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branch of the Sciurognathi. We increased to three classes either

Correspondences have confirmed, by the cloning by a LWS gene duplication [7] or
and in vitro expression of the polymorphism [8]. The SWS1
diurnal grey tree squirrel (Sciurus pigments have peak sensitivities
Shortwave visual carolinensis) SWS1 opsin coding λmax) from 355 nm in the
(λ
sensitivity in tree sequence, that a VS pigment is
indeed present in this species,
ultraviolet to >430 nm in the violet
region of the spectrum. Within the
and flying and have established by site- rodents, UVS pigments are found
directed mutagenesis that the mainly in the Sciurognathi, in
squirrels reflects tuning to longer wavelengths has species such as the house mouse,
been achieved by the single rat, Siberian hamster, pocket
changes in amino acid substitution of Phe to gopher and gerbil, but two
lifestyle Tyr at site 86 (rod opsin
numbering), convergent therefore
species of the Hystricognathi, the
cururo and degu, also have UVS
with the evolution of VS pigments pigments [9], demonstrating that
Lívia dos S. Carvalho, in the cow, pig [4,5] and wallaby the ancestral UVS pigment is
Jill A. Cowing, Susan E. Wilkie, [6]. The same Tyr86 substitution is retained in these lineages (Figure
James K. Bowmaker and present in the SWS1 gene of two 1A). VS pigments are also found in
David M. Hunt species of nocturnal flying both orders, in the guinea pig
squirrel, the Siberian flying (Hystricognathi) and several
The order Rodentia is subdivided squirrel, Pteromys volans, and the species of ground and tree
into two suborders, the Northern flying squirrel, squirrels (Sciurognathi), although
Sciurognathi and the Glaucomys sabrinus, from North the evidence for VS pigments in
Hystricognathi. Within the America, but in each case the the latter species comes from
Sciurognathi, the shortwave- gene is rendered non-functional indirect methods [1,2] and the
sensitive (SWS1) class of visual by deletion. Flying squirrels from yellow pigmented lens in these
pigments is ultraviolet-sensitive both continents have therefore species may have biased the
(UVS) amongst the largely dispensed with colour vision, estimate towards longer
nocturnal murine species, most probably as a result of the wavelengths. The λmax of the
whereas violet-sensitive (VS) switch from the diurnality of their SWS1 pigment may be therefore
pigments are thought to be immediate tree squirrel ancestor significantly shorter. Vision in
present in diurnal ground and tree to a nocturnal lifestyle. these diurnal species is highly
squirrels [1,2]. As the ancestral In mammals, colour vision is adapted to a diurnal lifestyle, with
mammalian pigment is most likely achieved by two classes of cone a cone-dominated retina that
UVS [3] and UVS pigments are photoreceptors that contain either contrasts with the rod-dominated
retained in many rodent species, a shortwave- (SWS1) or long retinae of most other mammals.
the evolution of VS pigments must wave-sensitive (LWS) visual The full length SWS1 coding
have occurred within the squirrel pigment. Only in primates is this sequence was amplified by PCR

A B C
1 3
Degu , Cururo Tyr86Phe
0.03 λmax 364 nm Wild type
Hystricognathi λmax 440 nm

Guinea pig1
VS 0.01
Absorbance difference

UVS
Rodentia
1 1 1
House mouse , Rat , Gerbil ,
Siberian hamster1, Pocket gopher3
Sciurognathi –0.01

Ground and Tree squirrels1

VS
–0.03

–0.05
350 400 450 500 550 600
Wavelength (nm)
Current Biology

Figure 1. Rodent SWS1 opsins.

(A) Cladogram showing evolution of VS pigments (dashed line) in both rodent lineages. Only species with confirmed UVS or VS pig-
ments are included. 1Nocturnal; 2diurnal; 3burrowing/fossorial. (B) Difference spectra for recombinant wild-type and Tyr86Phe mutant
grey squirrel SWS1 opsins. The Tyr86Phe mutant pigment was generated by site-directed mutagenesis in vitro. Both wild-type and
mutant pigments were expressed, purified and regenerated as described previously [4]. The difference spectra were obtained by sub-
tracting the dark spectra from the spectra after either hydroxylamine (VS pigments) or acid (UVS pigments) treatment. The λmax for
each pigment was then determined by fitting a Govardovskii standard curve. (C) Amino acid sequence of grey squirrel SWS1 opsin
aligned with mouse, guinea pig and bovine SWS1 opsins. Matching residues for the latter three sequences are identified by dots, gaps
are denoted by dashes. The seven α helices’ transmembrane regions are boxed and an arrow indicates site 86. The grey squirrel
SWS1 opsin sequence has been deposited in GenBank (accession number DQ302163).
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A Siberian flying squirrel,

B C
Pteromysvolans

9 bp deletion
0.05
Wild type

0.04

Absorbance
0.03

0.02
Northern flying squirrel,

Glaucomyssabrinus
0.01

0.00

350 400 450 500 550 600

Wavelength (nm)

Current Biology

Figure 2. SWS1 opsins of flying squirrels.

