JOURNAL OF CRUSTACEAN BIOLOGY, 28(1): 180–184, 2008
GONOMERY AND CHROMATIN DIMINUTION IN MESOCYCLOPS LONGISETUS (COPEPODA)
Ellen M. Rasch and Grace A. Wyngaard
(EMR, correspondence) Department of Anatomy and Cell Biology, James H. Quillen College of Medicine,
East Tennessee State University, Johnson City, Tennessee 37614-0582, ([email protected]);
(GAW) Department of Biology, James Madison University, Harrisonburg, Virginia 22807, ([email protected])
ABSTRACT
The segregation of progenitor somatic cells from those of the primordial germ cells during early cleavage divisions occurs in certain
copepods exhibiting the phenomenon of chromatin diminution during early embryogenesis. These species provide an interesting
alternative to the usual reproductive strategy of other species of freshwater cyclopoid copepods. Levels of DNA for the soma and germ
cells of Mesocyclops longisetus have been determined for individual nuclei by using Feulgen-DNA cytophotometry to monitor changes of
DNA amounts during gametogenesis and early cleavage stages of embryogenesis. Germ cell nuclei of both female and male adults contain
marked elevations of DNA, far in excess of expected 4C DNA level for their replication prior to meiosis. The elevated amounts of DNA in
these germ cells are equivalent to the elevated DNA content found during the gonomeric divisions observed in embryos. Following the
gonomeric divisions there is roughly a 40% loss of germ cell heterochromatin during the chromatin diminution stages of embryogenesis.
The role of this excised DNA remains unclear.
KEY WORDS: Copepoda, developmental biology, DNA, Mesocyclops
somes relative to the postdiminuted somatic chromosomes is
additional evidence of the large prediminuted genome and
may provide a means for associating the elevated DNA in
the germ line nuclei of adults with the germ line limited
DNA that is excised from the presomatic line of embryonic
cells during these processes.
Here we report on quantitative changes in DNA levels
that occur during gametogenesis in adults and during
diminution in early embryos in a Louisiana population
of the copepod Mesocyclops longisetus (Thiébaud, 1912).
DNA-Feulgen cytophotometry was used to determine the
DNA content of somatic cell nuclei and germ cell nuclei
of adult males and females, of mitotic gonomeric chromosomes in embryos prior to germ line-soma differentiation, and anaphase chromosomes in the process of
chromatin diminution, which marks separation of the germ
and somatic lineages. This first report of the DNA contents
of gonomeric figures allow us to follow the changes that
accompany gametogenesis and early cleavage stages including the process of chromatin diminution and infer the
role that gonomery may serve in species that undergo
chromatin diminution.
INTRODUCTION
There is a considerable body of literature on the phenomenon of chromatin diminution in cyclopoid copepods,
initially recognized and described by Beermann (1959) for
Cyclops furcifer (Claus, 1857) and subsequently identified
to occur during early embryogenesis in certain other copepod species (Terpilowska, 1971; Beermann, 1959, 1966,
1977; Einsle, 1993; Grishanin and Akifiev, 1993; Grishanin
et al., 1994; Rasch and Wyngaard, 1997, 2006a; Wyngaard
and Rasch, 2000). The primary features of chromatin
diminution are the selective exclusion of certain kinds of
DNA from the progenitor somatic cells and the retention
and perpetuation of this DNA by germ cell primordia. The
process of programmed fragmentation of chromosomes and
the excision of specific heterochromatin segments during
early embryogenesis results in the loss of large amounts of
DNA from the somatic cell genome (Beermann, 1959, 1966;
Rasch and Wyngaard, 2001, 2006a). A similar pattern of
chromatin elimination has been described for ascarid
nematodes, hypotrichous ciliates, mycetophylid dipterans,
Japanese hagfish and some marsupials (Tobler et al., 1992;
Kloc and Zagrodzinska, 2001 for review). Despite recent
surveys of genome size and the presence or absence of
chromatin diminution in copepods (Rasch and Wyngaard,
2006b), quantitative data on the amounts of excised DNA
are reported for only a few species (Beermann, 1966).
