Naturwissenschaften (2013) 100:1099–1101 DOI 10.1007/s00114-013-1100-z
COMMENTS & REPLIES
Luminescent system of Lucihormetica luckae supported by fluorescence lifetime imaging Peter Vršanský & Dušan Chorvát
Received: 1 July 2013 / Revised: 10 September 2013 / Accepted: 14 September 2013 / Published online: 5 November 2013 # Springer-Verlag Berlin Heidelberg 2013
The presence of bioluminescent system in cockroaches (Zompro and Fritzche 1999) and in Lucihormetica luckae in particular has been contested in reply by Merritt (2013), based on the absence of direct evidence of bioluminescence in the original article (Vršanský et al. 2012). Main counterarguments were based on (1) a single anecdotal reference to the presence of luminescence among cockroaches, (2) lack of the manifestation of the difference in fluorescence spectra recorded from lanterns and the rest of the body and (3) lack of luminescence among larvae in cultures. The first and last objections in the comment of Merritt (2013) can be true as a single observation reports alight Lucihormetica verrucosa larvae, and, in culture, the adult apparently loses the capability of bioluminescence after some time period (M. Bartos, personal communication 2012). Our effort since the publication of our original article, when we have collected about a hundred of anecdotal records to observations of luminescence of diverse species of Lucihormetica spread throughout the northern half of South America, including two records from Ecuador and one record likely corresponding to a closely related species of L. luckae from Brazil (Curuça River, B. Pawlikowska, personal communication 2013), is also
Communicated by: Sven Thatje This is the authors' reply to the comment of Merritt (Standards of Evidence for Bioluminescence in Cockroaches, Naturwissenschaften 100(7): 697–698, 2013). P. Vršanský (*) Geological Institute, Slovak Academy of Sciences, Dubravská cesta 9, P.O. Box 106, 840 05 Bratislava, Slovakia e-mail: [email protected] D. Chorvát International Laser Centre, Ilkovicova 3, 841 04 Bratislava, Slovakia e-mail: [email protected]
irrelevant because such massive evidence should result in a measured data of bioluminescence. Nevertheless, it is important to note that many of these observations were reported by trustworthy researchers and could not be ignored. Also, in the course of the present research, we discovered Lucihormetica with four luminescent lanterns—an addition to 107 species (40 indigenous) recently reported from continental Ecuador (Vidlička 2013a, b; unpublished) and also a brand new group of luminescent cockroaches. Focus on plausibility of bioluminescence in L. luckae holotype thus lies on autofluorescence (AF) measurements. It is necessary to highlight that we agree with the author that, principally, there is no way to prove the bioluminescence in the species that might be extinct, except for finding a living alight individual. In the course of the original research, we performed measurements which were not included in the figures, but were mentioned in the text and form a base for our interpretations. For the purpose of clarity, we follow the suggestion of Merritt (2013) and provide herein (Fig. 1a–c) a figure explicitly showing a difference in spectra and time-resolved fluorescence characteristics of the holotype. We compare the signals gathered from the lanterns (Fig. 1b, spot, area 1) vs. the rest of the pronotum, which hypothetically might contain a luciferin but not the enzyme (thorax, areas 2 and 3), vs. elytra (area 4). In fluorescence contrast, all the luminescent areas were visually unrecognizable from the lantern system. We agree with Merrit (2013) that an animal body may contain several substances with overlapping emission spectra and of variable concentration. In such complex system, simple fluorescence spectroscopy is not able to properly distinguish differences without biochemical extraction and appropriate numerical methods. As any invasive technique was not possible to apply on the holotype, this was the main reason why we used the most advanced fluorescence recording technique available, based on simultaneous measurement of both spectral and
