Reference: Biol. Bull. 229: 47–57. (August 2015)
© 2015 Marine Biological Laboratory
Aquaporins in the Antarctic Midge, an Extremophile
that Relies on Dehydration for Cold Survival
SHIN G. GOTO1,*, RICHARD E. LEE, JR.2, AND DAVID L. DENLINGER3
Graduate School of Science, Osaka City University, Osaka, Japan; 2Department of Biology, Miami
University, Oxford, Ohio; and 3Departments of Entomology and Evolution, Ecology and Organismal
Biology, Ohio State University, Columbus, Ohio
1
metabolous insect (Fig. 1). This species has a patchy but
locally abundant distribution along the western coast of the
Antarctic Peninsula and South Shetland Islands. It is commonly observed in moderately damp sites, especially those
enriched by vertebrate feces (Strong, 1967; Edwards and
Baust, 1981; Sugg et al., 1983). Particularly dense aggregations of larvae are associated with the nitrophilous alga
Prasiola crispa. Larvae appear to be non-selective feeders
(Baust and Edwards, 1979) capable of feeding on dead plant
material, algae, and microorganisms including fungi
(Strong, 1967; Edwards and Baust, 1981). Many details of
the life cycle of B. antarctica have been described, especially features of the 2-year period of larval development
(Sugg et al., 1983; Usher and Edwards, 1984; Convey and
Block, 1996). The wingless adults, approximately 3 mm in
length, emerge during the summer after a brief pupal period.
Adults live fewer than 10 d (Harada et al., 2014), and in this
short period mating occurs in aggregations of flightless
males, like the male swarms of winged midges in temperate
and arctic regions. Females lay a cluster of eggs within a
day or so after mating and then die; first instar larvae hatch
within a few weeks (Harada et al., 2014).
Although ambient air temperatures may reach – 40 °C
during the winter at Palmer Station (Anvers Island), larvae
are not particularly cold hardy and cannot survive at temperatures below –15 °C (Baust and Lee, 1987). This is a
relatively modest level of cold tolerance compared with
many alpine and polar insects. Due to oceanic thermal
buffering, as well as thermal buffering by overlying snow
and ice, temperatures in the midge’s subnivean microhabitat
remain rather constant between –2 °C and –7 °C during
winter (Kawarasaki et al., 2014). Summer temperatures are
considerably higher and sometimes exceed 10 °C or 15 °C,
but temperatures can abruptly decrease to subzero levels
Abstract. The terrestrial midge Belgica antarctica relies
extensively on dehydration to survive the low temperatures
and desiccation stress that prevail in its Antarctic habitat.
The loss of body water is thus a critical adaptive mechanism
employed at the onset of winter to prevent injury from
internal ice formation; a rapid mechanism for rehydration is
equally essential when summer returns and the larva resumes the brief active phase of its life. This important role
for water movement suggests a critical role for aquaporins
(AQPs). Recent completion of the genome project on this
species revealed the presence of AQPs in B. antarctica
representing the DRIP, PRIP, BIB, RPIP, and LHIP families. Treatment with mercuric chloride to block AQPs also
blocks water loss, thereby decreasing cell survival at low
temperatures. Antibodies directed against mammalian or
Drosophila AQPs suggest a wide tissue distribution of
AQPs in the midge and changes in protein abundance in
response to dehydration, rehydration, and freezing. Thus
far, functional studies have been completed only for PRIP1.
It appears to be a water-specific AQP, but expression levels
are not altered by dehydration or rehydration. Functional
assays remain to be completed for the additional AQPs.
The Antarctic Midge
The midge Belgica antarctica Jacobs, 1900 (Diptera:
Chironomidae) is the southernmost, free-living, holoReceived 27 November 2014; accepted 20 February, 2015.
* To whom correspondence should be addressed. E-mail: shingoto@sci.
osaka-cu.ac.jp
Abbreviations: 2ME, ␤-mercaptoethanol; AQP, aquaporin; BIB, Big
Brain protein; DRIP, Drosophila intrinsic protein; DVIP, Dermacentor
variabilis integral protein; HgCl2, mercuric chloride; LHIP, Lygus hesperus integral protein; PRIP, Pyrocoelia rufa integral protein; RPIP, Rhodnius prolixus integral protein.
47
48
S. G. GOTO ET AL.
seawater during and after storms. As these pools of seawater
dry, the midge larvae are exposed to increasing concentrations of seawater, resulting in a reduction of body water
content. Larvae of B. antarctica successfully manage such
daily and seasonal osmotic perturbations, and fully tolerate
a loss of up to 70% of their body water (Benoit et al., 2007).
The midge larvae can survive in fresh water for 28 d, and in
seawater for 10 d (Baust and Lee, 1987; Elnitsky et al.,
2009).
Dehydration for Survival
Figure 1. Adults (upper panel) and fourth (final) instar larvae (lower
panel) of the Antarctic midge Belgica antarctica. Adults are approximately
3 mm in length and the fourth instar larvae are approximately 6 mm in
length. Adult female (left) is larger than adult male (right).
even during midsummer. Thus, unlike most temperate insects that markedly increase their cold tolerance in preparation for winter, B. antarctica retains a modest level of
freeze tolerance year round (Baust and Lee, 1987). The
midge larvae are common near Adélie penguin rookeries,
and in midsummer the midge microhabitat can change dramatically as extensive deposition of guano causes the microhabitat pH to decrease to as low as 4 in vernal pools
containing this effluent. Indeed, larvae show an ability to
survive for more than 2 weeks over a range of pH 3-12
(Baust and Lee, 1987). Depending on microbial activity and
ice cover in specific microhabitats, B. antarctica can be
exposed to anoxic conditions during winter. Larvae have an
impressive ability to withstand such severe conditions for
nearly a month (Baust and Lee, 1987).
