Free orbicules of Cupressaceae in daily aerobiological samples

Introduction: Pollen grains of Cupressaceae are a major cause of pollinosis, especially in Mediterranean countries (1,2), and a strong risk factor for allergic asthma (2, 3). Pollen and orbicules of Cupressaceae carry a carbohydrate epitope recognised by human IgE (4). However, while airborne pollen grains of Cupressaceae are commonly observed in aerobiological monitoring, free orbicules were never reported in aerobiological samples. We report (a) the presence of orbicules in Calocedrus decurrens (Torr.) Florin (Cupressaceae) and (b) the observation of free orbicules belonging to Cupressaceae in daily aerobiological samples. Materials and Methods: Dried microsporophylls of Calocedrus decurrens (Torr.) Florin (Cupressaceae) were fixed to aluminum stubs with double adhesive carbon tape, sputter-coated with gold, and observed and photographed with a SEM (7) (JEOL JSM 5410, Jeol Ltd, Tokyo, Japan). Daily aerobiological samples were collected in Pisa, Italy with a “Lanzoni VPPS 2000” pollen trap installed in an urban area of Pisa about 17 m from the road pavement (Google maps coordinates 43.718343° N, 10.395110° E) and equipped with pre- siliconed sampling tape Silkostrip (Lanzoni). Sections corresponding to 24-hour intervals were cut from the tape, placed on microscope slides and stained with fuchsin jelly (5). The slides were then examined with optical (Leica Diaplan) and confocal microscope (Nikon A1plus) at 400x (6). Results and discussion: The inner side of the tapetum and the outer side of the pollen exine of Calocedrus decurrens are covered by free, not embedded, orbicules ranging from 0.6 to 0.7 μm. Our observations are consistent with other researches supporting the hypothesis that orbicules may become airborne as loose particles when pollen grains are released from pollen sacs (8). Under the optical microscope, daily aerobiological samples sometimes exhibit clusters of very small, well stained dots, around Cupressaceae pollen grains. When the same samples are observed with the confocal microscope, the cluster is resolved in submicronic particles, ranging from 0.5 to 0.6 μm, with the same autofluorescence emission as the exine of the pollen grains, which leads us to identify them as orbicules (7). We were able to observe a large number of orbicules both on the exine of pollen grains of Cupressaceae and as loose bodies around them. Conclusions: For the first time, we proved that orbicules of Cupressaceae can be detected as loose particles in aerobiological samples at the same time of the “parental” pollen grains but vastly outnumbering them. On account of their smaller size, free orbicules can reach – and carry their allergenic load (4) - much deeper in respiratory systems than pollen grains. Although further cross-disciplinary research is needed, we hypothesize that free orbicules contribute to the strong correlation of airborne pollen grains of Cupressaceae with pollinosis