Research Article |
Corresponding author: Vladimír Šustr ( sustr@upb.cas.cz ) Academic editor: Jasna Štrus
© 2015 Andrei Giurginca, Vladimír Šustr, Karel Tajovsky, Maria Giurginca, Iulia Matei.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Giurginca A, Šustr V, Tajovský K, Giurginca M, Matei I (2015) Spectroscopic parameters of the cuticle and ethanol extracts of the fluorescent cave isopod Mesoniscus graniger (Isopoda, Oniscidea). In: Taiti S, Hornung E, Štrus J, Bouchon D (Eds) Trends in Terrestrial Isopod Biology. ZooKeys 515: 111–125. https://doi.org/10.3897/zookeys.515.9395
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The body surface of the terrestrial isopod Mesoniscus graniger (Frivaldsky, 1863) showed blue autofluorescence under UV light (330–385 nm), using epifluorescence microscopy and also in living individuals under a UV lamp with excitation light of 365 nm. Some morphological cuticular structures expressed a more intense autofluorescence than other body parts. For this reason, only the cuticle was analyzed. The parameters of autofluorescence were investigated using spectroscopic methods (molecular spectroscopy in infrared, ultraviolet-visible, fluorescence, and X-ray fluorescence spectroscopy) in samples of two subspecies of M. graniger preserved in ethanol. Samples excited by UV light (from 350 to 380 nm) emitted blue light of wavelengths 419, 420, 441, 470 and 505 nm (solid phase) and 420, 435 and 463 (ethanol extract). The results showed that the autofluorescence observed from living individuals may be due to some β-carboline or coumarin derivatives, some crosslinking structures, dityrosine, or due to other compounds showing similar excitation-emission characteristics.
Mesoniscus graniger , autofluorescence, molecular spectroscopy, β-carboline and coumarine derivatives
Among arthropods, the fluorescence of body surface was firstly reported in scorpions. The intensity of the fluorescence increased with the hardening of the cuticle (
Scorpions emit visible light (400–700 nm) under UV radiation (
It is assumed that more than one fluorescent compound may be present in scorpions. 7-hydroxy-4-methylcoumarin was detected as another fluorescent compound in an extract of scorpion cuticle by
The autofluorescence of the cuticle of the cave isopod Mesoniscus graniger (Frivaldsky, 1863) was found during analysis of the content of its digestive tract under fluorescent microscope (
Living as well as individuals of M. graniger preserved in ethanol, were used in our study. Living animals were sampled for epifluorescent microscopy in the Slovak Karst National Park (Domica and Ardovská caves). The individuals stored in ethanol used for spectroscopic analyses were collected in the Romanian Karst: the Cernişoara Valley, 20 individuals corresponding to the subspecies M. graniger graniger (Frivaldsky, 1863) (labelled in the following analyses as G) and from the Sighiştelului Valley, 16 individuals corresponding to the subspecies M. graniger dragani Giurginca, 2003 (labelled in the following analyses D). In order to assess if the autofluorescence is present in the entire range of M. graniger, we used individuals from the Petnička Pećina (Valjevo, Serbia) and for assessing the presence of this feature in both species of the genus Mesoniscus, we tested the individuals of Mesoniscus alpicola (Heller, 1858) from the Falkensteinhöhle (Niederösterreich, Austria).
Photographs of living fluorescent individuals of M. graniger were recorded with the Olympus XZ61 stereomicroscope equipped with the Olympus DP20 camera and the Hoya UV (0) photographic filter using the Helling UV-Inspector 385 lamp (365 nm) as a source of excitation light. Animals were placed in a refrigerator for a minute to reduce their movement before taking pictures. Images obtained in different focal planes were stacked by the Helicon Focus 5.3 software (Helicon Soft, Ltd.) to obtain a large depth of focus for the resulting photos. Details of fluorescent body surface of M. graniger were documented on the Olympus BX 60 fluorescent microscope equipped with the Olympus DP50 camera. The Olympus U-MWU mirror unit (330-385 nm exciter filter and BA420 barrier filter) was used.
Under field conditions, the autofluorescence of living animals was documented with the Canon EOS 600D camera under the excitation light of the Helling UV-Inspector 385 lamp in the Ardovská Cave (Slovakia).
