Evolution of novel functions: cryptocyanin helps build new exoskeleton in Cancer magister
1 Oregon Institute of Marine Biology, University of Oregon, Box 5389,
Charleston, OR 97420, USA
2 Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672,
USA
* Author for correspondence (e-mail: nterwill{at}darkwing.uoregon.edu)
Accepted 26 April 2005
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Summary |
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Key words: Cancer magister, cryptocyanin, hemocyanin, ecdysis, exoskeleton, Crustacea
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Introduction |
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Hemocyanin, the blue blood protein of arthropods and molluscs, reversibly
binds oxygen at its highly conserved copper-binding sites and supplies tissues
with oxygen (Van Holde and Miller,
1995). In C. magister, the hemocyanin circulates as
two-hexamer 25S and one-hexamer 16S oligomers in the hemolymph, and the
oligomers are composed of a total of six unique polypeptide subunits
(Ellerton et al., 1970
;
Larson et al., 1981
). The
hemocyanin undergoes an ontogenic change in subunit composition during
development from megalopa to adult crab, which is accompanied by corresponding
changes in oxygen affinity (Terwilliger
and Brown, 1993
; Terwilliger
and Ryan, 2001
; Terwilliger
and Terwilliger, 1982
). Three of the subunits, Hc subunits 1, 2
and 3, are constitutive, while Hc subunits 4, 5 and 6 are progressively
recruited and upregulated during development. Cryptocyanin has a similar
sequence and hexameric quaternary structure and is phylogenetically closely
related to hemocyanin (Burmester,
1999
; Terwilliger et al.,
1999
). Cryptocyanin lacks several of the six critical
copper-binding histidines, however, and has lost the ability to bind oxygen.
Despite this loss of function, cryptocyanin continues to be synthesized in
high concentrations. Its presence in the hemolymph of the crab, particularly
at specific times in the molt cycle, indicates that it has been exploited to
carry out new functions.
Ecdysis, or molting of the exoskeleton, is a challenging requirement for
growth in the members of the Ecdysozoa. The process requires periodic and
precise control to simultaneously form a new exoskeleton and remove the old
one (Skinner, 1985). To
understand how cryptocyanin is involved in the molting process, we
investigated where and when this member of the hemocyanin gene family is
synthesized and explored its function. We compared the patterns of protein and
mRNA expression of hemocyanin and cryptocyanin in cohorts of juvenile C.
magister as the individual crabs progressed from ecdysis to postmolt,
intermolt, premolt and the next ecdysis through multiple molt cycles. We also
carried out eyestalk ablation experiments to assess the hormonal regulation of
cryptocyanin production. The early juvenile stages of C. magister
molt quickly and thus provide insights into the functions of these proteins
over a brief time frame, in contrast to the annual molt cycle of the adult
crab. The results provide a marked example of physiological evolution, where a
gene duplication and subsequent mutation has resulted in two proteins with
dynamically different patterns of regulation and distinct functions.
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Materials and methods |
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Hemolymph samples for analysis of cryptocyanin and hemocyanin levels were
drawn three times a week from each crab, starting at day 0, 2nd instar. One
µl of hemolymph was electrophoresed on non-dissociating, non-denaturing 5%
polyacrylamide gels, pH 7.4 (pH 7.4 PAGE;
Terwilliger and Terwilliger,
1982). The protein bands were quantified by video gel analysis and
imaging software (Jandel Scientific, San Rafael, CA, USA). Standard curves
were obtained by first lyophilizing purified cryptocyanin, two-hexamer 25S
hemocyanin and one-hexamer 16S hemocyanin
(Terwilliger, 1999
;
Terwilliger et al., 1999
) and
determining their extinction coefficients. Purified proteins were quantified
spectrophotometrically, and a dilution series of each was run on pH 7.4 PAGE.
Because the duration of the molt cycle increases and becomes progressively
asynchronous with age of the crab, experimental results were graphed as a
percentage of the molt cycle; increasing molt cycle duration is represented on
the x-axis (Fig. 1).
