J OURNAL OF C RUSTACEAN B IOLOGY, 33(5), 620-626, 2013
FLUORESCENCE IN ARTHROPODA INFORMS ECOLOGICAL STUDIES IN
ANCHIALINE CRUSTACEANS, REMIPEDIA, AND ATYIDAE
Dawn Glenn 1 , M. Joey Pakes 1,2,∗ , and Roy L. Caldwell 1,2
1 Department
of Integrative Biology, University of California, Berkeley, CA 94720, USA
of Paleontology, University of California, Berkeley, CA 94720, USA
2 Museum
ABSTRACT
Autofluorescence is pervasive throughout the natural world and occurs in many animals, plants, and minerals. Fluorescent properties vary
predictably over molt cycles of arthropod exoskeletons. This study examines intrinsic fluorescence in scorpions and crustaceans using
novel fluorometric analyses and microscopy. Anchialine cave crustaceans, which we report here autofluoresce, are generally sparsely
populated, and difficult for researchers to access. Therefore, little is known about the growth, molting, reproduction, and life history
of their endemic inhabitants. Methods proposed here for measuring autofluorescence under near-ultraviolet wavelengths are valuable in
informing techniques for optimal in situ observation and location of new larval forms, exuviae, spermataphores and eggs. We compare
the intensity and emission breadth of fluorescent compounds extracted from California woodland scorpions (Uroctonus mordax) and
California bay shrimp (Crangon franciscorum) to determine the effect of preservation duration on autofluorescent properties. Fluorescence
data were obtained from anchialine cave crustaceans collected from Cenote Crustacea, Quintana Roo, Mexico and Dan’s Cave, Abaco
Island, Bahamas. They exhibit peak emission wavelengths ranging from 429 nm to 515 nm and interspecific variation in emission peaks.
Traits relating to emission spectra, such as breadth and intensity, may correlate with a variety of ecological parameters, such as sex, age,
and reproductive state, allowing for potential field and laboratory-based applications.
K EY W ORDS: anchialine cave crustaceans, scorpions, fluorescence
DOI: 10.1163/1937240X-00002170
I NTRODUCTION
Autofluorescence, sometimes referred to as intrinsic or natural fluorescence, is ubiquitous among animals, plants, and
minerals throughout marine and terrestrial habitats. This pervasiveness may result from the role of autofluorescence in
animal communication and UV protection (Lim et al., 2007;
Kloock, 2010) or because it is an intrinsic property of arthropod exoskeletons (Stachel et al., 1999; Mazel, 2007). While
we know many aromatic molecules associated with this
property (Rost, 1995), the identity and ontological variations
of these biologically fluorescent compounds remain a mystery. Most autofluorescence research focuses on the scorpion
cuticle (Stachel et al., 1999; Kloock, 2010; Gaffin et al.,
2012), which varies in fluorescence intensity over a molt cycle (Stachel et al., 1999). The first instar and exoskeleton immediately following a molt do not fluoresce and subsequent
instars fluoresce with increasing intensity (Stachel et al.,
1999). The fluorescent compounds that slowly leach into the
alcohol of preserved scorpion specimens (Lawrence, 1954)
are most often used in fluorescence studies. Despite leaching
and possible photo-bleaching, both ethanol preserved and
dried scorpion specimens over 100 years old have retained
their fluorescence, demonstrating the persistence of these
compounds (Pavan, 1954; Stahnke, 1972). Without systematic studies into preservation effects, comparison of fluorescence datasets is impossible.
∗ Corresponding
Although autofluorescence has been observed in marine
and freshwater arthropods (Galassi et al., 1998; Mazel,
2007; Michels, 2007), no studies of this property occur
in anchialine crustaceans. Anchialine caves are densitystratified systems in which one or more layers of less saline
water overlay a marine layer, creating discrete haloclines and
stable physico-chemical gradients (Seymour et al., 2007).
This unique habitat has led to the evolution of numerous
endemic fauna, such as species of ostracodes, isopods and
atyid shrimp, as well as the enigmatic class of Crustacea,
Remipedia (Yager, 1981). As these systems are sparsely
populated and difficult to access, little is known about the
growth, reproduction, and life history of their inhabitants
(Koenemann et al., 2007). For example, molting has not been
described for many anchialine crustaceans (Carpenter, 1999;
Koenemann et al., 2007); the fluorescent properties of these
animals may aid in their in situ observation.
