Limb.
Oceanogr., 38(6), 1993, 1193-l 199
0 1993, by the American
Society of Limnology
Heritable
variation
and Oceanography,
in carotenoid
Inc.
content in Dhphnia
magna
Luc De Meester and Natalie Beenaerts
Laboratory
of Animal
Ecology, University
of Ghent, K. L. Ledeganckstraat
35, 9000 Gent, Belgium
Abstract
Phenotypic variability in carotenoid content in a group of 28 Daphnia magna clones derived from two
populations is shown to have a significant, albeit rather low, genetic component (hR2 = 0.23). To test the
idea that pigmentation and vertical migration are two alternative mechanisms to reduce photodamage,
we checked for a positive correlation between carotenoid content and phototactic behavior. Although no
significant correlation was observed for the total group of clones, a significant and positive relation was
found for the clones of one of the two populations. These results suggest that, in particular populations,
depth during the day and carotenoid pigmentation can bc co-selected.
ited to clear or shallow waters (e.g. mountain
lakes, Ringelberg et al. 1984), restricting the
power of the hypothesis to vertical migration
patterns under specific conditions. Second, it
has been suggested that an important function
of carotenoids could be energy storage (Ringelberg 1980). Higher survival under high light
intensity of more heavily pigmented animals
may then result from higher stress tolerance,
and the correlation between pigmentation and
vertical distribution
(Hairston 1980a) might
reflect only feeding history (Ringelberg 1980).
The strength of this argument is, however,
weakened because the function of carotenoids
in energy storage has not been shown experimentally, whereas their effect in reducing mortality upon exposure to high light intensity is
well established (see also Hairston 1980b).
In recent years, evidence has accumulated
that vertical migration may act as a (visual)
predator avoidance
mechanism
(Lampert
1989), often directly induced by predator-mediated chemicals (Ringelberg 199 1). As evidence supporting the predator avoidance hypothesis accrued, attention was drawn from
other hypotheses that may account for the evolution of vertical migration. Yet there is no
reason why variability
in vertical migration
patterns should be determined by only one
evolutionary factor. Field studies with electrophoretic techniques (Weider 1984) and laboratory observations on phototactic behavior
(De Meester 199 1) suggest that, at least in
Daphnia, migration patterns may be genotypeAcknowledgments
dependent. The observation of genotypc-deH. J. Dumont critically read and edited the manuscript.
pendent migration behavior implies that the
A review by N. G. Hairston, Jr., is gratefully a&nowlmigration pattern of a particular population or
edged. L. D. M. is a postdoctoral researcher with the National Fund for Scientific Research (Belgium).
subpopulation can have adaptive significance
Among diaptomid
copepods, individuals
with a high concentration of carotenoid pigments have been shown to survive natural intensities of visible and ultraviolet light better
than less pigmented individuals
(Hairston
1976, 1979; Byron 1982). The experiments
performed by Siebeck (1978) with different
Daphnia species from mountain puddles and
lakes also showed that the more pigmented
species (Daphnia pulex obtusa) was more tolerant of UV-B radiation than the less pigmented ones (Daphnia galeata and Daphnia
longispina). Hairston (1980a) observed that
the carotenoid content of Diaptomus sicilis and
Diaptomus nevadensis was correlated with
daytime vertical distribution
in two lakes and
hypothesized that diurnal vertical migration
in zooplankton may have evolved as a mechanism to avoid photodamage. According to
this hypothesis, zooplankters have two alternative ways of protecting themselves against
the deleterious effects of bright sunlight: produce photoprotective
pigments or perform a
nocturnal vertical migration, staying lower in
the water during the day than at night. The
better option would then be heavy pigmentation when predation pressure is weak and vertical migration when predation pressure is
strong.
Two critiques of this hypothesis have been
put forward. First, the deleterious effects of UV
radiation and high light intensities seem lim-
1193
1194
De Meester and Beenaerts
other than predator avoidance (Dumont and
De Meester 1990).
Irrespective of the possible role of photodamage as a selective force underlying evolutionary development of nocturnal migration
behavior, reduction of the deleterious effects
of light may be an important consequence of
living at a deeper depth in the daytime. Provided that the making and storage of carotenoids is costly, it is expected that animals that
do migrate to deeper water during the day
would tend to have a lower carotenoid content
than animals that stay in shallow water.
