1. Genetics and Genomics
  2. Neuroscience
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Fruitless mutant male mosquitoes gain attraction to human odor

  1. Nipun S Basrur  Is a corresponding author
  2. Maria Elena De Obaldia
  3. Takeshi Morita
  4. Margaret Herre
  5. Ricarda K von Heynitz
  6. Yael N Tsitohay
  7. Leslie B Vosshall  Is a corresponding author
  1. Laboratory of Neurogenetics and Behavior, The Rockefeller University, United States
  2. Kavli Neural Systems Institute, United States
  3. Howard Hughes Medical Institute, United States
Research Article
Cite this article as: eLife 2020;9:e63982 doi: 10.7554/eLife.63982
5 figures, 1 table, 1 data set and 3 additional files

Figures

Sex-specific mosquito attraction to humans and fruitless splicing.

(A, B) Arm-next-to-cage assay schematic A and image B with female (top) and male (bottom) Aedes aegypti mosquitoes. (C) Percent mosquitoes next to arm measured every 30 s. Data are mean ± s.e.m., n = 6 trials, n = 20 mosquitoes/trial; *p<0.05, Mann-Whitney test for each time point. (D, E) Schematic of Aedes aegypti fruitless genomic locus D and sex-specific splicing region with RNA-seq read evidence E. (F) Phylogeny of mosquito species with outgroup Drosophila melanogaster, with conserved fruitless exon structure inferred from de novo transcriptome assembly. In E, F, coding and non-coding exons are represented by filled and open dashed bars, respectively. Toxorhynchites rutilus and Culex salinarius images were used to represent Toxorhynchites amboinensis or Culex quinquefasciatus, respectively. See acknowledgments for photo credits. Cartoons indicate blood-feeding (blood drop) and non-blood-feeding (flower) species. (G, H) Aedes aegypti fruitless exon usage based on male and female RNA-seq data (normalized counts) from the indicated tissue plotted for each exon G and m, f, and c1 exons H n = 3–4 independent RNA-seq replicates (Matthews et al., 2016). (I) Schematic of generation of fruitless∆M and fruitless∆M-tdTomato mutants.

Figure 2 with 1 supplement
Expression of fruitless in the mosquito brain.

(A–F) Confocal images of brains of the indicated genotypes showing fruitless >tdTomato (green) and Brp (magenta) expression. Scale bars, 20 µm. (D–F) Top-to bottom images are optical sections of the same brain, arranged from anterior to posterior.

Figure 2—figure supplement 1
Expression of fruitless in the female mosquito ventral nerve cord.

(A–B) Confocal images of ventral nerve cords of the indicated genotypes showing fruitless >tdTomato (green) and Brp (magenta) expression. All scale bars, 20 µm.

Expression of fruitless in the mosquito olfactory system.

(A) Schematic of antennal olfactory sensory neurons and their projections to the antennal lobe of the mosquito brain. (B–D) Confocal images of antennae of the indicated genotypes with fruitless >tdTomato (green) and Orco (magenta) expression. (E) Confocal image of orco mutant antenna, as negative control for Orco and tdTomato antibodies, with DAPI (blue). (F) Number of antennal lobe glomeruli labeled by fruitless >tdTomato in the indicated genotypes. (G–I) Confocal images of antennal lobes of the indicated genotypes with fruitless >tdTomato (green) and Brp (magenta) expression. (J–L) Confocal images of antennal lobes of the indicated genotypes showing fruitless >tdTomato (green) and Brp (magenta) expression. Top-to bottom images are optical sections of the same lobes, arranged from anterior to posterior. All scale bars, 20 µm.

Male fruitless mutant mosquitoes gain attraction to a live human host.

(A) Feeding assay schematic. (B) Feeding on indicated meal (n = 8 trials/meal; n = 20 mosquitoes/trial). (C) Insemination assay schematic. (D) Insemination of wild-type females by males of the indicated genotype (n = 6 trials/male genotype, n = 20 females/trial; *p=0.0022, Mann-Whitney test). (E) Schematic of Quattroport assay, highlighting ability to run multiple stimuli and genotypes simultaneously. (F) Side-view schematic of Quattroport, highlighting close-range (attraction) and long-range (activation) metrics (G) Percent activated animals, n = 8–14 trials/group, n = 17–28 mosquitoes/trial. (H, J) Quattroport assay schematic for nectar-seeking H and live human host seeking J. 1% CO2 is added to the airstream in the live human host seeking assay J. (I, K) Percent of attracted animals (n = 8–14 trials per group, n = 17–28 mosquitoes/trial). Data in B, D, G, I, K are mean ± s.e.m. In B, I, G, K, data labeled with different letters are significantly different from each other (Kruskal-Wallis test with Dunn’s multiple comparisons, p<0.05). In B, comparisons are made between genotypes for each meal. In I, K, comparisons are made between all genotypes and stimuli.

Figure 5 with 2 supplements
Olfactory cues selectively drive male fruitless mutant attraction to humans.

(A) Heat-seeking assay schematic. A 20 s pulse of 10% CO2 is added to the assay. (B) Percent of animals on Peltier. Data are mean ± s.e.m., n = 6 trials/temperature, n = 50 mosquitoes/trial. Data labeled with different letters are significantly different from each other, within each temperature. (C) Schematic of human odor host-seeking assay (left) and stimuli (right). (D) Percent of attracted animals. Data are mean ± s.e.m., n = 8–14 trials/group, n = 17–28 mosquitoes/trial. (E–F) Summary of results and model of gain of fruitless function in Aedes aegypti. Photo credit: Aedes aegypti (Alex Wild); D. melanogaster (Nicolas Gompel). In B, D, data labeled with different letters are significantly different from each other (Kruskal-Wallis test with Dunn’s multiple comparisons, p<0.05). In B, comparisons are made between genotypes at each temperature. In D, comparisons are made across all genotypes and stimuli.

