Reducing scorpion fluorescence via prolonged exposure to ultraviolet light.
Abstract: A simple technique is presented for reducing the fluorescence of living scorpions by prolonged exposure to UV light. Scorpion's fluorescence peak can be eliminated by a 1-mo exposure to low intensity UV light. Although the fluorescence peak returns within 1 wk after removal from UV light exposure, the magnitude remains reduced. This technique potentially opens up new options for testing a variety of hypotheses about possible functions of scorpion fluorescence including potential effects on cuticle strength, visual responses, predation, cannibalism, and mating.

Keywords: Spectra, spectroscopy
Article Type: Report
Subject: Fluorescence (Physiological aspects)
Scorpions (Physiological aspects)
Ultraviolet radiation (Usage)
Author: Kloock, Carl T.
Pub Date: 09/01/2009
Publication: Name: Journal of Arachnology Publisher: American Arachnological Society Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2009 American Arachnological Society ISSN: 0161-8202
Issue: Date: Sept, 2009 Source Volume: 37 Source Issue: 3
Geographic: Geographic Scope: United Kingdom Geographic Code: 4EUUK United Kingdom
Accession Number: 253537146
Full Text: There is no known function of scorpion fluorescence (Brownell 2001; Kloock 2008). Although it is certainly possible that fluorescence has no function, it is only by testing and falsifying potential functions that they can be eliminated from consideration. In order to test potential functions of scorpion fluorescence, having scorpions with reduced fluorescence could be a powerful tool. Several methods for reducing fluorescence exist, but those currently available are problematic. Kloock (2005) removed fluorescence from preserved scorpions by applying a varnish that blocked ultra-violet light (UV), and fluorescence can also be eliminated or reduced either by not supplying UV light or blocking it with filters. Use of coatings limits experiments because live, non-fluorescing scorpions cannot be used. In addition, coating scorpions introduces experimental complications, given the different chemicals needed to either block or allow fluorescence and secondary coatings to remove these effects dim the fluorescence of controls. Detecting fluorescence under natural conditions, if possible, requires very sensitive visual senses that scorpions may possess (Kloock 2008), but any dimming could prevent detection, and thus affect experimental attempts to demonstrate detection. Eliminating or blocking UV light with filters prevents fluorescence of living specimens, but also introduces a necessary experimental complication: effects of fluorescence and UV light cannot be separated by techniques that simply eliminate UV light. Given that scorpion eyes (Machan 1968) and their extra-ocular light sense (Zwicky 1970) are sensitive to both UV light and light near the fluorescence peak (~500 nm), this is a serious complication. Modifying scorpions so that they can be exposed to UV light without fluorescing can remove this problem, although possible behavioral and physiological side effects of any such manipulation could introduce new difficulties.

Anecdotal accounts suggest that long-term exposure to ultraviolet radiation may reduce scorpion fluorescence (Wankhede 2004). This has not previously been quantitatively demonstrated. I present here evidence that long-term exposure to UV light significantly reduces scorpion fluorescence.

Paruroctonus becki (Gertsch & Allred 1965) (Vaejovidae) were collected July-September 2008 in Kern County, California (voucher specimens deposited at the California Academy of Sciences), and housed in small, foam-plugged plastic vials. Sixteen females were randomly chosen from this population for the experimental manipulation and placed in small open-topped plastic arenas (13 cm long x 10 cm wide x 7 cm high) with 50 ml of soil collected from the same site as the scorpions. The soil helped to absorb and retain moisture, but was not deep enough to allow scorpions to bury themselves to escape irradiation. Scorpions were exposed 24 hrs/day for 32 days to two 40 W fluorescent blacklights (GE F40BLB) at a constant distance of 75 cm. This resulted in scorpions constantly receiving 11 [micro]W/[cm.sup.2] of UV light energy (measured with a Mannix UV-340 light meter: range = 290-390 nm). Control scorpions were kept without significant UV light exposure. They were exposed to light from white fluorescent lights ([less than or equal to]1 [micro]W/cm2 UV) daily. Once a week, each scorpion was fed a single mealworm larva (Tenebrio sp.) and provided water by spraying the soil surface with a mister. I selected only females to simplify analysis and because females were more readily available. I see no reason to expect major differences between genders, but this should be tested in the future.

