KARYOLOGICAL STUDIES ON URUGUAYAN SPIDERS
I. BANDING PATTERN IN CHROMOSOMES OF LYCOSA SPECIES (ARANEAE-LYCOSIDAE)
N.BRUM-ZORRILLA & A. POSTIGLIONI
Division Citogenetica, Instituto de Investigaciones Biologicas 'Clemente Estable', Av. Italia 3318, Montevideo,
Uruguay
Received October 30, 1979/Accepted April 15, 1980
C-, G- and Q-banding patterns of mitotic and meiotic
chromosomes of Lycosa malitiosa Tullgren, Lycosa
thorelli Keyserling and Lycosa sp. were studied.
Small C-bands are localized in the centromeric re-
gions of the three species. Brightly fluorescent
blocks were detected with Hoechst 33258 (0.5
ttg/ml) stain, in mitotic chromosomes (centrome-
ric regions). In only some chromosomes of one spe-
cies, Lycosa sp. positive fluorescence was found at
their telomeric regions. G-banding is consistently
positive in L. thorelli. The chromomere pattern ofbi-
valents at pachytene and differential distribution in
the three species are discussed.
Introduction
The techniques for karyological research developed
in the last few years have permitted a more accurate
knowledge of spider karyotypes (Brum-Zorrilla &
Cazenave, 1974; Matsumoto, 1977; Postiglioni &
Brum-Zorrilla, 1977).
Giemsa stain has been used to reveal a variety of
banding pattems in eukaryotic organisms. However
this methodological approach has rarely been applied
to spider chromosomes. The interest in performing
studies in this field has been enhanced by recent re-
ports including for instance the one by Benavente &
Wettstein (1977), who studied the sex-determining
mechanism in Lycosa malitiosa, at the ultrastructural
level. Similarly the extensive ecological, ethological
and systematic research carried out by Capocasale &
Costa (1975) and Costa (1975) on Uruguayan Lyco-
sidae spiders stimulated our interest to perform cy-
Genetica 54, 149-153 (1980). 0016-6707/80/0542-0149 $1.00.
© Dr. W. Junk B.V. Publishers,The Hague. Printed in The Netherlands.
togenetic studies in the same species investigated by
these authors, which results will be published in a se-
ries of papers. The first reports the organization of
heterochromatin in the chromosomes of three species
of the genus Lycosa." L. malitiosa TuUgren, L. thorelli
Keyserling, and Lycosa sp. The main purpose of this
work is to determine whether or not heterochromatin
is a discrete and constant component of the genome
of these species, and to recognize the chromosome
regions in which it is located.
Material and methods
Adult specimens (female and male), of the three spe-
cies of the genus Lycosa: Lycosa malitiosa TuUgren
(25 males), L. thorelli Keyserling (15 males and 10 fe-
males) and Lycosa sp. (18 males and 4 females) were
processed. Taxonomic determinations of these spec.
imens were made by the staff of the Division of Ex-
perimental Zoology of our Institute (IIBCE).
The material was collected in the field or raised in
laboratory cages. The females were injected with 0.1
ml. of a 0.04% colchicine solution. After an exposure
of 20 hours some drops of haemolymph were re-
moved from the tarsal articulations, and dispersed in
5 ml. of balanced saline solution (BSS) and centri-
fuged at 800 rpm for 10 minutes. Hypotonic treat-
ment (BSS/distiUed water 1:1) was performed for 20
minutes before inmersion in methanolacetic acid
(3:1). Testes were removed and fixed after trypsin
treatment following the procedure published by Drets
& StoU (1974). In other cases testicular tubules were
inmersed in hypotonic solution (BSS/distilled water,
149
1:1), for 30 minutes. The suspension of the fixed
cells (3:1) was placed into 60% acetic acid, centri-
fuged immediately and transferred to fresh fixative
for 10 minutes. Each drop of the cell suspension was
placed on a pre-cleaned slide dipped previously in
70% methanol and then flame.dried.
treatment of Sumner et al., (1971), yielded effective
results (Fig. 3). The chromosomes of the other spe-
cies treated under the same conditions showed some
dark bands which can not be considered as G-bands
(Fig. 4). This fact made it difficult to make a precise
identification of the chromosome pairs.

