Abstract
Some species of springtail (Collembola) are luminous, but it is not known whether light emitted by springtail is due to self-luminescence, feeding on luminous fungi, or accidental infection by luminous bacteria. To address this question, we characterized the luminescence of a luminous springtail, Lobella sp. (family Neanuridae) discovered in Tokyo, Japan. The emitted light was yellowish-green (540 nm) and was found to originate from tubercles on the thorax (segments II and III) and abdomen (segments I–VI) using a low-light imaging system. The luminescence persisted for several seconds but showed occasional oscillations in a laboratory environment. We also observed fat bodies containing eosin-positive granules under the integument of the tubercles in the tergum by hematoxylin and eosin (HE) staining that were not present in a nonluminous springtail (Vitronura sp.). The fat bodies in Lobella sp. are presumably photocytes analogous to the firefly lantern, and the eosin-positive granules are the likely source of bioluminescence, which implies that springtails are self-luminescent.
Keywords
- luminous springtail
- Lobella
- light organ
- fat body
- histology
1. Introduction
Some species of springtail (Collembola) are luminous.
One of authors (Sano) discovered a luminous springtail,
2. Materials and methods
2.1 Springtails
2.2 Luminescence imaging
The luminescence of the specimen was captured on Fujicolor 1600 film (Fujifilm, Tokyo, Japan) with a Nikon FM camera (Nikon, Tokyo, Japan) with Nicol 50-mm (F1.4) and 2× teleconverter lenses. Luminescence images were also acquired with a luminescence microscope using a short focal length lens system [7, 8] equipped with an ImagEM electron-multiplying charge-coupled device (CCD) camera (C9100-13; Hamamatsu Photonics, Hamamatsu, Japan). The total magnification was reduced from 4× (UPLSAPO4× objective lens; Olympus, Tokyo, Japan) to 0.8× using the short focal length imaging lens (f = 36 mm, NA = 0.2) in order to capture low intensity light.
Video recording of the luminous specimen was performed using a C2400 high-resolution SIT video camera system (Hamamatsu Photonics) equipped with a Zuiko auto-zoom 35–105 mm (F3.5–4.5) lens (Olympus). The video was converted to an avi format file, and time-lapse image analysis was performed using TiLIA software [9] in order to determine the time course of luminescence intensity in a region of interest.
2.3 Spectroscopy
The luminescence spectrum of the specimen was determined using a U-2900 spectrometer (Hitachi High-Technologies, Tokyo, Japan) under the following conditions: transmittance mode without incident light; scan range, 450–650 nm in 5-nm steps; scan speed, 240 nm/min; and response time, 2 s.
2.4 Histology
The whole body of each specimen was fixed in alcohol Bouin’s solution and stored at room temperature. The sample was dehydrated in a graded series of ethanol, embedded in paraffin, and sectioned at a thickness of 5 μm with a microtome (Microm HM310; Leica Biosystems, Nussloch, Germany). The sections were stained with Delafield’s hematoxylin and eosin (HE). A BX53 microscope (Olympus) with a DP74 color CCD camera (Olympus) was used for light microscopy observation.
3. Results
3.1 Habitat description
3.2 Luminescence
In a field observation at the Yokosawa site, we observed flashes of light on the ground when lifting the shiitake logs. However, we were unable to discern the color of the light by the naked eye since it was too weak. Figure 4 shows
To identify the luminescent region of the body, luminescence image of the specimen was captured by microscopy (Figure 6). The luminous spots corresponded to tubercles on the thorax (segments II and II) and abdomen (segments I through VI). Time-lapse image analysis showed that the luminescence persisted for several seconds (less than half a minute) in the laboratory environment with occasional oscillations with a 3.0-s flash interval, 2.1-s pulse duration, 0.9-s inter-pulse duration, and 3.0-s oscillatory peak interval (determined from the average of four peaks in Figure 7).
3.3 Histology
HE staining of a cross section of the second abdominal segment of
4. Discussion
The light emitted by
There have been few studies on luminous springtails, and most of these have been review articles [2, 3, 4, 5]. Some reasons for the lack of research are the difficulty of species identification by entomologists who do not study Collembola and the small size of specimens, which makes biochemical analyses challenging. The present findings provide a basis for future studies on the mechanisms as well as the evolution of bioluminescence in insects using a variety of experimental approaches—e.g., molecular modeling, genetic engineering, and omics technology with artificial intelligence processing—that are applicable to small-sized organisms and wild specimens [16, 17, 18, 19]. Additionally, springtail displays a unique mating behavior where the male deposits spermatophores on the ground or close to a female and keeps other males away through jostling [20, 21]. It is possible that the bioluminescence of springtail plays an important role in mating, as is the case for firefly and other luminous organisms [22].
Acknowledgments
We thank the late Prof. Ryosaku Ito (Showa University, Japan) for identifying the luminous springtail used in this study and the late Dr. Masayasu Konishi (Koganei City, Tokyo, Japan) for enabling our communications with Professor Ito and for valuable discussion.
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