Ethics statement

Work on vertebrate animals in this study was governed by and in compliance with the Animal Welfare Act (enforced by the USDA) and the Public Health Services Policy (enforced by the Office of Laboratory Animal Welfare) as well as New York State regulations. In addition, The Rockefeller University is an AAALAC accredited research facility committed to upholding the care standards provided by the 8th edition of the Guide for the Care and Use of Laboratory Animals. All blood-feeding procedures with mice were approved and monitored by The Rockefeller University Institutional Animal Care and Use Committee (approved protocol #11487). Work with normal human volunteers was conducted in compliance with the Declaration of Helsinki Principles. All blood-feeding and behavioral experiments with human volunteers were approved by the Institutional Review Board of The Rockefeller University Hospital (approved protocol LVO-0652). All human subjects gave their written informed consent to participate in these experiments.

Mosquito maintenance

Ae. aegypti mosquitoes (Orlando strain) were maintained at 25–28°C with 70–80% relative humidity under a 14 hour light: 10 hour dark cycle (lights on 8 am). Eggs were hatched in de-oxygenated, deionized water containing powdered Tetramin tropical fish food (Tetra, Melle, Germany). Larvae were cultured in deionized water and fed Tetramin tablets as needed. Adults were given unlimited access to 10% sucrose solution. Adult females were blood-fed on mice for stock maintenance and on human arms and legs for isolation of mutants, egg-laying, and host-seeking behavior experiments.

Bioinformatics

The 49 predicted class A rhodopsin-like GPCRs in the “peptide” and “orphan” receptor categories described in the Ae. aegypti genome publication [11] were analyzed for similarity to D. melanogaster and An. gambiae NPYLRs. From this group, eight candidate npylr genes were identified. Further analysis revealed that two of the eight were duplicate gene predictions that we combined to construct complete annotations for six candidate npylr genes. NCBI BLAST, Genewise (http://www.ebi.ac.uk/Tools/psa/genewise), and HMMER (http://hmmer.janelia.org/) bioinformatics tools were used to identify two additional npylr genes from published raw genomic sequence reads, returning the total candidate list to eight. All protein alignments and phylogenetic analyses were performed using default ClustalW and neighbor-joining tree-building methods in MacVector (http://www.macvector.com). Snake plots of predicted receptor topology were created using toppred (http://mobyle.pasteur.fr/).

A candidate Head Peptide gene from the tick, Ix. scapularis, was identified with tblastn searches of Ae. aegypti Head Peptide-I protein sequence against the trace reads archive available at NCBI in 2013 (ftp://ftp-private.ncbi.nlm.nih.gov/pub/TraceDB/ixodes_scapularis); trace identifier (TI Number): 1355069083, trace name: 1101271482505, center: J. Craig Venter Institute (http://gsc.jcvi.org/projects/msc/ixodes_scapularis/ixodes_scapularis/index.shtml).

Molecular biology

RNA was isolated using RNeasy Mini Kits (Qiagen, Valencia, CA, USA) from various tissues as noted. DNA isolation from whole mosquitoes was carried out with the Qiagen DNAeasy Blood & Tissue Kit, with samples homogenized with 2 mm glass beads (Sigma Aldrich Cat#Z273627-1EA, St. Louis, MO, USA) using a Qiagen TissueLyzer II at 1800 rpm for 1 min. Unless otherwise noted, synthesis of cDNA was performed using SuperScript III Reverse Transcriptase (Invitrogen, Grand Island, NY, USA) and polymerase chain reactions (PCR) were performed using EMD Millipore KOD polymerase (Billerica, MA, USA). All DNA sequencing was performed by Genewiz (South Plainfield, NJ, USA).

Receptor cloning

Full-length cDNAs for Ae. aegypti npylr2, npylr5, npylr6, and npylr8 were obtained by reverse transcriptase coupled to the polymerase chain reaction (RT-PCR) with primers located at the 5′ and 3′ ends of the predicted open reading frame from purified Ae. aegypti female head and body RNA. Full-length cDNAs for Ae. aegypti npylr1 (alleles a, b, c, d), npylr3, npylr4, npylr7 (alleles a and b) were obtained by RT-PCR using Advantage 2 PCR polymerase (Clontech/Takara Bio, Mountain View, CA, USA) based on 5′ and 3′ sequences confirmed by rapid amplification of cDNA ends (RACE, Clontech/Takara Bio).

