Journal of Experimental Marine Biology and Ecology 367 (2008) 164–173
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Journal of Experimental Marine Biology and Ecology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j e m b e
Thermal ecophysiology of gametophytes cultured from invasive Undaria pinnatifida
(Harvey) Suringar in coastal California harbors
Sarah K. Henkel ⁎, Gretchen E. Hofmann
Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, CA 93106 USA
a r t i c l e
i n f o
Article history:
Received 22 February 2008
Received in revised form 16 September 2008
Accepted 16 September 2008
Keywords:
Heat shock
Hsp70
Kelp
Photosynthesis
Seaweed
Temperature
a b s t r a c t
Given the ecological and economic impact of invasive species, knowledge of traits that allow the invasive
kelp Undaria pinnatifida (Harvey) Suringar to expand its range is important for developing prevention and
eradication efforts. Physiological mechanisms of thermotolerance may be important as they can influence
both local and regional patterns of distribution and abundance. An established measure of thermotolerance is
the heat shock response where heat shock proteins (Hsps) are produced in response to high temperature and
protect cellular proteins from misfolding and degradation. It is hypothesized that Hsp production varies with
developmental stage in many species, and U. pinnatifida, gametophytes have been shown to have a broader
temperature range for growth and survival than sporophytes. Thus, we examined up-regulation of the hsp70
gene in gametophytes of invasive U. pinnatifida and compared it to values previously measured for
sporophytes. Fertile sporophytes were collected from harbors in Monterey, Santa Barbara, Los Angeles, and
San Diego, California, and gametophytes were cultured from them in the laboratory. After being grown in
controlled light and temperature conditions, gametophyte tissue was exposed to a temperature gradient and
quantitative real-time PCR was used to determine relative amounts of hsp70 transcript. Expression of hsp70
was extremely low in gametophytes. An investigation of gametophyte thermal ecophysiology using oxygen
evolution measures and pulse-amplitude-modulated (PAM) fluorometry at a subset of temperatures revealed
that U. pinnatifida gametophytes decrease dark-adapted yield with increasing temperature from 17 to 31 °C.
Photosynthetic rates declined but remained positive at all incubation temperatures, while respiration
increased with increasing temperatures up to 26 °C and then declined. Thus, U. pinnatifida gametophytes
remained metabolically active at temperatures well above normal environmental conditions, indicating they
are broadly thermotolerant. This tolerance, however, likely is conferred by mechanisms other than upregulation of the hsp70 gene as no significant increases in expression related to temperature were observed
in this study.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
Regarded as one of the “100 worst invasive alien species” by the
International Union for Conservation of Nature and Natural Resources,
the Asian kelp, Undaria pinnatifida (Harvey) Suringar, was first
observed in California in 2000. Currently the invader is found in
harbors from Monterey Bay to San Diego Bay, California, and has
established benthic populations at Catalina Island, California, and
Todos Santos Island, Baja California, México (Aguilar-Rosas et al.,
2004; Silva et al., 2002). Invasive U. pinnatifida populations also are
present in the Mediterranean Sea, Atlantic Europe, Australia, New
Zealand, Tasmania, and Argentina, occurring from the low intertidal to
at least 25 m depth. Given its wide geographic and depth distributions, it appears that this species has a broad or flexible temperature
⁎ Corresponding author. Department of Ecology, Evolution, and Marine Biology, Mail
Code 9610, University of California, Santa Barbara, Santa Barbara, CA 93106-9610 USA.
Tel.: +1 805 893 6176; fax: +1 805 893 4724
E-mail address: [email protected] (S.K. Henkel).
0022-0981/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2008.09.010
tolerance. Sporophyte growth has been reported from 3 to 20 °C in the
laboratory, and sporophytes have been observed in habitats ranging
from 0 to 27 °C (Saito, 1975; Sinner et al., 2000a; Skriptsova et al.,
2004). The small, filamentous gametophytes can grow in temperatures of 8 – 28 °C and can recover from conditions of -1 to 33.5 °C
(Peters and Breeman, 1992; Saito, 1975; Sanderson, 1990; Sinner et al.,
2000b; Wallentinus, 1999), although few studies have investigated
gametophyte performance across a range of temperatures, and
possible mechanisms for how U. pinnatifida gametophytes cope with
temperature stress have not been identified. The extensive survival
and growth capability of the gametophyte as well as its small size
relative to the sporophyte suggest that it may be able to persist
through tropical passage attached to ship hulls or in ballast water,
making it the likely life-history stage for introduction. The goal of this
study was to investigate physiological and organismal responses of
invasive U. pinnatifida gametophytes across a temperature gradient.
Like their terrestrial counterparts, marine invasive species can alter
ecosystem structure, function, and processes. Invasive seaweeds can
have especially strong impacts by changing light availability, nutrient
S.K. Henkel, G.E. Hofmann / Journal of Experimental Marine Biology and Ecology 367 (2008) 164–173
cycling, and food availability for herbivores (Britton-Simmons, 2004;
Sanchez et al., 2005; Torres et al., 2004). Additionally, they can result
in economic damage to fisheries, aquaculture, and SCUBA-based
tourism (Ribera and Boudouresque, 1995; Verlaque, 1994). Even
introduced species that are typically non-competitive can become
“invasive” and proliferate when native species are in decline due to
anthropogenic effects or climate regime shifts (Ambrose and Nelson,
1982; Valentine and Johnson, 2004; Walker and Kendrick, 1998). A
trait that has been suggested to be important to rapid spread of an
invading species is a plastic ecophysiology (reviewed in (Schaffelke
et al., 2006) or a broadly tolerant physiology to persist in a range of
conditions, including those encountered during transport. In the case
of kelps, the biology and survival of the gametophyte is equally, and
perhaps more, important than the sporophyte to potential establishment and successful invasion (Inderjit et al., 2006).
