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-Ethinylestradiol Reduces the Competitive Reproductive Fitness of the Male Guppy (Poecilia reticulata)1
Department of Zoophysiology, Institute of Biological Sciences, University of Aarhus, DK-8000 Aarhus C, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ethinylestradiol (EE2) from birth to adulthood. Subsequently, an exposed male competed against an unexposed male for the opportunity to fertilize a receptive female. The successful males siring the majority of the offspring in each brood were then identified using microsatellites in genetic paternity analysis. Only the highest dose of EE2 produced harmful effects with a significantly female-biased sex ratio, significant reductions in male sperm count, testis weight, body coloration and courtship behavior, and a significant increase in body size. These feminizing effects were translated into a highly significant reduction in fertility, where only 1 of the 17 exposed males sired offspring in competition with unexposed males. The evidence suggests that EE2-treated males have reduced reproductive fitness compared with untreated males, possibly the result of EE2 effects on multiple fitness traits. To our knowledge, this is the first study providing evidence of endocrine disruption at the population level that has included the ecologically highly relevant effect of sexual competition on male reproductive fitness.
behavior, male sexual function, sperm, stress, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were infertile [10], providing further evidence that estrogens play important roles in males.
An estrogenic chemical suspected of playing an important role in the feminizing effect of sewage water is the synthetic estrogen, 17-ethinylestradiol (EE2) [11]. EE2 is the main active ingredient in many contraceptives and is excreted by women in a conjugated form, which can then be deconjugated in sewage treatment works [12] and discharged in the activated state. EE2 is a highly potent estrogen and is reported to induce vitellogenin (yolk protein precursor) in male rainbow trout (Oncorhynchus mykiss) [13] and intersex gonads in fathead minnows (Pimephales promelas) [14] and Japanese medaka (Oryzias latipes) [15] at environmentally realistic doses of a few nanograms per liter [16]. However, endocrine disruption at these lower levels of biological organization does not necessarily have adverse effects on population health. This is clearly demonstrated by a recent study by Balch et al. [15], showing that 2 and 10 ng EE2/L induced intersex gonads in male Japanese medaka without leading to a significant difference in fertility of intersex males and nonintersex males. Although other studies have shown a more direct relationship between intersex and infertility [2], the study by Balch et al. [15] clearly illustrates the great challenge of translating the subtle functional deficits within individuals into population-level effects [17]. Direct measurement of harmful effects on reproductive success is therefore widely accepted as a key parameter [14], and the potential of EE2 to reduce reproduction has now been confirmed in a number of studies. In Japanese medaka [15, 18–20], rainbow trout [21], and zebrafish (Danio rerio) [22], EE2 is reported to significantly decrease reproduction at concentrations as low as 10 ng/L.
In studies previously conducted in our laboratory, the natural estrogen 17ß-estradiol and the estrogenic octylphenol polyethoxylate metabolite 4-tert-octylphenol [23] were observed to significantly feminize a whole suite of male guppy (Poecilia reticulata) sexual characteristics, including sperm count, body coloration, and courtship behavior [4, 5, 24]. Similar demasculinizing effects have also been observed following exposure to antiandrogenic chemicals [25, 26]. The secondary sexual characteristics advertise the fitness of guppy males toward potential female partners, which exercise a degree of choice and both precopulatory intermale competition and postcopulatory sperm competition caused by multiple matings in females is consequently intense [27]. It is therefore assumed that such feminizing and demasculinizing effects are propagated into a reduced reproduction [4, 26], an assumption that was confirmed in a recent study by Bayley et al. [28], where the antiandrogenic vinclozolin (fungicide) metabolites significantly reduced male reproduction when mated separately with unexposed females. However, the degrees of endocrine disruption at the cellular, organ, and individual levels that actually translate into an impaired reproduction in a natural mating environment cannot be reliably assessed without including the effect of intermale competition. Male competition for mating and sperm competition are important factors affecting reproductive fitness in many species, including the guppy [29], and the consequences of feminization or demasculinization may thus be more serious under natural mating conditions [2]. Accordingly, the harmful effects of endocrine disruption are likely to be underestimated when using experimental designs that do not include competition [2]. To our knowledge, however, no study to date providing evidence of population-level effects of endocrine disrupters has included this ecologically highly relevant parameter.
