Short-term high fat feeding induces inflammatory responses of tuft cells and mucosal barrier cells in the murine stomach.

Feeding mice with a high fat diet (HFD) induces inflammation and results in changes of gene expression and cellular composition in various tissues throughout the body, including the gastrointestinal tract. In the stomach, tuft cells expressing the receptor GPR120 are capable of sensing saturated long chain fatty acids (LCFAs) and thus may be involved in initiating mechanisms of mucosal inflammation. In this study, we assessed which cell types may additionally be affected by high fat feeding and which candidate molecular mediators might contribute to mucosa-protective immune responses. A high fat dietary intervention for 3 weeks caused an expansion of tuft cells that was accompanied by a higher frequency of mucosal mast cells and surface mucous cells which are a known source of the insult-associated cytokine interleukin 33 (IL-33). Our data demonstrate that both brush and mucosal mast cells comprise the enzyme ALOX5 and its activating protein FLAP and thus have the capacity for synthesizing leukotriene (LT). In HFD mice, several tuft cells showed a perinuclear colocalization of ALOX5 with FLAP which is indicative of an active LT synthesis. Monitoring changes in the expression of genes encoding elements of LT synthesis and signaling revealed that transcript levels of the leukotriene C4 synthase, LTC4S, catalyzing the first step in the biosynthesis of cysteinyl (cys) LTs, and the cysLT receptors, cysLTR2 and cysLTR3, were upregulated in mice on HFD. These mice also showed an increased expression level of IL-33 receptors, the membrane-bound ST2L and soluble isoform sST2, as well as the mast cell-specific protease MCPT1. Based on these findings it is conceivable that upon sensing saturated LCFAs tuft cells may elicit inflammatory responses which result in the production of cysLTs and activation of surface mucous cells as well as mucosal mast cells regulating gastric mucosal function and integrity.

SNMP1 and odorant receptors are co-expressed in olfactory neurons of the labial and maxillary palps from the desert locust Schistocerca gregaria (Orthoptera: Acrididae).

In insects, pheromones are detected by olfactory sensory neurons (OSNs) of the antennae that co-express pheromone receptors (PRs) and the "sensory neuron membrane protein 1" (SNMP1). Beyond its relevance for pheromone detection via the antenna, little is known about a potential expression and functional role of SNMP1 in cells of other chemosensory appendages. Here, we report that in the desert locust Schistocerca gregaria, SNMP1 is also expressed in the labial and maxillary palps of the mouthparts. In the palps, the SNMP1-positive cells were situated next to the so-called terminal sensilla that are considered as chemosensory. Moreover, the SNMP1-positive cells of the palps expressed the "odorant receptor co-receptor" (Orco), a marker for OSNs endowed with odorant receptors (ORs), suggesting that these cells are olfactory. With respect to an olfactory function of the SNMP1-positive cells, further analyses examining a possible expression of ORs (notably putative PRs) in the labial and maxillary palps revealed that several members of a particular OR subfamily from S. gregaria, the b-OR group, are co-expressed with SNMP1 in cells of the palps. Interestingly, b-OR types co-expressed with SNMP1 in antennal OSNs were also co-expressed with SNMP1 in cells of the palps, indicating a specific pairing in the expression of SNMP1 and given ORs in both antennae and palps. The co-expression of SNMP1 and certain b-ORs that are regarded as candidate PRs opens up the possibility that chemosensory cells on the palps of the desert locust may contribute to pheromone detection.

A Subset of Odorant Receptors from the Desert Locust Schistocerca gregaria Is Co-Expressed with the Sensory Neuron Membrane Protein 1.

In the desert locust Schistocerca gregaria (S. gregaria), pheromones are considered to be crucial for governing important behaviors and processes, including phase transition, reproduction, aggregation and swarm formation. The receptors mediating pheromone detection in olfactory sensory neurons (OSNs) on the antenna of S. gregaria are unknown. Since pheromone receptors in other insects belong to the odorant receptor (OR) family and are typically co-expressed with the "sensory neuron membrane protein 1" (SNMP1), in our search for putative pheromone receptors of S. gregaria, we have screened the OR repertoire for receptor types that are expressed in SNMP1-positive OSNs. Based on phylogenetic analyses, we categorized the 119 ORs of S. gregaria into three groups (I-III) and analyzed a substantial number of ORs for co-expression with SNMP1 by two-color fluorescence in situ hybridization. We have identified 33 ORs that were co-expressed with SNMP1. In fact, the majority of ORs from group I and II were found to be expressed in SNMP1-positive OSNs, but only very few receptors from group III, which comprises approximately 60% of all ORs from S. gregaria, were co-expressed with SNMP1. These findings indicate that numerous ORs from group I and II could be important for pheromone communication. Collectively, we have identified a broad range of candidate pheromone receptors in S. gregaria that are not randomly distributed throughout the OR family but rather segregate into phylogenetically distinct receptor clades.

