Effects of Chytrid and Carbaryl
Exposure on Survival, Growth and
Skin Peptide Defenses in Foothill
C A R L O S D A V I D S O N , *, †
M I C H A E L F . B E N A R D ,‡
H . B R A D L E Y S H A F F E R ,‡
J O H N M . P A R K E R ,§C H A D R I C K O ’ L E A R Y ,⊥
J . M I C H A E L C O N L O N ,|A N D
L O U I S E A . R O L L I N S - S M I T H⊥
Environmental Studies Program, San Francisco State
University, 1600 Holloway Avenue, San Francisco CA 94132,
Section of Evolution and Ecology, and Center for Population
Biology, University of California, Davis, California 95616,
Office of Laboratory Animal Care, Northwest Animal Facility,
University of California, Berkeley, California 94720-7150,
Departments of Microbiology and Immunology and of
Pediatrics, Vanderbilt University Medical Center, Nashville,
Tennessee 37232, Department of Biochemistry, Faculty of
Medicine and Health Sciences, United Arab Emirates
University, 17666 Al-Ain, United Arab Emirates
contribute to amphibian population declines. Sub-lethal
levels of contaminants can suppress amphibian immune
defenses and, thereby, may facilitate disease outbreaks. We
foothill yellow-legged frogs (Rana boylii) to determine
whether sublethal exposure to the pesticide carbaryl would
increase susceptibility to the pathogenic chytrid fungus
Batrachochytrium dendrobatidis that is widely associated
with amphibian declines. We examined the effect of
carbaryl alone, chytrid alone, and interactions of the two
We found no effect of chytrid, carbaryl, or their interaction
on survival. However, chytrid infection reduced growth
growth in post-metamorphic amphibians due to infection
growth in vitro, which may explain why chytrid exposure
did not result in significant mortality. Skin peptide defenses
were significantly reduced after exposure to carbaryl
suggesting that pesticides may inhibit this innate immune
defense and increase susceptibility to disease.
to be a primary cause of amphibian declines for nearly a
decade (1-3), yet there has been surprisingly little ecotoxi-
is strongly implicated in declines worldwide, with the
discovery of a previously unknown chytrid fungus (Batra-
associated with field mortality in Australia, North, South,
and Central America, and Europe. As the research and
regulatory communities recognize that pesticides, disease,
about population declines, experimental work on multiple
direction for amphibian decline studies.
In California, declines of four frog species (Rana boylii,
R. cascadae, R. draytonii, and R. muscosa) are strongly
associated with the amount of upwind pesticide use (4).
Furthermore, for these four species and the Yosemite toad
(Bufo canorus), upwind use of cholinesterase-inhibiting
types of pesticides (4). However, documented field levels of
pesticides are generally several orders of magnitude below
lethal concentrations as determined in laboratory studies
likely not due to direct lethal effects, but rather through
sublethal effects and interactions with other stressors (5).
Recent studies by several research groups suggest that
susceptibility (6-10, reviewed in ref 11), suggesting that a
A disease that may interact with pesticides to promote
declines in amphibian populations is chytridiomycosis (the
disease caused by B. dendrobatidis). Amphibians with
include at least 93 species (www.jcu.edu.au/school/phtm/
been found on a number of amphibian species, including
Rana boylii. Two non-mutually exclusive explanations may
account for the sudden increase in chytrid frequency.
Batrachochytrium dendrobatidis may be a novel pathogen
attacking a defenseless host (12). Alternatively, some aspect
of the environment may have changed, increasing the
susceptibility of amphibians to the disease (13). The pos-
sibility that the susceptibility of amphibian populations to
the chytrid fungus has increased as a result of pesticide-
induced immune suppression is consistent with field ob-
servations and the second explanation, but has yet to be
Our research was designed to examine the effects of
pesticide and chytrid interactions on frogs. We conducted
foothill yellow-legged frog (Rana boylii) susceptibility to
chytrid fungus. We exposed juvenile R. boylii to a single
sublethal dose of carbaryl, followed by exposure to chytrid
fungus. The animals were followed for 2 months to evaluate
exposure on the production or release of antimicrobial skin
chytrid fungus (14-17). Finally, we examined the ability of
R. boylii skin peptides to inhibit growth of chytrid cultures
in vitro to assess their potential role in providing protection
Materials and Methods
Choice of Species. We worked with foothill yellow-legged
* Correspondingauthorphone: 415-405-2127;fax: 415-338-2880;
†San Francisco State University.