One of the most remarkable abilities of this species is
tolerance to water perturbation. During winter, larvae are
surrounded by ice and snow, and access to biologically
available water may be limited. However, in early summer,
melting snow and ice flood their microhabitats with fresh
water. The flooding may last from a few days to weeks
depending on the location. Later, the microhabitat may dry
or leave isolated pools with high salinity. Thus, many microhabitats are terrestrial sometimes and aquatic at other
times. The midges inhabit small islands that are frequently
close to the coast, and therefore larvae can be submerged in
Although larvae have an intrinsically high level of dehydration tolerance, this ability can be enhanced further by
several environmental triggers that affect their body water
content. Larvae that are slowly dehydrated under high relative humidity (RH) are slightly, but significantly, more
tolerant of desiccation than larvae that are rapidly dehydrated under low RH (Hayward et al., 2007). In addition,
survival is higher when rehydration (i.e., hydration after
dehydration) is performed at 100% RH rather than by direct
contact with water (Hayward et al., 2007). These results
indicate that the rate of water efflux/influx is a pivotal point
for their tolerance. Benoit et al. (2007) found that larvae
held in groups coiled around neighboring larvae and retained water more effectively than did solitary larvae, i.e.,
group effect under dehydrating conditions. Thus, larvae can
manage dehydration/rehydration rates behaviorally by seeking suitable microhabitat and by clustering with neighbors
to regulate their tolerance.
Dehydration at a slow rate confers cross-tolerance to cold
in larvae of Belgica antarctica (Hayward et al., 2007).
Nearly all larvae that were desiccated for 2 d at 98.2% RH
withstood a 3-day exposure at –10 °C, whereas fewer than
10% of the fully hydrated larvae survived 2 d at –10 °C
(Fig. 2A). None of the fully hydrated larvae survived a
15-min exposure to –15 °C, whereas 10% of the desiccated
larvae survived this treatment (Hayward et al., 2007). Exposure of B. antarctica larvae to hyperosmotic seawater
reduced their body water content, a treatment that also
increased freeze tolerance relative to freshwater-acclimated
larvae (Elnitsky et al., 2009; Fig. 2B). Fewer than 65% of
freshwater-acclimated larvae survived –12 °C and fewer
than 15% survived –15 °C. In contrast, nearly 95% and 55%
of larvae acclimated to seawater survived freezing at –12 °C
and –15 °C, respectively. Even when frozen at –20 °C,
nearly 15% of seawater-acclimated larvae survived. This
finding contrasts with freshwater acclimation, after which
no larvae survive freezing at –20 °C. In addition, survival of
seawater-acclimated larvae rehydrated for 24 h, during
which their water content was restored to pre-acclimation
levels, was significantly greater than that of freshwateracclimated larvae (Fig. 2-B). Furthermore, seawater exposure increased subsequent tolerance of larvae to dehydration
AQUAPORINS IN THE ANTARCTIC MIDGE
A
100
Survival (%)
Desiccated
80
60
40
Intact
20
0
0
12
24
48
72
Time at -10°C (h)
B
100
Survival (%)
Rehydration
80
Seawater
acclimation
60
40
Freshwater
acclimation
20
0
-20
-15
-12
-10
Temperature (°C)
Figure 2. Effects of dehydration on cold tolerance in Belgica antarctica. (A) Survival of intact larvae and larvae dehydrated at 98.2% RH for
48 h after exposure to –10 °C for various durations. After Hayward et al.
(2007). (B) Survival of larvae acclimated to osmotic challenges. Larvae
were acclimated to either seawater (⬃1000 mOsm kg–1) or fresh water
(⬃0 mOsm kg–1) for 3 d prior to assessment of freeze tolerance. A third
group of larvae (rehydration) were acclimated to seawater for 3 d followed
by rehydration for 24 h in freshwater. Larvae were frozen in ⬃100 ␮l of
freshwater for 6 h. After Elnitsky et al. (2009). Error bars indicate standard
error (SE).
(Elnitsky et al., 2009). Such cross-tolerance is also reported
in some other invertebrates (Bayley et al., 2001; Everatt et
al., 2014). This relationship indicates that both stressors
result in similar injuries and physiological challenges, including reduction of the stability and function of plasma
membranes, impairment of protein folding, and increase of
pH and osmolality of cellular fluid (reviewed by Everatt et
al., 2015).
Overwintering larvae undergo another distinct form of
dehydration, known as cryoprotective dehydration (Holmstrup et al., 2002). Small invertebrates with high integumental permeability will dehydrate when exposed to an
environment at equilibrium with the vapor pressure of ice,
owing to the vapor pressure difference between supercooled
water and ice at the same temperature. Such water loss
continues until, at equilibrium, vapor pressure of the body
49
fluids equals that of the surrounding ice. At this time, risk of
inoculative freezing is eliminated because the melting point
of the animal’s body fluids equals ambient temperature.
Equilibration of the body fluid melting point with that of the
environment is also facilitated by accumulation of sugars
and sugar-alcohols, i.e., cryoprotectants. B. antarctica larvae are among the few species capable of resisting inoculative freezing by undergoing cryoprotective dehydration, a
feat they achieve by water loss and accumulation of glucose
and trehalose (Elnitsky et al., 2008; Kawarasaki et al.,
2014).
Dehydration induces many transcriptomic, metabolomic,
and proteomic changes (Michaud et al., 2008; Li et al.,
2009; Lopez-Martinez et al., 2009; Teets et al., 2013).
Recent RNA sequencing of B. antarctica (Teets et al., 2012)
revealed that cellular recycling pathways, including the
ubiquitin-mediated proteasome and autophagy, are up-regulated, and concurrently genes involved in general metabolism and ATP production are down-regulated in response to dehydration. Metabolomics reveals shifts in
metabolite pools that correlate closely with observed
changes in gene expression, indicating that coordinated
changes in gene expression and metabolism are a critical
component of the dehydration response. Thus, the dehydration
response is not simply passive, but includes active processes
that regulate the physiology of B. antarctica. Cryoprotective
dehydration also triggers several distinct gene cascades, but a
majority of the differentially expressed genes are shared between dehydration and cryoprotective dehydration treatments
(Teets et al., 2012).
Significance of Aquaporins for Dehydration in
Belgica antarctica
Under dehydrating conditions, larvae of B. antarctica
lose water at an exceptionally high rate (⬎10%/h) (Benoit et
al., 2007). In water, larvae can become overhydrated, with
their water content increasing to 120% within 2 h (Yi et al.,
2011). The capacity of the species to quickly lose and regain
water suggests a possible role for aquaporins (AQPs).
AQPs, channel proteins that are critical for the movement of
water across the cell membrane, have been described in
various organisms including insects (Campbell et al., 2008).
