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First published online February 1, 2008
Journal of Experimental Biology 211, 510-523 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.008755
Production of different phenotypes from the same genotype in the same environment by developmental variation
1 Zoological Institute and Museum, University of Greifswald,
Johann-Sebastian-Bach-Straße 11/12, D-17487 Greifswald, Germany
2 Biology 1, University of Regensburg, Universitätsstraße 31, D-93040
Regensburg, Germany
3 Department of Analytical Chemistry, University of Wuppertal,
Gauss-Straße 20, D-42119 Wuppertal, Germany
4 Department of Mathematics and Computer Science, Jahnstraße 15a,
University of Greifswald, D-17487 Greifswald, Germany
* Author for correspondence (e-mail: gunter.vogt{at}web.de)
Accepted 29 November 2007
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSPERSPECTIVESReferences |
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Key words: genotype, phenotype, variation, marbled crayfish, development, growth, colour, reproduction, behaviour, sense organs
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSPERSPECTIVESReferences |
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;
Dow, 2007;
Hallgrímsson and Hall,
2005; Pigliucci et al.,
2006; West-Eberhard,
2003). Phenotypic variation can be produced by genetic
differences, environmental influences and stochastic developmental events.
Whilst the genetic and environmental components are rather well investigated,
developmental variation (DV), also called `intangible variation' or
`developmental noise', remained a largely untouched field of research
(Astauroff, 1930;
Falconer and Mackay, 1996;
Gärtner, 1990;
Peaston and Whitelaw, 2006;
Veitia, 2005).
With the exception of fluctuating asymmetry, the right–left
difference of a trait (Dongen,
2006; Møller,
2006; Nijhout and Davidowitz,
2003), DV has rarely been directly investigated, mainly because
suitable laboratory models are not at hand
(Peaston and Whitelaw, 2006).
However, population genetic calculations
(Falconer and Mackay, 1996)
and some studies with parthenogenetic aphids and daphnids
(Lajus and Alekseev, 2004;
Warren, 1902), polyembryonic
armadillos (Loughry and McDonough,
2002), human monozygotic twins
(Falconer and Mackay, 1996),
inbred lines of Drosophila, guinea pigs and mice
(Astauroff, 1930;
Gärtner, 1990;
Peaston and Whitelaw, 2006;
Wright and Chase, 1936), and
cloned mammals and plants (Archer et al.,
2003a; Archer et al.,
2003b; Jaenisch and Bird,
2003; Scowcroft,
1985) revealed that DV often accounts for more than half of the
total variation of a trait. Moreover, DV was assumed to contribute
significantly to differences in aging and carcinogenesis of humans
(Aranda-Anzaldo and Dent, 2003;
Kirkwood et al., 2005) and to
severely hamper standardization of test animals
(Gärtner, 1990).
An ideal laboratory model for investigation of DV should produce high
numbers of isogenic progeny, possess a variety of morphological traits that
are easy to analyse, and enable rearing in simple environments. The
Marmorkrebs or marbled crayfish, a varico-coloured parthenogenetic all-female
species detected by us a few years ago
(Scholtz et al., 2003),
fulfils all of these requirements, and has further advantages such as easy
accessibility to all life stages, adaptability to a wide spectrum of
experimental conditions, indeterminate growth, direct development, stereotyped
cell lineage in early development, broad behavioural repertoire and, most
importantly, stepwise alteration of the phenotype by moulting
(Alwes and Scholtz, 2006;
Dohle et al., 2004;
Seitz et al., 2005;
Vogt and Tolley, 2004;
Vogt et al., 2004). Moreover,
the exuviae produced by moulting yield permanent records of the morphological
traits, permitting accurate analysis under the microscope and comparison of
character states of individual crayfish throughout life.
The marbled crayfish, which appeared first in the mid 1990s in the German aquarium trade, is apparently a parthenogenetic strain of the North American cambarid Procambarus alleni (Faxon 1884), as revealed by analysis of the 16S hypervariable region of the mitochondrial genome (Keith Crandall, personal communication). Under optimal conditions it has a generation time of 5–6 months and clutch sizes of 50–400 eggs, depending on age. The eggs and the first two lecithotrophic juvenile stages are permanently carried under the mother's abdomen and are thus exposed to the same environment. Stage-3 juveniles, the first feeding stage, and also sometimes stages 4 and 5, adhere to the maternal pleopods to rest but leave them regularly for feeding. The late embryonic stages and first juvenile stages can be raised in very simple in vitro systems, facilitating standardization of the environmental conditions. Moreover, all live stages can be fed with the same pellet food as sole food source.
In the present study we have investigated DV in many hundred marbled
crayfish of all ages with respect to development and growth, life-span,
reproduction, coloration, number of olfactory and gustatory sense organs,
behaviour, and the epigenetic markers fluctuating asymmetry and global DNA
methylation. The emphasis of our experiments was on the analysis of variation
within batches, which were shown to be genetically identical in the marbled
crayfish (Martin et al.,
2007), the batch-mates being exposed at any time to the same
environmental and nutritional conditions. Food was generally given in excess.