The consequences of different stressors and the tolerance
mechanisms of algae, as well as the impact of these stresses on the
physiology of vegetative tissue and as a function of developmental
stage, are all poorly understood (Coelho et al., 2000). The presence of
heat shock proteins (Hsps), which protect cellular proteins from
misfolding and degradation, and their induction by environmental
stress have been shown to provide a central tolerance mechanism in
many organisms (Feder and Hofmann, 1999). The temperature at
which the genes encoding Hsps are induced is unique to each
organism and its growth conditions (typically 5 to 10 °C above normal
growing temperatures; Kimpel and Key, 1985; Lindquist, 1986), and
the ability to alter the temperature at which the response is induced is
used as an indicator of a plastic ecophysiology. If producing Hsps is
costly for organisms and natural selection has worked towards
165
optimizing the cost/benefit ratio of Hsps, then their production and/
or induction also may vary as a function of developmental stage
(Coleman et al., 1995). A variety of strategies involving heat shock
proteins for dealing with temperature stress have been reported in
early life-history stages of other metazoans. Inducible defenses
against temperature stress may be conferred by up-regulation of
hsp70 gene transcription to produce Hsp70, translation of pre-existing
hsp70 transcript, enhanced synthesis of constitutive Hsps in the
70 kDa range, transcription of smaller molecular weight Hsps, or
phosphorylation of constitutively expressed heat shock proteins. For
this study, we focused on up-regulation of hsp70 gene transcription as
a possible mechanism for thermotolerance in gametophytes of the
invasive kelp Undaria pinnatifida.
To date, studies have been conducted on hsp70 gene expression in
the red alga Plocamium cartilagineum (Vayda and Yuan, 1994) and the
sporophyte stage of U. pinnatifida and Egregia menziesii (Henkel and
Hofmann, 2008). Additionally there have been investigations of heat
shock protein (Hsp70) production in embryos of the rockweed Fucus
gardini (Li and Brawley, 2004) and in other species of algae (Ireland
et al., 2004). However, hsp70 gene expression has not been linked to
growth or physiological performance in kelps. Comparisons of the
heat shock response and photosynthetic performance in the gametophyte stage of U. pinnatifida may elucidate the potential for this
species to survive transport and become established in novel habitats.
This is important as early developmental stages often are considered
to be most critical in determining overall spatial and temporal success
of a species in natural environments. Furthermore, studies on
ecological developmental biology are timely as they may lend
researchers the ability to investigate the potential for species to
Fig. 1. Undaria pinnatifida collection sites.
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S.K. Henkel, G.E. Hofmann / Journal of Experimental Marine Biology and Ecology 367 (2008) 164–173
Table 1
Average daily minimum and maximum recorded temperatures in April 2006 in the
harbors where Undaria pinnatifida was collected
Collection Site
Temperature
MB (Monterey Harbor)
SB (Santa Barbara Harbor)
LA (Cabrillo Harbor, Los Angeles)
SD (Coronodo Island, San Diego Bay)
12.9–13.3 °C
12.7–14.0 °C
13.1–13.9 °C
16.3–17.8 °C
SD harbor temperatures are from Navy Pier in San Diego Bay, approx 1.5 km N of the
collection site on Coronado Island.
tolerate anthropogenically induced changes in the short term as well
as their evolutionary potential to adapt to altered environments in the
long term (Sultan, 2007).
The objective of this study was to investigate a possible mechanism
for the apparently broad temperature tolerance of invasive Undaria
pinnatifida gametophytes by examining heat shock protein gene
induction. Additionally, we evaluated photosynthesis and respiration
across a range of temperatures with the aim of linking the molecular
heat shock response with a more organismal-level physiological
response. We hypothesized that gametophytes would induce hsp70 at
temperatures higher than that previously observed for sporophytes.
Further, we hypothesized that the gametophytes would maintain
robust photosynthetic performance at temperatures preceding the
induction of hsp70, but that those rates would decline as the heat
shock response was mounted and cellular processes switched over to
the “protect and repair” response. Finally, we made qualitative
observations about degradation and recovery of gametophytes after
longer episodes of heat stress followed by a return to benign
conditions to obtain an overall, organismal level response to increased
temperatures.
2. Materials and methods
2.1. Gametophyte culture
Fertile Undaria pinnatifida were collected from four invaded
harbors in California: Monterey Bay (MB), Santa Barbara (SB), Los
Angeles (LA), and Coronado Island in San Diego Bay (SD) (Fig. 1) in
order to sample the population across its introduced range and
determine if any local adaptation to temperature has occurred since its
invasion, as previous work (Uwai et al., 2006) has indicated that at
least the Monterey Bay (MB), Santa Barbara (SB), Los Angeles (LA)
populations are all the same haplotype. Individuals were collected
from floating docks or pilings and were found growing at or just below
the zero tide line on pilings. All samples for gene expression were
collected in March or April of 2006. A second collection was made in
April 2007 from Santa Barbara Harbor to culture additional gametophytes for photosynthesis and recovery experiments. Temperature
data for harbor sites were obtained from SCOOS (Southern California
Ocean Observing System; http://www.sccoos.org/) and NOAA (http://
www.noaa.gov/) (Table 1).