Some AQPs are water-specific, but others exhibit aquaglyceroporin activity, transporting not only water but other
small molecules including glycerol and urea (see Kataoka et
al., 2009; Wallace et al., 2012). In various insect species,
physiological roles of AQPs, i.e., water absorption and
elimination, have been extensively studied (e.g., Herraiz et
al., 2011; Wallace et al., 2012; Nagae et al., 2013; Staniscuaski et al., 2013). Recent studies have examined the role
of AQPs in insects that experience extremes in body water
content. Freeze-tolerant insects routinely survive the freezing of 60% or more of their body water. Treatment with
50
S. G. GOTO ET AL.
140
120
100
80
60
40
20
Cell viability (%)
E
l2
H
gC
l2 +
H
2M
tr
Dehydration
B
gC
ol
E
on
C
H
gC
l2 +
2M
ol
C
H
on
gC
tr
in
al
S
l2
0
e
Relative water content (%)
A
Overhydration
100
80
ethanol (2ME), which counters the blocking effect of
HgCl2, is added to the solution, tissue desiccation is induced
under dehydrating conditions. When midgut samples are
moved to hydrating conditions (diluted saline solution),
tissue water content increases rapidly (overhydration). Under these hydrating conditions, HgCl2 reduces the rapid
influx of water into the tissues. However, this protective
effect is eliminated by adding 2ME to the solution. Yi et al.
(2011) also investigated freeze tolerance in several specific
tissues. When the larval fat body, midgut, and Malpighian
tubules are exposed to low temperatures (–10 °C or –20 °C),
viability of the tissues is slightly reduced. However, viability is greatly reduced when these tissues are treated with
HgCl2. When 2ME is added to the solution, the negative
effects of HgCl2 disappear (Fig. 3-B). These results suggest
a function of AQPs in regulating tissue water content in B.
antarctica, and underscore their significance in water management and, consequently, their role in retention of cellular
viability during freezing.
60
40
Belgica antarctica Aquaporins
20
The next question is, which of the AQPs are involved in
water management during the processes of dehydration/
rehydration? It is well known that a single insect species
possesses multiple AQPs and that these AQPs show tissuespecific expression (Drake et al., 2010; Marusalin et al.,
2012; Benoit et al., 2014; Fabrick et al., 2014). Studies with
antibodies against mammalian and Drosophila AQPs suggest a wide distribution of AQP-like proteins in various
tissues of B. antarctica (Yi et al., 2011). Western blotting
indicates that AQP2-immunoreactive (-ir) protein levels in
larvae increase in response to dehydration, rehydration, and
freezing. On the other hand, levels of AQP3-ir proteins are
greatly reduced by these treatments. Drosophila intrinsic
proteins (DRIP)-ir become more abundant in response to
dehydration and rehydration. These results imply a pivotal
role for these AQP-like proteins in water relations and
freeze tolerance in B. antarctica (Yi et al., 2011). However,
it is still unclear precisely what was detected by these
antibodies because mammalian AQP2 and AQP3 are not
closely related to insect AQPs (see fig. 2 in Goto et al.,
2011).
Although Goto et al. (2011) first identified an AQP gene
from B. antarctica using classic cloning techniques, recent
transcriptomic and genomic information on B. antarctica
(Teets et al., 2012; Kelley et al., 2014) reveals four additional AQPs and their relatives. Based on the nucleotide
sequences available, we designed several primers and obtained full cDNAs by rapid amplification of cDNA ends
(RACE) from RNA derived from whole bodies of nonstressed larvae, and sequenced the obtained DNA fragments
(Table 1). RACE revealed that some of the genes produce at
least two transcript variants encoding different amino acid
Midgut
H
gC
l2 +
2M
E
l2
gC
ol
H
tr
on
C
2M
E
l2
gC
gC
l2 +
H
H
C
on
tr
ol
0
Malpighian
tubule
Figure 3. Significance of aquaporins on water management and freeze
tolerance in Belgica antarctica. (A) Water content of the midgut tissue
after a 10-min incubation in dehydrating and overhydrating media. Midgut
tissue was held in Coast’s solution (a Ringer solution developed by G. M.
Coast (1988)), dehydrated in 4⫻ Coast’s solution (dehydration), or overhydrated in 1/4⫻ Coast’s solution (overhydration). (B) Cell viability of
tissues following exposure to –10 °C. All samples were held in Coast’s
solution. Control, treatment with neither 0.2 mmol l–1 mercuric chloride
(HgCl2) nor 2 mmol l–1 ␤-mercaptoethanol (2ME); HgCl2, treatment only
with HgCl2; HgCl2⫹2ME, treatment with both HgCl2 and 2ME. After Yi
et al. (2011). Error bars indicate standard error (SE).
mercuric chloride (HgCl2), a known inhibitor of some AQPs
(Preston et al., 1993), results in higher cellular mortality
during freezing in the rice stem borer Chilo suppressalis
(Izumi et al., 2006) and in the goldenrod gall fly Eurosta
solidaginis (Philip et al., 2008). These results suggest that
AQPs play an essential role in the acquisition of freeze
tolerance by facilitating cellular water loss as ice forms in
the hemolymph (Izumi et al., 2006; Philip et al., 2008).
Yi et al. (2011) also used HgCl2 to explore physiological
roles of AQPs in B. antarctica (Fig. 3-A). Water content of
the midgut tissue after incubation in dehydrating conditions
(concentrated solution) rapidly decreases. However, treatment with HgCl2 prevents tissue desiccation under dehydrating conditions. On the other hand, when ␤-mercapto-
51
AQUAPORINS IN THE ANTARCTIC MIDGE
Table 1
Aquaporins of Belgica antarctica
Transcript
variant
DRIP1
PRIP1
RPIP1
RPIP2
LHIP1
BIB1
A
B
A
B
A
B
GenBank
accession No.
No. amino acid
residues
pI
MW (kDa)
Reference
AB985768
AB602340
AB602341
AB986065
AB986066
AB985770
AB985769
AB986245
AB986246
252
291
270
283
240
282
262
639
459
6.40
6.22
6.70
5.76
7.03
6.50
5.24
9.43
9.65
26.3
30.7
28.4
30.8
26.1
30.9
28.8
70.9
51.7
Present study
Goto et al. (2011)
Goto et al. (2011)
Present study
Present study
Present study
Present study
Present study
Present study
pI, isoelectric point.
sequences, possibly due to alternative splicing (Table 1).
The B. antarctica AQPs show high homology with AQPs in
other insect species (data not shown) and are classified into
either DRIP, Pyrocoelia rufa integral proteins (PRIP), Big
Brain proteins (BIB), Rhodnius prolixus integral proteins
(RPIP), or Lygus hesperus integral protein (LHIP) subfamilies in the phylogenetic tree (Fabrick et al., 2014; Fig. 4).
