Wong Ze-Lin, Serena Teo Lay Ming - Tropical Marine Science Institute

Contributions to Marine Science 2012
Contributions to Marine Science 2012: 135–143
Date of Publication: 29 Sep.2012
© National University of Singapore
FLUORESCENT PATTERNS IN SOME PORTUNUS SPECIES
(CRUSTACEA: BRACHYURA: PORTUNIDAE)
Wong Ze-Lin
Department of Biological Sciences, National University of Singapore,
14, Science Drive 4, Singapore 117543.
Ng Ngan-Kee
Department of Biological Sciences, National University of Singapore,
14, Science Drive 4, Singapore 117543.
Email: dbsngnk@nus.edu.sg
S. L-M Teo
Tropical Marine Science Institute, National University of Singapore,
18 Kent Ridge Road, Singapore 119227.
Email: tmsteolm@nus.edu.sg
Fernando J Parra-Velandia
Tropical Marine Science Institute, National University of Singapore,
18 Kent Ridge Road, Singapore 119227.
Email: tmsfjp@nus.edu.sg
ABSTRACT. — Portunus is a large genus of swimming crabs, which has been observed to have fluorescing
patterns in certain species, including commercially important ones. We examined these patterns for their
potential usefulness as a diagnostic character in species identification. Specimens of various Portunus
species were photographed under normal light and UV light. It was observed that fluorescent patterns varied
inter- and intra-specifically. The patterns were consistent between males and females of each species, but
not amongst juveniles. Fluorescent colour patterns were most pronounced in fresh specimens, and may be
useful as a complementary taxonomic character for species identification.
KEY WORDS. — Structural colour, fluorescence, iridescence, crustacean, Portunus
INTRODUCTION
The detection of electromagnetic (EM) radiation is important
in the natural world as many organisms rely on their sense
of sight to gather information about their environment
and communicate with conspecifics and other organisms.
It plays an important role in inter- and intra-specific
communication, protection (camouflage and aposematism),
sexual selection and foraging (Altshuler, 2001; Lim et al.,
2007; Stevens & Marilaita, 2009). As vision is widely
used, many marine organisms use colour as cues. With
the exception of bioluminescence, most organisms require
that electromagnetic radiation be reflected off its body to
appear visible and in colour to others; this can be achieved
by the use of microstructures (i.e. structural colours) or, the
commonest way, by using pigments which absorb specific
electromagnetic waves of the spectrum (normally between
100-1200 nm), and reflect also a specific wavelength with
lower energy than that of the absorbed (Britton, 1983; Parker,
2005) producing a colour.
The term fluorescence applies when the absorbed wavelength
is in the ultraviolet band range of the spectrum (200-400 nm)
and normally the re-emitted electromagnetic wave is of a
longer, and often, visible wavelength; it has been observed in
aquatic and terrestrial invertebrates such as corals, scorpions
and spiders (Stachel et al., 1999; Mazel & Fuchs, 2003; Lim
et al., 2007). However, besides all the information on animal
interaction using fluorescence, the exact mechanism is poorly
known or seldom discussed.
The genus Portunus Weber, 1795, is a large genus of
swimming crabs comprising 93 species (Ng et al., 2008),
many of which are commercially important (FAO fishery
statistics, 2012) and occur mainly in the Indo-west Pacific
(Davie, 2002). Portunus pelagicus has been found to be
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Fluorescent patterns in Portunus crabs
a species complex comprising four species: P. pelagicus
(Linnaeus, 1758), P. segnis (Forskål, 1775), P. reticulatus
(Herbst, 1799) and P. armatus (A. Milne-Edwards, 1861)
(Lai et al., 2010)
The authors of the present study noted that some carapaces
of these crabs fluoresce and appear covered with elaborate
granulated fluorescent patterns under ultraviolet (UV) light
(Fig. 1). We examined the carapace patterns of eleven species
to investigate if these fluorescent patterns can be used as a
simple method to distinguish species.
