( Maeda et al., 2015 ) may be linked to Kenyon cells in the mushroom body, given the immunolocalization of RYamide and the high enrichment of RYaR1 in the brain ( Hixson et al., 2022 ) (see Supplementary Fig. S3A). Taken together, this suggests that the brain is one of the main sources of RYamide in mosquitoes. Furthermore, RYamide has been reported near the anterior edge of the brain in mated, previtellogenic female Ae. aegypti ( Dou et al., 2024 ), supporting our findings. Our study strengthens the evidence for the RYamide localization through RNAi-mediated knockdown, offering a more detailed view of the specific clusters of RYamide-producing neurons and confirming that both sexes exhibit the same expression pattern. Furthermore, the results are generally consistent with RYamide expression reported in the adult B. mori brain, where RYamide-expressing cells include a pair of protocerebral posterior dorsomedial (PDM) and protocerebral anterolateral (PAL) neurons ( Roller et al., 2016 ). Unlike the two RYamide expressing posterior ventrolateral (PVL) neurons observed at the base of optic lobes in B. mori , strong RYamide immunoreactivity in processes was detected in the medulla of the optic lobes in adult mosquitoes. In addition to the brain, RYamide transcript was enriched in the carcass, comprised of the fat body, epidermis, cuticle, and abdominal ganglia. Notably, a pair of neurons in the anteromedial region of the terminal abdominal ganglion exhibited intense RYamide immunoreactivity ( Fig. 4D-F ). These findings indicate that the ventral nerve cord, a key part of the mosquito nervous system, is also a source of RYamide. In support of this observation, the presence of RYa1 in the terminal abdominal ganglion in Ae. aegypti was confirmed by an earlier peptidomic study through mass spectrometry ( Hauser et al., 2010 ). On the other hand, the two RYamide-expressing cells detected in the larval and pupal stages of B. mori were no longer observed in the adult ( Roller et al., 2016 ), implying that RYamide produced in the terminal abdominal ganglia of adult mosquitoes may serve a unique role. The terminal abdominal ganglion in mosquitoes is a neural structure that regulates reproductive ( Gwadz, 1972 ) and hindgut-related functions ( Van Handel and Klowden, 1996 ). For example, surgically removing the terminal abdominal ganglion of a post-blood meal female mosquito results in the accumulation of waste products from digested blood, including uric acid and hematin ( Van Handel, 1975 ), in the gut lumen ( Van Handel and Klowden, 1996 ), while also significantly reduced water loss. In addition to the detection of RYamide immunoreactive neurosecretory cells in the terminal abdominal ganglion of adult mosquitoes, prominent axonal projections originating from these neurons were observed extending towards the rectal pads (or rectal papillae) within the rectum along with processes extending over length of the ileum and terminating over the pyloric valve forming a circular plexus. The rectal pads are the primary site responsible for actively reabsorbing water and ions from the gut lumen to the haemolymph, helping to achieve a key function of the hindgut in excreting nitrogenous waste ( Donini and O’Donnell, 2005 ; Phillips et al., 1987 , 1987 ; Ramsay, 1950 ). Previous studies noted that the ultrastructure of rectal papillae in 0-6-hour post-blood meal female mosquitoes exhibits more elaborate infolding on the apical membrane, suggesting increased activity within the rectal papillae during the peak diuresis period ( Hopkins, 1967 ; Sohal and Copeland, 1966 ). In both larval and adult mosquito rectal papillae, P-type Na + /K + -ATPase and V-type H + -ATPase have been immunolocalized on the basal and apical surfaces, respectively ( Patrick et al., 2006 ). Reabsorption of K + is mediated by V-type H + -ATPase, while Na + reabsorption is coupled to the apical Na + /H + exchangers and basolateral P-type Na + /K + ATPases ( Andersen et al., 2017 ). Interestingly, available RNA-seq data indicates that RYaR2 is highly enriched in the hindgut of sucrose-fed female mosquitoes ( Hixson et al., 2022 ) (see Supplementary Fig. S3B). In support of this insight from the public database, a recent preprint study demonstrated that RYaR2 (referred therein as NPYLR7) is expressed exclusively in rectal pad cells of the hindgut, which were found to elicit calcium signaling in response to RYamide peptide ( Greppi et al., 2025 ). Interestingly, deletion of RYaR2 disrupts egg viability and protein provisioning after a blood meal, but it did not impair overall fluid regulation following blood-feeding, a simulated isotonic saline meal or sucrose feeding by female Ae. aegypti ( Greppi et al., 2025 ). In line with these recent findings, RYaR2 activation has been previously shown to induce suppression of host-seeking behaviour ( Duvall et al., 2019 ). However, considering the enrichment of RYamide signaling in males as well as females, including the prominent immunoreactive staining associated with the hindgut, the current findings provide evidence that RYamide may participate in excretory functions at times not requiring a pronounced diuresis. In conclusion, this study expands our understanding of RYamide and its receptors in Ae. aegypti , one of the most successful human disease vectors. By characterizing the developmental and tissue-specific expression of the RYamide transcript, along with the distribution of RYamide immunoreactivity, we demonstrate that the brain and ventral nerve cord serve as the primary sites of RYamide production, while the rectum appears as a key target of RYamide and RYaR2 signaling. The close association between RYamide immunoreactive processes and the rectal pads in adult male and female mosquitoes suggests a potential role in ionoregulatory processes, presenting a promising direction for further investigation. Currently, only a limited number of RYamide receptors have been deorphanized in arthropods, and we functionally characterized two GPCRs in Ae. aegypti , RYaR1 (AAEL017005) and RYaR2 (AAEL019786), as bona fide RYamide receptors. Both receptors exhibit high specificity for the endogenous Ae. aegypti RYamides, while other tested neuropeptides with a similar RF/Yamide C-terminal motif were several orders of magnitude less effective in activating them. NPY-related signaling pathways are closely associated with foraging and feeding-related behaviors across both invertebrates and vertebrates, including insects, mice, and humans ( Colmers and Wahlestedt, 1993 ; de Bono and Bargmann, 1998 ; Wu et al., 2003 ). Reflecting this functional conservation, the sequences of vertebrate NPYRs and invertebrate NPYLRs exhibit a high degree of similarity. It was previously reported that invertebrate NPYLRs share approximately 60% sequence similarity with vertebrate NPY Y2 receptors ( Nässel and Wegener, 2011 ). Interestingly, NPYLR7 (now known as Ae. aegypti , RYaR2) has been shown to suppress host-seeking behavior in female mosquitoes upon activation with small molecule agonists ( Duvall et al., 2019 ). Herein, NPYLR7 has been deorphanized and renamed as RYaR2, which provides new insights and directions for the RYamide story. First, our findings indicate that RYamide and RYaR2 signaling are strongly associated with the rectal pads, with RYaR2 shown recently to be localized to a non-neuronal population of cells ( Greppi et al., 2025 ) within the rectal pads, suggesting a potential role in iono- and/or osmoregulation activity by the rectum of adult mosquitoes. This raises intriguing questions about whether the suppression of host-seeking behavior and osmoregulatory functions are interconnected. Additionally, it remains unclear whether NPYLR7/ RYaR2 mediates distinct physiological roles in male mosquitoes given the enrichment of RYamide transcript in males and the strong immunoreactivity associated with the hindgut originating from RYamide producing neurons within the terminal abdominal ganglion, matching the distribution observed in female mosquitoes. Secondly, small-molecule agonists targeting NPYLR7/ RYaR2 have been developed that suppress host-seeking behavior in mosquitoes. Now that RYamide has been identified as the endogenous ligand of NPYLR7/ RYaR2, this offers valuable insights for the structure-guided design of novel agonists with greater potency and selectivity. Given the importance NPYLR7/ RYaR2 in driving the suppression of host-seeking, biting and attraction to human hosts ( Dou et al., 2024 ; Duvall et al., 2019 ; Greppi et al., 2025 ), further studies on this signaling pathway could yield innovative control strategies aimed at preventing mosquito biting behavior and, as a result, mitigate disease transmission. Funder Information Declared Natural Sciences and Engineering Research Council, https://ror.org/01h531d29 , RGPIN-2020-06130 References ↵ Andersen , M.K. , MacMillan , H.A. , Donini , A. , Overgaard , J ., 2017 . Cold tolerance of Drosophila species is tightly linked to the epithelial K + transport capacity of the Malpighian tubules and rectal pads . Journal of Experimental Biology 220 , 4261 – 4269 . doi: 10.1242/jeb.168518 OpenUrl Abstract / FREE Full Text ↵ Bradley , T.J ., 1987 . Physiology of osmoregulation in mosquitoes . Annual Review of Entomology 32 , 439 – 462 . doi: 10.1146/annurev.en.32.010187.002255 OpenUrl CrossRef PubMed Web of Science ↵ Brown , M.R. , Crim , J.W. , Arata , R.C. , Cai , H.N. , Chun , C. , Shen , P ., 1999 . Identification of a Drosophila brain-gut peptide related to the neuropeptide Y family . Peptides 20 , 1035 – 1042 . doi: 10.1016/S0196-9781(99)00097-2 OpenUrl CrossRef PubMed Web of Science ↵ Brown , M.R. , Crim , J.W. , Lea , A.O ., 1986 . FMRFamide- and pancreatic polypeptidelike immunoreactivity of endocrine cells in the midgut of a mosquito . Tissue and Cell 18 , 419 – 428 . doi: 10.1016/0040-8166(86)90061-3 OpenUrl CrossRef PubMed Web of Science ↵ Caminade , C. , McIntyre , K.M. , Jones , A.E ., 2019 . Impact of recent and future climate change on vector-borne diseases . Annals of the New York Academy of Sciences 1436 , 157 – 173 . doi: 10.1111/nyas.13950 OpenUrl CrossRef PubMed ↵ Carvalho , F.D. , Moreira , L.A ., 2017 . Why is Aedes aegypti Linnaeus so successful as a species? Neotropical Entomology 46 , 243 – 255 . doi: 10.1007/s13744-017-0520-4 OpenUrl CrossRef PubMed ↵ Cassenaer , S. , Laurent , G ., 2012 . Conditional modulation of spike-timing-dependent plasticity for olfactory learning . Nature 482 , 47 – 52 . doi: 10.1038/nature10776 OpenUrl CrossRef PubMed Web of Science ↵ Christ , P. , Hill , S.R. , Schachtner , J. , Hauser , F. , Ignell , R ., 2018 . Functional characterization of mosquito short neuropeptide F receptors . Peptides 103 , 31 – 39 . doi: 10.1016/j.peptides.2018.03.009 OpenUrl CrossRef ↵ Christ , P. , Reifenrath , A. , Kahnt , J. , Hauser , F. , Hill , S.R. , Schachtner , J. , Ignell , R. , 2017 . Feeding-induced changes in allatostatin-A and short neuropeptide F in the antennal lobes affect odor-mediated host seeking in the yellow fever mosquito, Aedes aegypti . The Public Library of Science One 12 , e0188243 . OpenUrl ↵ Collin , C. , Hauser , F. , Krogh-Meyer , P. , Hansen , K.K. , de Valdivia , E.G. , Williamson , M. , Grimmelikhuijzen , C.J.P. , 2011 . Identification of the Drosophila and Tribolium receptors for the recently discovered insect RYamide neuropeptides . Biochemical and Biophysical Research Communications 412 , 578 – 583 . doi: 10.1016/j.bbrc.2011.07.131 OpenUrl CrossRef PubMed ↵ Colmers , W.F. , Wahlestedt , C. , 1993 . The Biology of Neuropeptide Y and Related Peptides. Springer Science & Business Media . ↵ Cook , B.J. , Wagner , R.M. , Peterson , T.L ., 1991 . The hindgut muscularis of the stable fly, Stomoxys calcitrans : Some of its structural, motile and pharmacological properties . Journal of Insect Physiology 37 , 635 – 645 . doi: 10.1016/0022-1910(91)90040-7 OpenUrl CrossRef ↵ de Bono , M. , Bargmann , C.I. , 1998 . Natural Variation in a Neuropeptide Y Receptor Homolog Modifies Social Behavior and Food Response in C. elegans . Cell 94 , 679 – 689 . doi: 10.1016/S0092-8674(00)81609-8 OpenUrl CrossRef PubMed Web of Science ↵ Dickerson , M. , McCormick , J. , Mispelon , M. , Paisley , K. , Nichols , R ., 2012 . Structure–activity and immunochemical data provide evidence of developmental- and tissue-specific myosuppressin signaling . Peptides 36 , 272 – 279 . doi: 10.1016/j.peptides.2012.05.002 OpenUrl CrossRef PubMed ↵ Donini , A. , O’Donnell , M.J ., 2005 . Analysis of Na+, Cl-, K+, H+ and NH4+ concentration gradients adjacent to the surface of anal papillae of the mosquito Aedes aegypti: application of self-referencing ion-selective microelectrodes . Journal of Experimental Biology 208 , 603 – 10 . doi: 10.1242/jeb.01422 OpenUrl Abstract / FREE Full Text ↵ Dou , X. , Chen , K. , Brown , M.R. , Strand , M.R ., 2024 . Reciprocal interactions between neuropeptide F and RYamide regulate host attraction in the mosquito Aedes aegypti . Proceedings of the National Academy of Sciences 121 , e2408072121 . doi: 10.1073/pnas.2408072121 OpenUrl CrossRef PubMed ↵ Drake , L.L. , Rodriguez , S.D. , Hansen , I.A. , 2015 . Functional characterization of aquaporins and aquaglyceroporins of the yellow fever mosquito, Aedes aegypti . Scientific Reports 5 . doi: 10.1038/srep07795 OpenUrl CrossRef PubMed ↵ Duvall , L.B. , Ramos-Espiritu , L. , Barsoum , K.E. , Glickman , J.F. , Vosshall , L.B ., 2019 . Small-molecule agonists of Ae. aegypti neuropeptide Y receptor block mosquito biting . Cell 176 , 687 – 701 .e5. doi: 10.1016/j.cell.2018.12.004 OpenUrl CrossRef PubMed ↵ Farris , S.M ., 2008 . Tritocerebral tract input to the insect mushroom bodies . Arthropod Structure & Development 37 , 492 – 503 . doi: 10.1016/j.asd.2008.05.005 OpenUrl CrossRef PubMed Web of Science ↵ Galizia , C.G. , Rössler , W ., 2010 . Parallel Olfactory Systems in Insects: Anatomy and Function . Annual Review of Entomology 55 , 399 – 420 . doi: 10.1146/annurev-ento-112408-085442 OpenUrl CrossRef PubMed Web of Science ↵ Godoy , R.S.M. , Barbosa , R.C. , Procópio , T.F. , Costa , B.A. , Jacobs-Lorena , M. , Martins , G.F ., 2021 . FMRF-related peptides in Aedes aegypti midgut: neuromuscular connections and enteric nervous system . Cell and Tissue Research 385 , 585 – 602 . doi: 10.1007/s00441-021-03462-3 OpenUrl CrossRef PubMed ↵ Greppi , C. , Frank , K. , Saltz , V. , Duvall , L.B ., 2025 . A Signaling Hub in the Mosquito Rectum Coordinates Reproductive Investment After Blood Feeding . bioRxiv 2025 . 08 . 25 .672111. doi: 10.1101/2025.08.25.672111 OpenUrl Abstract / FREE Full Text ↵ Guo , D. , Zhang , S. , Zhang , Y.-J. , Ma , J.-Y. , Gao , C.-F. , Wu , S.-F ., 2020 . Sulfakinin inhibits activity of digestive enzymes in the brown planthopper, Nilaparvata lugens . Journal of Asia-Pacific Entomology 23 , 1073 – 1082 . doi: 10.1016/j.aspen.2020.09.004 OpenUrl CrossRef ↵ Guo , F. , Yi , W. , Zhou , M. , Guo , A ., 2011 . Go Signaling in Mushroom Bodies Regulates Sleep in Drosophila . Sleep 34 , 273 – 281 . doi: 10.1093/sleep/34.3.273 OpenUrl CrossRef PubMed Web of Science ↵ Guo , Z. , He , X. , Jiang , C. , Shi , Y. , Zhou , N ., 2021 . Activation of Bombyx mori neuropeptide G protein–coupled receptor A19 by neuropeptide RYamides couples to Gq protein-dependent signaling pathways . Journal of Cellular Biochemistry 122 , 456 – 471 . doi: 10.1002/jcb.29874 OpenUrl CrossRef PubMed ↵ Gwadz , R.W ., 1972 . Neuro-hormonal regulation of sexual receptivity in female Aedes aegypti . Journal of Insect Physiology 18 , 259 – 266 . doi: 10.1016/0022-1910(72)90126-6 OpenUrl CrossRef PubMed Web of Science ↵ Hanrahan , J.W. , Phillips , J.E ., 1983 . Cellular mechanisms and control of KCl absorption in insect hindgut . Journal of Experimental Biology 106 , 71 – 89 . doi: 10.1242/jeb.106.1.71 OpenUrl Abstract / FREE Full Text ↵ Harbach , R.E ., 2007 . The Culicidae (Diptera): a review of taxonomy, classification and phylogeny . Zootaxa 1668 . doi: 10.11646/zootaxa.1668.1.28 OpenUrl CrossRef ↵ Hauser , F. , Neupert , S. , Williamson , M. , Predel , R. , Tanaka , Y. , Grimmelikhuijzen , C.J.P ., 2010 . Genomics and peptidomics of neuropeptides and protein hormones present in the parasitic wasp Nasonia vitripennis . Journal of Proteome Research 9 , 5296 – 5310 . doi: 10.1021/pr100570j OpenUrl CrossRef PubMed Web of Science ↵ Heuer , C.M. , Kollmann , M. , Binzer , M. , Schachtner , J ., 2012 . Neuropeptides in insect mushroom bodies . Arthropod Structure & Development 41 , 199 – 226 . doi: 10.1016/j.asd.2012.02.005 OpenUrl CrossRef PubMed ↵ Hixson , B. , Bing , X.L. , Yang , X. , Bonfini , A. , Nagy , P. , Buchon , N ., 2022 . A transcriptomic atlas of Aedes aegypti reveals detailed functional organization of major body parts and gut regional specializations in sugar-fed and blood-fed adult females . eLife 11 . doi: 10.7554/eLife.76132 OpenUrl CrossRef PubMed ↵ Hopkins , C.R ., 1967 . The fine-structural changes observed in the rectal papillae of the mosquito Aedes aegypti , L. and their relation to epithelial transport of water and inorganic ions. Journal of the Royal Microscopical Society 86 , 235 – 52 . doi: 10.1111/j.1365-2818.1967.tb00585.x OpenUrl CrossRef PubMed ↵ Ida , T. , Takahashi , T. , Tominaga , H. , Sato , T. , Kume , K. , Ozaki , M. , Hiraguchi , T. , Maeda , T. , Shiotani , H. , Terajima , S. , Sano , H. , Mori , K. , Yoshida , M. , Miyazato , M. , Kato , J. , Murakami , N. , Kangawa , K. , Kojima , M ., 2011 . Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila . Biochemical and Biophysical Research Communications 410 , 872 – 877 . doi: 10.1016/j.bbrc.2011.06.081 OpenUrl CrossRef PubMed ↵ Inui , A ., 1999 . Feeding and body-weight regulation by hypothalamic neuropeptides—mediation of the actions of leptin . Trends in Neurosciences 22 , 62 – 67 . doi: 10.1016/S0166-2236(98)01292-2 OpenUrl CrossRef PubMed Web of Science ↵ Iredale , P.A. , Hill , S.J ., 1993 . Increases in intracellular calcium via activation of an endogenous P 2 -purinoceptor in cultured CHO-K1 cells . British Journal of Pharmacology 110 , 1305 – 10 . doi: 10.1111/j.1476-5381.1993.tb13960.x OpenUrl CrossRef PubMed Web of Science ↵ Jansen , C.C. , Beebe , N.W ., 2010 . The dengue vector Aedes aegypti : what comes next . Microbes and Infection 12 , 272 – 9 . doi: 10.1016/j.micinf.2009.12.011 OpenUrl CrossRef PubMed Web of Science ↵ Jékely , G ., 2013 . Global view of the evolution and diversity of metazoan neuropeptide signaling . Proceedings of the National Academy of Sciences 110 , 8702 – 8707 . doi: 10.1073/pnas.1221833110 OpenUrl Abstract / FREE Full Text ↵ Jones , D.T. , Taylor , W.R. , Thornton , J.M ., 1992 . The rapid generation of mutation data matrices from protein sequences . Bioinformatics 8 , 275 – 282 . doi: 10.1093/bioinformatics/8.3.275 OpenUrl CrossRef PubMed ↵ Juliano , S.A. , Philip Lounibos , L ., 2005 . Ecology of invasive mosquitoes: effects on resident species and on human health . Ecology Letters 8 , 558 – 574 . doi: 10.1111/j.1461-0248.2005.00755.x OpenUrl CrossRef PubMed Web of Science ↵ Keene , A.C. , Waddell , S ., 2007 . Drosophila olfactory memory: single genes to complex neural circuits . Nature Reviews Neuroscience 8 , 341 – 354 . doi: 10.1038/nrn2098 OpenUrl CrossRef PubMed Web of Science ↵ Kotsakiozi , P. , Gloria-Soria , A. , Caccone , A. , Evans , B. , Schama , R. , Martins , A.J. , Powell , J.R. , 2017 . Tracking the return of Aedes aegypti to Brazil, the major vector of the dengue, chikungunya and Zika viruses . The Public Library of Science Neglected Tropical Diseases 11 , e0005653 . OpenUrl ↵ Kozak , M ., 1986 . Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes . Cell 44 , 283 – 92 . doi: 10.1016/0092-8674(86)90762-2 OpenUrl CrossRef PubMed Web of Science ↵ Kraemer , M.U.G. , Sinka , M.E. , Duda , K.A. , Mylne , A. , Shearer , F.M. , Brady , O.J. , Messina , J.P. , Barker , C.M. , Moore , C.G. , Carvalho , R.G. , Coelho , G.E. , Van Bortel , W. , Hendrickx , G. , Schaffner , F. , Wint , G.R.W. , Elyazar , I.R.F. , Teng , H.-J. , Hay , S.I. , 2015 . The global compendium of Aedes aegypti and Ae. albopictus occurrence . Scientific Data 2 , 150035 . doi: 10.1038/sdata.2015.35 OpenUrl CrossRef PubMed ↵ Kumar , S. , Stecher , G. , Li , M. , Knyaz , C. , Tamura , K ., 2018 . MEGA X: molecular evolutionary genetics analysis across computing platforms . Molecular biology and evolution 35 , 1547 – 1549 . OpenUrl CrossRef PubMed ↵ Lajevardi , A. , Paluzzi , J.-P.V ., 2020 . Receptor characterization and functional activity of pyrokinins on the hindgut in the adult mosquito, Aedes aegypti . Frontiers in Physiology 10 . doi: 10.3389/fphys.2020.00490 OpenUrl CrossRef ↵ Larkin , M.A. , Blackshields , G. , Brown , N.P. , Chenna , R. , McGettigan , P.A. , McWilliam , H. , Valentin , F. , Wallace , I.M. , Wilm , A. , Lopez , R. , Thompson , J.D. , Gibson , T.J. , Higgins , D.G ., 2007 . Clustal W and Clustal X version 2.0 . Bioinformatics 23 , 2947 – 2948 . doi: 10.1093/bioinformatics/btm404 OpenUrl CrossRef PubMed Web of Science ↵ Leader , D.P. , Krause , S.A. , Pandit , A. , Davies , S.A. , Dow , J.A.T ., 2018 . FlyAtlas 2: a new version of the Drosophila melanogaster expression atlas with RNA-Seq, miRNA-Seq and sex-specific data . Nucleic Acids Research 46 , D809 – D815 . doi: 10.1093/nar/gkx976 OpenUrl CrossRef PubMed ↵ Lee , D.-W ., 2022 . Identification of neuropeptide receptors from the brain of the bean pod borer, Maruca vitrata . Journal of Asia-Pacific Entomology 25 , 101845 . doi: 10.1016/j.aspen.2021.11.006 OpenUrl CrossRef ↵ Li , Y. , Strausfeld , N.J ., 1999 . Multimodal efferent and recurrent neurons in the medial lobes of cockroach mushroom bodies . Journal of Comparative Neurology 409 , 647 – 663 . doi: 10.1002/(SICI)1096-9861(19990712) 409:4%253C647::AID-CNE9%253E3.0.CO;2-3 OpenUrl CrossRef PubMed Web of Science ↵ Liesch , J. , Bellani , L.L. , Vosshall , L.B ., 2013 . Functional and genetic characterization of neuropeptide Y-like receptors in Aedes aegypti . PLOS Neglected Tropical Diseases 7 , e2486 . doi: 10.1371/journal.pntd.0002486 OpenUrl CrossRef PubMed ↵ Loh , K. , Herzog , H. , Shi , Y.