The autofluorescence of M. graniger from Serbia and that of M. alpicola was confirmed under the Bactericide Lamp LBA 55W (253.7 nm) and the First Light Illuminator-System Biodoc (302 nm). No spectral analyses were performed on the samples of M. graniger from Serbia and on the samples of M. alpicola.
Sample preparation:
The samples preserved in 75% ethanol were filtered in order to separate the solid from the liquid phase (ethanol extract). The solid phase was air dried and stored in Petri-type laboratory vessels; the liquid phase was kept in Erlenmayer-type laboratory vessels.
Apparatus and investigation methods:
For the analyses of samples (G solid phase, G ethanol extract, D solid phase, and D ethanol extract) we used molecular spectroscopy techniques in the infrared (IR) (middle – MID and near – NIR), ultraviolet-visible (UV-VIS) and fluorescence (FP) range. In addition, a part of each sample was analyzed by X-ray fluorescence spectroscopy (XRF).
For the IR analysis, we used the Bruker Optics Tensor 27 spectrometer, with Opus 4.2 specialized software, in the 500–4000 cm-1 range. The analysis used the spectral KBr technique with a device for micro-pellets. The IR analysis was used for the solid samples and the ethanol extracts.
For the UV-VIS and NIR analysis, we used the UV-VIS-NIR-620 apparatus (Jasco, Japan) with 10 ml quartz cells for the liquid phase and with the ILN-725 diffuse reflection accessory for the solid phase, in the 200–2500 nm range. The apparatus has a monochromator and photoelectric cells corresponding to the investigated domains (UV = 200–400 nm, VIS = 400–800 nm and NIR = 800–2000 nm). Although the NIR region is a part of the IR spectroscopy, for constructive reasons it was included in this apparatus, the energy source being more powerful than that used for the IR range. The UV-VIS and NIR analysis was used for the solid samples and the ethanol extracts.
For the molecular fluorescence analysis, we used the FP6500 and FP6300 spectrofluorimeters (Jasco, Japan) using 10 ml quartz cells for the liquid phase and special tanks with quartz window for the solid phase, in the 200–800 nm range. Specific wavelengths were used for excitation in the UV-VIS range, with sources specific to each spectral region (UV and VIS) and the emission spectra were registered. The FP analysis was used for the solid samples and the ethanol extracts.
For the XRF (X-ray fluorescence) analysis, a part of each sample was grounded in an agate mortar and, subsequently, loaded into small plastic cylinders and XRF-analyzed on a Horiba XGT-7000 X-ray Analytical Microscope. The XRF analysis was used only for the solid samples.
Mesoniscus graniger body surface shows a blue auto-fluorescence when excited with UV light at a wavelength of 365 nm (Fig.
Autofluorescence was present in all tested specimens of M. graniger collected from different localities inhabited by this species from the Slovak to the Serbian karst regions. Both subspecies of M. graniger from Romania (M. graniger graniger and M. graniger dragani) show the same intensity of autofluorescence, which is also found in M. alpicola, the second species of the genus.
Following observations made under laboratory conditions, we tried to document the autofluorescence under field conditions (See Suppl. material
The FT-IR (Fourier Transform Infrared) analysis of the solid phase of both subspecies (Fig.
The FT-IR spectra of the sample of the subspecies M. graniger graniger (Fig.
The FT-IR analysis of the ethanol extract of the subspecies M. graniger graniger showed bands belonging to aromatic fragments and some oxidation compounds (carbonyl group νC = O at 1725 cm-1), pointing to a break in the amidic chain proved by the absence of the 1240 cm-1 band (amide III). The absence of the 1240 cm-1 band might be due to the insolubility of some compounds (Fig.
The presence of Ca, already inferred by the IR analysis, was confirmed by the XRF analysis, the Ca content (weight %) being 43.83% in sample G and 16.25% in sample D (expressed as Ca2+).
The analysis in the UV-VIS-NIR domains of the samples solid phase undertaken on the material of the subspecies M. graniger dragani only (D samples) showed several characteristic bands (Fig.
The UV-VIS-NIR analysis of the ethanol extracts (Fig.