The purpose of the present paper is to determine whether, under standardized laboratory conditions, there exists heritable variation
in carotenoid content in Daphnia magna. If
so, this information
can be used to test the
idea that pigmentation
and diurnal vertical
migration behavior are related adaptations. It
has been shown that heritable differences in
phototactic behavior in D. magna result in different vertical distribution patterns (De Meester 1993). In outdoor containers, animals of a
positively phototactic clone have a shallower
depth during the day than animals from a negatively phototactic clone, with intermediately
phototactic clones showing an intermediate
distribution (De Meester 1993). By looking for
a significant and positive correlation between
carotenoid content and phototactic behavior
of clones, we attempt to test a basic prediction
of the photodamage hypothesis. If genotypes
with different phototactic behavior are due to
selection by visual predation in the way proposed by the photodamage hypothesis, we expect phototactic behavior and carotenoid content to be co-selected (i.e. positively phototactic
clones should have a higher carotenoid content
than negatively phototactic ones).
Methods
Origin of the clones - We used 28 clones (Table 1): three (C,, C3, and C,) isolated from a
single natural population (a fishless city pond
in Gent), and 25 sexual offspring clones (intraand interclonal crosses) of these three clones
as well as of a clone (Pi) isolated from another
pond (Driehoekvijver,
Heusden). In the latter
pond, predation pressure by fish is variable but
always low (mainly R&us rutilus and Gasterosteus aculeatus). The method of obtaining
sexual offspring of known origin is described
by De Meester (199 1). The intraclonal sexual
offspring clones were: 4 C, x , 4 C3 x , 1 C, x
(C,56), 2 Pr X , and 4 P, 32 x clones (i.e. intraclonal offspring of clone P, 32, which is one of
the P1 x clones that was analyzed). In addition,
10 offspring clones from interclonal crosses
were analyzed: 4 P, 12C, x (resulting from
crossing P, 12 and C,, with clone C1 as the
father clone), 4 Pi 8 1Ci x , 1 P1 12P1 32, and 1
Pi 8 1Pi 12. We worked with these clones because they encompass clones with very different phototactic behavior (De Meester 199 1;
see Table I) and represent two different populations. The study of sexual offspring of known
origin enables us to estimate heritability under
sexual reproduction.
Culture conditions-Clones were cultured in
l-liter jars at moderate density (25-35 adult
females liter-i),
20+ 1°C and long-day photoperiod (14 : 10 L/D). Light in the culture
room was provided
by fluorescent lamps
(“cool-white”),
the light intensity at the level
of the culture bench was 103 x 10e3 W m-2.
The culture medium was dechlorinated tapwater, enriched with Scenedesmus acutus
grown in batch cultures. A food concentration
of -4 x 1O5S. acutus cells ml- l was restored
3 times a week. To avoid gradual accumulation
of minor differences in culture conditions,
which can originate due to characteristics of
the different clones per se (De Meester pers.
obs.), we mixed the medium of all cultures
when we added food and fresh medium. Redistributing the medium was done as follows:
a common batch of fresh food-enriched medium was distributed over all cultures, 0.25
liter per culture. The animals were transferred
to the fresh medium, the “old” medium was
filtered over 60 pm and mixed, after which it
was redistributed over all cultures (0.75 liter
per culture). At - 1-week intervals, the culture
jars were thoroughly cleaned to prevent accumulation of detritus and algae on the walls
and bottom.
Analysis of carotenoid content -Only animals cultured for at least two generations under the preset conditions were used for analysis, to minimize maternal effects (Falconer
198 1). Animals were inoculated when <24-h
old and analyzed when carrying their second
brood. The method for extracting carotenoids
was similar to that of Herring (1968). As a rule,
10 animals were analyzed at a time. They were
1195
Photoprotection in Daphnia
Table 1. Analysis of carotenoid content of several Daphnia magna clones. Number of analyses-n;
average (-I- SD)
OD standardized to dry weight (g) with species-specific length-weight regression, with OD the average of measurements
between wavelengths 450 and 480-OD
g-l; wavelength at which maximal OD was observed most often (in parentheses:
average (k SD) pg carotenoids per gram
number of observations with maximal OD at the given wavelength)-&,;
dry weight, calculated under the assumption of a specific extinction coefficient in ethanol of 2,100-fig
g-l; average
value of index of phototactic behavior, with characterization
of behavior as positive (+), intermediate (x), or negative
(-) (see De Meester 199 1)--I. Brackets indicate that an equal number of observations showed maximal OD at 462 nm.