Sensilla-Specific Expression of Odorant Receptors in the Desert Locust Schistocerca gregaria.

The desert locust Schistocerca gregaria recognizes multiple chemical cues, which are received by olfactory sensory neurons housed in morphologically identifiable sensilla. The different sensillum types contain olfactory sensory neurons with different physiological specificities, i.e., they respond to different categories of chemical signals. The molecular basis for the sensilla-specific responsiveness of these cells is unknown, but probably based on the endogenous receptor repertoire. To explore this issue, attempts were made to elucidate whether distinct odorant receptors (ORs) may be expressed in a sensilla-specific manner. Analyzing more than 80 OR types concerning for a sensilla-specific expression revealed that the vast majority was found to be expressed in sensilla basiconica; whereas only three OR types were expressed in sensilla trichodea. Within a sensillum unit, even in the multicellular assembly of sensilla basiconica, many of the OR types were expressed in only a single cell, however, a few OR types were found to be expressed in a consortium of cells typically arranged in a cluster of 2-4 cells. The notion that the OR-specific cell clusters are successively formed in the course of development was confirmed by comparing the expression patterns in different nymph stages. The results of this study uncover some novel and unique features of locust olfactory system, which will contribute to unravel the complexity of locust olfaction.

Odorant Binding Proteins of the Desert Locust Schistocerca gregaria (Orthoptera, Acrididae): Topographic Expression Patterns in the Antennae.

Odorant binding proteins (OBPs) enriched in the sensillum lymph are instrumental in facilitating the transfer of odorous molecules to the responsive receptors. In Orthopteran locust species, an in-depth understanding of this important soluble protein family is still elusive. In a previous study, we have demonstrated that the repertoire of locust OBPs can be divided into four major clades (I-IV) on the phylogenetic scale and for representatives of subfamily I-A and II-A a distinct sensilla-specific expression pattern was determined. In this study, by focusing on a representative locust species, the desert locust Schistocerca gregaria, we have explored the antennal topographic expression for representative OBPs of other subfamilies. First, subtypes of subfamily III-A and III-B were exclusively found in sensilla chaetica. Then, a similar expression pattern in this sensillum type was observed for subfamily I-B subtypes, but with a distinct OBP that was expressed in sensilla coeloconica additionally. Moreover, the atypical OBP subtype from subfamily IV-A was expressed in a subpopulation of sensilla coeloconica. Last, the plus-C type-B OBP subtype from subfamily IV-B seems to be associated with all four antennal sensillum types. These results profile diversified sensilla-specific expression patterns of the desert locust OBPs from different subfamilies and complex co-localization phenotypes of distinct OBP subtypes in defined sensilla, which provide informative clues concerning their possible functional mode as well as a potential interplay among OBP partners within a sensillum.

Access to the odor world: olfactory receptors and their role for signal transduction in insects.

The sense of smell enables insects to recognize and discriminate a broad range of volatile chemicals in their environment originating from prey, host plants and conspecifics. These olfactory cues are received by olfactory sensory neurons (OSNs) that relay information about food sources, oviposition sites and mates to the brain and thus elicit distinct odor-evoked behaviors. Research over the last decades has greatly advanced our knowledge concerning the molecular basis underlying the reception of odorous compounds and the mechanisms of signal transduction in OSNs. The emerging picture clearly indicates that OSNs of insects recognize odorants and pheromones by means of ligand-binding membrane proteins encoded by large and diverse families of receptor genes. In contrast, the mechanisms of the chemo-electrical transduction process are not fully understood; the present status suggests a contribution of ionotropic as well as metabotropic mechanisms. In this review, we will summarize current knowledge on the peripheral mechanisms of odor sensing in insects focusing on olfactory receptors and their specific role in the recognition and transduction of odorant and pheromone signals by OSNs.

Distinct Subfamilies of Odorant Binding Proteins in Locust (Orthoptera, Acrididae): Molecular Evolution, Structural Variation, and Sensilla-Specific Expression.

