X-linked heterochromatin distribution in the holocentric chromosomes of the
green apple aphid Aphis pomi
A. Criniti1, G. Simonazzi1, S. Cassanelli1, M. Ferrari1, D. Bizzaro2 & G. C. Manicardi1,*
1Dipartimento di Scienze Agrarie, Universita` di Modena e Reggio Emilia, Reggio Emilia, Italia; 2Istituto di
Biologia e Genetica, Universita` Politecnica delle Marche, Ancona, Italia; *Author for correspondence (Phone:
+39-522-522059; Fax: +39-522-522053; E-mail: manicardi.giancarlo@unimore.it)
Received 27 September 2004 Accepted 23 December 2004
Key words: A. pomi, AgNORs, aphid, cytogenetics, heterochromatin, holocentric chromosomes
Abstract
Chromatin organization in the holocentric chromosomes of the green apple aphid Aphis pomi has been
investigated at a cytological level after C-banding, NOR, Giemsa, fluorochrome staining and fluorescent
in situ hybridization (FISH). C-banding technique showed that heterochromatic bands are exclusively
located on X chromosomes. This data represents a peculiar feature that clearly contradicts the equilocal
distribution of heterochromatin typical of monocentric chromosomes. Moreover, silver staining and
FISH carried out with a 28S rDNA probe localized rDNA genes on one telomere of each X chro-
mosome; CMA3 staining reveals that these silver positive telomeres are the only GC-rich regions among
A. pomi heterochromatin, whereas all other C-positive bands are DAPI positive thus containing AT-
rich DNA.
Introduction
To date, almost all studies concerning chromatin
structure and organization have been focused on
monocentric chromosomes whereas data regarding
holocentric ones have been neglected. These
chromosomes are also termed holokinetic because,
during mitotic anaphase, they behave as if the
spindle attachment is not localized, so that chro-
matids move apart in parallel and do not form the
classical V shaped figures usually observed during
the movement of monocentric ones (Blackman,
1987). Aphids, because of the easiness with which
mitotic chromosomes could be obtained from
embryonic tissues, represent a useful tool for a
better understanding of holocentric/holokinetc
chromosomes architecture, in order to work out
differences and/or similarities to monocentric
ones. The identification of chromosomal markers
in organisms possessing holocentric chromosomes
is extremely important since the lack of a primary
conscription together with the difficult in obtaining
a clear-cut banding pattern have greatly hampered
cytogenetic studies in species possessing such a
peculiar chromatin organization (Blackman 1987,
Hales et al., 1997, Manicardi et al., 2002). The
interest of a cytogenetic approach towards this
taxon is also emphasized by the lymph-sucking
feeding of these insects which represent a serious
problem for agriculture, not only in view of their
parasitic action against crops, but also because
they represent active vectors of crop viruses.
This work is aimed to search for the localization
and the DNA composition of heterochromatin in
the holocentric chromosome of the green apple
aphid Aphis pomi a small yellow-green aphids
widely distributed in Europe, Middle East,
throughout North America. Apple and Pear are the
primarily host (Blackman&Eastop, 1984) onwhich
A. pomi can cause direct damage to apple fruit
(Oatman & Legner, 1961), but in most cases the
damage is indirect through reductions in general
Genetica (2005) 124:93–98
Springer 2005
DOI 10.1007/s10709-004-8154-y

vigor of the apple tree (Hamilton, Swift & Marini,
1986). Green apple aphids are therefore important
indirect pests of apple that require monitoring and,
often, insecticide treatments (Carroll & Hoyt, 1984,
Pfeier, Brown & Varn 1989).
Material and methods
Colonies of Aphis pomi were collected on apple
trees close to Reggio Emilia (Northern Italy).
Chromosome spreads of embryo cells obtained
from parthenogenetic females were prepared as
previously described by Manicardi et al. 1996.
C-banding treatment was performed according
to Sumner’s technique (1972). After the treat-
ment, some slides were stained with 5% Giemsa
solution in Soerensen buffer pH 6.8, for 10 min.
Chromomycin A3 (CMA3) staining was made in
accordance to Schweizer (1976), whereas 4¢-6¢-
diamidino-2-phenylindole (DAPI) treatment was
carried out as described by Danlon and Magenis,
(1983). Silver staining of nucleolar organizing
regions (NORs) was performed following the
technique of Howell and Black, (1980).
DNA extraction from aphid embryos was car-
ried out as described in Bizzaro, Manicardi &
Bianchi, 1996. The 28S rDNA probe was obtained
by PCR amplification of A. pomi genomic DNA
carried out using two primers, F (5¢-AACAAAC-
AACCGATACGTTCCG) and R (5¢-CTCTGT-
CCGTTTACAACCGAGC), designed according
to the coding 28S sequence of the aphid Acyrtho-
siphon pisum (GenBank X66419) (Amako, Kwon
& Ishikawa, 1996). The amplification mix con-
tained 100 ng genomic DNA, 1 lM of each pri-
mer, 200 lM dNTPs and 2 U of DyNAZyme II
DNA polymerase (Finnzymes Oy). The amplifi-
cation was performed with a thermocycler Hybaid
at an annealing temperature of 60C for 1 min and
extension at 72C for 1 min. Probe labelling and
fluorescent in situ hybridisation (FISH) were per-
formed according to Bizzaro, Manicardi &
Bianchi, 1996.
Results
Aphis pomi metaphases, obtained from partheno-
genetic females, revealed a chromosome number of
2n=8 (Figure 1(a)). Occasionally plates containing
7 or 9 chromosomes have been observed (Figure 1(b
and c)). These aneuploidies have never been ob-
served in a whole specimen but they were always
surrounded by plates containing the classical 2n=8
karyotype. Moreover, in some cases polypolid
plates have been evidenced (Figure 1(d)).
Giemsa staining of C-banded chromosomes put
in evidence four heterochromatic bands limited to
the X chromosomes both at telomeric and inter-
stitial sites, whereas autosomes lack any kind
of differential staining (Figure 2(a)). Staining of
C-banded chromosomes with fluorochromes
shows that most of the A. pomi heterochromatin
was brightly fluorescent after DAPI staining and
therefore AT rich (Figure 2(b)), whereas only the
C-band located at one telomere of each X chro-
mosome contains CMA3 positive, GC rich DNA
(Figure 2(c)). Banding pattern on X chromosomes
is always visible independently from the degree of
X chromosome condensation even if, in more
condensed metaphases, X chromatin results gen-
erally heavily stained in respect to the autosome
one (Figure 2(e)).
The CMA3 positive X telomeres were also
argentophilic after AgNO3 staining (Figure 2(g))
and brightly fluorescent after FISH experiments
carried out using a 28S rDNA as a probe
(Fig. 2(h)), thus demonstrating that these telo-
meres are the NORs, containing actively tran-
scribed rDNA genes. A variable heteromorphism
between homologous NORs was observed with all
the technique utilised (Figure 2(c, g and h)) and,
occasionally, plates in which the rDNA genes
result fully concentrated on only one of the X
chromosomes were observed (Figure 2(i)).
In addition to the NORs, silver staining also
revealed the presence of axial structures, running
parallel along the chromatid axes, without point of
intersection, as expected by the holocentric nature
of aphid chromosomes (Figure 2(g)). This peculiar
feature is highlighted in late metaphase plates in
which the chromatids do not form the classical V
shaped figures usually observed during the move-
ment of monocentric chromosomes (Figure 2(f)).
Discussion
The foremost result evidenced in this paper
regards the X restricted localization of all
heterochromatic bands to A. pomi X chromo-
some. A preferential localization of C-positive
94