Collected samples were brought back fresh from the field and
held overnight in recirculating seawater tanks at 10 °C (the lowest
temperature seen by any population; Santa Barbara Harbor, 10.2 °C)
to allow recovery from transport stress. Fertile sporophylls were
excised from the thallus, placed in a 2% bleach solution for 5 minutes,
and rinsed in autoclaved seawater. Sporophylls then were placed in
an antibiotic solution of Penicillin G (622.5 mg/L), Streptomycin
(250 mg/L), and Chloramphenical (100 mg/L) for 2 h followed by
another autoclaved seawater rinse. Following dark incubation at 4 °C
overnight, modified autoclaved seawater (Anderson et al., 1997) with
vitamin solution (Table 2), was added to sporophylls to induce spore
release. Spore solutions were put in flasks and placed in a 15 °C
culture chamber. After spore settlement, water was decanted from
flasks and new modified autoclaved seawater plus vitamins was
added to cultures. Each culture thus consisted of masses of
vegetative filamentous gametophytes germinated from the spores
of one individual. Gametophytes were allowed to grow for 9 months
at 15 °C and 12:12 h (light:dark) under 15 W cool white fluorescent
bulbs; light intensity in the incubator ranged from 10 to 20 μmol
photons· m-2 · s-1.
2.2. hsp70 expression
For the heat stress experiment, 150–200 μl of filamentous
gametophyte tissue from 4 cultures from each site was placed in
1.5 mL centrifuge tubes filled with filtered seawater and placed in an
aluminum gradient heat block at 12, 17, 22, 26, 31, 33, and 36 °C ± 1 °C
for 1 h; thus at every temperature n = 4 for each site. Light levels were
b5 μmol photon · m-2 · s-1 inside the tubes inside the aluminum block.
After thermal incubation, samples were removed from heat, tubes
were centrifuged, and seawater was pipetted off tissue. Samples were
then stored at -80 °C until extraction.
For analysis of hsp70 gene induction under the different temperature exposures, total RNA was extracted from the frozen gametophyte
tissue following a modified protocol for pine needles (Chang et al.,
1993). Samples were sonicated in 600 μl of extraction buffer (prewarmed at 65 °C: 2% CTAB (w/v); 100 mM Tris-HCl (pH 8.0); 25 mM
EDTA; 2 M NaCl; 2% PVP (w/v); 0.05% spermidine (w/v); 2% ßmercaptoethanol (v/v)). Tubes were centrifuged at 12000 g for 15 min
at 10 °C, and supernatant was subjected to two extractions with equal
volume of 24:1 chloroform:isoamyl. RNA was precipitated from the
final supernatant overnight at 4 °C using 1/4 volume of 10 M LiCl. RNA
was collected by centrifugation at 12000 g for 20 min at 10 °C. The
pellet was then washed twice with cold (-20 °C) 70% EtOH in DEPCH2O using 10 min 12000 g spins at 10 °C, dried under vacuum, and
resuspended in DEPC-treated water. Total RNA (600 ng) was
transcribed into cDNA using Stratascript cDNA synthesis kit (Stratagene) with oligo dT priming.
Quantitative PCR was conducted by amplifying cDNA synthesis
products (2 μl) with primers for the response gene, hsp70 (1.0 μM), and
the internal control, ribulose (1.0 μM), (Henkel and Hofmann, 2008) in
20 μl SYBR green supermix (Bio-Rad) reactions (run in duplicate).
Primer concentrations were empirically determined based on lowest
Ct values and highest efficiencies. A single cDNA standard was run in
duplicate on each plate to allow comparison across plates. Four 10-fold
serial dilutions of the standard sample from each PCR plate were used
as a standard curve to calculate PCR efficiency. Fluorescence threshold
values were set at levels to maximize PCR efficiency (between 90 and
110%), and melt curve analysis was performed to confirm that only a
single product was amplified. To estimate genomic DNA (gDNA)
contamination, non-transcribed RNA from representative samples
was also used as template for real-time PCR. Amplification showed
that gDNA contamination constituted no more than 1/1000 of the
amplification observed with transcribed cDNA samples.
2.3. Photosynthetic measures
Oxygen evolution rates and chlorophyll fluorescence at 17, 22, 26 and
31 °C were determined for cultured gametophyte tissue from the four
Table 2
Protocol for vitamin solution
Quantity
Compound
1L
500 mg
10 mg
20 mg
500 mg
100 mg
dH2O
Thiamine
Vitamin B12
Biotin
Niacinamide
p-aminobenzoic acid
1 mL added per L of modified, autoclaved seawater.
S.K. Henkel, G.E. Hofmann / Journal of Experimental Marine Biology and Ecology 367 (2008) 164–173
167
oven at 60 °C overnight. Samples were weighed again to determine
dry weight, and the FW:dwt ratio was obtained.
2.6. Survivorship and recovery
Finally, to evaluate organismal level and longer term effects of heat
stress on U. pinnatifida gametophytes, cultured gametophytes from
the Santa Barbara Harbor population (n = 4) were placed in 20 mL
scintillation vials in an aluminum gradient heat block and subjected to
the same 7 incubation temperatures and low light (b5 μmol photons·
m-2·s- 1) used in the gene expression experiments (12, 17, 22, 26, 31, 33,
and 36 ± 1 °C). Samples were heat stressed for 4 h, qualitative
observations of condition were made, and samples were then
returned to the 15 °C incubator under ca. 20 μmol photon · m- 2 · s- 1
and observed for continuing degradation or recovery for three weeks.
