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First published online August 3, 2006
Journal of Experimental Biology 209, 3155-3163 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02318
Age determination in individual wild-caught Drosophila serrata using pteridine concentration
1 School of Tropical Biology, James Cook University, Townsville QLD 4812,
Australia
2 School of Integrative Biology, Queensland University, St Lucia 4067,
Australia
* Author for correspondence (e-mail: Simon.Robson{at}jcu.edu.au)
Accepted 9 May 2006
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2=14.3 and 20.4%,
respectively) suggests that the calibration of the age prediction equation for
field populations would be significantly improved when combined with
fine-scaled studies of habitat temperature and light conditions. The ability
to determine relative age in individual wild-caught D. serrata
presents great opportunities for a variety of evolutionary studies on the
dynamics of natural populations.
Key words: age determination, pteridine, Drosophila serrata, survivor function
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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), despite the large potential benefits of knowing field
survival probabilities in an organism that is also amenable to genetic
experiments (Johnston and Ellison,
1982). Estimates in the field using data from capture-release
experiments suggest modal longevities of a week or less in a number of
Drosophila species (reviewed by
Powell, 1997). A direct method
of age determination in Drosophila has been developed
(Johnston and Ellison, 1982)
using counts of growth layers in internal thoracic muscle attachments
(apodemes). This method was used to age field-caught D. mercatorum
(Templeton et al., 1993), and
it was found that the modal age class in two replicate years was 0-3 days old,
with few individuals over 12 days old. Although this method may have
application for aging younger field-caught flies, the maximum number of growth
layers (<18) obtainable (Johnston and
Ellison, 1982) may not allow for complete characterisation of the
age structure of a population in many instances. Cytoplasmic incompatibility
caused by Wolbachia infection in D. simulans decreased with male age
(Turelli and Hoffman, 1995),
which was used to infer that matings in natural populations must involve males
at least 2 weeks old, although this does not supply a method of age
determination that could be generally applied to other Drosophila
species.
A number of biochemical techniques have also been used to predict the age
of individual insects. Pteridines, a group of fluorescent chemicals derived
from a pyrimidine-pyrazine ring structure
(Ziegler and Harmsen, 1969),
increase with chronological age in populations of various dipteran taxa
populations reared under laboratory conditions
(Langley et al., 1988;
Mail and Lehane, 1988;
McIntyre and Gooding, 1995).
The relatively widespread occurrence of pteridines in insects
(Ziegler and Harmsen, 1969),
the ability to detect and characterize the small quantities found in
individuals via fluorescence spectrophotometry
(Lehane and Mail, 1985), and
the relative ease of sample preparation [field samples can be desiccated prior
to analysis (Lehane and Mail,
1985)] make quantifying these chemicals a potentially useful
technique for age determination of field populations.
Here, we examine of the utility of 6-biopterin concentration to predict the
age in individual Drosophilidae using Drosophila serrata. D. serrata
and its close relatives have been adopted as a model system for investigating
the evolution of stress resistance
(Hoffmann et al., 2003),
morphology (Hoffmann and Shirriffs,
2002) and mate choice (Higgie
et al., 2000; Hine et al.,
2004), but in natural populations such studies are limited by a
lack of detailed knowledge of the age of individuals. We present the results
of three experiments. Firstly, we determine how pteridine levels increase with
age for males and females under laboratory conditions. Secondly, we examine
the interaction of temperature and light intensity on individual pteridine
levels, which may vary under field conditions. Finally, we explore the utility
of this technique for predicting age in individual wild-caught flies.
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Materials and methods |
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) (S. K. A. Robson, unpublished).
