Chitosan/siRNA nanoparticle targeting demonstrates a requirement for single-minded during larval and pupal olfactory system development of the vector mosquito Aedes aegypti

Abstract

Background: Essentially nothing is known about the genetic regulation of olfactory system development in vector mosquitoes, which use olfactory cues to detect blood meal hosts. Studies in Drosophila melanogaster have identified a regulatory matrix of transcription factors that controls pupal/adult odorant receptor (OR) gene expression in olfactory receptor neurons (ORNs). However, it is unclear if transcription factors that function in the D. melanogaster regulatory matrix are required for OR expression in mosquitoes. Furthermore, the regulation of OR expression during development of the larval olfactory system, which is far less complex than that of pupae/adults, is not well understood in any insect, including D. melanogaster. Here, we examine the regulation of OR expression in the developing larval olfactory system of Aedes aegypti, the dengue vector mosquito.

Results: A. aegypti bears orthologs of eight transcription factors that regulate OR expression in D. melanogaster pupae/adults. These transcription factors are expressed in A. aegypti larval antennal sensory neurons, and consensus binding sites for these transcription factors reside in the 5' flanking regions of A. aegypti OR genes. Consensus binding sites for Single-minded (Sim) are located adjacent to over half the A. aegypti OR genes, suggesting that this transcription factor functions as a major regulator of mosquito OR expression. To functionally test this hypothesis, chitosan/siRNA nanoparticles were used to target sim during larval olfactory development. These experiments demonstrated that Sim positively regulates expression of a large subset of OR genes, including orco, the obligate co-receptor in the assembly and function of heteromeric OR/Orco complexes. Decreased innervation of the antennal lobe was also noted in sim knockdown larvae. These OR expression and antennal lobe defects correlated with a larval odorant tracking behavioral defect. OR expression and antennal lobe defects were also observed in sim knockdown pupae.

Conclusions: The results of this investigation indicate that Sim has multiple functions during larval and pupal olfactory system development in A. aegypti.

Figure 1

Transcription factor expression in the A. aegypti larval antenna. Expression of the eight indicated transcription factors is detected in A. aeygpti fourth instar larval antennae (A-H). Each transcription factor is expressed in a subset of A. aegypti larval antennal sensory neurons, and expression levels of the transcription factors vary from neuron to neuron within this subset. The proximal ends of antennae are oriented upward in all panels. Scale bars = 25 microns.

Figure 2

Expression and siRNA-nanoparticle mediated knockdown of sim in the larval olfactory system.sim is expressed in the antennae (A) and brain (A1) of wildtype L4 larvae. Two sim siRNAs, sim⁴³⁰(C, C1) and sim⁷¹⁸(D, D1), were found to effectively knockdown (KD) sim in both L4 antennae (C, D) and brains (C1, D1) when delivered to A. aegypti larvae via chitosan nanoparticle feeding. Control siRNA feedings did not impact sim expression (antenna in B; brain in B1). In wildtype (A1) and control-fed animal (B1) L4 brains, clusters of cells expressing sim transcripts (red asterisks) are detected adjacent to the antennal lobe, the boundaries of which are marked by the yellow dotted circles in panels A1-D1. The overall reduced number of antennal sensory neurons targeting to the antennal lobes in sim knockdown animals does not appear to result from cell death, as evidenced by the lack of cleaved-caspase 3 staining in the L4 antennae of sim knockdown animals (E3,4; compare to wildtype and control-fed animals in E1,2; nuclei are marked by TO-PRO in E). Note that while no cleaved-caspase 3 was detected in the antennae shown, positively labeled cells were detected in other wild-type A. aegypti pupal tissues (not shown), suggesting that the reagent employed in these studies is effective in A. aegypti. Acetylated tublin staining (green, F) revealed normal antennal sensory neuron axon bundles in sim⁴³⁰ and sim⁷¹⁸ knockdown animals (compare to wildtype and control-fed antennae in F1 and F2, respectively). The proximal ends of antennae are oriented upward in panels A-D, E, and F. Dorsal is oriented upward in panels A1-D1. Scale bars = 25 microns.

Figure 3

sim is required for OR gene expression in the A. aegypti larval antenna.orco and ORs 9, 10, 16, 49, 60, 78, 89, 90, and 100 transcripts are detected in wildtype (A1-J1, respectively) and control-fed (A2-J2, respectively) L4 antennae. Downregulated expression of orco and ORs 9, 10 16, 49, 60, 78, 89, and 100, all of which have adjacent Sim consensus binding sites, is observed in animals that were fed with chitosan/siRNA sim⁴³⁰ (A3-H3, J3 respectively) or sim⁷¹⁸ (A4-H4, J4, respectively). orco(A3, A4), OR9(B3,B4), OR10(C3, C4), OR49(E3,E4), and OR89(H3, H4) expression is not detectable in sim knockdown individuals, while expression of ORs 16(D3, D4), 60(F3, F4), 78(G3, G4), and 100(J3, J4) is reduced in comparison to wildtype (A1-H1, J1) and control-fed (A2-H2, J2) larvae. Expression of OR90(I1-I4), which lacks adjacent Sim consensus binding sites, is unchanged in knockdown animals (I3, I4). The proximal ends of antennae are oriented upward in all panels. Scale bars = 25 microns.

