Drosophila simulans Lethal hybrid rescue mutation (Lhr) rescues ...

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RESEARCH ARTICLE

Drosophila simulans Lethal hybrid rescue mutation (Lhr) rescues inviable hybrids by restoring X chromosomal dosage compensation and causes fluctuating asymmetry of development R. N. CHATTERJEE*, P. CHATTERJEE, A. PAL and M. PAL-BHADRA Department of Zoology, University of Calcutta, 35 B. C. Road, Kolkata 700 019, India

Abstract The Drosophila simulans Lhr rescues lethal hybrids from the cross of D. melanogaster and D. simulans. We describe here, the phenotypes of Lhr dependent rescue hybrids and demonstrate the effects of Lhr on functional morphology of the salivary chromosomes in the hybrids. Our results reveal that the phenotypes of the ‘Lhr dependent rescued’ hybrids were largely dependent on the genetic background and the dominance in species and hybrids, and not on Lhr. Cytological examination reveal that while the salivary chromosome of ‘larval lethal’ male carrying melanogaster X chromosome was unusually thin and contracted, in ‘rescued’ hybrid males (Cmel Xmel Ysim ; Amel Asim ) the X chromosome showed typical pale staining, enlarged diameter and incorporated higher rate of 3 H-uridine in presence of one dose Lhr in the genome. In hybrid males carrying simulans X chromosome (Cmel Xsim Ymel ; Amel Asim ), enlarged width of the polytene X chromosome was noted in most of the nuclei, in Lhr background, and transcribed at higher rate than that of the single X chromosome of male. In hybrid females (both viable, e.g., Cmel Xmel Xsim ; Amel Asim and rescued, e.g., Cmel Xmel Xmel ; Amel Asim ), the functional morphology of the X chromosomes were comparable to that of diploid autosomes in presence of one dose of Lhr. In hybrid metafemales, (Cmel Xmel Xmel Xsim ; Amel Asim ), two dose of melanogaster X chromosomes and one dose of simulans X chromosome were transcribed almost at ‘female’ rate in hybrid genetic background in presence of one dose of Lhr. In rescued hybrid males, the melanogaster-derived X chromosome appeared to complete its replication faster than autosomes. These results together have been interpreted to have suggested that Lhr suppresses the lethality of hybrids by regulating functional activities of the X chromosome(s) for dosage compensation. [Chatterjee R. N., Chatterjee P., Pal A. and Pal-Bhadra M. 2007 Drosophila simulans Lethal hybrid rescue mutation (Lhr) rescues inviable hybrids by restoring X chromosomal dosage compensation and causes fluctuating asymmetry of development. J. Genet. 86, 203–215]

Introduction Genetic basis of hybrid inviability was investigated in Drosophila, using different genetic crosses and hybrid-rescue mutations (Hutter and Ashburner 1987; Hutter 1997; Orr and Irving 2000; Presgraves et al. 2003; Sawamura et al. 2004). There are large bodies of data that have been accumulated with hybrid lethality in the melanogaster species complex indicated that the hybrid females carrying melanogaster X chromosome and simulans X chromosome in presence of simulans egg cytoplasm are embryonic lethal (Hadorn 1961; see Sawamura et al. 1993a,b,c). On the other hand, hybrid males not carrying simulans X are larval lethal *For correspondence. E-mail: [email protected]ffmail.com.

(Sturtevant 1920; Sawamura et al. 1993a; Barbash et al. 2000). As homogametic F1 hybrids have two sets of complete haploid genome from both parental species, one X to one haploid set of autosomes is balanced. Heterogametic hybrids lack an X chromosome that is incompatible in hybrid genetic environment, since Y chromosome of either of these species does not involve inviability of hybrid males (Hutter et al. 1990; Orr et al. 1997). Earlier, Dobzhansky (1940) and Muller (1940) suggested that a group of complementary genes is involved in hybrid lethality. Recently, Barbash et al. (2000) claimed that the genetic factor(s) underlying hybrid inviability within D. melanogaster complex are mainly due to the D. melanogaster Hmr+ and D. simulans Lhr+ . Watanabe (1979) and Takamura and Watanabe (1980) noted that the Lethal hybrid rescue mutation (Lhr), of D.

Keywords. Lethal hybrid rescue gene; Drosophila melanogaster; D. simulans; Dosage compensation; polytene chromosome. Journal of Genetics, Vol. 86, No. 3, December 2007

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R. N. Chatterjee et al. simulans can rescue adult hybrids of the both sexes when crossed to D. melanogaster. This rescue mutation is located in the right arm of the second chromosome (2-95) (Watanabe 1979). Hutter and Ashburner (1987) discovered another X linked mutation, Hybrid male rescue (Hmr), mapped on 9D19E4 (31.84) site, of the D. melanogaster X chromosome. They noted that when the strain is crossed to its relatives D. simulans, D. mauritiana and D. sechellia, ‘rescued’ an otherwise lethal male hybrids. Sawamura et al. (1993a,b) identified Zygotic hybrid rescue (Zhr) gene of D. melanogaster and maternal hybrid rescue (mhr) gene of D. simulans that are responsible for hybrid lethality at different phase of development. The existence of the distinct sets of ‘rescue’ mutations in the population of the species complex lead us to suggest that two or more independent mechanisms of lethality are operated in melanogaster–simulans hybrids (Sawamura et al. 1993b; Barbash et al. 2000; Presgraves et al. 2003). Two of the rescue mutations have been shown to be loss of function mutation (Sawamura and Yamamoto 1993; Barbash et al. 2000, 2003). Lhr also acts as a loss of function mutation (Watanabe 1979). However, absence of Lhr phenotype in D. simulans has impeded the characterization and isolation of Lhr+ and therefore less is known about Lhr. Earlier Dobzhansky (1957); Lakhotia et al. (1981); Mutsuddi et al. (1984), have shown that the X chromosome of male is hyperactive in ‘viable’ hybrids of different species of Drosophila. On the other hand, there are some reports of improper dosage compensation in ‘lethal’ hybrids of Drosophila (Meer 1976; see Sawamura 1999). Hutter et al. (1990) noted that the salivary gland chromosomes of melanogaster–simulans ‘larval lethal’ male hybrids are unusually thin with the X chromosome especially contracted. An alteration of the sequence of DNA replication in interspecific hybrids of Drosophila was also reported by Meer (1976). However, so far our knowledge is concerned, very few attempts have been made to analyse and compare critically the functional activities of the X chromosome of ‘viable’ and ‘Lhr dependent’ rescued hybrids. In view of these reasons, an attempt has been made in this paper to perform cytological examinations of salivary gland X chromosomes of ‘viable’ and ‘Lhr dependent rescued’ hybrids. The phenotypes of Lhr dependent-rescued hybrids were also analysed to know the developmental defects of the hybrids caused by maternal /zygotic incompatibilities. Our results reveal that ‘Lhr’ has epistatic effect on X chromosomal activity pattern for restoring dosage compensation in hybrids and cause fluctuating, asymmetry of development in the hybrids.