It was the aim of our experiments to test the suitability of the marbled
crayfish for investigation of DV and to provide baseline data for future
research on the relationship of epigenetics and phenotype and on environmental
epigenomics.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSPERSPECTIVESReferences |
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To exclude non-DV sources of variation we took the following measures in
addition to the use of a genetically uniform test species: (1) analysis of
batches to minimize potential influences of mutations and seasonal rhythms;
(2) rearing of animals under identical laboratory conditions to standardize
macro-environmental parameters; (3) use of simplified rearing systems to
minimize micro-environmental influences; (4) feeding of all life stages with
the same pellet food as sole food source in excess, to exclude any influences
of difference in food quality; (5) focussing on juvenile stages 1–6, as
these stages can be determined reliably and are particularly peaceful; (6)
regular inspection of the stock population for crayfish diseases
(Vogt, 1999); (7) performance
of the experiments and collection of all morphological and life history data
by the same experienced person (G.V.). Responsibilities: G.V., idea,
experiments, morphological and life history analyses; M.H. and C.D.S.,
analysis of microsatellites; M.T. and O.J.S., analysis of DNA methylation; and
G.v.d.B., statistical analysis of meristic and metric traits.
Genetic analyses
To verify clonality of our experimental animals we chose the highly
sensitive microsatellite technique. The following specimens were analysed: (1)
the founder females A and B of the two laboratory lineages used for the
experiments, (2) nine offspring of dam A from the same batch, (3) seven
specimens of the oldest known German aquarium lineage established in 1995, and
(4) three siblings of another German aquarium population established in 1998.
For comparison, we analysed an aquarium-reared batch of the sexually
reproducing Procambarus clarkii, which is closely related to the
Marmorkrebs (Scholtz et al.,
2003).
Genomic DNA was obtained from ethanol-preserved tissue from walking legs or the pleon, using the Puregene Kit (Gentra Systems, Minneapolis, MN, USA). PCR were performed in a final volume of 20 µl with annealing temperatures varying between 46°C and 58°C and 30 cycles. Successfully amplified products were ethanol-precipitated and cycle-sequenced in the automated sequencer ABI Prism 310 (Applied Biosystems, Darmstadt, Germany). The sequences obtained were then aligned and compared with the corresponding published sequences of Procambarus clarkii from GenBank prior to the use of 5'-labeled primers for fragment length analysis.
Ten microsatellite primer combinations, nine designed for the crayfish
Procambarus clarkii (PclG-03, PclG-04, PclG-07, PclG-08, PclG-15,
PclG-26, PclG-27, PclG-32, PclG-37)
(Belfiore and May, 2000) and
one for Orconectes placidus (locus 2.6)
(Walker et al., 2002), were
tested for potential use with the marbled crayfish. Out of these, two primer
combinations rendered successfully amplified DNA with microsatellites in the
marbled crayfish: PclG-04 and PclG-26. The primers used were TAT ATC AGT CAA
TCT GTC CAG (forward) and TCA GTA AGT AGA TTG ATA GAA GG (reverse) for PclG-04
and CTG TAG GCC TTC ATG GAC TTC TTG (forward) and TGT TCA CAT CAG CAG GAG ATA
ACT A (reverse) for PclG-26. The latter primers were newly designed by us,
based on the sequences of Procambarus clarkii and the marbled
crayfish.
Investigation of life history parameters, coloration, and morphological traits
Development and growth, life-span, reproduction, coloration, and
morphological characters were investigated in several batches and monitored
for a maximum of 910 days. Variation of development and growth was
investigated in batches from dam A, dam B, daughters B1,
B3, B4 and B5 and granddaughter
B3-1 in different environments, e.g. maternal pleopods, 12-well
micro-plates, 18x6 cm net culture systems and 30x20 cm and
60x30 cm aquaria. The aquaria were equipped with a thin layer of gravel
and stones as shelter. Feeding was started when all juveniles of a batch had
moulted into stage 3. The only food source for all life stages was TetraWafer
Mix, which was fed once a day ad libitum. The water was completely
changed weekly, and the aquaria were carefully cleaned at these occasions.
Excess food was siphoned out daily. In the smaller culture vessels water was
replaced as necessary. The water source (tapwater) was the same for all
experiments, as were temperature (room temperature) and light conditions
(natural light rhythm) at a given time.
Long-term variation of growth and reproduction was studied in eight offspring of dam B (B1–B8), that were communally reared in a 60x30 cm aquarium until death, in 20 offspring of dam B3-1 (groups C and D) that were communally reared in 30x20 cm aquaria for 365 days, and four individually raised offspring from dam B3-1 (F1–F4) that were reared in 30x20 cm aquaria for 350 days. In these experiments temperature fluctuated between 18°C and 23.5°C but was the same for each group member at a given time. Further details of the various growth experiments are found in the respective paragraphs of the Results.
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). The differences in the means of carapace length (CL) and numbers of aesthetascs (Ae) and corrugated setae (CS) among batches and among juvenile stages were analysed by a bifactorial analysis of variance (ANOVA) with interaction, and differences in variation of the same parameter among batches and juvenile stages by Bartlett tests. Both methods rely on approximate normality of the residuals.
Modular relationships between traits were analysed in the same specimens by
comparison of the coefficients of relative variation of CL and numbers of Ae
and CL based on confidence intervals. We selected a relative measure of
variation to ensure comparability among different juvenile stages. In order to
provide precise confidence intervals, we used the geometric standard deviation
sg=exp{sd[log(x)]} as a measure of relative variation
rather than the more traditional coefficient of variation
CV=sd(x)/mean(x). For small relative variations – as
in our case – both are approximately related as sg=1+CV.