In the present study, a new approach in the evaluation of population health effects of environmental estrogens is presented where male ability to sire offspring in competition with another male is used as a measure of endocrine disruption at the population level. A male guppy exposed to EE2 throughout its lifetime competed against an unexposed male for the opportunity to fertilize a receptive female. Five offspring from each brood were then assigned to one of the males using paternity analyses with microsatellites as genetic markers. The relative frequency of control males and exposed males siring the majority of the broods was then used as a direct measure of male reproductive success in a competitive environment. A suite of male guppy sexual characteristics that is routinely quantified in our laboratory and previously shown sensitive to estrogen exposure [4] was furthermore included in this study.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fish used in this experiment were wild-type guppies (P. reticulata) imported from Lagos, Nigeria, 6 mo before the experiment was started and stocked in a 500-L stainless steel tank. For details on stock aquarium conditions, see [26]. The experiments were carried out according to Danish legislation concerning the use of experimental animals.
Six hundred newborn juveniles were collected in the stock aquarium and randomly distributed into four 30-L glass aquaria containing 12-L of water. Juveniles in the four aquaria were exposed to nominal concentrations of 0, 10, 50, and 200 ng EE2 (Sigma Chemical Co., St Louis, MO) per liter in a continuous flow-through system. The actual concentrations were, in all cases, lower than or approximately equal to the intended nominal values; see below for measurements of actual concentrations. The control aquarium received acetone only. Stock solutions of EE2 in acetone were prepared every second day and then continuously dosed at a rate of 2.8 ml/day to the inflowing water hose via Hamilton syringes driven by electric clockworks. The flow rate of the peristaltic pump that provided the inflow water was 35 L/day. The fish were fed daily with newly hatched Artemia salina and commercial TetraMin flake food, and all aquaria received equal amounts of food. The exposure lasted for a total of 108 days before the competition experiments were started.
Measurement of EE2 Concentrations
An Agilent Technology LC-MSD system consisting of a series 1100 HPLC and a G1946A MSD quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface was used for separation and quantification of EE2 in the aquarium water. Extraction and quantification of EE2 were performed as described by Rose et al. [30]. Measurements were performed on one water sample from each treatment group.
Competition Experiment and Male Sexual Behavior
Two hundred fifty juveniles were isolated from the stock aquarium and virgin females were separated from the males before adulthood. This procedure was performed because female guppies can store sperm from a single mating for a number of reproductive cycles [27].
Sixty-nine virgin females were randomly distributed into sixty-nine 10-L glass aquaria and acclimated for a minimum of 2 days before they were transferred to a 2-L glass jar a few minutes before the two competing males were introduced. These 2-L glass jars contained stock aquarium water, which is assumed to contain pheromones from postparturition females. These pheromones have a stimulating effect on male sexual activity [31].
A control male and an exposed male were randomly chosen for each of the 23 replicates for the three doses of EE2, and their coloration patterns noted for their subsequent identification. The males were then transferred to the 2-L glass jar containing the virgin female and acclimated for 30 min before the sexual behavior of the two competing males was quantified. The number of gonopodial thrusts (forced copulations) and the number and duration of sigmoid displays (courtship behavior) were then measured by visual inspection for 10 min. Thereafter, the three fish were transferred to the female's 10-L aquarium, where they were left for 24 h, at which time the males were removed, identified as either control or exposed male by their color pattern, and placed individually in 2-L glass jars. Exposure to EE2 then continued for 2–3 days in these individual aquaria until all replicates were collected, at which time the sperm count and morphological measurements were performed. The two competing males in each replicate were always measured simultaneously.
The aquaria containing females were checked daily for offspring, which were collected for the paternity analysis. Pregnancy in female guppies usually lasts for 3–4 wk [27], but to leave time for all fertilized females to produce offspring, the aquaria were inspected for 72 consecutive days before all females were humanely killed.