Materials and methods

melanogaster, and (c) y2 mutant strain. We also used the same Oregon R stock used previously by Lee (1978 ) to measure viability in D. simulans female hybrids who found it to be strong biased against hybrid viability. Our results reveal that the Oregon R strain, maintained in our laboratory is not very different from the Oregon R stock used previously by Lee (1978). (ii) Three D. simulans strains, (a) Lhr (K18 strain), (b) yellow (y), and (c) wild type strain of D. simulans (California 0251.163 strain) were used for present investigation. Flies were raised on standard Drosophila food medium containing corn-meal – molasses – yeast – agar-agar. Propionic acid was added as a mold inhibitor. Adults and all developmental stages were reared at 24◦ ±1◦ C and 80% relative humidity, unless otherwise specified. All flies were kept in uncrowded condition and the culture medium was changed for every 10 to 15 days. All D. melanogaster marker mutations and aberrations are described in Lindsley and Zimm (1992). Genetic crosses

For all crosses, just eclosed virgin females were collected, checked for the sex under a binocular microscope, crossed to appropriate young males (4 to 5 days old) in 15 : 20 proportion in culture bottles. The flies were transferred to fresh food bottles after 4–5 days. After 3 to 4 such changes, the parental flies were discarded and F1 progenies (including larvae and pupae) were scored and examined under a dissecting binocular microscope for sex and markers. The crossing schemes in figure 1a–c were used to generate hybrids for present investigation. The cross of D. melanogaster and D. simulans usually produce sterile unisexual hybrids (Sturtevant 1920). However, when D. simulans Lhr males were crossed to D. melanogaster females, both males and females were generated more or less at equal frequency. These males were as vigorous as their sister although they were completely sterile. The reciprocal cross D. melanogaster) produces hy( D. simulans Lhr × brid males and females. ‘Partial’ hybrid females and metafemales were produced by crossing Lhr males to four to five day old C(1) RM, yv f := females of D. melanogaster. This cross yielded among others, ‘partial’ hybrid females (Cmel Xmel Xmel Y sim ; Amel A sim ) and hybrid metafemale (Cmel Xmel Xmel X sim ; Amel Amel ) (see figure 1c). It may be noted here that from this cross, the number of ‘viable’ adult females progenies were small (i.e. partially rescued hybrids). The melanogaster X chromosome bearing females (Cmel Xmel Xmel Y sim ; Amel A sim ) larvae were also identified by yellow colour mouth parts.

Fly stocks

Nomenclature

(i) The following D. melanogaster stocks were used, (a) Oregon R strain of D. melanogaster, maintained in our laboratory since 1956, (b) C(1)RM, yv f /O :=, XY of D.

Chromosomes from the melanogaster and simulans species are indicated by the subscript mel and sim respectively. The cytoplasm is abbreviated as ‘C’.

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Drosophila simulans Lethal hybrid rescue

Figure 1. Experimental protocol to generate ‘viable’ and ‘rescued’ hybrids between D. melanogaster and D. simulans. (a) D. melanogaster females crossed with D. simulans males (b) D. simulans females crossed with D. melanogaster males and (c) compound X D. melanogaster females crossed with D. simulans males. A bar represent X chromosome; J-shaped bar represent Y chromosome and V-shaped bar represent compound X. The species origin and maternal cytoplasm of the two species are indicated by the boxes. Shaded box represents maternally supplied cytoplasm from D. simulans. The rescue effect of Lhr mutation in an inviable hybrids is also shown. Preparation of the specimen for microscopy

Morphological measurements

Hybrid progenies that were collected from different crosses were either kept in a mixture of ethanol/glycerol (3:1 v/v) for some days or processed as necessary. For light microscopy, the flies were prepared as described by Szabad (1978) and mounted in DPX. The external terminalia and sex combs were examined under a compound microscope using X 10 ocular and X 40 objective lenses. For scanning electron microscopy, the flies were sputter coated with gold–palladium and viewed under 25 kV with a Hitachi S 530 microscope (Acharyya and Chatterjee 2002). Photomicrographs were taken whenever necessary.