Confidence limits for sg can be computed easily based on the
2-confidence limits for the estimated standard deviation
sd[log(x)]. Again, the method relies on approximate normality. The
statistical computations were done with the statistics software R
(R Development Core Team,
2007).
Investigation of fluctuating asymmetry
Fluctuating asymmetry (FA), the deviation from perfect symmetry to the left
(L) or right (R), was individually determined for the number of corrugated
setae on pereiopods 1–3 and the number of aesthetascs. FAs were
calculated according to the formula 100x(R–L)/(R+L)x
for 168 stage-2 to stage-6 juveniles from dams A, B, B1 and
B3. Longitudinal development of FA was investigated by using the
exuviae of offspring of dam B reared individually over a maximum of eight
moulting stages.
Investigation of DNA methylation
Methylation of the DNA was measured in the hepatopancreas and abdominal
musculature of four communally reared adolescent batch-mates and three
communally reared adult batch-mates. Analyses were performed with capillary
electrophoresis, using a novel and highly sensitive sample preparation
technique (Schiewek et al.,
2007). Each sample was measured at least 20 times.
Genomic DNA was isolated from frozen samples of the hepatopancreas and abdominal musculature with Qiagen Genomic-tip 20/G according to the supplier's instructions (Qiagen, Hilden, Germany). Only the last eluation step was changed: destilled water was used instead of TE-buffer. For hydrolysis and derivatization 1 µg DNA was diluted in 5 µl water and hydrolyzed by incubation for 3 h at 37°C with 4.2 µl of an enzyme mixture composed of micrococcal nuclease (MN) (150 mU µl–1)/spleen phosphodiesterase (SPD) (12.5 mU µl–1) and 0.8 µl buffer (100 mmol l–1 CaCl2 in 250 mmol l–1 Hepes, pH 6.0). To the hydrolysate were added 1.8 mol l–1 1-ethyl-3-(3'-N,N'-dimethylaminopropyl)-carbodiimid (EDC) (15 µl in 50 mmol l–1 Hepes, pH 6.4), 27 mmol l–1 of the fluorescent dye Bodipy FL EDATM (15 µl in 50 mmol l–1 Hepes, pH 6.4) and 15 µl Hepes (50 mmol l–1, pH 6.4). The sample was then incubated in the dark for 21 h at 25°C. For reduction of Bodipy and salt content 55 µl of the derivatised sample were transferred into a 15-ml cap and diluted with 425 µl water. To the solution was slowly added 52.5 mmol l–1 sodium tetraphenylborate (425 µl in 1 mmol l–1 sodium phosphate buffer, pH 6.0). After mixing, 11 ml methylene chloride was added to the solution, which was mixed again and centrifuged for 4 min at 1912 g. The aqueous phase was isolated and analyzed by capillary electrophoresis.
Analysis with capillary electrophoresis was carried out on a PACETM
MDQ system with a laser-induced-fluorescence detector (argon-ion laser with
em=488 nm) from Beckman Coulter (Munich, Germany). The
fused-silica capillary used was purchased from CS-Chromatography-Service
(Langerwehe, Germany) and had a total length of 50 cm (with the detection
window at 40 cm) and an inner diameter of 50 µm. The separations were
achieved with an electrolyte consisting of 90 mmol l–1 SDS in
a solution of 90% v/v sodium phosphate buffer (20 mmol l–1,
pH 9.0) and 10% v/v methanol as organic modifier (5s-sample injection at 0.5
p.s.i. at 20°C and an applied voltage of 18 kV). The cathode was the
outlet in all runs. For conditioning, the capillary was rinsed with 1 mol
l–1 NaOH (15 min), 1 mol l–1 HCl (15 min), 1
mol l–1 NaOH (15 min), water (5 min) and electrolyte (10
min). Before each run it was rinsed with 200 mmol l–1 sodium
dodecyl sulphate (1 min), 1 mol l–1 NaOH (1.5 min), water (1
min) and finally with electrolyte (2 min).
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RESULTS AND DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSPERSPECTIVESReferences |
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Genetic identity of the marbled crayfish
Marbled crayfish reproduce by apomictic thelytoky, the development of
female offspring from unfertilized eggs without meiosis, as revealed by
cytological investigation of the oocytes from pre-vitellogenesis until
cleavage (Vogt et al., 2004).
The progeny of such apomictic parthenogens is generally regarded as being
genetically uniform and identical to the mother, with the exception of random
mutations. The clonal nature of the Marmorkrebs was recently demonstrated
(Martin et al., 2007) using
the highly sensitive microsatellite technique. By sequencing six
micro-satellite loci, three of them being heterozygous, the authors revealed
that batch-mates were genotypically identical with each other and the mother.
They further revealed allelic identity in individuals from various generations
of their laboratory population. In this study, the marbled crayfish were
analysed using primers developed for Procambarus clarkii (see
Belfiore and May, 2000), a
species closely related to the Marmorkrebs
(Scholtz et al., 2003). The
size ranges and sequences of respective loci were different in the marbled
crayfish and Procambarus clarkii [compare Belfiore and May
(Belfiore and May, 2000) with
Martin et al. (Martin et al.,
2007)], excluding cross-contamination.