This study uses novel techniques to examine the variations
in intraspecific emission peak, breadth, and intensity, as well
as preservation duration of fluorescent properties in California woodland scorpions (Uroctonus mordax, Thorell, 1876)
and California bay shrimp (Crangon franciscorum, Stimpson, 1856). To ascertain whether ecological parameters correlate with emission spectra, scorpion and bay shrimp data
were also compared with the emission spectra of anchialine cave crustaceans (ostracode, isopod, amphipod, thermosbaenacean, atyid shrimp, and remipede specimens) col-
author; e-mail: [email protected]
© The Crustacean Society, 2013. Published by Brill NV, Leiden
DOI:10.1163/1937240X-00002170
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lected from Cenote Crustacea, Quintana Roo, Mexico and
Dan’s Cave, Abaco Island, Bahamas. Additionally, epifluorescence microscopy allowed observation of external and
internal morphology not normally visible under white light.
M ATERIALS AND M ETHODS
Cave Crustacean Collection and Fixation
Between June 24-27, 2010 we collected in Cenote Crustacea, Quintana Roo,
Mexico as follows: (1) the amphipod Mayaweckelia cenoticola Holsinger,
1977 and the thermosbaenacean Tulumella unidens Bowman and Iliffe,
1988 from brackish water layers (8-17 ppt); (2) the remipede Speleonectes
cf. tulumensis (Yager, 1987), an isopod Metacirolana mayana (Bowman,
1987), and the ostracode Danielopolina mexicana Kornicker and Iliffe,
1989 from the marine layer (34-35 ppt); and (3) the atyid caridean Typhlatya
pearsi Creaser, 1936 from brackish and marine water layers. In addition,
Speleonectes lucayensis (Yager, 1981) was collected from the marine
layer in Dan’s Cave, Abaco Island, Bahamas in 2006, which exhibits
similar salinity gradients. All specimens were collected on SCUBA and
immediately preserved. One S. cf. tulumensis individual was preserved in
RNAlater and used for microscopy studies only. All other specimens were
preserved in 95% ethanol and used for both fluorometric analyses and
microscopy.
Preparation of Scorpions and Bay Shrimp
California woodland scorpions (U. mordax) were collected locally from
the East Bay hills February 20-26, 2011. Within 48 hours of collection,
live specimens were frozen at −20°C in individual 15 ml Falcon tubes
(specimens < 0.5 g) or 50 ml Falcon tubes (specimens > 0.5 g). Prior to
preservation, specimens of U. mordax were rinsed with ethanol to remove
sediment. California bay shrimp (C. franciscorum) were obtained from San
Francisco Estuary via a commercial bait shop on February 8 and March 2,
2011 and held in a seawater system (15°C, approx. 33 ppt salinity). Twelve
juvenile and twelve gravid female individuals between 43-62 mm were
selected from this group and frozen live in 15 ml Falcon tubes at −20°C.
All specimens were weighed and measured (from prosoma to tip of telson
for scorpions and tip of rostrum to tip of telson for shrimp).
Three individuals each of U. mordax, juveniles of C. franciscorum, and
gravid females of C. franciscorum were preserved in 95% ethanol (20 ml
EtOH for U. mordax >0.5 g, 10 ml EtOH for U. mordax <0.5 g and all
specimens of C. franciscorum) for the following time periods: two, four,
and eight weeks. All preserved specimens were stored in the dark at ambient
temperature. We obtained five additional female U. mordax, preserved in
95% ethanol between 1948-1961, from the collections of the University of
California Berkeley, Essig Museum of Entomology to compare long-term
preservation effects of fluorescence properties.
At the end of each trial, 6 ml of ethanol was removed from each
specimen and placed in three 2 ml tubes for fluorometric analysis.
Fluorometric Analysis
Ethanol from C. franciscorum, U. mordax and cave animals, in addition
to an ethanol control, were centrifuged for 30 minutes at 12 000 g
at 4°C. Supernatant was transferred into new 2 ml tubes. Following
standard protocols adapted from Palmer et al. (2003), peak excitation and
emission wavelengths were then measured in Fisherbrand® 1.5 ml disposable polystyrene cuvettes and a HORIBA Jobin Yvon FluoroLog® -3
spectrofluorometer. Breadth of emission spectra was calculated as Width at
Ninety percent Peak Intensity (WNPI; Kloock, 2008) (Fig. 1). All statistical
analyses were performed in R (R Development Core Team, 2010).
