116 J. Eur. Opt. Society-Rapid Publ. 22, 12( 2026)
2 Materials and methods
In this study, we combined specimen collection and taxonomic identification with detailed morphological and optical analyses, as well as optical simulations. Structural features of the abdominal cuticle were examined by optical and electron microscopy, while spectral reflectance was measured to characterise the insects’ optical responses. Chromaticity and photoreceptor quantum catch calculations were used to assess the visibility of the colours to conspecifics and predators. Finally, the observed structures were modelled to link physical structure to optical behaviour.
2.1 Specimen identification
Samples of C. vicina and L. richardsi were collected either naturally dead or alive in the province of Namur, Belgium and the city of Exeter, UK between 2013 and 2017. The specimens that were collected alive were chemically euthanized in a jar containing ethyl acetate. All samples were kept without any further conservation methods in room conditions of temperature and humidity. They were identified using identification keys [ 45 – 48 ].
2.2 Photonic structure morphology
Optical microscopy was performed using an Olympus BX61( Tokyo, Japan) microscope, an Olympus XC50 camera and a halogen Osram HLX 64625( Munich, Germany) visible white light source. The dipteran abdomens were observed in the reflection mode of the microscope. The abdomens of dead dipterans were dissected with a scalpel and placed on microscope slides covered by black paper. The flatter areas of the samples were observed.
The morphology of the dipteran abdomens’ structures was investigated using a JEOL 7500F( Tokyo, Japan) field emission scanning electron microscope( SEM) and a JEOL JEM-1400 transmission electron microscope( TEM). For SEM analysis, abdomens were cut into pieces of about 5mm 5 mm. Some samples were immersed into liquid nitrogen immediately before being broken. This process increased the probability of a neat fracture. All SEM samples were attached to the sample mount by conducting double-sided adhesive tape and sputter-coated with 10 nm of gold. The dipteran abdomens were prepared following a standard TEM sample preparation method [ 49 ]. 80 nmthick cross sections were ultramicrotomed and transferred onto TEM analysis grids. The layer thicknesses were measured along a line perpendicular to the multilayers, using image processing software ImageJ [ 50 ]. Measurements were taken at multiple positions in each selected micrograph. For C. vicina, 8 SEM images were analysed, while 5 TEM images were used for L. richardsi.
2.3 Optical characterisation
The normalised reflection spectra, also called reflection factor, R( k)={ I( k) � B( k)}/{ W( k) � B( k)}, i. e., the ratio between the spectral intensities I( k) and W( k) reflected by the sample and by an Ocean Optics WS-1( Dunedin, Florida, USA) white reference, respectively, including noise corrections( namely, spectral intensity in darkness) B( k), were measured using an Avantes AvaSpect-2048( Apeldoorn, The Netherlands) spectrophotometer, unpolarised visible light from an Ocean Optics DH-2000-BAL source and optical fibres in specular configuration( i. e., incidence and detection angles with the same magnitude). The analysed spot size extended over a few square millimetres. A fibre holder allowed measurements to be performed for angles ranging from �75 ° to 75 ° with respect to normal incidence, in steps of 15 °.
From the reflection spectra, CIE 1931 coordinates( x, y, z) were calculated [ 51 – 53 ]. They allow the quantification of the colours perceived by the human eye and the two independent coordinates( x, y) are presented on a 2-degree observer chromaticity diagram. In these calculations, the spectrum of the incident light source was assumed to be the CIE standard illuminant D 65, corresponding to the average daylight emitted by the Sun( considered as a 6500 K black body) and received in Northern Europe.
Such a chromaticity diagram is however not valid for other species’ colour perception. The spectral sensitivities of the photoreceptors located in eyes differ indeed from one species to another. In addition, some species have colour vision involving a different number of photoreceptors, such as some birds and some insects with tetrachromatic vision, i. e., four different types of photoreceptors, unlike human vision, which is trichromatic.
To gauge the visibility of the species to predators and conspecifics, the quantum catches of the photoreceptors associated with Calliphoridae species and three of their known predators, namely Parus caeruleus Eurasian blue tit, Zootoca vivipara common lizard and Vespula vulgaris common wasp, were calculated using the method suggested by Vorobyev et al. [ 54 – 56 ] These quantities correspond to the relative amount of photons absorbed by each type of photoreceptor related to a given species. In addition to the three kinds of photoreceptors, namely the short wavelength sensitive( SWS), the middle wavelength sensitive( MWS) and the long wavelength sensitive( LWS) cone photoreceptors, involved in the colour vision of these four selected species, the blow flies, the Eurasian blue tit, and the common lizard possess a fourth type of photoreceptors, namely very short wavelength sensitive( VSW) cone photoreceptors. The calculation of the related quantum catches requires the spectral sensitivities of these photoreceptors. This data was taken from the literature [ 19 – 21, 57 – 60 ].
2.4 Photonic model of the blow fly cuticle and numerical methods
A conventional one-dimensional transfer-matrix( 1D-TM) method [ 61 ] was used to simulate the spectral reflectance from the structures observed in the dipteran integuments. This method rigorously solves Maxwell’ s equations in each layer of a photonic multilayer structure for both transverse electric( TE) and transverse magnetic( TM) polarisation of electromagnetic waves propagating through such layered media. In this formalism, the electromagnetic field wave is