Measurement of Male Sexual Characteristics
Two to three days after being isolated from the females, the males were anaesthetized in a benzocaine solution, lightly dried on filter paper, and weighed. Each male was then placed on a reversed Petri dish under a dissection microscope, where the gonopodium was swung forward and a picture taken following the procedure described in detail by Bayley et al. [26]. These pictures were later analyzed for gonopodium length, body length without the caudal fin, body area minus fins, and area of the orange coloration on the body minus the fins using Image-Pro Plus 4.5 (Media Cybernetics, Inc., Silver Spring, MD). The gonopodium index was then calculated as the gonopodium length as a percentage of the body length and the coloration index was calculated as the area of orange coloration as a percentage of the body area. Immediately after the image was recorded, the sperm count was measured. The male was stripped by stroking the abdomen with a tiny metal rod toward the gonopodium and sperm cells collected and counted following the procedure described by Toft and Baatrup [24]. Following the sperm count, the testes were dissected out, dried gently with filter paper, and weighed. The gonadosomatic index was then calculated as the testis weight as a percentage of the body weight.
Paternity Analysis
Genomic DNA from caudal fin tissue of adult fish (
0.2 mg wet weight) and whole bodies of juveniles was extracted using the CTAB method with chloroform/isoamylalcohol (24:1) extraction, isopropanol precipitation, and TE-buffer (10 mM Tris-Cl and 1 mM EDTA) dissolution [32].
Previous experiments examining sperm competition in guppies have shown that all juveniles in a brood are almost entirely sired by one male [33]. In a previous experiment in our laboratory (unpublished data), on average 94% of the juveniles in a single brood were sired by the successful male (the male siring the majority of the juveniles in a brood) and a correct identification of the successful male therefore only requires analysis of a fraction of the brood. In rare cases, however, each of the competing males sired approximately half the brood. To obtain a probability of at least 95% of correct identification of the successful male in all broods, five juveniles were randomly chosen for paternity analysis. If there was multiple paternity in these five offspring, the entire brood was analyzed for paternity.
A maximum of 12 loci (Table 1) were included in each paternity analysis and each PCR was performed in a volume of 6 µl containing 1 µl template DNA solution, 0.2 mM of each dNTP, 3 pmol of each primer (forward primer Cy5-end labeled), 0.3 U Taq DNA Polymerase (Amersham Pharmacia Biotech) and the PCR buffer supplied with the Taq DNA Polymerase (Amersham Pharmacia Biotech). Thermocycling conditions were 94°C for 3 min for initial denaturation followed by 40 cycles of 94°C for 30 sec, Ta (annealing temperature; Table 1) for 40 sec and 72°C for 1 min, ending with a final extension step at 72°C for 7 min. Fragment lengths were measured on an ALFexpress sequencer (Amersham Pharmacia Biotech) following the manufacturer's recommendations. After gel electrophoresis, alleles were sized in relation to internal and external standards by using ALFwin Fragment Analyser 1.00.32 (Amersham Pharmacia Biotech).
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Statistical Analyses
Differences between males from the control group and the three exposure groups in body weight; testis weight; gonadosomatic index; body length; gonopodium length; gonopodium index; body area; orange coloration area; coloration index; sperm count; number of gonopodial thrusts, if they were performed; and number and duration of sigmoid displays, if they were performed (individuals not performing the given sexual behavior were excluded from this analysis) were detected using unpaired t-test where model assumptions were satisfied. Where data complied with normality but no simple transformation resulted in homoscedasticity, the approximate t-test was used. Where no simple transformation resulted in normality of data, the nonparametric Mann-Whitney test was used.
Data for sex ratio and frequency of males performing gonopodial thrusts and sigmoid displays were analyzed for heterogeneity among groups using the 
2-test. For sexual behavior data, pairwise 2-tests between the control group and the three exposure groups were performed where significant differences were detected. For sex ratio data, the four groups were individually compared with an expected sex ratio of 50:50 using the 2-test, which is equivalent to the null hypothesis that EE2 had no effect on sex ratio.
Paternity data from the five assigned juveniles (or whole brood if analyzed) was used to classify each male as successful (sired >50% of the brood) or unsuccessful (sired <50% of the brood). It was then tested if P(control male successful) was significantly different from P = 0.50, which is equivalent to the null hypothesis that EE2 had no effect on paternity, by analyzing each of the three EE2 concentrations as a binomial experiment.