The list of traits that were chosen for analysing the characters in hybrids is shown in table 1. The external nonsexual traits were tibia and wing length. The external sexual traits were the area of posterior lobe and genital arch. The testis length was used as the internal sexual counterpart. The testis was dissected out from each individual fly on a slide containing a drop of Drosophila Ringer’s solution (Ashburner 1989). The genital arches were flattened by covering them with an 18 mm2 coverslip and heating the slide until the solution is boiled. Testis were transferred and stretched in a drop of glycerol medium. An Olympus camera (Camedia C-5060)

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R. N. Chatterjee et al. Table 1. Morphological traits used in present investigations. Traits

Sexual

External

Genital arch area Sex comb tooth number Vaginal tooth number

Internal

Testis length

Nonsexual Tibia length Wing length

10 min (sp. activity 17.500 mCi/mM and 18,500 mCi/mM respectively). Cytological preparations of the pulse-labeled glands were made as described above. Cover glasses were removed by freezing on dry ice or by immersing in acetic acid: ethanol (1:1) mixture. Slides were processed for autoradiography following usual methods (see Chatterjee and Mukherjee 1981). The exposure time was 10 days.

Results connected to Olympus Zoom microscope and a compaq computer were used to capture images of the different preparations. Image analysis software of Olympus was used in image capturing and for measurements of different morphological traits. Measurements were taken by using either a straight or free hand, mouse controlled-line selection. Tibia length was measured as in Long and Singh (1995). The posterior lobe of the genital arch was demarcated by a line across its base, and its area was assessed (see Coyne 1983). Testis length was measured as Civetta and Singh (1998). Data were obtained in pixel and then converted into millimeters by scaling with micrometer. Measurements were taken from both sides of a trait where necessary and the mean was used as an individual score. Analysis of dominance and asymmetry

D. melanogaster, D. simulans and their respective interspecific hybrids were assessed for levels of dominance and asymmetry of sexual and nonsexual internal and external morphological traits. Tibia length, wing length, the area of the posterior lobe of genital arch and testis lengths were analysed. Some times the two sides of internal traits could not be distinguished as left or right and therefore, all comparisons of trait asymmetry were made by using the absolute difference between sides of a trait. Three replicate measurements of each side of a trait were taken. Cytological preparation of chromosomes and autoradiography

For cytological preparations of chromosomes, well nourished third instar larvae or prepupae as appeared appropriate were dissected out by hand using buffered Ringer solution at pH 7.2 (Ashburner 1989). Thereafter, the salivary glands were fixed in aceto-ethanol (1:3 v/v) and stained in aceto-orcein for 10 min and washed in 50% acetic acid and squashed on a drop of lacto-aceto-orcein for temporary preparation. Autoradiographic procedure

In vivo transcription of polytene chromosomes were made by conventional autoradiography as described by Chatterjee and Ghosh (1985), using 3 H-uridine or 3 H-thymidine, obtained from Bhabha Atomic Research Center, Trombay, Mumbai. The excised glands were incubated in 10 µl of Drosophila Ringer containing 4uCi 3 H-UR or 3 H-TdR for

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Morphological studies and Lhr effect

When morphological phenotypes of male’s external terminalia of D. melanogaster and D. simulans were compared considerable differences in shape and size of genital arch, posterior lobe and bristle patterns were noted (figure 2a,b). There was a higher variation of sexual traits than nonsexual traits between two species (tables 2 and 3). Generally, nonsexual traits were higher in mean values for D. melanogaster than D. simulans, for e.g., the posterior lobe area showed lower mean values in D. melanogaster, while sexual traits were higher mean values in D. simulans (table 2). On the other hand, the female external terminalia of D. melanogaster and D. simulans were morphologically very similar (figure 2c,d). As reported earlier (Watanabe 1979), morphologically D. simulans Lhr males and females were comparable to D. simulans Lhr+ males and females. In fact, Lhr has almost no influence in the phenotypes of sexual and nonsexual traits (figure not included). Hybrid viability and Lhr

As reported earlier (Hutter et al. 1990; Orr et al. 1997), so here, the hybrid males from the cross of D. melanogaster females and D. simulans males X chromosome (Cmel Xmel Y sim ; Amel A sim ) are larval lethal (table 4). These larvae exhibited delayed larval development, very sluggish in nature, followed by death at late larval or prepupal stages. In reciprocal cross, nearly 33% hatched eggs were unable to reach at larval or prepupal stage of development (table 4). Of the sample of 237 hatched eggs, only 158 eggs developed into adults. When D. melanogaster females were crossed to D. simulans males carrying Lhr, nearly 86% of the hybrid progenies were survived up to adult stages. Of the sample of 465 hatched eggs 396 flies were emerged. The rescued males from the cross were also as vigor as their sisters. It, therefore, appears that Lhr can suppress the larval male lethality caused by presence of only melanogaster X chromosome in the hybrid genome. In reciprocal cross (i.e., when Lhr strain of simulans females were used) some hybrid females (escapers) were produced (only 14% of total progeny). When attached-X melanogaster females (XX/Y yv f :=) were crossed to normal simulans males, normally hybrid males were generated. Very few hybrid females were also recovered from the cross. They never

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Drosophila simulans Lethal hybrid rescue

Figure 2. Photomicrographs showing the terminalia of male and female Drosophila (a) the terminalia of a D. melanogaster male, (b) the terminalia of a D. simulans male, (c) the terminalia of a female D. melanogaster, (d) the terminalia of a female D. simulans. Note the posterior lobe area of D. simulans male. ap, anal plate; ga, genital arch; pl, posterior lobe; vp, vaginal plate; lp, lateral plate; p, penis apparatus; cl, clasper.

Table 2. Morphological trait values of the male D. melanogaster, D. simulans and their hybrids. Mean area of posterior lobe with S. E. (×10 mm2 )

Mean number of sex comb tooth with S. E.

Mean testis length with S. E.

Mean tibia length with S. E. (mm)

Mean wing length with S. E.