We have also tested the genetic uniformity of our experimental animals by
investigation of nuclear microsatellite loci, using some of the primer
combinations developed for Procambarus clarkii. Of the primer
combinations tested, two (PclG-04 and PclG-26) yielded heterozygous alleles,
which were investigated in depth and sequenced. All of the 21 marbled crayfish
examined from our livestock and two further German populations showed the same
allelic pattern, namely alleles of 156 bp and 172 bp at locus PclG-04 (repeat
motif: TCTA) and alleles of 186 bp and 188 bp at locus PclG-26 (repeat motif:
CA). The PCR products of our marbled crayfish were compared to the sequences
of Procambarus clarkii (see
Belfiore and May, 2000)
(GenBank accession numbers AF290921 and AF290931, respectively), confirming
that they differed in the sequences of the flanking regions as well as in the
number of repeats. This way we can exclude possible cross-contamination. A
batch of the sexually reproducing congeneric Procambarus clarkii,
which served for comparison, included 11 homozygotes (229 bp/229 bp alleles)
and 9 heterozygotes (193 bp/229 bp alleles) for 20 full siblings in the
PclG-04 locus, which is in sharp contrast to our marbled crayfish.
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) that
batch-mates of the marbled crayfish are genetically identical. They further
demonstrate genetic uniformity of our laboratory lineages and suggest genetic
identity with the oldest German lineage from which the founder female of our
laboratory population originated.
Variation of development and growth
The life cycle of the marbled crayfish can be subdivided into embryonic,
juvenile, adolescent and adult periods
(Vogt et al., 2004). At
20°C the embryonic period lasts ca. 20–25 days and is finished by
hatching. In the following, the first eight postembryonic stages with
prevailing spotted colour patterns are called juveniles and the later
non-reproducing stages with increasingly marmorated patterns are referred to
as adolescents. The adult period starts at the earliest at stage 15 with the
first egg laying. Postembryonic growth occurs in discrete steps by moulting
and proceeds until death, usually comprising more than 20 stages. Growth of
crayfish has a genetic component but is greatly influenced by the rearing
temperature and the food (Jones et al.,
2000; Reynolds,
2002). It is regulated by several growth promoting and inhibiting
hormones, among them ecdysone, moult inhibiting hormone and methyl-farnesoate
(Laufer et al., 2002;
Vogt, 2002).
In our stock population, the speed of embryonic development was in some clutches homogeneous, in others heterogeneous, even under the same environmental conditions. For our experiments we used only specimens from homogeneously developing clutches, so the speed of development was always rather uniform during late embryonic development and also during the non-feeding juvenile stages 1 and 2, but became diverse after onset of feeding in stage 3, either on the maternal pleopods (Fig. 1A), in net-culture systems (Fig. 1B) or in micro-plates (Fig. 1E). For instance, in a natural mother–offspring association of dam A reared in a 30x20 cm aquarium, all juveniles passed stage 1 in 3–4 days and stage 2 in another 6–8 days. Stage 3, in contrast, was passed in 10 days by the fastest grower but only in 27 days by the slowest grower, although food was available in excess. Consequently, at day 35 after hatching 5 of 32 specimens were in stage 3 (Fig. 1C), 19 in stage 4, and eight already in stage 5 (Fig. 1D).
A similarly broad range of variation in development and growth was obtained
when batch-mates were individually raised in the wells of a micro-plate
(Fig. 1E), the simplest
environment possible, or communally reared in net-culture systems. In the
latter system we placed excess amounts of food at different sites of the
vessels to guarantee that all juveniles had unlimited access to the food.
Moreover, food uptake was regularly monitored by inspection of the stomach and
gut through the rather transparent epidermis. We wish to emphasize that
juvenile marbled crayfish are not filter feeders like the larvae of many
decapod crustaceans (Anger,
2001). Instead, they take macroscopic food particles with their
pereiopods from the ground and deliver them to the mouth like adult crayfish.
Consequently, potential irregular distribution of microscopic debris in the
water body is not expected to have an influence on the amount of food
internalized.
The range of variation of growth usually increased in the period of adolescence among both communally (Fig. 1G and Fig. 2A,B) and individually (Fig. 2D) reared batch-mates. Again, by taking the measures listed above we could ensure that each individual had unlimited access to the food. Variation of growth was measured by determination of carapace length, total length and mass, but is best illustrated with respect to mass. For instance, in one of our experiments the heaviest individual of a communally reared group weighed approximately 20 times more than the lightest specimen at day 164 after hatching (Fig. 2B), although food was available in excess at any time. The broadest spectrum of variation of growth was obtained when adolescent batch-mates were reared without shelters, i.e. under conditions of social stress (Fig. 1F) (for details see Behaviour section below).
When batch-mates were divided into equal groups on the first day of stage 3 (i.e. before onset of feeding) and then raised in identical aquaria under the same environmental and nutritional conditions, the developing spectra of growth variation of the groups were usually not the same (Fig. 2A,B), indicating that group dynamics develop differently, despite the genetic identity of the group members and uniformity of the environment.
Increase of size and mass was also quite variable in the adult life period
in both communally reared batch-mates (Fig.
