Journal of Experimental Marine Biology and Ecology
290 (2003) 133 – 146
www.elsevier.com/locate/jembe
Effect of temperature, salinity and delayed
attachment on development of the solitary
ascidian Styela plicata (Lesueur)
Vengatesen Thiyagarajan, Pei-Yuan Qian *
Department of Biology, The Hong Kong University of Science and Technology (HKUST),
Clear Water Bay, Kowloon, SAR Hong Kong, China
Received 7 November 2002; received in revised form 23 January 2003; accepted 6 February 2003
Abstract
The solitary ascidian Styela plicata (Lesueur) is a common member of epibenthic marine
communities in Hong Kong, where seawater experiences extensive seasonal changes in temperature
(18 – 30 jC) and salinity (22 – 34x
). In this investigation, the relative sensitivity of different
developmental stages (i.e., duration of embryonic development, larval metamorphosis and postlarval growth) to various temperature (18, 22, 26 and 30 jC) and salinity (22x, 26x
, 30xand
34x
) combinations is reported. Fertilized eggs did not develop at lower salinities (22xand 26x
).
At higher salinities (30xand 34x
), the duration of embryonic development increased with
decreasing temperature (18 jC: 11.5 F 0.3 h; 30 jC: 8.5 F 0.3 h). More than 50% of larvae
spontaneously attached and metamorphosed at all the levels of temperature and salinity tested. At
higher temperatures (22, 26 and 30 jC) and salinities (30xand 34x
), functional siphon developed
in about 72 h after hatching, whereas at low temperature (18 jC), siphon developed only in < 30% of
individuals in about 90 h. However, none of the metamorphosed larvae developed subsequently at
low salinity (22x
). When forced to swim (or delayed attachment), larvae lost about 0.27 mJ after 48
h (about 22% of the stored energy). Such a drop in energy reserves, however, was not strong enough
to cause a significant impact on post-larval growth. This study suggests that temperature and salinity
reductions due to seasonal monsoon may have significant effect on the embryo and post-larval
growth of S. plicata in Hong Kong.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Ascidians; Delayed attachment; Embryonic development; Larvae; Metamorphosis; Styela plicata
* Corresponding author. Tel.: +852-2358-7331; fax: +852-2358-1559.
E-mail address: [email protected] (P.-Y. Qian).
0022-0981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0022-0981(03)00071-6
134
V. Thiyagarajan, P.-Y. Qian / J. Exp. Mar. Biol. Ecol. 290 (2003) 133–146
1. Introduction
Solitary ascidians, especially Styela plicata (Lesueur), are conspicuous components in
epibenthic (fouling) marine communities in warm and temperate regions (Yamaguchi,
1975; West and Lambert, 1976; Fisher, 1976, 1977; Abbott and Newberry, 1980; Lapointe
and Bourget, 1999; Lam, 2000; Glasby, 2001). Recently, we studied the seasonal
variations of S. plicata recruitment in subtropical waters of Hong Kong (Qiu et al.,
2003). The recruitment of this species occurs mainly during winter and spring when
seawater temperature is low (about 18– 24 jC) and salinity is high (about 33– 34x).
More importantly, complete recruitment failure occurred during summer when seawater
temperature is high (about 26– 30 jC) and salinity is low (about 22 –30x). This variation
in temporal recruitment pattern suggests that some developmental stages of S. plicata in
Hong Kong waters may be sensitive to environmental factors specific to this area.
However, the interactive effects of two most important environmental factors, i.e.,
temperature and salinity, on development of ascidians and subsequently their recruitment
have yet been investigated.
Recruitment rates of ascidians may depend not only on extrinsic factors (e.g., temperature and salinity) but also on intrinsic factors, such as extended swimming period and
energy content at metamorphosis (Svane and Young, 1989). For instance, extending the
larval swimming period of the colonial ascidian Diplosoma listerianum for as short as 3 h
resulted in slower metamorphosis and reduced post-metamorphic growth (Marshall et al.,
2003). Delayed attachment, in other words ‘extended larval swimming’, can thus
ultimately impact recruitment (Pechenik, 1990, 1999).
