Females smell differently: characteristics and significance of the most common olfactory sensilla of female silkmoths.

In the silkmoth Bombyx mori, the role of male sensilla trichodea in pheromone detection is well established. Here we study the corresponding female sensilla, which contain two olfactory sensory neurons (OSNs) and come in two lengths, each representing a single physiological type. Only OSNs in medium trichoids respond to the scent of mulberry, the silkworm's exclusive host plant, and are more sensitive in mated females, suggesting a role in oviposition. In long trichoids, one OSN is tuned to (+)-linalool and the other to benzaldehyde and isovaleric acid, both odours emitted by silkworm faeces. While the significance of (+)-linalool detection remains unclear, isovaleric acid repels mated females and may therefore play a role in avoiding crowded oviposition sites. When we examined the underlying molecular components of neurons in female trichoids, we found non-canonical co-expression of Ir8a, the co-receptor for acid responses, and ORco, the co-receptor of odorant receptors, in long trichoids, and the unexpected expression of a specific odorant receptor in both trichoid sensillum types. In addition to elucidating the function of female trichoids, our results suggest that some accepted organizational principles of the insect olfactory system may not apply to the predominant sensilla on the antenna of female B. mori.

Evidence for a role of SNMP2 and antennal support cells in sensillum lymph clearance processes of moth pheromone-responsive sensilla.

In insect antenna, following the activation of olfactory sensory neurons, odorant molecules are inactivated by enzymes in the sensillum lymph. How the inactivation products are cleared from the sensillum lymph is presently unknown. Here we studied the role of support cells (SCs) and the so-called sensory neuron membrane protein 2 (SNMP2), a member of the CD36 family of lipid transporters abundantly expressed in SCs, in sensillum lymph clearance processes in the moths Heliothis virescens and Bombyx mori. In these species, the sex pheromone components are inactivated to long-chain fatty acids. To approach a role of SNMP2 in the removal of such inactivation products, we analyzed the uptake of a fluorescent long-chain fatty acid analog into a newly generated HvirSNMP2-expressing cell line. We found an increased uptake of the analog into SNMP2-cells compared to control cells, which could be blocked by the CD36 protein inhibitor, SSO. Furthermore, analyses of sensilla from antenna treated with the fatty acid analog indicated that SNMP2-expressing SCs are able to take up fatty acids from the sensillum lymph. In addition, sensilla from SSO-pretreated antenna of B. mori showed reduced removal of the fluorescent analog from the sensillum lymph. Finally, we revealed that SSO pretreatment of male silkmoth antenna significantly prolonged the duration of the female pheromone-induced wing-fluttering behavior, possibly as a result of impaired lymph clearance processes. Together our findings in H. virescens and B. mori support a pivotal role of olfactory SCs in sensillum lymph maintenance processes and suggest an integral role of SNMP2 in the removal of lipophilic "waste products" such as fatty acids resulting from sex pheromone inactivation.

A chemical defense deters cannibalism in migratory locusts.

Many animals engage in cannibalism to supplement their diets. Among dense populations of migratory locusts, cannibalism is prevalent. We show that under crowded conditions, locusts produce an anticannibalistic pheromone called phenylacetonitrile. Both the degree of cannibalism and the production of phenylacetonitrile are density dependent and covary. We identified the olfactory receptor that detects phenylacetonitrile and used genome editing to make this receptor nonfunctional, thereby abolishing the negative behavioral response. We also inactivated the gene underlying phenylacetonitrile production and show that locusts that lack this compound lose its protection and are more frequently exposed to intraspecific predation. Thus, we reveal an anticannibalistic feature built on a specifically produced odor. The system is very likely to be of major importance in locust population ecology, and our results might therefore provide opportunities in locust management.

The specific expression patterns of sensory neuron membrane proteins are retained throughout the development of the desert locust Schistocerca gregaria.