MATERIAL AND METHODS
Ethanol preserved specimens from the Raffles Museum of
Biodiversity Research Zoological Reference Collection,
(ZRC) National University of Singapore were examined.
All the catalogued Portunus spp. specimens with the widest
geographic distribution were screened to ensure that they had
a clean carapace (i.e. free of epibionts) and they exhibited
complete fluorescing patterns.
Only species with five or more specimens available were
considered. Ten specimens from the species Portunus
rubromaginatus (Lanchester, 1900), P. tenuipes (De Haan,
1835), P. sanguinolentus (Herbst, 1783), and P. tweediei (Shen,
1937) were selected to study adult interspecific variations.
For the species Portunus pelagicus (Linnaeus, 1758); P.
segnis (Forskål, 1775); P. reticulatus (Herbst, 1799), and P.
armatus (A. Milne-Edwards, 1861), only five specimens were
examined as no distinct fluorescing patterns were observed on
the carapace. Three fresh specimens of Portunus pelagicus
were obtained from Sheng Siong Supermarket, Singapore.
These specimens were photographed immediately to preserve
its natural state. These fresh specimens were also viewed
under the SEM (see below).
Before photographs were taken, the specimen catalogue
number, the gender and carapace width and length (in mm)
were recorded. The examination of the fluorescing patterns
on the crab’s bodies was carried out through the comparison
of the photographs of specimens under white light and
under two different commercially available UV lights (JML
1197UV range 350 nm - 400 nm, 370 nm peak; UV torch
range 390 nm - 450 nm, 405 nm peak; emitted wavelengths
measured using an Ocean Optics USB400 spectrometer).
Photographs of the crabs were taken using a mounted Nikon
D7000 camera with an AF Micro Nikkor 60 mm 1:2.8D
lens, against a black background to reduce any reflections
or shadows. Each specimen was cleaned and gently dried
before photography (Felgenhauer, 1987). Photography of the
complete specimen (dorsal view and ventral view), dorsal
view of carapace, left and right chela, left and right natatory
leg were taken. Photographs were classified and labelled for
easy referencing.
The fluorescing patterns and microstructures of the carapace
(GPF) were compared specifically for inter- and intravariations
to check for an identification based on fluorescing
patterns basis. Crab morphology was determined following
Carpenter & Niem (1998). The images taken under UV light
were split into RGB channels using ImageJ (Abramoff, 2004).
The green and blue channels from the UV torch were then
merged with the green channel of the UV lamp, giving the
overall fluorescing image for the specimen. Finally to extract
the overall pattern, Adobe Illustrator CS5.1 was used to trace
out the patterns on the crabs’ carapace. All resultant images
were cleaned using Adobe Photoshop CS5.1.
RESULTS
Based on a total of 7,406 photographs taken from a total
of 131 specimens, the carapaces of all 11 species examined
were fluorescent. However, fluorescing patterns and
microstructures (GPF) were observed only in 6 of them.
The fluorescent parts of the carapace also corresponded
to the exposed parts of the carapace, i.e. portions of the
carapace not covered by setae. Photographs revealed that
Fig. 1. (A) Portunus gracilimanus carapace under UV lamp. (B) Illustration of GFP on carapace. Scale = 10 mm.
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from the 11 species studied, six species belonging to the
subgenera Lupocycloporus (Portunus gracilimanus, Fig. 1),
Monomia (P. gladiator, Fig. 2; P. rubromarginatus, Fig. 3) and
Xiphonectes (P. tenuipes, Fig. 4; P. hastatoides, Fig. 5 and P.
tweediei, Fig 6) could be discerned, based on their carapace
granulated fluorescing patterns (GFP) visible under UV light
(Figs. 8–11); while the other five species from the subgenus
Portunus (P. sanguinolentus, Fig. 7; P. pelagicus, Fig. 8; P.
segnis, P. reticulatus, P. armatus) do not have distinct GFP.