Quantitative descriptions of chromatin diminution are
needed before we can observe trends and pose hypotheses
of causal relationships between the amounts of discarded
DNA and the general biology of copepods.
In several copepod species for which chromatin diminution is known, gonomery has also been observed (Häcker,
1895; Beermann, 1977). During the early embryonic cleavage divisions just prior to the diminution, there is a
separation of maternal and paternal sets of chromosomes
on the spindle. The large size of the gonomeric chromo-
MATERIALS AND METHODS
Animals were collected from Joe Brown lagoon, Orleans parish, Louisiana
in 1993, maintained in a modified Provosoli medium (Wyngaard and
Chinnappa, 1982) and fed Cryptomnas ozolini (UTEX-LB2194), supplemented with Artemia salina nauplii. After identification for sex, each male,
female, or female carrying external egg sacs was placed on a gelatin-subbed
slide in a drop of methanol/acetic acid (3:1, v/v) for 2-3 min, softened in
45% acetic acid for 1-2 min, and then squashed under a siliconized
coverslip. After freezing in liquid N2, the preparation was thawed in
absolute ethanol and air dried. The protocol for the Feulgen reaction for
DNA has been described elsewhere (Rasch, 2003). Each set of copepod
preparations for staining included a slide carrying a thin film of chicken
blood cells to serve as an internal reference standard of 2.5 pg DNA per
nucleus to allow us to express values obtained as relative absorbance units
by cytophotometry in terms of estimates of levels of DNA pg for individual
copepod nuclei or division figures (Hardie et al., 2002; Rasch, 2003).
180
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RASCH AND WYNGAARD: GONOMERY AND CHROMATIN DIMINUTION
Table 1. DNA content (pg DNA per nucleus) from somatic and
differentating germ cells of female and male Mesocyclops longisetus.
Somatic nuclei
Specimen
#
#
#
#
#
#
#
#
#
#
715
716
717
718
719
725
726
752
753
754
Prediminution nuclei
Sex
DNA (pg)
6 SE
n
DNA (pg)
6 SE
n
$
$
$
$
$
#
#
#
#
#
1.84
1.86
1.78
1.76
1.73
1.76
1.72
1.78
1.81
1.83
0.055
0.050
0.074
0.045
0.046
0.025
0.076
0.060
0.027
0.025
50
25
50
70
50
40
30
50
40
50
12.20
10.86
9.940
11.45
11.73
11.90
11.76
10.32
10.26
10.32
0.431
0.240
0.224
0.110
0.266
0.097
0.076
0.421
0.276
0.416
50
25
20
50
50
50
40
25
25
25
Feulgen-DNA amounts for individual nuclei were obtained with a
Vickers M86 scanning and integrating microdensitometer (York, UK) at
560 nm with the highest resolution pattern of this instrument by using
a 1003 apochromatic lens, N.A. 1.32 and a 0.5 lm scanning spot. The
subtractive method advocated by Bedi and Goldstein (1976) was used for
all measuring. Preparations were mounted in refractive index liquids to
minimize light loss due to scatter (Rasch, 2003).
DNA contents of somatic and germ cells prior to diminution (PD) nuclei
were measured in interphase nuclei of adults because mitotic divisions are
not typically observed in adult somatic tissues. Gonomeric division figures
from embryos were scanned as an entire configuration and also as separate
components, i.e., the separate halves of the chromosomal groups moving
to the spindle poles during anaphase. In cases of chromatin diminution
the each group of daughter chromosomes at anaphase was scanned and the
chromatin fragments were measured separately with a small adjustable
mask to get estimates of the amounts of discarded chromatin.
