Chromosoma (Berl.) 52, 27--35 (1975) �9 by Springer-Verlag 1975

Interphase Chromosome Arrangement in Anopheles atroparvus

G. Diaz 1 and K. R. Lewis 2

llstituto di Art~tomia Umbria, Universitk di Cagliari, Via Porcell 2, 09100 Cagliari, Italy and 2Botany School, South Parks Road, Oxford, OX1 31~A, England

Abstract. The arrangement of chromosomes in interphase nuclei of Anopheles atroparvus has been inferred from an analysis of: 1. The early stages of mitosis as seen following Quina- crine staining, and 2. The reversible effects on the chromatin pattern obtained following the treatment of living cells with various NaC1 solutions, and the following conclusions have been reached: (a) The chromatin is connected to the nuclear membrane, (b) Homologous chromo- somes show close side-by-side somatic pairing, (c) The long arms of the sex chromosomes form a fluorescent peripheral body, (d) The autosomes are strongly reflexed at the centromeres, (e) The autosomal ecntromeric regions are polarized towards the peripheral body, (f) The telomeric regions of all the autosomes are closely apposed. - - A ring-shaped pattern of inter- phase chromatin is constantly and reversibly induced by NaC1 0.15 to 0.18 M solutions. - - These relationships indicate a peripheral arrangement of the interphase somatic complement.- The distribution of the chromosomes in polytene nuclei and at the beginning of meiosis resembles that suggested above for somatic interphase cells. This distribution may apply more widely in the Diptera. (Summary see p. 34.)

Introduction

The following observations indicate t ha t the distr ibution of chromosomes in certain interphase nuclei, at least, is no t at r andom: 1. Somatic pairing between homologues (Kitani, 1964; Maguire, 1967; Halfer and Barigozzi, 1972; Fe ldman and Avivi, 1973). 2. Non- random distr ibution of heterologous chromosomes (Miller et al., 1963; Wagenaar , 1969). 3. Non- random distr ibution of induced chromosome aberrat ions (Sax, 1940; Evans, 1961; Fox, 1966; K u m a r and Nata- rajah, 1966). 4. Non- random distr ibution of somatic ehiasmata (German, 1964).

The ar rangement of chromosomes at interphase can be inferred, to some extent, f rom the pa t te rn observed during mitosis, especially its early stages. I t has also been studied by in vivo observations on the reversible changes induced in the pa t te rn of chromat in by variat ions in the ionic s t rength and p H of saline media (Ris and Mirsky, 1949 ; Anderson and Norris, 1960 ; Brasch and Setterfield, 1974).

Bo th approaches have been adopted in the present s tudy of the interphase a r rangement of chromosomes in Anopheles atroparvus. Ear ly stages of first meiotic prophase were also examined in order to compare the interphase arrange- ments which precede the two kinds of nuclear division.

The results are considered in relation to those obtained for other Diptera in an a t t empt to formulate a model of chromosome arrangement of genera] applicabil i ty to the group.

The somatic complement of Anopheles atroparvus (2n = 4 + X X / X Y ) consists of two pairs of metacentr ic autosomes and a pair of subtelocentric sex chromo-

28 G. Diaz and K. R. Lewis

somes the heterologous members of which differ only in the size of the sholt arm (Tiepolo et al., 1975). h te rphase nuclei of both males and females include a Feulgen-positive peripheral body (Frizzi, 1947).

Materials and Methods Brain ganglia, testes and salivary glands were removed from fourth instar larvae of a

strain reared in the Institute of Genetics, University of Cagliari. Brain ganglia: after dissection in 1% sodium citrate, some ganglia were transferred to

aqueous NaC1 solutions of different molar concentrations (NaC1 0.03, 0.07, 0.15, 0.18, 0.30 M) for 5 minutes and then gently squashed for observations under phase contrast. Others were fixed in methanol-acetic acid (3 : 1), transferred to 40 % acetic acid and squashed. After remo- val of coverslips, the specimens were stained with Quinacrine DC1 (2 mg/ml) in SSrensen's buffer at pH 6.8 for 10 minutes.

Testes and salivary glands, dissected in Shen's solution, were stained with acetic orcein, squashed and observed under a phase contrast microscope.