(A) Siberian and Northern flying squirrels. (B) Partial sequences of the SWS1 gene and sequence chromatograph alignments for grey
squirrel, S. carolinensis, Siberian flying squirrel, P. volans, and Northern flying squirrel, G. sabrinus (GenBank accession numbers
DQ302164 and DQ302165). The deletions found in flying squirrel sequences are indicated by dashes. The deleted amino acids are
shown below the grey squirrel sequence using bovine rod opsin numbering. The amino acids below G. sabrinus sequence show a
short stretch of substitutions arising from the frame shift mutation at position 300 (grey squirrel SWS1 opsin numbering). (C) The dark
spectra for grey squirrel wild-type and the 9 base-pair deletion mutant showing failure of the latter to form a pigment. (Photograph
for P. volans was kindly provided by Ilpo Hanski, and the photograph of G. sabrinus is from the Animal Picture Archive.)

from grey squirrel retinal cDNA
(Supplemental Table 1 available
on-line). In vitro expression and
regeneration of the pigment with
11-cis-retinal [4] gave a λmax at
440 nm (Figure 1B), identifying
therefore a VS pigment. Tyr is
present at site 86, as found in the
VS pigments of the cow, pig and
Tamar wallaby (Figure 1C). The
role of Tyr in tuning the grey
squirrel pigment was confirmed
by replacement with Phe, which
resulted in a 76 nm shortwave
shift into the UV. The use of Tyr86
for the longwave shift is
evolutionarily convergent
therefore with these other three
species and contrasts with Val86
in the guinea pig [10]. The
mechanism for VS pigment
evolution thus differs in the two
rodent suborders.
Flying squirrels in contrast are
nocturnal, raising the question of
whether they have a UVS
pigment. Partial SWS1 gene
sequences that included codon
86 were obtained from genomic
DNA (Table S1 in the
Supplemental Data) for the
Siberian flying squirrel, Pteromys
volans, and the Northern flying
squirrel, Glaucomys sabrinus
(Figure 2A). As shown in Figure
2B, both species have Tyr86,
implying that a UVS pigment is
not present in flying squirrels. But
in P. volans, a 9 base-pair
deletion removes residues 93–95
in α helix II. To confirm that this
deletion would preclude pigment
formation, it was introduced into
the grey squirrel opsin for in vitro
expression: as shown in Figure
2C, no absorbance peak is
present after the addition of 11-
cis-retinal. The opsin gene for G.
sabrinus also has a deletion,
removing residues 77 and 78, plus
a single nucleotide frameshift
deletion at base-pair 300.
Both genes are therefore non-
functional, indicating that flying
squirrels lack colour vision. None
of the observed mutations in the
SWS1 gene is shared between the
two flying squirrel species so it is
possible that the loss of function
arose separately in the two
species after geographic
isolation. Common changes may,
however, be present in other
regions of the gene. Flying
squirrels have a very rod-
dominated retina and, consistent
with the above observations, the
only cones that have been
detected by ERG flicker
photometry have a peak
sensitivity at about 512 nm
(compared to 543 nm in the tree
squirrel) with no evidence for a
SWS class (reviewed in [11]).
The absence of a functional
SWS1 pigment in mammals is not
unique: it is very common in
marine mammals and is also
found in two species of primate,
three species of carnivore and
several species of rodent [9].
Interestingly, all the terrestrial
mammals that have lost the SWS1
opsin are nocturnal, which
suggests that colour vision is not
critical for fitness. The loss of the
SWS1 pigment appears linked
therefore to nocturnality, but the
reverse is not true since most
nocturnal animals retain a
functional SWS pigment.
Acknowledgements
This work was funded by a Leverhulme
Trust grant to D.M.H. and J.K.B. We are
grateful to John Gurnell, for the grey
squirrel tissue, and to Ilpo Hanski,
Matthew Wheatley and Karl Larsen for
flying squirrel tissue. We are indebted
to Rosalie Crouch for the gift of 11-cis-
retinal. We are also grateful to Wayne
Davies for a set of degenerate PCR
primers and for helpful comments.
Supplemental Data
A supplemental table providing details
of primers used is available at
http://www.current-biology.com/
cgi/content/full/16/3/R81/DC1/
References
1. Jacobs, G.H. (1976). Wavelength
discrimination in gray squirrels. Vision
Res. 16, 325–327.
2. Jacobs, G.H. (1978). Spectral sensitivity
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and colour vision in the ground-dwelling
sciurids: results from golden mantled
ground squirrels and comparisons for
five species. Anim. Behav. 26, 409–421.
3. Hunt, D.M., Wilkie, S.E., Bowmaker, J.K.,
and Poopalasundaram, S. (2001). Vision