RESULTS
The DNA levels of somatic cell and germ cell nuclei differ
greatly in Mesocyclops longisetus. The diploid somatic cell
DNA contents for male and female adults are approximately
1.7-1.8 pg DNA (Table 1, Fig. 1A), whereas the DNA
contents of the large, densely stained germ cell nuclei that
occur in both male and female adults are 9.9-12.2 pg DNA
(Table 1, Fig. 1B). The elevated DNA content of these large
nuclei far exceeds the doubling projected for a 4C level of
DNA that would be expected for germ cell nuclei that have
simply replicated the somatic genome prior to the onset of
meiosis. The differences observed for the large nuclei
amount to about a 6-fold increase in the amount of DNA
found for somatic cell nuclei of the same specimen (Table 1,
Fig. 1A). These unusual nuclei are readily recognized
because of their large size and staining intensity. They have
a nuclear core of highly compacted heterochromatin, often
with a rim of thin Feulgen-positive strands surrounding the
core (Fig. 1B). These nuclei are identified here as prediminution or PD nuclei and are characteristic of germ cells
in adults of this species.
In the early cleavage stages of embryogenesis obtained
from females carrying egg sacs, mitotic configurations of
gonomeric chromosomes are clearly evident as a consequence of the relatively large size of the chromosomes and
because of the separation of maternal and paternal sets on
the spindle, a process known as gonomery (Fig. 1C, Häcker,
1896). Levels of DNA for these mitotic divisions are similar
to those found for PD nuclei (Tables 1 and 2). Elevated
levels of DNA regularly occur in embryos at early cleavage
stages during the gonomeric divisions. This process char-
Fig. 1. Nuclei of the copepod Mesocyclops longisetus stained with
Feulgen reaction for DNA. A, Somatic cells from an appendage; B,
Prediminution germ cell nuclei from an adult female showing large
heterochromatic cores with wispy veils of thin chromatin strands; C, A pair
of anaphases moving in synchrony toward the poles of the spindle reveal
segregation of maternal and paternal chromosomes in a gonomeric mitosis
from a 8-16 cell stage embryo; D, Chromatin diminution showing the
heterochromatin fragments left at the equator during anaphase and the sets
of presumptive somatic chromosomes moving to the poles of the spindle.
(Reprinted by permission from Blackwell Publishing, from Rasch and
Wyngaard, 2006a).
acteristically shows that maternal and the paternal chromosomes remain as separate entities. During the earliest
cleavages these nuclei appear as a pair of metaphases or
as two parallel pairs of anaphases moving toward the poles
on a common spindle (Fig. 1C). The DNA contents of the
gonomeric chromosomes at metaphase or in anaphase are
approximately 11-12 pg DNA (table 2). The large size of the
Table 2. DNA content of gonomeric mitotic figures from Mesocyclops
longisetus embryos at 16 cell cleavage stage and somatic and prediminuted
nuclei of their mother.
DNA content (pg)
Cell type/mitotic stage
Mean
6 SE
n
Somatic cell interphase
Prediminuted nucleus
Whole metaphase
Half metaphase
Whole anaphase
Half anaphase
1.81
11.58
12.64
6.31
12.24
5.78
0.022
0.134
0.327
0.091
0.208
0.166
178
98
12
11
22
22
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 28, NO. 1, 2008
Table 3. DNA levels of discarded DNA remaining in the spindle
during the anaphase of chromatin diminution in embryos of Mesocyclops
longisetus.
Embryo
number
Anaphase
a (pg)
Anaphase
b (pg)
Anaphase
sum (pg)
Excised
DNA (pg)
Total
DNA (pg)
DNA %
Excised
HD-1
HD-2
HD-3
HD-4
3.54
3.74
3.67
3.44
3.53
4.98
2.74
3.31
7.07
8.72
6.41
6.75
4.46
5.07
5.54
4.89
11.53
13.79
11.95
11.64
38.6
36.8
46.4
42.0
individual chromosomes in these mitotic configurations
(Fig. 1C) and the intensity of their staining facilitate their
identification (Fig. 1C).
