Two bioluminescent diptera: The North American Orfelia fultoni and the Australian Arachnocampa flava. Similar niche, different bioluminescence systems

Photochemistry and Photobiology, Jan 2002 by Viviani, Vadim R, Hastings, J Woodland, Wilson, Therese

ABSTRACT

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 ((Delta^sub 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 -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.

INTRODUCTION

Among dipterans, bioluminescence has been reported only in Mycetophilidae (fungus gnats). Harvey listed three genera, Arachnocampa, Orfelia (previously Platyura) and Keroplatus (1). Of these, Arachnocampa is most widely known for the brilliant displays its larvae create on the roofs of caves in New Zealand and Australia. The almost unknown Orfelia of North America, discovered by Fulton only in 1941 (2), shares many of the outward characteristics of Arachnocampa. At first glance, the two brownish larvae look much alike, being on average 10-20 mm long and 1-2 mm in diameter. To catch prey, both build remarkable webs in similar environments, such as crevices on damp stream banks (3); but unlike Arachnocampa, Orfelia is seldom found on the roofs of caves and does not use "fishing lines" as does Arachnocampa. Both Arachnocampa and Orfelia are carnivorous, even cannibalistic; in spite of the name, there is little indication that fungi are part of the diets of either of these gnats.

Here, however, the similarities end. The bioluminescence of Orfelia is blue, and that of Arachnocampa is blue-green. The light-emitting organs are different. Arachnocampa has only one caudal lantern derived from the malpighian tubules (4), whereas Orfelia larvae have very unusual, bilateral, anterior and posterior lanterns (2). They are characterized by two rows of black bodies consisting of large binucleated cells filled with dark granules, seemingly budding out of mitochondria (5). This report also suggested that the chemistry of the bioluminescence of Orfelia differed from that of Arachnocampa in at least one significant aspect: adenosine triphosphate (ATP) enhances the luminescence of crude extracts of Arachnocampa (6,7), whereas it has no effect on that of Orfelia (5).

Considering the well-known dependence of the bioluminescence of Coleoptera on ATP (8), the difference between Arachnocampa and Orfelia with respect to ATP seemed significant and intriguing. Our results support the conclusion that the chemistries of bioluminescence of Orfelia fultoni and Arachnocampa flava from Australia are distinct; there are no cross-reactions of enzymes or substrates.

MATERIALS AND METHODS

Reagents. Coenzyme A (CoA), dithiothreitol (DTT), ethylenediamine tetra-acetic acid (EDTA), D-luciferin, ATP, reduced flavin mononucleotide (FMNH,), ascorbic acid, glutathione, nicotinamide adenine dinucleotide (reduced form) (NADH), carbonic anhydrase, bovine serum albumin, alcohol dehydrogenase, beta-amylase, apoferritin, tyroglobulin and pyrophosphate were obtained from Sigma Chemical Co. (St. Louis, MO). Triton X-100 was obtained from Fisher Scientific (Pittsburgh, PA), Sephacryl S-300 and S-400 from Pharmacia (Piscataway, NJ).

Insects. Orfelia fultoni larvae were collected from May through June in 1999, 2000 and 2001 at the Shenandoah National Park. (Virginia), from early April through August at the Highlands Biological Station (Highlands, NC), Cashiers (North Carolina) and Glenville (North Carolina), usually among moss on stream banks, in exposed cavities under stones along streams or under decaying logs. In Pickett State Park (Tennessee) they were found in May 2001 at the base of sandstone caves. Larvae were located at night by their bioluminescence and collected by hand; usually about 20-30 larvae per night. They were transferred to plastic jars with moist soil and moss from their habitat. Some larvae were kept alive for as long as 3 weeks on a diet of Drosophila. Most were immediately rinsed with water, transferred to Eppendorf tubes, frozen in liquid nitrogen, then stored at -80 deg C. Arachnocampa flava larvae were collected at Springbrook National Park (Queensland, Australia) during spring and summer of 2000 and 2001, on damp banks similar to those rich in Orfelia but with a high density of trapdoor spiders; the larvae were shipped to our laboratory in liquid nitrogen.