‡University of California, Davis.
§University of California, Berkeley.
⊥Vanderbilt University Medical Center.
|United Arab Emirates University.
Environ. Sci. Technol. 2007, 41, 1771-1776
10.1021/es0611947 CCC: $37.00
Published on Web 01/23/2007
2007 American Chemical SocietyVOL. 41, NO. 5, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY91771
with upwind pesticide use (4). Additionally, as a member of
the boylii group of ranid frogs, R. boylii shares phylogenetic
affinities with other declining ranid frogs in California, and
our results should be relevant to declines of these species.
We collected young R. boylii metamorphs from the Garcia
River (Mendocino County, CA), October 6, 2002. See Sup-
porting Information for details of animal care.
We choose the pesticide carbaryl (1-naphthyl N-methyl
carbamate) for our experiments because it is widely used in
California and the United States. It is also a strong cho-
linesterase inhibitor and thus may be representative of
and carbamate pesticides). We used commercially available
carbaryl (Monterey brand “7” manufactured for Lawn and
Garden Products, Fresno CA). All other chemicals were
purchased from Sigma Chemical (St. Louis, MO) unless
to estimate the 24-hour minimum lethal concentration of
carbaryl for R. boylii metamorphs (See Supporting Informa-
tion for details).
Culture of Chytrid and Exposure of Frogs to the
Pathogen. We used chytrid strain “LJR119” isolated from R.
at the University of California, Berkeley. The chytrid was
raised on 1% tryptone agar plates. Zoospores were washed
a hemocytometer counting chamber, and diluted to a
concentration of 1.9 × 105zoospores per mL. Animals were
placed in one-liter beakers for 24 h and exposed to 9.4 × 106
zoospores per frog in a volume of 50 mL of water. Control
was flushed from sterile agar plates.
Experimental Design. We tested for an effect of chytrid,
pesticides, and their interaction on survival, individual
growth, and quantity of skin peptide secretions. We used a
two-way, full factorial design, with chytrid or no chytrid
crossed with no carbaryl or a carbaryl dose of 0.48 mg/L.
gravity), and then diluted 1000-fold. The resulting carbaryl
dose (0.48 mg/L) was 10% of the estimated minimum lethal
represented a sublethal level. This level is lower than that
locations. Beginning on day 0, frogs were exposed to
pesticides for 24 h in one-liter jars containing 70 mL of the
pesticide/artificial pond water solution. At the same time,
the no-carbaryl control treatment animals were placed in
identical jars with 70 mL of artificial pond water for 24 h. At
the end of the exposure period all animals were returned
to individual containers with clean water. Twenty-four h
chytrid treatments began on day 3. Each animal was housed
and treated separately, and therefore, each constituted an
independent replicate. The experiment was run for 70 days.
Animals were monitored daily, and any dead animals were
preserved in 10% buffered formalin. At the end of the
experiment, all surviving animals were weighed, then eu-
samples on day 19 caused the unexpected death of five
animals were excluded from all statistical analysis.
survival with a chi square test on proportion surviving per
treatment. First, to maximize power, we tested for an effect
of each factor separately (i.e., carbaryl or chytrid exposure),
by pooling treatments to produce a one-factor test. Then,
using ANCOVA with log transformed final weight as the
dependent variable and log transformed initial weight as a
covariate. Unless otherwise noted, a p-value of e0.05 was
Evaluation of Carbaryl Exposure. To allow comparison
between our carbaryl exposures and any future sampling of
four additional animals that were not part of our main
experiment to the pesticide treatment. Following the expo-
sure, they were euthanized, frozen at -20 °C, and tested for
carbaryl levels in body tissue. Testing was done by the
Geochemical and Environmental Research Group (Texas
A&M University, College Park, TX). Sample extracts of body
under selective ion monitoring.
determined by histological examination of formalin-fixed
of the two chytrid treatments (total ) 14 frogs), and two
randomly selected frogs from each of the two non-chytrid
infection. Skin scraped from the superficial ventral integu-
preparation. As a further check, tissue from animals that
were identified as chytrid-positive by skin scraping were
stained with hematoxylin and eosin, and examined for
whether treatments would affect the quantity and composi-
tion of recoverable skin peptides, frogs were induced to
secrete peptides at day 3 (after the pesticide treatment had
been applied, but immediately preceding the application of
the chytrid treatment), and again at days 7, 18, 33, and 49.