For both npylr1 and npylr7, we isolated multiple alleles from the Orlando strain. These alleles contained sequence polymorphisms that produced changes to the protein coding sequence of the corresponding receptor as follows: NPYLR1A: D225E, 413ΔT, N487T relative to the genome reference NPYLR1B; NPYLR1B: genome reference sequence with GenBank accession KC439529 and found on Vectorbase supercontigs AAEL013505 and AAEL007924; NPYLR1C: D225E, N487T relative to the genome reference NPYLR1B; NPYLR1D: D225E, N487T, L495F relative to the genome reference NPYLR1B; NPYLR7A: genome reference sequence with Genbank accession KC439537 and associated with Vectorbase supercontigs AAEL008296 and AAEL015418; NPYLR7B: R14Q, L161F, Y189H, R250Q relative to genome reference NPYLR7A. This allele also has an insertion of a T residue at amino acid position 22. The numbering of polymorphisms C-terminal to this residue refers to NYPLR7A numbering.

An. gambiae snpfr was amplified from RNA derived from antennal tissue of the G3 strain based on published sequences by Garczynski and colleagues [12]. D. melanogaster snpfr clone GH23382 was obtained from the Drosophila Genomics Resource Center (https://dgrc.cgb.indiana.edu/). Ix. scapularis I.sca-npylr1a and I.sca-npylr1b were identified in the draft Ix. scapularis genome (IscaW1.2) in Vectorbase (http://www.vectorbase.org) using blastp. Both receptor cDNAs were cloned using RT-PCR from whole tick tissue provided by Rick Ostfeld (Cary Institute, Millbrook, NY). We were unable to determine if I.sca-npylr1a and I.sca-npylr1b are encoded by two distinct genes or are variant alleles produced by the same gene.

The sequences of all oligonucleotide primers used in this study are listed below.

All receptor cDNAs were cloned into Invitrogen's TOPO-TA Cloning system for propagation in TOP10 or DH5alpha cells. For the cell-based assay, all receptor cDNAs were recloned into the XhoI-NotI sites of the mammalian expression vector pME18S [13] except for A.gam-snpfr and D.mel-snpfr, which were cloned into EcoRI-NotI sites of the same vector.

Genbank accession numbers for the cDNAs used in this study are as follows: Ae. aegypti genes: npylr1a KC439528, npylr1b KC439529, npylr1c KC439530, npylr1d KC439531, npylr2 KC439532, npylr3 KC439533, npylr4 KC439534, npylr5 KC439535, npylr6 KC439536, npylr7a KC439537, npylr7b KC439538, and npylr8 KC439539.

Ix. scapularis genes: I.sca-npylr1a KC439540 and I.sca-npylr1b KC439541.

Primers

The following oligonucleotide primers used in this study were synthesized by Integrated DNA Technologies (Coralville, IA, USA): Primers for receptor cDNA cloning: A.aeg-npylr1a,b,c,d forward, 5′-ATGGCCATAACGATGTCATCACG-3′; A.aeg-npylr1a,b,c,d reverse, 5′-TTACAGTATCTCCGGCAGCTTGG-3′; A.aeg-npylr2 forward, 5′-ATGCTGGCAAGTACCGCTAAGAC-3′; A.aeg-npylr2 reverse, 5′-TTACAAACGTGTAATGTCTTCTTGGAAGC-3′; A.aeg-npylr3 forward, 5′-ATGAAGTCCAAGGAGACCGCGTCGGATGC-3′; A.aeg-npylr3 reverse, 5′-CTCGCCCGTAATCTTTGGCACCGC-3′; A.aeg-npylr4 forward, 5′-CGTTGTCAGCTTCGACGATGAGTGT-3′; A.aeg-npylr4 reverse, 5′-CGCCAGGAAACGTGCAGCTTCG-3′; A.aeg-npylr5 forward, 5′-ATGAGCGGCGCGCCATTCACGGTC-3′; A.aeg-npylr5 reverse, 5′-TCACCGTAGCAGGGACGTTTCCGT-3′; A.aeg-npylr6 forward, 5′-CACGCCACAATGGATTACCC-3′; A.aeg-npylr6 reverse, 5′-CATCACTTGAACAGGATCCGC-3′; A.aeg-npylr7a,b forward, 5′-GCGATGAACTTCACTGCCGAGTT-3′; A.aeg-npylr7a,b reverse, 5′-CTACAACCCCTTCCGGCACCACT-3′; A.aeg-npylr8 forward, 5′-ATGGACGTGGTCCTGTCCAGGCTG-3′; A.aeg-npylr8 reverse, 5′-TCACGGCATGAGCTCGGTAAGC-3′; Agam-snpfr forward, 5′-TTATAGAATAGCGGGCACTTTCGAGTC-3′; Agam-snpfr reverse, 5′-GACGCCTCGGAATGCTGACG-3′; I.sca-npylr1a forward, 5′-AACCCAAGCTTGTTCAATCC-3′; I.sca-npylr1a reverse, 5′-ATGCTGACATCTGGGGGTAG-3′; I.sca-npylr1b forward, 5′-TTCTTGCAGATGTCGGATCA-3′; I.sca-npylr1b reverse, 5′-TTTCTCCATGTTGCAGTGCT-3′; D.mel-snpfr76f forward, 5′-ATGGCCAACTTAAGCTGGCTGAG-3′; D.mel-snpfr76f reverse, 5′-CCTATCTCAGTTGATTCGCCTC-3′. Primers for qPCR: A.aeg-npylr1 forward, 5′-GCTATCTGCTACATCTGTGTGTCAA-3′; A.aeg-npylr1 reverse, 5′-GTCCGAGTAGAAGTCGTTGCTCAT-3′; A.aeg-rpl8 forward, 5′-TCACTGCCCACACCAAGAAGCG-3′; A.aeg-rpl8 reverse, 5′-CGGCAATGAACAACTGCTTGCG-3′. Primers for A.aeg-npylr1 homologous recombination arms: Left arm forward, 5′-TGCTGGCGTTACGGCAAACTGATTC-3′; Left arm reverse, 5′-GAACGTCACATTAACAGCGTCGCTG-3′; Right arm forward, 5′-GGTCAAGCCTTGATGCAGGACAATAC-3′; Right arm reverse, 5′-AGTATCTCCGGCAGCTTGGTGTCG-3′. Primers for non-homologous end-joining genotyping: A.aeg-npylr1 forward, 5′-CGGAACTTACGAAGCATTCAGCGAC-3′; A.aeg-npylr1 reverse, 5′-GAACACTACGTAGCATACCAACACG-3′. Primers for homologous recombination genotyping: A.aeg-npylr1 forward, 5′-TAATCGTGTGGACTAGAAGAGGG-3′; A.aeg-npylr1 reverse, 5′-AGCTCTTCGCAGTAGAATGTACG-3′.