Pteridine levels in individual fly heads were determined using methods
modified from Mail and Lehane (Mail and
Lehane, 1988). Pteridines were first extracted from individual
heads by grinding the tissue in liquid nitrogen for 60 s, then in a solution
of 0.5 ml chloroform:methanol (2:1) on ice for another 60 s, followed by
sonnication for 3 min. A solution of 0.75 ml 0.1 mol l-1 NaOH
adjusted to pH 10 with glycine (ca. 11.5 g l-1) was then added to
optimize pteridine extraction, vortexed for 10 s at 1800 r.p.m. then
centrifuged for 5 min at 5000 g at 4°C to remove any
remaining particulate material. Pteridine concentrations were determined by
taking a 0.7 ml sample of the supernatant, and analyzing with a Perkin Elmer
Luminescence Spectrometer LS50B (Beaconsfield, Bucks, UK). Samples were
excited with a wavelength of 355 nm, the emission spectra recorded between 300
and 600 nm, and the greatest intensity of the spectra (which corresponds to an
emission wavelength of 445 nm) recorded in arbitrary units. Spectral intensity
at this wavelength is proportional to 6-biopterin concentration
(Mail and Lehane, 1988).
Confirmation of pteridine detection and quantification protocols
A series of serial dilutions using 6-biopterin (Sigma-Aldrich: Sydney, NSW,
Australia), a principle component of dipteran pteridines
(Mail and Lehane, 1988;
Penilla et al., 2002;
Wu and Lehane, 1999), were
used to confirm that the protocols used were capable of accurately detecting
and quantifying the levels of pteridines found in individual D.
serrata. Firstly, 0.7 ml of a stock solution of 6-biopterin (10 000 µg
l-1) was treated as a tissue sample, extracted and characterized
using the methods outlined above. The shape of the emission spectra was then
compared to that obtained from individual flies using the same extraction
method. To confirm our ability to quantify the pteridine levels found in
individual flies, the stock solution of 6-biopterin was serially diluted with
samples analysed at each step, until there were no longer any changes in the
intensity of the emission spectra. These results were also compared with a
control (extract solution only) and the data from individual flies to ensure
that individual pteridine levels could be accurately quantified.
Assay of laboratory-reared D. serrata of known age
To determine the association between pteridine content and age, virgin male
and female D. serrata from the Forster stock described previously
(Higgie et al., 2000) were
sexed and placed in standard food vials, five individuals of the same sex to a
vial, with three replicate vials for each sex. This procedure was conducted at
18 time periods spanning 48 days, spaced from 2 to 5 days apart, resulting in
108 vials at the end of the experiment containing flies aged between 2 and 48
days. Vials were changed every 3 days to maintain the flies in good condition,
and were kept at a constant temperature of 25°C in a room with a 12 h:12 h
light:dark cycle at 170 lux. After 48 days, 335 flies had their heads removed,
weighed, and then prepared for the pteridine content assay. Some of the
cohorts of older flies suffered significant levels of mortality by the end of
the experiment. Only vials that had at least two individuals surviving were
used in the analysis and vial means were taken to be used as independent
data.
The analysis of pteridine content was conducted using the linear model for
an analysis of covariance (ANCOVA) testing for homogeneity of slope:
 | (1) |
where age (Age) was treated as a continuous variable, sex (Sex) was a fixed factor, subscripts i and j refer to the sex and age of the particular individual Y, µ is the population mean pteridine content, and the interaction term Sex*Age tested for a difference between sexes in the slope of the relationship between age and pteridine content. In a second ANCOVA, head mass replaced age in Eqn 1 to test for the effect of head mass on pteridine content.
Effect of temperature and light intensity on pteridine content
To determine how temperature and light intensity may affect pteridine
content we conducted a two-way factorial experiment using five temperatures
(19, 21, 23, 25 and 27°C) and four light intensities. Light intensity was
manipulated by covering the glass vials containing adult flies with neutral
density polyester filters (Lee Filters, Andover, UK). Three grades of filter
(209 0.3, 210 0.6, 211 0.9) were used to reduce light intensity to
approximately 50%, 25% and 12.5% of ambient across a wavelength range of 400
to 700 nm, respectively. The fourth light intensity used was ambient in the
temperature cabinets used for the experiment, which was generated by three 8-W
fluorescent tubes. Five replicate vials, each containing five virgin 1-day-old
males, were placed in each of the 20 treatment combinations, and left for 8
days. At the end of 8 days, flies had their heads removed and were prepared
for the pteridine assay as above.
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 | (2) |
where light intensity (Light) and temperature (Temp) were fixed effects,
and subscripts i and j refer to the light intensity and temperature
environments of the particular individual Y and µ is the
population mean pteridine content. The effect size of each factor with
pteridine content was determined by calculating 2, an
estimate of the degree of association between each of these two fixed factors
and pteridine in the population
(Tabachnick and Fidell,
1989).