Additional cohorts have been raised and monitored through the 12th instar,
with consistent patterns of hemocyanin and cryptocyanin fluctuations during
the molt cycle.
|
Cryptocyanin was amplified with non-degenerate primers based on the
sequence of C. magister cryptocyanin (GenBank AF091261;
Terwilliger et al., 1999);
cryptocyanin (Cc) sense AGA CCC AGA TTG CCG AAG G and Cc antisense GCA CGC CTG
GGG GAG TAT C. Hemocyanin primers were degenerate and based on C.
magister N-terminal sequence for hemocyanin (Hc) subunits 1 and 2
(Durstewitz and Terwilliger,
1997a
) and the crustacean hemocyanin Copper A site; Hc sense CAY
MGN CAR CAR GCN GTB AAY MG and Hc antisense CGV CAN GTN ARY TGR TGR TGV ACC
CA. For normalization, a set of degenerate primers for arthropod
tubulin was designed: tubulin sense ACK GCY GTB GAR CCS TAC and tubulin
antisense TCV ARS GCR GCC ARR TCY TC.
Real-time quantitative PCR was carried out at Mount Desert Island Biological Laboratory using a Mx4000 Multiplex Quantitative PCR System and QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA, USA). For each of the three target mRNAs, a reference cDNA from intermolt hepatopancreas was serially diluted to show the empirical relationship between threshold cycle (Ct) and template abundance. This relationship was then used as the basis for calculating relative mRNA expression in the test samples. Tubulin mRNA in hepatopancreas tissue was constant throughout the molt cycle, and mRNA levels for hemocyanin and cryptocyanin were normalized to that of tubulin mRNA.
Sequence of C. magister hemocyanin subunits 1 and 2
The sequences of hemocyanin subunits 1 and 2 of C. magister were
determined in order to develop hemocyanin-specific in situ probes.
C. magister hepatopancreas cDNA was prepared, and a 700 bp PCR
product was amplified using the Hc subunits 1 and 2 sense and antisense
primers as described above. Based on the sequence of this product,
gene-specific primers were designed for amplification of the 3' and
5' ends using the SMARTTM RACE cDNA Amplification Kit (BD
Biosciences-Clontech, San Jose, CA, USA). Products were either directly
sequenced or cloned into a pCR4-TOPO vector (TOPO TA cloning kit by
Invitrogen). DNA was sequenced by the University of Oregon Sequencing Facility
and the Oregon State University Center for Gene Research and Biotechnology.
This resulted in the complete cDNA sequence of C. magister Hc subunit
1 (GenBank AY861676).
The sequence of C. magister Hc subunit 2 was obtained by using a set of degenerate hemocyanin primers designed against the conserved amino acid sequences MNEGEFVYA and QGDPHGKF. A 750 bp product was cloned, and a number of the resulting colonies were sequenced. One was very similar to C. magister Hc subunit 1 but different enough to allow the design of gene-specific primers for 3' and 5' RACE. This yielded the complete sequence of C. magister Hc subunit 2 (GenBank AY861677).
In situ hybridization
Gene-specific sense and antisense oligonucleotide probes were designed and
synthesized by GeneDetect
(http://www.GeneDetect.com).
The cryptocyanin-specific probe hybridized to nucleotides 16501697 of
C. magister cryptocyanin
(Terwilliger et al., 1999).
The hemocyanin-specific probes were a mixture of Hc subunits 12
(described above) and Hc subunit 6
(Durstewitz and Terwilliger,
1997a
) specific probes. The subunit 12 probe was generated
against nucleotides 159206, a region where the two subunits are
identical, and the subunit 6 probe was designed against nucleotides
17591806 (GenBank U48881). A poly-dT probe was used as a control for
mRNA quality. The probes were 3' labeled with the DIG Oligonucleotide
Tailing Kit (Roche, Indianapolis, IN, USA).