Staining method C-banding

In order to stain C-bands, the Arrighi & Hsu (1971) For the demonstration of C-bands the chromosome
procedure was used as follows: (1) 0.2 N HC1, 10 preparations were denatured in NaOH solution accor-
minutes; (2) NaOH in BSS (1:3), 1 minute; ( 3 ) 2 x ding to the method of Arrighi & Hsu (1971). It was
SSC, 10 minutes; (4) 2x SSC at 65°C overnight. seen that the denaturation with a solution of NaOH
Slides were then dried and stained for 20 minutes in balanced saline solution (BSS), 1:3 during 1 minute
in citrate-phosphate buffered Giemsa solution at pH is a prerequisite for the optimal staining of hetero-
6.8. Several G-banding methods (Seabright, 1971; chromatic regions. Following this procedure, small
Wang & Federoff, 1971) were carried out, but the on- pos_itive blocks were detected in the Centromeric re-
ly One that yielded successful results was the one pro-
posed by Sumner et al. (1971). Hoeehst 33258
(0.05/ag/ml. and 0.5 ttg/ml, in Hank's solution at pH
7) was used to detect fluorescent chromosome bands.
Examination of slides was carried out in a Zeiss pho-
tomicroscope fitted with epi-fluorescence condenser
III RS (excitation filter BG12, barrier filter 50) and
photographed on Tri-X-film. C and G banded prepara-
tions were observed under bright field and photo-
graphed on Kodak High Contrast Copy.

Results

Chromosome complement

An identical karyotype was found in the three species

studied (Lycosa malitiosa, L ycosa thorelli and Lycosa
sp.): 20 autosomes with an X;X2 sex-chromosome
constitution were found in males and 20 autosomes
plus X1 X1X2X2 in females. All chromosomes (auto-
somes and sex-chromosomes) are uniarmed with a
terminal centromere. At somatic metaphase, the auto-
somes and the X's cannot be distinguished. In both
sexes all chromosomes have similar sizes and shapes
(Figs. 1,2).
Figs. 1-6. Mitotic metaphases ofLycosa sp. (1,5,6, sperma-
G-banding togonia), L. thorelli (2, 9 haematocyte; 3, spermatogonium)
and L. malitiosa (4, spermatogonium): -(1-2 Giemsa;- (3-4)
G-banding; - (5) C-banding, note positively heteropycnotic
In spite of several G-banding methods being used XIX2; - (6) Hoechst 0.5 #g/ml, arrows indicate centro-
(Seabright, 1971; Wang & Federoff, 1971) only some meric and telomeric fluorescent blocks, - Bars represent 10
preparations of L. thorelli treated with the G-banding ~m.

150
gions of the autosomes of all species. Figure 5 illus-
trates a spermatogonial metaphase plate in which posi-
tive blocks can be seen. This particular plate was se-
lected on account of the X's presenting a strongly
positive heteropycnosis which does not occur in the
autosomes.
Hoechst 33258 staining
As stated in the preceding chapter, two concentra-
tions of Hoechst 33258 were used to stain the meta-
phase chromosomes of all three Lycosa species. It was
observed that the fluorescence pattern of the chromo-
somes changed with the concentration of the Hoechst
33258. At a low concentration (0.05/lg/ml) all chro-
mosomes showed an intense and homogeneous fluo-
rescence, but at a higher concentration (0.5 /~g/ml)
small fluorescent blocks were located in the centro-
meric regions. Lycosa sp. also shows telomeric
fluorescent blocks in some chromosomes of its
complement, with a higher concentration of the
dye (Fig. 6).
meres which appear irregularly distributed. In Lycosa
sp. (Fig. 8) coalescence between chromomeres was ob-
served. L. thorelli (Fig. 9) was the only species that
showed a large number of separate chromomeres,
with a regular distribution along the bivalents. When
the pachynema cells are stained with Hoechst 33258
(0.5 /ag/ml) bright fluorescent blocks were observed
(Fig. 10) corresponding to the positive chromomeres
observed with saline treatment.
C-bands were visible in meiotic cells using the
Arrighi & Hsu (1971 ) technique, under the same tech-
nical conditions as mentioned above for mitotic cells.
Positive, small, heterochromatic blocks located in the
centromeric regions were detected. Figure 11 illustra-
tes a diplotene cell with these small positive blocks.
After Hoechst 33258 staining (0.5 /~g/ml) non-
fluorescent bands in diplonema were found. At dia-
kinesis when the chromosomes are higllly condensed

Meiotic chromosomes

The meiotic process follows a similar pattern in the

three species. At early prophase both X's (XlX2)are
easily recognized by their positive heteropycnosis.
They are close together and remain that way after
migration to the same pole at the first meiotic divi-
sion. At diplotene each autosome bivalent has one
chiasma, but occasionally two chiasmata could be
observed. A more detailed study of the behaviour and
evolution of the sex chromosomes system with sta-
tistical analysis is reported in the next papers of this
series (Postiglioni & Brum-Zorrilla, 1980).

Meiotic chromosome banding

At pachynema the autosome bivalents of these spe-

cies are characterized by the presence of chromo-
meres whose number and size are varied. When the
slides are treated for 3 hours with 2x SSC at 60°C,
the two strands of the homologous pairs are clearly
recognizable and their chromomeres and interchromo-
mere spaces are symmetrical. The distribution of the
clusters of chromomeres varies with each species
showing a different pattern. In L. malitiosa (Fig. 7) the
treatment accentuates the staining of certain chromo-
Figs. 7-12. Pachytene (Figs. 7-10), diplotene (Fig. 11) and
diakinesis (Fig. 12) in species of Lyeosa: (7, 10) L. malitiosa
G-banding and Hoechst 0.5 ~g/ml, respectively; - (8, 11)
Lycosa sp., G-banding and C-banding, respectively, in (11)
one bivalent enlarged, arrows indicate positive C-bal~ds; -
(9, 12) L. thorelli, G-banding and Hoechst 0.5 t~g/ml,respec-
tively, homogeneous fluorescence of all chromosomes. Bars
represent 10 ~m.