No protein belonging to the Dermacentor variabilis integral
proteins (DVIP) subfamily was found.
We concentrate our discussion on the five “regular”
AQPs, excluding BIB because it does not have a role in
water and ion transport in Drosophila (Tatsumi et al., 2009).
Two Asn-Pro-Ala (NPA) motifs or derivatives of the NPA
motif, which form a single aqueous pathway in the narrowest region of the pore (Spring et al., 2009), are found in
these AQPs (Fig. 5). Like other members of the AQP
superfamily (Murata et al., 2000), six transmembrane helices are detected by TMPred (Hofmann and Stoffel, 1993)
and by I-TASSER (Zhang, 2008; structure modeling is
shown in Fig. 6). It is important to note that the functional
classes, i.e., aquaporin and aquaglyceroporin, do not necessarily correspond to phylogenetic classifications. For example, some members of the PRIP subfamily function to
transport water and glycerol/urea and hence are aquaglyceroporins. Yet, not all members of the PRIP subfamily
transport glycerol and therefore not all members of this
subfamily are functional aquaglyceroporins (PvAQP2 in
Polypedilum vanderplankii and LhAQP4 in L. hesperus)
(Kikawada et al., 2008; Fabrick et al., 2014). Although
molecular modeling allows us to predict pore size of the
AQPs, and thus their function (Wallace et al., 2012; Staniscuaski et al., 2013), experimental assays including the
Xenopus oocyte swelling assay (e.g., Philip et al., 2011) and
the yeast complementation assay (e.g., Staniscuaski et al.,
2013) are needed to verify function. Amongst B. antarctica
AQPs, functional assays have been completed only for
PRIP1; the assay verified its water-specific transporting
ability (Goto et al., 2011). B. antarctica PRIP1 mRNA was
detected in various organs under non-stressed conditions,
suggesting that this AQP plays a fundamental role in cell
physiology. Unexpectedly, the PRIP1 transcriptional expression was not affected by either dehydration or rehydration (Goto et al., 2011; see next section). Functional analyses of other B. antarctica AQPs under normal and
dehydration stresses remain to be completed.
Although direct investigation (e.g., northern hybridization or qPCR) of the expression of B. antarctica AQPs in
response to dehydration has not yet been performed, except
for PRIP1 (Goto et al., 2011), Teets et al. (2012) measured
their gene expression levels in response to desiccation and
cryoprotective dehydration by RNA sequencing. In their
experiments, control larvae were held at 4 °C and 100% RH
(fully hydrated), while experimental larvae were exposed to
two forms of desiccation: desiccation at a constant temperature of 4 °C and 93% RH for 5 d (resulting in about 40%
water loss), and cryoprotective dehydration, in which larvae
were gradually chilled over 5 d from ⫺0.6 °C to ⫺3 °C at
vapor pressure equilibrium with surrounding ice and then
held at ⫺3 °C for 10 d (also yielding about 40% water loss).
The RNA sequencing dataset detected all five AQP genes,
but DRIP1 was divided into two genes (Gene model ID:
B648_07068 and B648_07070). These two gene models
correspond to the first and second half of the DRIP1 sequence, respectively, indicating that the sequence data of
DRIP1 is not well integrated in the analyses. Figure 7 shows
fold change in gene expression of B. antarctica AQPs in
response to desiccation and cryoprotective dehydration.
DRIP1 is strongly, and PRIP1 is weakly, up-regulated under
desiccation and cryoprotective dehydration, whereas RPIP1
and RPIP2 are weakly down-regulated. LHIP1 expression is
severely suppressed, especially under desiccating conditions. This fact suggests that some AQPs are likely upregulated to enhance water loss during dehydration, others
are down-regulated to conserve water in specific tissues, and
still others remain unchanged (Fig. 8). Investigation into
tissue specificity of these AQPs and their roles during
52
S. G. GOTO ET AL.
92
60
58
72
76
99
87
99
99
99
82
53
53
99
85
94
99
56
78
72
55
88
71
93
99
XP 001865732.1 Culex quinquefasciatus
Q9NHW7.2 Aedes aegypti
XP 319584.4 Anopheles gambiae
ABV60346.1 Lutzomyia longipalpis
Ba-DRIP1
NP 001036919.1 Bombyx mori
BAH47554.1 Grapholita molesta
BAG72254.1 Coptotermes formosanus
XP 624531.3 Apis mellifera
XP 001607940.2 Nasonia vitripennis
AHI85743.1 Lygus hesperus
Q25074.1 Haematobia irritans exigua
ADD19102.1 Glossina morsitans morsitans
ADD20045.1 Glossina morsitans morsitans
AAM68740 Drosophila melanogaster
XP 002425393.1 Pediculus humanus corporis
Q23808.1 Cicadella viridis
ABW96354.1 Bemisia tabaci
XP 972862.1 Tribolium castaneum
ACO10737.1 Caligus rogercresseyi
NP 001139377.1 Acyrthosiphon pisum
NP 001139376.1 Acyrthosiphon pisum
XP 394391.1 Apis mellifera
XP 001607929.1 Nasonia vitripennis
AAL09065.1 Pyrocoelia rufa
LhAQP4B Lygus hesperus
AHI85750.1 Lygus hesperus
XP 002429480.1 Pediculus humanus corporis
XP 968342.1 Tribolium castaneum
NP 001153661.1 Bombyx mori
NP 610686.1 Drosophila melanogast er
ADD19396.1 Glossina morsitans morsitans
BAF62090.1 Polypedilum vanderplanki
BAI60044.1 Anopheles gambiae
XP 001865728.1 Culex quinquefasciatus
XP 001656932.1 Aedes aegypti
XP 002433221.1 Pediculus humanus corporis
XP 001948407.1 Acyrthosiphon pisum
XP 396705.4 Apis mellifera
XP 001604170.1 Nasonia vitripennis
XP 968782.1 Tribolium castaneum
NP 476837 Drosophila melanogaster
Ba-BIB1-A
Ba-BIB1-B
99
71
96
BIB
XP 314890.4 Anopheles gambiae
XP 001866597.1 Culex quinquefasciatus
XP 001649747.1 Aedes aegypti
AHI85749.1 Lygus hesperus
XP 970728.2 Tribolium castaneum
EGI59562.1 Acromyrmex echinatior
EFN76752.1 Harpegnathos saltator
XP 001601231.2 Nasonia vitripennis
NP 001106228.1 Bombyx mori
Ba-RPIP1-A
Ba-RPIP1-B
Ba-RPIP2
95
PRIP
Ba-PRIP1-A
Ba-PRIP1-B
BAF62091.1 Polypedilum vanderplanki
NP 788433.2 Drosophila melanogaster
99
DRIP
XP 002430355.1 Pediculus humanus corporis
XP 001650168.1 Aedes aegypti
NP 001232971.1 Acyrthosiphon pisum
CAC13959.1 Rhodnius prolixus
AHI85748.1 Lygus hesperus
AHI85747.1 Lygus hesperus
AHI85746.1 Lygus hesperus
AHI85745.1 Lygus hesperus
AHI85744.1 Lygus hesperus
NP 611810.1 Drosophila melanogaster
XP 002430403.1 Pediculus humanus corporis
ABI53034.1 Dermacentor variabilis
XP 002424369.1 Pediculus humanus corporis
AHI85752.1 Lygus hesperus
AAF58409.2 Drosophila melanogaster
Ba-LHIP1
RPIP
DVIP
LHIP
Figure 4. Phylogenetic inference and classification of Belgica antarctica and other arthropod aquaporins.