Within 2 wk, UV-exposed scorpions exhibited visibly reduced fluorescence, particularly on the dorsal surfaces. The thinner, more flexible portions of the exoskeleton (i.e., carapace and mesosoma) experienced a larger fluorescence reduction than the thicker regions (pedipalps, metasoma). After 32 days, fluorescence on the dorsal surfaces was no longer visible, though ventral surfaces and chelicerae still fluoresced dimly, in a pattern consistent with the effects of shading. At this point, the 12 surviving UV-exposed scorpions and 12 randomly selected control scorpions were measured with a reflectance spectrometer using a UV light source to stimulate fluorescence.

Emission spectra measurements were taken with a BW-Tek BRC111A CCD Spectrometer using BWSpec version 2.24 software with an integration time of 100 ms and 20 averages per sample. A single UV LED with peak emission at 390 nm and transmitted via a fiber-optic cable directly to the reflectance probe supplied excitation energy. A light-absorbing fabric (Edmund scientific #3060068) served as a dark reference, and I used the dark subtraction method for all measurements. The spectra were analyzed in raw form, without smoothing techniques (e. g., Fourier or running averages) applied to remove noise from the spectra.

All spectrum measurements occurred inside a light-tight box enclosing both the scorpion and reflectance probe. A squeeze cage, as described in Kloock (2008), immobilized scorpions during measurement. The reflectance probe was lowered until it touched the carapace at a point just behind the medial eyes. The order of measurements was randomized to reduce potential bias.

Fig. 1 provides a visual demonstration of the results of the manipulation. More importantly, spectrometer measurements showed large effects (Fig. 2, Table 1). The peak seen in each spectrum at ~400 nm (Fig. 2) results from reflection of the UV light source (peak emission at 390 nm) and is not the result of fluorescence. To avoid this artifact, all measurements were made over the range of 450-700 nm.

Thirty-two days of UV exposure produced a significant reduction in peak power (Table 1; t-test assuming unequal variance, t = 8.88, P = 2.39 x [10.sup.-6], df = 12) and a significant increase in the wavelength of peak power (t-test assuming unequal variance, t = 4.48, P = 0.0007, df = 12). The emission spectra for the UV-exposed scorpions show no clear peaks (Fig. 2). An F-test for variance ratios (Zar 1996) shows that the variance in the peak wavelength of the UV-exposed scorpions far exceeded the variance for control scorpions (UV-exposed scorpions s2 = 655, control scorpions [s.sup.2] = 19.0, F = 34.5, P = 6.46*[10.sup.-7]). This and the wide range of values (Table 1) indicate that the wavelength of peak power was essentially random for the UV-exposed scorpions.

[FIGURE 1 OMITTED]

After measurement, both UV exposed and control scorpions were placed in covered containers with soil and a shelter (a fragment of terra cotta pot) and maintained on a 13 hours white light: 11 hours dark cycle, with feeding and watering schedules maintained as above. One week later, spectra were remeasured. Table 1 shows that in one week without UV exposure, significant recovery of fluorescence had occurred, with consistent peaks again evident in the spectra and no difference between groups in wavelength of peak power (t-test assuming unequal variance, t = 1.32, P = 0.204, df = 12). Although the relative intensity of the fluorescence was still significantly reduced compared with controls (t-test assuming unequal variance, t = 4.60, P = 6.06 x [10.sup.-4], df = 12.), fluorescence intensity increased significantly within the UV-exposed group over the one-week recovery period (t-test assuming unequal variance, t = 3.56, P = 1.56 x [10.sup.-4], df = 12). Significant differences in the variance between controls and UV-exposed scorpions still existed for peak intensity (F = 35.2, P = 5.79 x [10.sup.-7]) and for peak wavelength (F = 3.28, P = 0.030). Preserved UV-reduced specimens showed no signs of recovery, indicating that active metabolic processes are responsible for recovery. Determining just what those processes are will require further study.

Photobleaching, the loss of fluorescence due to prolonged exposure to excitation wavelengths, is commonly encountered in fluorescence microscopy, which focuses on methods of preventing it (Deschenes & Vanden Bout 2002). Photobleaching is probably caused by a variety of different mechanisms (Georgakoudi et al. 1997), so more detailed study will be needed to determine a mechanism of the photobleaching observed here. Two molecules responsible for scorpion fluorescence have been identified: [beta]-carboline (Stachel et al. 1999) and 4-methyl-7-hydroxycoumarin (Frost et al. 2001), which may aid future investigations into this phenomenon. Similarly, the mechanism of recovery has not yet been investigated, and possible side effects of the treatment, including the potential of retinal or other tissue damage and effects on behavior of long-term exposure, need to be investigated and controlled in any future experiments using this technique.