HEK293T cell-based calcium imaging assay

HEK293T cells were cultured using standard protocols in a Thermo Scientific FORMA Series II – Water Jacketed CO₂ incubator (Waltham, MA, USA). Lipofectamine 2000 (Invitrogen) was used for transient transfection of 1 µg pME18S>Candidate receptor and 1 µg pME18S>murine G_qα15 [14]. Transfected cells were loaded with the calcium sensitive dye Fura-2 (Invitrogen) according to product instructions. Cells were imaged on a Nikon Eclipse TE-2000-U microscope (Melville, NY, USA) using fluorescent excitation from a Lambda DG-4 (Sutter Instruments Co., Novato, CA, USA). Bath application of ∼300 µl solutions containing peptides diluted in Ringer's solution (0.14 M NaCl, 5.6 mM KCl, 5 mM HEPES, 2 mM pyruvic acid, 2 mM CaCl₂, 2 mM MgCl₂, 0.15% Glucose, 1.25 mM KH₂PO₄, pH 7.4) were delivered using a diaphragm pump (Gilson Minipuls3, Middleton, WI, USA) with real-time recording and analysis in Metafluor software (Version 7.1.2.0; Molecular Devices, Sunnyvale, CA, USA). Data from a complete peptide dilution series were collected from eight responding cells within each plate of three independent transfections. Data were normalized by setting the highest fluorescent response within each experiment to 100%.

The variant alleles of NPYLR1 (alleles A–D) and NPYLR7 (alleles A–B) were tested in the cell-based assay as follows: NPYLR1A, NPYLR7A, and NPYLR7B were tested against the entire panel of peptides. NPYLR1B, NPYLR1C, and NPYLR1D were tested against Head Peptide-I and sNPF-3 only.

Behavior assays

Fluorescent measurements

Frozen mosquitoes were loaded individually into wells of a 96-well plate (Biorad, catalogue #HSP-9661) containing 100 µl phosphate-buffered saline (PBS) (prepared as a 1× solution from 10× PBS without Ca²⁺ or Mg²⁺; Lonza, BioWhittaker; Walkersville MD) plus one 2 mm glass bead (Sigma Aldrich, Cat#Z273627-1EA) and covered with PCR Sealers (BioRad, catalogue #MSB1001, Hercules, CA, USA). Control wells containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 µl of 0.02% fluorescein were combined with unfed control female mosquitoes to create a reference dilution curve. Plates were homogenized using a Qiagen TissueLyzer II at 1800 rpm for 1 minute. Homogenized plates were taken to the Rockefeller University High-Throughput Screening Resource Center, where 15 µl of homogenized solution from each well was transferred to a 384 well plate (Greiner Bio One, catalogue #784201, Monroe, NC, USA) alongside 15 µl of a 1∶10 dilution in PBS dispensed using a Thermo Multi-Drop Combi and Perkin Elmer JANUS Mini (Waltham, MA, USA). Samples were vortexed briefly and fluorescent intensity for each well was measured using a Biotek Synergy NEO plate reader (Winooski, VT, USA). Using the reference dilution curve, fluorescent measurements were converted back to volume (µl) of solution ingested.