Figure 4

Larval antennal lobe defects in sim knockdown animals. In wildtype (A1) and control-fed (B1) L4 larvae, D7162 dye-filled antennal sensory neurons innervate the antennal lobe (highlighted by yellow dots throughout the figure), which is labeled by mAb nc82 (A3, B3). Serotonergic projection neurons are labeled by anti-5HT staining in the antennal lobes of these individuals (A2, B2). An overlay of the three labels is shown in panels A4 and B4 (as well as C4, and D4). sim⁴³⁰(C1-C4) and sim⁷¹⁸(D1-D4) animals show a reduction in the number of antennal sensory neurons (D7162 fills in C1, D1) targeting the antennal lobe. The neuropil (nc82 label in C3, D3) and projection neurons (5HT label in C2, D2) are substantially reduced in the antennal lobes of sim knockdown (KD) animals. A subset of antennal sensory neurons which are believed to be gustatory receptors [2] pass through the antennal lobe and project to the subesophageal ganglion (A1, B1; yellow arrowhead). In sim knockdown animals (C1, D1; yellow arrowhead) these neurons are substantially reduced in number. Dorsal is oriented upward in all the panels. Scale bars = 25 microns.

Figure 5

sim deficient larvae have a decreased yeast odorant attractant response. Individual sim⁴³⁰ or sim⁷¹⁸ knockdown (KD) vs. control-fed L4 animals were assessed for their response to a yeast odorant attractant. In each assay (replicate one = red, replicate two = orange) individual control-fed animals that were attracted to the yeast were awarded a score of 1, while animals that were not attracted to the yeast received a score of 0. Average scores for control vs. knockdown animals are plotted for each replicate experiment, for which n numbers are indicated; error bars represent standard error (panel A1). The mean score for knockdown animals fed with either sim⁴³⁰ or sim⁷¹⁸ was significantly lower than that of control-fed animals in both replicate experiments (p < 0.001, A1). Levels of sim RNA were severely reduced in the antennae (B2, B3) and brains (C2, C3) of sim knockdown animals (sim⁴³⁰ in B2, C2; sim⁷¹⁸ in B3, C3) that failed to respond to the yeast, while normal levels of sim transcript were observed in control-fed antennae (B1) and brains (C1; red asterisks mark clusters of sim expression) of animals that were attracted to the yeast. The proximal ends of antennae are oriented upward in panels B1-B3. Dorsal is oriented upward in C1-C3, in which the antennal lobe is marked by yellow dots. Scale bars = 25 microns.

Figure 6

Expression and knockdown of sim in pupae.sim is expressed in the antennal lobe (A, B) and antenna (A1, B1) of wildtype (A, A1) and control-fed (B, B1) 36 hr APF pupae. Cell clusters of sim expression surrounding the antennal lobe (A, B) are highlighted by red dots, while clusters of sim expression are marked by red arrowheads in the antenna (A1, B1). Animals fed with knockdown (KD) siRNAs sim⁴³⁰(C, C1) and sim⁷¹⁸(D, D1) lack sim expression in the antennal lobe region (C, D) and antenna (C1, D1). Antennal lobes are denoted by yellow-dotted circles. Dorsal is oriented upward in panels A-D. The proximal ends of antennae are oriented upward in A1-D1. Scale bars = 25 microns.

Figure 7

Sim is required for OR gene expression in pupae.orco and OR 9, 10, and 16 transcripts are detected in wildtype (A1-D1, respectively) and control-fed (A2-D2, respectively) 36 hr APF pupal antennae. However, orco(A3, A4), OR9(B3,B4), OR10(C3, C4), and OR16(D3,D4) expression is not detectable in sim⁴³⁰(A3-D3) or sim⁷¹⁸(A4-D4) knockdown 36 hr APF pupal antennae. The proximal ends of antennae are oriented upward in all panels. Scale bars = 25 microns.

Figure 8

sim knockdown pupal antennal lobe phenotypes. In 36 hr APF wildtype (A1-A3) and control-fed (B1-B3) pupae, ORNs have innervated the antennal lobe and targeted specific glomeruli within the lobe (D7162 dye fills in A1, B1; nc82 staining in A2, B2; overlays are shown in A3 and B3). Distinct glomerular structures are fully formed by this time point (yellow dotted circles in A1, A2, B1, B2). D7162 fills (C1, D1) of pupal ORNs and nc82 staining of the synaptic neuropil (C2, D2; overlays in C3, D3) in Aae sim knockdown pupae (sim⁴³⁰ in C1-C3, sim⁷¹⁸ in D1-D3) revealed sparser and more disorganized ORNs and a collapse in glomerular structure in the antennal lobe. ORNs in sim knockdown animals (C1, D1) fail to converge on discrete glomeruli. Dorsal is oriented upward in all panels. Scale bars = 25 microns.