[FIGURE 2 OMITTED]

With this important caveat, the ability to reduce fluorescence potentially opens up a broad variety of new experiments. For example, Camp & Gaffin (1999) and Blass & Gaffin (2008) suggest that fluorescence may function as a light amplifier, or aid in detecting UV light. If true, then scorpions with reduced fluorescence should display an altered light avoidance response. Tests can also be designed to test ecological hypotheses of function (summarized in Kloock 2008). For example, experiments could be designed to test whether scorpions with reduced fluorescence experience different levels of predation, cannibalism, prey capture or mating success than fluorescent scorpions under different lighting conditions in both natural and laboratory settings.

It is also possible that fluorescence is a byproduct of a molecule whose primary function is unrelated to fluorescence itself. For example, Stachel et al. (1999) suggested that the fluorescent molecule functions in sclerotization. To test this hypothesis, experiments can be designed to test the effects of fluorescence reduction on cuticle strength. Similarly, the fluorescent molecule may function in reducing water loss (Lourenco & Cloudsley-Thompson 1996), and experiments could be designed to determine the effect of fluorescence reduction on the rate of water loss.

Caution must be exercised in experimental design because we have, as yet, no information on side effects of the technique of fluorescence reduction itself on scorpion behavior or physiology, which could complicate future experiments. The challenge of experimental design using this technique will be to adequately control for potential side effects of fluorescence reduction. One way to accomplish this will be to cross UV presence and fluorescence reduction: if fluorescence reduction has an effect unrelated to fluorescence, similar differences should be observed between fluorescence-reduced and control scorpions regardless of the presence of UV light. Of course, the details of any experiment may require more complicated controls to be developed. Provided adequate controls are used, this technique makes possible future experiments designed to determine whether or not scorpion fluorescence (or the fluorescent molecules) serves specific functions. Even if fluorescence serves no function, such experiments can enhance our understanding of scorpion ecology, physiology, and behavior.

ACKNOWLEDGMENTS

A CSUB University Research Council Grant supplied funding. J. Mohamed provided the photograph and Dr. P. Mickler allowed use of his photographic equipment. I thank the reviewers for their comments, which improved the manuscript.

Manuscript received 15 October 2008, revised 1 April 2009.

LITERATURE CITED

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Brownell, P. 2001. Sensory ecology and orientational behaviors. Pp. 159-183. In Scorpion Biology and Research. (P. Brownell & G.A. Polis, eds.). Oxford University Press, Oxford, UK.

Camp, E.A. & D.D. Gaffin. 1999. Escape behavior mediated by negative phototaxis in the scorpion Paruroctonus utahensis (Scorpiones, Vaejovidae). Journal of Arachnology 27:679-674.

Deschenes, L.A. & D.A. Vanden Bout. 2002. Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis. Chemical Physical Letters 365:387-395.

Frost, L.M., D.R. Butler, D. O'Dell & V. Fet. 2001. A coumarin as a fluorescent compound in scorpion cuticle. Pp. 365-368. In Scorpions 2001: in Memoriam, Gary A. Polis. (V. Fet & P.A. Seldon, eds.). British Arachnological Society, Burnham Beeches, Buckinghamshire, UK.

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Carl T. Kloock: Department of Biology, California State University, Bakersfield, 9001 Stockdale Highway, Bakersfield, California, 93311, USA. E-mail: ckloock@csub.edu
Table 1.--Data from fluorescence spectra of control and UV-
exposed scorpions after 1 mo of exposure, and repeated after 1 wk
of recovery. n = 12 for each treatment. Peak values determined
over the range 450-700 nm.

                                               Mean
                               Mean peak    wavelength      Range:
                               relative      of peak      wavelength
                               intensity    intensity,      of peak
Measurement      Treatment       (SE)        nm (SE)     intensity, nm

After exposure   Control       608 (120)    501 (1.26)      496-511
                 UV-exposed   72.1 (10.2)   535 (7.39)      497-576

After recovery   Control      482 (77.2)    501 (2.16)      492-519
                 UV-exposed   122 (13.0)    508 (3.91)      490-533
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