Fig 1.
CRISPR-mediated construction of AalbOrco-QF2 driver and AalbQUAS-mCD8:GFP effector lines.
Homology-assisted CRISPR knock-in strategy. The T2A-QF2-3xP3-DsRed element (2549-bp) was inserted into the fourth exon of AalbOrco via CRISPR-mediated homologous recombination. PCR validation utilized primers AalbOrco-F/AalbOrco-R and produced a 552-bp wild-type amplicon and a 3,071-bp mutant-specific product in homozygous individuals. (B) Primers AalbOrco-F and AalbOrco-R were designed to amplify the region outside the homology arm. This ensured allele-specific amplification, as the wild-type genome produced only the 552 bp fragment, while the knock-in allele generated the 3,071 bp band. (C) Agarose gel electrophoresis successfully confirmed knock-in integration in transgenic mosquitoes. The 3,071-bp band exclusively appeared in homozygous AalbOrcoDsRed/DsRed individuals, while wild-type controls showed only the 552-bp fragment. (D) The 15xQUAS-GFP cassette (5,447-bp) was inserted into random TTAA sites in the genome via PiggyBac system. (E) In AalbOrco-QF2 driver lines, DsRed fluorescence localized specifically to the eyes (scale bar: 300 μm), indicating functional promoter activity. (F) AalbQUAS-mCD8:GFP effector lines exhibited ECFP fluorescence in the eyes, demonstrating transposon-mediated cassette insertion (scale bar: 300 μm).
Fig 2.
Localization of Orco-expressing neurons in the late embryo of Ae. albopictus.
(A) & (B) Representative Orco-expressing neurons in the antenna-like tissue of the embryo at sample 1 (A) and sample 2 (B) 72 hours post-egg laying.
Fig 3.
Localization of Orco-expressing neurons in the larval antenna of Ae. albopictus.
(A - D) Maximum intensity projections of z-stack images showing GFP-labeled neurons in the antennae of 1-4 instar larvae. Scale bar: 20 μm. (E) Quantitative analysis of Orco+ neurons in 1-4 instar larvae. The number of Orco+ neurons increases with developmental stage, NL1 = 5.25 ± 1.40 (n = 8); NL2 = 7 ± 1.10 (n = 6); NL3 = 11 ± 0.89 (n = 6); NL4 = 11.93 ± 1.49 (n = 14).
Fig 4.
Localization of Orco in olfactory appendages of Ae. albopictus.
(A and B) Representative confocal z-stack of Orco-expressing neurons in the antenae of female (A) and male (B) mosquitoes which exhibits sexual dimorphism with Orco expressed in all 13 segments of the female antennae while only in the distal two segments of the male antennae. (C) A representative confocal z-stack of a whole-mount female labellum showing Orco is expressed in T2 sensilla but not T1 sensillum (highlighted by arrows; scale bars, 10 μm). (D and E) Representative confocal z-stack of Orco-expressing neurons in the capitate peg sensilla of maxillary palp of both female (D) and male (E) mosquito.
Fig 5.
Comparison of expression of Or tuning receptors between the wild-type and AalbOrcoDsRed/DsRed homozygous mutant mosquitoes.
Heatmap showing the average antennal expression in FPKM for each of the Or genes in wild-type and AalbOrcoDsRed/DsRed mosquitoes. (B) Transcriptional level of Orco gene in wild-type and AalbOrcoDsRed/DsRed mosquitoes. (C) Top 20 most highly expressed Or genes in wild-type were significantly reduced in AalbOrcoDsRed/DsRed mosquitoes. (D) Expression in FPKM for detected IR tuning receptor and co-receptor genes in wild-type and AalbOrcoDsRed/DsRed mosquitoes. (E) Expression in FPKM for detected GR genes in wild-type and AalbOrcoDsRed/DsRed mosquitoes. (F) Expression of all the two upregulated Or tuning receptors genes in the AalbOrcoDsRed/DsRed mosquitoes. Mann-Whitney U test was applied in the statistical analysis, statistical significance is presented as P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001(****) and P > 0.05 (ns).
Fig 6.
EAG responses of wild-type and AalbOrcoDsRed/DsRed Ae. albopictus to a broad panel of human odorants.
(A) Representative EAG response traces of wild-type and AalbOrcoDsRed/DsRed mosquito to different odorants; (B) Comparison of EAG responses of wild-type and AalbOrcoDsRed/DsRed Ae. albopictus to 50 odorants in different chemical classes (n = 7). EAG responses (∆mV) for each odorant at a 10−1 dilution were normalized to the solvent control (Paraffin oil and DMSO were used as solvents. Indole and skatole were dissolved in DMSO, while the other 48 compounds were dissolved in Paraffin oil) by subtracting the solvent-induced EAG value. Mann-Whitney U test was applied in the statistical analysis, with P ≥ 0.05 indicating no significance (ns), and P < 0.05 (*) as significant difference.
Fig 7.
Responses of ORNs of different trichoid sensillum of wild-type and Orco mutant mosquito to odorants.
(A) & (B) Heatmaps showing averaged neuronal responses to a panel of 34 odorants from individual sensilla randomly sampled across the antenna of wild-type (left) and AalbOrcoDsRed/DsRed (right) Ae. albopictus. (C) Representative response traces of SST and SBTI sensillum to selected chemical componds (diluted to 1% concentration in paraffin oil or DMSO). There were two neurons in most of the sensilla with large amplitude (blue traces) representing the response of ‘A’ neuron and small amplitude (red traces) representing the response of ‘B’ neuron. (D) Averaged female SST sensillum responses in wild-type and AalbOrcoDsRed/DsRed mosquitoes. Mann-Whitney U test suggested neuronal responses to selected compounds in Orco mutants were significantly reduced compared to the wild-type (n = 7). (E) Averaged female SBTI sensillum responses in wild-type and AalbOrcoDsRed/DsRed mosquitoes. Mann-Whitney U test suggested neuronal responses to fenchone and camphor in Orco mutants were significantly reduced than wild-type (n = 6). (F) Averaged female CP sensillum responses in the the maxillary palp of wild-type and AalbOrcoDsRed/DsRed mosquitoes. Mann-Whitney U test suggested neuronal responses to 1-octen-3-ol in Orco mutants were significantly reduced compared to the wild-type (n = 6), while no significant differences were observed in neuronal responses to CO2 between wild-type and Orco mutants (n = 6). Statistical significance was presented as P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****).
Fig 8.
Orco knockout-induced changes in the mosquito blood-feeding and host-seeking behaviors.
(A) Schematic picture of setting of blood-feeding assay. (B) Comparison of feeding success between AalbOrcoDsRed/DsRed and wild-type mosquitoes. The successful blood-feeding rate of the AalbOrcoDsRed/DsRed mosquito was significantly lower than that of wild-type mosquitoes on mice (P < 0.05). (C) Schematic picture of setting of two-choice host preference assay. (D) Comparison of host preference between AalbOrcoDsRed/DsRed and wild-type mosquitoes. AalbOrcoDsRed/DsRed mutant mosquitoes showed reduced preference for human hand (P < 0.0001). Statistical analysis was done using GraphPad Prism 5. Data are presented as mean ± s.e.m. Mann-Whitney U test was used to compare two sets of data with the significance set to a P-value < 0.05.