151
only a homogeneous fluorescence is visible along the
autosome bivalents, while the X's display a bright
fluorescence along their entire length (Fig. 12).
Discussion
Chromosome banding
C-banding
The pattern of C-banding is similar to that reported
earlier in Lycosa malitiosa (Brum-Zorrilla & Cazenave,
1974) and Polybetes pithagoricus (Olivera, 1978). It
is interesting to point out that the chromosomes of
all spiders studied by us have a small amount ofhetero-
chromatin.
It has been postulated that while euchromatic re-
gions of the karyotype remain conservative during the
evolution of mammalian species, a marked change
could be produced in the heterochromatic regions by
deletion or addition of heterochromatin material (Hsu
& Arrighi, 1971; Pathak et al, 1973). It is possible
that the small amount detected by C-banding in the
species reported in this paper, could act as a barrier
preventing chromosome recombination in interspecif-
ic hybrids, thus preserving the stability of the karyo-
type through evolution.
One of the factors in relation to the production
of C-bands appears to be the formation and further
persistence of the chromatin structure in C-band
regions which provides sites of optimal dye binding
absent in other chromosome regions (Comings et
al, 1973; McKay, 1973). A selective loss of material
from other non-C-band regions may be an other im-
portant factor in determining the C-banding pattern
(Comings et al, 1975). In our observations a deconden-
sation of nonC-band regions was apparent after C-
banding treatment, suggesting that a loss of DNA
may occur. However, in our experiments it is very dif-
ficult to draw any definitive conclusion on the nature
of heterochromatin found with C.banding methods,
because there is, as yet, no information on the mole-
cular organization of spider chromosomes. It is neces-
sary to effect a biochemical investigation of DNA in
these species ha order to interpret the role of hetero-
chromatin in speciation.
Hoechst 33258 banding
With Hoechst 33258 banding no differential staining
can be observed in any of the three species studied
152
with low (0.05 /~g/ml) concentration of the dye.
However, at a higher concentration (0-5/ag/ml) small
fluorescent blocks appeared in the centromeric re-
gions, similar to positive C-bands. Telomeric fluores-
cent blocks were found in some chromosomes of the
complement of Lycosa sp. which were not revealed
with C-banding methods. This fact would suggest the
existence of more than one type of heterochromatin
in the chromosomes of these spiders. Gatti et al.
(1976) also found variations in the fluorescence
pattern of Drosophila chromosomes in accordance
with the concentration of Hoechst 33258, which they
ascribed to different types of heterochromatin. Fur-
ther research on other spider species and/or use of
other fluorochromes may help to clear this point.
G-banding
In spite of the fact that the experimental conditions
were similar in the three species studied, only Lycosa
thorelli showed consistent G-banding. G-bands have
been reported in the chromosomes of some inverte-
brates including Diptera species: e.g. Aedes albopinc.
tus (Steiniger & Mukherjee, 1975); Heteropeza pig-
maea (Fantes & Camenzind, 1975). In other groups,
such as the Orthoptera, reproducible G-bands were
only observed in supernumerary chromosomes (Gal-
lagher et al., 1973; Webb, 1976). Comparing these re-
suits it seems that the G-banding method does not
give consistent results in invertebrate chromosomes.
McKay (1973) postulated that a differential distribu-
tion of chromosome material can be involved in the
production of G-banding patterns. As different chro-
mosome reactions were found in the three species of
spiders studied it can be inferred that a certain hetero-
geneity exists in the distribution of chromosome ma-
terial. This inference is also supported by the differ-
ences in the chromomere patterns of the autosome
bivalents at pachynema. A correlation between G-
band and chromomere pattern was demonstrated for
mammalian chromosomes (Okada & Coming, 1974;
Pathak et al., 1976) and more recently for human
chromosomes (Hungerford & Hungerford, 1978).
The fact that Lycosa thorelli has shown G-banding
and a consistent chromomere pattern suggests that
the condensation of chromatin (chromomeres)
facilitates the detection of G-banding in these species.
We thank Prof. R. Caiaocasale and Prof. F. Costa from
the Division of Experimental Zoology of (IIBCE) for
providing the specimens and their systematic classifi-
cation, Dr. R. Sotelo for critical reading of the manus-
cript and Mrs. A.M. Cazenave for her excellent tech-
nical assistance.
This work was supported by the 'Programa de De-
sarrollo Cientffico y Tecnol6gico (OAS)' and Ministe-
rio de Educaci6n y Cultura, Montevideo, Uruguay.
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