2A,C) and siblings raised individually under the same conditions
(Fig. 2D). In this period of
life the situation is complicated by reproduction, as most females diminish
food uptake during the breeding period, which starts with the development of
externally visible glair glands. These glands produce the cement that is used
to attach the eggs to the pleopods (Vogt
and Tolley, 2004). Because of this close interdependence of growth
and reproduction the range of variation can considerably fluctuate among adult
batch-mates, either kept communally (Fig.
2C) or individually (Fig.
2D). Consequently, the relative group position of an individual
can change considerably with time (Fig.
2C,D).
The grade of variation in development and growth differed considerably between rearing systems even when food and water conditions were kept constant. This became particularly apparent in an experiment with progeny of dam B, which were taken from the pleopods in stage 2 and then raised at constant water temperature of 21°C in three different rearing systems, a 60x30 cm aquarium equipped with shelter, a 30x20 cm aquarium equipped with a net only, and three 11x8 cm net culture vessels. At day 25 after hatching variation was highest in the aquarium with shelter: of the 51 sibs two were in stage 3, 24 in stage 4, 16 in stage 5 and nine in stage 6. In the smaller aquarium eight of 14 sibs were in stage 4 and six in stage 5, and in the net culture systems eight of 18 sibs were in stage 3 and ten in stage 4.
Our data suggest that development and growth can vary considerably among isogenic batch-mates even when reared in the same macro-environment with excess availability of food. These differences cannot be attributed to potential accumulation and defence of the food by one or more individuals of a group, as this behaviour was never observed. Moreover, such an argument would not explain the differences observed among individually raised batch-mates. Since variation in growth and development broadens markedly after onset of feeding, we assume that the individual decision, how much to feed and how often to feed and probably also slight differences in metabolism, which increase with time, are the main causes for this phenomenon. Our data also show that the range of variation can be modulated by differences in macro-environmental factors like space and shelter but also by group dynamics in the same environment.
Variation of life span and reproduction
A broad range of variation was also noted with respect to life-span and
reproduction. Basic information on these traits is found elsewhere [for
crayfish in general (Reynolds,
2002; Vogt, 2002)
and for marbled crayfish (Vogt et al.,
2004)]. The data given below refer to days after hatching.
The communally reared batch-mates B1–B8 had life-spans of 437, 626, 610, 571, 910, 568, 626 and 626 days, respectively. All specimens died during moulting, with the exception of B2, B7 and B8, which were sacrificed for determination of DNA methylation. The range of the life-span of sibs died of natural causes varied between 437 and 910 days. The maximum age of a marbled crayfish so far recorded in our laboratory is 1154 days.
Female B1 reproduced three times at days 157, 267 and 375, B2 three times at days 160, 369 and 497, B3 three times at days 168, 392 and 502, B4 three times at days 183, 390 and 516, B5 five times at days 315, 394, 507, 643 and 850, B6 twice at days 328 and 507, B7 once at day 500 and B8 also once at day 531. Thus, the time from hatching to first spawning varied among these batch-mates between 157 and 531 days. Differences in reproduction become even more evident by comparing the reproductive success at a given day of life. For instance, at the age of 430 days female B1 had completed three reproductive cycles with a total of 219 stage-3 juveniles, the first independent life stage. Her sibs B2, B3, B4 and B5 had finished two reproductive cycles in the same period resulting in a total of 66, 153, 73 and 92 juveniles, respectively, whereas batch-mate B6 had produced only one clutch of 52 juveniles. Sibs B7 and B8 had not yet reproduced at that time. The highest number of stage-3 juveniles so far produced in our laboratory by a single female per clutch is 421.
Our results demonstrate that life-span, onset of reproduction, frequency of
reproduction and total reproductive success can vary considerably between
batch-mates even when reared in the same aquarium and fed with a single food
source in excess, suggesting that epigenetic phenomena can considerably
influence fitness. Variation in reproductive traits was also observed among
batch-mates of the parthenogenetic Daphnia pulicaria
(Lajus and Alekseev, 2004). In
these water fleas, some clone-mates produced resting eggs in a standardized
environment but others did not.
Variation of coloration
The most extreme range of variation was recorded with respect to the
eponymous coloration of the marbled crayfish
(Fig. 3B), which is produced by
two types of chromatophores and a diffuse background tanning. The red
chromatophores appear at about 80% of embryonic development and contain red
astaxanthin and, in later stages, also green and blue protein conjugates of
astaxanthin. The white pigment cells appear first in stage-3 juveniles and
include pteridines. The early juvenile stages display spotted motifs composed
of individual pigment cells (Fig.
3A,C–J), which are transformed into marmorated motifs later
on by multiplication of the chromatophores
(Fig. 3B,K–Q). We would
like to emphasize that crayfish cannot change their colour coat rapidly within
seconds, minutes or hours by a `physiological' colour change, which is typical
of some shrimps or squids. Instead, they display a `morphological' change of
pigmentation, which involves quantitative changes of the chromatophores and
qualitative changes of the pigments over weeks and months
(Rao, 1985;
Vogt, 2002). In early juvenile
stages of the marbled crayfish the cuticle is transparent, and therefore, the
pigment spots seen are the chromatophores of the epidermis. From approximately
stage 7 the pigmentation pattern is more and more imprinted into the
cuticle.