Peptide synthesis

Head Peptide-I (pERPhPSLKTRFa), Head Peptide-III (pERPPSLKTRFa), and Head Peptide-I [Cys10] (pERPhPSLKTRC) were synthesized by the Rockefeller University Proteomics Resource Center. sNPF-1 (KAVRSPSLRLRFa), sNPF-1(4-11) (SPSLRLRFa), sNPF-2+4 (APQLRLRFa), and sNPF-3 (APSQRLRWa), NPF (SFTCARPQDDPTSVAEAIRLLQELETKHAQHARPRFa), human NPY (YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRYa), and human PYY (IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRYa) were synthesized by Bachem Bioscience Inc. (King of Prussia, PA, USA).

Peptide injections

Solutions of Head Peptide-I, sNPF-3 and Head Peptide-I [Cys10] were dissolved in saline (0.1M NaCl, 4 mM KCl, 2 mM CaCl₂) at a concentration of 4 and 10 mM for injection. ∼20 female mosquitoes aged 5 to 14 days were anesthetized on ice for 3 min, moved individually onto a chill-plate (BioQuip catalogue #1429, Rancho Dominguez, CA, USA), and injected with 200 nl of desired solution using a Drummond Nanoject II (Drummond catalogue # 3-000-204, Broomall, PA, USA) attached to 3.5″ needles (Drummond, catalogue #3-000-203-G/X) shaped on a needle puller (Sutter Instruments Co., Model P-97). Solutions were injected into the abdomen by piercing the second abdominal tergite from the ovipositor. Injected mosquitoes were placed in loaders and allowed one hour to recover from injection before being tested for host-seeking behavior in the uniport olfactometer.

Quantitative PCR (qPCR)

RNA was purified from whole bodies of female mosquitoes before and 24, 48, and 72 hours after blood-feeding. Purified RNA was converted to cDNA and aliquoted at 250 ng/µl RNA equivalents for each qPCR reaction. Reactions were prepared as instructed by BioRad iQ SYBR Green SuperMix (catalogue #170-8882) in BioRad iQ 96-well Plates (catalogue #223-9441) covered with BioRad Microseal “B” Adhesive Seals (catalogue #MSB-1001) to be run on a iQ5 iCycler (BioRad). Primers were designed to meet the following criteria: 90–110% efficiency and an R² above 0.98 in control reactions. Three biological replicates of each sample were performed in triplicate for every condition.

Targeted mutagenesis with zinc-finger nucleases

Zinc-finger nucleases targeting Ae.aegypti npylr1 were designed using the CompoZr Custom ZFN technology by Sigma-Aldrich Life Science (St. Louis, MO, USA). Genetic Services Inc. (Cambridge, MA, USA) injected purified npylr1 ZFN mRNA into ∼3000 pre-blastoderm stage Ae. aegypti Orlando embryos (three batches of 1000, 800, and 1200 eggs each) at a concentration of 200 ng/µl plus a homologous recombination vector at 850 ng/µl using embryo preparation methods described previously [16]. The homologous recombination vector contained 1319 and 1451 bp of homology to the left and right flanking sequence of the ZFN cut site and were obtained by PCR reactions from Ae. aegypti Orlando genomic DNA. Left and right arms were cloned into SalI and NotI sites, respectively, of the homologous recombination donor plasmid pSL1180-HR-PUbECFP (a gift of Conor McMeniman and deposited in Addgene, plasmid number 47917) that contains the Ae. aegypti polyubiquitin promoter [17] driving expression of ECFP, for a total insert size of 2565 bp.

36% of injected embryos hatched and were sexed as male and female prior to eclosion. 94% of the hatched individuals developed into adults and each injection batch was inter-crossed after reaching sexual maturity (∼2 days). Each of the three inter-crossed groups was considered to contain independent ZFN events. Potential mutants carrying the polyubiquitin-ECFP cassette were isolated by aliquoting 3-day old generation 1 (G1) larva into 96-well plates (VWR International, catalogue #29444-018) and screening for ECFP fluorescence on a Nikon SMZ1500 excited by an Intensilight C-HGFI. Of an estimated 55,000 total larvae screened, 27, 6, and 18 larvae were ECFP positive in each respective batch.

Non-homologous end-joining mutants were isolated a few months later from a small number of unhatched eggs stored from the third injection batch of G1 mosquitoes. Seven G1 larvae hatched and were outcrossed to wild-type Ae. aegypti Orlando. After successful mating, genomic DNA was prepared from each of the seven individuals and npylr1 PCR amplicons were sequenced for ZFN activity. Small deletions were detected in two individuals. PCR clones for the two positive individuals were Sanger sequenced by Genewiz to confirm 4 and 8 bp deletions, respectively.