The level of significance was 0.05. 2-Tests were performed with MS Excel 2000 (Microsoft Corporation). All other statistical tests were performed with SPSS 10.0 for Windows (SPSS Inc., Chicago, IL). Data are presented as mean ± SEM unless otherwise specified.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ethinylestradiol at the highest dose tested (nominal 200 ng/L) significantly female biased the sex ratio, feminized the males and almost completely eliminated reproduction. No significant effects on reproduction were detected at the two lower doses (nominal 10 and 50 ng/L). Concentration of EE2 in the Water
The actual concentrations of EE2 in the water of the three nominal concentrations of 10, 50, and 200 ng/L were 10.5, 44.4, and 112 ng/L, respectively. No EE2 was detected in the control aquaria receiving acetone only.
Mortality and Sex Ratio
The fish in all aquaria appeared to be healthy and no differences in swimming behavior or feeding activity were detected by visual inspection. Mortality in the four groups was low (less than 10%) and no differences were detected between the groups. All treatments were therefore considered to be sublethal.
Analysis of the phenotypic sex ratio in the four treatment groups showed a significant treatment effect of EE2 (2-test, P < 0.001). The group exposed to 112 ng/L was significantly female biased (2-test, P < 0.001), with only 28.6% of the individuals being sexed as males (Table 2). Furthermore, many of the individuals classified as males because their gonopodium was slightly differentiated were in fact extremely feminized (see below), thereby making the effect on sex ratio even more pronounced.
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Male Sexual Behavior
The frequency of courtship behavior (sigmoid displays) was significantly different in the four male groups (2-test, P = 0.035) and pairwise comparisons of control and exposed groups showed a significant decrease at the highest EE2 dose (Table 2). Only 1 of the 23 males in this group performed sigmoid displays and the effect of EE2 on the number and duration of this behavior was therefore not tested statistically. However, the frequency of males that attempted to mate the female via forced gonopodial thrusts and the number of gonopodial thrusts were unaffected by the 112 ng/L EE2 exposure, with 17 of the 23 males performing this behavior (Table 2). It was furthermore observed that a number of the males performing gonopodial thrusts had only slightly differentiated anal fins that clearly did not represent a functional copulatory organ. No significant effects were detected in any of the five behavior parameters in the 10.5 and 44.4 ng/L groups, with the exception of a significantly decreased duration of the sigmoid displays in the 10.5 ng/L group (Table 2).
Male Morphological Characteristics
The group exposed to 10.5 ng/L EE2 had a significantly higher testis weight (t-test, P = 0.006), gonadosomatic index (t-test, P = 0.005), and sperm count (t-test, P = 0.001) than the control group, but no differences were detected between the control group and the group exposed to 44.4 ng/L EE2 (Fig. 1). Conversely, the effect of 112 ng/L EE2 on male sexual development was highly significant (Fig. 1). Body size (t-test; P < 0.001 [body weight], P < 0.001 [body length], and P < 0.001 [body area]), testis weight (t-test, P = 0.048), and gonopodium length (t-test, P < 0.001) were significantly increased. However, there was a clear nonsignificant tendency toward a decrease in gonadosomatic index (t-test, P = 0.051), showing that the testis weight corrected for the significant increase in body size was in fact decreasing (Fig. 1). Furthermore, the body carotenoid coloration area (Mann-Whitney test, P < 0.001), coloration index (Mann-Whitney test, P < 0.001), and sperm count (t-test, P < 0.001) were significantly reduced, thus producing a significant feminization of almost the entire suite of male sexual characteristics by 112 ng/L EE2 (Fig. 1).
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Male Reproduction
The feminizing effect of 112 ng/L EE2 was clearly translated into a significantly reduced male ability to compete with control males for siring juveniles, whereas reproduction at the two lower doses was unaffected.
In total, 46 of the 69 females (15 in the 10.5-ng/L experiment, 14 in the 44.4-ng/L experiment, and 17 in the 112-ng/L experiment) produced a total of 763 offspring in their first broods between the 21st and 54th days after the males were introduced. The remaining 23 females did not produce any offspring. Brood sizes ranged from 1 to 31, with an average of 16.6 ± 1.1 juveniles per brood.