Patterns exhibited by males and females’ specimens were
similar, and pattern discernibility often depended on the
condition of the carapace.
Of those species with GFP, only adults displayed fluorescing
patterns while the juveniles did not. In juveniles, patterns
were observed on the protogastric and mesogastric regions
of the carapace, while the areas posterior of the metagastric
and branchial regions on the carapace were without patterns.
One species without GFP, P. sanguinolentus (Fig. 7) has three
distinct dark spots on the posterior edge of the carapace visible
under normal and UV light, which separated it from other
species. For species belonging to P. pelagicus complex (P.
pelagicus, P. segnis, P. reticulatus, P. armatus), there were
no fluorescing patterns distinguishable from normal light
pigmentation that can be used as a diagnostic character.
Interestingly, the fresh specimens of P. pelagicus displayed
greater contrast under UV light compared to preserved
specimens.
The fluorescing patterns were observed on the carapace of
only six species. Carapace patterns could be broadly divided
into four groups, which are consistent with the subgenera
Lupocycloporus, Monomia, Xiphonectes and Portunus.
Observations showed that Portunus (L.) gracilimanus has
distinct horizontal bands throughout the carapace. Specimens
from the subgenera Monomia and Xiphonectes display
circular or ovate clustered granules. Species from these
subgenera can be further differentiated by the GFP found
specifically at the mesogastric and protogastric regions.
Fig. 2. (A) Portunus gladiator carapace under UV lamp. (B) Illustration of GFP on carapace. Scale = 10 mm.
Fig. 3. (A) Portunus rubromarginatus carapace under UV lamp. (B) Illustration of GFP on carapace. Scale = 10 mm.
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Fluorescent patterns in Portunus crabs
In Portunus (L.) gracilimanus the granulation occurred
in horizontal bands, parallel to the posterior edge of the
carapace. The longest band comprise granules which form a
ridge along the branchial groove and anterior of the cervical
groove. Granules tend to diffuse towards the posterior margin
(Fig. 1B).
In Portunus (M.) gladiator (Fig. 2B) such granules aggregated
in oval or circular clusters. Granules on the branchial groove
form a slightly diffuse thin band. Small patches of granules
also occurred along the anterolateral margin. Crescent-shaped
granule clusters in the protogastric region were observed
surrounding the mesogastric region. Mesogastric granules
align to form a t-shaped cluster, this cluster distinguishes
P. (M.) gladiator from P. (M.) rubromarginatus (Fig. 3B)
and a disjointed Y-shaped cluster distinguishes P. (M.)
rubromarginatus from P. (M.) gladiator. The granules
in P. (M.) rubromaginatus (Fig. 3B) occurred in oval or
circular clusters. Another characteristic feature of P. (M.)
rubromarginatus is the two conspicuous small clusters of
granules at the epigastric region; these clusters are not as
pronounced in P. (M.) gladiator (Fig. 2B). The defined
crescent-shaped cluster separates this subgenus from
Xiphonectes.
In Portunus (X.) tenuipes (Fig. 4B), granules form ovate
clusters or bands. Granules on the branchial groove form a
thick band. Distinct clusters of granules at the protograstric
and mesogastric regions. Clusters at the mesogastric regions
form a disjointed Y shaped similar to P. (M.) rubromarginatus,
except that the ovate clusters here are broader. These small
distinct clusters at these regions are characteristic of this
species. Two small clusters of granules accompanied by a
row of granules at the epigastric region are also distinct to
this species.
In P. (X.) hastatoides (Fig. 5B), the protogastric and
mesogastric clusters are more diffused and somewhat merged
together. Granules on the branchial groove line the ridge, but
more diffused than ridge on P. (X.) tenuipes. Crescent-shaped
granule clusters in the protogastric region surrounding the
mesogastric region merge with t-shaped granulations on
mesogastric regions forming a large irregular shaped pattern
characteristic of this species.