Odorant binding proteins (OBPs) play an important role in insect olfaction, facilitating transportation of odorant molecules in the sensillum lymph. While most of the researches are concentrated on Lepidopteran and Dipteran species, our knowledge about Orthopteran species is still very limited. In this study, we have investigated OBPs of the desert locust Schistocerca gregaria, a representative Orthopteran species. We have identified 14 transcripts from a S. gregaria antennal transcriptome encoding SgreOBPs, and recapitulated the phylogenetic relationship of SgreOBPs together with OBPs from three other locust species. Two conserved subfamilies of classic OBPs have been identified, named I-A and II-A, exhibiting both common and subfamily-specific amino acid motifs. Distinct evolutionary features were observed for subfamily I-A and II-A OBPs. Surface topology and interior cavity were elucidated for OBP members from the two subfamilies. Antennal topographic expression revealed distinct sensilla- and cellular- specific expression patterns for SgreOBPs from subfamily I-A and II-A. These findings give first insight into the repertoire of locust OBPs with respect to their molecular and evolutionary features as well as their expression in the antenna, which may serve as an initial step to unravel specific roles of distinct OBP subfamilies in locust olfaction.

In Search for Pheromone Receptors: Certain Members of the Odorant Receptor Family in the Desert Locust Schistocerca gregaria (Orthoptera: Acrididae) Are Co-expressed with SNMP1.

Under given environmental conditions, the desert locust (Schistocera gregaria) forms destructive migratory swarms of billions of animals, leading to enormous crop losses in invaded regions. Swarm formation requires massive reproduction as well as aggregation of the animals. Pheromones that are detected via the olfactory system have been reported to control both reproductive and aggregation behavior. However, the molecular basis of pheromone detection in the antennae of Schistocerca gregaria is unknown. As an initial step to disclose pheromone receptors, we sequenced the antennal transcriptome of the desert locust. By subsequent bioinformatical approaches, 119 distinct nucleotide sequences encoding candidate odorant receptors (ORs) were identified. Phylogenetic analyses employing the identified ORs from Schistocerca gregaria (SgreORs) and OR sequences from the related species Locusta migratoria revealed a group of locust ORs positioned close to the root, i.e. at a basal site in a phylogenetic tree. Within this particular OR group (termed basal or b-OR group), the locust OR sequences were strictly orthologous, a trait reminiscent of pheromone receptors from lepidopteran species. In situ hybridization experiments with antennal tissue demonstrated expression of b-OR types from Schistocerca gregaria in olfactory sensory neurons (OSNs) of either sensilla trichodea or sensilla basiconica, both of which have been reported to respond to pheromonal substances. More importantly, two-color fluorescent in situ hybridization experiments showed that most b-OR types were expressed in cells co-expressing the "sensory neuron membrane protein 1" (SNMP1), a marker indicative of pheromone-sensitive OSNs in insects. Analyzing the expression of a larger number of SgreOR types outside the b-OR group revealed that only a few of them were co-expressed with SNMP1. In summary, we have identified several candidate pheromone receptors from Schistocerca gregaria that could mediate responses to pheromones implicated in controlling reproduction and aggregation behavior.

Identification and Characterization of Two "Sensory Neuron Membrane Proteins" (SNMPs) of the Desert Locust, Schistocerca gregaria (Orthoptera: Acrididae).

Pheromone-responsive neurons of insects not only require specific receptors but in addition several auxiliary components, including the "sensory neuron membrane protein," SNMP. Accordingly, SNMP is considered as a marker for neurons responding to pheromones. For the desert locust Schistocerca gregaria, it is known that the behavior, including aggregation behavior and courtship inhibition, is largely controlled by pheromones. However, little is known about pheromones, their receptors, and the pheromone-responsive cells in locusts. In this study, we have identified two SNMP subtypes, SNMP1 and SNMP2, and compared their phylogenetic relationship and primary structure motifs with SNMPs from other species. Both SNMPs were found in chemosensory tissues, especially the antennae. Employing double in situ hybridization, we identified and localized the SNMP-expressing cells in the antennae. Cells expressing SNMP1 were localized to sensilla trichodea but also to sensilla basiconica, which in locust respond to pheromones. One or a few cells express SNMP1 within the multineuron clusters from sensilla basiconica, whereas the SNMP2 subtype was expressed in cells surrounding the neuron clusters, possibly supporting cells. Based on the finding that SNMP1 is expressed in distinct neurons under chemosensory sensilla, it is conceivable that these cells may represent pheromone-responsive neurons of the desert locust.

Expression of a GABA(B) - receptor in olfactory sensory neurons of sensilla trichodea on the male antenna of the moth Heliothis virescens.