Amino acid sequences were aligned with MUSCLE (web service from EMBL-EBI; http://www.ebi.ac.uk/Tools/
msa/muscle/) and their evolutionary history was inferred by neighbor-joining (Saitou and Nei, 1987) with the
Poisson model by MEGA 6.06 (Tamura et al., 2013). Bootstrap (Felsenstein, 1985) values of more than 50%
(500 pseudoreplications) are shown. Classification of AQP subfamilies is based on Fabrick et al. (2014).
53
AQUAPORINS IN THE ANTARCTIC MIDGE
DRIP1
PRIP1-A
PRIP1-B
RPIP1-A
RPIP1-B
RPIP2
LHIP1
M
M
M
-
T
S
S
-
D
I
E
M
E
E
A
A
S
R
N
L
K
M
M
E
I
N
G
T
T
D
D
D
M
M
M
L
L
L
K
K
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E
Figure 5. Deduced amino acid sequences of Belgica antarctica aquaporins. Amino acid sequences were
aligned with MUSCLE (web service from EMBL-EBI; http://www.ebi.ac.uk/Tools/msa/muscle/). Asterisk (*),
conserved amino acid; colon (:), strong positive residue; dot (.), weaker positive residue; dash (–), alignment gap.
The NPA motifs and derivatives of the NPA motifs are shown as white characters in the black boxes. Amino
acids corresponding to the aromatic/arginine (ar/R) constriction region involved in size selectivity by forming a
pore are indicated by boxes.
dehydration and rehydration are needed to develop a more
comprehensive understanding of their specific functions.
AQPs in Other Extremophilic Animals and Future
Directions in Belgica antarctica Aquaporin Research
Although many detailed studies of insect AQPs have
been performed, such work on extremophilic insects or
other animals is limited. The only other example in Insecta
is the African sleeping chironomid Polypedilum vanderplanki. This species is a good example of anhydrobiosis (life
with no water). P. vanderplanki inhabits shallow, exposed,
and temporary rain-filled granite rock pools in semiarid
regions of Africa. Larvae in these pools live within a tubular
nest made from mud and saliva. Larvae within the tubular
54
S. G. GOTO ET AL.
PRIP1-A
DRIP1
C-score= 1.13
Estimated TM-score = 0.87±0.07
Estimated RMSD = 3.6±2.5Å
RPIP1-A
C-score= -0.21
Estimated TM-score = 0.69±0.12
Estimated RMSD = 6.6±4.0Å
RPIP2
C-score= -0.90
Estimated TM-score = 0.60±0.14
Estimated RMSD = 8.1±4.4Å
C-score= -0.48
Estimated TM-score = 0.65±0.13
Estimated RMSD = 7.1±4.2Å
LHIP1
C-score=0.34
Estimated TM-score = 0.76±0.10
Estimated RMSD = 5.0±3.3Å
Figure 6. 3D modeling of the aquaporins of Belgica antarctica as constructed by I-TASSER (Zhang, 2008).
Only LHIP1 possesses the signal peptide, and it was removed when the model was constructed. N-terminus is
shown in dark blue. I-TASSER identified human AQP2 (PDB, 4nefA) as the best template for all AQPs.
nest tolerate complete desiccation by entering an anhydrobiotic state and subsequently recover upon rehydration, but
neither eggs, pupae, nor adults have this capacity (reviewed
by Cornette and Kikawada, 2011). This species possesses at
least two AQPs, PvAqp1 and PvAqp2; the former belongs to
the PRIP subfamily and the latter, to the RPIP subfamily
(Fig. 4). Both are water-specific AQPs, but their tissue
specificity is distinct: PvAqp1 is ubiquitous while PvAqp2 is
fat body-specific. Interestingly, in response to dehydration,
expression of PvAqp1 is greatly induced while expression of
PvAqp2 is suppressed (Kikawada et al., 2008). These results
suggest that PvAqp1 is involved in the removal of water
during induction of anhydrobiosis, whereas PvAqp2 may
help to prevent water loss from the fat body. B. antarctica
PRIP1 is closely related to PvAqp1, but expression of B.
antarctica PRIP1 was unaffected by dehydration or rehydration (Goto et al., 2011). Thus, in spite of the fact that
both chironomid species exploit dehydration to enhance
55
AQUAPORINS IN THE ANTARCTIC MIDGE
Fold change in expression
4.0
Desiccation
Cryoprotective dehydration
3.0
2.0
Up-regulation
1.0
Down-regulation
6)
8)
7)
LH
IP
2
1
(B
(B
64
64
8_
8_
07
06
16
24
24
7)
06
05
64
(B
IP
1
R
P
IP
P
R
R
P
8_
07
8_
64
(B
1
IP
IP
R
D
D
R
IP
1
1
(B
(B
64
64
8_
8_
07
07
06
07
8)
0)
0.0
Figure 7. Fold change in expression of Belgica antarctica aquaporins
in response to desiccation and cryoprotective dehydration. Data are from
RNA sequencing datasets in Teets et al. (2012). Genbank accession Nos.
of DRIP1 (Gene model ID: B648_07068), DRIP1 (B648_07070), PRIP1
(B648_07057), RPIP1 (B648_06247), RPIP2 (B648_06248), and LHIP1
(B648_07166) are GAAK01006849, GAAK01006851, GAAK01006838,
GAAK01006050, GAAK01006051, and GAAK01006945, respectively.
their ability to withstand environmental challenges, they
may use distinct AQPs to remove their body water.