In P. (X.) tweediei (Fig. 6B), clusters are diffused and
dispersed. Granules on the branchial groove line the ridge
and spreads out towards the protogastric regions. Crescentshaped
granule clusters in the protogastric region surrounding
Fig. 4. (A) Portunus tenuipes carapace under UV lamp. (B) Illustration of GFP on carapace. Scale = 10 mm.
Fig. 5. (A) Portunus hastatoides carapace under UV lamp. (B) Illustration of GFP on carapace. Scale = 10 mm.
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the mesogastric region merge with t-shaped granulations on
mesogastric regions forming a large irregular shaped pattern.
However there is a distinct separation between protrusions in
pattern. This demarcation line is unique in this species.
No conspicuous granulated fluorescing patterns were
observed under UV light on the carapace of Portunus
sanguinolentus. However, the carapace has three large
defined darkened pigmented spots near the posterior margin
(Fig. 7B). Similarly, no conspicuous granulated fluorescing
patterns on the carapace of P. pelagicus (Fig. 8), P. segnis,
P. reticulatus and P. armatus were observed. The carapaces
for these species show the same pigmented patterning under
normal and UV light.
Portunus gladiator, P. sanguinolentus, P. pelagicus and
P. reticulatus also showed iridescence. Iridescence in P.
gladiator was only found in the 2 nd abdominal segment and
anterior ridges of the chela. Both P. sanguinolentus and
P. pelagicus displayed iridescence mainly on the carapace
and occasionally in the other parts such as the ventral side
of the carapace, the merus of 2 nd , 3 rd and 4 th perieopods. P.
reticulatus exhibited iridescence throughout its entire body.
It was observed that iridescence in these species became
evident only when the alcohol on the specimens dried up.
On the other hand the same patterns were observed in live
specimens of P. pelagicus, even when submerged in water
while in holding tanks of the supermarket.
The chelipeds of the crabs examined were also fluorescent,
but no clear distinguishable pattern was observed from
these structures that can be used for species identification.
Fluorescing patterns on the chela follow the granulation or
ridges found on each cheliped. While there were differences in
cheliped architecture, the overall patterns follow three general
trends. Species from subgenera Monomia and Xiphonectes
have similar GFP, which is consistent with the ridges and
exposed parts of the carapace (Fig 9). Portunus gracilimanus
also display fluorescing patterns along the ridges and exposed
parts of the carapace, with the exception of the tip of the
chelipeds and spines, which are darkened (Fig. 9B, C).
Specimens from the subgenus Portunus also exhibit darkened
tips but only in the dactylus and propodus of the chelipeds.
However, the entire chela of these specimens fluoresces as
oppose to the chelipeds of other species which only exhibit
fluoresce at exposed areas of the carapace.
Demarcation line
Fig. 6. (A) Portunus tweediei carapace under UV lamp. (B) Illustration of GFP on carapace. Scale = 10 mm.
Fig. 7. (A) Portunus sanguinolentus carapace under UV lamp. (B) Illustration of spots on carapace. Scale = 10 mm.
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Fig. 8. (A) Portunus pelagicus carapace under UV lamp. (B) Illustration of mottled patterns on carapace. Scale = 10 mm.
Fig. 9. Cheliped of Portunus rubromaginatus (left) and P. gracilimanus (right). The tips of the spines, dactylus and propodus of P.
gracilimanus are darkened (circled in red). Under (A) normal light, (B) UV torch and (C) UV lamp. Scale = 10 mm.