For each non-homologous end-joining mutant, G1 individuals were inter-crossed for four generations to establish homozygous lines. Genotyping of individual non-homologous end-joining mutants was carried out by Genewiz using a capillary gel electrophoresis DNA analyzer (ABI3730xl, Applied Biosystems, Carlsbad, CA, USA) to analyze PCR amplicons from 6-FAM fluorescently labeled primers spanning the npylr1 ZFN cut site. Data were analyzed using Peak Scanner software (Applied Biosystems). Homologous recombination mutants were out-crossed to Ae. aegypti Orlando for five generations prior to establishing homozygous lines. Mutant individuals carrying the integrated homologous recombination cassette were genotyped by gel electrophoresis of PCR amplicons spanning the inserted DNA. Heterozygous control lines for behavioral experiments were generated by crossing homozygous mutant lines to the Ae. aegypti Orlando strain, which expresses a mixture of the four functionally equivalent npylr1 alleles.

Statistics

Statistical tests were performed as indicated in each figure legend using Graph Pad PRISM (La Jolla, CA, USA) except for statistical analysis of qPCR results, which also used BioRad iQ5 software.

NPYLR1 identified as a receptor sensitive to Head Peptide-I and sNPFs

Head Peptide-I shows sequence similarity to short neuropeptide-F peptides (sNPFs) that have been implicated in feeding behaviors [18]–[20] and are known to signal through Neuropeptide Y (NPY)-Like Receptors (NPYLRs) [21]. We identified eight npylr genes in the Ae. aegypti genome (Figure 2A), all of which are orphan receptors that have not been linked to a candidate peptide ligand. To deorphanize these receptors and identify those that respond to Head Peptide-I, we transfected each into HEK293T cells along with murine G_qα15, a promiscuous Gα subunit capable of coupling diverse G protein-coupled receptors to a phospholipase C stimulated calcium signaling pathway [14]. We screened each receptor in a cell-based calcium imaging assay for sensitivity to a panel of Ae. aegypti peptides including Head Peptide-I [8], [10], behaviorally inactive Head Peptide-III [10], inactive Head Peptide-I [Cys10] [10], sNPF-1 [22], sNPF-1(4-11) [22], sNPF-2+4 [22], sNPF-3 [22], and NPF [22](Figure 2B). We also used human Neuropeptide Y (NPY) and Peptide YY (PYY) [23](Figure 2B). Cells transiently co-transfected with a given receptor and G_qα15 were loaded with the calcium-sensitive dye Fura-2 and imaged in response to bath application of peptides (Figure 2C, D, and E).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Deorphanization of NPY-Like receptors.

(A) Phylogram depicting the genetic relationships of eight Ae. aegypti NPY-Like Receptors (NPYLR1, red; NPYLR2-8, black) as well as NPYLR1 homologues An. gambiae-sNPFR (blue), Ix. scapularis I.sca-NPYLR1A and I.sca-NPYLR1B (light and dark green), and D. melanogaster-sNPFR (magenta). (B) Peptides used in the HEK293T cell-based calcium imaging assay displayed with single letter amino acid codes and these variants: pE: pyroglutamic acid, hP: hydroxyproline, a: amidation, sNPF: Ae. aegypti short-Neuropeptide-F, NPF: Ae. aegypti Neuropeptide-F, Human NPY: Neuropeptide Y, Human PYY: Peptide YY. (C) Top: Representative images of HEK293T cells transiently transfected with Ae. aegypti npylr1a, illustrating calcium flux after application of indicated peptides. Bottom: Individual traces of fluorescent ratios representing Ca²⁺ flux for the same HEK293T cells following application of the indicated peptides. (D) Head Peptide-I dose-response curves and EC50 values for NPYLR1 homologues in Ix. scapularis, An. gambiae, and D. melanogaster. (E) Summary of results for the cell-based calcium imaging screen. NPYLR2, NPYLR 3, NPYLR 4, and NPYLR 6 did not respond to any peptides tested (HP = Head Peptide).

https://doi.org/10.1371/journal.pntd.0002486.g002

Of the eight receptors screened, NYPLR1 was most sensitive to all four sNPFs (EC50: 35–75 nM), but also responded to Head Peptide-I, albeit at much higher concentrations (EC50: 823 nM). Head Peptide-I [Cys10] evoked no response at concentrations up to 50 µM (Figure 2C). The Ae. aegypti Orlando strain expresses four npylr1 alleles, which were functionally indistinguishable in the cell-based assay (data not shown).