All solutions were prepared with DEPC-treated water. Freshly dissected tissues were fixed in R-F Fixative (Hasson, 1997) that included 1x PBS. The tissues were dehydrated, embedded in Paraplast-Plus and sectioned at 5 µm. Rehydrated slides were postfixed in 4% paraformaldehyde, acetylated and treated with proteinase K. Approximately 20 ng of probe was added to the prehybridization buffer for each slide, and hybridization was carried out overnight in a humid chamber at 37°C. Post-hybridization washes of 1x SSC and 0.5x SSC were done at 50°C. The DIG-labeled oligonucleotides were detected with an anti-DIG-alkaline phosphatase antibody. The slides were stained with NBT/BCIP staining solution containing 1 mmol l-1 levamisole.
To test for specificity of probe binding, both sense and antisense gene-specific oligonucleotide probes were used. As a control to ensure that the positively reacting antisense probes were specifically binding to RNA, RNase treatment of sections was done prior to hybridization with the antisense probe.
Immunohistochemistry
Crabs were fixed in Bouin's solution overnight at room temperature,
embedded in Paraplast-Plus and sectioned at 10 µm. After rehydration,
sections were blocked with 5% milk and incubated with either a hemocyanin- or
cryptocyanin-specific monoclonal antibody
(Terwilliger et al., 1999),
which had been prepared at the University of Oregon Monoclonal Antibody
Facility. Reaction with a biotinylated anti-mouse secondary antibody (Sigma,
St Louis, MO, USA) was followed by detection with the peroxidase Vectastain
Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA), used according to
the recommended procedures. The sections were then stained with the DAB
Substrate Kit (Vector Laboratories). After staining, the sections were washed
and lightly counterstained with hematoxylin.
Monoclonal antibody specificity was determined by western blot analysis
against a range of purified proteins, including C. magister
hemocyanin and cryptocyanin, plus crab hemolymph
(Terwilliger, 1999;
Terwilliger et al., 1999
).
Antibody specificity was tested on tissue sections by preadsorption of each of
the monoclonal antibodies, anti-hemocyanin and anti-cryptocyanin, with both
purified antigens, hemocyanin and cryptocyanin, before incubation with tissue
sections (Beltz and Burd,
1989
). Preadsorption of each antibody with its corresponding
antigen eliminated immunoreactivity, while preadsorption with the alternate
protein gave a positive reaction, confirming specificity of each antibody.
Eyestalk ablation
The eyestalks were surgically removed from juvenile 6th instar C.
magister 2 days postmolt. The animals were maintained in running seawater
and fed as previously described. Hemolymph samples were taken every other day
and electrophoresed on pH 7.4 PAGE. Hemolymph proteins were quantified as
described above.
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Results |
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To better understand the pattern of regulation of these two proteins, we
analyzed the abundance of cryptocyanin and hemocyanin mRNA in three tissues of
juvenile C. magister during a complete molt cycle using real-time
quantitative PCR. In hepatopancreas tissue, expression levels of hemocyanin
mRNA were relatively constant, with slight increases in postmolt and premolt
crabs (Fig. 2A). The
differences were approximately onefold, similar in magnitude to those reported
for Callinectes sapidus hemocyanin mRNA
(Brouwer et al., 2002). By
contrast, cryptocyanin mRNA in the same C. magister crabs was
expressed at levels more than 20-fold higher than hemocyanin
(Fig. 2B). Cryptocyanin mRNA
levels were low immediately postmolt and gradually increased. Cryptocyanin
mRNA was consistently high during the mid third of the molt cycle and then
decreased during the latter third. There was essentially no expression of
either cryptocyanin or hemocyanin mRNA in epidermis or leg muscle over the
molt cycle (data not shown).
|
To identify which cells in the hepatopancreas tubules or surrounding
connective tissue were synthesizing hemocyanin or cryptocyanin, we carried out
in situ hybridization studies. We determined the cDNA sequences of
hemocyanin subunits 1 and 2, both constitutive subunits that are expressed in
hemocyanins from megalopas, juveniles and adult C. magister, and used
those as well as the sequence of hemocyanin subunit 6, a developmentally
expressed subunit (Durstewitz and
Terwilliger, 1997b) to design the hemocyanin-specific probes.