Survival analysis of laboratory populations
Estimates of survival were generated from the data collected during the
assay of laboratory populations of known age, in order to provide a reference
in which to assess the predicted ages of individual wild-caught flies. Data on
the presence or absence of individual flies collected when vials were sampled
represent left- and right-censored estimates of individual survival times,
respectively (Allison, 1995).
These estimates were converted into interval-censored data
(Klein and Moeschberger, 2003)
and nonparametric survival curves generated using the %ICE Macro
(Peto, 1973) with SAS V8.
Differences in survival between sexes was tested with the SAS procedure PROC
LIFEREG with the distribution model optimized by minimizing the Akaike's
information criterion (Collet,
1994).
Assay of field-caught D. serrata
Field-caught males of D. serrata were collected on the St Lucia
campus of the University of Queensland on 20 February 2003 using banana bait,
and 40 males were weighed and then prepared for the pteridine content assay on
the same day. Brisbane has an average temperature of 25.3°C at 09:00 h and
27.4°C at 15:00 h during this month of the year.
To predict the ages of field-caught males, the data for laboratory males
were initially placed into a multiple linear regression model, where pteridine
content was predicted by age and head mass. Vial means for individual cohorts
of laboratory males were used as independent units. As the beta coefficient
for head mass was not significant (t=0.98, P=0.33) this term
was removed and the model reduced to:
 | (3) |
This significant model (F1,39=51.0,
P<0.001, r2=0.57) was then modified to form
the following predictive equation for individual age of wild flies, based on
pteridine content:
 | (4) |
The inverse confidence limits for the ages of individual wild-caught males
predicted using inverse regression Eqn 4, were calculated by deriving the 95%
fiducial limits (Draper and Smith,
1998). The derivation equation included the term
`g'.
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Fig. 2 shows the relationship between 6-biopterin concentration and emission intensities at 445 nm, for a subset of the range of concentrations examined. There was a significant linear relationship between the two variables in the range of 10 000 µg l-1 down to 1.2 µg l-1 (corresponding to an emission level of 0.01 arbitrary units, Pearson r2=0.99, N=18). Lower concentrations of 6-biopterin (0.6-0.08 µg l-1) and the control solution produced no emissions at 445 nm.
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Assay of laboratory-reared D. serrata of known age
Fig. 3 displays the increase
in pteridine content over the 48 days of the experiment for both sexes.
Pteridine content increased in both sexes by approximately 75% over the 48-day
period. ANCOVA determined that pteridine content was strongly affected by age
(F1,87=153.97, P<0.001), and the linear
relationship between pteridine content and age did not differ in slope between
the sexes (F1,87=0.16, P=0.688). Head mass was
positively associated with pteridine content, but not significantly so
(F1,87=2.03, P=0.158), and the relationship
between head mass and pteridine content did not differ significantly between
the sexes (F1,87=0.92, P=0.341).
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Effect of temperature and light intensity on pteridine content
Two-way ANOVA indicated that temperature had a significant effect on
pteridine content (F4,73=4.41, P=0.003),
increasing with temperature under all four light intensities
(Fig. 4). Light intensity also
had a significant effect on pteridine content
(F3,73=11.45, P<0.001), with increasing levels
found under higher light intensity. There was no evidence for an interaction
between these two factors on pteridine content
(F12,73=0.94, P=0.509). Estimates of the
association between temperature and pteridine and light intensity and
pteridine (population effect size 2) were 14.3% and 20.4%,
respectively.
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2=4.3, P<0.05, d.f.=1). Although it
was not possible to estimate the maximum longevity of females and males,
individuals of both sex remained alive at the final census date, 48 days.
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).
Pteridines have now been detected in 28 species from at least eight dipteran
families (e.g. Table 1).
Confirming detectable levels of pteridines in D. serrata is a
necessary step in evaluating their suitability as age predictors, for although
pteridine concentrations can usually be measured in single individuals, this
is not always the case. Lardeux et al.
