A NEW TECHNIQUE FOR EXAMINING SURFACE MORPHOSCULPTURE OF SCORPIONS

2005·5.5 MB·English

by  Erich S Volschenk

2005. The Journal of Arachnology 33:820-825
A NEW TECHNIQUE EOR EXAMINING SUREACE
MORPHOSCULPTURE OF SCORPIONS
Erich S. Volschenk': Curtin University of Technology, School of Environmental
Biology, GPO Box U 1987, Perth, Western Australia 6845, Australia; Western
Australian Museum, Francis Street, Perth 6000, Western Australia, Australia; and
Queensland Museum, Box 3300, South Brisbane, Queensland, 4101, Australia.
ABSTRACT. A new technique forexamining the exomorphology ofthe scorpion epicuticleis described
that utilizes the fluorescent property of scorpion cuticle. Fluorescence of the scorpion exoskeleton under
longwave ultraviolet light is a well known property previously only utilized for the capture orobservation
of scorpions at night. Fluorescence is an energy emission that is analogous to the secondary electron
emissions utilized in electron microscopy to provide information about surface detail. This new technique
is fast, inexpensive and non-destructive, and provides an alternative means of documenting of surface
macrosculpture for the description and identification of scorpion species.
Keywords: Fluorescence, scorpion, epicuticle, exomorphology, images
Among the many unique and unusual fea- The intensity of scorpion fluorescence varies
tures that scorpions exhibit, arguably the most among species and the time elapsed since the
curious is their fluorescence on exposure to last molt, and non-fluorescent scorpions are
long wavelength UV (ultraviolet light). Fluo- unknown (Stahnke 1972). Despite a reason-
rescence is an energy (light) emission that re- able understanding of the origins of the fluo-
sults from the excitation ofelectrons in certain rescence in scoipions, there is still no consen-
compounds by light of specific wavelengths. sus as to why this phenomenon exists. The
Once excited by a photon, the electrons of fluorescent property has to date been exploited
these compounds almost immediately return most successfully as a tool in their observa-
to their previous energy state, and simulta- tion, detection or capture (Stahnke 1972).
neously a lower level energy emission (visible Scorpions are predominantly nocturnal and,
light) results. equipped with a blacklight, a researcher can
UV
Pavan (1954a) first demonstrated that find many more specimens, as well as species,
light of wavelength 366.3 nm causes maxi- at night than is possible during the day with
mum
fluorescence of scorpion epicuticle. an equivalent searching effort (Honetschlach-
Scorpion fluorescence has captivated the in- er 1965; Lamoral 1979; Sissom et al. 1990;
terest of researchers (Honetschlacher 1965; Williams 1980).
Lawrence 1954; Williams 1980) since it was Images of biological specimens made from
revealed that they exhibit this curious phe- SEM (scanning electron microscopes) pre-
nomenon. Pavan (1954a, 1954b) found that dominantly utilize the detection of emission
the fluorescence emanates from the outermost of SEs (secondary electrons) to form digital
layer ofthe cuticle, the epicuticle, but was un- images. Secondary electrons result when a fo-
able to discover the fluorescent compound or cused beam of electrons of sufficient accel-
compounds. More recently, Stachel and
eration voltage passes over a suitable (con-
Stockwell (1999) isolated the fluorescentcom- ductive) subject, typically of high molecular
pound, (3-carboline, from scorpion epicuticle. mass. High energy electrons from the primary
beam displace loosely bound outer-orbital
b'rCaotrereZsopoloongdyi,ngAmaedrdriecsasn: MDuespaerutmmeonftNaotfurIanlveHritse-- electrons in the subject, and SEs are emitted
tory, 79th street @ Central Park West, New York, from the surface of the specimen. These SEs
NY. 10024, United States of America. E-mail: have a different voltage (energy) to that ofthe
[email protected] electron beam that scans the surface of the
820
VOLSCHENK—IMAGING SCORPION FLUORESCENCE 821
— —
Figure 1. Carapace of Lychas sp.I. from Aus- Figure 2. Carapace of Lychas sp.l. from Aus-
tralia (WAM; conventional image (scale bar = tralia, fluorescence image (scale bar = 1mm).
1mm).
