Giant wood spider Nephila pilipes alters silk protein in response to prey variation
1 Department of Life Sciences, Tunghai University, Taichung 407,
Taiwan
2 Center for Tropical Ecology and Biodiversity, Tunghai University, Taichung
407, Taiwan
* Author for correspondence (e-mail: spider{at}mail.thu.edu.tw)
Accepted 13 December 2004
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Summary |
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Key words: spider silk, dragline, major ampullate gland, Nephila
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Introduction |
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Recent research has revealed that dragline silks are composed of the
products of at least two genes: major ampullate spidroin 1 (MaSp1;
Xu and Lewis, 1990) and major
ampullate spidroin 2 (MaSp2;
Hinman and Lewis, 1992
). The
MaSp1 protein exhibits poly (GA), poly (A) and poly (GGX) motifs (G, glycine;
A, alanine; X, any amino acid; Xu and
Lewis, 1990
). Among these motifs, poly (GA) and poly (A) are major
components of the ß-sheet crystal structure
(Gosline et al., 1999
) and
they are regarded as being responsible for the tensile strength of the silk
(Winkler and Kaplan, 2000
).
The GGX repeat regions might form helix structures that function as linkages
between crystalline and non-crystaline portions of the molecule, or function
to help align protein molecules in the silk
(Hayashi et al., 1999
). By
contrast, the MaSp2 protein exhibits GPGXX and GPGQQ motifs (P, proline; Q,
glutamine; Hayashi et al.,
1999
). These motifs formed ß-turn spirals of the dragline
silk (Hayashi et al., 1999
)
and, thus, are responsible for the extensibility of the silk. The relative
composition of amino acids and secondary structures determines the strength
and extensibility of the silk, which in turn will greatly affect the type and
efficiency of the prey trapped by the web
(Olive, 1980
; Craig,
1987
,
1992
). In this study we
investigated whether spiders will physiologically adjust the protein of silk
when they encounter changes in prey type.
Recently, orb-weaving spiders of the genus Nephila have been
extensively used in silk-related studies ranging from spinning process
(Vollrath and Knight, 2001),
physical properties (Vollrath,
1999
,
2000
), molecular biology
(Winkler and Kaplan, 2000
) to
genetic engineering (Fahnestock et al.,
2000
). An understanding of whether or not Nephila spiders
alter silk protein in response to prey variation will provide important
insights to future silk-related studies. In the present study, we first
conducted field surveys to see whether a spatial variation in silk amino acid
percentages and prey compositions existed in wild populations. Secondly, we
conducted feeding manipulations by giving spiders different types of prey
(dipterans vs orthopterans) to see if these treatments would cause
changes in amino acid percentages and secondary structures of the silk.
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Materials and methods |
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Survey of prey composition of wild populations
This part of our study aimed to understand whether or not N.
pilipes exhibited spatial variation in the composition of prey consumed.
We recorded the foraging histories of N. pilipes populations in Lanyu
in 1999, in Taipei and Fouyenshan in 2000, and in Fushan in 2001. All these
censuses lasted about 10 days and were conducted in August of each year, at
which time N. pilipes were the most abundant. During the censuses,
the web sites of about 20 female spiders were marked and monitored for trapped
prey hourly between 8:00 h to 18:00 h. The number and taxonomic orders of the
trapped prey were recorded. A pair-wise comparison of ratios of different
orders of insects caught by N. pilipes in these localities was
performed using 2 tests of homogeneity to see whether prey
composition of this spider in Taiwan exhibits spatial heterogeneity.
Effect of diet on dragline amino acid compositions
In this part of our study, we used female N. pilipes (body length
15-20 mm) to examine the effect of manipulating diet on silk amino acid
percentages. Spiders were collected from secondary forests in central Taiwan
and were reared in large cages made of wooden frames and screens
(40x40x30 cm). We kept N. pilipes in large cages to
facilitate their normal web building and recycling behaviors. The caged
spiders were placed in an outdoor screenhouse (5x5x3 m) thus the
spiders were kept in physical conditions similar to those of their normal
habitats. Craig et al. (2000)
demonstrated that Argiope keyserlingi significantly altered the
amount of serine in dragline when the diet was switched from fly (Diptera) to
crickets (Orthoptera). Therefore, we randomly assigned 20 caged spiders into
two groups (N=10 each) and fed them with either crickets or flies of
equal biomass. Before the food manipulation all the spiders were first fed
with mealworms till they had constructed three orbs. Before these spiders
received food manipulation their draglines were force-reeled. The dragline
amino acid percentages of spiders designated to be fed with either cricket or
fly were compared to make sure that these spiders exhibited similar dragline
composition before the food treatment. Spiders in the first group were fed
with one cricket each day and in the second group spiders were fed with equal
weights of flies (the weight of one cricket was about that of three flies).