Skin peptides were induced by immersion in 200 µM
norepinephrine hydrochloride, following Woodhams et al.
(16) (See Supporting Information for details). The total
concentration of skin peptides recovered was determined
by Micro BCA Assay (Pierce, Rockford, IL) following manu-
(Sigma Chemical, St. Louis, MO) was used to establish a
measurement. After pooling, we had two peptide samples
to the day 33 sampling resulted in only one peptide sample
in the no-carbaryl, no-chytrid treatment on day 33 and day
49. For day 3 peptide samples (in which there were only
carbaryl and no carbaryl treatments), we tested differences
in total recovered peptides using a t-test. For all subsequent
affected total recovered peptides, and whether these effects
changed over time using a repeated measures design. The
pesticide treatment and chytrid treatment were considered
we were missing two data points, and thus used Satterth-
of animals was log transformed to meet normality assump-
tions. Our analyses were conducted with SAS statistical
Evaluation of Antimicrobial Peptide Defenses against
Chytrid. Six new peptides with antimicrobial activity (brevi-
nin-1BYa, brevinin-1BYb, brevinin-1BYc, ranatuerin-2BYa,
ranatuerin-1BYb, and temporin-1BYa) were previously iso-
17729ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 5, 2007
(22). We tested four of these peptides in purified form for
growth inhibition of chytrid zoospores. In addition to the
purified peptides, we tested partially purified peptide mix-
tures collected from the skin secretions of our experimental
zoospores. Growth inhibition assays were conducted as
for details). Chytrid growth with peptides was compared to
positive controls (no peptides) and negative controls (0.4%
if chytrid growth with each of the different concentrations
of skin peptides was significantly different from the chytrid
growth observed in the positive control. To accomplish this,
we conducted an ANOVA on chytrid culture growth, where
each concentration of skin peptides was a treatment (rep-
licated 3-6 times). We tested whether the chytrid growth
Dunnett’s t-test, which controls the error rate for multiple
comparisons between a single control treatment and ad-
ditional treatments. The minimal inhibitory concentration
(MIC) was determined as the lowest concentration at which
chytrid growth was not significantly greater than that
observed for negative controls.
Carbaryl Body Levels and Chytrid Infection. Of the four
frogs tested for carbaryl in body tissue, one had 0.9 µg/gbw
carbaryl (µg carbaryl/g frog body weight), one had 0.6 µg/
gbw carbaryl, and two had levels below the 0.1 µg/gbw
detection limits. Of the 18 frogs tested for chytrid infection,
animal was in the carbaryl, chytrid treatment, and had died
3 days after the initiation of chytrid exposure. Histological
examination showed heavy chytrid infection. The lack of
than 3 days after chytrid exposure may be due to infected
animals clearing their infections in the course of the
experiment (see below).
Survival. There was low mortality (14-20% mortality)
across all treatments (Table 1), with no significant effect of
treatment on survival (Pearson Chi-square, p ) 0.98). There
was no significant effect on survival of either chytrid or
pesticide treatments tested separately (pooled chytrid treat-
ments, Chi-square, p ) 0.68; pooled carbaryl treatments,
in the chytrid treatments (Table 1).
S1). There was no pesticide treatment effect on final mass
(ANCOVA, df ) 1, F ) 0.5, p ) 0.51). However, chytrid
treatment had a significant negative effect on final mass
(ANCOVA, df ) 1, F ) 18.3, p < 0.001). Rana boylii exposed
to chytrid (Table 1, Figure 1). Initial mass was positively
between chytrid and carbaryl exposure on growth.