Ae. aegypti NPYLR1 is homologous to the sNPF receptors (sNPFR) of D. melanogaster [21], [24] and An. gambiae [12]. Contrary to previous reports in An. gambiae [12], we found that NPYLR1 orthologues from both insects responded to Ae. aegypti Head Peptide-I (Figure 2D), despite no evidence for a Head Peptide gene in either insect. Both orthologues were also reported to lack sensitivity to Head Peptide-III [12], [21], [24], but we found that NPYLR1 was equally sensitive to both Head Peptide-I and Head Peptide-III. We have no explanation for these discrepancies, but note that since D. melanogaster and An. gambiae lack a Head Peptide gene, the physiological relevance for the response or non-response of these orthologues to Head Peptides is unclear.

We identified a candidate Head Peptide gene in the incomplete genome of the Lyme disease vector, Ixodes scapularis, which like Ae. aegypti displays intermittent blood-feeding [25], [26]. The candidate Ix. scapularis Head Peptide gene would encode two copies of a pro-peptide with sequence ERPPPQKIRF, which is 70% similar and 60% identical to the pro-peptide form of Ae. aegypti Head Peptide-I: QRPPSLKTRF. We did not validate this prediction by RNA analysis in ticks nor did we functionally test whether this peptide, predicted solely based on in silico analysis, exists in vivo or would activate any of the NPYLRs tested here. We found no evidence for Head Peptide gene candidates in any other currently available genome except Aedes aegypti.

Because ticks have an apparent Head Peptide gene, we searched the genome for NPYLR1 orthologues and found two candidate genes. We cloned both tick NPYLR1 genes and demonstrated that each was sensitive to Ae. aegypti Head Peptide-I (Figure 2D). These results are the first evidence for Head Peptide-I signaling outside of Ae. aegypti.

In summary, four of eight Ae. aegypti NPYLRs responded to one or more of the peptide ligands (Figure 2E). We examined two npylr7 alleles, and while NPYLR7A did not show any responses, NPYLR7B responded to three of ten peptides tested. The four NPYLRs that failed to respond to our panel of peptides (NPYLR2, NPYLR3, NPYLR4, and NPYLR6) may respond to other ligands not tested in our assay or may not be functionally expressed in our assay.

With functional evidence that NPYLR1 responds to both Head Peptide-I and sNPFs, we tested whether sensitivity of the receptor to sNPFs indicated a role for these neuropeptides in host-seeking inhibition. To begin, we replicated previously published experiments that reported Head Peptide-I inhibition of host-seeking behavior by injection into non-bloodfed females [9] (Figure 3A). We then found that injection of sNPF-3 also inhibited host-seeking behavior in non-blood-fed females, whereas control injections of buffer or inactive Head Peptide-I [Cys10] had no effect (Figure 3A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Injection of Head Peptide-I or sNPF-3 inhibits host-seeking behavior and npylr1 expression is up-regulated after blood-feeding.

(A) Percent attraction of non-blood-fed female mosquitoes to human host stimuli after injection of buffer (open bar) or 4 and 10 mM of the indicated peptide. (n = 6; ∼20 females per trial, ANOVA with Bonferroni correction for multiple comparisons; ** = p<0.01). (B) qPCR analysis of npylr1 transcripts in total RNA extracted from female whole body tissue at the indicated times after a blood meal. (n = 3, ANOVA-Tukey's Multiple Comparison of ΔCt values). All data are plotted as mean±SEM.

https://doi.org/10.1371/journal.pntd.0002486.g003

Given earlier observations that Head Peptide-I titers increase in hemolymph at 48 hours after blood-feeding [10], we asked if npylr1 gene expression was similarly controlled by feeding state. Using qPCR analysis of RNA derived from female whole body tissue, we found that expression of npylr1 is up-regulated for at least three days following a blood meal and peaks with a 15-fold increase at 48 hours (Figure 3B). This peak in npylr1 expression is coincident with the time of maximal host-seeking inhibition (Figure 1B).

Targeted mutagenesis of npylr1 with zinc-finger nucleases

To ask if NPYLR1 is necessary for blood-feeding-induced host-seeking inhibition, we generated npylr1 null mutant mosquitoes using targeted mutagenesis with zinc-finger nucleases (ZFNs). ZFNs are fusion proteins of a sequence-specific DNA binding protein and a fokI-nuclease that induces mutagenic double-stranded breaks [27], [28]. To prepare for mutagenesis, we clarified the gene structure of npylr1. The current draft of the Ae. aegypti genome (AaegL1.4) has two gene entries for npylr1, suggestive of a gene duplication (AAEL013505 and AAEL007924). We used Southern blotting and PCR-based genotyping to confirm that these two annotated genes are in fact two alleles of a single npylr1 gene locus that show normal Mendelian segregation (data not shown).