AQPs are also found in the tardigrade Milnesium tardigradum (Mali et al., 2010; Schokraie et al., 2012), a species
that also undergoes anhydrobiosis to survive environmental
extremes. Eleven AQP genes are present in this species, and
their expression is up- or down-regulated by desiccation and
rehydration (Grohme et al., 2013). However, relative expression changes are modest, just more or less than twofold, fold differences that are far lower than those found for
PvAqp1 in Polypedilum vanderplanki. This result possibly
indicates that AQPs play a minor role during anhydrobiosis
AQPs
DRIP1
PRIP1
RPIP1
RPIP2
LHIP1
Water loss
Water conservation
Water regulation
Dehydration
Cellular recycling
Downregulation of energy metabolism
Membrane modification
Desiccation responsive metabolites
Cytoskeleton reorganization
Oxidative damage repair
Figure 8. Major events occurring during dehydration in larvae of
Belgica antarctica. Dehydration signals induce or suppress expression of
genes and proteins involved in various cascades. This species has at least
five aquaporins (AQPs); during dehydration some are likely up-regulated
to enhance water loss, others down-regulated to conserve water in specific
tissues, and still others remain unchanged to maintain regular water movement. The functional roles of most of these AQPs remain to be investigated.
in this species. Alternatively, water transport may be efficiently fine-tuned by small changes in expression of the
respective proteins (Grohme et al., 2013), or a large AQP
repertoire itself might act as an adaptive tool kit, as suggested in Caenorhabditis elegans (Huang et al., 2007).
The Arctic springtail (Collembola) Megaphorura arctica
species exploits cryoprotective dehydration to enhance its
cold tolerance (Holmstrup et al., 2002). This springtail has
at least three AQPs: AQP A, AQP B, and AQP C (Clark et
al., 2009). AQP A is down-regulated in response to moderate dehydration but is up-regulated by severe dehydration.
In contrast, AQP C is up-regulated during the preliminary
stages of cold dehydration, and AQP B is consistently
down-regulated in response to dehydration and cryoprotective dehydration. The significance of these expression
changes remains unknown.
With the exception of work on B. antarctica, none of the
studies on extremophilic animals to date has demonstrated
the significance of AQPs in dehydration, rehydration, or
cryoprotective dehydration. Experiments with HgCl2 have
suggested the importance of AQPs in water management
and also for freeze tolerance in B. antarctica (Yi et al.,
2011). Although HgCl2 is an easy tool to clarify the role of
AQP, it is not always definitive because some AQPs are
HgCl2-insensitive (Ishibashi et al., 1997) and some channels other than AQPs are also affected by HgCl2 (Zeng et al,
2012; Hayoz et al., 2013). Recent studies with RNA interference (RNAi) successfully clarified the significance of
AQPs for certain physiological processes in some insect
species (Drake et al., 2010; Liu et al., 2011; Benoit et al.,
2014). In B. antarctica, it is important to clarify the function
of the newly identified AQPs and their patterns of tissue
distribution. Their significance for dehydration should also
be examined by RNAi. Preliminary experiments verified
that RNAi is effective in B. antarctica, and double-stranded
RNA can be delivered to this species by rehydration, as
developed for use in larvae of the mosquito Culex pipiens
(Lopez-Martinez et al., 2012). We anticipate that these
approaches will unveil the significance of AQPs in adapting
to life in the cold and desiccating environment of Antarctica
(Fig. 8).
Acknowledgments
This work was funded by NSF grants (NSF PLR 1341385
to REL, and NSF PLR 1341393 to DLD).
Literature Cited
Baust, J., and J. S. Edwards. 1979. Mechanisms of freezing tolerance
in an Antarctic midge Belgica antarctica Physiol. Entomol. 4: 1–5.
Baust, J. G., and R. E. Lee, Jr. 1987. Multiple stress tolerance in an
antarctic terrestrial arthropod: Belgica antarctica. Cryobiology 24:
140 –147.
56
S. G. GOTO ET AL.
Bayley, M., S. O. Petersen, T. Knigge, H.-R. Köhler, and M. Holmstrup. 2001. Drought acclimation confers cold tolerance in the soil
collembolan Folsomia candida. J. Insect Physiol. 47: 1197–1204.
Benoit, J. B., G. Lopez-Martinez, M. R. Michaud, M. A. Elnitsky, R. E.
Lee, Jr., and D. L. Denlinger. 2007. Mechanisms to reduce dehydration stress in larvae of the Antarctic midge, Belgica antarctica.
J. Insect Physiol. 53: 656 – 667.
Benoit, J. B., I. A. Hansen, G. M. Attardo, V. Michalková, P. O. Mireji,
J. L. Bargul, L. L. Drake, D. K. Masiga, and S. Aksoy. 2014.
Aquaporins are critical for provision of water during lactation and
intrauterine progeny hydration to maintain tsetse fly reproductive success. PLoS Negl. Trop. Dis. 8: e2517.
Campbell, E. M., A. Ball, S. Hoppler, and A. S. Bowman. 2008.
Invertebrate aquaporins: a review. J. Comp. Physiol. B 178: 935–955.
Clark, M. S., M. A. S. Thorne, J. Purać, G. Burns, G. Hillyard, Ž. D.
Popović, G. Grubor-Lajšić, and M. R. Worland. 2009. Surviving
the cold: molecular analyses of insect cryoprotective dehydration in the
Arctic springtail Megaphorura arctica (Tullberg). BMC Genomics 10:
328.
Coast, G. M. 1988.
Fluid secretion by single isolated Malpighian
tubules of the house cricket, Acheta domesticus, and their response to
diuretic hormone. Physiol. Entomol. 13: 381–391.
Convey, P., and W. Block. 1996. Antarctic Diptera: ecology, physiology and distribution. Eur. J. Entomol. 93: 1–13.
Cornette, R., and T. Kikawada. 2011. The induction of anhydrobiosis
in the sleeping chironomid: current status of our knowledge. IUBMB
Life 63: 419 – 429.
Drake, L. L., D. Y. Boudko, O. Marinotti, V. K. Carpenter, A. L.
Dawe, and I. A. Hansen. 2010. The aquaporin gene family of the
yellow fever mosquito, Aedes aegypti. PLoS One 5: e15578.
Edwards, J. S., and J. Baust. 1981. Sex ratio and adult behavior of the
Antarctic midge Belgica antarctica (Diptera, Chironomidae). Ecol.