(Lardeux et al., 2000) were
unable to detect pteridine in individuals of two very small dipteran species,
Aedes polynesiensis and Culex quinquefasciatus
(Table 1), and were forced to
pool samples among individuals to examine the relationship between pteridine
concentrations and age. The inability to detect pteridines at the individual
level in these two species probably reflects in part the reduced levels found
in these species and the limitations of the extraction and detection
techniques used. Wu and Lehane (Wu and
Lehane, 1999), for example, were able to detect pteridines in
similarly sized Anopholes gambiae and A. stephensi by using
reverse-phase HPLC.
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Pteridine levels in D. serrata are positively correlated with
individual age, rearing temperature and rearing light intensity conditions,
but do not differ significantly between males and females. Increasing
pteridine levels with age is commonly found within the Diptera, with a few
exceptions in the Culicidae and Glossinidae
(Table 1). Species within the
Culicidae show either a negative relationship between age and pteridine levels
(e.g. Anopheles albimanus, A. gambiae, A. stephensidipterans) or no
relationship at all (e.g. Aedes polynesiensis), while in contrast to
five other glossinid species, pteridine levels in Glosinna austensi
show no relationship to individual age. Explanations for the lack of a
positive relationship between pteridine levels and age in these taxa are
currently lacking, and the low r2 values in the studies
using G. austensi indicate that pteridine levels can be poor
predictors of individual age, even when a significant relationship exists
between the two variables (Penilla et al.,
2002). In our case, over 70% of the variation in pteridine levels
was explained by variation in age in both sexes under laboratory conditions,
suggesting that this approach is more promising in Drosophila.
The influence of sex on pteridine levels also appears variable within the
Diptera, with sex effects being detected in four of the seven studies designed
to test for it (Table 1).
Temperature effects on pteridine levels are much more consistent. Increased
adult rearing temperatures resulted in elevated pteridine levels in eight of
the nine species in which this was examined
(Table 1). The lack of a
temperature effect in Simulium sirbanum is puzzling, given the
predicted positive effect of temperature on pteridine levels based on enzyme
kinetics (Moon and Krafsur,
1995).
The significant effect of light intensity on the pteridine levels of adults
increases our understanding of the factors influencing the deposition of
pteridines. Pteridines are typically concentrated in the heads of insects
where they function as visual pigments
(Ziegler and Harmsen, 1969).
Yet although previous studies have claimed that pteridines accumulate in
insects when stimulated by sunlight
(Hanser, 1948), cited by
Penilla et al. (Penilla et al.,
2002) there have been limited tests of this relationship. No
differences were noted in the pteridine levels of adults hatched under
different ultra-violet conditions (Langley
et al., 1988), but details on the duration of the exposure to the
modified lighting conditions were not provided. Longer durations such as those
utilized in this study of D. serrata may have produced comparable
light intensity effects.
Numerous factors influence the level of pteridines in individual dipterans,
in addition to the previously discussed factors of age, sex, rearing
temperature and light intensity. Different genetic strains of Drosophila
melanogaster accumulate pteridines at different rates as they age
(McIntyre and Gooding, 1996)
and diet and mating frequency may also effect pteridine accumulation. The
pteridine levels of parous female Culicoides variipennis sonorensis
that have fed on blood are less than in those of nulliparous females that have
not, while pteridine levels in female Anopheles albimanus increase
with the number of blood meals (Penilla et
al., 2002). Although studies of the presence of particular
pteridines in insects and the biochemical pathways associated with their
accumulation continue (e.g. Fan et al.,
1976; Parisi et al.,
1976; Silva et al.,
1991), the link between these processes and the behavioral and
ecological factors that modify them unfortunately remains obscure.
How suitable are pteridine levels as predictors of age in individual
wild-caught flies, particularly in light of the variety of internal and
external factors that influence individual pteridine levels and the difficulty
in measuring the exposure of wild individuals to these variables? Wild
populations exist in a world of numerous microhabitats, for example, each with
their own temperature and light intensity regimes that vary on a daily and
seasonal basis (Endler, 1993).
In some cases researchers have measured hourly temperatures, the number of
sunlight hours and the roosting behaviour of field populations and used this
to increase the predictive power of their equations (e.g.