The molecular fluorescence analysis (FP) of the samples of solid phase of M. graniger dragani obtained by excitation at 265 nm (Fig.
The FP analysis with excitation at 380 nm (Fig.
The molecular fluorescence analysis of the ethanol extracts were obtained by excitation at 280, 350 and 380 nm, the colour of the emission being light blue - blue (see Table
D | G | |
---|---|---|
λ excitation (nm) | λ emission (nm) | λ emission (nm) |
280 | 317 | 313 |
350 | 420 | 435 |
380 | 435 | 463 |
The bands from 313 and 317 nm are attributed to the presence of tyrosine and some aromatic structures with hydroxyl groups (λex = 280 nm) (
The differences between the emission bands (excitation at 380 nm) of the solid samples and ethanol extracts might be due to the formation of hydrogen bonds with the involvement of the OH groups of the ethanol, emphasizing the influence of the reaction environment, but also its interactions with the chitin and the traces of conjugated lipids. Also, the different solubility in alcohol of the various compounds leads to differences between the emission bands. The molecular fluorescence (FP) tests confirm the data provided by the autofluorescence microscopy, allowing the identification of the β-carboline (beside other aromatic compounds) as the main source of the fluorescence.
The investigations by fluorescence microscopy and by spectroscopic molecular analysis showed the presence of fluorescence in the 330–385 nm excitation domains due to aromatic structures, most probably belonging to the β-carboline type, and changes in the polyamide structure at ageing, changes recorded in M. graniger graniger and M. graniger dragani.
The autofluorescence is characteristic for all observed individuals without respect to their geographic origins. It was confirmed in all tested specimens from the entire area inhabited by M. graniger, both subspecies showing the same intensity, and it was found also in M. alpicola from elsewhere. Furthermore it was recorded in animals observed in caves as well as in individuals kept in laboratory. The individuals stored for long periods in ethanol in collections retain this property. Accidental contamination of M. graniger by any fluorescent compounds from the food or by fluorescent microorganisms restricted to certain caves, is challenged by the universal presence of the autofluorescence in all tested populations collected from different caves in various geographic areas.
The very intensively fluorescent structures on the body surface of M. graniger seem to roughly correspond to some of the structures we observed previously on the body surface of this species using scanning electron microscopy (
Both the solid and ethanol extract samples contain proteins (polypeptides), chitin (N-acetyl glucosamine) and calcite also identified spectrally in FT-IR and by XRF. It corresponds with general information about the body composition of terrestrial isopods (
Our observations underline mainly changes of the polypeptides structure by chain alteration (the disappearance of amide III in the case of G sample), crosslinking and the forming of dityrosine and other polycondensated compounds, among which β-carboline due to oxidative processess. We have to stress that β-carboline is present in the body of living animals as a result of their natural ageing and it is not only the result of ageing of material stored in alcohol.
The microscopically observed blue fluorescence of M. graniger as a response to excitation UV light (about 350 nm) corresponds to the wavelengths range of the blue colour (approximately 450–495 nm after
The functional advantage of invertebrate fluorescence is not yet known regardless of many hypotheses discussed in literature (see
The authors are greatly indebted to Dr. Andreea Ţuţulan-Cuniţă, “Victor Babes” National Institute of Pathology (Bucharest) and to Dr. Daniela Dimofte, Faculty of Geology and Geophysics, University of Bucharest for their kind assistance. Authors thank Dr. V. Stagl and Dr. J. Gruber (Naturhistorisches Museum, Vienna, Austria) for the material of M. alpicola and Dr. B. M. Mitič and Dr. S. E. Makarov (University of Belgrade, Serbia) for M. graniger samples from Serbia. This paper was supported by Programme 1, Project 1 of the “Emile Racovitza” Institute of Speleology of the Romanian Academy. Colleagues from P. J. Šafárik University in Košice (Slovakia), Ľubomír Kováč and Peter Ľuptáčik assisted in recording the field movie.
Autofluorescence of living Mesoniscus graniger
Data type: MPEG video file
Explanation note: Living M. graniger individuals recorded by Canon EOS camera on the cave sediment inside Ardovská Cave (Slovak Karst, Slovakia) under white LED lamp and UV lamp consecutively.