Results

Brain Ganglia: Arrangement o[ Chromosomes during Mitosis as Found by Quinacrine Fluorescence

The peripheral body of the resting nuclei of Anopheles atroparvus is strongly fluorescent and has the same appearance in males and females (Fig. 1). With the approach of mitosis, there is a gradual increase in distance between the more highly fluorescing parts of the peripheral body. The pattern thus produced (Fig. 1) is very similar to that seen at mctaphase in the long arms of the sex chromosomes which the body presumably represents (Figs. 7-9). This initial change in the peripheral body is not accompanied by modifications in the non- fluorescing chromatin. Subsequently, the latter shows a series of morphological changes in which three principal stages can be recognised: 1. The chromatin acquires the appearance of an interlaced rope, one end (proximal) of which is connected to the fluorescent body (Fig. 2). 2. The chromatin rope divides longi- tudinally into two long cords. This process proceeds from the proximal end to form a Y-shaped structm'e (Fig. 3) but stops at the distal end, giving rise to a ring of chromatin (Fig. 4). 3. Each of the two cords, in turn, uncoil into two filaments to give, in all, four filaments of chromatin which are joined to the fluo- rescent body at their proximal ends (Fig. 5). Later, these filaments, two-by-two, part from the fluorescent body, thus allowing the recognition of individual chromosomes (Fig. 6).

Following these changes, the four autosomes can be clearly seen to form two somatic pairs each of which is strongly reflexed at the centromere which also proves to be the point of attachment to the peripheral body. By this stage it is also clear that the fluorescent body largely represents the paired sex chromosomes (Fig. 7).

All the above changes occur during early prophase. Subsequently, the auto- somes straighten and, following contraction, the three groups of somatically paired chromosomes show two main arrangements: end-to-end (Fig. 8) and

Interphase Chromosome Arrangement in Anopheles 29

Fig. 11. The appearance of the fluorescent body in resting nuclei (upper arrow) and those committed to mitosis (lower arrow)

Figs. 2--6. Changes in nuclear morphology during early mitotic prophase. Fig. 2. A rope of chromatin is linked to the fluorescent body at one end (proximal), the other (distal) being free. Fig. 3. The rope of chromatin unwinds progressively from the proximal end to give a Y-shaped configuration (arrow). Fig. 4. The unwinding of chromatin stops at the distal end (arrow) to produce a ring of chromatin. Fig. 5. Further uncoiling of the two cords reveals four filaments of chromatin, all connected to the fluorescent body at their proximal ends. Residual attach- ments between the distal ends are also apparent (arrow). Fig. 6. The fluorescent body (sc), which represents the long arms of the X and Y, separates from the autosomes which are

sharply reflexed at the centromeres and form two distinguishable somatic pairs (a,a)

1 The bars represent 10 ~m.

30 G. Diaz and K, R. Lewis

Figs. 7--10. Mid-prophase to late anaphase. Fig. 7. Mid-prophase. The chromosomes are in close somatic pairing. The two pairs of autosomes (a,a) are polarized. The long arms of the sex chromosomes (sc) are already condensed, while the short arms are isochromatic with the auto- somes. Fig. 8. Late prophase. End-to-end configuration. Arrows indicate the three centro- meric regions. The long arms of the sex chromosomes (sc) terminate the chromosomal chain. Fig. 9. Late prophase. Radial configuration. Fig. 10. Late anaphase. The fluorescing telomeric regions (arrows) of the sex chromosomes are recognizable. Strong convergence of all centro- meres and a side-by-side approach of homologous chromosomes are seen in the nucleus on

the right

radial (Fig. 9). I n the end- to-end configurat ions the long arms of the sex chromo- somes are always located at one end of the chromosomal chain: they are never associated with the telomeres of the other chromosomes.

The d is rupt ion of these associations between heterologous chromosomes coincides with the par t ing of homologues at prometaphase. I n anaphase, however, a rapid coavergence of all centromeres and a side-by-side approach of homologous

chromosomes can be observed (Fig. 10).

Brain Ganglia: Salt-induced Patterns o/Chromatin

In terphase nuclei of Anopheles atroparvus show var ious reversible chromat in patterIls following t r ea tmen t with salt solutions at different concentrat ions .

Interphase Chromosome Arrangement in Anopheles 31

Figs. 11--15. Phase contrast micrographs of interphase nuclei, in vivo, showing changes in the pattern of chromatin, following variations in the salt concentration of the medium. All • 2, 400. Fig. 11. NaC1 0.0375 M: the peripheral body is condensed while the autosomal chromatin appears to be homogeneously distributed. Fig. 12a and b. NaC1 0.075 M: (a) the chromatin shows a reticulate pattern and (b) clear connections with the nuclear membrane. Fig. 13. NaC1 0.15 3/h a ring of ehromatin, enclosing the peripheral body, is apparent. Attachments of the chromatin to the nuclear membrane are also seen (arrows). Fig. 14. NaC1 0.18 M: the chromatin is again ring-shaped but shows fewer connections to the nuclear membrane. Fig. 15. NaC1 0.30 M: the autosomal chromatin is tightly packed, attached to the peripheral body