After the caged spiders had constructed seven more webs, dragline silks were
force-reeled, and amino acid percentages were analyzed and statistically
compared.
Effect of diet on dragline secondary structures
Fourier Transform Infrared (FTIR) spectroscopy is a powerful tool in
studying the folding conformation of peptide chains and secondary structures
of proteins (Goeden-Wood et al.,
2003; Sethuraman et al., 2003) and this method had been used in
spider silk researches (Wilson et al.,
2000
; Chen et al.,
2002
). In this study, the FTIR spectra of draglines produced by
N. pilipes fed with different prey types were analyzed to compare the
percentages of various secondary structures. To obtain dragline silk FTIR
spectra, an ATR-Perkin Elmer Spectrum GX FTIR spectroscopy equipped with MCT
detector was used. The resolution of MCT detector was 4 cm-1, and
the operation condition of scan number was 128. We used the program Peakfit
4.11 to facilitate the separation of peaks and assignment of the amide I bands
(1600-1700 cm-1). The proportion of secondary structures was
calculated by integrating the area of each peak and then normalizing to the
total area under the spectral curve. A MANOVA test was used to compare the
percentages of various secondary structures in draglines of N.
pilipes fed with different prey.
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Results |
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Prey composition of wild populations
Nephila pilipes populations in four localities in Taiwan varied
considerably in composition of prey ingested. In all four localities, insects
of the order Diptera, Hymenoptera and Coleoptera were the major prey
(Fig. 2). Compared with other
insect orders, Orthoptera was less represented in the diet. Although insects
of these four orders were the major prey in all four populations, results of
pairwise 2 tests of homogeneity showed that the relative
proportion of different insect orders differed significantly among these
populations (Table 2).
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Effect of diet on dragline amino acid compositions
After N. pilipes fed with mealworms built three webs, dragline
collected from 20 individuals designated to fed with different prey type did
not show significant variations in amino acid percentages (F=0.002
for glutamine, 0.431 for serine, 3.111 for glycine, 0.785 for alanine, 0.176
for proline, all of them P>0.05). This result indicated that
before the spiders received different type of prey, they exhibited similar
amino acid composition in their dragline. At the end of food manipulation,
complete data were available from eight spiders fed with flies and six spiders
fed with crickets. Results of the MANOVA test showed that the food
manipulation used in this study generated a significant variation in dragline
amino acid percentages. The means and standard errors of percentages of the
main dragline amino acids of spiders of the two groups are given in
Table 3. With the exception of
glycine, the percentages of the major amino acids differed significantly
between spiders fed with different prey. Compared with N. pilipes fed
with flies, those fed with cricket exhibited 40% higher glutamine and proline
but 20% lower alanine. Among the silk samples collected from spiders subjected
to feeding treatments, the abundance relationships between various major amino
acids differed (Fig. 3). A
significantly positive relationship was found between percentages of glutamine
and proline (r2=0.94, P<0.001;
Fig. 3A). Conversely,
significantly negative relationships were found between percentages of glycine
and serine (r2=0.54, P<0.001;
Fig. 3B) and between those of
alanine and proline (r2=0.88, P<0.001;
Fig. 3C).
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Effect of diet on dragline secondary structures
According to peak separation analyses conducted via Peakfit 4.11,
the FTIR spectra of N. pilipes draglines were separated into five
major peaks. We used the results of previous studies to assign these peaks to
various secondary structures. The peak at 1612.5±0.6 cm-1
was assigned as protein side chains that contained amide groups or aromatic
rings generating vibration at this region
(Tatulian et al., 1998;
Iconomidou et al., 2000
). Chen
et al. (2002
) suggested that
some helical conformations, such as
-helix and 310-helix,
had absorption bands similar to those of random coil (unordered structure) and
were sometimes not well resolved. Therefore, they assigned the absorption
peaks around 1647 cm-1 to random coil and/or helical conformation.
We followed the suggestion of Chen et al.
(2002
) and assigned the peak of
1646.8±0.4 cm-1 as random coil/helix. The peaks at
1663.5±0.2 cm-1 and 1682.1±0.3 cm-1 were
both assigned as ß-turn structures according to Huang et al.
(2003
) and the percentages
were combined in the MANOVA analysis. The peak at 1628.6±0.4
cm-1 was assigned as ß-sheet according to Goeden-Wood et al.
(2003
) and Sethuraman et al.
(2004
). Results of the MANOVA
test comparing percentages of secondary structures also showed a significant
effect by food manipulation. The means and standard errors of percentages of
secondary structures were given in Table
4. Draglines from spiders fed with crickets contained a
significantly higher percentage of ß-turn and marginally significantly
(P=0.06) lower percentage of ß-sheet structures
(Table 3).