Investigation of the pigmentation pattern of the areola, a precisely lined area of the dorsal cephalothorax (Fig. 3A), in 20 offspring of dams A and B1 from hatching to stage 7 revealed that each specimen has a unique pattern of chromatophores (Fig. 3C–F). This individual pattern is already well expressed in hatchlings and can be tracked through later life stages, although it becomes increasingly complex with time (Fig. 3G–J). Addition of both types of chromatophores to the existing pattern becomes particularly obvious from stage 5 on (compare Fig. 3H,J). The individuality of coloration is also maintained in adults, as shown by analysis of the posterio-lateral area of the carapace (Fig. 3B), the most intensely marmorated part of the adult body. Individual tracking of ten adults for a maximum of eight moulting cycles and 880 days revealed that during growth given motifs are enlarged in size whilst only moderately altered in structure by addition of chromatophores (Fig. 3K–M). Striking dissimilarities of the analysed area between dam B (Fig. 3N) and her mature offspring (Fig. 3O–Q) suggest that the marbling pattern is not inherited.
These results and the inspection of many more individuals indicate that
marbled crayfish are individually identified throughout life, despite their
clonal nature, principally permitting kin and group recognition. The extreme
variation in marmoration is comparable only with the variability of leopard
spots, zebra stripes, or human fingerprints and irises
(Daugman and Downing, 2001;
Murray, 2003), and may be best
explained by Turing reaction–diffusion or Murray–Oster
mechanochemical patterning mechanisms
(Murray, 2003). This idea is
supported by regularly observed differences in marmoration between right and
left body sides (Fig. 6A),
which is a typical outcome of those mathematical models. Uniformly colored
marbled crayfish have not yet been found, suggesting that marmoration as such
is genetically determined whilst the spatial distribution of the
chromatophores is epigenetically regulated.
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;
Vogt, 2002). The aesthetascs
are located on the outer branches of the 1st antennae
(Fig. 1D). Each aesthetasc
includes more than 100 dendrites of olfactory receptor neurons
(Sandeman and Sandeman, 2003;
Vogt, 2002). Aesthetascs and
CS appear first in juvenile stage 2. They are not only distinct morphological
and functional units involved in perception of environmental information and
probing of the food, but are also distinct evolutionary modules, as the
aesthetascs are found in all crustaceans
(Hallberg et al., 1992)
whereas the CS are confined to the Decapoda (G.V., unpublished).
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).
Differences in the numbers of aesthetascs among batch-mates are related
both to different numbers of aesthetascs per article and to different numbers
of aesthetasc-bearing articles per antenna. The distal-most article always
carries three unpaired aesthetascs, which is not varied. The following
articles carry usually one pair of aesthetascs in the juvenile stages
2–4 and a maximum of two pairs in the following stages, including the
adults (Fig. 4A)
(Vogt and Tolley, 2004). In
these articles the number of aesthetascs can be varied from one to four.
Variation of the number of CS is mainly related to differences in length of
the rows of these sensory structures on the propodus and dactylus of each of
the pereiopods 1–3 (Fig.
4B). Interestingly, differences in the numbers of aesthetascs
between individuals are expected to have considerable consequences for brain
structure, because each of the many chemoreceptors of an aesthetasc is
individually integrated into the olfactory lobe of the deutocerebrum,
resulting in enlargement of this brain area
(Mellon and Tewari, 2000;
Sandeman and Sandeman, 2003).
Such brain differences may finally lead to differences in individual
performance.
In order to analyse the dependence of the traits CS, aesthetascs (Ae) and carapace length (CL) from juvenile stage and batch in more detail, a bifactorial ANOVA with interaction was applied. The idea behind the model is that the influence of stage and of batch could combine multiplicatively if size and therefore the numbers of sensory organs were determined by an early factor such as yolk supply. The results are presented in Table 1 (these log-based values are very similar to corresponding results obtained without using logarithms). The data indicate that for each of the three variables both main effects and interaction term are highly significant. This means that there is a difference among the juvenile stages, which is trivial, that there is a significant multiplicative difference in the means between the different batches, which may be caused by differences in yolk supply, feeding behaviour or group dynamics among batches, and that these two effects do not fully explain the observed dataset, as the interaction term is also significant. Different batches show a varying deviation from the mean in the different juvenile stages. These differences cannot be explained by a single additive or multiplicative effect, but suggest an unpredictable and/or nonlinear mechanism for generation of variability.
|
The variances of the 20 sub-samples (different stages and batches) were compared by a Bartlett test for log(CL) and log(CS) and were shown to be highly significant [P=0.002247 for log(CL) and P=7.415x10–5 for log(CS)]. Fig. 5 shows the estimated geometric standard deviations with their corresponding 95%-confidence limits. The confidence limits overlap in most cases so that no individual differences can be established. However for CL and CS the relative variation seems to be larger in all stages for batch B1. We therefore conclude that there is a maternal influence on the development of the genetically identical progeny but that this influence is not deterministic but rather expressed in the variation of the offspring. On the other hand the relative errors show no obvious change throughout the different juvenile stages. Based on all data the geometric standard deviation was estimated by transforming MSE of the corresponding ANOVA models to be sg(CS)=1.049, sg(Ae)=1.060 and sg(CL)=1.041, and thus the within-batch variation is about 5% for all three parameters.
|
|
We suppose that the variation in the numbers of aesthetascs and corrugated
setae among batch mates are caused by slight differences in the expression and
reaction–diffusion patterns of morphogenetic factors. This idea is
inferred from results by Dohle et al.
