INTRODUCTION

Bioluminescence, or ability to produce biological light, among insects is found in three orders: Coleoptera, Collembola, and Diptera. While the mechanisms of Coleoptera (beetle) bioluminescence are thoroughly studied, the molecular basis of Diptera light emission still has to be investigated. Diptera order includes only one group with bioluminescent species—fungus gnats from family Keroplatidae, comprising almost 100 genera and ~1000 species [1]. Among them only few have the ability to glow: species of the genus Arachnocampa (Edwards, 1924) from subfamily Arachnocampinae (Australia and New Zealand) [2], Orfelia fultoni (Fisher, 1940) from subfamily Keroplatinae, tribe Orfeliini (North America) [3], and, finally, species of the genus Keroplatus (Bosc, 1792) (Europe and northern Asia) and recently discovered Neoceroplatus betaryensis (Falaschi, Johnson & Stevani, 2019) (Brazil) from subfamily Keroplatinae, tribe Keroplatini [4].

The light production in Keroplatidae is based on the oxidation of a luciferin molecule with the help of a luciferase as a catalyzing enzyme [5, 6], however, there is little similarity in morphology, physiology and chemical nature of bioluminescence between Diptera species belonging to different subfamilies [7]. Arachnocampa spp. larvae use their Malpighian tubules to produce light and emit it from the end of the abdomen [8]. Bioluminescence system of Arachnocampa luminosa is ATP-dependent and the luciferin molecule probably comprises tyrosine and xanthurenic acid residues [9]. O. fultoni larvae possess light-emitting structures only in their five anterior segments and in the region near the abdominal tip [10], luminescence reaction is cofactor-independent and possibly shares the mechanism with Keroplatini species [11]. In Keroplatus genus species, the entire larval body glows due to specialized proteinaceous granules of the fat body [12]. Its larvae construct a slime web underneath tree fungi and emit blue light (λmax = 460 nm) [13]. Bioluminescence nature of Keroplatus larvae was discovered in the middle of the 19-th century [14] in K. tipuloides (Bosc, 1792) (=K. sesioides Wahlberg, 1839) and then confirmed several times later [3, 15, 16]. Until now biochemistry of Keroplatus bioluminescence as well as its biological and ecological function are still among the least studied aspects for glowing insects. Here we report riboflavin (vitamin B2) as one of the fluorescent compounds found in relatively high concentration [17] in Keroplatus testaceus larvae. We suppose that riboflavin may play a role in bioluminescence chemistry of this Keroplatidae species.

RESULTS AND DISCUSSION

For the present study Keroplatus larvae were collected from the undersurface of bracket fungi in the Kivach Nature Reserve, Karelia region, Russia (Fig. 1) in June–July 2020. To preserve the components of the bioluminescence system the biomaterial was frozen in aluminium foil on dry ice immediately after collection and then transferred to the –70°C laboratory freezer for long term storage. A few living larvae were transferred to the laboratory facilities to identify the species. As we expected, the collected larvae belonged to K. testaceus, the most common species in the region.

Fig. 1.
figure 1

Keroplatus spp. larva on the undersurface of a bracket fungus (scale bar 20 mm).

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For the primary investigation of Keroplatus bioluminescence system we focused on isolation of fluorescent components from larvae extracts, which could contain possible oxyluciferin candidates [18]. Among a number of extracts (ethanol, acetone, hexane, aqueous buffers extracts etc.), prepared from frozen K. testaceus larvae, aqueous extracts were the most intensely fluorescent (Fig. 2a, inset). Optimization of the extraction conditions (including the choice of the optimal temperature and buffer composition) allowed to obtain a fluorescent extract, which was then filtered with an ultrafiltration membrane to discard the macromolecular impurities. As a result, only the permeate was fluorescent, and then the obtained sample was applied to the reverse phase HPLC for further purification (Fig. 2a). Fractions, containing fluorescent compounds, were identified by fluorescence and absorbance measurement assays at 366 nm, collected separately, lyophilized and used for analysis. A consecutive series of chromatographic experiments allowed the isolation of ~3.5 a.u. of an individual fluorescent compound from K. testaceus larvae biomass. The sample of the obtained compound was analysed by high-resolution NMR and HRMS. A combination of NMR experiments (Fig. 2b; Fig. S1 and Table S1 in Supplementary Information) and HPLC-analysis of the lyophilized sample in comparison with a commercially available riboflavin (vitamin B2) confirmed the identity of these substances. HRMS also showed the presence of riboflavin molecular ions in positive and negative modes. The concentration of riboflavin in Keroplatus testaceus larvae biomass is quite high ~3.4 a.u. per 1 g, which corresponds to 83 µg of riboflavin per 1 g of biomass.

