HighlightsSimple and rapid smFISH protocol suitable for medium throughput.Sensitive mRNA detection deep in whole-mount larval and adult Drosophila brains.Multiplexed detection of RNA in combination with antibody staining.Quantitation of primary transcription and post-transcriptional mRNA levels.Reliable cell type markers in a whole-mount brain complementary to antibody markers.

a b s t r a c tSteinernema carpocapsae can be effective against root-feeding insects despite its reputation as a sedentary ambusher. In pot experiments, using twigs as surrogate roots and pine weevil larvae as targets, we tested the hypothesis that roots serve as physical routeways and conduits of feeding-associated stimuli, thus enhancing the success of S. carpocapsae applied at the surface against subterranean hosts. Insect mortality was lowest (25%) in the absence of plant material, increased to 48% when twigs linked nematodes and insects, and further increased to 69% when the insects were allowed feed on the twigs. This is the first experimental support for the root-routeway hypothesis.

Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains, thereby generating the asymmetric protein distributions necessary for cellular and developmental polarity. However, the full range of transcripts that are asymmetrically distributed in specialized cell types, and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system, to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins, and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. We propose that the transport of mRNAs encoded by these genes into the dendrites allows their expression to be regulated on a local scale during the dynamic developmental processes of dendrite outgrowth, branching, and/or remodeling.

While post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap protein trap lines across the intact Drosophila nervous system. 98% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualisation tools for post-transcriptional regulation.

The large pine weevil Hylobius abietis is a serious pest of reforestation in northern Europe. Development takes place in the stumps of felled conifer trees and emerging adults feed on and kill newly planted trees. Application of entomopathogenic nematodes around tree stumps has been shown to reduce the emergence of adult weevils. In order to target application at the most susceptible stage, the susceptibility of larvae and pupae to Heterorhabditis downesi and Steinernema carpocapsae was compared in a close-contact assay on filter paper. An average of 95.8 % of larvae were killed by H. downesi and 82.1 % by S. carpocapsae while only 16.3 and 15.0 % of pupae were killed by these two species, respectively. However, many of the H. abietis that were exposed as pupae died after metamorphosis to callow adult, with mortality of pupae and callow adults combined reaching 62.5 % for H. downesi and 69.9 % for S. carpocapsae. For both nematode species significantly more insects died as larvae than as either pupae or pupae/callow adults. When pupae were exposed to infective juveniles (IJs) for 2 days and were then washed while still pupae to remove surface IJs, adults were later found to be infected indicating that IJs can infect pupae, survive metamorphosis and subsequently kill adults.

Monoterpenes, source of the distinctive odor of conifers, are generally considered plant defensive compounds. However, they are also known to act as long-range insect attractants, as they are volatile and permeate forest airspaces. Moreover, they are lipid soluble and can be absorbed into plant epicuticular waxes. We test their role in short-range host plant choice by both adult females and larvae of a folivorous forest pest (Choristoneura fumiferana). We conducted laboratory assays testing the responses of Eastern spruce budworm to an artificial monoterpene mix (α-pinene, β-pinene, limonene, myrcene) and to white spruce (Picea glauca) epicuticular waxes in closed arenas. Ovipositing females preferred filter paper discs treated with P. glauca waxes to controls, and preferred the waxes + monoterpenes treatment to waxes alone. However, females showed no preference between the monoterpene-treated disc and the control when presented without waxes. Feeding larvae prefered wax discs to control discs. They also consumed discs treated with realistic monoterpene concentrations and wax preferentially over wax-only discs, but showed no preference between extremely high monoterpene concentrations and wax-only controls. In an insect-free assay, P. glauca epicuticular wax decreased monoterpene volatilization. These results suggest that P. glauca waxes and realistic concentrations of monoterpenes are stimulatory to both egg-laying females and feeding larvae, and that their effects are synergistic.

While post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap lines across the intact Drosophila nervous system. 97.5% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia, and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualization tools for post-transcriptional regulation.

RNA in situ hybridization can be a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. Here, we describe the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.e., exons, 3'UTR, and introns, we show examples of a gene that is regulated post-transcriptionally and one that is regulated at the level of transcription. We also show that the method can be used to co-visualise a variety of different transcripts and proteins in neuronal stems cells as well as deep brain structures such as mushroom body neuropils. Finally, we introduce the use of smFISH as asensitivealternative to conventional antibody labelling to mark specific neural stem cell populations in the brain.