Antimicrobial Peptide Defenses against Chytrid. Skin
peptides collected from control (no carbaryl, no chytrid)
chytrids (Figure 2b) at concentrations of 6.25 µg/mL and
above (Figure 2; Zoospores: ANOVA, df ) 8, 35, 12.6, F )
171, p < 0.0001; Mature Chytrids: ANOVA, df ) 8, 36, 12.6,
F ) 95.28, p < 0.0001). The MIC against mature chytrid cells
was 50 µg/mL (Figure 2b) and against zoospores was 25 µg/
mL (Figure 2a). These observations suggest that R. boylii
secretes potent antifungal peptides. All four of the purified
peptides previously isolated from R. boylii significantly
inhibited growth of zoospores at concentrations above 6.25
µM (Supporting Information, Figure S1). The MICs were 6
µM for brevinin-1BYc; 12.5 µM for brevinin-1BYa and
Thus, all four peptides were potent inhibitors of growth of
chytrid, as was the cocktail of total skin peptides from wild-
Effects of Carbaryl and Chytrid on Skin Peptides. The
concentration of total skin peptides was estimated by Micro
BCA Assay using bradykinin as a standard. Because the
mixture of skin peptides is diverse and contains peptides
with variable numbers of the amino acids cysteine, cystine,
measure. However, it is the most sensitive measure of total
peptides recovered under different treatment protocols.
Using this assay, total peptide concentrations recovered at
greater than in frogs exposed to carbaryl (t-test on peptide
concentrations at day 3: df ) 6, t ) 2.797, p < 0.031) (Figure
over time (days 7, 18, 33, and 49) found no effect of carbaryl
treatments on skin peptides of frogs through the remainder
of the experiment (ANOVA, df ) 1, 21.3, F ) 2.56, p < 0.12).
df ) 1, 21.3, F ) 0.92, p < 0.35) on peptide concentrations,
but the peptide concentrations steadily increased through
(day X pesticide: ANOVA, df ) 1, 20, F ) 1.55, p < 0.23; day
X chytrid ANOVA, df ) 1, 20, F ) 0.77, p < 0.39; pesticide X
chytrid X day: ANOVA, df ) 1, 20, F ) 0.17, p < 0.68).
TABLE 1. Mortality and Growth of Juvenile R. Boylii Exposed
to Chytrid and Carbaryla
treatmentmortality dead/total (%) growth mean (SD)
no carbaryl, no chytrid
carbaryl, no chytrid
no carbaryl, chytrid
aGrowth is final weight (g) of surviving animals on day 70, minus
FIGURE 1. Growth of individual R. boylii metamorphs as a function
of pesticide and chytrid exposure. Growth was calculated as (final
weight minus initial weight).
VOL. 41, NO. 5, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY91773
for samples obtained at days 3, 7, and 18. Although carbaryl
exposure affected the amount of recoverable skin peptides
on day 3, it did not appear to affect the ability of peptides
to inhibit chytrid growth in vitro. Regardless of whether the
preparations inhibited in vitro chytrid growth. The MICs for
day 3 samples were 12.5-25 µg/mL for all groups tested
against chytrid zoospores and 50-60 µg/mL against mature
chytrids (Table S2). For samples collected at day 7, MICs
were about 12.5-50 µg/mL against zoospores and 25-500
µg/mL against mature chytrids. Samples collected at day 18
were slightly less potent than those collected at the earlier
time points. The MICs against zoospores ranged from 12.5-
500 µg/mL (Table S2).
Our experimental results indicate that wild-caught, post-
metamorphic juveniles of R. boylii may be well-protected
against chytridiomycosis-induced mortality. Exposure to
chytrid resulted in dramatically reduced growth (Figure 1)
but had no effect on mortality. Previous studies found that
tadpoles of Gray treefrogs (Hyla chrysoscelis) and Fowler’s
and smaller weight at metamorphosis (25). This, however,
is the first report of suppressed development in post-
metamorphic frogs due to infection with chytrids.
Rana boylii’s ability to fend off the lethal consequences
of chytrid infection may be due to a strong complement of
at least four peptides that are individually capable of
suppressing chytrid growth in vitro. Exposure to carbaryl
decreased recoverable peptide levels for at least 3 days.
Decreased amounts of antimicrobial peptides could poten-
suppresses immune defenses, and therefore, it may interact
with disease to cause amphibian population declines.
