PRIMARY CULTURES OF ANTENNAL CELLS OF MAMESTRA BRASSICAE (LEP., NOCTUIDAE). BIOSYNTHESIS OF PBPs IN VITRO.

Philippe LUCAS, Patricia NAGNAN-LE MEILLOUR
INRA, Unite de Phytopharmacie et des Mediateurs Chimiques, Route de Saint Cyr, 78026 Versailles Cedex, France
(plucas@versailles.inra.fr)

Moth sex pheromones are encoded by specialized olfactory receptor neurons (ORNs) that innervate scnsilla trichodea found on the antennae. The stimulus-reception process and the first steps of olfactory transduction are thought to take place at the membrane of the outer dendritic segments of ORNs, which are bathed by the sensillum Iymph. The sensillum Iymph is particularly enriched in small soluble protehis called pheromone binding proteins (PBPs). To help address the question of involvement of PBPs in early steps of pheromone discrimination in Lepidoptera and draw the electrophanmacological profile of ORNs, we have developed procedures for primary cell cultures of antennal cells dissociated from antennal flagella of Mamestra brassicae pupae.

Antennal flagella were dissected from 3-day-old pupae, and were grown after dissociation in a mixture of Leibovitz's 1.15 medium and Grace's medium. In the first two days of culture, cells differentiated in at Icast 5 types:

• Neuronal cells (type A) were characterized morphologically and immunocytochemically. Neurons extruded thin processes withh1 a few hours afler plating that could reach several hundred microns after a week in the culture. They differentiated into monopolar or bipolar cells and could survive for more than a month in our culture conditions. They were either isolated, clustered in small groups, or present in undissociated antennal explants. On the basis of the diameter of their cell body, we distinguished large (10-16 µm) and small neurons (5-7 µm).
• Type B consisted of large (approx. 20 µm diam.) and flat cells, with a veil-like cytoplasm and were usually grouped. They could survive for more than a month. Very similar cells were previously suggested to be either plasmocytes or epidermal cells.-1
• Type C were large and flat cells, highly variable in size and shape. They closely resembled insect glial cells.
• Type D were large cells that grew thick processes, until 100µ~m long. These differentiations resembled olfactory hairs, were not observed on female antennal cultures and thus, could bc grown by trichogen cells.
• Type E were flat spindle-shaped cells with an elongated phase-bright cell body (approx. 20 µm long) and processes at each end. These cells were present in low number and did not survive beyond 2 days in the cultures. High proportion of similar cells in the hemolymph of pupae suggests that they could bc hemocytes.

The presence of PBPs in the cell cultures was detected using a mixture of monoclonal antibodies raised against PBPI-Mbra and PBP2-Mbra. The immunodetection of PBPs in the culture medium of antennal cultures indicates that accessory cells followed at least partially their normal course of differentiation in vitro. Thus, our culture system provides a good in vitro model to study perireceptor events (importance and specificity of sensillar proteins in the activation of the dendritic receptor sites) as well as clectrophysiological properties of pheromone receptor neurons.

1. Stengl, M. and Hildebrand, J.G. J. Neurosci., 10, 837 (1990).