In this paper, the sensitivity of various development stages of S. plicata (i.e., embryos,
larvae and juveniles) to various temperature (18, 22, 26 and 30 jC) and salinity (22x,
26x, 30xand 34x) combinations was tested under laboratory conditions. In addition,
we determined the effect of delayed attachment on post-larval growth and the energy
changes that occur during embryonic development and forced larval swimming. The
results of this study may provide some insight on how extrinsic (e.g., temperature and
salinity) and intrinsic (e.g., delayed attachment) factors affect the embryonic and postlarval growth of S. plicata.
2. Materials and methods
2.1. Embryo culture
Adult S. plicata were collected from subtidal rocks in Hong Kong (22j19VN,
114j16VE) and were used for experiments within 2– 3 days. Embryos were cultured
according to Young (1982). Briefly, oocytes were obtained by pressing and washing
dissected ovaries from 5 to 15 adults with the help of a 250-Am nylon mesh. Oocyte
suspensions were washed several times in seawater. Sperm were stripped from testes as
above and used to fertilize oocytes by adding f 1% by volume of faint milky sperm
suspension. Excessive sperm were washed away from cultures after 10 min with the
help of a 100-Am nylon mesh. Embryos were cultured in polystyrene Petri dishes
V. Thiyagarajan, P.-Y. Qian / J. Exp. Mar. Biol. Ecol. 290 (2003) 133–146
135
(FalconR no. 1006, diameter 60 mm) at 25 jC. Seawater was changed every 3 h until
hatching.
2.2. Biochemical analyses
Total lipids were extracted according to Mann and Gallager (1985) and quantified by
the sulfuric acid-charring technique of Marsh and Weinstein (1966) with tripalmitin as a
standard. A microanalytical fractionation scheme was adopted from Mann and Gallager
(1985) to simultaneously quantify protein and carbohydrate. Total proteins were quantified
in a sample of aqueous homogenate as trichloroacetic acid (TCA) precipitated protein by
the method of Lowry et al. (1951). Total carbohydrates were extracted from the
homogenized samples by cold 5% TCA and quantified by the phenol – sulfuric acid
procedure, using D-glucose as a standard (Dubois et al., 1956). All these measurements
were made for six replicate samples with 50– 200 individuals per replicate.
2.3. Experiment 1: effect of temperature and salinity on the duration of embryonic
development
To examine the combined effect of temperature and salinity on the duration of
embryonic development, fertilized eggs obtained according to the Section 2.1 were
transferred into polystyrene Petri dishes (FalconR no. 1006, diameter 35 mm) with 5
ml of 0.22-Am-filtered seawater. The cultures were maintained in the experimental
salinities and temperatures. The experimental range of temperature and salinity was
selected to cover the ranges that embryos of S. plicata would likely experience in Hong
Kong waters. The experiment was set up in an orthogonal two-factorial design, using four
levels of temperature (18, 22, 26 and 30 jC) and 4 levels of salinity (22x, 26x, 30x
and 34x) for a total of 16 treatments. Each treatment consisted of six dishes with 10 –25
fertilized eggs per dish. Different temperatures and salinities were obtained by keeping
cultures in biological incubators set for testing temperature (Powers Scientific SD33SE)
and diluting 0.22-Am-filtered seawater (34x) with double-distilled water, respectively.
Development of embryos through various stages were recorded at 30-min intervals until
most of the eggs hatched into free-swimming tadpoles in 26 jC – 34xtreatments; in all
other treatments, only the time of tadpole emergence were recorded. For all treatments,
percentage of eggs hatched was recorded. We changed the water every 3 h of the
experiment, which continued until the fertilized eggs developed into tadpoles or for 24 h.
The experiment was repeated six times during March –June 2001 and May –July 2002
using gametes obtained from different specimens. Results are expressed as mean ( F S.D.)
of six replicate cultures.