in the ultraviolet. Cell. Mol. Life Sci. 58,
1583–1598.
4. Cowing, J.A., Poopalasundaram, S.,
Wilkie, S.E., Robinson, P.R., Bowmaker,
J.K., and Hunt, D.M. (2002). The
molecular mechanism for the spectral
shifts between vertebrate ultraviolet-
and violet-sensitive cone visual
pigments. Biochem. J. 367, 129–135.
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D.D. (2002). Spectral tuning in the
mammalian short-wavelength sensitive
cone pigments. Biochemistry 41,
6860–6865.
6. Deeb, S.S., Wakefield, M.J., Tada, T.,
Marotte, L., Yokoyama, S., and Marshall
Graves, J.A. (2003). The cone visual
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Sequence, spectral tuning, and
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7. Nathans, J., Thomas, D., and Hogness,
D.S. (1986). Molecular genetics of
human color vision: the genes encoding
blue, green, and red pigments. Science
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8. Mollon, J.D., Bowmaker, J.K., and
Jacobs, G.H. (1984). Variations of colour
vision in a New World primate can be
explained by polymorphism of retinal
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Biol. Sci. 222, 373–399.
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photoreceptor properties: Adaptations
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Bowmaker, J.K., and Hunt, D.M. (2004).
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rodent violet-sensitive visual pigment.
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UCL Institute of Ophthalmology,
University College London, 11-43 Bath
Street, London EC1V 9EL, UK.
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Satiation gives
krill that sinking
feeling
Geraint A. Tarling1 and
Magnus L. Johnson2
The daily vertical migration of
pelagic animals to and from the
upper ocean layers is the most
widespread and coordinated
movement of biomass on Earth.
The behaviour is a major pathway
for carbon to reach the ocean’s
interior as organisms eat at the
surface and release their waste at
depth. Estimates of carbon
sequestration via this ‘active flux’
of pelagic animals assume that
the behaviour is performed just
once every 24 hours [1], but there
is indirect evidence that there may
be a continuous movement
between the upper and lower
layers, with individuals ascending
when hungry and descending
once satiated [2,3]. We found that
Antarctic krill, one of the most
abundant pelagic animals found in
a region known to be an important
carbon sink [4], exhibited a
sinking response when replete
that could contribute significantly
to carbon sequestration.
Many animals perform daily
vertical migration, indicating that
it confers selective advantages.
The behaviour minimises visibility
to predators whilst satisfying the
need to exploit the food-rich
surface layers. However, daily
vertical migration patterns are not
always up at night and down in
the day, because there is
evidence that many populations
divide into deep and shallow
layers at the same time [2].
Individuals found in deeper layers
can contain food types that could
only have been eaten at the
surface [3]. This suggests a
pattern of swimming to the
surface, sinking when full and
migrating up again when
digestion is complete. Such
behaviour has yet to be directly
observed in individual pelagic
animals.
Amongst pelagic animals,
Antarctic krill has a considerable
biomass and profoundly
Figure 1. In situ images of krill
swimming (upper) and parachuting
(lower). The pictures are provided by
Uwe Kils, who used a free-falling
autonomous imaging system [11].
influences the ecology of the
Southern Ocean. Krill remain
pelagic for their entire life cycle
with 73% of their total daily
metabolism being used to fuel
swimming [5]. These animals
must overcome their negative
buoyancy through the
continuous generation of upward
thrust from specialised
swimming legs on the abdomen,
called pleopods. Krill sink
immediately when inactive,
although the rate of descent may
be controlled by fanning out the
pleopods in a horizontal plane to
form a parachute [6] (Figure 1)
To measure krill swimming
characteristics in laboratory
experiments, it is necessary to
use some sort of tethering device
to prevent them from hitting
container walls. Such studies
have found that gender, size and
moult-stage affect maximum
power output [7]. The use of free-
falling cameras in situ have
revealed that krill adopt a variety
of swimming patterns, from the
continuous beating of pleopods
to regular switching between
beating and parachuting [6]. No
studies of krill have yet been able
to relate swimming behaviours to
internal states, such as levels of
hunger and satiation.