Comparison of pore water samplers and cryogenic distillation under laboratory and field conditions for soil water stable isotope analysis.

We used pore water samplers (PWS) to sample for isotope analysis (1) only water, (2) soil under laboratory conditions, and (3) soil in the field comparing the results with cryogenic extraction (CE). In (1) and (2), no significant differences between source and water extracted with PWS were detected with a mean absolute difference (MAD) always lower than 2 ‰ for δ2H and 1 ‰ for δ18O. In (2), CE water was more enriched than PWS-extracted water, with a MAD respect to source water of roughly 8 ‰ for δ2H and 4 ‰ for δ18O. In (3), PWS water was enriched relative to CE water by 3 ‰ for δ2H and 0.9 ‰ for δ18O. The latter result may be due to the distinct water portions sampled by the two methods. Large pores, easily sampled by PWS, likely retain recent, and enriched, summer precipitation while small pores, only sampled by CE, possibly retain isotopically depleted water from previous winter precipitation or irrigation inputs. Accuracy and precision were greater for PWS relative to CE. PWS is therefore suggested as viable tool to extract soil water for stable isotope analysis, particularly for soils used in this study (sandy and silty loams).

Potencies of effector genes in silencing odor-guided behavior in Drosophila melanogaster.

The genetic toolbox in Drosophila melanogaster offers a multitude of different effector constructs to silence neurons and neuron populations. In this study, we investigated the potencies of several effector genes - when expressed in olfactory sensory neurons (OSNs) - to abolish odor-guided behavior in three different bioassays. We found that two of the tested effectors (tetanus toxin and Kir2.1) are capable of mimicking the Orco mutant phenotype in all of our behavioral paradigms. In both cases, the effectiveness depended on effector expression levels, as full suppression of odor-guided behavior was observed only in flies homozygous for both Gal4-driver and UAS-effector constructs. Interestingly, the impact of the effector genes differed between chemotactic assays (i.e. the fly has to follow an odor gradient to localize the odor source) and anemotactic assays (i.e. the fly has to walk upwind after detecting an attractive odorant). In conclusion, our results underline the importance of performing appropriate control experiments when exploiting the D. melanogaster genetic toolbox, and demonstrate that some odor-guided behaviors are more resistant to genetic perturbations than others.

Intracellular regulation of the insect chemoreceptor complex impacts odour localization in flying insects.

Flying insects are well known for airborne odour tracking and have evolved diverse chemoreceptors. While ionotropic receptors (IRs) are found across protostomes, insect odorant receptors (ORs) have only been identified in winged insects. We therefore hypothesized that the unique signal transduction of ORs offers an advantage for odour localization in flight. Using Drosophila, we found expression and increased activity of the intracellular signalling protein PKC in antennal sensilla following odour stimulation. Odour stimulation also enhanced phosphorylation of the OR co-receptor Orco in vitro, while site-directed mutation of Orco or mutations in PKC subtypes reduced the sensitivity and dynamic range of OR-expressing neurons in vivo, but not IR-expressing neurons. We ultimately show that these mutations reduce competence for odour localization of flies in flight. We conclude that intracellular regulation of OR sensitivity is necessary for efficient odour localization, which suggests a mechanistic advantage for the evolution of the OR complex in flying insects.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm.

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system. To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution. Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal´s dynamic behavioral reaction.

Pheromones mediating copulation and attraction in Drosophila.

Intraspecific olfactory signals known as pheromones play important roles in insect mating systems. In the model Drosophila melanogaster, a key part of the pheromone-detecting system has remained enigmatic through many years of research in terms of both its behavioral significance and its activating ligands. Here we show that Or47b-and Or88a-expressing olfactory sensory neurons (OSNs) detect the fly-produced odorants methyl laurate (ML), methyl myristate, and methyl palmitate. Fruitless (fru(M))-positive Or47b-expressing OSNs detect ML exclusively, and Or47b- and Or47b-expressing OSNs are required for optimal male copulation behavior. In addition, activation of Or47b-expressing OSNs in the male is sufficient to provide a competitive mating advantage. We further find that the vigorous male courtship displayed toward oenocyte-less flies is attributed to an oenocyte-independent sustained production of the Or47b ligand, ML. In addition, we reveal that Or88a-expressing OSNs respond to all three compounds, and that these neurons are necessary and sufficient for attraction behavior in both males and females. Beyond the OSN level, information regarding the three fly odorants is transferred from the antennal lobe to higher brain centers in two dedicated neural lines. Finally, we find that both Or47b- and Or88a-based systems and their ligands are remarkably conserved over a number of drosophilid species. Taken together, our results close a significant gap in the understanding of the olfactory background to Drosophila mating and attraction behavior; while reproductive isolation barriers between species are created mainly by species-specific signals, the mating enhancing signal in several Drosophila species is conserved.

Compound valence is conserved in binary odor mixtures in Drosophila melanogaster.

Most naturally occurring olfactory signals do not consist of monomolecular odorants but, rather, are mixtures whose composition and concentration ratios vary. While there is ample evidence for the relevance of complex odor blends in ecological interactions and for interactions of chemicals in both peripheral and central neuronal processing, a fine-scale analysis of rules governing the innate behavioral responses of Drosophila melanogaster towards odor mixtures is lacking. In this study we examine whether the innate valence of odors is conserved in binary odor mixtures. We show that binary mixtures of attractants are more attractive than individual mixture constituents. In contrast, mixing attractants with repellents elicits responses that are lower than the responses towards the corresponding attractants. This decrease in attraction is repellent-specific, independent of the identity of the attractant and more stereotyped across individuals than responses towards the repellent alone. Mixtures of repellents are either less attractive than the individual mixture constituents or these mixtures represent an intermediate. Within the limits of our data set, most mixture responses are quantitatively predictable on the basis of constituent responses. In summary, the valence of binary odor mixtures is predictable on the basis of valences of mixture constituents. Our findings will further our understanding of innate behavior towards ecologically relevant odor blends and will serve as a powerful tool for deciphering the olfactory valence code.