Since there are many areas of overlapping similarity between hemocyanin and
cryptocyanin, the probe sequences were carefully chosen from unique regions of
each cDNA. To ensure specificity of each probe, we tested it as a PCR primer
along with a gene-specific primer for either hemocyanin or cryptocyanin. In
each case, we obtained a single product only with the corresponding
gene-specific primer. In situ results using the hemocyanin antisense
probes localized hemocyanin mRNA to the tubules of the hepatopancreas
(Fig. 3B). It was expressed in
the basal portions of the R cells, but was not evident in B cells or F cells.
Cryptocyanin mRNA expression was also found only in R cells in the tubules of
the hepatopancreas (Fig. 3C).
The cryptocyanin in situ reaction was much stronger than that of
hemocyanin. No reactivity was observed when the tissues were incubated with
hemocyanin or cryptocyanin sense probes
(Fig. 3B,C) or when the tissues
were preincubated with RNase prior to exposure to the antisense probes (data
not shown). No other tissues, including muscle, connective tissue, reserve
cells, and epidermis, showed any reaction to the hemocyanin or cryptocyanin
antisense probes, indicating that both hemocyanin and cryptocyanin mRNA
expression are specifically localized in the same cell type, the R cells of
the hepatopancreas.
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During premolt, large, rounded `protein cells' or `reserve cells'
(Cuenot, 1893;
Johnson, 1980
;
Travis, 1965
) accumulated in
the connective tissue, especially below the epidermis. In early premolt, these
cells did not react with cryptocyanin antibody, but immediately before and
during ecdysis, the reserve cells became positive, staining golden and then
dark brown (Fig. 5). In the
next few days postmolt, as hemolymph cryptocyanin decreased, the reserve cells
were smaller, irregularly shaped and markedly fewer in number. By 3 days
postmolt, no cryptocyanin was visible anywhere, nor were reserve cells
apparent. At 6 days postmolt, cryptocyanin synthesis resumed, as evidenced by
its reappearance in hepatopancreas cells and hemolymph. The protein in the
hemolymph gradually increased in staining intensity until the next ecdysis at
day 16 into 2nd instars.
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The apparent hormonal regulation of cryptocyanin production and utilization
was further explored through eyestalk ablation experiments on juvenile C.
magister. Crustacean molt inhibiting hormone (MIH) is synthesized and
stored in the X organ/sinus gland complex in the eyestalks of a crab, and
removal of the eyestalks stimulates premature ecdysis
(Chang et al., 1993;
Skinner, 1985
;
Zeleny, 1905
). Eyestalks were
removed from 6th-instar juvenile crabs two days after they had molted from the
5th instar. The eyestalk-ablated juvenile crabs produced typical hemolymph
patterns of hemocyanin and cryptocyanin and underwent premature ecdysis in the
first post-ablate molt cycle (Fig.
6A). In the second post-ablate molt cycle (7th instar), hemolymph
levels of hemocyanin in the ablated crabs returned to normal, but cryptocyanin
did not reappear. Unablated 7th-instar control crabs underwent normal
expression of cryptocyanin and hemocyanin
(Fig. 6B). These results
indicate that molting hormones have a major regulatory effect on cryptocyanin
expression and catabolism.
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Discussion |
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The amounts of cryptocyanin and hemocyanin in the hemolymph during the molt
cycle differ both temporally and quantitatively. Although some dilution of
total hemolymph protein occurs immediately after molting due to uptake of
water (Mykles, 1980), the
changes in concentration of cryptocyanin in the hemolymph from premolt to
postmolt are much greater than those of hemocyanin. In addition, after
ecdysis, cryptocyanin levels remain low longer than hemocyanin levels.
Therefore, the differences between the two proteins are not due to a simple
dilution effect. Early juvenile crabs molt at least 10 times in the first year
and have brief intermolt periods, so the crabs undergo quick transitions from
postmolt to premolt. Their postmolt levels of hemolymph cryptocyanin are low,
while in the long intermolt period of the adult crab, cryptocyanin is not
detectable. These patterns of protein levels in the hemolymph reflect the
abundance and fluctuations of cryptocyanin and hemocyanin mRNA during the molt
cycle as well.