Four of the 46 broods were composed of less than five juveniles, with two consisting of one juvenile and two consisting of four juveniles. All juveniles in these four broods were assigned to fathers and five juveniles in the remaining 42 broods were randomly chosen for paternity analysis. Mixed paternity occurred in five of the 42 brood fractions and the rest of the juveniles in these five broods were therefore included in the analysis (with no change in the classification of successful male, however). A total of 263 juveniles were included in the paternity analysis and only two were not assigned to fathers because of a lack of genetic variation between the competing males. These two juveniles were excluded from further analysis.
The control male was successful in 16 out of the 17 fertile competition interactions (P(control male successful) = 0.94) at the highest EE2 dose (Fig. 2), and this almost total elimination of reproduction was highly significant (binomial test, P < 0.001). In contrast, no significant effect on reproduction was detected at the two lower doses (binomial test: 10.5 ng/L, P = 0.42; 44.4 ng/L, P = 0.42). The control male was successful in 9 out of 14 replicates in the 10.5-ng experiment (P(control male successful) = 0.64) (a replicate was excluded because the control male and exposed male sired five juveniles each and a successful male could therefore not be determined) and in 5 out of 14 replicates in the 44.4-ng/L experiment (P(control male successful) = 0.36).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ethinylestradiol were highly feminized, with a significantly increased body size and significantly decreased sexual coloration, sperm count, and frequency of courtship behavior. These effects were propagated into an almost total elimination of reproduction with only 1 of 17 exposed males fathering offspring in competition with unexposed males. Reproduction is considered to be a key parameter when evaluating the harmful potential of endocrine disrupters [14]. Generally, the numbers of eggs spawned, hatching, or reaching a certain developmental stage [14, 18, 19, 21] have been compared between control and EE2-exposed groups, but these measures can be relatively insensitive because of a high level of variation in fecundity, as seen in many batch-spawning cyprinids [14]. This problem is eliminated in the new approach for evaluating population-level effects of environmental estrogens presented here, where the classification of males as either successful or unsuccessful is independent of variation in female fecundity. More importantly, this design also includes the effects of competition between males unlike previous studies of endocrine disruption at the population level. Intermale competition is intense in guppies and the consequences of, e.g., reduced sperm count, body coloration, and sexual behavior may therefore be more serious under natural mating conditions [2] because selection against feminized males is assumed to be stronger than in experimental designs where competition is not included. In this study, exposed males competed against unexposed males for fertilizing an unexposed female. Jobling et al. [1] report that "the degree of exposure to estrogen(s) may vary tremendously, even within populations of fish that were sampled from the same site" based on observations of highly fluctuating intersex indices in roach (Rutilus rutilus) sampled in UK rivers. Factors causing such differences include variation in time/timing of exposure and fish movement [1] and competition between males receiving different levels of exposure is therefore assumed to be environmentally realistic. To what extent population health is affected by the observed reduction in the ability of exposed males to compete with unexposed males for fathering offspring is difficult to predict using this design alone. The considerable feminization of the 112-ng/L EE2-exposed males in the present study is clearly assumed to produce harmful effects on population health parameters, such as population size and genetic variation, but a more precise quantification of long-term adverse effects will in general have to rely on data from experiments resembling natural mating environments to an even higher degree.
The onset of adverse effects in the present study was very abrupt, with the feminizing effect of 112 ng/L being in clear contrast with the complete lack of even a nonsignificant tendency toward a feminization of any of the 17 parameters included in this study at the concentration of 44.4 ng/L. This demonstrates that the sensitivity of male guppy ability to sire offspring in competition with other males as an endpoint is very similar to the biomarkers at the organ level, such as body coloration and gonopodium length. The no-observable-effect concentration (NOEC) and lowest-observable-effect concentration (LOEC) values of 44.4 and 112 ng EE2/L in this study are in good agreement with a study by Scholz and Gutzeit [19], where reproduction and testis morphology in male medaka (O. latipes) exposed to 10 ng EE2/L were unaffected, whereas 100 ng EE2/L produced complete sex reversal. However, several studies report of endocrine disruption at considerably lower doses, indicating species differences in sensitivity to EE2, despite dissimilarities in exposure regimes. Lange et al. [14] reported NOEC and LOEC values in the fathead minnow (P. promelas) in a full life-cycle study including the embryonic stages to be 1.0 and 4.0 ng/L, respectively, and male rainbow trout (O. mykiss) exposed for 62 days as adults to 10 ng EE2/L had a significantly reduced fertilization success in vitro [21].