2.7. Data analysis
Fig. 2. Relative levels of hsp70 expression of gametophytes cultured from four harbor
populations of Undaria pinnatifida at 7 heat shock temperatures. Error bars represent
+1 S.E. MB = Monterey Harbor, SB = Santa Barbara Harbor, LA = Cabrillo Harbor in Los
Angeles, SD = Coronado Island in San Diego Bay.
sites to estimate physiological performance. Unfortunately, after the
gene expression experiments, only enough tissue from a single Los
Angeles culture remained to conduct physiological experiments; thus,
n = 4 at each temperature for San Diego, Santa Barbara, and Monterey, but
n = 1 at each temperature for Los Angeles. Gametophyte tissue from the
cultures (~0.05 g FW) was placed in 1.5 mL centrifuge tubes and placed in
water baths at experimental temperatures for 1 h prior to assessment.
Algae were then transferred to a Clark-type oxygen electrode system
(Rank Brothers, Bottisham, England) at the same temperature. Darkadapted yield (Fv/Fm) values were obtained using a PAM fluorometer
(Model 210, Walz, Effeltrich, Germany). Dark respiration was determined
by recording changes in dissolved oxygen for 30 min; the chamber was
then illuminated using a Fiber-Lite® High Intensity Illuminator (DolanJenner, Boxborough, MA) and oxygen evolution was measured for 20 min
to determine net photosynthesis (Pnet). The mean light intensity (±1 S.D.)
inside the incubation chamber was 91 ± 38 μmol photon · m- 2 · s- 1,
measured using a fiber-optic quantum sensor of the PAM fluorometer.
Net photosynthesis of Macrocystis pyrifera gametophytes has been
shown to be inhibited at irradiances ≥140 μmol photon · m- 2 · s- 1 (Fain
and Murray, 1982), so we believe these values to be saturating but not
limiting. To correct for bacterial respiration and electrode oxygen
consumption, control incubations were performed at the start of each
temperature treatment. Following photosynthesis measurements,
gametophyte tissue was blotted dry, weighed to determine fresh weight
(FW) and frozen at -20 °C.
Reported relative mRNA levels were calculated in the following
fashion: (1) hsp70 and ribulose Ct values were first normalized to that
of the corresponding product of the cDNA standard from each plate,
(2) resultant hsp70 values were then normalized to the resultant ribulose values for the each sample. The ribulose gene was deemed to be an
appropriate internal control because the ribulose mRNA levels did not
differ significantly across treatments (Temperature: F(6,536.9) = 0.217,
P = 0.970, Site: F(3,1737.3) = 1.403, P = 0.248).
Each gene expression data set was examined for variance homogeneity using Cochran's test; because ANOVA is robust to non-normality
2.4. Photosynthetic pigment composition
Chlorophyll a was extracted from frozen tissue in 1.5 mL centrifuge
tubes using plastic pestles and 90% acetone solution. After dark
incubation overnight at 4 °C, extract was collected and remaining
tissue was subjected to a second 90% acetone extraction, which was
combined with the initial extract. Absorbance of the pigment extract
was quantified at 730, 664, 647 and 630 nm using a BioSpec-1601
spectrophotometer (Shimadzu Corporation, Columbia, MD), and
chlorophyll a concentration was calculated according to the equations
of Jeffrey and Humphrey (1975).
2.5. Fresh weight: dry weight ratio determination
Additional fresh gametophyte tissue from the culture flasks was
used to determine the fresh weight (FW) to dry weight (dwt) ratio of
U. pinnatifida gametophytes. Samples ranging from 0.025 to 0.100 g
were blotted dry and weighed. Samples were then placed in a drying
Fig. 3. (A) Undaria pinnatifida gametophyte hsp70 expression relative to hsp70
expression at 12 °C. (B) U. pinnatifida sporophyte hsp70 expression relative to 12 °C,
data re-analyzed from Henkel and Hofmann (2008). Error bars represent +1 S.E.
168
S.K. Henkel, G.E. Hofmann / Journal of Experimental Marine Biology and Ecology 367 (2008) 164–173
(Underwood, 1997) departures were not considered reason to reject
parametric procedures. Two-way ANOVA models were employed to
analyze the effects of the fixed factors temperature and site on the
relative hsp70 expression data. To evaluate the effects of temperature
and site on Pnet, respiration, and dark-adapted yield, a two-way ANOVA
model was used only for the 3 sites where n = 4 (SD, SB, and MB). Where
differences were significant, a Student-Newman-Keuls' multiple comparison test was used to facilitate interpretation of ANOVA results.
3. Results
3.1. hsp70 expression
In order to assess a molecular response of Undaria pinnatifida
gametophytes to temperature, we measured levels of transcript for a
thermally sensitive gene, hsp70. In general, hsp70 expression was
distinctly different in gametophytes than that previously observed for
sporophytes. Expression of hsp70 relative to ribulose was very low
(0.00023–0.07424 fold) in the gametophyte tissue with no evidence of
induction and no significant effects of temperature (F6,0.00282 = 0.240,
p = 0.962) or site (F3,0.00524 = 0.891, p = 0.450) (Fig. 2). When hsp70
expression for each temperature treatment was determined relative to
expression of the same individual at 12 °C (fold induction), there was
still no pattern related to incubation temperature. Fold expression
values for gametophytes (0.5–6 fold over 12 °C) were similar to those
previously observed for sporophyte tissue at temperatures where
hsp70 was not induced (0.1–2 fold over 12 °C), while sporophyte tissue
that induced hsp70 (incubated at 22 and 26 °C) exhibited 6–27 fold
expression over that at 12 °C (Fig. 3A,B).