Hemocyanin and cryptocyanin mRNA are both expressed in the hepatopancreas
tissues of C. magister
(Durstewitz and Terwilliger,
1997b; Terwilliger et al.,
1999
). Our preliminary histochemistry had localized cryptocyanin
protein in connective tissue cells surrounding the hepatopancreas tubules and
below the epidermis, as well as in the hemolymph. In the real-time PCR
studies, therefore, we assayed hepatopancreas tissue, epidermis and, as a
control, leg muscle. Cryptocyanin mRNA was not amplified in epidermis tissue,
indicating that cryptocyanin is not synthesized in epidermal cells or
underlying connective tissue. It is clear that hepatopancreas cryptocyanin
synthesis is markedly upregulated during early premolt and is downregulated as
the crab approaches ecdysis, whereas hemocyanin seems to be synthesized at a
low, relatively constant level. The decrease in cryptocyanin mRNA during late
premolt preceded the decrease in hemolymph protein, consistent with
translational regulation of cryptocyanin synthesis and/or a fairly long
half-life of cryptocyanin protein. Therefore, we find major differences in
tissue-specific abundance and timing between cryptocyanin and hemocyanin mRNA
and protein during the molt cycle.
We had suspected that different cell types in the hepatopancreas tubules or
surrounding connective tissue would be responsible for cryptocyanin and
hemocyanin synthesis because of the differences in expression patterns. The
crab hepatopancreas or digestive gland is composed of two major branches that
originate laterally at the juncture between the cardiac and the pyloric
stomach. Each branch further subdivides into three lobes, which continue
branching into blind-ended tubules and fill the body space on either side of
the stomach. The hepatopancreas tubules are loosely held together and
enveloped in webby strands of cellular connective tissue
(Fig. 3A). The tubule walls are
composed of multiple cell types, R (resorptive), F (fibrillar), B
(blister-like), M (midget) and E (embryonic), that have been characterized on
the basis of morphology, position and histochemistry
(Al-Mohanna and Nott, 1989;
Jacobs, 1928
). Multiple
functions have been attributed to the hepatopancreas and its cell types,
including synthesis and secretion of digestive enzymes, absorption of
nutrients and storage of heavy metals. It is not certain whether the cell
types represent stages of differentiation or unique cell lines
(Gibson and Barker, 1979
;
Icely and Nott, 1992
;
Vogt, 1985
).
Our in situ results identify the R cells of the hepatopancreas as
the site of synthesis of both cryptocyanin and hemocyanin. We suggest that the
two proteins are secreted basally into the blood vessels located between and
surrounding the hepatopancreas tubules in C. magister, an idea
supported by ultrastructural evidence for pinocytotic trafficking at the basal
surface of the R cell in Penaeus semisulcatus
(Al-Mohanna and Nott, 1987).
The cryptocyanin in situ reaction was much stronger than that of
hemocyanin, consistent with the higher levels of cryptocyanin mRNA in the
real-time PCR studies (Fig. 2B)
and higher protein levels in the hemolymph
(Fig. 1) of the premolt
crab.
The rapid disappearance of cryptocyanin from the hemolymph at ecdysis
stimulated us to see how the protein is utilized by the crab. We found that
cryptocyanin plays a significant role in formation of the new exoskeleton.
Secretion of a crab's new exoskeleton across the epidermis begins during
premolt, shortly after apolysis of the old exoskeleton is initiated but before
the old exoskeleton is shed (Skinner,
1962; Williams et al.,
2003
). Formation of the new exoskeleton occurs in layers, with
first a thin epicuticle secreted into the extracellular space between
epidermis and old exoskeleton, then a thicker exocuticle layer appearing
beneath the new epicuticle during late premolt. Immediately after ecdysis,
while the new exoskeleton is still soft, unfolding and expanding to a larger
size than the old exoskeleton had been, the crab continues to secrete more
exocuticle. This is the period when the fluid-inflated soft exoskeleton serves
as a hydrostatic skeleton (deFur et al.,
1985
; Taylor and Kier,
2003
). Within hours postmolt, secretion of the endocuticle layer
begins beneath the new exocuticle. This layer continues to increase in
thickness, while calcification and sclerotization of the new exoskeleton
progress, until the crab enters intermolt.