Fig. 2.
figure 2

Extraction and structural elucidation of a fluorescent compound—riboflavin (vitamin B2)—from Keroplatus spp. larvae: (a) chromatographic profile of aqueous extract from lyophilized Keroplatus spp. larvae. Fluorescent fraction is marked with red arrow. Inset—fluorescence spectrum of aqueous extract from frozen Keroplatus spp. larvae (excitation at 366 nm); (b) Multiplicity-edited 1H-13C HSQC NMR spectrum of fluorescent compound from Keroplatus larvae (red and magenta), overlaid with NMR of standard sample of riboflavin (blue and cyan) at the same conditions. Inset—conditions of NMR spectra acquisition (see “Experimental” for more details) and chemical structure of riboflavin with atom numbering (green), coincident with peak labels and chemical shifts in table S1 (see Supplementary Information).

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Biological and ecological function of Diptera bioluminescence is still unclear. Arachnocampa luminosa larvae are predators that live in dark caves and caverns. Presumably, they use light emission to attract prey into sticky fishing lines [19]. Сarnivorous Orfelia fultoni also use light to attract and capture prey into sticky webs [20].

Meanwhile few is known about biology and especially about biochemistry and ecological function of Keroplatus bioluminescence. The bioluminescence of Keroplatus was discovered over a century ago [14], and species of the genus are quite rare. Currently there are suggestions to use Keroplatus as a bioindicator of state of forest ecosystems, including for the tracking of changes in the ecological state of specific biotopes and nature conservation [21]. Keroplatus spp. larvae feed on fungal spores and small invertebrates [22]. They emit visible light with the shortest wavelength of all terrestrial arthropods. The light may be used to attract potential prey, as in other Keroplatidae. There is also a hypothesis that the light may have a defensive function, due to the fact that larvae tend to avoid sunlight, or it could possibly be aimed at the fungus to produce more spores [23]. Keroplatus bioluminescence system is unique and one of the least studied among all terrestrial species. Any new data may be useful for further understanding of Keroplatidae light emission nature and chemistry.

Riboflavin (vitamin B2) is a starting compound in the biosynthesis of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Biosynthesis of riboflavin takes place in bacterial, fungal and plant cells, but not in animal cells [24]. High level of riboflavin in Keroplatus larvae indicates that the species accumulate it for a certain reason. As a vitamin, riboflavin is essential for the transformation of the insect from larva to imago stage. We also suppose that it might be important for Keroplatus bioluminescence, however, to determine the exact role of riboflavin in larvae luminescence, it is necessary to establish the structures of all compounds that take part in this process. As it was previously reported, riboflavin and FMN act as light emitters in bacterial bioluminescence [25–27] and their emission spectra are modified by covalent binding to a protein. Interestingly, recent experiments with the related Orfelia fultoni bioluminescence system showed the presence of a green-yellow fluorescent pigment of an unknown structure with fluorescence maximum at 543 nm in “hot” extracts [11], which might as well be riboflavin, but this assumption requires further confirmation. To establish the potential role of riboflavin or its metabolites in Keroplatus bioluminescence reaction further investigations are necessary.

EXPERIMENTAL

Sample collection. Keroplatus testaceus larvae were collected from tree fungi in the forests of Kivach State Nature Reserve with the permission of the administration. Kivach State Nature Reserve is located at a distance of 27 km from Kondopoga and 80 km from Petrozavodsk (62.3°N, 33.9°E). Address: 186220, Zapovednaya street, 14, Kivach village, Kondopozhsky district, Republic of Karelia, Russia. Collected larvae were coiled into aluminum foil (2-3 larvae per piece 12–14 cm2), frozen on dry ice and transferred to the laboratory facilities for long term storage in the lab freezer at –70°C. A few living larvae were transferred to the laboratory facilities to identify the species.

Species identification. Three species from Keroplatus genus (K. testaceus Dalman, 1918, K. dispar Dufour, 1839, and K. tuvensis Zaitzev, 1991) are known from Karelia [28]. All have similar appearance and can be reliably differentiated by details of male terminalia, especially the shape of the ventral medial process of gonocoxites [22]. We expected that the collected larvae belonged to K. testaceus, the most common species in the region. Reliable identification by preimaginal stages in this group of species is hardly possible. To confirm the determination, we placed several full-grown larvae into the rearing chamber on June 28, 2020. As a result, one adult female emerged on July 9, 2020. Females of K. testaceus can be distinguished from K. dispar by the wing venation characters (subcosta ending approximately opposite the end of radial-median fusion) and the shape of cerci (widest in the middle) [29, 30]. The obtained female was identified using the key by Matile [29] and found to belong to K. testaceus (Fig. S1 in Supplementary Information). Considering the fact that the larvae were collected in a rather limited area on similar substrates and at the same time, we assume that they all belong to the same species.

