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.

Expressional and functional comparisons of two general odorant binding proteins in Agrotis ipsilon.

Insect general odorant binding proteins (GOBPs) have been long thought to bind and transport host plant volatiles to the olfactory receptors on the dendrite membrane of the olfactory neurons. Recent studies indicate that they can also bind female sex pheromones. In present study, two GOBP genes, AipsGOBP1 and AipsGOBP2 were cloned from the adult antennae of Agrotis ipsilon. Tissue expression profiles indicated that both of them are antennae-specific and more abundant in the female antennae than in the male antennae. Temporal expression profiles showed that both AipsGOBP1 and AipsGOBP2 began to express in antennae 3 days prior to adult emergence from pupae, and reached their highest expression level 3 and 4 days after adult emergence, respectively. Mating increased their expression in the female antennae but reduced their expression in the male antennae. In situ hybridization and immunolocalization demonstrated that both AipsGOBP1 and AipsGOBP2 are expressed and co-localized in sensilla basiconica and sensilla trichodea of both sexes. AipsGOBP2 exhibited a high binding affinity in vitro with the two major sex pheromone components Z7-12:Ac and Z9-14:Ac and the four plant volatiles cis-3-hexen-1-ol, oleic acid, dibutyl phthalate and ß-caryophyllene with Ki values less than 5 µM. AipsGOBP1, on the other hand, showed medium binding affinities with the five A. ipsilon sex pheromones and six plant volatiles. AipsGOBP2 also showed a broader ligand-binding spectrum and a greater ligand-binding affinity than AipsGOBP1 with the tested aldehyde and alcohol sex pheromones of Lepidoptera species. Taken together, our results indicate that AipsGOBP2 may play greater roles than AipsGOBP1 does in binding sex pheromones and host plant volatiles.

The sensory neurone membrane protein SNMP1 contributes to the sensitivity of a pheromone detection system.

Male moths detect female-released sex pheromones with extraordinary sensitivity. The remarkable sensory ability is based on a cooperative interplay of pheromone binding proteins in the lymph of hair-like sensilla trichodea and pheromone receptors in the dendrites of sensory neurones. Here we examined whether in Heliothis virescens the so-called 'sensory neurone membrane protein 1' (SNMP1) may contribute to responsiveness to the pheromone component, (Z)-11-hexadecenal (Z11-16:Ald). By means of immunohistochemistry and in situ hybridization we demonstrated that SNMP1 is in fact present in cells expressing the Z11-16:Ald receptor HR13 and the dendrites of sensory neurones. To assess a possible function of SNMP1 we monitored the responsiveness of cell lines that expressed HR13 alone or the combination SNMP1/HR13 to stimulation with Z11-16:Ald by calcium imaging. It was found that SNMP1/HR13 cells were 1000-fold more sensitive to pheromone stimulation compared with HR13 cells. In contrast, cells that expressed HR13 and the non-neuronal SNMP2-type showed no change in pheromone sensitivity. Overall, our reconstitution experiments demonstrate that the presence of SNMP1 significantly increases the HR13-based responsiveness of cells to Z11-16:Ald, suggesting that SNMP1 also contributes to the response of the antennal neurones and thus to the remarkable sensitivity of the pheromone detection system.