-C ., 2015 . Regulation of energy homeostasis by the NPY system . Trends in Endocrinology & Metabolism 26 , 125 – 135 . doi: 10.1016/j.tem.2015.01.003 OpenUrl CrossRef PubMed ↵ Lorenz , C. , Alves , J.M.P. , Foster , P.G. , Suesdek , L. , Sallum , M.A.M ., 2021 . Phylogeny and temporal diversification of mosquitoes (Diptera: Culicidae) with an emphasis on the Neotropical fauna . Systematic Entomology 46 , 798 – 811 . doi: 10.1111/syen.12489 OpenUrl CrossRef Luong , T.D.N. , 2023 . Characterization of RYamide neuropeptides and their Receptor in the Disease Vector Mosquito, Aedes aegypti. York University, Electronic Theses and Dissertations . ↵ Maeda , T. , Nakamura , Y. , Shiotani , H. , Hojo , M.K. , Yoshii , T. , Ida , T. , Sato , T. , Yoshida , M. , Miyazato , M. , Kojima , M. , Ozaki , M ., 2015 . Suppressive effects of dRYamides on feeding behavior of the blowfly, Phormia regina . Zoological Letters 1 , 35 . doi: 10.1186/s40851-015-0034-z OpenUrl CrossRef PubMed ↵ Mains , J.W. , Kelly , P.H. , Dobson , K.L. , Petrie , W.D. , Dobson , S.L ., 2019 . Localized control of Aedes aegypti (Diptera: Culicidae) in Miami, FL, via inundative releases of Wolbachia -infected male mosquitoes . Journal of Medical Entomology 56 , 1296 – 1303 . doi: 10.1093/jme/tjz051 OpenUrl CrossRef PubMed ↵ Marusalin , J. , Matier , B.J. , Rheault , M.R. , Donini , A ., 2012 . Aquaporin homologs and water transport in the anal papillae of the larval mosquito , Aedes aegypti. Journal of Comparative Physiology B 182 , 1047 – 1056 . doi: 10.1007/s00360-012-0679-2 OpenUrl CrossRef PubMed ↵ Masek , P. , Scott , K ., 2010 . Limited taste discrimination in Drosophila . Proceedings of the National Academy of Sciences 107 , 14833 – 14838 . doi: 10.1073/pnas.1009318107 OpenUrl Abstract / FREE Full Text ↵ Matsumoto , S. , Kutsuna , N. , Daubnerová , I. , Roller , L. , Žitňan , D. , Nagasawa , H. , Nagata , S. , 2019 . Enteroendocrine peptides regulate feeding behavior via controlling intestinal contraction of the silkworm Bombyx mori . PLoS One 14 , e0219050 . doi: 10.1371/journal.pone.0219050 OpenUrl CrossRef PubMed ↵ Matthews , B.J. , McBride , C.S. , DeGennaro , M. , Despo , O. , Vosshall , L.B ., 2016 . The neurotranscriptome of the Aedes aegypti mosquito . BMC Genomics 17 , 32 . doi: 10.1186/s12864-015-2239-0 OpenUrl CrossRef PubMed ↵ McCormick , J. , Nichols , R ., 1993 . Spatial and temporal expression identify dromyosuppressin as a brain-gut peptide in Drosophila melanogaster . Journal of Comparative Neurology 338 , 279 – 288 . doi: 10.1002/cne.903380210 OpenUrl CrossRef ↵ McGregor , B.L. , Connelly , C.R ., 2021 . A Review of the control of Aedes aegypti (Diptera: Culicidae) in the continental United States . Journal of Medical Entomology 58 , 10 – 25 . doi: 10.1093/jme/tjaa157 OpenUrl CrossRef PubMed ↵ Mirabeau , O. , Joly , J.-S ., 2013 . Molecular evolution of peptidergic signaling systems in bilaterians . Proceedings of the National Academy of Sciences 110 , E2028 – E2037 . doi: 10.1073/pnas.1219956110 OpenUrl Abstract / FREE Full Text ↵ Mizunami , M. , Weibrecht , J.M. , Strausfeld , N.J ., 1998 . Mushroom bodies of the cockroach: Their participation in place memory . Journal of Comparative Neurology 402 , 520 – 537 . doi: 10.1002/(SICI)1096-9861(19981228) 402:4%253C520::AID-CNE6%253E3.0.CO;2-K OpenUrl CrossRef PubMed Web of Science ↵ Morrison , A.C. , Zielinski-Gutierrez , E. , Scott , T.W. , Rosenberg , R ., 2008 . Defining challenges and proposing solutions for control of the virus vector Aedes aegypti . PLOS Medicine 5 , e68 . doi: 10.1371/journal.pmed.0050068 OpenUrl CrossRef PubMed ↵ Nässel , D.R. , Wegener , C ., 2011 . A comparative review of short and long neuropeptide F signaling in invertebrates: Any similarities to vertebrate neuropeptide Y signaling? Peptides 32 , 1335 – 1355 . doi: 10.1016/j.peptides.2011.03.013 OpenUrl CrossRef PubMed ↵ Ohno , H. , Yoshida , M. , Sato , T. , Kato , J. , Miyazato , M. , Kojima , M. , Ida , T. , Iino , Y ., 2017 . Luqin-like RYamide peptides regulate food-evoked responses in C. elegans . eLife 6 , e28877 . doi: 10.7554/eLife.28877 OpenUrl CrossRef PubMed ↵ Paluzzi , J.