Mail et al., 1983). Individual
pteridine levels have also been combined with other morphological factors such
as the stage of ovarian development to improve predictive power
(Wall et al., 1991).
Alternatively, others have made no attempt to incorporate temperature effects
into their predictive equations, based on the rationale that the actual body
temperatures of wild flies could differ significantly from ambient
temperatures anyway (e.g. Langley et al.,
1988).
The relatively high number of flies predicted to have negative ages in this
study (Fig. 6) indicates that
the predictive equation derived from laboratory populations, which does not
include information on rearing temperatures and light exposures, currently
underestimates the age of wild-caught flies. The significant effects of
temperature and light on individual pteridine intensity demonstrated in the
laboratory study (14.3% and 20.4%, respectively), however, strongly suggest
that the detection of the temperature and light conditions experienced by
individual flies and the incorporation of this information would significantly
improve age determination in wild-caught flies. Such improvements have been
achieved in some studies (e.g. Mail et
al., 1983), but in general we have a very limited understanding of
the variation in temperature and light conditions experienced in wild
populations of Drosophila (Feder,
1996). The small size of adult Drosophila
(Feder et al., 2000) and the
potential for dispersal over many km (Coyne
and Milstead, 1987), currently preclude the direct detection of
the microhabitats inhabited by individuals. However, indirect evidence
suggests the realized effect of individual variability in temperature and
light exposures in field populations may not be as significant as suggested by
laboratory studies. For although numerous laboratory-based studies have
demonstrated the significant effects of temperature on many aspects of the
developmental biology of Drosophila (e.g.
Hoffman, 1995;
Partridge et al., 1995), the
ability of free-living individuals to actively select and inhabit a limited
range of microhabitats, and hence the `ecological relevance' of such factors
in free-living populations, is less clear
(Feder et al., 2000). Indirect
estimates involving the expression of two temperature-sensitive reporter
genes, for example, suggest that free-living D. melanogaster
experience temperature stress relatively infrequently
(Feder et al., 2000) and so the
individual variability in temperature and light microhabitats may be less than
that of the environment as a whole. Ultimately, the ability to determine the
temperature and light conditions experienced by individual flies may depend on
a combination of technological advances, the extent to which temperature and
light influence individual pteridine levels and the specific ecology of the
target species. Rainforest specialists such a D. serrata, inhabiting
environments with relatively reduced variability in microhabitat conditions,
may prove to be more appropriate species for the calibration of real age
predictive equations.
The utility of the pteridine method for age determination in individuals
from the wild therefore depends on the type of questions asked and the degree
of precision required. Most pteridine studies have involved species of
significant agricultural or medical importance due to their role as vectors of
animal or human disease (e.g. Langley et
al., 1988; Tomic-Carruthers et
al., 1996; Wu and Lehane,
1999). The goal of these studies has therefore been to determine
the exact age of individuals in the wild to better understand life history and
transmission dynamics of the diseases they carry. In these studies the
influence of such factors as ambient temperature, sex and light on absolute
pteridine levels can make predicting absolute age difficult and require the
formulation of species-specific predictive equations.
For many general evolutionary questions in such areas as mate choice and life-history evolution, however, where individuals choose from available options, it is knowledge of an individual's relative rather than its absolute age that is important. In these cases, the pteridine methods outlined here represent a significant improvement on other approaches. Even if temperature and light regimes differ on a daily basis, the linear and non-interacting effect of these two variables means that the pteridine levels of older flies should be greater than that of younger flies.
Greater certainty may also be obtained by `trading-off' the ability to predict the age of single versus a group of individuals against the certainty of these predictions. The confidence limits associated with the inverse regression predictions decrease significantly when the dependent variable used to predict the independent variable, in this case pteridine concentration predicting age, is based on the mean of a sample of individuals rather than a single individual. In some cases it might be worth combining the pteridine concentrations of subgroups of individuals, in order to calculate predicted ages with greater certainty.
The age of Drosophila in the field has typically been suggested to
be less than 2 weeks in a variety of species
(Powell, 1997). The
possibility that flies under field conditions survive for over 4 weeks, as
suggested by our analysis, suggests that quantifying life-history traits and
mating success at ages greater than those commonly used in laboratory studies
may be important.
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