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Discussion |
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Results of food manipulation showed that N. pilipes altered
dragline silk amino acid when they encounter a stable temporal variation of
prey type. The dragline silks produced by N. pilipes fed with
crickets had significantly higher percentages of glutamine and proline but
lower alanine. Conversely, the dragline produced by N. pilipes fed
with flies exhibited the opposite pattern. All these amino acids are major
components of various motifs of the silk protein. According to series of
studies conducted on a South American Nephila species, N.
clavipes, dragline silk is composed of the products of at least two
genes, MaSp1 and MaSp2
(Xu and Lewis, 1990;
Hinman and Lewis, 1992
), and
each exhibits different amino acid compositions and motifs. The MaSp1 protein
contains higher proportion of ß-sheet crystals comprising poly (GA) and
poly (A) motifs, both of which are regarded as responsible for the tensile
strength of the dragline. Conversely, MaSp2 is unique in containing
ß-turn structures composed of poly (GPGXX) or poly (GPGQQ) motifs, which
are related to the extensibility of dragline. While alanine is the major
component of MaSp1, proline and glutamine are relatively unique to MaSp2
proteins (Hinman and Lewis,
1992
). Currently, the full length DNA sequences of MaSp1
and MaSp2 are not available. However, based upon the published
partial sequences (
2 kb) the percentages of alanine, proline and
glutamine in MaSp1 are 28.0, 0.0 and 10.2
(Xu and Lewis, 1990
;
Hayashi and Lewis, 1998
). The
corresponding percentages in MaSp2 are 22.3, 15.5 and 12.8 respectively
(Hinman and Lewis, 1992
;
Hayashi and Lewis, 1998
).
Although these data are estimated from dragline of N. clavipes,
results of a recent study conducted by Tai et al.
(2004
) showed that the
MaSp1 sequences of N. pilipes were very similar to those of
N. clavipes. Therefore, the percentages of major amino acids in
dragline of N. pilipes should be similar to those of N.
clavipes. In addition to significant amino acid percentage differences
between two N. pilipes groups, the abundance relationships between
various major amino acids also suggested that our observed results were
generated by differential expressions of MaSp1 and MaSp2
gene products. A significantly positive relationship was found between
percentages of glutamine and proline, and significantly negative relationships
were found between glycine and serine and between those of alanine and
proline. Moreover, in all three amino acid abundance relationships in
Fig. 3, individual N.
pilipes were separated into two distinct groups according the prey type
they ingested. Such results provided supports for our hypothesized model of
differential expression of two dragline silk genes. In addition, two N.
pilipes fed with flies adopted the same proportion of amino acids as
those fed with crickets (Fig.
3). The reason for such a phenomenon might be that these two
individuals, for some reason, were not responsive to the flies they had
ingested and therefore exhibited the same gene expression and thus amino acid
abundance patterns with those fed with crickets.
Significantly higher proline and glutamine percentages in dragline of
N. pilipes fed with crickets suggested that in these silks there were
relatively higher proportion of MaSp2, and consequently more ß-turn
structures and higher extensibility. Conversely, a significantly higher
proportion of alanine in dragline of N. pilipes fed with flies
indicated a relatively higher proportion of MaSp1 protein, more ß-sheet
crystals and, thus, a higher tensile strength. Results from FTIR spectroscopy
were congruent with these predictions. Draglines from spiders fed with
crickets contained a higher percentage of ß-turn and a lower percentage
of ß-sheet. Previous studies have demonstrated that silk properties are
vital for successful prey catching (Craig,
1987,
1992
). Silks suitable for
catching large orthopterans and flying insects differ in physical properties,
and Nephila spiders may have evolved genetic plasticity of the silk
protein to cope better with both prey types. As flying insects are intercepted
by the web, they tend to pull perpendicularly away from the web. Therefore,
webs with stronger radial and spiral silks will better retain flying insects
(Olive, 1980
). Conversely,
intercepted large orthopterans tend to rip down along the panel of the web,
accumulating more and more of the sticky spiral. Orbs with higher numbers of
spirals, a larger catching area and more elastic silk will better retain this
type of prey (Olive, 1980
).
Results of this and other studies
(Robinson and Robinson, 1970
;
Nyffeler and Breene, 1991
;
Craig et al., 2001
) indicated
that the prey of orb-weaving spiders varies both spatially and temporally,
therefore, they may have evolved the ability to adjust silk protein according
to the presence of different prey type. We suggest that the relative abundance
of different prey types in the diet can induce orb-weaving spiders to adjust
relative strength and extensibility of silk by altering silk protein to
enhance the catching success.
