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Larvae of  O. fultoni  (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are  located mainly in the anterior and posterior parts of the body. Although closely related to  Arachnocampa spp  (Keroplatidae: Arachnocampininae),  O.fultoni  has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related  Neoceroplatus spp , shares the same kind of luciferin-luciferase system of  Orfelia fultoni . However, the molecular properties, identities and functions of luciferases, SBF and luciferin of  Orfelia fultoni  and other  luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using  O. fultoni  as a source of luciferase, and the recently discovered non-luminescent cave worm  Neoditomiya  sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of  O. fultoni  is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in  Orfelia fultoni  and in smaller dark granules in  Neoditomiya sp , consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name  keroplatin .

A new brilliantly blue-emitting luciferin-luciferase system from Orfelia fultoni and Keroplatinae (Diptera)