D. melanogaster Cmel Xmel Ymel ; Amel Amel

4.22 ± 0.08 (35)

9.89 ± 0.13 (35)

1.93 ± 0.03 (35)

0.46 ± 0.04 (35)

1.70 ± 0.35 (35)

D. simulans Csim Xsim Ysim ; Asim Asim ; Lhr+ /Lhr+

11.66 ± 0.06 (40)

10.79 ± 0.10 (40)

1.34 ± 0.02 (40)

0.44 ± 0.03 (40)

1.36 ± 0.22 (40)

D. simulans Csim Xsim Ysim ; Asim Asim ; Lhr/Lhr

11.73 ± 0.05 (30)

10.77 ± 0.08 (30)

1.43 ± 0.02 (30)

0.43 ± 0.05 (30)

1.43 ± 0.11 (30)

× sim mel Cmel Xsim Ymel ; Amel Asim ; +/Lhr+

7.78 ± 0.17 (30)

9.98 ± 0.13 (30)

1.18 ± 0.02 (30)

0.44 ± 0.05 (30)

1.55 ± 0.21 (30)

mel × sim Cmel Xmel Ysim ; Amel Asim ; +/Lhr

6.1 ± 0.23 (33)

9.23 ± 0.12 (33)

0.74 ± 0.09 (33)

0.44 ± 0.08 (33)

1.41 ± 0.23 (33)

× sim mel Cmel Xsim Ymel ; Amel Asim , +/Lhr

8.81 ± 0.11 (30)

10.28 ± 0.10 (30)

1.08 ± 0.02 (30)

0.48 ± 0.07 (30)

1.62 ± 0.22 (30)

Species and hybrids (with genotype)

*Significantly different from parental species. Numbers in parenthesis represent the number of observations

metamorphosed as adults. When simulans Lhr males were crossed to attached-X females of D. melanogaster, some females were ‘rescued’. Out of 231 adults, only 27 adult females were produced. They were ‘partial’ hybrid fe-

males (Cmel Xmel Xmel Y sim ; Amel A sim ), confirmed by cytological analysis (see figure 6b). A few larvae and/or pharate adults were also recovered from the cross. Of the sample of 231 hatched eggs three such larvae were recovered.

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R. N. Chatterjee et al. Table 3. Sexual phenotypes of the female D. melanogaster, D. simulans and their hybrids.

Genotype

Abdominal No. of tergite bristles observations T7 T8

Abdominal Range of sternite bristles vaginal tooth Mean tooth S6 S7 number per row no. with S. E.

Cmel Xmel Xmel ; Amel Amel Csim Xsim Xsim ; Asim Asim ; Lhr+ Csim Xsim Xsim ; Asim Asim ; Lhr Cmel Xmel Xsim ; Amel Asim ; Lhr+

25 26 25 25

(20–35) (22–40) (22–39) (25–38)

(8–10) (5–8) (7–9) (5–7)

13–16 11–13 11–16 11–14

14.7 ± 0.9 12.1 ± 0.7 12.1 ± 0.8 12.4 ± 0.7

Cmel Xmel Xsim ; Amel Asim ; Lhr Cmel Xmel Xmel Ysim ; Amel Asim ; Lhr

25 12

(20–38) (5–8) (13–20) (6–10) (18–32) (5–7) (12–17) (5–8)

11–13 8–12

15.0 ± 0.8 10.6 ± 0.7

(5–6) (6–8) (5–8) (6–7)

(18–22) (12–19) (14–21) (11–19)

Orientation of vaginal tooth one row one row one row one row, few are at different rows one row two rows, abnormal

Table 4. Analysis of lethal phase of hybrids at 24◦ ± 1◦ C.

Cross mel; +/ + × mel; + sim; Lhr + /Lhr + × sim; Lhr + /Lhr + mel; +/ + × sim; Lhr + /Lhr + sim; Lhr + /Lhr + × mel; +/+ mel +/ + × sim; Lhr/Lhr sim; Lhr/Lhr × mel; +/+ mel; +/+ (XX, y pr v/Y) × sim; Lhr + /Lhr+ mel; + (XX, y pr v/Y) × sim; Lhr/Lhr

Hatched eggs

Prepupae

891 789 365 259 495 309 293 342

758 (85.1) 653 (82.8) 237 (64.9) 162 (62.5) 465 (94.0) 236 (76.4) 183 (62.5) 276 (80.7)

Adults

Hybrid female

723 (81.1) 357 616 (78.1) 301 158 (66.2) 156 151 (58.3) 0 396 (80.0) 207 193 (62.4) 24 127 (43.3) 5∗ 231 (67.5) 27 + 3∗= 30

Hybrid male

Relative viability F/M

366 315 2 151 189 169 122 201

0.98 0.96 78 0 1.10 0.14 0.04 0.15

*Pharate adults; figures in the parentheses represent percentages; for prepupae and adults, the percentages were calculated by taking the number of hatched eggs as 100%

Chromosomal analysis of these individuals (see below) indicated that they were triplo X females (Cmel Xmel Xmel /X sim ; Amel A sim ) (see figure 6c). These females looked feeble and died predominantly as pharate adult or after eclosion or at pupal stage. These results together lead us to suggest that hybrid lethality due to presence of melanogaster X chromosome in the hybrid genome can be rescued by Lhr. This is true for both male and female.

hybrids, were compared with ‘Lhr dependent’ male hybrids, a clear difference in testes length was noted. In rescued hybrid males, the testis length was considerably reduced (see table 2). It, may imply that this internal sexual trait is also under the influence of

Hybrid morphology

In hybrids, the morphological traits (either sexual or nonsexual) were either dominant or additive. The tibia length, showed a consistent pattern of over dominance in hybrids than both parents (table 2). On the other hand, the genital arch area, and the number of male sex comb teeth show intermediate value in the hybrids between two parental species (see table 2). When the morphological phenotypes of male terminalia of ‘Lhr independent’ and ‘Lhr dependent’ hybrids were compared, a clear difference in the genital arch structure, posterior lobe area, and bristle patterns between two types of hybrids were noted (table 2; figure 3a,b). The sexual asymmetries were significantly higher in‘rescued’ hybrids than ‘viable hybrids’. Such a result may not be surprising, since hybrid incompatibilities are by definition, negative interactions between alleles at different loci. When the testes length of the ‘Lhr independent’ male 208

Figure 3. Examples of morphological phenotypes of the terminalia of the hybrids (a) a viable hybrid male, and (b) a rescued hybrid male. Symbols as in figure 2.