Figure 5—figure supplement 1
No significant blood-feeding or mating defects in fruitless∆M females.

(A) Percent of females crossed to indicated male genotype blood-feeding on a live human arm. (B) Percent of females crossed to indicated male genotype laying eggs. Data in A, B, are mean ± s.e.m., n = 3–10 trials/group, n = 12–27 mosquitoes/trial. Data labeled with different letters are significantly different from each other (Kruskal-Wallis test with Dunn’s multiple comparisons, p<0.05). Comparisons were made across all genotypes. (C) Number of inseminated females by males of indicated genotype. n = 10 females.

Figure 5—figure supplement 2
Female fruitless∆F mutant mosquitoes have blood-feeding and egg-laying defects.

(A) Schematic of Aedes aegypti fruitless genomic locus. (B) Sex-specific fruitless transcripts and generation of fruitless∆F mutant, with effect of splicing on Fruitless protein. (C) Crossing scheme to generate female mutants and potential male recombinants. (D) Blood-feeding assay schematic. (E) Feeding on live human arm; p<0.0001, Chi-square test. (F) Insemination assay schematic. (G) Insemination of females of indicated genotype by wild-type males; p=0.5464, Fisher’s exact test. (H) Egg-laying assay schematic. (I) Egg-laying by females of indicated genotype; p<0.0001, Fisher’s exact test.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Aedes aegypti)fruitlessRefSeqLOC5567734
Gene (Anopheles gambiae)fruitlessVectorBaseAGAP000080
Gene (Culex quinquefasciatus)fruitlessRefSeqCPIJ010853
Gene (Wyeomyia smithii)fruitlessThis paperFigure 1—source data 1
Gene (Toxorhynchites amboinensis)fruitlessThis paperFigure 1—source data 1
Strain, strain background (Aedes aegypti, both sexes)LVP-IB12BEI resourcesMRA-735
Strain, strain background (Anopheles gambiae, both sexes)G3Flaminia Catteruccia (Harvard University)
Strain, strain background (Culex quinquefasciatus, both sexes)JHBBEI resourcesNR-43025
Strain, strain background (Wyeomyia smithii, both sexes)PBWilliam Bradshaw and Christina Holzapfel (University of Oregon)
Strain, strain background (Toxorhynchites amboinensis, both sexes)Laurence Zwiebel (Vanderbilt University)
Genetic reagent (Aedes aegypti, both sexes)fruitless∆MThis paperfruitless mutant strain; eggs available on request
Genetic reagent (Aedes aegypti, both sexes)fruitless∆M-tdTomatoThis paperfruitless mutant strain; eggs available on request
AntibodyMouse monoclonal anti-Apocrypta bakeri Orco antibodyVanessa Ruta (The Rockefeller University) (PMID:30111839)15B2IF(1:50)
AntibodyRabbit polyclonal anti-RFP antibodyRocklandCat# 600-401-379, RRID:AB_2209751IF(1:200)
AntibodyMouse monoclonal anti-Brp antibodyDSHBCat# nc82, RRID:AB_2314866IF(1:5000)
AntibodyGoat polyclonal anti-mouse Alexa Fluor 488 antibodyThermo FisherCat# A-11001, RRID:AB_2534069IF(1:500)
AntibodyGoat polyclonal anti-mouse Alexa Fluor 555 Plus antibodyThermo FisherCat# A32732, RRID:AB_2633281IF(1:500)
AntibodyGoat polyclonal anti-mouse Alexa Fluor 647 polyclonal antibodyThermo FisherCat# A-21235, RRID:AB_2535804IF(1:500)
Recombinant DNA reagentFruitless∆-T2A-QF2 (plasmid)This paperPlasmid used to generatefruitless∆M strain
Recombinant DNA reagentFruitless∆-T2A-CsChrimson-TdTomato (plasmid)This paperPlasmid used to generatefruitless∆M-tdTomato strain
Commercial assay or kitNEBuilder HiFi DNA AssemblyNEBNEB:E5520S
Commercial assay or kitHiScribe Quick T7 kitNEBNEB:E2050S
Peptide, recombinant proteinCas9PNABioPNABio:CP01-200
Sequence-based reagentsgRNA_forThis paperPCR primer for sgRNAGAAATTAATACGACTCACTATAGCACCGAAGGTATGTTGAGGTTTTAGAGCTAGAAATAGC
Sequence-based reagentsgRNA_revThis paperPCR primer for sgRNAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC
Software, algorithmSTAR v 2.5.2aRNAseq alignment
Software, algorithmTrinity v 2013-03-25De novo assembly
Software, algorithmPrism v8GraphPadPrism 8statistics
Software, algorithmMacVector v15.0.3MacVectorMacVectorPlasmid cloning
OtherDAPI stainSigmaD95421:10,000
OtherQuattroportThis paperhttps://github.com/VosshallLab/Basrur_Vosshall2020; Basrur, 2021; (copy archived atswh:1:rev:529b0ba393bac09207d298e73e425b452338e876)

Data availability

All raw data are provided in the accompanying source data files. Plasmids are available at Addgene (#141099, #141100). RNA-seq data are available in the Short Read Archive at Genbank (Bioproject: PRJNA612100). Details of Quattroport fabrication and operation are available at Github: https://github.com/VosshallLab/Basrur_Vosshall2020 (copy archived at https://archive.softwareheritage.org/swh:1:rev:529b0ba393bac09207d298e73e425b452338e876).

The following data sets were generated
  1. 1
    NCBI BioProject
    1. N Basrur
    (2020)
    ID PRJNA612100. Sex-specific mosquito brain transcriptomes.

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