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Several distinct patterns were observed on the natatory legs
of all these species. Fluorescing patterns on the natatory legs
can be categorised into two groups: (a) those with venation,
and (b) those without venation. Portunus gracilimanus, P.
gladiator, P. rubromarginatus; P. hastatoides, P. tenuipes,
P. tweediei displayed conspicuous fluorescing venation
patterns throughout the natatory legs (Fig. 10). Portunus
rubromaginatus can be further differentiated through the
single vein found in the posterior edge of the dactylus; the
other species shows two veins (Fig. 10). With the lack of
venation and setae, fluorescence was observed throughout
the natatory legs of P. sanguinolentus, P. pelagicus, P. segnis,
P. reticulatus and P. armatus.
DISCUSSION
Carapaces of six Portunus species showed distinguishable
granulated fluorescing patterns that can be used to differentiate
specimens to species. While the other structures such as the
chela and the natatory legs display patterns, they were not
suitable as diagnostic characteristic features. The comparison
of carapace under normal and UV light sources revealed
patterns, which are more conspicuous under UV light
especially under 350 nm – 400 nm, but these patterns were
also visible under normal light, and thus conferred little
advantage for separating samples using UV. As the alcohol
that these crabs were preserved in fluoresced green under
UV light, it may be that the crabs do contain fluorescing
proteins, possibly metabolites, though the localization of
the protein within the carapace is unknown (Gentien, 1981).
The mild fluorescence detected might be due to duration
and medium of preservation, as alcohol has been known to
extract the fluorescent compounds (Stachel et al., 1999), and
what was observed would be residual fluorescence observed
in the preserved specimens due to underlying proteins within
the carapace, in the matrix of the endocuticle. It was noted
that for the fresh P. pelagicus specimen photographed under
normal light and UV light, the carapace showed greater
contrast and vividness between mottled patterns and areas
Fig. 10. Natatory leg of Portunus rubromarginatus (left) and P. hastatoides (right). The natatory leg fluorescing patterns of subgenera
Lupocycloporus, Xiphonectes and Monomia is similar to P. hastatoides, P. rubromarginatus is different with a single vein at the posterior
edge (circled in red). Under (A) normal light, (B) UV torch and (C) UV lamp. Scale = 10 mm.
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Fluorescent patterns in Portunus crabs
without patterns, and regions of the carapace without patterns
seem to fluoresce brighter compared to those of preserved
specimens. Thus the use of preserved samples may have
confounded the results.
There was very little variation observed between male and
female conspecifics. Juveniles often displayed incomplete
patterns on the carapace. The juveniles seem to develop GFP
at the protogastric and mesograstric regions first, followed
by the metagastric region during early instars. However the
exact stage or size cannot be determine from these number
of specimens, though a rough estimate from P. gracilimanus
shows that crabs fully develop patterns between 11 x 7
mm and 15 x 10 mm. This evidence suggests that carapace
complexity increases as the crabs mature.
The genus Portunus comprises five subgenera with a total of
93 species. The current study considered the patterns for only
11 species (12%) from four subgenera. The results indicate
that to effectively use carapace colour as a complementary
diagnostic character for sorting samples of Portunus, fresh
specimens should be examined and as such, the method is
perhaps only useful for sorting of specimens in the field or
in fisheries (Fig. 11). Structural colour patterns, which would
be less impacted by chemical preservation methods used in
taxonomy, only occurred in a few species (unpublished data)
and as such, have limited use. Further analyses, including an
assessment of fresh specimens encompassing more species
from other regions, the identification of the fluorescent
pigment and its localization on the carapace structure, are
needed to characterize and accurately evaluate the utility of
this character in the taxonomy of Portunus species.
ACKNOWLEDGEMENTS
This work was supported in part by the Raffles Museum of
Biodiversity Research, the Tropical Marine Science Institute,
and to the Innovative Marine Antifouling Solutions for High
Value Applications project (SERC Grant No. 102-166-0102).
The authors thank Prof Peter Ng, Assoc Prof Peter Todd,
Assoc Prof Li Daiqin, Drs Joelle Lai, J.C. Mendoza, Diego
Pitta de Araujo, Mr Tommy Tan, Rueben Yue, Mdm Wong,
Gerald Liew, Henrietta Woo, Low Bi Wei and Su Shiyu, Paul
Chen, Son and Marcus Chua for their assistance.
Fig. 11. Species dichotomous key using fluorescent patterns based on the Portunus carapace.
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