Biomass extraction. Two-three frozen K. testaceus larvae (~0.2 g) were ground in a mortar in liquid nitrogen. Frozen powder was added to 1 mL of TBS buffer (50 mM Tris-HCl, 2 mM EDTA, pH 7.5) and incubated for 30 min with ice bath cooling and stirring. Then the mixture was centrifuged at 15 000 g (4°C) for 15 min. The obtained supernatant, containing substances of interest, was collected in a separate tube and lyophilized and stored at –70°C or applied to the ultrafiltration filter unit before separation and purification procedure.

Ultrafiltration. The biomass extract solution was filtered on a 3kDa Amicon® Ultra centrifugal filter unit (Merck Millipore, Germany) according to the manufacturer’s instructions. Fluorescence was measured for the concentrated retentate and the permeate. It was found that only the permeate was fluorescent. The resulting sample was further used for the reverse-phase chromatography.

HPLC analysis. Reverse-phase chromatography was performed using a Luna 5 µм C18(2) 100 Å (4.6 × 250 mm; Phenomenex, USA) with a Shimadzu chromatography system (Shimadzu Corporation, Japan). The following solvents were used as a mobile phase: solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.08% trifluoroacetic acid in acetonitrile with a 25–45% B gradient over 20 min and a 1 mL/min flow rate. During the purification procedure, absorbance of the solution leaving the column was monitored at 222 and 366 nm. Fractions, containing substances of interest, were collected separately and lyophilized.

Fluorescence spectra. Fluorescence spectra were acquired with Cary Eclipse Fluorescence Spectrometer (Agilent Technologies, USA). For each measurement lyophilized sample was dissolved in a TBS buffer (50 mM Tris-HCl, 2 mM EDTA, pH 7.5) and 1 mL probe was used. Measurements were corrected for background fluorescence based on monitoring fluorescence of TBS-buffer solution.

NMR spectroscopy. NMR spectra were acquired at Bruker Avance III 800 MHz NMR spectrometer (Bruker, Germany) equipped with 5 mm CPTCI cryoprobe. Lyophilized fraction with observable fluorescence was dissolved in D2O (330 μL), the pH value 8.9 was measured by Orion 2-star benchtop pH-meter (Thermo Scientific, USA). The sample was placed in the 5 mm Shigemi NMR tube to obtain the largest concentration and NMR sensitivity. The 1D and 2D NMR spectra were measured at 20°C. We acquired 1D 1H-NMR spectrum with water presaturation, 2D multiplicity edited 1H-13C HSQC (Fig. 2b) and 2D DQF-COSY spectra (Fig. S1 in Supplementary Information). The obtained data were sufficient to unambiguously identify riboflavin as the only chemical substance in the sample. Its identity was verified by comparison with riboflavin standard (Sigma-Aldrich, USA), the similar NMR spectra at the same pH 8.9 and temperature 20°C were acquired in D2O on the Bruker Avance III 600 MHz NMR spectrometer (Bruker, Germany) equipped with 5 mm CPTXI cryoprobe. The chemical shifts were referenced to the HOD signal (4.826 ppm at 20°C [31]). NMR signal assignment of riboflavin coincided with the data fetched from the SDBS (Spectral Database for Organic Compounds), compound number 2254 (https://sdbs.db.aist.go.jp).

HRMS analysis. High-resolution mass-spectra of purified compounds were obtained on QExactive Plus Orbitrap (Thermo Fisher Scientific, USA) with ESI source, connected to Ultimate 3000 RSLCnano HPLC system (Thermo Fisher Scientific, USA). Samples were separated on Agilent Zorbax C8 2 × 150 mm column (Agilent, USA) in linear gradient from 15 to 99% of solvent B in 8 min at 200 μL/min: solvent A—0.1% formic acid, 10 mM ammonium formate in water, solvent B—0.1% formic acid, 10 mM ammonium formate in 90% acetonitrile, 10% H2O. MS data were collected in DDA mode with the spectra recorded in Positive and Negative modes with 250–2000 mass range at 35K resolution for MS1. MS2 spectra were recorded at 17.5K resolution with stepped (N)CE 15, 30, 45 and 1.4 m/z isolation window. HRMS for purified compound from biomass: found m/z 377.1470 ([M + H]+) and 375.1285 ([M – H]–), calculated for C17H21N4O6 ions ([M + H]+) m/z = 377.1456 and ([M – H]–) m/z = 375.1310.

CONCLUSIONS

Fungus gnats, such as Keroplatus spp., are difficult to study due to their rarity, generally small size and cryptic mode of life, and very few facts of their bioluminescence biochemistry are known to date. We analyzed aqueous extracts from K. testaceus larvae and found that one of the purified fluorescent substances was riboflavin (vitamin B2). We suggest that riboflavin may play a role in the bioluminescence of this species of Keroplatidae, however, the exact role of the compound in larval bioluminescence is still to be established.