1911) (S58519). Images were taken with a
specimen and are principally utilized in the Leica DC100 digital camera attached to a Lei-
production of images of surface detail from ca MZ6 stereo dissection microscope, fitted
electron microscopes. The production of the with an iris diaphragm. Standard illumination
SE emission in an electron microscope is anal- was provided from a Leica light source. Ul-
ogous to the light emission called fluores- traviolet illumination was provided from mod-
cence, and prompted the author to considered ified portable blacklight units normally uti-
experimentation with imaging ofcuticular sur- lized in the field detection and collection of
face detail from the fluorescence ofscorpions. scorpions. Each unit consisted of a portable
The surface sculpturing (granulations and ca- 12V fluorescent light fixture, fitted with two
rinae) ofscorpions is frequently utilized in the black light tubes (National, FL8 BL-B), and
identification of species (Lamoral 1979; Sis- powered by a 12V rechargeable lead-acid bat-
som 1990), however, this useful character can tery (Panasonic, LC-R127R2P). When fluo-
be difficult to examine as it is often obscured rescence images were being taken, the two
by complex color patterns lying beneath the blacklights were placed on either side of the
cuticle. Images made using this fluorescence specimen. Specimens were imaged at night to
technique were recently published by Prendini minimize extraneous light. Conventional illu-
(2003a, 2003b, 2003c) and further exemplify mination was used, with the iris diaphragm
its usefulness. fully constricted to provide maximum depth
of field, to position and focus the image, after
METHODS
which the blacklights were switched on and
The specimens used to exemplify this tech- all other sources of illumination (except com-
nique are lodged in the Western Australian puter monitor which was turned away from
Museum: Lychas sp. 1 (ESV2255), Lychas the microscope) were switched off. All images
sp.2 (T56392) and Lychas variatus (Thorell were taken with the slow imaging option of
1876) (WAM 97/1226); and the Queensland the DC100 software, owing to the lower in-
Museum: Hemilychas alexandrinus (Hirst tensity of the fluorescence, longer periods (up
822 THE JOURNAL OF ARACHNOLOGY
—
froFmiguAruestr3a.l—iaM,escoonsvoemnatilontaelrgiitmesagoef(Lsyccalheasbasrp.2=. froFmiguAruestr4a.liaMn,esfolsuoomreaslcetnecregitiemsagoef (Lsyccahleasbasrp.2=.
mm).
1mm). 1
to 10 seconds) were required per image cap- eton. Conventional imaging under ethanol al-
ture. most completely obscures the surface sculp-
&
For imaging, each specimen was placed turing in images, (Figs. 1, 3, 5 7) but
into a small glass petri dish with enough 90- provides accurate documentation ofcolor pat-
100% ethanol to just cover it. The clarity of terning. Surface detail revealed in the fluores-
the image deteriorated considerably as the cent images mimics those obtained from scan-
depth of ethanol above the specimen in- ning electron microscopes except that setae
creased. More dilute concentrations ofethanol and macrosetae are not revealed, and the
were also trialed, however 70-80% mixtures depth of field is relatively shallow.
developed a faint scum over the surface that Figures 1 and 2 depict the carapace of Ly-
decreased the clarity of the images. chas sp. 1, an undescribed species from Aus-
Images of epicuticle fluorescence were in tralia. The specimen had become badly dis-
shades of blue, and these were converted to torted during or following preservation (tissue
greyscale in the graphics editing package Cor- displacement was evident beneath the cuticle).
el® Photo-Paint (version 7). Some images Consequently, under conventional illumina-
were also enhanced for publication by making tion the carapace had a glassy semitransparent
minor improvements to levels of brightness appearance, resulting from light reflecting
and contrast, the same adjustments typically from beneath the carapace. The fluorescent
being conducted on images made using a image reveals only the surface detail and this
SEM. The technique described in this contri- detail could accurately be compared with
bution is exemplified using four different spe- more intact specimens or similarly damaged
cies of Australian buthids. specimens to facilitate the identification of
more intact specimens of this species. Figures
RESULTS
3 and 4 depict the mesosomal tergites of Ly-
Fluorescence images (Figs. 2, 4, 6 & 8) re- chas sp. 2, another undescribed Lychas spe-
veal grey scale surface detail and structure of cies from Australia. Figure 5 depicts the or-
the external sculpturing of scorpion exoskel- nately patterned carapace of Lychas variatus
VOLSCHENK—IMAGING SCORPION FLUORESCENCE 823
—
Figure 5. Carapace ofLychas variatus (Thorell —
1876), conventional image (scale bar = 1mm). Figure 6. Carapace ofL. variatus, fluorescence
image (scale bar = 1mm).
