Examination of the genetic basis for sexual dimorphism in the Aedes aegypti (dengue vector mosquito) pupal brain

BACKGROUND: Most animal species exhibit sexually dimorphic behaviors, many of which are linked to reproduction. A number of these behaviors, including blood feeding in female mosquitoes, contribute to the global spread of vector-borne illnesses. However, knowledge concerning the genetic basis of sexually dimorphic traits is limited in any organism, including mosquitoes, especially with respect to differences in the developing nervous system. METHODS: Custom microarrays were used to examine global differences in female vs. male gene expression in the developing pupal head of the dengue vector mosquito, Aedes aegypti. The spatial expression patterns of a subset of differentially expressed transcripts were examined in the developing female vs. male pupal brain through in situ hybridization experiments. Small interfering RNA (siRNA)-mediated knockdown studies were used to assess the putative role of Doublesex, a terminal component of the sex determination pathway, in the regulation of sex-specific gene expression observed in the developing pupal brain. RESULTS: Transcripts (2,527), many of which were linked to proteolysis, the proteasome, metabolism, catabolic, and biosynthetic processes, ion transport, cell growth, and proliferation, were found to be differentially expressed in A. aegypti female vs. male pupal heads. Analysis of the spatial expression patterns for a subset of dimorphically expressed genes in the pupal brain validated the data set and also facilitated the identification of brain regions with dimorphic gene expression. In many cases, dimorphic gene expression localized to the optic lobe. Sex-specific differences in gene expression were also detected in the antennal lobe and mushroom body. siRNA-mediated gene targeting experiments demonstrated that Doublesex, a transcription factor with consensus binding sites located adjacent to many dimorphically expressed transcripts that function in neural development, is required for regulation of sex-specific gene expression in the developing A. aegypti brain. CONCLUSIONS: These studies revealed sex-specific gene expression profiles in the developing A. aegypti pupal head and identified Doublesex as a key regulator of sexually dimorphic gene expression during mosquito neural development

siRNA-Mediated Gene Targeting in Aedes aegypti Embryos Reveals That Frazzled Regulates Vector Mosquito CNS Development

Although mosquito genome projects uncovered orthologues of many known developmental regulatory genes, extremely little is known about the development of vector mosquitoes. Here, we investigate the role of the Netrin receptor frazzled (fra) during embryonic nerve cord development of two vector mosquito species. Fra expression is detected in neurons just prior to and during axonogenesis in the embryonic ventral nerve cord of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector). Analysis of fra function was investigated through siRNA-mediated knockdown in Ae. aegypti embryos. Confirmation of fra knockdown, which was maintained throughout embryogenesis, indicated that microinjection of siRNA is an effective method for studying gene function in Ae. aegypti embryos. Loss of fra during Ae. aegypti development results in thin and missing commissural axons. These defects are qualitatively similar to those observed in Dr. melanogaster fra null mutants. However, the Aa. aegypti knockdown phenotype is stronger and bears resemblance to the Drosophila commissureless mutant phenotype. The results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, suggest that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance have evolved in distantly related insects

Semaphorin-1a Is Required for Aedes aegypti Embryonic Nerve Cord Development

Although mosquito genome projects have uncovered orthologues of many known developmental regulatory genes, extremely little is known about mosquito development. In this study, the role of semaphorin-1a (sema1a) was investigated during vector mosquito embryonic ventral nerve cord development. Expression of sema1a and the plexin A (plexA) receptor are detected in the embryonic ventral nerve cords of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector), suggesting that Sema1a signaling may regulate mosquito nervous system development. Analysis of sema1a function was investigated through siRNA-mediated knockdown in A. aegypti embryos. Knockdown of sema1a during A. aegypti development results in a number of nerve cord phenotypes, including thinning, breakage, and occasional fusion of the longitudinal connectives, thin or absent commissures, and general distortion of the nerve cord. Although analysis of Drosophila melanogaster sema1a loss-of-function mutants uncovered many similar phenotypes, aspects of the longitudinal phenotypes differed between D. melanogaster and A. aegypti. The results of this investigation suggest that Sema1a is required for development of the insect ventral nerve cord, but that the developmental roles of this guidance molecule have diverged in dipteran insects

Quantification of antennal lobe defects in <i>sema1a</i> knockdown animals.

<p>The above set of data represents a compiled summary of results obtained for control-fed vs. <i>sema1a</i> knockdown (KD) animals from a total of eight replicate experiments performed in both larvae (left) and pupae (right). The total number of animals assessed (n) and numbers/percentages of animals displaying wildtype morphology vs. antennal lobe (AL) defects (olfactory sensory neuron targeting defects, lack of neuropil and glomeruli formation) are indicated. Knockdown was immunohistochemically confirmed through anti-Sema1a antibody staining in a subset of animals (15 larvae and 10 pupae) displaying the most severe defects (denoted with *). All KD animals assessed in this manner were found to have reduced levels of Sema1a, while Sema1a levels in control-fed animals were normal (examples are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002215#pntd-0002215-g002" target="_blank">Figures 2</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002215#pntd-0002215-g007" target="_blank">7</a>).</p

A decreased response to yeast odorant attractant is observed in <i>sema1a</i> knockdown animals.