The DNA content of a pair of metaphases or the DNA
amount found for a pair of anaphases correspond with the
DNA amount found for the PD nuclei of mature adults
(Tables 1 and 2). The DNA content of paired metaphases
and whole anaphases are also equivalent to the DNA levels
of nuclei in the process of chromatin diminution (Table 3,
Fig. 1D). Fragments of heterochromatic DNA remain at in
the spindle at the equator of the configuration. Approximately 40% of the DNA of the whole anaphase figure is
discarded in this division. The resultant groups of daughter
anaphase chromosomes contain more than the 2C amount
of DNA expected for somatic nuclei. This excess of the
expected value of 1.8 pg DNA is taken to indicate that
another round of mitotic divisions follows the initial chromatin diminution stage observed here in order to restore the
2C DNA level of the progenitor cells of the soma.
DISCUSSION
In comparing the DNA levels of embryonic division figures
(Table 2) with the DNA content of diploid somatic cell
nuclei and PD nuclei (Table 1), it is evident that the germ
cells carry a sizeable load of genetic material in the form
of large amounts of heterochromatin that are retained and
perpetuated only in the germ line cells. An estimate of the
amount of expendable DNA that is left in the spindle during
chromatin diminution was obtained by measuring total
diminution figures and then measuring just the DNA left
in the spindle (Table 3). The discarded DNA accounts for
roughly 40% of the total DNA content of both the PD nuclei
and of the DNA levels observed during the gonomeric
divisions at the earliest embryonic cleavage stages (Tables
1, 2 and 3). Gonomery has been observed during early
embryonic cleavage divisions in four cyclopoid species
that posses chromatin diminution (Beermann, 1977; Rasch
and Wyngaard, 2006a), but not in species that lack chromatin diminution (personal observations). The only other
copepod for which gonomery is known is the harpactioid
Tigriopus californicus Baker, 1912, but gonomery is
restricted to the zygotes (Ar-hishdi, 1963).
Direct measurements of the nuclear DNA contents of
chromatin diminution figures and gonomeric figures prior to
diminution, as well as germ cell and somatic nuclei (Tables
1, 2 and 3), form the basis of our suggestion that the large
amount of heterochromatin of the PD germ cells is equivalent to the elevated DNA content observed during the
gonomeric divisions. The subsequent sequence of chromatin
diminution events would then be a case of the reorganization of the genome by the exclusion of chromosomal
fragments that apparently are not necessary for the functions
of the somatic cells of this species.
The heterochromatin occurring in the PD nuclei would
appear to be the antecedent of the elevated DNA level of
gonomeric stages which occur prior to the excision of
chromatin. The chromatin diminution process restores the
DNA content of the daughter cells to the lower levels
observed in the soma. The ‘‘extra’’ DNA observed when
making comparisons of somatic cell DNA levels with the
amount of DNA that accumulates as heterochromatin in PD
nuclei is remarkably similar in size to its subsequent elimination from the progenitor somatic cells by the diminution
process (Table 3). The observed variability of the DNA
content of PD nuclei is to be expected since they synthesize
the dense cores of heterochromatin during gametogenesis.
The quantitative behavior of DNA and the sequence of
events indicate that this heterochromatin may function
in epigenetic events during early embryogenesis. Further
studies to characterize this DNA will be needed to clarify its
functional role in development.
The pattern of findings here is very similar to that reported
for M. edax (Forbes, 1890) during gametogenesis and early
embryogenesis (Rasch and Wyngaard, (2001, 2006a). The
identification of the PD nuclei as germ line derivatives that
proceed to undergo gonomery followed by chromatin
diminution during early cleavage stages has not been
described or discussed by previous workers on the subject
of chromatin diminution in Cyclops (Stich, 1962; Beermann,
1966, 1977; Grishanin et al., 1994), but has been described
for Mesocyclops edax, Brazilian populations of M. longisetus
curvatus and Metacyclops mendocinus (Wierzejski, 1892)
and a German population of Cyclops strenuus (Fischer,
1851) cited by Rasch and Wyngaard, (2006a). The drawings
of differentiating germ cells in female gonads of Cyclops
strenuus by Lerat (1905) depict cells with nuclei that look
like the PD nuclei described here, although no particular
comments are given in this older paper.
