The absorption of visible light by matter is largely due to electronic transitions. Aside from a few highly colored pigments and chromophores, molecular components of living things are mainly transparent to visible light. Visible light ranges in wavelength from 700 to 380 nm, from red to blue, corresponding to a photon energy of 1.8 to 3.3 eV (eV). The electronic band-gap of proteins and nucleic acids is of the order of 5 eV [1]. Therefore, most of the contents of a fly are transparent to visible light. Furthermore, most of the molecules in living systems are closed-shell molecules hence invisible to ESR.

Drosophila melanogaster was chosen as a subject for our study because it is small enough to fit in an X-band ESR and is easy to produce and manipulate. Electron spin measurements in live flies were performed in our lab with ESR before [28]. ESR measurements with alive and intact animals larger than flies are not easy, partly because larger animals require L-band (≈1GHz) measurements to avoid intense microwave absorption by water, with attendant loss of sensitivity compared to X-band. Absorption of X-band microwaves by flies limits the number of flies to about 30. Several studies have been reported on the spin in living systems with ESR [7,8,14,26].

The first barrier to visible light entering a fly is its cuticle. The composition and the chemical interactions that make the insect cuticle are not fully understood. However, it is known that the fly cuticle is layered [19]. The outermost layers of the cuticle commonly contain lipids, specifically wax, in keeping with its purpose of water conservation. Deeper layers incorporate pigments, proteins, and carbohydrates, respectively. Chitin, one of the main biopolymers of the cuticle and the second most abundant polysaccharide on earth [18], is transparent to visible light [20]. The only known molecular category that exclusively provides colour due to its chemical structure via visible light absorption in flies is melanin [29]. The other key polymer in flies is sclerotin. Sclerotins are known to function to provide structural strength [22]. Interestingly, biochemical pathways of melanin and sclerotin intersect (Fig. 2). While N-acetyl dopamine (NADA) sclerotin is reported to be colorless, N-β-alanine dopamine (NBAD) sclerotin exhibits yellowish hues [31].

Melanin is an amorphous polymer extensively produced by Drosophila melanogaster. The pigment is primarily located on the cuticle in the form of black (eumelanin) and brown melanin. The differences in the production and regulation of melanin types are partially known (Fig. 2). Apart from the cuticular melanins, there is some evidence for the existence of molecular machinery to produce neuromelanin in drosophila [3,13]. Neuromelanin is also a catecholamine-based pigment [6] that is found in some catecholaminergic nuclei of humans and some other mammals' brains. Neuromelanin is well known for its association with Parkinson's Disease. Accumulation of neuromelanin in the brain is considered to be age dependent [32]. Hence, for animals with a short life span like fruit flies, neuromelanin production seems to be absent.

Yellow and ebony genes play pivotal roles in determining the cuticle colour of flies. The effects of the genes are easily observable in the phenotype (Fig. 1). The genes are required in the synthesis of eumelanin (black melanin) and NBAD sclerotin (Fig. 2). In the case of ebony knock-out flies, they are not able to form the link between dopamine and β-alanine [10] to synthesize yellow sclerotin and have a very dark cuticle coloration. In yellow knock-out flies, the conversion of dopachrome into 5,6- dihydroxyindole for eumelanin production is hindered, leading to their distinctive yellow cuticle coloration.

The majority of ESR signals come from the cuticle. The ESR spectrum of fruit flies has been shown to depend on the presence of melanin and sclerotin [12]. The effect of light on both synthetic and natural melanin has been described previously in vitro [2,4,9,17,24]. One of the studies [17] hypothesized that the constituents of eumelanin comprised carbon-centered radicals and semiquinones, with semiquinones being implicated in the light response. Our objective was to investigate the light response of fruit flies with varying levels of black melanin content.