Clone
G
C,36
C,56
C,61
c,so
n
OD g- ’
6
2
56.93k9.87
53.98k6.83
50.11-1-12.16
55.97k7.87
52.23* 10.27
9
3
3
6
6
45.08& 10.54
43.8Ok5.45
47.53fl6.13
42.33k3.44
48.5725.32
2
G
Gl
w
w
C,lO
x msx
Pgg
I
’
0.92
0.48
0.95
0.80
0.93
(+)
(x)
(+)
(i-)
(+)
1460(3)1
1460(3)1
322.Ok75.3
312.9k38.9
339.5* 115.0
302.5k24.7
336.9k55.4
-0.92
-1.00
- 1.00
-0.97
-0.99
(-)
(-)
(-)
(-)
(-)
-0.33
-0.80
(x)
(x)
460(4)
460(3)
460(4)
460(4)
406.5k70.4
385.5k48.9
357.9f86.8
399.9k56.1
374.2k73.4
460(7)
460(2)
460(2)
WWI
6
4
52,2Ot-7.0 1
40.7Ok4.68
460(5)
464(3)
372.9f50.0
291.Ok33.4
P,l2
3
60.2Ok9.53
464(2)
429.9f68.3
-0.96
(-)
P,32
P, 32,48
P, 32,70
P,32,85
P,32,180
4
3
7
4
3
57.lOkl5.39
48.50f5.88
59.29k8.5 1
55.03zk9.69
64.00f 11.33
464(3)
464(2)
464(4)
MU;
407.9f 110.0
346.6k41.8
423.4k60.9
393.1269.3
457.2k80.6
-0.38
- 1.00
-0.20
0.94
-0.81
(x)
(-)
(x)
(+)
(x)
P, 32P, 12,l
6
47.48 k7.68
464(4)
339.Ok55.0
-0.92
(-)
P,8lP,
7
37.0027.04
464(5)
264.7k50.3
-0.85
(-)
6
5
4
5
48.67k6.35
34.98k8.36
53.4Ok5.59
59.52k20.30
WW;
347.6k45.3
249.9t-59.8
381.4k39.7
425.2f 145.2
0.60
0.09
-0.34
-0.76
(x)
(x)
(x)
(x)
5
6
6
5
43.22k3.70
50.5515.35
61.92-+10.41
56.26f5.81
308.6k26.4
349.3250.1
442.1 -t-74.4
401.7f41.4
-0.06
-0.60
-0.60
-0.19
(x)
(x)
(x)
(x)
G
C,56
P,
P,
P,
P,
12,2
12c, ,2
12c, ,3
12c,,9
12C,,lO
P,8lC,,l
P,81C,,4
P,8lC,,5
P,8lC,,6
kept for 3-4 h in dechlorinated tapwater enriched with baker’s yeast (0.3 g liter-‘) to promote replacement of the algae in the intestine
with yeast. The animals were measured (length
from top of eye to base of tail spine), dried
slightly on filter paper, and homogenized in
1.5 ml of 90% ethanol. Extraction was completed by placing the samples in the dark at
40°C for 1 h, after which they were centrifuged
for 10 min at 1,000 r-pm. The absorption spectrum of the supernatant between 400 and 500
nm was determined with a Pye Unicam SP 1800
spectrophotometer.
OD was measured at lonm intervals. In calculations, we used the average OD between 450 and 480 nm because
the absorption peak of carotenoids is in this
region (Herring
1968; Davies 1976). The
1464(2)1
460(3)
460(3)
460(4)
146(X3)1
460(3)
wavelength of maximal absorbance in the 450480-nm range was then determined with a precision of 2 nm.
Length-weight
regressions -Carotenoid
content was expressed relative to dry weight.
We determined a length-weight
relation for
animals cultured under the same conditions as
for the analysis. Length-weight regressions were
determined as
W = bLa,
in which W is weight in pg, and L is length in
pm (Dumont et al. 1975). Average length and
weight were determined in three groups of eight
individuals
(primiparae, animals with a second brood, and animals with a third brood)
for each clone. Thus, we obtained three data
De Meester and Beenaerts
1196
Table 2. Heritability
in the broad sense (hB2) of carotcnoid content, measured as ODC450-4803
g I dry wt, for all
28 clones as well as for the clones derived from the two
populations separately. Number of clones-N,
number of
analyses- n.