Entomol. 6: 239 –243.
Elnitsky, M. A., S. A. L. Hayward, J. P. Rinehart, D. L. Denlinger, and
R. E. Lee, Jr. 2008. Cryoprotective dehydration and the resistance
to inoculative freezing in the Antarctic midge Belgica antarctica. J.
Exp. Biol. 211: 524 –530.
Elnitsky, M. A., J. B. Benoit, G. Lopez-Martinez, D. L. Denlinger, and
R. E. Lee, Jr. 2009. Osmoregulation and salinity tolerance in the
Antarctic midge, Belgica antarctica: seawater exposure confers enhanced tolerance to freezing and dehydration. J. Exp. Biol. 212: 2864 –
2871.
Everatt, M. J., P. Covey, J. S. Bale, M. R. Worland, and S. A. L.
Hayward. 2015.
Responses of invertebrates to temperature and
water stress: a polar perspective. J. Therm. Biol. (In press). doi:
10.1016/j.jtherbio.2014.05.004.
Everatt, M. J., P. Convey, M. R. Worland, and S. A. L. Hayward. 2014.
Contrasting strategies of resistance vs. tolerance to desiccation in two
polar dipterans. Polar Res. 33: 22963.
Fabrick, J. A., J. Pei, J. J. Hull, and A. J. Yool. 2014. Molecular and
functional characterization of multiple aquaporin water channel proteins from the western tarnished plant bug, Lygus hesperus. Insect
Biochem. Mol. Biol. 45: 125–140.
Felsenstein, J. 1985. Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39: 783–791.
Goto, S. G., B. N. Philip, N. M. Teets, Y. Kawarasaki, R. E. Lee, Jr.,
and D. L. Denlinger. 2011. Functional characterization of an aquaporin in the Antarctic midge Belgica antarctica. J. Insect Physiol. 57:
1106 –1114.
Grohme, M. A., B. Mali, W. Wełnicz, S. Michel, R. O. Schill, and M.
Frohme. 2013. The aquaporin channel repertoire of the tardigrade
Milnesium tardigradum. Bioinform. Biol. Insights 7: 153–165.
Harada, E., R. E. Lee, Jr., D. L. Denlinger, and S. G. Goto. 2014. Life
history traits of adults and embryos of the Antarctic midge Belgica
antarctica. Polar Biol. 37: 1213–1217.
Hayoz, S., L. Cubano, H. Maldonado, and R. Bychkov. 2013. Protein
kinase A and C regulate leak potassium currents in freshly isolated
vascular myocytes from the aorta. PLoS One 8: e75077.
Hayward, S. A. L., J. P. Rinehart, L. H. Sandro, R. E. Lee, Jr., and D
.L. Denlinger. 2007. Slow dehydration promotes desiccation and
freeze tolerance in the Antarctic midge Belgica antarctica. J. Exp. Biol.
210: 836 – 844.
Herraiz, A., F. Chauvigné, J. Cerdá, X. Bellés, and M.-D. Piulachs.
2011. Identification and functional characterization of an ovarian
aquaporin from the cockroach Blattela germanica L. (Dictyoptera,
Blattellidae). J. Exp. Biol. 214: 3630 –3638.
Hofmann, K., and W. Stoffel. 1993. TMBASE: A database of membrane spanning protein segments. Biol. Chem. Hoppe-Seyler 374: 166.
Holmstrup, M., M. Bayley, and H. Ramløv. 2002.
Supercool or
dehydrate? An experimental analysis of overwintering strategies in
small permeable arctic invertebrates. Proc. Natl. Acad. Sci. USA 99:
5716 –5720.
Huang, C. G., T. Lamitina, P. Agre, and K. Strange. 2007. Functional
analysis of the aquaporin gene family in Caenorhabditis elegans.
Am. J. Physiol. Cell. Physiol. 292: 1867–1873.
Ishibashi, K., M. Kuwahara, Y. Gu, Y. Kageyama, A. Tohsaka, F.
Suzuki, F. Marumo, and S. Sasaki. 1997. Cloning and functional
expression of a new water channel abundantly expressed in the testis
permeable to water, glycerol, and urea. J. Biol. Chem. 272: 20782–
20786.
Izumi, Y., S. Sonoda, H. Yoshida, H. V. Danks, and H. Tsumuki. 2006.
Role of membrane transport of water and glycerol in the freeze tolerance of the rice stem borer, Chilo suppressalis Walker (Lepidoptera:
Pyralidae). J. Insect Physiol. 52: 215–220.
Kataoka, N., S. Miyake, and M. Azuma. 2009. Aquaporin and aquaglyceroporin in silkworms, differently expressed in the hindgut and
midgut of Bombyx mori. Insect Mol. Biol. 18: 303–314.
Kawarasaki, Y., N. M. Teets, D. L. Denlinger, and R. E. Lee, Jr. 2014.
Alternative overwintering strategies in an Antarctic midge: freezing vs.
cryoprotective dehydration. Funct. Ecol. 28: 933–943.
Kelley, J. L., J. T. Peyton, A. S. Fiston-Lavier, N. M. Teets, M. C. Yee,
J. S. Johnston, C. D. Bustamante, R. E. Lee, Jr., and D. L. Denlinger. 2014. Compact genome of the Antarctic midge is likely an
adaptation to an extreme environment. Nat. Commun. 5: 4611.
Kikawada, T., A. Saito, Y. Kanamori, M. Fujita, K. Snigorska, M.
Watanabe, and T. Okuda. 2008. Dehydration-inducible changes
in expression of two aquaporins in the sleeping chironomid, Polypedilum vanderplanki. Biochim. Biophys. Acta 1778: 514-520.
Li, A., J. B. Benoit, G. Lopez-Martinez, M. A. Elnitsky, R. E. Lee, Jr.,
and D. L. Denlinger. 2009. Distinct contractile and cytoskeletal
protein patterns in the Antarctic midge are elicited by desiccation and
rehydration. Proteomics 9: 2788 –2797.
Liu, K., H. Tsujimoto, S.-J. Cha, P. Agre, and J. L. Rasgon. 2011.
Aquaporin water channel AgAQP1 in the malaria vector mosquito
Anopheles gambiae during blood feeding and humidity adaptation.
Proc. Natl. Acad. Sci. USA 108: 6062– 6066.
Lopez-Martinez, G., J. B. Benoit, J. P. Rinehart, M. A. Elnitsky, R. E.