-P. , Vanderveken , M. , O’Donnell , M.J ., 2014 . The heterodimeric glycoprotein Hormone, GPA2/GPB5, regulates ion transport across the hindgut of the adult mosquito, Aedes aegypti . PLoS One 9 , e86386 . doi: 10.1371/journal.pone.0086386 OpenUrl CrossRef PubMed ↵ Paluzzi , J.P. , Young , P. , Defferrari , M.S. , Orchard , I. , Carlini , C.R. , O’Donnell , M.J ., 2012 . Investigation of the potential involvement of eicosanoid metabolites in anti-diuretic hormone signaling in Rhodnius prolixus . Peptides 34 , 127 – 34 . doi: 10.1016/j.peptides.2011.10.025 OpenUrl CrossRef PubMed ↵ Patrick , M.L. , Aimanova , K. , Sanders , H.R. , Gill , S.S ., 2006 . P-type Na + /K + -ATPase and V-type H + -ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti . Journal of Experimental Biology 209 , 4638 – 4651 . doi: 10.1242/jeb.02551 OpenUrl Abstract / FREE Full Text ↵ Patrick , M.L. , Richard J. Gonzalez , Chris M. Wood , Rod W. Wilson , Timothy J. Bradley , Adalberto L. Val , 2002 . The characterization of ion regulation in Amazonian mosquito larvae: evidence of phenotypic plasticity, population-based disparity, and novel mechanisms of ion uptake . Physiological and Biochemical Zoology 75 , 223 – 236 . doi: 10.1086/342002 OpenUrl CrossRef PubMed Web of Science ↵ Peters , R.S. , Meusemann , K. , Petersen , M. , Mayer , C. , Wilbrandt , J. , Ziesmann , T. , Donath , A. , Kjer , K.M. , Aspöck , U. , Aspöck , H. , Aberer , A. , Stamatakis , A. , Friedrich , F. , Hünefeld , F. , Niehuis , O. , Beutel , R.G. , Misof , B ., 2014 . The evolutionary history of holometabolous insects inferred from transcriptome-based phylogeny and comprehensive morphological data . BMC Evolutionary Biology 14 , 52 . doi: 10.1186/1471-2148-14-52 OpenUrl CrossRef PubMed ↵ Phillips , J.E. , Hanrahan , J. , Chamberlin , M. , Thomson , B ., 1987 . Mechanisms and control of reabsorption in insect hindgut, in: Evans, P.D., Wigglesworth, V.B. (Eds.), Advances in Insect Physiology . Academic Press , pp. 329 – 422 . ↵ Picinic , B. , Paluzzi , J.-P.V. , Donini , A ., 2024 . Protein localization of aquaporins in the adult female disease vector mosquito , Aedes aegypti. Frontiers in Insect Science 4 . doi: 10.3389/finsc.2024.1365651 OpenUrl CrossRef ↵ Qi , Y. , Lee , N.J. , Ip , C.K. , Enriquez , R. , Tasan , R. , Zhang , L. , Herzog , H ., 2022 . NPY derived from AGRP neurons controls feeding via Y1 and energy expenditure and food foraging behaviour via Y2 signalling . Molecular Metabolism 59 , 101455 . doi: 10.1016/j.molmet.2022.101455 OpenUrl CrossRef PubMed ↵ Ramsay , J.A ., 1950 . Osmotic regulation in mosquito larvae . Journal of Experimental Biology 27 , 145 – 157 . doi: 10.1242/jeb.27.2.145 OpenUrl Abstract / FREE Full Text ↵ Reidenbach , K.R. , Cook , S. , Bertone , M.A. , Harbach , R.E. , Wiegmann , B.M. , Besansky , N.J ., 2009 . Phylogenetic analysis and temporal diversification of mosquitoes (Diptera: Culicidae) based on nuclear genes and morphology . BMC Evolutionary Biology 9 , 298 . doi: 10.1186/1471-2148-9-298 OpenUrl CrossRef PubMed ↵ Reinhold , J.M. , Lazzari , C.R. , Lahondère , C ., 2018 . Effects of the environmental temperature on Aedes aegypti and Aedes albopictus mosquitoes: a review . Insects 9 . ↵ Roller , L. , Čižmár , D. , Bednár , B. , Žitňan, D., 2016 . Expression of RYamide in the nervous and endocrine system of Bombyx mori . Peptides 80 , 72 – 79 . doi: 10.1016/j.peptides.2016.02.003 OpenUrl CrossRef PubMed ↵ Schildberger , K ., 1984 . Multimodal interneurons in the cricket brain: properties of identified extrinsic mushroom body cells . Journal of Comparative Physiology A 154 , 71 – 79 . doi: 10.1007/BF00605392 OpenUrl CrossRef ↵ Schroll , C. , Riemensperger , T. , Bucher , D. , Ehmer , J. , Völler , T. , Erbguth , K. , Gerber , B. , Hendel , T. , Nagel , G. , Buchner , E. , Fiala , A ., 2006 . Light-Induced Activation of Distinct Modulatory Neurons Triggers Appetitive or Aversive Learning in Drosophila Larvae . Current Biology 16 , 1741 – 1747 . doi: 10.1016/j.cub.2006.07.023 OpenUrl CrossRef PubMed Web of Science ↵ Schwaerzel , M. , Monastirioti , M. , Scholz , H. , Friggi-Grelin , F. , Birman , S. , Heisenberg , M. , 2003 . Dopamine and Octopamine Differentiate between Aversive and Appetitive Olfactory Memories in Drosophila . J. Neurosci . 