Group
of clones
Method*
N
n
h,,’
Sign.
All
A
B
28
28
146
120
0.223
0.144
P < 0.001
P < 0.025
C clones
P clones
A
A
12
8
67
37
0.154
0.091
P < 0.05
ns
* A-OD
standardized
to dry weight through species-specific
relationship.
B-OD
standardized
to dry weight through
length-weight
relationship.
length-weight
family-specific
points for each clone. We could therefore determine the length-weight
relation for the
complete data set as well as for each family
(i.e. parent + offspring clones) separately. Although the latter has the advantage of taking
genotype-dependent differences in the lengthweight relationship into account, we mainly
used the length-weight
relationship obtained
for the total set of clones (28 x 3 data points)
to standardize OD values. We did this because
the family-specific
length-weight
regressions
were based on too few data points (15 in most
cases). The length-weight
relationship as determined here includes the weight of the brood
because carotenoid content was also determined for animals carrying their brood.
Heritability estimates-Heritability
in the
broad sense was determined by a clonal repeatability analysis (Falconer 198 1; De Meester 199 1). After testing for homogeneity of
variances and normality,
the between-genotype variance component was weighed against
the within-genotype
variance component in a
one-way ANOVA, and hB2was estimated according to Lessels and Boag (1987). The resulting estimate is inflated by maternal and
other common environmental
effects. However, both were minimized in the present study,
as all analyses were done with animals from
different generations and the culture medium
was redistributed three times a week.
We also estimated heritability in the narrow
sense through an offspring-midparent
regression (Falconer 198 1). The average of the OD
g-l value of the two parent clones was used as
midparent value. The slope of the regression
is an estimate of the heritability in the narrow
sense (h2). Its value estimates the proportion
of the phenotypic variance that is determined
by additive gene effects and therefore measures
heritability
under sexual reproduction.
Results
Length-weight
relationship - Primiparae
measured between 2.37 and 2.77 mm, adults
carrying their second brood between 2.73 and
3.17 mm, and adults carrying their third brood
between 2.78 and 3.41 mm. For all 28 clones
combined, the length-weight relationship was
w = 1. 174 x 10-7L2.63 .
IIeritability of carotenoid content -The average carotenoid content of animals from the
different clones, standardized to dry weight, is
reported in Table 1. In all analyses, peak OD
was observed between 460 and 464 nm. The
heritability
estimates obtained by clonal repeatability analyses are given in Table 2. For
the complete set of 28 clones, a significant genetic component to phenotypic variability in
carotenoid content, with hB2 = 0.23, was obtained when OD was standardized with the
length-weight
relationship
obtained for the
same group of clones. When the results of the
different clones were standardized to pg dry
weight with the length-weight relationship obtained for each family separately, the resulting
heritability estimate was lower (hB2= 0.14) but
still significant (P < 0.025). Significant differences in carotenoid content between sister
clones were observed in one family (P, 8 1C, X )
but not in the others. Analyzing the data for
the clones derived from the two habitats separatcly (C and P clones, respectively, excluding
P1 x C1 crosses) showed that variability in carotenoid content had a significant between-genotype component in the C population (hB2=
0.15), whereas differences between genotypes
were insignificant for clones derived from the
P population.
Figure 1 shows a scattergram in which the
value for OD g- l of the average offspring clone
was plotted against the average value of the
parent clones for the three families of intraclonal and the two families of interclonal origin (each family consists of four offspring
clones). An offspring-on-midparent
regression
on the results of all intraclonal crosses (13)
yields a heritability estimate (narrow sense) of
0.8 1. Although this estimate is significant (P
< 0.025), the confidence interval is broad
1197
Photoprotection in Daphnia
Table 3. Correlation (Kendall’s r and significance level) between carotenoid content (OD,,,,,
g-l) and phototactic behavior (r). Number of clones--n.
Group
of clones
n
7
Sign.
All
C clones
P clones
28
12
8
0.232
0.708
0.143
ns
P < 0.05
ns
Discussion
I
. . . . . . . . . . . . . . . . . . . . . . . . . .