However, in our experiments we found no interaction
between chytrid and pesticide exposure on mortality or
growth suggesting that under these laboratory conditions,
reduced skin peptide levels did not increase the effects of
chytrid infections on R. boylii. These results raise a number
Why Did Exposure of R. boylii to Chytrid Fail to Induce
Mortality? There have been a limited number of published
studies describing experimental infections of amphibians
with chytrid, and they demonstrate a wide range of suscep-
tibilities among anuran species and life history stages (26-
28). In many cases, exposure to 103chytrid zoospores per
frog was sufficient to cause high mortality. In the only other
work on R. boylii, Elizabeth Davidson et al. (29) found that
exposure of postmetamorphic juveniles to a dose of 8.5 ×
103or 8.5 × 102zoospores per mL of water did not result in
animals that were initially infected with chytrid based on
examination of sloughed skin had no identifiable infection
determined by histology at later times, suggesting that R.
boylii may be able to eliminate infections.
Based on experimental work with other anuran species,
the zoospore exposure used in the current study (9.4 × 106
zoospores per frog) should have been sufficient to induce
to chytrid-induced mortality. Our finding of heavy chytrid
dramatically reduced growth rates for chytrid-exposed
animals suggests that all chytrid-exposed animals were
infected, but that most individuals were able to control
the infection, resulting in chytrid-negative skin scrapes two
months after exposure. These observations suggest that R.
boylii has effective defenses that prevent death (but not
reduced growth) from chytridiomycosis, at least under our
defenses that may protect postmetamorphic juveniles from
infection. Six antimicrobial peptides have previously been
isolated from this species (22), and the four individual
peptides that we tested in this study against zoospores of
µM, Figure S1). Overall, the skin peptides that we collected
from our study animals were also quite effective in growth
inhibition of chytrid (Figure 2 and Table S2).
To evaluate whether frogs actually produce a sufficient
quantity of skin peptides to deter chytrids, we compared
peptide concentrations found to be effective in our chytrid
growth assays to the estimated total skin peptides produced
control frog produced about 230 µg/gbw of peptides as
determined by Micro BCA assay. A frog weighing about 1.6
g (an average weight for animals early in the study) has an
about 28.6 µg/cm2. If the mucous layer is 500 µm thick, then
the fluid over 1 cm2of skin is equal to 50 µL, resulting in
about 28.6 µg/50 µL of mucous. This amount is equivalent
to 572 µg/mL of mucous. Our experiments showed that the
MICs for skin peptides from R. boylii against chytrid were
about 25-500 µg/mL, suggesting that R. boylii has excellent
FIGURE 2. Growth inhibition of chytrid by R. boylii skin peptides.
The y-axis indicates chytrid culture growth as measured by
increased optical density at 492 nm. Top panel: zoospores after 7
days of culture. Bottom panel: Mature chytrid cells after 4 days of
culture. In both cases, incubation occurred in the presence of skin
secretions from experimental animals that had not been exposed
to chytrid or carbaryl. Each data point represents the mean (
standard error (SE) of three to six replicate wells. If no error bar
paraformaldehyde (PF). Asterisk means significantly less growth
than positive controls (Dunnett’s t-test, p e 0.05). MIC is the lowest
concentration at which growth was not significantly greater than
in negative controls.
17749ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 5, 2007
of skin peptides, may not be very susceptible to chytridi-
omycosis. Whether this represents a species-wide response
or a more localized resistance that evolved locally in north-
from the Sierra Nevada.
Why was There No Apparent Interaction of Carbaryl
and Chytrid to Increase Mortality? Our analysis of total
that the concentration of norepinephrine-induced skin
peptides was dramatically reduced in comparison with
exposed to chytrids. One reasonable possibility is that the
skin peptide concentrations that remain following carbaryl
treatment (approximately 45 µg/gbw), although much re-
Using the same calculations as above, 45 µg/gbw of skin
peptide translates to a concentration of peptides in the
zoospores were significantly inhibited by concentrations of
skin peptides at 12.5 µg/mL or higher, although the greatest
peptides in carbaryl-treated frogs was significantly reduced,
it still may have been sufficient to inhibit zoospore coloniza-
tion. It is also possible that a one-time, as opposed to
continuing, carbaryl exposure allowed frogs to recover
peptide defenses in time to defend against the effects of an
initial chytrid infection. In the field, animals may receive
multiple exposures to carbaryl or other pesticides. Finally,
it is possible that while carbaryl suppressed skin peptide
levels, other aspects of the immune system may have
protected R. boylii against chytrid infection.