23 , 10495 . doi: 10.1523/JNEUROSCI.23-33-10495.2003 OpenUrl Abstract / FREE Full Text ↵ Sohal , R.S. , Copeland , E ., 1966 . Ultrastructural variations in the anal papillae of Aedes aegypti (L.) at different environmental salinities . Journal of Insect Physiology 12 , 429 – 434 . doi: 10.1016/0022-1910(66)90006-0 OpenUrl CrossRef PubMed Web of Science ↵ Song , F. , Li , H. , Jiang , P. , Zhou , X. , Liu , J. , Sun , C. , Vogler , A.P. , Cai , W ., 2016 . Capturing the Phylogeny of Holometabola with Mitochondrial Genome Data and Bayesian Site-Heterogeneous Mixture Models . Genome Biology and Evolution 8 , 1411 – 1426 . doi: 10.1093/gbe/evw086 OpenUrl CrossRef PubMed ↵ Sprenger , P.R.D. , Reiter , D ., 1987 . The used tire trade: a mechanism for the worldwide dispersal of container breeding mosquitoes . Journal of the American Mosquito Control Association 3 , 494 . OpenUrl PubMed Web of Science ↵ Stanek , D.M. , Pohl , J. , Crim , J.W. , Brown , M.R ., 2002 . Neuropeptide F and its expression in the yellow fever mosquito, Aedes aegypti . Peptides 23 , 1367 – 1378 . doi: 10.1016/S0196-9781(02)00074-8 OpenUrl CrossRef PubMed Web of Science ↵ Strand , M.R. , Brown , M.R. , Vogel , K.J. , 2016 . Chapter Six - Mosquito Peptide Hormones: Diversity, Production, and Function, in: Raikhel, A.S. (Ed.), Advances in Insect Physiology . Academic Press , pp. 145 – 188 . doi: 10.1016/bs.aiip.2016.05.003 OpenUrl CrossRef ↵ Tan , J. , Neupert , S. , Paluzzi , J.-P ., 2024 . Functional characterization of CCHamides and deorphanization of their receptors in the yellow fever mosquito, Aedes aegypti . General and Comparative Endocrinology 359 , 114618 . doi: 10.1016/j.ygcen.2024.114618 OpenUrl CrossRef PubMed ↵ Tussey , D.A. , Morreale , R. , Carvalho , D.O. , Stenhouse , S. , Lloyd , A.M. , Hoel , D.F. , Hahn , D.A ., 2023 . Developing methods for chilling, compacting, and sterilizing adult Aedes aegypti (Diptera: Culicidae) and comparing mating competitiveness between males sterilized as adults versus pupae for sterile male release . Journal of Medical Entomology 60 , 1038 – 1047 . doi: 10.1093/jme/tjad079 OpenUrl CrossRef PubMed ↵ Van Handel , E. , 1975 . Direct determination of uric acid in fecal material . Biochemical medicine 12 , 92 – 3 . doi: 10.1016/0006-2944(75)90099-X OpenUrl CrossRef PubMed ↵ Van Handel , E. , Klowden , M.J. , 1996 . Defecation by the mosquito, Aedes aegypti , is controlled by the terminal abdominal ganglion . Journal of Insect Physiology 42 , 139 – 142 . doi: 10.1016/0022-1910(95)00089-5 OpenUrl CrossRef ↵ Veenstra , J.A. , Khammassi , H ., 2017 . Rudimentary expression of RYamide in Drosophila melanogaster relative to other Drosophila species points to a functional decline of this neuropeptide gene . Insect Biochemistry and Molecular Biology 83 , 68 – 79 . doi: 10.1016/j.ibmb.2017.03.001 OpenUrl CrossRef PubMed ↵ Wahedi , A. , Paluzzi , J.-P. , 2018 . Molecular identification, transcript expression, and functional deorphanization of the adipokinetic hormone/corazonin-related peptide receptor in the disease vector, Aedes aegypti . Scientific Reports 8 , 2146 . doi: 10.1038/s41598-018-20517-8 OpenUrl CrossRef PubMed ↵ Wei , Z. , Baggerman , G. , J. Nachman , R. , Goldsworthy , G. , Verhaert , P. , De Loof , A. , Schoofs , L., 2000 . Sulfakinins reduce food intake in the desert locust, Schistocerca gregaria . Journal of Insect Physiology 46 , 1259 – 1265 . doi: 10.1016/S0022-1910(00)00046-9 OpenUrl CrossRef PubMed ↵ Wu , Q. , Wen , T. , Lee , G. , Park , J.H. , Cai , H.N. , Shen , P ., 2003 . Developmental Control of Foraging and Social Behavior by the Drosophila Neuropeptide Y-like System . Neuron 39 , 147 – 161 . doi: 10.1016/S0896-6273(03)00396-9 OpenUrl CrossRef PubMed Web of Science ↵ Yañez-Guerra , L.A. , Delroisse , J. , Barreiro-Iglesias , A. , Slade , S.E. , Scrivens , J.H. , Elphick , M.R ., 2018 . Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome . Scientific Reports 8 , 7220 . doi: 10.1038/s41598-018-25606-2 OpenUrl CrossRef ↵ Yañez-Guerra , L.A. , Elphick , M.R ., 2020 . Evolution and Comparative Physiology of Luqin-Type Neuropeptide Signaling . Frontiers in Neuroscience 14 . ↵ Zars , T ., 2000 . Behavioral functions of the insect mushroom bodies . Current Opinion in Neurobiology 10 , 790 – 795 . doi: 10.1016/S0959-4388(00)00147-1 OpenUrl CrossRef PubMed Web of Science ↵ Zeledon , E.V. , Baxt , L.A. , Khan , T.A. , Michino , M. , Miller , M. , Huggins , D.J. , Jiang , C.S. , Vosshall , L.B. , Duvall , L.B ., 2024 . Next-generation neuropeptide Y receptor small-molecule agonists inhibit mosquito-biting behavior . Parasites & Vectors 17 , 276 . doi: 10.1186/s13071-024-06347-w OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted October 17, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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