Our immunohistochemical results provide morphological evidence that
cryptocyanin is secreted into the hemolymph, becomes incorporated into the
extracellular matrix around the internal organs and also moves across the
epidermis into the extracellular matrix of the new exoskeleton in synchrony
with the molt cycle. Thus, cryptocyanin, synthesized in the hepatopancreas,
contributes to the structure of the exoskeleton, along with cuticular proteins
(Coblentz et al., 1998;
Compere et al., 2002
)
synthesized by the epidermis. When the postmolt portion of the exocuticle and
then the endocuticle layers appear immediately after ecdysis, they are
initially immunoreactive against cryptocyanin. The subsequent loss of
immunoreactivity in the exocuticle that we observe could be the result of
specific proteolytic cleavage, although it is more likely due to cross-linking
during sclerotization, since proteins are known to cross-link during this
process in both crustaceans and insects.
Insect hexamerins, additional members of the hemocyanin gene family,
resemble cryptocyanin in several ways
(Beintema et al., 1994;
Telfer and Kunkel, 1991
;
Terwilliger, 1999
). These
hexameric hemolymph proteins also lack copper and therefore do not participate
in oxygen transport or phenoloxidase activity. The concentrations of some
hexamerins fluctuate with molting cycles, and this is regulated by
ecdysteroids and juvenile hormones. In addition, while functional diversity of
hexamerins is high, it has been demonstrated in several insect species that
certain hexamerins are incorporated into the nascent cuticle
(Konig et al., 1986
;
Webb and Riddiford, 1988
).
Cryptocyanin that is not incorporated into the exoskeleton is removed from
the hemolymph by the reserve cells soon after molting. Earlier studies had
postulated that the reserve cells were the site of hemocyanin synthesis, based
on electron microscopy of large crystalline inclusions in the cells
(Ghiretti-Magaldi et al.,
1977). Our in situ and our immunohistology studies
indicate that reserve cells have neither hemocyanin nor cryptocyanin mRNA but
do contain cryptocyanin at ecdysis. The crystalline structures identified by
Ghiretti-Magaldi and others are probably cryptocyanin, not hemocyanin.
Endocytosis of cryptocyanin by the reserve cells at ecdysis may be the result
of a hormonally regulated appearance of cryptocyanin-specific receptors on the
reserve cells, in a manner similar to the uptake of hexamerins in certain
insects (Haunerland, 1996
). We
suggest that the reserve cells metabolize the cryptocyanin, whose amino acids
may be used for energy and tissue growth in the newly molted crab, and then
decrease in size and number until the next premolt.
Hormonal regulation of cryptocyanin production and utilization is supported by eyestalk ablation experiments on juvenile C. magister. The resumption of hemocyanin levels but not cryptocyanin in the hemolymph after the eyestalks were removed and the crabs underwent ecdysis point to a requirement for ecdysteroid or X organ/sinus gland peptidergic regulation of cryptocyanin expression and catabolism.
Are there other roles for cryptocyanin in the molting crab? Recent reports
indicate that the C-terminal domain of hemocyanin can be cleaved and can
function as a bacteriocidal peptide in shrimp and crayfish
(Destoumieux-Garzon et al.,
2001; Lee et al.,
2003
). Cryptocyanin may have a similar function, especially in the
soft postmolt exoskeleton. Cryptocyanin may have other functions as well,
including calcium uptake and/or an osmotic role during ecdysial water
uptake.
Hemocyanin and cryptocyanin are two closely related members of the same gene family that are synthesized in the same tissue-specific cells and are secreted into the hemolymph. Hemocyanin, carrying out its main role as oxygen transporter, is maintained at a relatively constant level in the hemolymph. Cryptocyanin, on the other hand, has evolved as a structural protein, and its dynamic regulation waxes and wanes with the molt cycle. The contrasts between the two gene products illustrate how a gene duplication of a copperoxygen protein and its subsequent mutation may work in concert with the evolution of new regulatory mechanisms, leading to the assumption of new functions.
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Acknowledgments |
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References |
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