Light Microscopy
Specimens used for fluorometric analysis and additional frozen U. mordax,
live C. franciscorum, and RNAlater preserved S. tulumensis were imaged
using a Zeiss Lumar v.12 epifluorescence stereo microscope to compare
external and internal structures under different wavelengths of light. Images
were captured with BioVision Ivision software using a QImaging chargecoupled-device camera fitted to the microscope under reflected light, DAPI
(excitation 359-371 nm; emission 379 nm) and GFP BP (excitation 450490 nm; emission 500-550 nm) filters.
Comparison of Juvenile and Female California Bay Shrimp Carapace
Thickness
The thickness of the carapaces of C. franciscorum was measured in order
to compare measurement against emission intensity for signs of sexual
differentiation. Five juveniles and five gravid females C. franciscorum were
obtained from the San Francisco Estuary via a commercial bait shop in
June, 2012 and held in a seawater system (15°C, approx. 33 ppt salinity).
Specimens were weighed and their lengths were measured from tip of
rostrum to tip of telson. Carapaces were cut cross-wise at the midpoint
between tip of telson and posterior portion of the carapace, allowing
removal of a section of cuticle. Detachment of adhering matter and internal
soft tissue was performed with a tweezer and scalpel and sections were
rinsed with ethanol. Section thickness was measured under a dissecting
microscope using an ocular micrometer at 20× magnification.
Fig. 1. Exemplar emission spectra showing emission curve with intensity measured in arbitrary units (a.u.). Breadth is calculated as the Width at Ninety
percent of Peak Intensity (WNPI) (Kloock, 2008). Here, WNPI = 57 844 × 0.9 = 52 059.6.
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R ESULTS
Variation in Autofluorescence with Preservation Duration,
Maturity, and Cuticle Thickness
Gravid female (43-60 cm; 0.91-2.76 mg) and juvenile (5062 cm; 1.61-2.43 mg) bay shrimp did not significantly differ
from one another in length (ANOVA; p > 0.05) or weight
(ANOVA; p > 0.05) by preservation time group. C. franciscorum did not differ in emission breadth, or intensity over
the trial period (Fig. 2). While females and juveniles did not
show a difference across all preservation periods in breadth
and intensity, these bay shrimp groups did differ by fluorescence peak emission with juveniles emitting higher peaks at
all time periods (see Table S1 in the online Appendix to this
article, which can be accessed via http://booksandjournals.
brillonline.com/content/1937240x) (F = 5.408, df = 7,
p = 0.05). Mean cuticle thickness did not vary significantly between juveniles and brooding females. Scorpions
preserved from 1948-1961 emitted greater fluorescence intensity (F = 67.730, df = 11, p < 0.001) at a greater
breadth (F = 33.430, df = 8, p < 0.001) and with greater
peak emission (F = 5.797, df = 12, p = 0.03) than those
preserved in this study (Fig. 2, Table S1). Yet, the emission
intensity and breadth did not vary between short-term trial
periods (Fig. 2, Table S1). Peak emission increased between
two and four weeks of preservation in ethanol for U. mordax,
but we did not see a further increase in this property between
8 weeks of preservation and 60 years (Fig. 2).
Differential Autofluorescence of Scorpions and Crustaceans
Use of DAPI (excitation 359-371 nm; emission 379 nm)
and GFP BP (excitation 450-490 nm; emission 500-550 nm)
filters allowed for the visualization of internal and external
anatomy not easily seen under visible light. For example, the
ventral nervous system of S. tulumensis (Fig. 3a) appeared
yellow and more pronounced under DAPI (Fig. 3b). Newly
hatched stage I zoel larva of T. pearsei (Fig. 3c) are
also visible with differential fluorescence under GFP BP
channel (Fig. 3d). Greater emission occurs in the cranial
region in near UV light (Fig. 3d) than under reflected light
(Fig. 3c). Vestigial eyes of M. cenoticola fluoresced with
greater intensity relative to surrounding areas under GFP BP
channel (Fig. 3e). Fat bodies of M. mayana were also better
seen under near UV light (Fig. 3f). Differential emission
between reflected light and near UV light of T. unidens
(Fig. 3g and 3h, respectively) and images of S. tulumensis
(Fig. 3a and 3b, respectively), as well as the GFP BP
channel image of M. cenoticola (Fig. 3e) illustrate increased
fluorescence at scleratonized mouthparts. Scleratonization
can also be seen at the adductor muscle attachment scars
Fig. 2. Female U. mordax mean emission breadth, mean peak emission, and emission curves illustrating intensity differences over short- and long-term
preservation time scales. Scorpions did not differ in a breadth or b emission peak within experimental preservation trials. Extractions from museum
collections preserved at least 60 years prior had greater a emission breadth and c intensity but not in b peak emission values than more recently preserved
specimens.