2.4. Experiment 2: effects of temperature and salinity on metamorphosis and post-larval
growth
Newly hatched tadpoles obtained according to the Section 2.1 were used in this
experiment. The experimental design was the same as in experiment 1. Twenty larvae per
dish were used for each treatment. After 24 h, larvae that had not attached and
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metamorphosed were decanted. Newly hatched larvae were placed in polystyrene Petri
dishes (FalconR no. 1006) (6– 10 larvae dish 1) along with 5 ml of 0.22-Am-filtered
seawater to monitor the post-metamorphic performance. In this study, metamorphosis is
defined as the sequence of morphological events that transform the larva into a sessile
juvenile (Cloney, 1982). These processes include attachment and resorption of the tail. The
post-larval growth was assessed based on the development of functional siphon. Duration
of metamorphosis, time to develop functional siphon after hatching and survivorship at
each stage of development were recorded. The experiment was repeated six times during
March – June 2001 and May –July 2002 using gametes obtained from different specimens.
Results are expressed as mean ( F S.D.) of six replicate cultures.
2.5. Experiment 3: effects of forced swimming on energy consumption, metamorphosis
and post-larval growth
Tadpole larvae obtained according to the procedure described in ‘embryo culture’ section
were divided into three groups of f 1000– 1500 larvae each (groups 1 –3). Larvae were
washed several times in 0.22-Am-filtered seawater in order to isolate them from conspecific
settlement inducing cues. Group 1 was used immediately for biochemical analysis and postlarval growth studies. The attachment and metamorphosis of remaining larvae (groups 2 and
3) was prevented by continuous exposure to bright fluorescent illumination ( f 83 Amol
m 2 s 1) accompanied by strong agitation ( f 150 rpm) at low temperature (10 jC) (Craig
M. Young, personal communication). After 24 and 48 h, the larvae of groups 2 and 3 were
subjected to both biochemical analysis and post-larval growth studies, respectively. Larvae
were placed in polystyrene Petri dishes (FalconR no. 1006) (6 –10 larvae dish 1) along with
5 ml of 0.22-Am-filtered seawater (salinity = 33x; temperature = 24 –26 jC) to monitor the
post-larval performance according to experiment 2.
Lipid, protein and carbohydrate content were quantified in both fertilized eggs and
larvae according to the Section 2.2. To provide a collective measure of the relative
importance of each biochemical constituent in the fertilized egg and tadpoles, each
constituent was converted to equivalent units of available energy using the energy
conversion factors 35.24, 18.00 and 17.16 kJ g 1 for lipids, proteins and carbohydrates,
respectively (Beukema and De Bruin, 1979). The experiment was repeated six times with
6– 12 dishes of embryo or larvae at each time during May – June 2002, using gametes
obtained from different specimens. Results are expressed as mean ( F S.D.) of six replicate
cultures.
2.6. Data analysis
Data were checked for normality with Shapiro – Wilk’s test and homogeneity of
variance with Cochran’s test (Underwood, 1997). In experiments 1 and 2, the duration
of embryonic development and the percentage of individuals completing embryonic
development or metamorphosis at different treatments were compared using two-way
ANOVA. When interaction between the factors was significant, one-way ANOVA was
used to analyze the effect of temperature at fixed levels of salinity and vice versa. The
relationship between temperature and duration of embryonic development was analyzed
V. Thiyagarajan, P.-Y. Qian / J. Exp. Mar. Biol. Ecol. 290 (2003) 133–146
137
Table 1
Development schedule of S. plicata at 26 jC and 34x
Events
Mean F S.D. (hours)
Cleavage
Gastrula
Tail rudiment formation
Tail fully developed
Hatching
Attachment
Reduction of tail
Ampulla development
Development of functional siphon
1.7 F 0.5
3 F 0.7
5.5 F 1
7.5 F 0.7
11 F 1
15.5 F 1.5
17.5 F 2.4
23 F 1.8
80 F 4.5
Each mean value was obtained from six replicate cultures.
by Pearson product-moment correlation coefficient at fixed levels of salinity. The effects of
delayed attachment on metamorphosis and post-larval growth was subject to analysis of
covariance (ANCOVA) using energy content of tadpole as a covariate.