Journal of Genetics, Vol. 86, No. 3, December 2007

Drosophila simulans Lethal hybrid rescue sexual selection. The squash preparation of the testis further indicated that hybrid sterility was due to loss of capacity to produce functional sperms in the hybrids or a failure to initialize sperm from sperm bundle or any of the various problem occurring during spermatogenesis. Analogous situation was also noted in female hybrids. The morphological phenotypes of female hybrids were almost intermediate (figure 4a). However, in ‘partial’ hybrid females (Cmel Xmel Xmel Y sim ; Amel A sim ), morphological defects were extreme in external terminalia. The genital appendages of these females were asymmetric and abnormal (figure 4b–d). In some females, the genital structures were so reduced that only traces of female genitalia were noted (figure 4c). In some flies, the anal plates were present, however their shape resembles neither the wild type nor the normal female appearance. The shape of the vaginal plates were abnormal (figure 4c). The vaginal tooth were organized in 2 or 3 rows (table 3). In fact, our data reveal that the development of sexual traits were diverged in ‘partial’ hybrid females, due to presence of only melanogaster X chromosomes in the genome. Higher level of asymmetry of the size, development of the ovaries was also noted in the females. These data together clearly suggest that although Lhr can rescue the lethality of the female hybrids higher divergent coadaptive gene complexes are confronted more in the female hybrids due to presence of only melanogaster X chromosome in the hybrid genome.

Polytene X chromosome(s) of hybrids

When the salivary gland chromosomes were prepared from ‘larval lethal’ male hybrids (Cmel Xmel Y sim ; Amel A sim ), it was observed that as it was noted earlier (Hutter et al. 1990) the X chromosome was unusually thin and diffused in comparison to autosomes (figure 5a). However, when the salivary gland chromosomes were prepared from rescued hybrid males (Cmel Xmel Y sim ; Amel A sim ; Lhr), the morphology of the single X chromosome of males was typically pale stained and enlarged its width throughout its length (figure 5b). The cross of D. melanogaster attached-X females with D. simulans males generates hybrid males generally. These males (Cmel X sim Ymel ; Amel A sim ) displayed typically pale stained and enlarged diameter in comparison to that of autosomes. Curiously, the polytene X chromosome of the hybrid males, generated from the cross of melanogaster attached-X females with simulans-Lhr males, (see Materials and methods), was more puffier than that of the autosomes (see figure 5c). This observation may well indicate that Lhr can alter the organization of X chromosome of hybrid males. Taken together these data clearly suggest that Lhr may have some role in organization of the X chromosome in hybrids in hybrid genetic environment. Our data further reveal that the salivary gland Xchromosomes of ‘viable’ hybrid females (Cmel Xmel X sim ; Amel A sim ) were comparable with autosomes, with respect

Figure 4. (a) A hybrid female with genotype (Cmel Xmel Xsim ; Amel Asim ), (b–d) Partial hybrid female with genotype (Cmel Xmel Xmel Ysim ; Amel Asim ). Note the asymmetrical development pattern of the terminalia of ‘partial’ hybrid females. Symbols as in figure 2.

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R. N. Chatterjee et al. lar situation was also noticed in ‘viable’ hybrids. When the simulans X chromosome of the hybrid males were puffy in

Figure 5. Photomicrographs showing the salivary X chromosomes of hybrid males with different genotypes, (a) X chromosome of a ‘larval lethal’ hybrid male (Cmel Xmel Ysim ; Amel Asim ), (b) X chromosome of a rescued hybrid male carrying melanogaster X chromosome in Lhr background (Cmel Xmel Ysim ; Amel Asim ) and (c) X chromosome of hybrid male carrying simulans X chromosome in presence of one dose of Lhr (Cmel Xsim Ymel ; Amel Asim ). Note the enlarged width of the simulans X chromosome in Lhr background. Xmel , X chromosome from D. melanogaster; Xsim , X chromosome from D. simulans. Amel , autosome from D. melanogaster; Asim , autosome from D. simulans. Bar represents 10 µm.

to their staining intensity and width (figure 6a). The polytene X chromosomes of the ‘partial’ hybrid females (Cmel Xmel Xmel Y sim ; Amel A sim ) were also organized at ‘female’ level in presence of Lhr (figure 6b). In hybrid metafemales, two doses of melanogaster X chromosomes and one dose of simulans X chromosome (Cmel Xmel Xmel X sim ; Amel A sim ) can coexist in the hybrid genetic environment in presence of one dose of Lhr (figure 6c). The width of the X-chromosomes of the metafemales were 1.5 times than that of the diploid autosomes (see figure 6c). These data together suggest that in rescued hybrid females the X chromosomes were organized in ‘female’ level in presence of Lhr. 3

H-uridine autoradiography

Autoradiogram of the salivary gland chromosomes of ‘rescued’ hybrid males (Cmel Xmel Y sim : Amel A sim ) showed that the width of the X chromosome had direct bearing on the transcriptive activity pattern of the chromosome. As it appears from figure 7a, the melanogaster X chromosome was typically hyperactive in presence of Lhr (figure 7a). Simi210

