The Chromosomes of the Cymothoid Igopod
Aniloora.
By
H. G. Calan
With 3 Text-figures.
MATERIAL AND METHOD.
A n i l o c r a m e d i t e r r a n e a M. Edw. is a fish-louse belonging to the Cymothoid group of Isopods, found abundantly
at Naples. In common with other fish-lice, A n i l o c r a is a
protandrie hermaphrodite, descriptions of its sexual organization having been given by Bullar (1876) and Mayer (1879). For
a cytological study of this animal I took individuals while in
the predominantly male phase, removing the paired testes and
ovaries, and fixed them in La Cour's 2BD (1937) made up
in sea-water. The gonads were fixed for one hour, hardened in
1 per cent, chromic acid for twenty-four hours, washed in
running tap-water for half an hour, and then dehydrated and
imbedded in wax in the usual manner. Sections were cut at
15 fj. and stained by the gentian violet-iodine method (see La
Cour, 1937).
I examined mitoses in spermatogonia and oogonia and
meioses, first and second, in the male germ-cell line. Fixation
of the material was satisfactory for general purposes, although
not for great detail, the fault lying not in the technique employed
but probably in the high degree of hydration of the chromosomes, for other fixatives tried, such as strong Flemming and
Bouin in sea-water, all gave indifferent results. In general,
mitoses fixed rather better than meioses, especially the prophase
stages.
OBSERVATIONS AND DISCUSSION.
The diploid chromosome number is twelve, eight of the
chromosomes having median (M) and four subterminal (S)
centromeres. This is a low chromosome number for a crustacean. No particular interest attaches to the mitoses except
328
H. G. CALLAN
the variability of the staining reaction, a point to which
reference will be made later.
The configurations of the bivalents at first meiotic metaphase
show that there is extreme localization of chiasmata in this
§
o
3.
TEXT-FIG. 1, somewhat diagrammatic.
1. Side view, and 2. polar view, of first meiotie metaphase, normal type.
3. Side view of exceptional configurations of M-chromosomes at first meiotio
metaphase.
species. The M-chromosomes regularly form two chiasmata in
each arm, one immediately adjacent to the centromere, and one
at or near to the end of the arm (see Text-figs. 1 and 2). Four
exceptional conditions have been observed: (a) where in one
arm a proximal chiasma has failed, (b) where both proximal
chiasmata have failed, (c) where in one arm a distal chiasma
has failed, and (d) where both distal chiasmata have failed
(see Text-figs. 1, 3).
Out of 365 M-bivalents examined at metaphase, 350 were of
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CHROMOSOMES OF ANILOCBA
Association of M-chromosomes
Type
Xta
Nm-nmt
TEXT-FIG. 2. Diagram showing association of M-chromosomes.
Xta, chiasmata.
the normal type, four of type a, four of type b, five of type c,
and two of type d (see Test-fig. 2). The mean chiasma frequency
in these Mvalents is thus 3-94. The variance from the mean can be
calculated from the formula F =
1 F
2
(Ya;) 2 !
-1% (x )—x^-J- I
where
330
H. G. CALLAN
V = variance, n = the number of M-bivalents, and x = the
chiasma frequency per bivalent. This has a value of 0-087,
which represents 0-022 of the mean (see Table I). Values approaching this latter figure have only been found in organisms
with extreme localization of chiasmata ( F r i t i l l a r i a r u t h e n i c a , Darlington (1936), M e c o s t e t h u s g r o s s u s and
M e t r i o p t e r a b r a c h y p t e r a , Callan, unpub., from data
of White (1936)). Haldane (1931) has shown that, where pairing
N u m b e r of M - b i v a l e n t s
.
0
N u m b e r of chiasmata realized
1
6
2
9 350 Mean
3
4
3-94
Variance
0-087
TABLE I.
and chiasma-formation are normal, the value of variance approaches the mean, though never reaching this value on account
of interference. The variance in this particular case is thus
extremely low.
In order to analyse the factors responsible for producing such
invariability I have made a study of the proportions in which
the various types of association of the M-bivalents occur, with
a view to establishing possible correlations. A table is appended
(see Table II) showing the number of chiasma failures observed
as against the numbers to be expected, assuming absence of
correlation between the failures.
The calculation proceeds as follows:
In the case of the proximal chiasmata, out of 730 possible,
2 x 4 + 4 = 12 failed.