3. Results
3.1. Experiment 1
The development schedule for S. plicata embryos at 26 jC (34x) is presented in
Table 1. Duration of embryonic development was significantly affected by both temperature and salinity (Table 2A). Salinity had a stronger effect on duration of the embryonic
period than temperature, as indicated by MS values in Table 2A. There was also an
Table 2
Summary of two-way ANOVA results on effect of temperature and salinity on duration of embryonic
development, metamorphosis and siphon development (A) and on survivorship (B)
Source
df
Experiment 1:
embryogenisis
MS
F
(A) Duration
Temperature
3
1327.2 101.31
Salinity
3 18,435.4 1407.34
Temperature 9
446.4
34.08
Salinity
Error
80
13.1
(B) Survivorship
Temperature
3
281.2
Salinity
3 18,457.8
Temperature 9
153.5
Salinity
Error
80
85.5
3.28
215.82
1.79
Experiment 2:
metamorphosis
P
MS
F
< 0.001
199.5 0.81
< 0.001 162,919.2 66.21
< 0.001
393.5 1.59
246.1
< 0.025
< 0.001
0.084
3008.8 6.39
5849.4 12.43
880.9 1.87
470.3
Experiment 2:
siphon development
P
MS
F
0.491
170.8
3.42
< 0.001 12,025.4 241.08
0.129
3344.3 67.04
P
< 0.021
< 0.001
< 0.001
49.9
< 0.001
5932.8 64.85
< 0.001 12,471.8 136.33
0.067
986.7 10.78
91.5
< 0.001
< 0.001
< 0.001
138
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interactive effect between these two factors. Fertilized eggs did not develop at lower
salinities (22xand 26x) (Fig. 1A). At higher salinities (30xand 34x), embryos took
longer to hatch at lower temperature (at 30x, correlation coefficient (r) = 0.92; at
34x, correlation coefficient (r) = 0.93). Survivorship ranged from 30% to 50% and was
not significantly affected by temperature at 30x(one-way ANOVA, F3,21 = 3.37, P>0.01,
Fig. 2A) and 34x(one-way ANOVA, F3,21 = 0.95, P>0.05, Fig. 2A). Similarly, salinity
Fig. 1. S. plicata: effects of temperature and salinity on the duration of embryonic development, hatching to
metamorphosis and metamorphosis to siphon development. Each data point represents the mean ( F S.D. of six
replicate cultures) time in which >50% of total (A) fertilized eggs developed into free-swimming larvae
(embryonic development), (B) larvae attached and completed tail resorption after hatching, and (C) larvae
attached and developed functional siphon after hatching. ND = 100% of eggs/larvae died.
V. Thiyagarajan, P.-Y. Qian / J. Exp. Mar. Biol. Ecol. 290 (2003) 133–146
139
Fig. 2. S. plicata: effects of temperature and salinity on survivorship during embryonic development, hatching to
metamorphosis and metamorphosis to siphon development. Each data point represents the mean ( F S.D. of six
replicate cultures) percentage of fertilized eggs that hatched into free-swimming larvae after 24 h (A), percentage
of larvae that metamorphosed after 24 h of hatching (B) and percentage of attached larvae that developed
functional siphon after 7 days of hatching (C). ND = 100 % of eggs/larvae died.
(between 30xand 34x) did not significantly affect the survivorship of embryos, at any
given temperature (one-way ANOVAs, P>0.05).
3.2. Experiment 2
The post-metamorphic processes such as attachment, tail absorption, ampulla and
functional siphon development occurred after about 4, 6, 15 and 80 h of fertilization,
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V. Thiyagarajan, P.-Y. Qian / J. Exp. Mar. Biol. Ecol. 290 (2003) 133–146
respectively. Some larvae also underwent metamorphosis without attachment and were
discarded. More than 50% of larvae spontaneously attached and metamorphosed at all
levels of experimental temperatures and salinities. At lower salinities (22xand 26x),
larvae took much longer to metamorphose (from free-swimming larva to tail reabsorption)
than at higher salinities (30xand 34x); however, temperature variation had no
Fig. 3. S. plicata: biochemical composition and energy equivalents of fertilized egg and tadpole larvae. Mean
(n = 6) lipid, protein and carbohydrate content of fertilized egg and 0, 24 and 48 h (i.e., extended larval swimming
duration) old tadpole larvae (A). Energy changes based on lipid, protein and carbohydrate content during
embryonic development and extended larval swimming duration (B). FE = fertilized egg.
V. Thiyagarajan, P.-Y. Qian / J. Exp. Mar. Biol. Ecol. 290 (2003) 133–146
141
significant effect (Fig. 1B; Table 2A). In fact, larvae successfully attached and metamorphosed at temperatures as low as 10 jC within 48 h after hatching (data not shown).