GLENN ET AL.: FLUORESCENCE IN ANCHIALINE CRUSTACEANS
623
Fig. 3. Images of intact anchialine specimens under visible light and various near-UV wavelengths. a and b, S. cf. tulumensis (remipede): a, ventral view of
head to somite two under visible light; b, with DAPI channel (white arrow point to increased fluorescence intensity at distal ends of mandibles and maxilla
indicating sclerotization of cuticle and yellow arrow points to differential light emission along the midline exhibiting ventral nervous system). c and d, T.
pearsei (atyid shrimp): c, naupliar larval stage carapace under visible light; d, GFP BP channel. e, M. cenoticola (amphipod) anterior view under GFP BP
channel (white arrow points to relative increase in fluorescence intensity at mouthparts and blue arrow points to vestigial eye). f, M. mayana (isopod) with
GFP BP channel, full dorsal (note strong fluorescence from fat bodies at posterior). g and h, T. unidens (thermosbaenacean): g, juveniles imaged under
visible light; h, with DAPI channel (white arrows note increased fluorescence in sclerotized mouthparts). i, D. mexicana (ostracode) GFP BP channel (white
arrow notes adductor muscle attachment scar). j, exoskeleton of developing T. pearsei eggs in pleopods with DAPI channel. S. cf. tulumensis preserved in
RNAlater. All other samples preserved in Ethanol. Scale bars in a-b, i = 500 μm; in c, d, f-h = 1 mm; e, f = 2 mm. This figure is published in colour in the
olnie edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/1937240x.
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Fig. 4. Inter- and intra-specific variation in fluorescence of anchialine cave fauna, U. mordax and C. franciscorum. Note grouping of T. pearsei, S. lucayensis
and C. franciscorum.
of D. mexicana (Fig. 3i). Fluorescence of growing cuticle
was visible under DAPI channel in developing embryos of
T. pearsei (Fig. 3j).
Inter- and Intra-specific Variation in Autofluorescent
Properties of Anchialine Crustaceans
Anchialine cave specimens sampled included remipedes, decapods, thermosbanaceans, and several peracarids, which
ranged in emission peak from 429.18-487.81 nm and in
emission breadth from 6-60 nm (Fig. 4; see also Table S2
in the online Appendix to this article, which can be accessed via http://booksandjournals.brillonline.com/content/
1937240x). In general, the decapod crustaceans C. franciscorum and T. pearsei from estuarine and anchialine habitats exhibit greater emission breadths than pericarid and
remipede crustaceans. Furthermore, of the specimens we
sampled, species emission breadth and peaks clustered by
species (Fig. 4; Table S2).
D ISCUSSION
Research in difficult to access environments such as caves
or the deep sea, in which animals are difficult to detect
and observation time is limited, might greatly benefit from
fluorescent techniques. The power of this tool will increase
as we understand how fluorescent properties vary with
preservation and organismal biology.
Preservation of Fluorescence and Variation over Ontogeny
Time of preservation, along with type of preservation (Stachel et al., 1999), are important factors determining emission
spectra of an organism. After leaching of fluorophores into
ethanol, fluorescent properties, including emission peak and
breadth, are retained over long time periods (Pavan, 1954;
Stahnke, 1972). These fluorophores may be derived from
the central nervous system (CNS) (Nicol, 1991) or externally in the cuticle, such as β-carboline (Stachel et al., 1999)
or 4-methyl, 7-hydroxycoumarin (Frost et al., 2001). Since
we preserved whole and unpunctured organisms, ethanolic
extracts likely contained fluorophores from cuticular surfaces that were in contact with preservative. The increases
in fluorescence intensity over time in C. franciscorum and
U. mordax might have been caused by gradual leaching of
fluorophores with time. Likewise, the broader fluorescence
emission peaks that we observed in museum preserved as
compared to more recently preserved U. mordax may be explained by a leaching of a greater diversity of fluorophores
into ethanolic extracts as cuticle degrades. The resultant
mixture of fluorophores with slightly different spectra may
have generated a greater overall emission breadth. Knowledge of how preservation affects emission spectra may be
useful for comparative studies.