The sperm count in the males exposed to the lowest EE2 dose of 10.5 ng/L was significantly increased. That estrogenic chemicals can increase the sperm count in guppies has previously been demonstrated with 17ß-estradiol and 4-tert-octylphenol in both juvenile [4] and adult guppies [24]. Toft and Baatrup [24] provided a number of possible explanations of this surprising, but nevertheless consistent, result in guppies. The natural female estrogen, 17ß-estradiol, generally inhibits male teleost sperm production [34], but higher gonodotropin secretion after 17ß-estradiol exposure has been observed in vitro in pituitaries from juvenile poeciliids [35], which could indirectly stimulate spermatogenesis by increasing testosterone production. It can also be argued that the result is a methodical artifact because accumulated spermatozoa in the testis and sperm duct hindered in natural release are driven out when the male is stripped, but no conclusive explanation seems to exist [24]. It was speculated whether the increased sperm count was actually beneficial for male fitness [4]. However, this study clearly demonstrates that the almost doubled sperm count and the increased testis weight in the males exposed to 10.5 ng/L EE2 did not translate into a fitness benefit, with only 36% of the exposed males being successful in competition with unexposed males.
The effect of 112 ng/L EE2 on the sex ratio was highly significant. Sex determination is genetic in guppies, but full sex reversal can be obtained through the administration of sex steroids [36]. The low mortality in the present study excludes male-specific mortality as an explanation of the skewed sex ratio. Only a number of genotypic males differentiating into phenotypic females can therefore account for the observed pattern. This result is in accordance with previous studies by Miyamori [37] and Takahashi [38], who also showed that juvenile EE2 exposure could induce complete sex reversal in guppies. Identification of the sex-reversed fish would make it possible to obtain data on their reproductive capabilities, which could provide further insight into the actual extent of population-level effects. This requires a technique for genetic sex determination, which is not at present available in guppies. Guppy females are homogametic (XX) and males heterogametic (XY) [36], and a PCR-based screening of a Y-chromosome-specific DNA sequence may offer a simple and efficient tool for genetic sexing. This common technique for genetic sexing has, to date, only been developed in a few fish species, including the platyfish (Xiphophorus maculatus) [39] and Chinook salmon (Oncorhynchus tshawytscha) [40].
Investigations measuring concentrations in sewage treatment work effluent in Germany and Canada detected EE2 concentrations up to 15 and 42 ng/L, respectively, with median values of 1 and 9 ng/L, respectively [16]. Also, EE2 was detected at concentrations up to 7.0 ng/L in British sewage water, again with much lower average values [41], and at 4.5 ng/L in Swedish sewage water [42]. However, surface water concentrations are normally considerably lower and Lange et al. [14] concluded that European surface waters generally contain less than 0.5 ng EE2/L and that concentrations in most cases can be expected to be below 0.1 ng/L. Although the lowest dose used in this study (10.5 ng/L) is within the range of the highest concentrations measured in sewage water, the doses cannot be considered environmentally relevant on a broader scale.
In conclusion, the highly feminizing effect caused by a measured concentration of 112 ng 17-ethinylestradiol/L on the almost entire suite of male guppy sexual characteristics was translated into a significantly reduced ability to sire young in competition with other males. The ecologically highly important intermale competition was included to simulate a more natural mating environment, and this study thereby represents a new and ecologically relevant approach in the evaluation of endocrine disruption at the population level.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Mark Bayley, Department of Zoophysiology, Institute of Biological Sciences, University of Aarhus, Building 135, DK-8000 Aarhus C, Denmark. FAX: 45 8612 5175; Mark.Bayley{at}biology.au.dk
Received: 9 June 2004.
First decision: 30 June 2004.
Accepted: 27 August 2004.
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