(Dohle et al., 2004), who
showed that during limb development morphogens can communicate information to
members of different cell lineages. In other words, gene expression is
independent of stereotyped differential cleavage in early development of
crayfish. Such differences in reaction–diffusion-like morphogenetic
mechanisms would also explain the semi-independent behaviour of the traits
investigated.
Variation of behaviour
Communally and also individually raised batch-mates displayed a broad range
of variation with regard to particular behavioural elements. Basic data on the
behaviour of crayfish are published elsewhere
(Gherardi, 2002;
Lundberg, 2004). For instance,
as soon as the juveniles could move, which was in stage 2, they developed
differences in locomotion. They even developed differences in social behaviour
since some specimens preferred to be solitary in net culture while others
tended to aggregate. Many other examples were observed in adolescents and
adults. For instance, some batch-mates moulted in the morning but others in
the evening or during the night. Most specimens rested under the shelters in a
sitting position but some preferred always to lie on the back. Oviposition
usually occurred during the night but some individuals preferred to lay the
eggs during the day. Some dams fed during the periods of breeding and brooding
of the juveniles but others did not touch the food at all. Most of the berried
females removed decaying eggs from the clutch but some did not. These
behavioural differences were quite obvious but we also assume variation in
other less easily visible aspects, such as quantity of food uptake as
discussed above. Since most of these behavioural variations are related to
differences in individual decisions rather than to social interactions and
since they are also found among individually raised batch-mates they are
treated as a part of DV rather than of the macro-environment.
The most dramatic establishment of differences in behaviour among
batch-mates was observed when groups of five freshly moulted and size-matched
stage-6 sibs with neutral agonistic behaviour were placed in 30x20 cm
aquaria without shelter (replicated 3 times). In the next 34 days a social
hierarchy was gradually established, beginning at stage 7, the first stage
with sclerotized claws suitable for agonistic interactions. This social
hierarchy finally included one dominant, one subdominant and three
subordinates or one dominant, two subdominants and two subordinates.
Establishment of dominance was paralleled by the development of increasingly
offensive and aggressive behaviours
(Lundberg, 2004) of the
dominant and increasingly defensive and avoiding behaviours of the
subordinates. Such pronounced hierarchies were not established when shelters
were available, corroborating the importance of shelters for the culture of
adolescent and adult crayfish (Gherardi,
2002).
Interestingly, the dominants grew much faster than the subordinates
(Fig. 1F), although all
specimens had unlimited access to the food and fed regularly, as revealed by
inspection of the stomach and intestine through the transparent epidermis.
These observations suggest that even in size-matched clone-mates, slight
initial physiological or behavioural differences can finally result in a broad
range of phenotypes, probably by self-reinforcing circuits involving
behaviour, sense organs, brain, hormone system and metabolism. This idea is
corroborated by Song et al. (Song et al.,
2007), who observed higher survival of brain cell precursors in
dominant crayfish compared to subordinates. The relationship of dominance and
faster growth is well known from bisexual crayfish species but is usually
attributed to genetic differences in food conversion and aggression
(Reynolds, 1989). Behavioural
variations among genetically identical organisms kept in a constant
environment were also observed for cloned pigs
(Archer et al., 2003b).
Variation of characters among body sides
Right–left differences are traditional organismic indicators of the
epigenetic proportion of a trait, as the genes are the same in both body
sides. The most frequently measured parameter is fluctuating asymmetry (FA),
the numerical difference of a trait between right and left side
(Debat and David, 2001;
Dongen, 2006;
Lajus et al., 2003;
Møller, 2006;
Nijhout and Davidowitz, 2003).
However, FA has to be regarded as a special aspect of DV, because right and
left body structures are linked to each other by the nervous system and
circulation, which may allow correction of asymmetries. In the marbled
crayfish right–left differences were observed for all traits
investigated, being most prominent for coloration. In each specimen examined
the marmoration patterns of both body sides were far away form being
mirror-symmetric (Fig. 6A),
emphasizing that body pigmentation has a high epigenetic component.
FA of the aesthetascs and corrugated setae varied considerably between
juvenile stages and also among batches
(Fig. 6B). It fluctuated
roughly between +3% and –3%, in extreme cases between +6 and –6%.
In the entire population of the 168 specimens investigated FA was close to
zero for both sense organs (aesthetascs: –0.34%; corrugated setae:
–0.32%), indicating the good state of health of our laboratory
population (Møller,
2006). Interestingly, FAs of both traits were not correlated
(Fig. 6B). Common to both
traits, however, was shifting of FA between body sides throughout development,
suggesting that asymmetries are indeed corrected when they exceed a certain
range. This idea is corroborated by longitudinal analyses of individual FAs,
as exemplified in Fig. 6C. In
that specimen, FA of the aesthetascs changed body sides twice between stages 6
and 14, as did FA of the CS of pereiopod 2.
Our data also demonstrate that the traits investigated behaved as
semi-independent modules. For example, in the individual analyzed in
Fig. 6C, growth increments of
the carapace were almost identical (1.5 mm versus 1.4 mm) in stages 9
and 10, whereas the increases in the numbers of aesthetascs (5 versus
11) and CS (16 versus 60) were quite different in the same periods.
