Insect odorant receptors (ORs) have a unique design of heterodimers formed by an olfactory receptor protein and the ion channel Orco. Heterologously expressed insect ORs are activated via an ionotropic and a metabotropic pathway that leads to cAMP production and activates the Orco channel. The contribution of metabotropic signaling to the insect odor response remains to be elucidated. Disruption of the G_q protein signaling cascade reduces the odor response (Kain et al., 2008). We investigated this phenomenon in HEK293 cells expressing Drosophila Orco and found that phospholipase C (PLC) inhibition reduced the sensitivity of Orco to cAMP. A similar effect was seen upon inhibition of protein kinase C (PKC), whereas PKC stimulation activated Orco even in the absence of cAMP. Mutation of the five PKC phosphorylation sites in Orco almost completely eliminated sensitivity to cAMP. To test the impact of PKC activity in vivo we combined single sensillum electrophysiological recordings with microinjection of agents affecting PLC and PKC function and observed an altered response of olfactory sensory neurons (OSNs) to odorant stimulation. Injection of the PLC inhibitor U73122 or the PKC inhibitor Gö6976 into sensilla reduced the OSN response to odor pulses. Conversely, injection of the PKC activators OAG, a diacylglycerol analog, or phorbol myristate acetate (PMA) enhanced the odor response. We conclude that metabotropic pathways affecting the phosphorylation state of Orco regulate OR function and thereby shape the OSN odor response.

Insect olfactory sensory neurons (OSN) express a diverse array of receptors from different protein families, i.e. ionotropic receptors (IR), gustatory receptors (GR) and odorant receptors (OR). It is well known that insects are exposed to a plethora of odor molecules that vary widely in both space and time under turbulent natural conditions. In addition to divergent ligand specificities, these different receptors might also provide an increased range of temporal dynamics and sensitivities for the olfactory system. To test this, we challenged different Drosophila OSNs with both varying stimulus durations (10–2000 ms), and repeated stimulus pulses of key ligands at various frequencies (1–10 Hz). Our results show that OR-expressing OSNs responded faster and with higher sensitivity to short stimulations as compared to IR- and Gr21a-expressing OSNs. In addition, OR-expressing OSNs could respond to repeated stimulations of excitatory ligands up to 5 Hz, while IR-expressing OSNs required ~5x longer stimulations and/or higher concentrations to respond to similar stimulus durations and frequencies. Nevertheless, IR-expressing OSNs did not exhibit adaptation to longer stimulations, unlike OR- and Gr21a-OSNs. Both OR- and IR-expressing OSNs were also unable to resolve repeated pulses of inhibitory ligands as fast as excitatory ligands. These differences were independent of the peri-receptor environment in which the receptors were expressed and suggest that the receptor expressed by a given OSN affects both its sensitivity and its response to transient, intermittent chemical stimuli. OR-expressing OSNs are better at resolving low dose, intermittent stimuli, while IR-expressing OSNs respond more accurately to long-lasting odor pulses. This diversity increases the capacity of the insect olfactory system to respond to the diverse spatiotemporal signals in the natural environment.

Selection of oviposition substrate is critical in holometabolous insects. Female stable flies, Stomoxys calcitrans, locate and select vertebrate herbivore dung in which they lay their eggs. However, the preference for vertebrate herbivore dung by S. calcitrans females, its fitness consequences for offspring, and the semiochemicals used to locate and select oviposition substrates remain unclear. Using oviposition choice tests and life table bioassays we found that gravid female S. calcitrans prefer to oviposit on donkey and sheep dung, which also improves the performance of their offspring. GC-MS analysis followed by random forest classification identified β-citronellene and carvone as the most important predictive volatile organic compounds of donkey and sheep dung, respectively. In multiple choice oviposition bioassays, S. calcitrans laid more eggs in wet sand containing β-citronellene and carvone than in other treatments. The attractiveness of these compounds was confirmed in a field trial, with traps baited with β-citronellene and carvone catching more S. calcitrans. We conclude that gravid female S. calcitrans use semiochemical cues to choose oviposition substrates that maximise offspring fitness.

Rumen fermentation, which depends on feed components and rumen microbes, plays a crucial role in feed conversion and the production of various metabolites important for the physiological functions, health, and environmental smartness of ruminant livestock, in addition to providing food for humans. However, given the complexity and variation of the rumen ecosystem and feed of these various livestock species, combined with inter-individual differences between gut microbial communities, how they influence the rumen secondary metabolites remains elusive. Using metagenomics and metabolomics approaches, we show that each livestock species has a signature microbe(s) and secondary metabolites. These findings may contribute toward understanding the rumen ecosystem, microbiome and metabolite networks, which may provide a gateway to manipulating rumen ecosystem pathways toward making livestock production efficient, sustainable, and environmentally friendly.

In insects, oviposition decisions may lead to egg deposition in substrates with different larval density and nutritional levels. Individuals developing in such substrates may present plasticity in their phenotype. Here, we investigated the effect of two factors related to oviposition decisions, namely larval density and substrate quality, on the wing size and wing shape of the stable fly, Stomoxys calcitrans L. (Diptera: Muscidae). We reared S. calcitrans larvae at different densities (5, 15 and 25) and on different substrates (camel, cow, donkey and sheep dung). For each fly that emerged, we recorded body weight, and detached, slide-mounted and photographed the right wing. Next, we collected 15 landmarks on each photographed wing, and applied geometric morphometric analysis to assess variation in wing size and wing shape of S. calcitrans across the different larval densities and substrate types. We observed that wing size and wing shape of S. calcitrans were affected by larval density and the nature of the developmental substrate. Flies reared in a group of 5 had larger wing centroid size, wing length, wing width, wing area and wing loading compared with those reared in a group of 25. Also, flies developed in donkey and sheep dung had larger wing centroid size, wing length, wing width, wing area and wing loading in comparison with those grown in camel and cow dung. Canonical variate analysis followed by discriminant analysis revealed significant wing shape variation in S. calcitrans across the different densities and substrates. Wing size had a significant but weak positive effect on wing shape. This study demonstrates the high sensitivity of S. calcitrans wings to variation in larval density and developmental substrate, and that use of landmark-based geometric morphometric analysis could improve our understanding of how flies of veterinary importance respond to environmental variability.

Trypanosomes are important global livestock and human pathogens of public health importance. Elucidating the chemical mechanisms of trypanosome-relevant host interactions can enhance the design and development of a novel, next-generation trypanosomosis diagnostics. However, it is unknown how trypanosome infection affects livestock volatile odors. Here, we show that Trypanosoma congolense and Trypanosoma vivax infections induced dihydro-β- ionone and junenol, while abundance of dihydro-α-ionone, phenolics, p-cresol, and 3-propylphenol significantly elevated in cow urine. These biomarkers of trypanosome infection are conserved in cow breath and the urine metabolites of naturally infected cows, regardless of population, diet, or environment differences. Furthermore, treating trypanosome-infected cows reduced the levels of these indicators back to the pre-infection levels. Finally, we demonstrated that the potential of some specific biomarkers of phenolic origin may be used to detect active trypanosome infections, including low-level infections that are not detectable by microscopy. The sensitivity and specificity of biomarkers detection are suited for rapid, robust, and non-invasive trypanosomosis diagnosis under field conditions.