12
Thus the chance of a chiasma failing =
= 0-0164 = q.
7oO
Thus the chance of a chiasma being realized = 1—0-0164 =
0-9836 = p.
The chances of none, one, or two chiasmata failing are, therefore, as j?2:2j)g:g2respectively,i.e. as 0-9674:0-03234: 0-0002702.
The corresponding chances for the distal chiasmata failing
are 0-9755 : 0-02435 : 0-0001520.
In the table the chances of the various possible combinations
of chiasma failure are worked out by multiplication together
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CHROMOSOMES OP ANILOCBA
of their respective components, and then expressed as absolute
numbers out of the observed total of 365. The significant
squares are those where both proximal chiasmata have failed,
the expected number being 0-10 and the observed 4; and where
both distal chiasmata have failed, the expected number being
0-05 and the observed 2. There is thus evidence of strong
positive correlation in the faffing of the two proximal chiasmata,
Expected
j
j
Failure of proximal chiasmata
in M-oivalents
Failure of distal chiasmata in M-bivalents
Realized \
0
1
\ 344-45
0 i
|
350
8-60
2
11-51
0-10
4
j 0-29
5
0-0-5
4
0-00
0
0-00
0
0-00
2
2
0
0
TABLE IE.
M-bivaients, chromosomes having median centromeres.
and in the failing of the two distal chiasmata. An idea of the
relative strengths of these correlations can be obtained from the
formula
° 2 , where nQ is the number of bivalents where none,
% where one, and n2 where two have failed. When there is
complete positive correlation this has a value of 00; when there
is no correlation, of 1; and when there is complete negative
correlation, of 0. For the proximal chiasmata the value is 357
and for the distal chiasmata 115, showing that the former
correlation is the stronger.
In the case of the correlation between the failing of the two
proximal chiasmata, it would appear that proximity on the
chromosome of the two chiasma-forming regions when pairing
takes place is the factor of prime importance. When one region
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H. G. CALLAN
has successfully paired there is an increased likelihood for the
nearby regions to pair up also, in accordance with the 'zip'
principle. That there should be, however, a correlation between
the failing of the two distal chiasmata cannot be accounted for
in this way. One may presume that the two free ends of the
zygotene chromosome lie close together, so that if pairing is
effected with one of them, the probability is that the other will
manage to pair also. Clearly this second correlation indicates
that interference plays no part in the restriction of chiasmata
distribution down the length of the chromosome: limitation of
the initiation of pachytene pairing to the proximal and distal
regions and the positions taken up by the zygotene threads
within the nucleus are the major factors which secure such
invariability and localization of chiasmata as are found in
Anilocra.
The S-chromosomes form two chiasmata in the long arm, one
proximal and one distal, and no exceptions have been observed;
while in the short arm a chiasma may or may not occur. Often
in the same metaphase one of the S-bivalents has the centromeres close together and retained by a chiasma in the short
arms, while the other has the centromeres much wider apart
and the short arms retracted and not in contact. Such is the
position taken up later by the S-bivalents at early anaphase,
whether or not a chiasma has occurred in the short arms.
In the S-chromosomes there is therefore a corresponding invariability about the association of the long arms such as occurs
in the M-chromosomes.
The conclusions drawn from the metaphase configurations
cannot be substantiated from a study of diplotene, for the
fixation of this stage is very poor. However, the fact that a
'bouquet' is formed at pairing stages probably indicates loop
formation by the M-chromosomes, an orientation which was
inferred from the consideration of the correlation of chiasma
failures.
At anaphase the separation of the S-chromosomes precedes
that of the M-chromosomes, as is to be expected, since the short
arms offer less resistance to separation than do the long arms.
The M-chromosomes separate quite regularly and in time with
CHEOMOSOMES OF ANILOCEA
383
one another, thus providing circumstantial evidence that
identical conditions obtain in each one.
The extreme form of chiasma localization found i n A n i l o c r a
implies a rigidity in the recombination mechanism which is
possibly to be correlated with this animal's parasitic mode of
life, and it may be taken to indicate that an evolutionary standstill has been reached.
A further interesting feature of the meiotic chromosomes is
their staining reaction. At full metaphase the bivalents have
a bloated appearance and stain lightly; the centromere, however,
is intensely stained and apparently separated from the main
body of the chromosome by a non-staining region. At this stage
it is triangular in outline, showing tension. During anaphase
the stainability of the non-centric regions diminishes, irregular
deposits of the stain being laid down on the surface. The stage
at which this occurs varies greatly in individual cells. Eventually,
soon after the completion of separation, only the centromeres
remain stained and six are visible at either pole (see Text-figs.