Figure 6. Photomicrographs showing the salivary X chromosomes of females with different genotypes (a) Salivary X chromosomes of viable hybrid female (Cmel Xmel Xsim ; Amel Asim ) (b) salivary X chromosome of partial hybrid female (Cmel Xmel Xmel Ysim ; Amel Asim ) (Note the complete synapsis of the two melanogaster X chromosomes) and (c) salivary X chromosome of hybrid metafemale. Note the increased in width of the X chromosome (nearly 1.5 × than the width of diploid autosomes). Bar represents 10 µm. Symbols as in figure 5.

appearance, it was transcribed at a higher rate than that of the autosomes (figure 7b; table 5). It also appeared from the photomicrograph, presented in figure 7b that the 7AB and 9BC sites of the X chromosome incorporated increased rate of 3 H-uridine than the control. In hybrid females, the paired X chromosomes were transcribed at ‘female’ rate (figure 7c). Similar situation was also noted in ‘partial’ hybrid females in presence of one dose of Lhr in hybrid genome (figure 7d). Table 5 shows the comparative account of the 3 H-uridine labelling pattern of the X and autosomes of wild type strains of D. melanogaster, D. simulans and their hybrids. In order to estimate the relative transcriptive activity of the Xchromosome of both male and female, the number of silver grains over the 1A–6F segment of X chromosome and 21A–24F segment of the 2L were counted from the autoradiograms. The labelling patterns and the variability of the relative transcriptive activity (X frac./ 2L segment) of the X

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0.90 0.91 0.93 0.91 0.86 0.85 0.89 0.87 0.83 0.91 0.89 0.83 1.27 ± 0.09 1.25 ± 0.06 1.31 ± 0.02 1.27 ± 0.05 1.41 ± 0.09∗ 1.48 ± 0.07∗ 1.23 ± 0.03 1.25 ± 0.06 1.11 ± 0.06∗ 1.18 ± 0.04 1.21 ± 0.05 1.49 ± 0.06∗ 136.98 ± 9.37 99.82 ± 5.28 147.80 ± 8.26 147.60 ± 17.32 139.91 ± 7.28 191.39 ± 10.95 170.79 ± 10.16 93.71 ± 8.04 120.25 ± 6.02 175.24 ± 8.37 151.45 ± 10.35 226.29 ± 14.20 173.50 ± 15.03 124.70 ± 6.47 197.10 ± 11.14 187.50 ± 27.92 197.30 ± 7.28 287.20 ± 14.50 130.90 ± 11.30 116.20 ± 8.04 132.50 ± 8.28 206.50 ± 11.60 178.69 ± 17.35 336.90 ± 22.40 *Significantly different from control male and female at 5% level.

21 20 20 12 17 11 22 20 11 19 23 33 Cmel Xmel Xmel ; Amel Amel , +/+ (D. melanogaster, female) Csim Xsim Xsim ; Asim Asim , Lhr+ /Lhr+ (D. simulans, female) Cmel Xmel Xsim ; Amel Asim , (mel–sim hybrid female) Cmel Xmel Xmel ; Amel Asim , +/Lhr (partial hybrid female) Cmel Xmel Xmel Xmel ; Amel Amel , +/+ (metafemale) Cmel Xmel Xmel Xsim ; Amel Asim , +/Lhr (hybrid metafemale) Cmel Xmel Ymel ; Amel Amel , +/+ (D. melanogaster, male) Csim Xsim Ysim ; Asim Asim , +/+ (D. simulans, male) Cmel Xmel Ysim ; Amel Asim , +/+ (mel–sim, hybrid male) Cmel Xmel Ysim ; Amel Asim , Lhr/+ (rescued hybrid male) Cmel Xsim Ymel ; Amel Asim , +/Lhr+ Cmel Xsim Ymel ; Asim Amel , Lhr/+ (hybrid male with Lhr)

Correlation coefficient (r) Number of nuclei observed

X-chromosome fra. (1A–6F)

2L-chromosome (21A–24F)

X/A ratio with S. E.

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Species, genotype and sex

chromosome in wild-type strain of D. melanogaster and D. simulans were used as control. Data reveal that in all control set of experiments, the relative transcriptive activity as measured by X/A grain ratios was not very different from hybrid males and females (see table 4). In ‘rescued’ male hybrids, X chromosome was twice as active as the individual X chromosome of the female. The statistical analysis of the data also showed that there were positive correlation between the grain numbers on the X chromosome(s) to that of 2L of both the sexes of two species. A greater range of variation in the frequency density of X/A grain ratios in male hybrids carrying simulans X chromosome (Cmel X sim Ymel ; Amel A sim ), in presence of one dose of Lhr, was noted (table 5). In ‘partial’ hybrid females the two melanogaster X chromosomes were transcribed at ‘female’ rate in Lhr background. In hybrid metafemales, the mean X/A grain ratio was approximately 1.25 times than that of diploid females (i.e., partially dosage compensated; see table 5). These data together suggested that, hybrids Lhr plays an important role in regulating

Mean grain number with S. E.

Figure 7. Autoradiogram showing the 3 H-uridine incorporation patterns on the polytene X chromosome(s) of ’viable’ and ’rescued’ hybrid males and females, (a) a male nucleus with a genotype, Cmel Xmel Ysim ; Amel Asim , showing 3 H-uridine incorporation pattern on melanogaster X and autosomes in presnece of Lhr, (b) a male nucleus with a genotype Cmel Xsim Ymel ; Amel Asim , showing simulans X chromosome and autosomes in Lhr background, (c) a female nucleus with a genotype, Cmel , Xmel Xsim ; Amel Asim , showing 3 H-uridine incorporation pattern on X chromosomes and (d) a female nucleus with the genotype, Cmel Xmel Xmel Ysim ; Amel Asim , showing 3 H-uridine incorporation pattern on X chromosomes and autosomes in Lhr background. Symbols as in figure 5. Bar represents 10 µm.