(arrow), but lacks visible connections with the nuclear membrane

At the lowest salt concentra t ion (NaC1 0.03 M) the autosomal chromat in appears to be uniformly dispersed, while the peripheral body remains ra ther condensed (Fig. 11). Wi th an increase in concentra t ion (NaC1 0.07 M) the auto- somal chromat in becomes reticulate and shows clear connections with the nuclear membrane (Fig. 12). At still higher concentra t ions (e.g. NaC1 0.15 to 0.18 M) a characterist ic circular a r rangement of autosoma] chromatin, enclosing the peri- pheral body, is obtained which is again associated with the nuclear membrane (Figs. 13 and 14). Finally, at the highest concentra t ion (NaC1 0.30 M), the auto- somal chromat in is closely packed and connected to the nuclear membrane only via its association with the peripheral body (Fig. 15).

32 G. Diaz and K. R. Lewis

Fig. 16. Early stage of male meiotic prophase I. A strong resemblance to polytene nuclei is apparent. Centric association is indicated by arrow. (a, autosomal arms; l and s, long and

short arms, respectively, of the pair of sex chromosomes)

Fig. 17. Salivary gland nucleus. Centrie association is indicated by arrow. (II L, I I 1~, I I I L, I I I R are the four autosomal arms). The short arms of the sex chromosomes cannot be distin-

guished

Testes and Salivary Glands: Comparison between Early Prophase I and Polytene Chromosomes

Male meiotic prophase I is characterized by an early stage in which the three homologous pairs are joined to each other in the region of the centromeres. This stage, with six chromosomal strands radiating from a single central region, is very similar to tha t observed in polytene salivary gland nuclei (Figs. 16 and 17), except that the lat ter show only five arms: the sixth, which represents the short arms of the sex chromosomes cannot be distinguished. Except in this regard, arm ratio estimates in the two cell types gave similar results.

Discussion

Chmiags (1968) po,sed four questions in relation to the arrangement of chromo- somes in ia~erphase nuclei of higher organisms: 1. "Is the chromatia at tached to the nuclear membrane ? 2. Are the areas of a t tachment to the nuclear membrane located at specific sites on the chromatin ? 3. Are the sibes of chromatin attach- men t placed at specific locations on the nuclear membrane ? 4. Is the chromatin arranged in an orderly fashion in the intel~phase nucleus ?"

This s tudy a t tempts to answer the first and last of these questions with regard to interphase nuclei of Anopheles atroparvus.

The NaC1 0.07 M-induced pat tern of ehromatin provides clear evidence for connections between the ehromatin and the nuclear membrane analogous to those described by various other workers (Gall, 1963; DuPraw, 1965; Beams and Mu~ller, 1970; Comings and Okada, 1970; Maul, 1970).

The condensation of chromatin, reversibly induced by saline media (l~is and Mirsky, 1949) has been considered by Swanson (1957) as evidence for the integrity of the chromosomes in resting nuclei. In A.atroparvus, NaC1 0.15 to 0.18 M con- sistently induces a reversible ring-shaped pat tern of chromatin in resting nuclei.

Interphase Chromosome Arrangement in Ano2~heles 33

Since this is the only orderly pat tern obtained following treatment with various concentrations of saline, it can be taken as an indication of a circular arrangement of chromosomes during interphase.

The earliest stages of the mitotic sequences in Diptera are not emenable to investigation by classical staining methods and they are not described in most previously published accounts. These stages can, however, be studied by Quina- crine fluorescence and, being closer in time to interphase, they should reflect more accurately the arrangement of the chromosomes in resting nuclei.

However, it would be unwise to ascribe to interphase all the chromosomal relations found in early prophase, even if, as is commonly held (Wagenaar, i969), the arrangement of chromosomes does not change during interphase itself. For example, the intensity of somatic pairing might be affected by the degree of chromosome condensation and could, therefore, be different between interphase and prophase. Consequently, the changes in chromosome arrangement which characterise prophase itself are not relevant to a s tudy of the interphase arrange- ment. Those which follow anaphase, on the other hand, are significant because these largely determine the chromosome pat tern in resting nuclei.

Thus, from our findings, the following conclusions can be drawn concerning the arrangement of chromosomes in somatic interphase nuclei of A. atroparvus: a) The chromatin is connected to the nuclear membrane, b) Homologous chromo- somes are closely aligned, side-by-side, c) Each pair of homologous autosomes is sharply reflexed at the centromere, d) The certtromeric regions of both pairs of autosomes are polarized towards the peripheral body, e) The telomeric regions of all the autosomes arc associated, and f) The peripheral body is formed by the long arms of the sex chromosomes.