The desert locust Schistocerca gregaria detects odorants through olfactory sensory neurons (OSNs) that are surrounded by non-neuronal support cells (SCs). OSNs and SCs are housed in cuticle structures, named sensilla found abundantly on the antenna in all developmental stages of the hemimetabolic insect. In insects, multiple proteins expressed by OSNs and SCs are indicated to play a pivotal role in the detection of odorants. This includes insect-specific members of the CD36 family of lipid receptors and transporters called sensory neuron membrane proteins (SNMPs). While the distribution pattern of the SNMP1 and SNMP2 subtypes in OSNs and SCs across different sensilla types has been elucidated for the adult S. gregaria antenna, their localization in cells and sensilla of different developmental stages is unclear. Here, we determined the SNMP1 and SNMP2 expression topography on the antenna of the first, third and fifth instar nymphs. Through FIHC experiments we found that in all developmental stages SNMP1 is expressed in OSNs and SCs of the trichoid and basiconic sensilla while SNMP2 is restricted to the SCs of the basiconic and coeloconic sensilla thus resembling the adult arrangement. Our results demonstrate that both SNMP types have defined cell- and sensilla-specific distribution patterns established already in the first instar nymphs and retained into the adult stage. This conserved expression topography underlines the importance of SNMP1 and SNMP2 in olfactory processes throughout the development of the desert locust.

The Sensilla-Specific Expression and Subcellular Localization of SNMP1 and SNMP2 Reveal Novel Insights into Their Roles in the Antenna of the Desert Locust Schistocerca gregaria.

Insect olfactory sensilla house olfactory sensory neurons (OSNs) and supports cells (SCs). The olfactory sensory processes require, besides the odorant receptors (ORs), insect-specific members of the CD36 family, named sensory neuron membrane proteins (SNMPs). While SNMP1 is considered to act as a coreceptor in the OR-mediated detection of pheromones, SNMP2 was found to be expressed in SCs; however, its function is unknown. For the desert locust, Schistocerca gregaria, we previously visualized mRNA for SNMP1 in OSNs and SNMP2 mRNA in cells associated with OSN clusters. Towards an understanding of their functional implication, it is imperative to explore the cellular and the subcellular localization the SNMP proteins. Therefore, we have generated polyclonal antibodies against SNMP1 and SNMP2 and used fluorescence immunohistochemistry (FIHC) to visualize the SNMP proteins. We found SNMP1 in the somata and respective dendrites of all OSNs in trichoid sensilla and in subsets of OSNs in basiconic sensilla. Notably, SNMP1 was also detected in SCs of these sensilla types. In contrast, SNMP2 protein was only visualized in SCs of basiconic and coeloconic sensilla, but not of trichoid sensilla. Exploring the subcellular localization by electron microscopy using anti-SNMP1-ab and anti-SNMP2-ab revealed an immunogold labelling of SC microvilli bordering the sensillum lymph. Together our findings suggest a dual role of SNMP1 in the antenna of S. gregaria, in some OSN subpopulations in odor detection as well as in functions of some SCs, whereas the role of SNMP2 is limited to the functions of support cells.

Competing beetles attract egg laying in a hawkmoth.

In nature, plant-insect interactions occur in complex settings involving multiple trophic levels, often with multiple species at each level.1 Herbivore attack of a host plant typically dramatically alters the plant's odor emission in terms of concentration and composition.2,3 Therefore, a well-adapted herbivore should be able to predict whether a plant is still suitable as a host by judging these changes in the emitted bouquet. Although studies have demonstrated that oviposition preferences of successive insects were affected by previous infestations,4,5 the underlying molecular and olfactory mechanisms remain unknown. Here, we report that tobacco hawkmoths (Manduca sexta) preferentially oviposit on Jimson weed (Datura wrightii) that is already infested by a specialist, the three-lined potato beetle (Lema daturaphila). Interestingly, the moths' offspring do not benefit directly, as larvae develop more slowly when feeding together with Lema beetles. However, one of M. sexta's main enemies, the parasitoid wasp Cotesia congregata, prefers the headspace of M. sexta-infested plants to that of plants infested by both herbivores. Hence, we conclude that female M. sexta ignore the interspecific competition with beetles and oviposit deliberately on beetle-infested plants to provide their offspring with an enemy-reduced space, thus providing a trade-off that generates a net benefit to the survival and fitness of the subsequent generation. We identify that α-copaene, emitted by beetle-infested Datura, plays a role in this preference. By performing heterologous expression and single-sensillum recordings, we show that odorant receptor (Or35) is involved in α-copaene detection.

A small number of male-biased candidate pheromone receptors are expressed in large subsets of the olfactory sensory neurons in the antennae of drones from the European honey bee Apis mellifera.