3.2. Photosynthetic performance
Using photosynthetic properties as indicators of performance, we
found that Undaria pinnatifida gametophytes appeared to be tolerant,
at least in the short term, of temperatures as high as 26 to 31 °C. Net
photosynthesis decreased with increasing temperature (Fig. 4A,B)
but still remained positive at all temperatures. When oxygen
evolution was calculated relative to tissue chlorophyll a content
temperature effects were significant with a decrease from 2.60 to
0.02 mg O2 · mg- 1 Chl a · h- 1 (17 to 31 °C), but there were no
significant differences among sites (Table 3). There were no
significant effects of temperature or site when Pnet was expressed
in terms of tissue fresh weight (Table 3). Because culture samples are
tufts of filaments, it was not possible to obtain tissue surface area;
thus we could not determine Pnet based on that metric.
Respiration rates increased from 17 to 26 °C (- 9.2 to - 44.9 mg
O2 · g- 1 FW · h- 1) and then declined again at 31 °C (Fig. 4C), possibly
enabling the positive net photosynthesis observed at that temperature. Both temperature and site were significant effects
(Table 3); gametophytes cultured from Monterey Bay (the most
northern site) had significantly greater oxygen consumption
(higher respiration) than those from Santa Barbara and San Diego.
A measure of the efficiency of photosystem II, dark-adapted yield
(Fv/Fm) declined significantly with increasing incubation temperature
(0.520 to 0.212; Fig. 4D), and there were no significant differences
among sites (Table 3). The average fresh weight to dry weight ratio
was determined to be 2.9 (S.E. = 0.08, R2 = 0.86) and also did not differ
among sites (p = 0.581).
3.3. Survivorship and recovery
The results of longer term incubation of gametophytes cultured
from Santa Barbara indicate that Undaria pinnatifida gametophytes can
experience temperatures of 31 °C or less without sustained damage
and that they may be able to tolerate 33 °C without irreparable harm.
However, 36 °C seemed to provide a thermal challenge that they were
not able to overcome. After 2 h of heat stress, one of the 36 °C samples
turned green. At the end of the 4 h incubation, there were still no
observable changes in any of the other samples at any temperature.
After having been returned to the 15 °C incubator under white
Fig. 4. Short-term temperature response of (A) net photosynthetic rates relative to tissue fresh weight, (B) net photosynthetic rates relative to tissue Chl a content, (C) respiration rates
relative to tissue fresh weight, (D) dark-adapted yield of Undaria pinnatifida gametophytes cultured from San Diego, Santa Barbara, Los Angeles, and Monterey sporophyte
populations. Error bars indicate ±1 S.E. from the mean (n = 4 at each site and temperature, except LA n = 1 at each temperature).
S.K. Henkel, G.E. Hofmann / Journal of Experimental Marine Biology and Ecology 367 (2008) 164–173
fluorescent light at ca. 20 μmol photon · m- 2 · s- 1 for 3 days, the 36 °C
sample that had shown degradation during incubation was white and
the other three 36 °C samples exhibited loss of accessory pigments
(edges of the tufts were green). After 6 days of recovery, two of the
33 °C samples also exhibited some greening. After 3 weeks in recovery,
there was no further degradation of any of the samples; however, there
did not appear to be reversal of the damage already sustained in the
36 °C and two of the 33 °C samples.
4. Discussion
Many studies have shown that it is in early developmental stages of
marine animals that environmental sensitivity is at its peak (Anger,
1996a,b); thus the success and distribution of such species depend on
the thermotolerance limits of these potentially sensitive life stages.
Alternatively, studies on kelp have suggested that the few-celled,
filamentous gametophyte stage may more tolerant than the large,
sporophyte stage as gametophytes seem to persist through stressful
environmental conditions (Ladah and Zertuche-González, 2007;
Ladah et al., 1999). In its native Japan and most areas of introduction,
Undaria pinnatifida sporophytes disappear when water temperatures
reach 20–25 °C (Hay and Villouta, 1993), and it is presumed that the
gametophytes remain throughout the summer to produce the next
generation of sporophytes when favorable temperature conditions
return. However, there is a paucity of information on the reactions of
algal early life history stages to environmental stressors (Coelho et al.,
2000).
It is hypothesized that Undaria pinnatifida was introduced to
Tasmania and New Zealand via the gametophyte stage in ballast water
(Sanderson, 1990; Stuart, 2004). In fact, U. pinnatifida was the only
macroalga listed as a high-risk ballast transported species in the
Global Ballast Water Management Programme (GloBallast), initiated
by the International Maritime Organization (IMO, 2006). Mediterranean studies on ballast water and organisms therein found temperatures of ballast water varied from 13 to 28 °C according to sampling
season (Flagella et al., 2007). It is also possible that gametophytes may
be transported while attached to the external hull; thus being directly
exposed to fluctuating water temperatures. Under both of these
scenarios, a high degree of tolerance to thermal variability would be
required to survive transit and establish populations in a novel
environment upon release. The results of our longer-term incubation
and recovery experiment indicated that U. pinnatifida gametophytes
have the ability to withstand temperatures that would be experienced
Table 3
Two-way ANOVA results, testing for the effects of temperature and collection site on
different metabolic indicators in gametophytes of Undaria pinnatifida collected from
San Diego, Santa Barbara, and Monterey
Df
F
P
Photosynthesis (FW)
Temperature
Site
Temperature x Site
3
2
6
2.018
0.416
0.406
0.129
0.663
0.870
Photosynthesis (chl a)
Temperature
Site
Temperature x Site
3
2
6
3.202
0.152
0.710
0.035
0.860
0.643
Respiration (FW)
Temperature
Site
Temperature x Site
3
2
6
6.375
5.526
1.187
b 0.001
0.008
0.335
Dark-adapted yield
Temperature
Site
Temperature x Site
3
2
6
10.168
2.171
0.235
b 0.001
0.129
0.962
169
in crossing tropical waters and exceeding those measured in ballast
tanks.