Table 5. Data on the 3 H-uridine labelling pattern of the X-chromosome segment (1A–6F) and autosomal segment (21A–24F) of different genotypes of Drosophila.

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R. N. Chatterjee et al. able’ and ‘rescued’ hybrid males, the X chromosomes replicated their DNA in faster rate than autosomes (table 6). Unfortunately, due to poor-polytene chromosome morphology of ‘lethal hybrid’ male nuclei, no data could be scored from them.

Discussion

Figure 8. 3 H-TdR labelled autoradiograms of salivary chromosomes of female hybrids (a,b) and male hybrids (c,d). The labelling patterns of the hybrid female nuclei show the synchronous labelling pattern on both X chromosomes and autosomes (a,b). On the other hand, in both ‘viable’ and ‘rescued’ hybrids the Xmel and Xsim show labelling on very few regions (faster replication) while autosome(s) show very few unlabelled gaps (c,d). Xmel , The melanogaster derived X and Xsim , simulans derived X. Symbols as in figure 5.

transcriptive activity pattern of the X chromosome(s) in the hybrids, for dosage compensation. 3

H-thymidine autoradiography

Our data on 3 H-thymidine autoradiograms further reveal that in both ‘viable’ and ‘rescued’ female hybrids, there was no asynchronous pattern of 3 H-TdR labelling pattern on both X chromosome and autosomes (figure 8a,b). On the other hand, like parental species, in both viable and ‘rescued’ hybrid males, the labelling pattern on the X chromosome was faster and asynchronous in comparison to that of autosomes in all the terminal phase of DNA replication (figure 8c,d). This asynchronous labelling was due to faster rate of replication of the X chromosome of the hybrid males. Data presented in table 6 further provides evidences that labelling frequencies of X and 2R segment of these two species were more or less comparable. Both the homologue of the 2R chromosome derived from the two species also maintained their autonomy of replicating behaviour in hybrid females and males. The labelling frequencies on the X and 2R segments were also comparable in the hybrid females with the exception of few sites (e.g., 1A and 6A sites). Thus a conserved nature of replication behaviour of these two species was noted in hybrid genetic environment, despite of their evolutionary divergence. As parental species, in both ‘vi212

Previous work has revealed a good deal about the genetic basis of hybrid inviability of melanogaster–simulans hybrids (Wu and Palopoli 1994; Hollocher and Wu 1996; True et al. 1996; Ting et al. 1998). Viability patterns of the hybrids reveal that the gene(s) on the D. melanogaster X is incompatible with autosomal factor(s) of D. simulans (Sturtevant 1929; Hutter et al. 1990). Recently, it has been suggested that an autosomal gene, Lhr+ of D. simulans or Hmr+ of D. melanogaster are required for hybrid lethality and these factors are recessive and zygotically acting. The functions of these genes are to prevent gene flow between species (Sturtevant 1920; Hadorn 1961; Orr and Presgraves 2000). The results of analysis of the morphological phenotypes of the hybrids clearly demonstrated that both ‘viable’ and ‘rescued’ hybrid displayed intermediate on average and asymmetric phenotypes. This observation prompt us to suggest that in observed traits, the polygenic system is at work (i.e., each gene for the trait has a minor effect and shows additive). This may well indicate that many of the allelic fixations that occur in diverging subpopulations do not contribute to hybrid incompatibilities. However, some evidence of dysfunctional interactions that result abnormal development in external terminalia in ‘partial’ female hybrids was also noted. In partial hybrids, when a fraction of genome of only one species is included within hybrid cells, divergent coadapted gene complexes are confronted more in the hybrids and result in formation of morphological diversity, especially in external terminalia (Sanchez and Santamaria 1997). This may imply that the genetic basis of hybrid incompatibilities results in a stronger disruption of the sexual traits morphology in interspecific hybrids due to incompatible gene interaction. Raff and Kauffman (1983) noted here, that evolutionary changes in morphology of the two sibling species do not usually involve the generation of noval cell types but rather changes in morphogenesis i.e., in the process that are responsible for the generation of three-dimentional arrangements of various cell types. The testes length of ‘viable’ hybrids males were intermediate or reduced. In ‘rescued’ hybrid males, the trait showed less consistent pattern in Lhr background. In fact, the testis morphology of ‘rescued’ hybrid males was atrophied and asymmetric in nature. Squash preparations of the testis indicated that the defect in meiosis was common during spermatogenesis. The hybrids female were also sterile in phenotypes. The extreme developmental pleiotropy of oogenesis in D. melanogaster suggests that the loci responsible for hybrid female sterility between sibling species

Journal of Genetics, Vol. 86, No. 3, December 2007

Drosophila simulans Lethal hybrid rescue Table 6. Analysis of the frequency of 3 H-thymidine labelling on different sites of the X chromosome and 2R of D. melanogaster, D. simulans and melanogaster–simulans ‘viable’ and ‘rescued’ hybrids with Lhr. melanogaster–simulans-hybrids Male (Xs Ym ; Am As )

Male (Xm Ys ; Am As )

Female

Female

‘viable hybrid’

‘rescued hybrid’

(Xm Xs ; Am As )

0.59 0.13 0.17 0.74 0.26 0.36 0.20 0.21 0.31 0.08 0.26 0.41 0.22 0.14 0.31 0.76

0.86 0.24 0.41 0.93 0.48 0.61 0.61 0.48 0.79 0.19 0.57 0.85 0.50 0.36 0.88 1.00

0.41 0.08 0.14 0.72 0.41 0.43 0.21 0.23 0.39 0.09 0.28 0.45 0.17 0.11 0.31 0.69

0.48 0.07 0.17 0.66 0.37 0.49 0.25 0.21 0.30 0.10 0.30 0.41 0.22 0.09 0.38 0.79

0.95∗ 0.22 0.47 0.93 0.49 0.71 0.63 0.50 0.87∗ 0.29 0.65 0.90 0.57 0.42 0.84 1.00