while the fluorescent image (Fig. 6) reveals
the poorly sculptured surface. Figures 7 and 8 of scorpion fluorescence are proposed to aug-
depict the lateral aspects of metasomal seg- ment line drawings for the documentation of
ment V of Hemilychas alexandrine, another surface sculpture in scorpions. This imaging
Australian buthid. The finely reticulate pattern protocol provides a much cheaper substitute
is seen in Fig. 7 using standard illumination, for SEM. Using a digital camera, mounted to
whereas punctated nature of this metasomal a dissection microscope, images can be taken
segment is revealed in the fluorescent image, as quickly or slowly as possible and adjust-
Fig 8. In this case the fluorescent image re- ment of the specimen can be made directly
vealed surface detail not associated with gran- and immediately. A particular advantage of
ulations, but with punctations. this technique over SEM is the ability to man-
age very large specimens. Many scorpions are
DISCUSSION
too large to be examined examined using
The technique described here, for imaging SEM without dissecting the specimen before
scorpions under UV light provides images mounting the area of interest onto a stub. The
with detail similar to those taken with a scan- fluorescent technique described here can be
ning electron microscope. Unlike specimens applied to large scorpions without dissecting
examined in a conventional SEM, those from the specimen.
which the fluorescence images were made Some drawbacks ofthis technique relate to
were not coated in conductive material and the low depth of field experienced at high
were taken under normal atmospheric condi- magnifications, and a slightly grainy appear-
tions. Fluorescence imaging is a non-destruc- ance to the images. The relatively large size
tive technique that can be applied to type of scorpions, compared with most other chel-
specimens. These images reveal information icerates, implies that low depth offield issues
about the surface sculpturing of the cuticle are not likely to be experienced unless im-
that may otherwise be obscured or over-en- aging the smallest ofscorpions, or very small
hanced by subcuticular pigmentation. Images structures such as chelicerae and tarsi. An ad-
824 THE JOURNAL OF ARACHNOLOGY
— —
Figure 7. Lateral aspect of metasoma V of Figure 8. Lateral aspect of metasoma V of H.
Hemilychas alexandrinus (Hirst 1911), convention- alexandrinus: fluorescence image, showing surface
al image showing color patterning and some seta- punctations (scale bar = 1mm).
tion (scale bar = 1mm).
LITERATURE CITED
ditional drawback is the non-fluorescent na- Hirst, S. 1911. Descriptions of new scorpions. An-
ture of setae and macrosetae, making this nals and Magazine ofNatural History (8) 8:462-
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setae and the areoles of trichobothria typi- ing scorpions. Turtox News 43:69-70.
cally fluoresce more brightly than the sur- Lamoral, B.H. 1979. The Scorpions of Namibia
rounding surfaces and this property can assist (Arachnida: Scorpionida). Annals of the Natal
Museum 23:497-784.
in the location of trichobothria. The quality
Lawrence, R.E 1954. Fluorescence in Arthropoda.
of digital images has improved considerably
Journal of the Entomological Society of South-
since this study was conducted, and smoother
ern Africa 17:167-170.
images are already characteristic of high res- Pavan, M. 1954a. Primi dati per la caraterizzazione
olution digital cameras. With the application della sostanza fluorescente nel tegumento degli
of fluorescent stains and histological prepa- scorpion!. Bolletino Societa Italiana Biologia
rations, this technique may be applicable to Sperimentale 30:803-805.
other organisms that do not naturally fluo- Pavan, M. 1954b. Studi sugli scorpion! I, unanuova
resce. caratteristicatipica del tegumentodegli scorpion!.
Italian Journal ofZoology 21:283-291.
ACKNOWLEDGMENTS
Prendini, L. 2003a. Discovery of the male of Par-
abuthiis muelleri, and implications for the phy-
This paper formed part of the author’s doc-
logeny of Parabuthus (Scorpiones: Buthidae).
toral thesis, which was supported by a Curtin American Museum Novitates 3408:1-24.
University Postgraduate Scholarship, and re- Prendini, L. 2003b. A new genus and species of
search grant from ABRS (Australian Biolog- bothriurid scorpion from the Brandberg Massif,
ical Resources Study). I thank Prof. Jonathan Namibia, with a reanalysis ofbothriurid phylog-
Majer (Curtin University of Technology) for eny and adiscussion ofthephylogeneticposition
the use of his digital camera and microscope ofLisposoma Lawrence. Systematic Entomology
set-up. Thanks are also given to Dr. Mark Har- 28:149-172.
vey and Dr. Bill Humphreys (Western Austra- Prendini, L. 2003c. Systematics and biogeogra-
lian Museum), Dr. Robert Raven (Queensland phy of the family Scorpionidae (Chelicerata:
Museum), Dr. Lorenzo Prendini (American Scorpiones), with a discussion on phylogenetic
methods. Invertebrate Systematics 17:185-
Museum of Natural History) and Dr. David
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two anonymous referees who provided con- paleontology. In G. A. Polls (ed) Systematics,
structive reviews ofearlier versions ofthis pa- biogeography, and paleontology. Stanford Uni-
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