<p>Control-fed and <i>sema1a</i> knockdown animals were assessed for their response to a yeast odorant attractant. In this assay, individual 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 (n = 86) vs. knockdown (n = 87) animals are plotted in A. The mean score for <i>sema1a</i> individuals (0.5) was significantly lower than that of control-fed (mean = 1) animals (p<0.001). Levels of <i>sema1a</i> were severely reduced in the antennae (D) and brains (G) of <i>sema1a</i> knockdown animals that failed to respond to the yeast; compare to <i>sema1a</i> transcript levels in control-fed antennae (C) and brains (F), which were similar to <i>sema1a</i> transcript levels found in wildtype animals (B,E). The proximal ends of antennae are oriented upward in B–D. Dorsal is oriented upward, and yellow arrowheads mark the antennal lobes in E–G.</p

<i>sema1a</i> expression correlates with antennal lobe development in <i>Ae.</i> aegypti.

<p>The presumptive antennal lobe (circles in A1–C1; one brain hemisphere shown) is marked by HRP and monoclonal antibody mAb nc82 staining in the region just ventral to the SuEG in L3 (A1), L4 (B1) and 24 hours APF (C1) animals. Insets in A1–C1 show high magnification images (and slightly different focal planes) of the same antennal lobes co-stained for HRP and mAb nc82. Individual glomeruli are visible at 24 hours APF (marked by mAb nc82 stain in C1). Expression of <i>sema1a</i> transcript (A2, B2, C2) and protein (A3, B3, C3) is detected in larval and pupal brains (A2, A5 = L3; B2, B5 = L4; C2, C5 = 24 hours APF). Arrowheads mark the antennal lobes in A2, B2, and C2, and high magnification views of antennal lobes (co-stained for Sema1a and mAb nc82) are shown in A3–A5 (L3), B3–B5 (L4), and C3–C5 (24 hours APF). Brains are oriented dorsal up in all panels. Scale bar = 100 µm in A1, B1, and C1 and 25 µm in A3–A5, B3–B5, and C3–C5.</p

Pupal ORN targeting defects correlate with <i>sema1a</i> knockdown.

<p>Severe ORN targeting defects (dye fills in B1; compare to control-fed animal in A1) correlate with loss of Sema1a protein expression at 24 hours APF (detected through lack of antibody staining in B2; compare to control-fed in A2). Overlays are shown in A3 and B3.</p

Larval antennal lobe defects in <i>sema1a</i> knockdown animals.

<p>In wildtype (A) and control-fed (B) L4 larvae, antennal sensory neurons innervate the antennal lobe (marked by dotted yellow circle throughout the figure), which is labeled by mAb nc82 staining (A3, B3). Serotonergic projection neurons are marked by 5HT staining in the antennal lobes of these animals (A2, B2; overlays of all three labels are shown in A4 and B4). <i>sema1a</i> knockdown animals (C–E) show a reduction in the number of antennal neurons (C1, D1, E1) targeting the antennal lobe, which is marked by reduced mAb nc82 synaptic neuropil staining (C3, D3, E3; weak levels are slightly increased in these panels so that the antennal lobe staining can be viewed). Reduced anti-serotonin 5HT staining of projection neurons is also observed in knockdown animals (C2, D2, E2; overlays of all three labels are shown in C4, D4, and E4). Comparable antennal lobe <i>sema1a</i> knockdown phenotypes are observed in animals fed with siRNA¹¹⁹⁸ (D), siRNA⁸⁹⁰ (E), or a combination of the two (<i>sema1a</i> KD in C). Dorsal is up in all panels. Scale bar = 25 µm.</p

Larval antennal neuron targeting defects in <i>sema1a</i> knockdown animals.

<p>In the larval antennal lobe, antennal gustatory neurons (red arrowhead) target the antennal lobe and project ventrally (oriented down in all panels) toward the subesophageal ganglion. These neurons target properly in <i>sema1a</i> knockdown animals (C–J). In wildtype (A) and control-fed (B) L4 larvae, a subset of antennal sensory neurons innervating the antennal lobe send projection neurons dorsally (oriented up in all panels) to a target region (marked by blue arrows) in the SuEG. These neurons do not target properly to the SuEG in <i>sema1a</i> knockdown animals (C–J). Phenotypic categories observed include: antennal neurons failing to innervate outside of the antennal lobe (C, D; 38% of phenotypes; n = 38), neurons (yellow arrowheads) stopping short of the SuEG target (E, F; 19% of phenotypes), extension beyond the target region (blue arrows) within the SuEG (G, H; 24% of phenotypes), and extraneous branching within the SuEG (I, J; 19% of phenotypes). All four phenotypes were detected in animals fed with siRNA⁸⁹⁰ alone (H, J), siRNA¹¹⁹⁸ alone (D, F), or a combination of the two siRNAs (referred to as <i>sema1a</i> KD in C, E, G, and I), although only a subset of these results are included here. These phenotypes were never observed in wildtype larvae (A) or those that were fed with control siRNA nanoparticles (B). Scale bar = 25 µm.</p

Levels of <i>sema1a</i> correlate with performance in yeast behavioral assays.

<p>The above set of data represents a compiled summary of results obtained from four replicate experiments in which control-fed vs. <i>sema1a</i> knockdown (KD) animals were tested in a yeast odorant attractant assay. The total number of animals (n) indicates the number of individuals that were assessed in these assays. The number of individuals (# Animals) that were attracted (left; animals that touched the yeast pellet and received a score of 1) or not attracted (right; animals that did not touch the yeast pellet and received a score of 0) under each condition (Control or KD) are indicated, and the percentages of total animals are reported after the raw numbers. The levels of <i>sema1a</i> were assessed in the brains and antennae of animals attracted (left) or not attracted (right) to the yeast. The raw number/percentage of # animals with Normal (wildtype <i>sema1a</i>), Null (no detectable <i>sema1a</i> transcript), or Moderate (reduced but not wildtype <i>sema1a</i>) levels are indicated. Loss of <i>sema1a</i> expression correlated well with a lack of attraction to the yeast.</p