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Additional file 1: Fig. S1. Confirmation of shRNA expression in recombinant yeast strain MtnB.496. PCR reactions performed with primers corresponding to the MtnB.496 shRNA transcript generated a ~ 100 bp amplicon (see DNA standard at far left; cDNA template was prepared from MtnB.496 yeast total RNA). Negative control PCR reactions included an amplification with cDNA prepared from non-transformed yeast (marked by – sign) and a reaction with no cDNA added (marked by knot symbol). A representative ethidium bromide-stained agarose gel from one of two comparable biological replicate experiments is shown.

Additional file 2. A lack of Sac1.1 and otk.16 yeast interfering larvicide activity in A. aegypti larvae. Graph depicting results from larvicide trials that demonstrated a lack of larvicidal activity for yeast interfering RNA larvicides Sac1.1 and otk.16 in A. aegypti larvae.

Despite the devastating impact of mosquito-borne illnesses on human health, very little is known about mosquito developmental biology, including development of the mosquito visual system. Mosquitoes possess functional adult compound eyes as larvae, a trait that makes them an interesting model in which to study comparative developmental genetics. Here, we functionally characterize visual system development in the dengue and yellow fever vector mosquito Aedes aegypti, in which we use chitosan/siRNA nanoparticles to target the axon guidance gene semaphorin-1a (sema1a).Immunohistochemical analyses revealed the progression of visual sensory neuron targeting that results in generation of the retinotopic map in the mosquito optic lobe. Loss of sema1a function led to optic lobe phenotypes, including defective targeting of visual sensory neurons and failed formation of the retinotopic map. These sema1a knockdown phenotypes correlated with behavioral defects in larval photoavoidance.The results of this investigation indicate that Sema1a is required for optic lobe development in A. aegypti and highlight the behavioral importance of a functioning visual system in preadult mosquitoes.

In Drosophila melanogaster, commissureless (comm) function is required for proper nerve cord development. Although comm orthologs have not been identified outside of Drosophila species, some insects possess orthologs of Drosophila comm2, which may also regulate embryonic nerve cord development. Here, this hypothesis is explored through characterization of comm2 genes in two disease vector mosquitoes.Culex quinquefasciatus (West Nile and lymphatic filiariasis vector) has three comm2 genes that are expressed in the developing nerve cord. Aedes aegypti (dengue and yellow fever vector) has a single comm2 gene that is expressed in commissural neurons projecting axons toward the midline. Loss of comm2 function in both A. aegypti and D. melanogaster was found to result in loss of commissure defects that phenocopy the frazzled (fra) loss of function phenotypes observed in both species. Loss of fra function in either insect was found to result in decreased comm2 transcript levels during nerve cord development.The results of this investigation suggest that Fra down-regulates repulsion in precrossing commissural axons by regulating comm2 levels in both A. aegypti and D. melanogaster, both of which require Comm2 function for proper nerve cord development.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol for whole-mount in situ hybridization can be used to analyze gene expression in Ae. aegypti embryos and larvae, a critical aspect of understanding developmental gene function in this vector mosquito.

To construct CyclinD (CycD) (-/-)/Patched (Ptc) (+/-) double knockout mice to study the relationship between Hedgehog and Rb signaling pathways.Amphimixis was performed between female heterozygote CycD knockout mice CycD (+/-) and male heterozygote Ptc knockout mice Ptc (+/-), thus CycD (+/-)/Ptc (+/-) double knockout mice were obtained. These mice underwent secondary matched and thus construct CycD (-/-)/Ptc (+/-) double knockout mice.CycD (-/-)/Ptc (+/-) double knockout mice, CycD (+/-)/Ptc (+/-) heterozygote double knockout mice, and CycD knockout mice CycD (-/-)/Ptc (+/+) were gained.The method of amphimixis can construct CycD (--)/Ptc (+/-) double knockout mice that can be used to disclose the regulation between Ptc gene and CycD gene and the relativity between Hedgehog and Rb pathways in mammals.

Although many similarities in arthropod central nervous systems (CNS) development exist, differences in midline cell formation and ventral nerve cord axonogenesis have been noted in arthropods. It is possible that changes in the expression of axon guidance molecules such as Netrin, which functions during commissural axon guidance in Drosophila and many other organisms, may parallel these differences. In this investigation, we analyze this hypothesis by examining Netrin accumulation during development of the brine shrimp Artemia franciscana, a branchiopod crustacean. An Artemia franciscana netrin (afrnet) orthologue was cloned. An antibody to the afrNet protein was generated and used to examine the pattern of afrNet accumulation during Artemia development. Despite differences between Drosophila and Artemia nerve cord development, examination of afrNet accumulation suggests that this protein functions to regulate commissure formation during Artemia CNS development. However, detection of afrNet at the midline and on commissural axons occurs at a relatively later time point in Artemia as compared with Drosophila. Detection of afrNet in a subset of midline cells that closely resemble Netrin-expressing cells at the Drosophila midline provides evidence for homology of midline cells in arthropods. Expression of Netrins in many other tissues is comparable, suggesting that Netrin proteins may play many conserved roles during arthropod development.