Temperature and salinity did not interact to affect both survivorship and duration of
metamorphosis (Table 2). In contrast, temperature and salinity significantly interacted to
affect both survivorship and duration required for development of functional siphon. The
Fig. 4. S. plicata: effect of delayed attachment on the duration of metamorphosis, ampulla and siphon
development (A) and on the percentage of larvae completing metamorphosis, develop ampulla and siphon after 1,
3 and 7 days, respectively (B). Data plotted as mean F S.D. of six replicate cultures.
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Table 3
Summary of ANCOVA results with duration of metamorphosis, ampulla and siphon development (A) and
survivorship (B) as dependent variable, tadpole swimming duration (delayed attachment) as independent variable
and energy content of the larvae as covariate
Source
df
Experiment 3:
metamorphosis
MS
F
P
Experiment 3:
ampulla development
Experiment 3:
siphon development
MS
MS
F
P
F
P
(A) Duration
Delayed attachment
Energy content
Error
2
1
14
0.054
0.038
0.088
0.613
0.438
0.578
0.537
0.078
0.047
0.263
0.296
0.178
0.756
0.690
3.534
5.092
3.415
1.035
1.491
0.421
0.276
(B) Survivorship
Delayed attachment
Energy content
Error
2
1
14
21.520
13.470
32.905
0.654
0.409
0.559
0.551
11.580
11.582
23.950
0.483
0.483
0.642
0.517
10.843
14.329
17.134
0.633
0.836
0.569
0.402
functional siphon developed faster at higher salinities (30xand 34x) and temperatures
(22, 26 and 30 jC) (Fig. 1C; Table 2) than at low temperature (18 jC), where functional
siphon developed only in < 30% of individuals after 90 h (Figs. 1C and 2C). Metamorphosed juveniles failed to develop further at low salinity (22x) and in temperature –
salinity combination of 26x–18 jC (Fig. 1C).
3.3. Experiment 3
The principle biochemical constituents such as lipids, proteins and carbohydrates were
ordered based on their quantity as: proteins>lipids>carbohydrates, both in fertilized eggs
and free-swimming larvae (Fig. 3A). The total cost of development from fertilized egg to
tadpole larva measured for six cultures averaged about 0.25 mJ; lipids and proteins were
the major source of energy (Fig. 3B). When forced to swim, larvae lost about 0.16 mJ after
24 h and about 0.27 mJ after 48 h. The ANCOVA results revealed that delayed attachment
(duration of forced larval swimming) did not significantly affect either the duration of
metamorphosis or the rate of development of ampulla and functional siphon (Fig. 4A;
Table 3A) or survivorship (Fig. 4B; Table 3B).
4. Discussion
Embryonic and post-larval growth pattern of S. plicata is similar to that of several other
ascidian species (e.g., Anderson et al., 1975; Cloney, 1982; Satoh, 1994; Young and
Vazquez, 1995; Degnan et al., 1996). The growth response of their embryo to temperature
variations was also similar to previous observations: within tolerance limits, increased
temperature accelerates development (reviewed by Svane and Young, 1989). Although
differences in salinity exerted minimal effect on the duration of metamorphosis, its effects
on the embryo and functional siphon development of S. plicata were substantial (Fig. 1).
Similarly, the larvae of colonial ascidians successfully attached at salinities as low as
V. Thiyagarajan, P.-Y. Qian / J. Exp. Mar. Biol. Ecol. 290 (2003) 133–146
143
22xbut failed to complete metamorphosis at these low salinities (Vazquez and Young,
2000). Our laboratory results may be used to predict the duration required for the
development of S. plicata embryo and larvae in the natural environment. At low
temperature (about 18 jC) and high salinity (about 34x), i.e., a typical winter condition
in Hong Kong, embryos took about 13 h for hatching; however, during typical spring
conditions (e.g., about 22 jC and 34x), embryonic development was shorten substantially (10 –11 h). During typical mid-summer conditions (e.g., about 28 jC and 22x),
fertilized eggs did not develop into larvae. Similarly, at winter conditions, siphon
contraction of early juveniles started about 120 h after metamorphosis; however, during
spring conditions, it took only about 80 h. Unlike embryos, juveniles grew normally at
26xat temperatures >22 jC but did not grow at very low salinity (22x) at any
temperature (Fig. 1C). Our results clearly indicate that temperature and salinity fluctuations due to seasonal monsoon conditions can have a tremendous influence on the
recruitment dynamics of S. plicata in Hong Kong, through their effect on embryonic and
post-larval growth.