ZFNs targeting the 5′ end of the coding sequence encoding the N-terminal region of NPYLR1 (Figure 4A) were injected into wild-type mosquito embryos together with a DNA vector that would act as a template for repair by homologous recombination (HR). Three independent mutation events (npylr1^HR1, npylr1^HR2, npylr1^HR3) were confirmed by integration of a marker cassette containing a broadly expressed fluorescent protein into the npylr1 locus (Figure 4B and 4C). Southern blotting and PCR analysis confirmed each event to be a single integration at the npylr1 locus (Figure 4D and data not shown). Additional injected progeny were analyzed to isolate two new alleles lacking the ECFP cassette and produced by error-prone non-homologous end-joining, npylr1⁴ and npyl1⁸, with deletions of 4 bp and 8 bp respectively (Figure 4B). All five mutant alleles are predicted to create truncated NPYLR1 protein (Figure 4E). Using these homozygous mutants, we generated heteroallelic npylr1 mutant combinations to control for independent background mutations for behavioral analysis.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. Targeted mutagenesis of npylr1 using zinc-finger nucleases.

(A) Snake plot of predicted NPYLR1 membrane topology with the corresponding protein region encoded by the npylr1 zinc-finger nuclease targeted DNA sequence colored in purple. (B) Illustration of the zinc-finger nuclease target region in the npylr1 gene with corresponding mutations generated by non-homologous end-joining (npylr1⁴ and npylr1⁸, red) and integration of a fluorescent marker by homologous recombination (npylr1^HR2, cyan). (C) Photographs of npylr1^HR2/+ and wild-type mosquito pupae illustrating broad expression of the ECFP marker. (D) PCR verification of homologous recombination into the npylr1 locus of the 2565 bp polyubiquitin>ECFP cassette. (E) Snake plots of predicted protein truncations for npylr1 mutant alleles. Filled purple circles indicate ZFN target region and outlined purple circles indicate resulting frame-shift mutations.

https://doi.org/10.1371/journal.pntd.0002486.g004

Locomotor and egg-laying behavior of npylr1 mutants

As a likely receptor for neuromodulation, NPYLR1 may be involved in broader physiological roles such as motor coordination or egg development [29]. We found that locomotor activity of npylr1^8/4 was indistinguishable from control lines (Figure 5A right). npylr1^HR2/HR3 showed a decrease in average daily activity in comparison to wild-type but was not significantly different from either npylr1^HR2/+ or npylr1^HR3/+ controls (Figure 5A left). Based on these data, we conclude that locomotor activity is generally normal in npylr1 mutants.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 5. Normal locomotor behavior and egg-laying in npylr1 mutants.

(A) Average daily activity counts for individual female npylr1^HR2/HR3 mutants (left, n = 14) and npylr1^8/4 mutants (right, n = 7). (B) Eggs deposited per individual female npylr1^HR2/HR3 (left, n = 18–20) and npylr1^8/4 (right, n = 9–28). All data are plotted as mean±SEM. 1-Way ANOVA with Bonferroni correction for multiple comparisons. * = p<0.05; ** = p<0.01, ns = not significant.

https://doi.org/10.1371/journal.pntd.0002486.g005

Quantification of deposited eggs from individual females blood-fed to completion on a human arm showed no differences in npylr1^HR2/HR3 (Figure 5B left). A slight decrease was observed for npylr1^8/4 compared to wild-type, but not to npylr1^8/+ or npylr1^4/+ controls (Figure 5B right). Based on these results, we conclude that egg-laying is unaffected in npylr1 mutants.

Sugar and blood feeding of npylr1 mutants

Beyond acting as a putative modulator of blood-feeding-induced host-seeking behavior, sNPFs and/or Head Peptide-I may communicate a general internal fed state [18]. Female mosquitoes do not feed only on blood, but also on floral nectars to satisfy metabolic needs [30]. It is not known whether mosquitoes independently regulate sugar- and blood-feeding, so we examined both in npylr1 mutants.

We studied sugar-feeding using the CAFE (Capillary Feeder) assay adapted from a similar assay originally designed for D. melanogaster [15]. In this assay, five female mosquitoes feed from a glass capillary filled with 10% sucrose, and the change in volume is measured to determine the amount of sugar solution ingested by the group over a two hour period (Figure 6A). To confirm that this assay measures sugar-feeding and not water-seeking, we carried out control experiments which indicated that fasted mosquitoes did not consume water alone from the capillary (data not shown). Non-fasted mosquitoes or those fasted for 12 hours consumed very little sugar in this assay (Figure 6B). After 24, 48, or 72 hours of fasting, there was an increase in sugar ingested (Figure 6B), but we observed high mortality after 72 hours of fasting. Based on these results, we analyzed sugar feeding in npylr1^HR3/HR1 mutants and associated genetic controls after 0 and 48 hours of fasting and found no difference between mutants and controls at either time point (Figure 6C).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 6. Fasting response, sugar-feeding and blood-feeding are normal in npylr1 mutants.