This arrangement implies a spherical, peripheral disposition of chromosomes which is consistent with the ring-shaped, salt-induced pat tern of chromatin observed in living cells. I t is also in agreement with the arrangement of interphase chromosomes inferred from studies on induced breakage. Thus Sax (19r in Tradescctntia, found that over 80% of radiation-induced exchanges between chromosomes and chromatids occurred at loci which were equidistant from their respective eentromeres. Further, the results of Evans (1961) and Fox (1966) on radiation-induced interchanges, in Vicia ]aba, and locust embryos, respectively, indicate a high polarization of chromosomes in interphase. Furthermore, the ratio of dieentrie to ring chromosomes per cell, obtained by Kumar and Natarajan (1966) following irradiation in barley, was such as to exclude a random arrange- ment of the arms of a chromosome. But the results were explicable on the basis of inter-arm proximity for each chromosome and strong centric polarization. In addition, proximity of homologous chromosomes has often been reported as an essential element of the interphase state (German, 1964; Kitani, 1964; Comings, 1968; Wagenaar, 1969; u and Yamaguchi, 1973).

In A. atroparvus there is also a close similarity between salivary gland chromo- somes and those at early meiotic prophase I. The chromosome arrangement observed in these different and highly differentiated cells is comparable to that inferred for somatic interphase. This suggests that cell differentiation does not involve gross changes in the interphase chromosome arrangement.

3 Chromosoma (Berl.), Bd. 52

34 G. Diaz and K. 1%. Lewis

Cytogenet ic d a t a on var ious o ther D i p t e r i a n species a re in accord wi th the in te rphase chromosome a r r angemen t p roposed here. Moens (1973), for example , who s tud ied the ax ia l cores of the~syaap tonemM complex in b lack f ly spe rmato- cytes , found an associa t ion of te lomeres wi th een t romeres a t the chromocent re . Fu r the r , Nov i t sk i (1964), in an a l t e rna t ive hypo thes i s to d i s t r i bu t ive pa i r ing , pos tu la t e s t h a t a eentr ic assoc ia t ion of al l chromosomes precedes meiosis in D. melanogaster. Moreover, the chromosome " b o u q u e t " a r rangement , found in the ea r ly meiot ic s tages of some D i p t e r i a n species, corresponds, accord ing to W h i t e (1954), to the adhes ion of he te rochromat i c segments in s a l i v a r y g l and nuclei . Wagen~a r (1969) emphas izes t h a t the p r o x i m i t y of the ecn t romercs dur ing in t e rphase is a common occurrence which m a y p rec ip i t a t e t he p roduc t i on of ehromocent res in po ly t ene nuclei . Likewise, Vosa (1970) recognizes the p rox imi ty of p rocen t r i c segments in in te rphase nucle i of D. melanogaster. The chromosomes of Drosophila hydei also show centr ic assoc ia t ion and v e r y f requen t adhes ion of te lomer ic regions in po ly t ene nucle i (Berendes and Meyer, 1968).

The foregoing observa t ions re la t ing to d i f ferent cell t y p e s in var ious d i p t e r a n species suggest t h a t the in te rphase a r r a n g e m e n t of chromosomes p roposed for Anopheles atroparvus m a y a p p l y more wide ly in th is class of insects .

Summary The following studies have been made on Anopheles atroparvus ( 2 n = 4 - ~ X X / X u

1. The mitotic cycle in brain ganglia as revealed by Quinacrine staining. 2. The effects of NaC1 solutions on the distribution of chromatin in living interphase nuclei as seen by phase contrast microscopy, and 3. The arrangement of chromosomes in polytcne and early meiotic nuclei following acetic orcein staining.

The observations indicate that interphase chromosomes assume a peripheral distribution which involves : - - a) Connections with the nuclear membrane, b) Close somatic pairing of all chromosomes, c) Autosomal centric polarization and telomeric association, and d) The forma- tion of a peripheral body by the long arms of the sex chromosomes.

This interphase organization, which may apply more widely in Diptera, appears to obtain in pre-polytene and pre-meiotic nuclei also. This suggests that cell differentiation does not involve gross changes in the interphase arrangement of chromatin.

Acknowledgements. The authors thank Miss L. Picci for her skilful technical assistance and Prof. A. Riva for his helpful comments. The Anopheles atroparvus larvae were obtained through the courtesy of Prof. G. Frizzi, Chairman of the Institute of Genetics, Cagliari.

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Received 5Iay 26, 1975 / Accepted May 27, 1975 by H. Bauer Ready for press May 30, 1975

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