When we subjected U. pinnatifida gametophytes to a range of
temperature treatments in this study, we did not observe induction of
hsp70 transcription at high temperatures; in fact, overall expression of
hsp70 was extremely low at all temperatures. We are certain that
gametophytes are capable of gene transcription, as expression of the
control gene, ribulose, was robust. These finding are in contrast to
previous studies on the sporophyte stage of U. pinnatifida which
exhibited a classic heat-shock response with induction of hsp70 at
temperatures above normal environmental conditions (Henkel and
Hofmann, 2008). Although direct comparisons of the magnitude of
responses between the gametophytes and sporophytes is not possible
due to the use of different internal standards and differences between
the life-history stages in ribulose expression, comparison of the
induction profile are possible to see if the gene is “turned on” at a
different temperatures. However, we did not see evidence of the
hsp70 gene being turned on at any temperature in the gametophyte
experiment, even when expression at higher temperatures was
compared to expression at the “unstressed” temperature of 12 °C.
Similar results have been found in other studies where gametophytic
tissue of angiosperms, gymnosperms, and ferns are found to display a
different response to heat-shock than sporophytic tissue (Cooper
et al., 1984; Dupuis and Dumas, 1990; Frova et al., 1991; Roy and
Raghavan, 1994).
The lack of hsp70 induction we observed is not unique, as low
transcription of hsp70 has been documented for other early life history
stages such as Mus musculus (mouse) (Hahnel et al., 1986) and Daucus
carrota (common carrot) (Zimmerman et al., 1989) embryos. In these
cases, inducible defenses against temperature stress may be conferred
by translation of pre-existing heat shock gene transcript (Bienz and
Gurdon, 1982; Pitto et al., 1983; Storti et al., 1980; Zimmerman et al.,
1989). For example, in Brassica napus microspore embryogenesis,
Hsp70 protein expression is increased after exposure to the induction
treatment (Cordewener et al., 1995; Testillano et al., 2000), but this is
likely produced by translating existing mRNAs. An alternative method
also may be activation of existing heat shock proteins by phosphorylation as has been hypothesized for sea urchins (Roccheri et al., 1995).
Some studies that have focused on protein rather than gene
expression have found high amounts of the inducible isoform of
Hsp70 in early developmental stages and suggest that constant
expression of Hsp70 during development may provide protection
from environmental stressors more quickly than a classic induction
response (Podrabsky and Somero, 2007). Killifish (Fundulus heteroclitus) eggs successfully hatch at temperatures ranging from 15 to
30 °C (Taylor, 1999), and F. heteroclitus adults (Koban et al., 1991) and
Austrofundulus limnaeus embryos (Podrabsky and Somero, 2007) have
been shown to possess Hsps at non-stress temperatures, which has
been viewed as pre-adaptation to the abrupt changes in temperature
found in their natural environments. Similarly, in horseshoe crabs
(Limulus polyphemus), which are subjected to temperature extremes
as they develop on the beach, there was no evidence for the synthesis
of any new isoforms of Hsp70 following heat shock in embryos or
larvae; however, heat shocked embryos demonstrated levels of
constitutive Hsp70 that were comparable to control embryos (Botton
et al., 2006), indicating a predisposition to temperature tolerance.
This strategy also has been suggested for marine intertidal
animals (Horowitz, 2001), and may be the mechanism employed by
U. pinnatifida gametophytes.
Organisms also use related protein members of the Hsp70 family.
For example, fern gametophytes enhance synthesis of three constitutive Hsps in the range of 63 to 86 kDa rather than inducing the
synthesis of specific “inducible” proteins in response to heat shock
(Roy and Raghavan, 1994). Finally, smaller molecular weight Hsps also
may be employed as has been detected in carrot (Daucus carota; Pitto
et al., 1983; Zimmerman et al., 1989), alfalfa (Medicago sativa;
170
S.K. Henkel, G.E. Hofmann / Journal of Experimental Marine Biology and Ecology 367 (2008) 164–173
Gyorgyey et al., 1991) and sea urchin (Paracentrotus lividus; Roccheri et
al., 1995) embryos. Thus, U. pinnatifida gametophytes may maintain
high levels of other Hsps, up-regulate transcription of predetermined
amounts of hsp70 RNA, or use different sets of proteins. Experiments
quantifying protein levels would be necessary to determine if
U. pinnatifida gametophytes utilize other proteins or methods for
induction of the heat shock response.