Our earlier field observations showed that the recruitment of S. plicata in Hong Kong is
apparently reduced during warm summer months (June –August) (Qiu et al., 2003).
Similar observations have been made along the east coast of North America (Fisher, 1977)
and in Japanese waters (Kazihara, 1964). Yamaguchi (1975) showed that S. plicata tolerate
a wide variation of salinity and temperature and noted that ascidians as a group might be
one of the most eurythermal animals with regard to reproduction. Fisher (1975) has shown
that S. plicata can produce viable gametes during warm (about 28 jC) summer months. In
fact, adult S. plicata collected during summer months in Hong Kong had mature gonads
and we successfully cultured their embryos in the laboratory. Therefore in Hong Kong
waters, the gonad maturity is not wholly responsible for the lack of recruitment of new
individuals, during summer. Apart from reproduction, all pelagic phases of the life cycle
(e.g., embryos and larvae) can substantially influence adult population dynamics (Svane
and Young, 1989). For instance, Fisher (1977) hypothesized that predation on larvae and
young adults might be responsible for the lack of S. plicata settlement during warm
summer months along the east coast of North America. According to our results, it seems
that the embryos and early juveniles are most critical phases in their life cycle. In addition
to environmental factors, other factors such as period of sexual reproduction (Carballo,
2000 and references therein), larval supply (reviewed by Underwood and Keough, 2001),
larval behavior during settlement (reviewed by Svane and Young, 1989), post-settlement
mortality and growth (Gotelli, 1987; Svane, 1987; Stoner, 1990) and distribution of
predators (Young, 1985) can also affect recruitment pattern, but these factors were not
addressed in this study.
Recent evidence suggests that there may be sublethal costs associated with the
increasing duration of larval swimming stage, especially for species with aplanktotrophic
larvae (reviewed by Pechenik, 1990, 1999). Since the larvae of S. plicata are aplanktotrophic, we predicted an inverse correlation between larval swimming duration and ability
to initiate and complete metamorphosis as well as time of ampulla and siphon development. However, our results showed that larvae of S. plicata could have an extended larval
swimming period for over 2 days without incurring a measurable cost to metamorphosis
and post-larval growth. These results differ from previous studies on the larvae of crab,
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barnacles, bryozoans, sponges, abalone and colonial ascidians, in which delayed attachment negatively affected both survival and growth rate during early post-larval growth
(Woollacott et al., 1989; Pechenik et al., 1993; Gebauer et al., 1999; Maldonado and
Young, 1999; Wendt, 2000; Roberts and Lapworth, 2001; Marshall et al., 2003;
Thiyagarajan et al., 2003). Our results were similar to the situation of the Chilean
bryozoan Celleporella hyaline (Orellana and Cancino, 1991), where post-larval growth
was not affected by 28 h of delayed attachment. Larvae of S. plicata that delayed their
attachment seem to bring fewer reserves to the juveniles than do young larvae, because 48
h of forced swimming consumed 22% of the stored energy. Such a drop in energy reserves,
however, is not strong enough to cause a significant impact on post-larval growth in S.
plicata.
Acknowledgements
We wish to thank Professor Jan Pechenik (Tufts University) for his valuable comments
and for critically reading the manuscript. Special thanks are dedicated to Professor Craig
M. Young (University of Oregon) for suggestions and productive discussions during the
course of this work. The authors are grateful to Drs. T. Harder, J.W. Qiu, S.C.K. Lau and
S. Dobretsov (HKUST) for their helpful comments. We also wish to thank the anonymous
referee, whose comments made a significant contribution to the final version of this paper.
This work was supported by a research grant from RGC grants (HKUST 6133/99 M and
HKUST 6119/01 M) to P.Y. Qian. [SS]
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