In the olfactory pathway of Drosophila, a GABAB receptor mediated presynaptic gain control mechanism at the first synapse between olfactory sensory neurons (OSNs) and projection neurons has been suggested to play a critical role in setting the sensitivity and detection range of the sensory system. To approach the question if such a mechanism may be realized in the pheromone recognition system of male moths in this study attempts were made to explore if moth's pheromone-responsive cells express a GABAB- receptor. Employing a combination of genome analysis, RT-PCR experiments and screening of an antennal cDNA library we have identified a cDNA which encodes the GABAB-R1 receptor of Heliothis virescens. Moreover, based on the HvirGABAB-R1 sequence we could predict a GABAB-R1 protein from genome sequences of the silkmoth Bombyx mori. To assess whether HvirGABAB-R1 is expressed in OSNs of male antenna we performed whole-mount in situ hybridization (WM-ISH) experiments. Several HvirGABAB-R1 positive cells were visualized under long sensilla trichodea, known to contain pheromone-responsive OSNs. In parallel it was shown that cells under long trichoid hairs were labelled with pheromone receptor specific probes. In addition, the HvirGABAB-R1 specific probe also labelled several cells under shorter olfactory sensilla, but never stained cells under mechanosensory/gustatory sensilla chaetica. Together, the results indicate that a GABAB receptor is expressed in pheromone-responsive OSNs of H. virescens and suggest a presynaptic gain control mechanism in the axon terminals of these cells.

The co-expression pattern of odorant binding proteins and olfactory receptors identify distinct trichoid sensilla on the antenna of the malaria mosquito Anopheles gambiae.

The initial steps of odorant recognition in the insect olfactory system involve odorant binding proteins (OBPs) and odorant receptors (ORs). While large families of OBPs have been identified in the malaria vector A. gambiae, little is known about their expression pattern in the numerous sensory hairs of the female antenna. We applied whole mount fluorescence in Situ hybridization (WM-FISH) and fluorescence immunohistochemistry (WM-FIHC) to investigate the sensilla co-expression of eight A. gambiae OBPs (AgOBPs), most notably AgOBP1 and AgOBP4, which all have abundant transcripts in female antenna. WM-FISH analysis of female antennae using AgOBP-specific probes revealed marked differences in the number of cells expressing each various AgOBPs. Testing combinations of AgOBP probes in two-color WM-FISH resulted in distinct cellular labeling patterns, indicating a combinatorial expression of AgOBPs and revealing distinct AgOBP requirements for various functional sensilla types. WM-FIHC with antisera to AgOBP1 and AgOBP4 confirmed expression of the respective proteins by support cells and demonstrated a location of OBPs within sensilla trichodea. Based on the finding that AgOBP1 and AgOBP4 as well as the receptor type AgOR2 are involved in the recognition of indole, experiments were performed to explore if the AgOBP-types and AgOR2 are co-expressed in distinct olfactory sensilla. Applying two-color WM-FISH with AgOBP-specific probes and probes specific for AgOR2 revealed a close association of support cells bearing transcripts for AgOBP1 and AgOBP4 and neurons with a transcript for the receptor AgOR2. Moreover, combined WM-FISH/-FIHC approaches using an AgOR2-specific riboprobe and AgOBP-specific antisera revealed the expression of the "ligand-matched" AgOBP1, AgOBP4 and AgOR2 to single trichoid hairs. This result substantiates the notion that a specific response to indole is mediated by an interplay of the proteins.

Plant odorants interfere with detection of sex pheromone signals by male Heliothis virescens.

In many insects, mate finding relies on female-released sex pheromones, which have to be deciphered by the male olfactory system within an odorous background of plant volatiles present in the environment of a calling female. With respect to pheromone-mediated mate localization, plant odorants may be neutral, favorable, or disturbing. Here we examined the impact of plant odorants on detection and coding of the major sex pheromone component, (Z)-11-hexadecenal (Z11-16:Ald) in the noctuid moth Heliothis virescens. By in vivo imaging the activity in the male antennal lobe (AL), we monitored the interference at the level of olfactory sensory neurons (OSN) to illuminate mixture interactions. The results show that stimulating the male antenna with Z11-16:Ald and distinct plant-related odorants simultaneously suppressed pheromone-evoked activity in the region of the macroglomerular complex (MGC), where Z11-16:Ald-specific OSNs terminate. Based on our previous findings that antennal detection of Z11-16:Ald involves an interplay of the pheromone binding protein (PBP) HvirPBP2 and the pheromone receptor (PR) HR13, we asked if the plant odorants may interfere with any of the elements involved in pheromone detection. Using a competitive fluorescence binding assay, we found that the plant odorants neither bind to HvirPBP2 nor affect the binding of Z11-16:Ald to the protein. However, imaging experiments analyzing a cell line that expressed the receptor HR13 revealed that plant odorants significantly inhibited the Z11-16:Ald-evoked calcium responses. Together the results indicate that plant odorants can interfere with the signaling process of the major sex pheromone component at the receptor level. Consequently, it can be assumed that plant odorants in the environment may reduce the firing activity of pheromone-specific OSNs in H. virescens and thus affect mate localization.