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Bioluminescence in Diptera is found in the Keroplatidae family, within Arachnocampininae and Keroplatinae subfamilies, with reported occurrences in Oceania, Eurasia, and Americas. Larvae of Orfelia fultoni, which inhabit stream banks in the Appalachian Mountains, emit the bluest bioluminescence among insects, using it for prey attraction, similarly to Arachnocampa spp. Although bioluminescence has a similar prey attraction function, the systems of Arachonocampininae and Keroplatinae subfamilies are morphologically/biochemically distinct, indicating different evolutionary origins. To identify the possible coding genes associated with physiological control, ecological adaptations, and origin/evolution of bioluminescence in the Keroplatinae subfamily, we performed the RNA-Seq analysis of O. fultoni larvae during day and night and compared it with the transcriptomes of Arachnocampa luminosa, and reanalyzed the previously published proteomic data of O. fultoni against the RNA-Seq dataset. The abundance of chaperones/heat-shock and hexamerin gene products at night and in luciferase enriched fractions supports their possible association and participation in bioluminescence. The low diversity of copies/families of opsins indicate a simpler visual system in O. fultoni. Noteworthy, gene products associated with silk protein biosynthesis in Orfelia were more similar to Lepidoptera than to the Arachnocampa, indicating that, similarly to the bioluminescent systems, at some point, the biochemical apparatus for web construction may have evolved independently in Orfelia and Arachnocampa., (Copyright © 2021. Published by Elsevier Inc.)

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.

Bioluminescence in Diptera is found in the family Keroplatidae, in the glowworms of the genera Arachnocampa, Orfelia and Keroplatus. Despite belonging to the same family, Arachnocampa spp. and Orfelia fultoni display morphologically and biochemically distinct bioluminescence systems: Arachnocampa spp. produce light by the terminal ends of Malpighian tubules using ATP, a luciferin and a luciferase, whereas Orfelia fultoni produces light by translucent areas associated with rows of black bodies in the anterior and posterior parts of the body, using a 140 kDa luciferase and a luciferin which do not cross-react with the Arachnocampa luciferin-luciferase system, and a substrate binding fraction (SBF) which apparently releases luciferin in the presence of reductants. While several other keroplatids are not luminescent, we recently discovered a non-luminescent web-constructing keroplatid larva living in the roofs of caves in the Atlantic rainforest in Brazil, which noteworthily has a compound with Orfelia luciferin-like activity and its associated binding protein (SBF). Both the Neoditomyia luciferin-like compound and SBF cross-react with purified Orfelia luciferase to produce light in the same blue region of the Orfelia luciferin-luciferase system (479 nm). We also checked for the presence of Orfelia-type luciferin in Arachnocampa luminosa and Aedes aegytpi larval bodies, but no traces were found. Molecular studies indicate that Neoditomyia sp. is phylogenetically closer to Keroplatus and Orfelia than to Arachnocampa species. The presence of luciferin and its associated binding protein in this non-bioluminescent keroplatid larva indicates that luciferin may display another important biochemical function in keroplatid larvae and suggests that bioluminescence could be a recently evolved trait in Keroplatidae.

Fungus-gnats of the genus Arachnocampa are unique among bioluminescent insects for displaying blue-green bioluminescence, and are responsible for one of the most beautiful bioluminescence spectacles on the roofs of the Waitomo Caves. Despite morphological studies showing that Arachnocampa larval lanterns involve specialization of the Malpighian tubules, the biochemical origin of their bioluminescence remains enigmatic. Using a cDNA library previously constructed from lanterns of the New Zealand glowworm A. luminosa, we carried out the first transcriptional analysis of ~ 500 expressed sequence tags (ESTs) to identify putative candidate proteins for light production, and to better understand the molecular physiology of the lanterns and their relationship with Malpighian tubule physiology. The analysis showed an abundance of hexamerin-like proteins, as well as luciferase-like enzymes, indicating a possible critical role for these proteins in bioluminescence. These findings were corroborated by proteomic analysis of lantern extracts, which showed the presence of hexamerins and luciferase-like enzymes. Other gene products typical of Malpighian tubules, such as detoxifying enzymes, were also found. The results support the existence of an evolutionary link between Malpighian tubule detoxification and the origin of bioluminescence in these Diptera., (Copyright © 2015 John Wiley & Sons, Ltd.)

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. Whereas the oxidative mechanism which leads to light emission is similar for most luciferases, these enzymes and their substrates are evolutionarily unrelated. Among all bioluminescent groups, insects constitute one of the most diverse in terms of biochemistry. In the fungus-gnats (Mycetophilidae: Diptera), for example, bioluminescence is generated by two biochemically distinct systems. Despite the diversity, investigations on insect luciferases and biochemistry have been conducted mostly with fireflies. The luciferases from the related phengodid beetles, which can produce green to red bioluminescence using the same chemistry as firefly luciferases, have been recently investigated. Beetle luciferases originated from ancestral acyl-CoA ligases. Present data suggest that conserved motifs among this class of ligases are involved in substrate adenylation. The three-dimensional structure of firefly luciferase was recently solved and mutagenesis studies have been performed identifying putative residues involved in luciferin binding and bioluminescence color determination in several beetle luciferases. The knowledge gained through these studies is helping in the development of useful reporter gene tools for biotechnological and biomedical purposes.

Orfelia fultoni is the only bioluminescent dipteran (Mycetophilidae) found in North America. Its larvae live on stream banks in the Appalachian Mountains. Like their Australasian relative Arachnocampa spp., they build sticky webs to which their bioluminescence attracts flying prey. They bear two translucent lanterns at the extremities of the body, histologically distinct from the single caudal lantern of Arachnocampa spp., and emit the bluest bioluminescence recorded for luminescent insects (lambda(max) = 460 nm versus 484 nm from Arachnocampa). A preliminary characterization of these two bioluminescent systems indicates that they are markedly different. In Orfelia a luciferin-luciferase reaction was demonstrated by mixing a hot extract prepared with dithiothreitol (DTT) under argon with a crude cold extract. Bioluminescence is not activated by adenosine triphosphate (ATP) but is strongly stimulated by DTT and ascorbic acid. Using gel filtration, we isolated a luciferase fraction of approximately 140 kDa and an additional high molecular weight fraction (possibly a luciferin-binding protein) that activated bioluminescence in the presence of luciferase and DTT. The Arachnocampa luciferin-luciferase system involves a 36 kDa luciferase and a luciferin soluble in ethyl acetate under acidic conditions; the bioluminescence is activated by ATP but not by DTT. The present findings indicate that the bioluminescence of O. fultoni constitutes a novel bioluminescent system unrelated to that of Arachnocampa.