0.99 0.97 0.25 0.79

1.00 0.93 0.30 0.86

0.97 0.95 0.28 0.81

0.94 0.92 0.27 0.79

0.98 0.96 0.37 0.83

Chromosomal

D. melanogaster

D. simulans

sites

Male

Female

Male

X chromosome 1A 1C 3A 3C 4A 4BC 5A 5D 6A 7D 8E 9A 9B 9D–F 10A 11A

0.51 0.06 0.14 0.79 0.20 0.40 0.27 0.20 0.48 0.12 0.31 0.43 0.20 0.10 0.31 0.87

0.89 0.20 0.37 0.94 0.41 0.68 0.68 0.50 0.86 0.25 0.60 0.74 0.47 0.48 0.75 0.98

Chromosome 2R 56F 58A 60A 60F

0.98 0.94 0.27 0.85

1.00 0.97 0.29 0.87

The labelling frequency of a site was determined by the number of times of the site was labelled among the 3D to 1D patterns of labelled nuclei examined (see Chatterjee and Mukherjee 1977). *Replicative behaviour was different in the two homologues of the hybrid female nuclei.

of this complex are not specific to oogenesis (Davis et al. 1994). This may imply that the failure of male and female germ cell development in the hybrids were due to the changes in the hybrid genome that have resulted changes in the coadaptive gene complexes responsible for soma-germ line interaction in these species following their divergent evolution after speciation. This may well indicate that signals from one genome and their receptors from another might no longer be normal in presence of Lhr. It is believed that some modifier genes are being functioned from their species-specific genetic control (Civetta and Singh 1998). In short, speciesspecific modifiers in sexually selected traits may hold the key for rapid evolution of such trait during speciation. Data presented in this paper also reveal that the Lhr mutation, mainly suppress the melanogaster X linked lethal genes and can allow to use an alternative metabolic pathway that steps aside the hybrid lethality. This view is greatly strengthened by the observations that the imaginal discs that were extremely underdeveloped in ‘lethal’ hybrids, can continue to develop in Lhr background (our unpublished data; Sanchez and Dubendofer 1983). Cytological data further reveal that the Lhr has epistatic

effect on X chromosomal activity pattern for restoring dosage compensation in the hybrids. The lethality interaction of melanogaster X chromosome in the hybrids males, were suppressed by Lhr and hyper transcriptive activity of the melanogaster X chromosome was restored. This view is strengthened by observation that melanogaster X bearing male also replicate its DNA in faster rate than autosomes (figure 8c,d). In ‘partial’ hybrid females also two melanogaster X chromosomes were transcribed at a ‘female’ rate in Lhr background. In metafemales two melanogaster X chromosomes and one simulans X chromosome were transcribed at ‘female’ level in hybrid cellular environment. Lhr also induced higher rate of transcription of the simulans male X chromosome in hybrid genetic background (see table 5). In this hybrid, the simulans X chromosome was nearly 1.30 times as much as puffy as the single X chromosome of male (Cmel X sim Ymel ; Amel A sim ) (see figure 7c). These data together clearly suggest that the epigenetic modification of the chromatin template activity of the X chromosome(s) of the hybrids was induced by Lhr. Observation of Coyne (1983) on the functions of melanogaster derived X chromosome (Xmel ) and simulans derived chromosome (X sim ) in presence

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R. N. Chatterjee et al. of one dose of Lhr in the development of genital process of melanogaster–simulans hybrid males may be relevant in this context. He noted that simulans derived X chromosome in the hybrid male contribute approximately twice as much as the genital area as that of melanogaster-X bearing hybrid males in presence of one dose of Lhr. Similar situation was also noted by Orr et al. (1997) who also showed that Lhr increases the size of somatic clones in hybrids. In summary, our results reveal that Lhr represents simply a ‘switch gene’ that can override the altered cellular environment of the hybrids due to deleterious interactions between maternal effects, sex chromosome and autosomes for reproductive isolation of this species. In particular, Lhr allows recovery of the deleterious effect of melanogaster X chromosome in the hybrids. The biochemical collapse of the melanogaster X chromosome(s) bearing hybrids, reversed by Lhr in hybrid genetic environment may indicate that Lhr prevents the death of Xmel Y sim /Xmel Xmel cells by remodelling of the X chromosome(s) in the hybrids for restoration of dosage compensation. In this context, it may be noted here that species specific transcription factor(s) and their binding sites including the tritation region will cause incompatibility between species. As DNA binding protein may play an important role in chromosome condensation and/or chromosome activation, it is predictable that a majority of genetic incompatibility may be results failure of transcription regulation of an entire X chromosome i.e., dosage compensation. Coyne and Orr (1998) suggested that inviability can be ‘rescued’ by single mutant implies that lethality probably has a fairly simple developmental basis and is therefore a simple genetic basis. We believe that Lhr may allow to modulate the X chromosome in hybrids for dosage compensation like other dosage compensation regulatory genes of D. melanogaster (see Baker et al. 1994). Further analysis of the Lhr+ gene at the molecular level should lead to finer understanding the role of the gene in reproductive isolation of the melanogaster species complex. Acknowledgements The works presented here have been supported by DBT research grant [BT/IS/04/001/2003 dt. 15. 1. 2004] for ‘Prof. S. P. Raychaudhuri, Drosophila Stock centre’. Some stocks were obtained from Blowling Green stock centre, USA.

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Received 12 September 2006

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