(A) Diagram of the CAFE assay. (B) Volume of sugar ingested for female mosquitoes in the CAFE after fasting for 0, 12, 24, 48, and 72 hours (n = 8–16, except 72 hours, n = 2; 5 mosquitoes per trial. Mean±SEM. 1-way ANOVA compared to 0 hours with Dunnett's multiple comparison test. * = p<0.05, *** = p<0.001, ns = not significant). (C) Sugar ingested by the indicated genotypes at 0 and 48 hours after fasting (n = 16; 5 females per trial. Mean±SEM, 2-way ANOVA with Bonferroni correction for multiple comparisons). (D) Diagram of the BUFFET assay. (E) Sugar ingested by the indicated genotypes at 48 hours after fasting (n = 64; 16 females tested in each of 4 independent trials). (F) Diagram of the membrane feeding assay. (G) Percent of mosquitoes for the indicated genotypes that blood-fed in the membrane feeding assay (n = 6, 15 females per trial). (H) The average blood meal sizes for the indicated genotypes. (n = 6, 1–12 females confirmed as fed per trial). Data in E, G, H are plotted as mean±SEM, 1-way ANOVA with Bonferroni correction for multiple comparisons.

https://doi.org/10.1371/journal.pntd.0002486.g006

To quantify sugar feeding by individual female mosquitoes more precisely, we designed the BUFFET assay. In this assay, fifty female mosquitoes were fasted for 48 hours and then allowed to feed on 10% sucrose+0.02% fluorescein presented in a 96-well micro-titer plate at the bottom of a mosquito cage (Figure 6D). After 2 hours, mosquitoes were individually homogenized to release the fluorescein from their gut, which was quantified by a fluorescent plate reader. Results from the BUFFET indicated that the amount of sugar ingested by individual npylr1^8/4 mutants did not differ from controls (Figure 6E).

To measure blood feeding, we used a membrane feeding assay with blood containing the fluorescent dye fluorescein. In this assay, fifteen female mosquitoes were given 15 minutes to feed from an artificial glass feeder containing warmed sheep blood mixed with 0.02% fluorescein (Figure 6F). Carbon dioxide (CO₂), a general activator of mosquito behavior [31], [32], was added to the test environment to induce blood-feeding. Similar to the BUFFET, individual mosquitoes were then homogenized to release the fluorescein for quantification on a fluorescent plate reader. In these experiments, approximately 40% of both control and npylr1 mutant mosquitoes successfully blood-fed (Figure 6G). We found no difference in the volume of blood ingested by npylr1 mutants compared to controls (Figure 6H). The average volume of blood ingested was different between npylr1^8/4 and npylr1^HR2/HR3 experiments, but we believe this is an effect of using a different batch of sheep blood for each experiment or experimental variation from carrying out these experiments 3 months apart, rather than an effect of npylr1 genotype on blood-meal size.

Host-seeking behavior of npylr1 mutants

Prior to a blood meal, female npylr1 mutants displayed robust host-seeking behavior indicating no baseline defect in their response to host stimuli (Figure 7A and B). Following a blood meal, inhibition of host attraction was observed at 24 hours as expected from abdominal distension (Figure 7A and B). At 48 and 72 hours post-blood meal—time points suspected to be under the influence of Head Peptide-I [4], [10]—npylr1^HR2/HR3 and npylr1^8/4 displayed inhibition of host-seeking behavior at similar levels to controls (Figure 7A and B). Like wild-type mosquitoes, npylr1 mutants recovered host-seeking behavior following egg deposition between 72 hours and 120 hours post-blood meal (Figure 7A and B). To ask whether npylr1 mutants would show defects immediately following the relief of inhibition by abdominal distension at ∼24 hours post-blood meal, we increased the temporal resolution of these experiments to test at 32 and 40 hours post-blood meal. These experiments showed strong host-seeking suppression in all genotypes at both time points (Figure 7C).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 7. Normal host-seeking behavior of npylr1 mutants throughout the gonotrophic cycle.

Host-seeking behavior of npylr1 (A) homologous recombination mutants and (B) non-homologous end-joining mutants with associated controls before and 24, 48, 72, and 120 hours after a blood meal. Egg-laying was observed after 72 hours and completed by 120 hours (n = 6–12, ∼20 females per trial). (C) Host-seeking behavior of npylr1 homologous recombination mutants and associated controls at 32 and 40 hours after a blood meal (n = 3, ∼20 females per trial). All data are plotted as mean±SEM. 1-way ANOVA with Bonferroni correction for multiple comparisons within each time point.

https://doi.org/10.1371/journal.pntd.0002486.g007