What is the Mechanism for Carbaryl Effects on Skin
Peptides? Discharge of skin peptides is dependent on the
natural release of epinephrine or norepinephrine at sym-
pathetic nerve terminals innervating the granular glands of
of norepinephrine on the sympathetic nervous system.
Evidence to support this hypothesis comes from the similar
(SCC) in frog skin. In the semiaquatic frog Leptodactylus
chaquensis, bathing isolated frog skin in a relatively low
been reported to have very similar effects on the SCC across
the skin of Rana esculenta (34, 35). Thus, it is possible that
carbaryl mimics norepinephrine-induced skin peptide dis-
charge. Alternatively, carbaryl may act as a nonspecific
stressor that activates an alarm response of the sympathetic
the reduced concentrations of skin peptides detected in
postmetamorphic R. boylii 3 days after carbaryl exposure
are consistent with the interpretation that peptides had
already been discharged at day zero when the carbaryl was
applied, and that levels had not yet fully recovered.
In summary, our research demonstrated that sublethal
carbaryl exposure inhibited peptide defenses in postmeta-
a strong inhibitory effect on chytrid in culture. Although
carbaryl exposure significantly reduced skin peptide levels,
the frogs were still able to defend against chytrid infection,
and we did not detect an interaction between carbaryl
Thus, for this well-defended anuran species, the reduced
was apparently still sufficient to ward off the lethal effects
in increased mortality, it strongly inhibited growth of
postmetamorphic R. boylii, demonstrating that chytrid
exposure can have negative effects on R. boylii populations
even if it is not through direct mortality. The consequences
of reduced growth have yet to be determined in R. boylii;
however, size at and time to sexual maturity are key life-
history parameter in anurans (36, 37) that may well be
reduced due to chytrid infection in this declining species.
we encourage additional experimental study of this poten-
need to better understand the effects of multiple and lower
pesticide exposures on amphibian immune defenses for a
reduced skin peptide levels, affect susceptibility to and the
outcome of chytridiomycosis. Last, we encourage compara-
infection across multiple sites with different exposure
We thank Michelle Boone, Christine Bridges, Michael Fry,
design, Emilio Laca for generous statistical advice, Jose
Sericano for carbaryl residue analysis, Douglas Woodhams
for assistance with mass spectrometry, Kim Cadacio and
Briggs for providing us with the chytrid fungus, Tom Near
for help with field work and Lee Berger and David Green for
help with histology. Animals were collected under Fish and
Game permit 801023-05 to C. Davidson. This research was
supported in part by NSF Integrated Research Challenges in
(James Collins, P.I.) and NSF grants IBN-0131184 and IOB-
from frogs by treatment by day. Day 3 peptide collections preceded
the start of the chytrid treatment, and therefore, peptide concentra-
samples each. On day 3, peptide concentrations were significantly
less for carbaryl-exposed animals then non-exposed animals (t-
test). For days 7 on, each data point represents the mean of two
samples. However, death of some animals prior to the day 33
treatment on day 33 and day 49. A repeated measures ANOVA for
days 7-49 indicated a significant effect of time, but no significant
chytrid, carbaryl, or interaction effects.
VOL. 41, NO. 5, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY91775
0520804 to L.R-S., a Rose Foundation for Communities and Download full-text
the Environment grant to C.D., NSF grant 0213155 to H.B.S.,
and the University of California Agricultural Experiment
Supporting Information Available
Details of methods for animal care, determining lethal
carbaryl levels, skin peptide collection, and chytrid growth
inhibition assays are shown. In addition, there is an ANOVA
table for treatment affects on frog growth, a table on chytrid
growth inhibition assays by treatment, and a figure showing
chytrid growth inhibition for purified skin peptides. This
material is available free of charge via the Internet at http://
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Received for review May 18, 2006. Revised manuscript re-
ceived November 30, 2006. Accepted December 8, 2006.
17769ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 5, 2007