Immunohistochemical localization of Papilio RBP in the eye of butterflies

Abstract

SUMMARY We recently identified a novel retinoid binding protein, PapilioRBP, in the soluble fraction of the eye homogenate of the butterfly Papilio xuthus, and demonstrated that the protein is involved in the visual cycle. We now have localized the protein in the Papilio eye by light and electron microscopic immunohistochemistry using a monospecific antiserum produced against artificially expressed Papilio RBP. We found strong immunoreactivity in the primary as well as secondary pigment cells and in the tracheal cells. The pigment cells have long been regarded as an important site of the visual cycle, and this view is further supported by the present result. Interestingly, the cytoplasm and nuclei of these cells were equally labeled, indicating that the protein exists in both the cytoplasm and the nucleus. We conducted a survey for the existence of the Papilio RBP-like proteins in other insects including several species of butterflies, dragonflies, cicadas, grasshoppers and honeybees. Anti-Papilio RBP immunoreactivity was confirmed in the proteins isolated only from butterflies belonging to the superfamily Papilionoidea and not from other species. In all insects tested, however, fluorescing proteins were clearly detected, suggesting that these insects also have similar retinol-binding proteins.

Conserved chemosensory proteins in the proboscis and eyes of Lepidoptera

“…no. AAO62632) had been reported in Drosophila 46-47 and a retinol-binding protein of 235 amino acids has been identified in the butterfly Papilio xuthus 48-49 (acc. no.…”

Beyond chemoreception: diverse tasks of soluble olfactory proteins in insects

“…However, other proteins also act in the eyes of insects as carriers for visual pigments. Two larger proteins, a retinoid-binding protein of 273 amino acids, called PINTA [prolonged depolarization afterpotential (PDA) is not apparent], and a retinol-binding protein of 235 amino acids were identified in D. melanogaster (Wang & Montell, 2005;Wang, Jiao & Montell, 2007) and in the butterfly Papilio xuthus (Wakakuwa, Arikawa & Ozaki, 2003;Wakakuwa, Ozaki & Arikawa, 2004), respectively. These proteins belong to different families and are completely unrelated to OBPs or CSPs.…”

Immunolocalization suggests a role of the histamine‐gated chloride channel PxHCLB in spectral opponent processing in butterfly photoreceptors

“…We also localized cell bodies by Nissl staining ( magenta in Figure b,d) after immunohistochemistry. The large cell bodies immediately proximal to the basement membrane are those of tracheal cells (Wakakuwa, Ozaki, & Arikawa, ). Medium‐sized cell bodies within the lamina may be those of glial cells, but they have not been properly identified.…”

“…AntiPxHCLA labeled the whole lamina (Figure 3a), while antiPxHCLB labeled the proximal retina, fenestrated layer, as well as lamina (Figure 3c). We also localized cell bodies by Nissl staining (magenta in Figure 3b,d) after immunohistochemistry.The large cell bodies immediately proximal to the basement membrane are those of tracheal cells(Wakakuwa, Ozaki, & Arikawa, 2004).Medium-sized cell bodies within the lamina may be those of glial cells, but they have not been properly identified. Cell bodies of LMCs are densely packed between the proximal portion of the fenestrated layer and the distal border of the lamina(Figure 3b,d, arrowheads; SDS-PAGE profile and immunoblot analysis of the antiPxHCL antisera.…”