The protandrous C. franciscorum exhibit greater fluorescence intensity in juveniles than in gravid females. Since cuticle thickness did not vary significantly between juveniles
and brooding females, and if fluorophores in ethanolic extract were mainly derived from cuticle, these data suggest a
greater accumulation of fluorescent compounds in juvenile
exoskeletons as compared to those of gravid females. We
now know cave crustacean emission spectra is species specific, but finer scale sampling is needed to determine whether
size, molt stage, and sex correlates with variations in intraspecific emission peak or intensity. The scarcity of these
animals inhibited our ability to determine such differences.
Lessons from Fluorescence Imaging
Brightly fluorescent areas have been found in the exoskeletons (Michels, 2007; Haug, 2011), and CNS, including the
nervous cord and brain, of various malacostracans (Armant
and Elofsson, 1976; Sheehy, 1990). Relative increases in
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fluorescence intensity in the nervous system of cave crustaceans, such as the naupliar larvae of T. pearsei and the central nervous cord of S. tulumensis might be due to concentrated lipofuscin, which is also thought to accumulate with
age in crustaceans (Sheehy, 1990). As characteristics of the
nerve cord have been found informative in crustacean phylogenies (Harszch and Waloszek, 2001), fluorescence properties of lipofuscin could provide a valuable character in phylogenetic analyses.
The vestigial eyes of M. cenoticola also fluoresced in near
UV light similarly to the functional eyes of other amphipods,
scorpions (Haug et al., 2011; blue light), and stomatopods
(Caldwell, unpublished). Whether this emission results from
enervation to the ocular region and resultant lipofuscin accumulation (Sheehy, 1990) or photopigment fluorescence as
occurs in stomatopod ommatidia (Caldwell, unpublished)
and spider eye ommochromes (Théry and Casas, 2009) requires further study. Enervation and accompanying fluorescence increases also occur at olfactory glands and antennae
bases (Sheehy, 1990).
Increased fluorescence at the distal portion of mouthparts
in remipedes, thermosbanaceans, and amphipods is likely
due to sclerotization. Similar increases in fluorescence at the
tips of amphipod maxillae (Haug et al., 2011) may result as
a byproduct of oxidative crosslinking as the cuticle hardens
(Stachel et al., 1999). Fluorescence techniques combined
with comparative morphology will allow us to induce the
feeding strategy in rare organisms.
Evolution of Fluorescence
Fluorescence could have been maintained for UV protection
(Théry and Casas, 2009), anti-predatory behavior (Kloock,
2010), or use in communication (Arnold et al., 2002;
Mazel et al., 2004; Lim et al., 2007; Mazel, 2007). While
this character might be held over from the UV-perceiving
ancestors of cave species, it is more likely that anchialine
crustaceans fluoresce as a result of the byproducts, i.e., βcarbolines, than of oxidative cross-linking that occur during
sclerotization (Stachel et al., 1999). Fluorescent lipofuscin
is found in various tissues and occurs as a result of cellular
metabolism during the lipid peroxidation processes.
Fluorescence in Arthropods as a Tool
The fluorescent properties described here will aid in future
studies of cave fauna and will be advantageous in studying
small organisms, minute features, or larval forms. In Confocal Laser Scanning Microscopy (CLSM), autoflourescence
can be used to examine morphology that is difficult to visualize using other methods without damaging specimens
for future analyses. This method was instrumental in the description of remipede larvae (Koenemann et al., 2007). Differential fluorescence of structures also increases the ease of
morphological coding for phylogenetic analysis and species
descriptions (Michels, 2007).
Fluorescence has already been exploited for many years
as a method for nocturnal scorpion collection and has also
been expanded to the collection of insects (Stahnke, 1972).