Lee, Jr., and D. L. Denlinger. 2009. Dehydration, rehydration, and
overhydration alter patterns of gene expression in the Antarctic midge,
Belgica antarctica. J. Comp. Physiol. B 179: 481– 491.
Lopez-Martinez, G., M. Meuti, and D. L. Denlinger. 2012. Rehydration driven RNAi: a novel approach for effectively delivering dsRNA
to mosquito larvae. J. Med. Entomol. 49: 215-218.
Mali, B., M. A Grohme, F. Förster, T. Dandekar, M. Schnölzer, D.
AQUAPORINS IN THE ANTARCTIC MIDGE
Reuter, W. Wełnicz, R. O. Schill, and M. Frohme. 2010. Transcriptome survey of the anhydrobiotic tardigrade Minesium tardigradum in comparison with Hypsibius dujardini and Richtersius coronifer.
BMC Genomics 11: 168.
Marusalin, J., B. J. Matier, M. R. Rheault, and A. Donini. 2012.
Aquaporin homologs and water transport in the anal papillae of the
larval mosquito, Aedes aegypti. J. Comp. Physiol. B 182: 1047–1056.
Michaud, M. R., J. B. Benoit, G. Lopez-Martinez, M. A. Elnitsky, R. E.
Lee, Jr., and D. L. Denlinger. 2008. Metabolomics reveals unique
and shared metabolic changes in response to heat shock, freezing and
desiccation in the Antarctic midge, Belgica antarctica. J. Insect
Physiol. 54: 645– 655.
Murata, K., K. Mitsuoka, T. Hirai, T. Walz, P. Agre, J. B. Heymann,
A. Engel, and Y. Fujiyoshi. 2000. Structural determinants of water
permeation through aquaporin-1. Nature 407: 599 – 605.
Nagae, T., S. Miyake, S. Kosaki, and M. Azuma. 2013. Identification
and characterization of a functional aquaporin water channel (Anomala
cuprea DRIP) in a coleopteran insect. J. Exp. Biol. 216: 2564 –2572.
Philip, B. N., S.-X. Yi, M. A. Elnitsky, and R. E. Lee, Jr. 2008.
Aquaporins play a role in desiccation and freeze tolerance in larvae of
the goldenrod gall fly, Eurosta solidaginis. J. Exp. Biol. 211: 1114 –
1119.
Philip, B. N., A. J. Kiss, and R. E. Lee, Jr. 2011. The protective role
of aquaporins in the freeze-tolerant insect Eurosta solidaginis: functional characterization and tissue abundance of EsAQP1. J. Exp. Biol.
214: 848 – 857.
Preston, G. M., J. S. Jung, W. B. Guggino, and P. Agre. 1993. The
mercury-sensitive residue at cysteine 189 in the CHIP28 water channel.
J. Biol. Chem. 268: 17–20.
Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406 –
425.
Schokraie, E., U. Warnken, A. Hotz-Wagenblatt, M. A. Grohme, S.
Hengherr, F. Förster, R. O. Schill, M. Frohme, T. Dandekar, and
M. Schnölzer. 2012. Comparative proteome analysis of Milnesium
tardigradum in early embryonic state versus adults in active and
anhydrobiotic state. PLoS One 7: e45682.
Spring, J. H., S. R. Robichaux, and J. A. Hamlin. 2009. The role of
aquaporins in excretion in insects. J. Exp. Biol. 212: 358 –362.
Staniscuaski, F., J.-P. Paluzzi, R. Real-Guerra, C. R. Carlini, and I.
57
Orchard. 2013. Expression analysis and molecular characterization
of aquaporins in Rhodnius prolixus. J. Insect Physiol. 59: 1140 –1150.
Strong, J. 1967. Ecology of terrestrial arthropods at Palmer Station,
Antarctic Peninsula. Pp. 357–371 in Entomology of Antarctica, J. L.
Gressit, ed. American Geophysical Union, Washington DC.
Sugg, P., J. S. Edwards, and J. Baust. 1983. Phenology and life history
of Belgica antarctica, an Antarctic midge (Diptera: Chironomidae).
Ecol. Entomol. 8: 105–113.
Tamura, K., G. Stecher, D. Peterson, A. Filipski, and S. Kumar. 2013.
MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol.
Biol. Evol. 30: 2725–2729.
Tatsumi, K., S. Tsuji, H. Miwa, T. Morisaku, M. Nuriya, M. Orihara,
K. Kaneko, H. Okano, and M. Yasui. 2009. Drosophila big brain
does not act as a water channel, but mediates cell adhesion. FEBS Lett.
583: 2077–2082.
Teets, N. M, J. T. Peyton, H. Colinet, D. Renault, J. L. Kelley, Y.
Kawarasaki, R. E. Lee, Jr., and D. L. Denlinger. 2012. Gene
expression changes governing extreme dehydration tolerance in an
Antarctic insect. Proc. Natl. Acad. Sci. USA 109: 20744 –20749.
Teets, N. M., Y. Kawarasaki, R. E. Lee, Jr., and D. L. Denlinger. 2013.
Expression of genes involved in energy mobilization and osmoprotectant synthesis during thermal and dehydration stress in the Antarctic
midge, Belgica antarctica. J. Comp. Physiol. B 183: 189 –201.
Usher, M. B., and M. Edwards. 1984. A dipteran from south of the
Antarctic Circle: Belgica antarctica (Chironomidae) with a description
of its larva. Biol. J. Linn. Soc. 23: 19 –31.
Wallace, I. S., A. J. Shakesby, J. H. Hwang, W. G. Choi, N. Matı́nková,
A. E. Douglas, and D. M. Roberts. 2012. Acyrthosiphon pisum
AQP2: a multifunctional insect aquaglyceroporin. Biochim. Biophys.
Acta 1818: 627– 635.
Yi, S.-X., J. B. Benoit, M. A. Elnitsky, N. Kaufmann, J. L. Brodsky,
M. L. Zeidel, D. L. Denlinger, and R. E. Lee, Jr. 2011. Function
and immune-localization of aquaporins in the Antarctic midge Belgica
antarctica. J. Insect Physiol. 57: 1096 –1105.
Zeng, B., G.-L. Chen, and S.-Z. Xu. 2012. Divalent copper is a potent
extracellular blocker for TRPM2 channel. Biochem. Biophys. Res.
Commun. 424: 279 –284.
Zhang, Y. 2008. I-TASSER server for protein 3D structure prediction.
BMC Bioinformatics 9: 40.