3’5
I
4b 4’5 $0 55 sb
ODg-1, midparent value
0 intraclonal
offspring
C, clones
0 intraclonal
offspring
PA32
w offspring P,xC, crosses
Fig. 1. Scattergram plotting carotenoid content (OD,,,,,
g- ’ dry wt) of average sexual offspring clone against the
average value of the parent clone(s) for the five families
for which four offspring clones were analyzed. Solid line regression line for results on intraclonal families 0, = 0.806~
+ 8.224; P < 0.025); broken line-regression
line for results on all families (y = 0.489~ + 23.558; P > 0.05).
(0.22-l .4) due to the limited data set. The heritability estimate is much lower (h2 = 0.49)
and not significantly different from zero when
the results of interclonal crosses are included
in the analysis (23 offspring clones in total;
95% C.I. of slope: -0.21-l.
19).
Carotenoid content and phototactic behavior-There
is no significant correlation between average carotenoid content and the value of the phototactic index Z (see Table I) for
the total set of 28 clones analyzed (Table 3).
A significant and positive correlation is, however, found when the clones derived from the
fishlcss city pond (Gent) are analyzed separately. The correlation is not significant for the
P clones.
The length-weight
relationship determined
in our study is in good agreement with others
in which the brood was included in the analysis
(Bottrell et al. 1976).
Our analysis does not permit us to identify
the carotenoids with certainty. However, the
omnipresence of absorption peaks at 460,462,
or 464 points to echinenone as an important
constituent. Echinenone shows maximum absorption at 462 nm when extracted in ethanol
(Davies 1976). Several others (Herring 1968;
Partali et al. 1985) found echinenone to be an
important component of the carotenoids in
Daphnia. The approximate values for the carotenoid content for the different clones, ranging from 249.9 to 457.2 bg g-l dry weight, are
intermediate to those obtained by Hairston
(1978) for weakly (230 pg g- l) and heavily (749
pg g-l) pigmented Diaptomus kenai. The values rcportcd by Hebert and Emery (1990), 7001,400 bg g- I, are much higher. The differences
between our results and theirs may be related
to our extraction time being shorter, the use
of different species (D. pulex and Daphnia middendorfiana vs. D. magna), and the different
origin of the material. Indeed, a number of the
ponds studied by Hebert and Emery contained
daphnids that were also characterized by melanin pigmentation, suggesting that photodamage is an important selective factor in these
habitats.
WC observed a significant genetic component to the variation in carotenoid content of
D. magna, although heritability was not high
(hB2 = 0.23). In at least one of the two populations from which clones were derived, we
observed an intrapopulation
genetic polymorphism for carotenoid content. The observation
that clones with a relatively high (low) carotenoid content tend to yield sexual offspring
with similarly high (low) carotenoid content at
1198
De Meester and Beenaerts
least qualitatively corroborates the heritability
of the interclonal differences. On the other
hand, the value of the heritability
(narrow
sense) estimate should be taken as indicative
only, due to the low number of families and
offspring clones available. Although the carotenoid content of the offspring clones of the
P18 lC, cross is close to expected under the
assumption of maximum heritability,
the carotenoid content of the other interclonal offspring clones is lower than expected (see Table
0
vations indicate that, where photodamage is
an important selective factor, less pigmentation is required to protect clones found at deeper daytime depths from photodamage.
We conclude that the photodamage hypothesis cannot account in a general way for differences in daytime vertical distributions
between D. magna (sub)populations.
However,
our results indicate that photodamage and
daytime vertical distribution may nevertheless
be linked, in that reduced damage by high light
intensity may be an important consequence of
spending the day at deeper depths. Under very
specific circumstances, photodamage may be
a direct cause for evolution of a deeper daytime
depth distribution.
Indeed, whenever the cost
of remaining deeper in the water column (during the day) is less than the cost of accumulating carotenoids, photodamage per se may
result in living at deeper depths during the day
as an alternative to increased pigmentation.
Carotenoid content in zooplankton has been
reported to be influenced by diet as well as by
light intensity (Green 1957; Herring 1968; Partali et al. 1985). As we worked under standardized conditions, we somehow maximized
chances of finding heritable differences in carotenoid content. In the case of population C,
however, the differences in carotenoid content
between positively and negatively phototactic
clones observed in the laboratory will tend to
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Submitted: 30 April 1992
Accepted: 11 January 1993
Revised: 3 February 1993