The modular architecture of animals has received great attention in the last
years, because developmental modules may be uncoupled under certain
circumstances and act as quasi-independent units of evolutionary
transformation, possibly leading to new phenotypes or even new body plans
(Franz-Odendaal and Hall,
2006; Schlosser and Wagner,
2004).
Variation of global DNA methylation
Recent studies on cloned and inbred mice and monozygotic human twins
revealed that the epigenotype, which includes DNA methylation, histone
modifications and modifications of regulatory proteins on the DNA, can vary
between genetically identical individuals and may be associated with
phenotypic differences (Fraga et al.,
2005; Ohgane et al.,
2001; Whitelaw and Whitelaw,
2006). In order to test if differently grown batch-mates of the
marbled crayfish also show differences in the epigenetic code, we measured
global methylation of cytosines in the DNA of the hepatopancreas and the
abdominal musculature, using an improved and highly sensitive analytical
technique recently developed by us
(Schiewek et al., 2007). This
method allows the determination of very low genome-wide methylation levels
(<1%) from DNA samples as small as 1 µg. The methylation level is
calculated with the equation [5mdC]/([5mdC]+[dC]).
The data obtained from two communally raised batches revealed that global DNA methylation varied among batch-mates and also among tissues (Fig. 6D). In four 188-day-old adolescents with total lengths of 3.2 cm to 4.2 cm (mean: 3.58 cm) and mass of 0.68 g to 1.71 g (mean: 1.01 g), methylation varied between 1.75% and 1.94% (mean 1.86%) in the hepatopancreas and between 1.96% and 2.09% (mean 2.01%) in the abdominal musculature. In three 626-day-old adults of 6.2 cm to 7.0 cm total length (mean: 6.7 cm) and mass of 6.04 g to 10.32 g (mean: 7.99 g) methylation varied between 1.52% and 1.78% (mean: 1.65%) in the hepatopancreas and between 1.77% and 1.92% (mean: 1.84%) in the musculature.
Our data demonstrate within-batch variation of global DNA methylation in
the hepatopancreas and abdominal musculature but do not show a correlation
with growth, although there seems to be a slight tendency that the fastest
growers have the lowest methylation values in the abdominal musculature. At
present, it is not even known whether methylation plays a similar role in
regulation of the genes in crayfish as in vertebrates. Our data clearly
indicate, however, that the DNA of crayfish is methylated not only during
juvenile development but also in reproducing adults, which is in contrast to
Drosophila, where the DNA is methylated in the embryos only. But even
in this fly, which is also an exceptional case with respect to other aspects
such as the size of the genome, DNA methylation was shown to play a key role
in differentiation (Mandrioli and
Borsatti, 2006).
We would also like to emphasize that small differences in global DNA
methylation can have great consequences for the phenotype. For instance, in
in vitro fertilized bovine fetuses fetal overgrowth and associated
endocrine changes were related to only 11.2% deviation from normal methylation
values of the liver (Hiendleder et al.,
2006). In the hepatopancreas of our crayfish, which is
functionally comparable to the liver of vertebrates
(Vogt, 2002), the difference
of methylation between the heaviest and lightest specimen was 17.1% in the
adult group and 11.3% in the adolescents. This first study on global DNA
methylation in the marbled crayfish and its variation should be followed by
more specific research on the methylation of genes that regulate growth in
crayfish (see Development and growth section) and on potential correlations
with growth differences.
|
CONCLUSIONS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSPERSPECTIVESReferences |
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PERSPECTIVES |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSPERSPECTIVESReferences |
|---|
Although we have no evidence so far that any of the recorded variations are
adaptive or heritable, we would like to end with some speculations in order to
stimulate further research on the role of DV in evolutionary biology. DV may
be of particular significance to clonal organisms and invasive pests, because
it increases their chance to persist in fluctuating or new environments by
a-priori provision of a broader spectrum of phenotypes. In other
words, DV may keep clonal species in the game when the environment changes.
Since the phenotype is the principal target of natural selection, DV may even
act as a general evolution factor by contributing to the production of a
broader range of phenotypes that may occupy different ecological micro-niches,
paving the way to speciation via differential mutagenesis or
epigenetic inheritance (Loxdale and
Lushai, 2003; Rakyan and Beck,
2006; Schlichting and
Pigliucci, 1998; Whitelaw and
Whitelaw, 2006; West-Eberhard,
2005; Zakharov,
1993).
Meanwhile, some further examples of developmental variation (DV) were found
among identically raised batch-mates of the marbled crayfish. These variations
concern the symmetry of internal organs, movement patterns and social
behaviours, and the response to environmental toxicants. For instance, the
sternal artery, which connects the heart to the ventral thoraco-abdominal
artery, can be either paired and bilateral symmetric, or unpaired and right or
left asymmetric, as revealed by serial sectioning of more than 100 juveniles
(Vogt et al., manuscript submitted for publication). Movement patterns and
social behaviours were shown to vary considerably among stage-2 batch-mates
raised from in vitro cultured eggs
(Vogt, 2008). And the duration
of embryonic development, hatching success, growth of the juveniles, and grade
of malformation of the appendages varied considerably among sibs exposed in
12-well micro-plates to 100 µg l–1 17
-methyl
testosterone (Vogt, 2007).
|
Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSPERSPECTIVESReferences |
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