We do not believe that the lack of observed heat shock response in
U. pinnatifida gametophytes was a result of acclimation to the stable
incubation temperatures in which they were raised. Although it has
been shown that the heat shock response is acclimatizable within a
species to different environmental conditions (Halpin et al., 2002;
Hamdoun et al., 2003; Helmuth and Hofmann, 2001; Sharp et al.,
1994), there is no evidence to date of a thermally tolerant species that
does not employ heat shock proteins. Furthermore, induction of hsp70
gene expression has been reported in bacterial, yeast, and Hela cell
cultures (Slater and Craig, 1987; Sorger et al., 1987; Zhang et al., 1998)
long maintained in the laboratory. Thus, we conclude that
U. pinnatifida gametophytes must be utilizing heat shock proteins
via a mechanism that is downstream of gene transcription.
In terms of our organismal assessments, U. pinnatifida gametophytes did appear to be broadly thermotolerant. The lack of any
qualitative observations of degradation at 31 °C or lower and minimal
damage at 33 °C indicates that this stage is extremely robust. Despite
decreasing oxygen evolution rates with increasing temperature, net
photosynthesis remained positive even at the highest temperature
sampled in our photosynthesis experiments (31 °C). While the typical
short-term response of light-saturated photosynthesis is to increase
progressively with temperature up to an optimum, beyond which it
declines rapidly (Kremer, 1981; Sukenik et al., 1987), U. pinnatifida
gametophytes in our study exhibited an average 58% reduction in
oxygen evolution rate on a fresh weight basis and an average 69%
reduction on a chlorophyll a basis from 17 to 31 °C. However,
experiments that report increasing algal photosynthetic rates followed by declines have been conducted mostly between 5 and 20 °C.
For example, cultivated U. pinnatifida sporophytes in Korea increased
photosynthetic rates as much as 7 fold when subjected to incubations
from 5 to 20 °C with the optima at 15 or 20 °C, depending on the
season (Oh and Koh, 1996). Studies on U. pinnatifida that have sampled
temperatures comparable to our study also have reported declining
photosynthetic rates. In Chinese cultivars, a 5% reduction from 15 to
25 °C was measured (Wu et al., 1984), and in wild New Zealand
populations a 60% reduction in Pmax from spring to summer (a
temperature increase from 15 to 18 °C; (Campbell et al., 1999) and an
80% reduction in Pmax from winter to summer (Dean and Hurd, 2007)
have been reported. The New Zealand the authors attribute these large
declines to changes in nutrient content, pigmentation, and developmental stage in addition to temperature (Campbell et al., 1999). Thus,
we estimate the optimum temperature for photosynthesis of
U. pinnatifida gametophytes is around our lowest incubation temperature of 17 °C, as other kelp species have been found to peak at
approximately 15 °C (Fain and Murray, 1982) and the optimum is
typically a range of several degrees rather than a single value for
macroalgae (Madsen and Maberly, 1990; Oates and Murray, 1983).
Our results indicate that gametophytes of invasive U. pinnatifida
may be more thermotolerant than gametophytes of the native giant
kelp Macrocystis pyrifera. While oxygen evolution dropped from 65 to
27 mg O2 · g-1 dwt h-1 from 17 to 31 °C in this study, M. pyrifera
gametophyte oxygen evolution dropped from approximately 25 to
-25 mg O2 · g-1 dwt h-1 from 15 to 30 °C (Fain and Murray, 1982).
Previous experiments on U. pinnatifida sporophytes indicate that
upper oxygen evolution rates are at least 10 times higher than those
recorded for other Laminariales (Campbell et al., 1999). This ability for
U. pinnatifida to maintain positive net photosynthesis even at high
temperatures may contribute significantly to its ability to cross oceans,
invade novel habitats, and eventually displace native species.
The ratio of variable to maximum fluorescence (Fv/Fm) has long
been used as an indicator of physiological status. Reduced efficiency of
photosystem II (PSII) caused by exposure to environmental stress
lowers Fv/Fm due to a decrease in Fm and an increase in F0, the latter
indicating damage to PSII reaction centers (Osmond et al., 1993). The
Fv/Fm of unstressed plants has been reported to be around 0.8 for most
terrestrial angiosperms (Björkman and Demmig, 1987), mosses
(Meyer and Santarius, 1998), and green algae (Franklin et al., 1992);
however, it appears that kelps and red algae may exhibit lower yield.
For example, under optimal light and temperature conditions Laminara saccharina (Bruhn and Gerard, 1996) and Laminaria japonica
(Pang et al., 2007) sporophytes exhibit maximum Fv/Fm around 0.7.
The dark-adapted yield of U. pinnatifida gametophytes in this study
ranged from an overall average high of 0.461, decreasing with
increasing temperature incubations to an average low of 0.250,
corresponding to a 60% reduction over the temperature treatments.
These values were quite similar to those observed for the temperate,
red seaweed Laurencia pacifica which ranged approximately 0.45–
0.25 in temperature treatments from 15 to 30 °C (Padilla-Gamino and
Carpenter, 2007). In mosses, a drop in Fv/Fm below about 0.2 indicated
irreversible thermal inactivation of net photosynthesis (Meyer and
Santarius, 1998); thus U. pinnatifida gametophytes in our study may
have been nearing upper thermal tolerances for maintaining intact
photosynthetic pigment apparati at the incubation temperature of
31 °C.