Because many arthropods and fish fluoresce (Mazel, 2007),
this technique will allow greater ease of collection for
scientific study under near UV light in other fields. Scorpion
exuviae (Stahnke, 1972; Stachel, 1999) and spermatophores
(Sissom et al., 1990), as well as shrimp eggs, fluoresce,
suggesting that molts and gametes will be more easily
observed in the field using fluorescent techniques.
Moreover, since the reproductive methods of many crustaceans are unknown and molting is often linked to reproductive receptivity, such techniques could provide advances
for systems inhabited by rare autofluorescent fauna (Mazel,
2007), such as those in anchialine caves. We now film remipedes in situ using lights equipped with near UV filters and
cameras with yellow filters specified to enhance the peak
emission of our focal animals. Furthermore, interspecific
rates of lipofuscin accumulation are reported to vary and
may be used to determine the age of individuals within a
population (Nicol et al., 1991). Should wavelengths similarly vary with molt stage or sex in focal organisms, in situ
submersible fluorometers could provide more complete census data concerning sex ratios and sexual receptivity in the
field. Fluorescence photography and videography have also
been suggested as a means to increase the accuracy of percent cover estimates for cryptic animals in marine systems
(Mazel, 2007).
ACKNOWLEDGEMENTS
The authors are grateful for the training and use of fluorescence microscopes by Dr. S. Ruzin and Dr. D. Schichnes, PMB, UC Berkeley. We thank
Dr. D. King of the HHMI Mass Spectrometry Laboratory Lab, UC Berkeley for his advice on the fluorometric process, and J. Zaks and The Fleming
Group, Dept. of Chemistry, UC Berkeley for use of their spectrofluorometer. We also wish to acknowledge the assistance of Dr. S. Koenemann for
providing Bahamian remipede information, to C. B. Barr, Collection Manager at The Essig Museum of Entomology, UC Berkeley, and to Dr. T. Iliffe
and B. Gonzalez for aiding in specimen collection. We are grateful to C.
Groth for assisting with C. franciscorum measurements. This research was
supported by UC Berkeley Museum of Paleontology and the Society of Integrative and Comparative Biology. Permits were issued by SEMARNAT,
the Ministry of Environment and Natural Resources, Mexico.
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R ECEIVED: 29 January 2013.
ACCEPTED: 22 April 2013.
AVAILABLE ONLINE: 17 May 2013.
Mean emission breadth, peak emission and intensity with standard error for Crangon franciscorum and Uroctonus mordax.
Crangon franciscorum gravid female (n = 9)
Crangon franciscorum juvenile (n = 9)
Uroctonus mordax female (n = 9)
Uroctonus mordax (Essig) female (n = 5)
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Mean breadth (nm)
Mean peak emission (nm)
Mean intensity (a.u.)
46.20 ± 2.21
44.51 ± 1.60
26.20 ± 5.46
57.84 ± 0.39
465.73 ± 1.90
467.50 ± 3.06
457.55 ± 7.68
467.62 ± 0.44
26 032 ± 104.65
34 650 ± 116.92
18 282 ± 89.84
49 989 ± 58.37
Cave crustaceans collected in Cenote Crustacea, Quintana Roo, Mexico and Dan’s Cave, Abaco, Bahamas.
Species
Mayaweckelia cenoticola
Metacirolana mayana
Metacirolana mayana
Speleonectes lucayensis
Speleonectes lucayensis
Speleonectes tulumensis
Tulumella unidens
Typhlatya pearsei
Typhlatya pearsei
Length
(mm)
Preservation time
in EtOH (days)
Peak
excitation
(nm)
Peak
emission
(nm)
Peak
intensity
(a.u.)
5
7.5
5
18.5
24.5
13
2.5
9
18
112
434
112
1456
1456
112
112
112
112
419
419
419
390
390
419
419
397
397
476.426
514.532
502.471
429.176
429.176
475.921
476.726
473.626
473.821
–
2558
–
–
–
–
2191
8571
11 692
Collection
date
Collection
location
24 June 2010
23 June 2010
23 June 2010
2006
2006
24 June 2010
27 June 2010
23 June 2010
24 June 2010
Cenote Crustacea
Cenote Crustacea
Cenote Crustacea
Dan’s Cave
Dan’s Cave
Cenote Crustacea
Cenote Crustacea
Cenote Crustacea
Cenote Crustacea