1100
Naturwissenschaften (2013) 100:1099–1101
Fig. 1 a Fluorescence spectra detected at different areas of the L. luckae holotype. b Fluorescence lifetime image of the holotype. Rainbow falsecolour representation was applied within the range of 0–1.5 ns to show the mean lifetime resulted from the two-exponential decay analysis. c Distributions of the mean lifetimes revealed in areas 1–4. Frequency scale of the graph represents absolute number of pixels with given mean
lifetime within the selected area. d Confocal laser scanning image showing two orthogonal slices rendered from 3D image stack of the lantern. Two-photon excited fluorescence (yellow) was combined with image showing second harmonic generation (SHG, cyan) after excitation by 1038 nm femtosecond laser
fluorescence decay time coordinate, together with comparison to the reference samples of known bioluminescent species (Lampyris, Pyrophorus), with the aim to find possible correlations and/or differences in recorded spectrochronograms. It can be seen that the fluorescence spectra (Fig. 1a) and especially the fluorescence lifetime patterns (Fig. 1c) show significant differences in all investigated areas. It is important to mention that the data presented in the panels a and c of Fig. 1 are complementing each other, as they in fact describe 2dimensional plots. In addition to the original article, we can now benefit from a newly available technique of fluorescence lifetime imaging (FLIM), providing the spatial mapping of mean fluorescence decay rates (Fig. 1b), which are usually more sensitive indicators of the fluorophore environment than the fluorescence spectra. Indeed, the autofluorescence from tissues is a very complex phenomenon which should be studied in insects in more details. Fortunately, recent advances in detection technology and signal analysis allow to identify the main sources of AF in tissues and use the derived information for studies at even clinical level (Chorvat and Chorvatora 2009 and references therein). Such rigorous analysis of the AF in insects is still lacking, but even at qualitative level, it can be observed that the time-resolved fluorescence fingerprints of the lanterns of Lucihormetica differ significantly from the rest of
the body, while they are similar to lanterns in fireflies and click beetles, as we reported in the original paper (Fig. 1c). Additional support comes from the sophisticated morphology of the lanterns (Fig. 1d; Vršanský et al. 2012, Fig. 1g, h, k, l), covered with thin, transparent, film-like reflector with ridges for light dispersion. Based on the evidence gathered so far, we think that there exist enough arguments to conclude that alight cockroaches do exist and might represent a remarkable example of frequency-dependent evolution. Extreme rarity (eleven of 15 species are known based on a single individual) is much more significantly influencing their evolutionary success, than their fitness, to prevent the predators to check whether under the luminescent dots are not present edible cockroaches, instead of toxic click beetles.
Acknowledgments We thank Vladimír Jánsky (Natural History Museum, Bratislava) for technical support. This work is supported by the United Nation Educational Scientific and Cultural Organisation project Amba (supporting grant of International Scientific and Technical Co-operation of the Slovak Academy of Sciences). This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0436-12 and by Slovak Agency for the Structural Funds of EU under the contract ITMS:26240120018 (project NanoNet2).