2 and 3). The outlines of the chromosomes then show only by
virtue of a difference in their refractivity from that of the
cytoplasm. The centromeres are now rounded off, showing no
tension; they are slightly under I/A in size. For comparison,
it may be noted that the M-chromosomes at metaphase have
a length of 7jn and width of 2 /A.
The stainability of the mitotie chromosomes and of the
second meiotic chromosomes is also variable. In the majority
of mitoses the anaphase chromosomes remain stained, though
sometimes the centromeres only. In the majority of second
meioses, even at metaphase, the centromeres only are stained,
and in fact the chromosome material remains invisible until
late in the maturation of the sperm head. It can thus be seen
that there is a progressive tendency through the chromosome
cycle, from the somatic divisions to the secondary spermatocyte, for the chromosomes to be unstainable.
Variation in staining reaction given by the same stages of
division cannot be the result of differences in fixation through
the material, for the unstainable divisions are not distributed
through the tissue with any reference to the boundaries of the
834
H. G. CALLAN
latter. Furthermore, when there is a certain staining reaction
given by one chromosome, the same reaction is given by all the
remaining chromosomes in the division. The differential
staining reaction is probably the consequence of labile differences in the character of the chromosome surface (gentian
violet being a surface stain). This is perhaps dependent on local
differences in pH, which would account for the bad fixation of
2.
—"^
3.
TEXT-FIG. 3, somewhat diagrammatic.
1. Side view of early anaphase of first meiosis.
2. Side view, and 3. polar view, of late anaphase of first meiosis, outlines of unstained chromosomes indicated by dotted lines.
the chromosomes, related as it is to their degree of hydration.
It is not the result of a differential distribution of nucleic acid
between centromere and non-centric regions. I destained a
gentian violet preparation clearly showing the differential
staining, and then restained, using the Feulgen technique: no
differential staining resulted.
What relation the centromeres, as seen fixed and stained,
bear to their size and shape in life is a question of interpretation.
It might be argued that what can be stained is really the region
of the chromosome near to and including the centromere, not
the latter alone. However, its discreteness in stainability and
its similar size and shape in all the chromosomes suggests that
the artefact is at least an artefact of the centromere itself, and
CHROMOSOMES OF ANILOCEA
335
it is of interest to find that in A n i l o c r a there is this clear-cut
evidence of a distinction between the centric and non-centric
regions of the chromosome.
The fixations were made at Naples during my tenure of the
Oxford Biological Scholarship at the Stazione Zoologica for the
year 1938/9. The further work was carried out at the John
Innes Horticultural Institution, Merton Park. I am much
indebted to Dr. Mather for help with the statistical treatment
of chiasma-frequency, and to Dr. Darlington for many criticisms
and suggestions in the preparation of the argument.
SUMMARY.
A n i l o c r a m e d i t e r r a n e a has a diploid chromosome
number of twelve, eight of the chromosomes having median,
and four subterminal centromeres.
The localization of chiasmata is extreme and is of a previously
undescribed type, combining the terminal and centric systems.
The centromeres stain differentially from the non-centric
regions of the chromosomes; they are the only parts stained at
anaphase of first meiosis, and at metaphase and anaphase of
second meiosis.
BIBLIOGRAPHY
Bullar, J. F., 1876.—"Generative Organs of the Parasitic Isopoda",
' Jburn. Anat. Physiol.', 11.
Darlington, C. D., 1936.—"External Mechanics of the Chromosomes",
'Proc. Eoy. Soc.', B, 121.
Haldane, J. B. S., 1931.—"Cytological Basis of Genetical Interference",
'Cytologia', 3.
La Cour, L., 1937.—"Improvements in Plant Cytological Technique",
'Botanical Review', 5.
Mayer, P., 1879.—"Carcinologische Mittheilungen, 6. Hermaphroditismus
bei einigen Isopoden", 'Mitt. Zool. Stat. Neapel.', 1.
White, M. J. D., 1936.—"Chiasma localisation in Mecostethus grossus,
and Metrioptera .brachyptera (Orthoptera)", 'Zeits. Zellf. u. mikr.
Anat.', 24.