It is difficult to evaluate the consequences of the short-term
response of photosynthesis to increasing temperature because in
general, temperature optima for photosynthesis are generally several
degrees higher than the optimum for growth. Indeed, positive net
photosynthesis (measured over minutes or a few hours) can occur at
temperatures well above the upper thermal limit for long-term
survival (Davison, 1987; Davison and Davison, 1987; Li and Morris,
1982). Short term stress events (minutes to hours) disturb photosynthetic performance while long term stress events (days to weeks)
result in a decline in chlorophyll content (Lichtenthaler and Miehé,
1997). Over the long term, phenotypic acclimation (i.e. reversible
metabolic compensation) allows algae to maintain positive photosynthesis and growth over a broad range of environmental conditions,
and counteracts the short-term effect of temperature on the enzymic
reactions of CO2 assimilation (Davison, 1991; Davison et al., 1991;
Dudgeon et al., 1990; Kubler and Davison, 1993, 1995; Machalek et al.,
1996; Major and Davison, 1998). While we did qualitatively observe
the loss of some accessory pigments in the 33 °C samples and the loss
of chlorophyll and cell death in the 36 °C samples, longer term
experiments and more detailed quantification of pigment and
enzymes levels will be necessary to fully evaluate U. pinnatifida
gametophyte ability to survive extended exposure to high temperature and recover to invade new habitats.
An alternative hypothesis for the absence of induction of heat
shock protein gene transcription in U. pinnatifida gametophytes may
be that rather than invest resources in that response, they become
reproductive and initiate development to the sporophyte stage.
Environmental conditions or stressors at competent larval stages
have been shown to cue metamorphosis or early settlement in some
species of invertebrates (Boettcher, 2005; Edmunds et al., 2001;
Gaudette et al., 2001; Lutz et al., 1970; Pechenik, 1984; Pennington
et al., 1999; Zacharia and Kakati, 2004), and the onset of heat-induced
metamorphosis has been correlated with the appearance of heatshock proteins in a hydroid (Kroiher et al., 1992). However, it seems
that for U. pinnatifida a drop in temperature rather than heat stress
cues reproduction. In invasive California populations, cultures from
Monterey zoospores raised at 13 °C had 3 times as many sporophytes
as those raised at 21 °C, those from Santa Barbara had 10 times as
many sporophytes at 13 °C than at both 17 and 21 °C, and recruitment
pulses in Santa Barbara Harbor happened when temps were 17–21 °C
and 12–17 °C (Thornber et al., 2004). It cannot be determined from
S.K. Henkel, G.E. Hofmann / Journal of Experimental Marine Biology and Ecology 367 (2008) 164–173
these observations if the greater success and recruitment pulses at
lower temperatures are due to spore competency or gametophyte
fertility. However, in China, zoospores are cultured at 20 °C,
gametophytes are held at 25 °C, and discharge of male and female
gametes takes place when temperatures are lowered to 20–22 °C (Wu
et al., 2004), and in Japan reproduction usually occurs below 20 °C and
does not occur higher than 23 °C (Saito, 1956), so it is likely that low
temperatures rather than high induce gametogenesis in this species.
Finally, U. pinnatifida gametophytes may survive high temperature
conditions by becoming dormant and developing a resting stage. In
Japanese cultures, when temperatures exceed 25 °C, small male and
female gametophytes adopted a phase characterized by thick-walled
spherical cells filled with chromatophores that withstand temperatures up to 30 °C (Hay and Villouta, 1993) (Akiyama 1965, Tamura
1966, Saito, 1975, Gong 1991), and in Port Phillip Bay Australia,
U. pinnatifida gametophytes are reported to go dormant from summer
to winter (Campbell et al., 1999).
5. Conclusion
In conclusion, our study investigating survivorship, metabolism,
and gene expression of Undaria pinnatifida gametophytes under heat
stress indicated that the microscopic, sexual life-history stage of this
global invader was extremely thermotolerant. The lack of upregulation in hsp70, positive photosynthetic rates, and body of
evidence that indicated gametophytes are commonly able to survive
temperatures up to 33 °C, suggests that the gametophytes were not
experiencing high levels of protein damage and degradation at the
incubation temperatures or duration used in this study. This tolerance
for a wide range of temperature conditions does not seem to be
conferred by traditional induction of the heat shock response via upregulation of the gene coding for heat shock protein 70. Rather,
gametophytes of U. pinnatifida may be less susceptible to damage by
high temperature, or they use alternative strategies for dealing with
temperature stress as has been observed other organisms. Recent
studies reviewed in (Hamdoun and Epel, 2007) indicate that
developmental stages may not be as sensitive as previously thought
(Anger, 1996a,b), and that a variety of cellular mechanisms such as
constitutive expression or alternate types of heat shock proteins and/
or suspension of development provide robustness and buffer embryos
from the environment.
Acknowledgements
This study was funded primarily by an EPA STAR grant to SKH.
Additional funds were obtained from a PISCO grant to GEH. The
authors wish to acknowledge the following individuals who facilitated
Undaria collections: Steve Lonhart of the Monterey Bay National
Marine Sanctuary, Monterey Harbor Master Steve Pryor, Marla
Ranelletti, Rachel Woodfield of Merkle and Associates, and Erin
Maloney of Moss Landing Marine Laboratories. The authors gratefully
acknowledge the assistance of Dr. Robert C. Carpenter (California State
University, Northridge) for the use of the Oxygen Electrode System
and diving PAM as well as laboratory space in which to conduct the
photosynthesis experiments. The authors would further like to
acknowledge R. Spencer McLintock for assistance with heat shocking
gametophyte tissue. This is contribution number 303 from PISCO, the
Partnership for Interdisciplinary Studies of Coastal Oceans funded
primarily by the Gordon and Betty Moore Foundation and David and
Lucile Packard Foundation. [SS]
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