Naturwissenschaften (2013) 100:1099–1101
References Jr Chorvat D, Chorvatova A (2009) Multi-wavelength fluorescence lifetime spectroscopy: a new approach to the study of endogenous fluorescence in living cells and tissues. Laser Phys Lett 6:175–193 Merritt DJ (2013) Standards of evidence for bioluminescence in cockroaches. Naturwissenschaften 100:697–698 Vidlíčka Ľ (2013a) Cockroaches (Blattaria) of Ecuador—checklist and history of research. Zootaxa 3599:401–445
1101 Vidlíčka Ľ (2013b) New species of Macrophyllodromia (Blattaria, Blattellidae) from Ecuador and a key to males of the genus. Zootaxa 3635:185–193 Vršanský P, Chorvát D, Fritzsche I, Hain M, Ševčík M (2012) Lightmimicking cockroaches indicate tertiary origin of recent terrestrial luminescence. Naturwissenschaften 99:739–749 Zompro O, Fritzsche I (1999) Lucihormetica n. gen. n. sp., the first record of luminescence in an orthopteroid insect (Dictyoptera: Blaberidae: Blaberinae: Brachycolini). Amazoniana 15:211– 219
COMMENTS & REPLIES
Luminescent system of Lucihormetica luckae supported by fluorescence lifetime imaging Peter Vršanský & Dušan Chorvát
Received: 1 July 2013 / Revised: 10 September 2013 / Accepted: 14 September 2013 / Published online: 5 November 2013 # Springer-Verlag Berlin Heidelberg 2013
The presence of bioluminescent system in cockroaches (Zompro and Fritzche 1999) and in Lucihormetica luckae in particular has been contested in reply by Merritt (2013), based on the absence of direct evidence of bioluminescence in the original article (Vršanský et al. 2012). Main counterarguments were based on (1) a single anecdotal reference to the presence of luminescence among cockroaches, (2) lack of the manifestation of the difference in fluorescence spectra recorded from lanterns and the rest of the body and (3) lack of luminescence among larvae in cultures. The first and last objections in the comment of Merritt (2013) can be true as a single observation reports alight Lucihormetica verrucosa larvae, and, in culture, the adult apparently loses the capability of bioluminescence after some time period (M. Bartos, personal communication 2012). Our effort since the publication of our original article, when we have collected about a hundred of anecdotal records to observations of luminescence of diverse species of Lucihormetica spread throughout the northern half of South America, including two records from Ecuador and one record likely corresponding to a closely related species of L. luckae from Brazil (Curuça River, B. Pawlikowska, personal communication 2013), is also
Communicated by: Sven Thatje This is the authors' reply to the comment of Merritt (Standards of Evidence for Bioluminescence in Cockroaches, Naturwissenschaften 100(7): 697–698, 2013). P. Vršanský (*) Geological Institute, Slovak Academy of Sciences, Dubravská cesta 9, P.O. Box 106, 840 05 Bratislava, Slovakia e-mail: [email protected] D. Chorvát International Laser Centre, Ilkovicova 3, 841 04 Bratislava, Slovakia e-mail: [email protected]
irrelevant because such massive evidence should result in a measured data of bioluminescence. Nevertheless, it is important to note that many of these observations were reported by trustworthy researchers and could not be ignored. Also, in the course of the present research, we discovered Lucihormetica with four luminescent lanterns—an addition to 107 species (40 indigenous) recently reported from continental Ecuador (Vidlička 2013a, b; unpublished) and also a brand new group of luminescent cockroaches. Focus on plausibility of bioluminescence in L. luckae holotype thus lies on autofluorescence (AF) measurements. It is necessary to highlight that we agree with the author that, principally, there is no way to prove the bioluminescence in the species that might be extinct, except for finding a living alight individual. In the course of the original research, we performed measurements which were not included in the figures, but were mentioned in the text and form a base for our interpretations. For the purpose of clarity, we follow the suggestion of Merritt (2013) and provide herein (Fig. 1a–c) a figure explicitly showing a difference in spectra and time-resolved fluorescence characteristics of the holotype. We compare the signals gathered from the lanterns (Fig. 1b, spot, area 1) vs. the rest of the pronotum, which hypothetically might contain a luciferin but not the enzyme (thorax, areas 2 and 3), vs. elytra (area 4). In fluorescence contrast, all the luminescent areas were visually unrecognizable from the lantern system. We agree with Merrit (2013) that an animal body may contain several substances with overlapping emission spectra and of variable concentration. In such complex system, simple fluorescence spectroscopy is not able to properly distinguish differences without biochemical extraction and appropriate numerical methods. As any invasive technique was not possible to apply on the holotype, this was the main reason why we used the most advanced fluorescence recording technique available, based on simultaneous measurement of both spectral and