The present study directly estimates the amount of
excised DNA during chromatin diminution in M. longisetus
to be about 40% by measuring the amount of DNA
remaining in the metaphase plate during the anaphase of
chromatin diminution. To our knowledge, the only other
species for which the eliminated DNA has been estimated in
this manner is M. edax which leaves about 75% of its DNA
at the metaphase plate (Rasch and Wyngaard, 2006a). It is
interesting to note that the adult diploid nuclear somatic size
of M. edax is 3.0 pg DNA, almost twice that of M.
longisetus. Whether the diploid genome size relates to the
amount of heterochromatin discarded is unclear, although
there is such a relationship between genome size and the
diminution of chromatin in ascarid nematodes (Niedermaier
and Moritz, 2000). The elegant study of ascarid nematodes
by these authors has clearly demonstrated the occurrence of
two highly repetitive sequences associated with the
programmed and selective elimination of telomeric tracts
during chromatin diminution in these species. Drouin (2006)
has recently reported on the presence of several repetitive
sequences in his comparison of DNAs from adults and
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RASCH AND WYNGAARD: GONOMERY AND CHROMATIN DIMINUTION
prediminuted embryos of Mesocyclops edax. A reasonable
next step is to identify the heterochromatic sequences in
M. longisetus and to compare them to those excised in M.
edax. Both quantitative and qualitative descriptions of this
excised DNA in different cyclopoid species should provide
insights into the roles of chromatin diminution in the
biology of copepods.
Increases in DNA levels in another crustacean, Daphnia,
demonstrated that endopolyploidy (increases in nuclear
DNA content without subsequent cell division) may act as
a developmental control center to modulate mitotic activity
(Beaton and Hebert, 1997; Beaton and Cavalier-Smith,
1999). The elevated DNA levels in the PD nuclei of
copepods do not appear to serve this function. Rather, it may
be a case of regulation of other embryonic events, since it
is only after this critical period that the DNA is discarded
from somatic cell lineages, which undergo many cycles of
cell division.
Because of the unusual nature of chromatin diminution,
the phenomenon of epigenetics and current research in this
field offers new ideas on subtle changes in heterochromatin.
There can be consequences of heritable changes in the state
of chromatin without changing the underlying DNA sequences. That is to say, there may be retention of the
essential genotype, but heritable alterations of the phenotype
occur by changes in the proteins associated with the DNA.
The components of the chromatin template, DNA and
histone proteins, are the main substrates that are subject to
alterations that lead to distinct chromatin states. These
changes are brought about by DNA methylation and variant
histone modifications such as acetylation, methylation,
and phosphorylation among other covalent changes in the
expression of gene activity (Goldberg et al., 2007). The
unusual behavior of chromatin in the case of certain cyclopoid copepods that increase their chromatin content
during gametogenesis and then discard it during early
embryonic life suggests that this requirement in the course
of reproduction reflects its role in epigenetic changes in
chromosome state during the earliest embryonic cleavages
(Goldberg et al., 2007).
The possibility of the alteration of chromosomes by
additional heterochromatic DNA at a critical stage of early
development is one possible correlate with implications for
the success of embryonic development and eventually the
adult lifestyle. The altered state of DNA or its essential
special sequences are heritable without changing the basic
genotype of the organism, since it accompanies the phenotype at the stage of development associated with gametogenesis and the most critical periods of early embryonic life.
It is possible that the increase in DNA levels at these times
in the life history of certain copepods is non-coding and may
function in determining genome size-cell volume relationships (Beaton and Cavalier-Smith, 1999). Additional studies
using molecular approaches to the basic questions here will
be necessary to determine the function of the special DNA
involved in chromatin diminution. Copepods may provide
a useful model system for such explorations.
ACKNOWLEDGEMENTS
We thank B. A. Connelly for expert assistance. G. Marten and M. Nguyen
for collecting Mesocyclops longisetus, J. Reid for identifying the species
and H. Doward for culturing the animals and making preparations. This
work was supported in part by research grants from the National Science
Foundation DEB 0080921 (EMR), DEB 0084854 (GAW), the James
H. Quillen College of Medicine and the Thomas and Kate Jeffress
Memorial Trust.
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RECEIVED: 2 February 2007.
ACCEPTED: 19 April 2007.