In this study we did not perform HPLC analysis by ourselves but three lines
of evidence suggested that the analyses were accurate. First, the amino acid
composition of dragline produced by N. pilipes in this study was very
similar to those of other Nephila spiders. Second, we sent in several
major ampullate silk samples collected from Cyrtophora moluccensis
for analysis. The amino acid percentages of these samples were quite different
from that of Nephila spiders but were very similar to those reported
in Craig et al. (1999). Third,
we simultaneously sent in samples obtained from the same silking process but
diluted in different quantity of HFIP (1x vs 5x). The
amino acid percentages of these two samples were almost identical and the
differences ranged only from 0.01 to 0.5%. All evidence indicated that our
data reflected the actual percentages of amino acids in the samples.
One potential mechanism of the observed spatial variation in N.
pilipes dragline protein might be the accumulation of mutations among
isolated populations. It is possible that the unusually long and repetitive
nature of dragline genes may constantly generate variations in both DNA and
protein levels in different N. pilipes populations. Dragline silks
are encoded by MaSp1 and MaSp2, both of which are unusually
long and repetitive (Winkler and Kaplan,
2000). The length of mRNA in major ampullate glands was reported
to range from 4.4-15.5 kb (Hayashi,
2002
). The whole sequence can be divided into repetitive and
non-repetitive regions (Beckwitt and
Arcidiacono, 1994
). The repetitive regions are composed of
glycine- or alanine-rich units and a comparison of repetitive units from
different portions of both genes showed that much variation existed
(Gatesy et al., 2001
). The
unusually long and repetitive nature of dragline genes may greatly enhance the
occurrence of point mutations, biased base composition, replication error and
unequal crossing-over (Beckwitt et al.,
1998
; Hayashi,
2002
). If N. pilipes populations are isolated, it is
likely that the aforementioned genetic events generating rapid evolution of
dragline genes would result in local divergences in dragline proteins.
However, a recent study by Lee et al.
(2004
) on population genetic
structure of N. pilipes in Taiwan and neighboring islands suggested
that the populations were not isolated. Instead, owing to the excellent aerial
dispersal by the ballooning ability of N. pilipes similar to those of
other orb weavers (Dean and Sterling,
1985
; Decae, 1987
;
Greenstone et al., 1987
),
there were strong gene flows even between populations across high mountain
ranges (4000 m) and oceans. Therefore, the level of gene flow should be rather
high and local population divergence is unlikely to occur. The observed
variation in dragline amino acid composition is probably generated by
differential expression of MaSp1/MaSp2 gene products in
response to local prey composition variations.
Orb-weaving spiders have long been known to be able to adjust dragline
properties behaviorally in response to different foraging conditions and
results of this present study demonstrated that they can also adjust silk
proteins. Several studies have already demonstrated that spiders can fine-tune
mechanical properties of webs without changing silk proteins. Vollrath and
Köller (1996) reported
that when the body weight of orb-weavers was artificially increased, the
spiders initially increased the diameter of the radial threads and
subsequently doubled or tripled the number of radial threads when building the
web. In addition, reeling speed was also shown to be a significant factor on
physical properties of dragline silk. As the reeling speed increased the
arrangement of microstructures become more oriented and so the strength of the
silk increased (Madsen et al.,
1999
; Riekel and Müller,
1999
; Knight et al.,
2000
). Madsen et al.
(1999
) also reported that food
quantity significantly affects the property of dragline by decreasing the
breaking elongation. Since Craig et al.
(2000
) reported that hungry and
satiated Argiope keyserlingi did not differ in dragline amino acid
composition, the findings of Madsen et al.
(1999
) might be generated
physiologically or behaviorally. Vollrath
(1999
) suggested that spider
silks involved in prey catching did not seem to be selected to maximize a
narrow set of physical properties but were products of compromises of
different selective forces. Therefore, given the complicated foraging
conditions faced by orb-weaving spiders, the ability to adjust silk properties
in response to different conditions will be quite adaptive. Concluding from
the results of this and previous studies, we suggest that in face of prey
variation orb-weaving spiders adjust the mechanical properties of the webs by
manipulating silk diameter, number of radial threads and also the protein of
the silk.
Concluding from the results of this and previous studies, Nephila
spiders seem to exhibit plasticity on a behavioral and a molecular level to
varying foraging conditions. However, how such plasticity is achieved remains
unclear. It is possible that differential expression of different silk genes
is induced by different chemical composition of dipteran and orthopteran prey.
According to Ramos-Elorduy et al.
(1997), these two orders of
insects differed significantly in percentages of major amino acids.
Conversely, the different intensity of mechanical stimuli exhibited by smaller
dipterans and heavier orthopterans might also be potential factors. Based on
the results of the present study, future investigations on how
Nephila spiders achieve differential expressions of various dragline
genes in response to prey variation will be quite valuable to the genetic
studies of spider silks. In addition, further studies on how a variation in
diet affects silk physical properties and the efficiency of prey capture will
provide a new insights into the understanding of foraging ecology using
orb-weaving spiders.
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Acknowledgments |
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