1100
Naturwissenschaften (2013) 100:1099–1101
Fig. 1 a Fluorescence spectra detected at different areas of the L. luckae holotype. b Fluorescence lifetime image of the holotype. Rainbow falsecolour representation was applied within the range of 0–1.5 ns to show the mean lifetime resulted from the two-exponential decay analysis. c Distributions of the mean lifetimes revealed in areas 1–4. Frequency scale of the graph represents absolute number of pixels with given mean
lifetime within the selected area. d Confocal laser scanning image showing two orthogonal slices rendered from 3D image stack of the lantern. Two-photon excited fluorescence (yellow) was combined with image showing second harmonic generation (SHG, cyan) after excitation by 1038 nm femtosecond laser
fluorescence decay time coordinate, together with comparison to the reference samples of known bioluminescent species (Lampyris, Pyrophorus), with the aim to find possible correlations and/or differences in recorded spectrochronograms. It can be seen that the fluorescence spectra (Fig. 1a) and especially the fluorescence lifetime patterns (Fig. 1c) show significant differences in all investigated areas. It is important to mention that the data presented in the panels a and c of Fig. 1 are complementing each other, as they in fact describe 2dimensional plots. In addition to the original article, we can now benefit from a newly available technique of fluorescence lifetime imaging (FLIM), providing the spatial mapping of mean fluorescence decay rates (Fig. 1b), which are usually more sensitive indicators of the fluorophore environment than the fluorescence spectra. Indeed, the autofluorescence from tissues is a very complex phenomenon which should be studied in insects in more details. Fortunately, recent advances in detection technology and signal analysis allow to identify the main sources of AF in tissues and use the derived information for studies at even clinical level (Chorvat and Chorvatora 2009 and references therein). Such rigorous analysis of the AF in insects is still lacking, but even at qualitative level, it can be observed that the time-resolved fluorescence fingerprints of the lanterns of Lucihormetica differ significantly from the rest of
the body, while they are similar to lanterns in fireflies and click beetles, as we reported in the original paper (Fig. 1c). Additional support comes from the sophisticated morphology of the lanterns (Fig. 1d; Vršanský et al. 2012, Fig. 1g, h, k, l), covered with thin, transparent, film-like reflector with ridges for light dispersion. Based on the evidence gathered so far, we think that there exist enough arguments to conclude that alight cockroaches do exist and might represent a remarkable example of frequency-dependent evolution. Extreme rarity (eleven of 15 species are known based on a single individual) is much more significantly influencing their evolutionary success, than their fitness, to prevent the predators to check whether under the luminescent dots are not present edible cockroaches, instead of toxic click beetles.
Acknowledgments We thank Vladimír Jánsky (Natural History Museum, Bratislava) for technical support. This work is supported by the United Nation Educational Scientific and Cultural Organisation project Amba (supporting grant of International Scientific and Technical Co-operation of the Slovak Academy of Sciences). This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0436-12 and by Slovak Agency for the Structural Funds of EU under the contract ITMS:26240120018 (project NanoNet2).
Naturwissenschaften (2013) 100:1099–1101
References Jr Chorvat D, Chorvatova A (2009) Multi-wavelength fluorescence lifetime spectroscopy: a new approach to the study of endogenous fluorescence in living cells and tissues. Laser Phys Lett 6:175–193 Merritt DJ (2013) Standards of evidence for bioluminescence in cockroaches. Naturwissenschaften 100:697–698 Vidlíčka Ľ (2013a) Cockroaches (Blattaria) of Ecuador—checklist and history of research. Zootaxa 3599:401–445
1101 Vidlíčka Ľ (2013b) New species of Macrophyllodromia (Blattaria, Blattellidae) from Ecuador and a key to males of the genus. Zootaxa 3635:185–193 Vršanský P, Chorvát D, Fritzsche I, Hain M, Ševčík M (2012) Lightmimicking cockroaches indicate tertiary origin of recent terrestrial luminescence. Naturwissenschaften 99:739–749 Zompro O, Fritzsche I (1999) Lucihormetica n. gen. n. sp., the first record of luminescence in an orthopteroid insect (Dictyoptera: Blaberidae: Blaberinae: Brachycolini). Amazoniana 15:211– 219