In the European honey bee (Apis mellifera), the olfactory system is essential for foraging and intraspecific communication via pheromones. Honey bees are equipped with a large repertoire of olfactory receptors belonging to the insect odorant receptor (OR) family. Previous studies have indicated that the transcription level of a few OR types including OR11, a receptor activated by the queen-released pheromone compound (2E)-9-oxodecenoic acid (9-ODA), is significantly higher in the antenna of males (drones) than in female workers. However, the number and distribution of antennal cells expressing male-biased ORs is elusive. Here, we analyzed antennal sections from bees by in situ hybridization for the expression of the male-biased receptors OR11, OR18, and OR170. Our results demonstrate that these receptors are expressed in only moderate numbers of cells in the antennae of females (workers and queens), whereas substantially higher cell numbers express these ORs in drones. Thus, the reported male-biased transcript levels are due to sex-specific differences in the number of antennal cells expressing these receptors. Detailed analyses for OR11 and OR18 in drone antennae revealed expression in two distinct subsets of olfactory sensory neurons (OSNs) that in total account for approximately 69% of the OR-positive cells. Such high percentages of OSNs expressing given receptors are reminiscent of male-biased ORs in moths that mediate the detection of female-released sex pheromone components. Collectively, our findings indicate remarkable similarities between male antennae of bees and moths and support the concept that male-biased ORs in bee drones serve the detection of female-emitted sex pheromones.

The role of SNMPs in insect olfaction.

The sense of smell enables insects to recognize olfactory signals crucial for survival and reproduction. In insects, odorant detection highly depends on the interplay of distinct proteins expressed by specialized olfactory sensory neurons (OSNs) and associated support cells which are housed together in chemosensory units, named sensilla, mainly located on the antenna. Besides odorant-binding proteins (OBPs) and olfactory receptors, so-called sensory neuron membrane proteins (SNMPs) are indicated to play a critical role in the detection of certain odorants. SNMPs are insect-specific membrane proteins initially identified in pheromone-sensitive OSNs of Lepidoptera and are indispensable for a proper detection of pheromones. In the last decades, genome and transcriptome analyses have revealed a wide distribution of SNMP-encoding genes in holometabolous and hemimetabolous insects, with a given species expressing multiple subtypes in distinct cells of the olfactory system. Besides SNMPs having a neuronal expression in subpopulations of OSNs, certain SNMP types were found expressed in OSN-associated support cells suggesting different decisive roles of SNMPs in the peripheral olfactory system. In this review, we will report the state of knowledge of neuronal and non-neuronal members of the SNMP family and discuss their possible functions in insect olfaction.

The expression patterns of SNMP1 and SNMP2 underline distinct functions of two CD36-related proteins in the olfactory system of the tobacco budworm Heliothis virescens.

In insects, male and female pheromone signals are detected by olfactory sensory neurons (OSNs) expressing the "sensory neuron membrane protein type 1". SNMP1 is supposed to function as a co-receptor involved in the transfer of pheromones to adjacent pheromone receptors. In the moth Heliothis virescens, we previously found OSNs that project their dendrites into pheromone-responsive trichoid sensilla and are associated with cells containing transcripts for the HvirSNMP1-related protein HvirSNMP2. Like HvirSNMP1, HvirSNMP2 belongs to the CD36-family of two-transmembrane domain receptors and transporters for lipophilic compounds, but its role in the olfactory system is unknown. Here, we generated polyclonal anti-peptide antibodies against HvirSNMP2 as well as HvirSNMP1 and conducted an in-depth immunohistochemical analysis of their subcellular localization in the antenna of both sexes. In line with a function in pheromone detection, HvirSNMP1 was immunodetected in the somata and the dendrites of distinct OSNs in subsets of trichoid sensilla. These trichoid sensilla contained only one α-SNMP1-positive OSN in males and clusters of 2-3 labeled cells in females. In contrast, experiments with α-SNMP2-antibodies revealed a broad labeling of non-neuronal support cells (SCs) that are associated with OSNs in likely all trichoid and basiconic sensilla of the antenna with no differences between sexes. Detailed confocal microscope examinations of olfactory sensilla revealed SNMP2-like immunoreactivity close to the apical membrane of SCs and interestingly inside the sensillum. Together, these findings indicate a potential function of SNMP2 in pheromone- as well as general odorant-responsive sensilla and a role fundamentally different from SNMP1.