© 2006 The Japan Mendel Society Cytologia 71(2): 189–196, 2006
An Analysis of Chromosomes in the Uzi Fly, Blepharipa zebina Walker (Diptera: Tachinidae) Using C- and Q-banding
Kanganahalli N. Venkatachalapathy and Hosagavi P. Puttaraju*
Department of Sericulture, Bangalore University, Jnana Bharathi, Bangalore-560056, India
Received March 8, 2006; accepted May 20, 2006
Summary Investigations on mitotic and meiotic metaphase chromosomes from brain, testes and ovaries of the uzi fly, Blepharipa zebina a tachinid endoparasite of the non-mulberry silkworm, An- theraea mylitta (Lepidoptera: Saturnidae) larvae are presented. The karyotype has 5 pairs of sub- metacentric chromosomes and are arranged in a graded series. All the chromosomal pairs are homo- morphic in both male and female. The karyotype of this species is compared with those of other cy- tologically known Dipterans, which exhibit heteromorphic chromosomal pairs. None of the pairs ex- hibited complete heterochromotic nature to mark the difference between sex chromosomes and auto- somes when C- and Q-banding technique was applied. This is in contrast to the karyotype of another uzi fly, E. sorbillans a tachinid endoparasite of mulberry silkworm, Bombyx mori and other cyclor- raphan insects.
Key words Blepharipa zebina, Exorista sorbillans, Antheraea mylitta, Sex chromosomes, C- and Q-banding, Heterochromatin.
Uzi flies belong to the family Tachinidae of the order Diptera, are of the most important among the insect pests, which attack the silkworm species. There are 4 species of uzi flies are known today viz., the Japanese uzi fly, Crossocosmia sericaria (Rodani); the Hime uzi fly, Ctenophora pavida (Meigen); the Tasar uzi fly, Blepharipa zebina (Walker) and the Indian uzi fly, Exorista sorbillans (Wiedemann). The former 2 lay micro type and later 2 lay macro type eggs on their host silkworm species. These pests are known to cause a great damage to sericulture industry. Because of their impact of parasitism at their maggot stage on silkworm, the uzi flies have been a subject of investigators for quite some time. Among the 4 species, the Tasar and Indian uziflies have been attracting quite a number of researchers mainly because, they causes a great loss to respective non mulberry and mulberry sericulture industry which are known to provide a gainful employment to weaker sections and earns high foreign exchange in many developing countries including India. Earliar Puttaraju and his group from this laboratory have provided detailed information on cytology (1992, 1995, 1997, 2000), embryology (Manjunatha 1993, 1996), morphology (Manjunatha 1993, Venkatachalapathy 2002) and control measures (1995a, b, 2005) (Narase Gowda 2002) for the Indi- an uzi fly. In this paper an attempt has been to provide some cytological information on the Tasar uzi fly. Blepharipa zebina (Walker), the tasar uzi fly belongs to the order-Diptera, family—Tachinidae, subfamily—Goniinae and Tribe—Sturminii attacks the tasar silkworms Anthereae mylitta and A. proyeli. However, in respect of the nomelclature of this fly appears to be a great deal of confusion. Deleiado and Hardy (1977) have synchronised, the tasar uzifly Blepharipa zebina (Walker) as Ble- pharipa sericariae (Rondoni), B. indica Brauer and Bergentamm, Crossocosmia indica Brauer and
* Corresponding author, e-mail: [email protected] 190 K. N. Venkatachalapathy and H. P. Puttaraju Cytologia 71(2)
Bergenstamm and Crossocosmia zebina (Walker). However, Crosskey (1976) and Joseph et al. (1983) have synonymized B. zebina as Sturmia sericariae. But Crossocosmia sericariae ( Sturmia sericariae) is reported to lay eggs on the host food plant of Bombyx mori i.e., mulberry, Morus spp. (Sasaki 1886, Anonymous 1972) and B. zebina, the tasar uzi fly, the subject insect of the present work is known to lay eggs directly on the host larvae, Antheraea mylitta and A. proyeli (Jolly et al. 1974, Patil and Savanurmath 1989b). Moreover, Drs. K. M. Harris and I. M. White of Common- wealth Institute of Entomology, Department of C.A.B., International Institute of Entomology, Lon- don (personal communication) have identified this insect as Blepharipa zebina. Therefore, the same nomenclature is used for the tasar uzi fly in the present work. It is well known that the application of differential chromosome banding technique demon- strates in understanding the taxonomic relationship of a large number of closely related species and helps in identifying the status of several doubtful species. Differential banding patterns are being in- creasingly used for identifications of the human and other mammalian chromosomes. In insects these studies are restricted to grasshoppers (Beermann 1977, King and John 1980, Channaveerappa and Ranganath 1992, 1993), Drosophila (Holmquist 1975, Gatti et al. 1977, Leumenier et al. 1978), Mosquitoes (Motara and Rai 1978) and some Calyptrate insects (Bedo 1980, 1987, 1991, Kaul et al. 1978, 1989). This restriction is mainly because these insects have longer chromosomes, which facilitated to study the banding pattern in detail. John and King (1977b) have clearly demonstrated the difference in C-band pattern between the races of Australian grasshopper, Cryptobothrus chrysophorus. Similar studies are also available on some insects and animals (Pimpinelli et al. 1976, Yosida and Sagai 1975). The present investigations analyses the chromosomes in B. zebina based on the karyotype, and application of differential banding technique.
Materials and methods Sources of materials and maintenance The strains of tasar uzi fly, Blepharipa zebina were collected from the uzi infested tasar silk- worm, Antheraeae mylitta larvae/cocoons which were brought from 2 different states of India viz., Basic seed multiplication and training centre (BSM & TC), Bastar in Madhya Pradesh and BSM & TC, Nowrangpur in Orissa. The uzi maggots as and when they pierced out of the host body, were collected and stored in cages of 30 30 30 cm fitted with wire mesh where in they metamorphose into pupae and then to adults. The emerged flies were provided with cotton balls wetted with 8% glucose and maintained for further rearing as described by Shivashankarappa (1997) for the Indian uzi fly. One to 2 days old fifth insart larvae of tasar silkworm, Antherareae mylitta; hosts of B. zebina were kept inside the cages separately to stimulate the uzi flies for oviposition on their body. The in- fested tasar silkworm larvae were taken out from the cages for further rearing following the stan- dard method of silkworm rearing technique (Jolly 1987). The young uzi maggots on hatching bore into the body of larvae and complete their endoparasitic mode of life (Datta and Mukherjee 1978a). The maggots of different instars were removed by dissecting the larvae at appropriate time and were used for present investigation.
Preparation of chromosomes Mitotic and meiotic chromosomes were prepared following the technique described by Manju- natha and Puttaraju (1992) with slight modification by increasing concentration of KCl. About 0.1 ml of 0.025% colchicine was injected into the body of healthy third instar maggots, pupae and adults. After 30 min, the injected maggots, pupae and adults were dissected for brain ganglia, testes and ovaries respectively. These tissues were treated with hypotonic solution of 0.60% KCl, for 30 min and this was followed by fixation in Carnoy’s solution (1 part acetic acid: 3 parts 2006 An Analysis of Chromosomes in the Uzi Fly 191 methanol) for 30 min. Then the tissues were transferred on to a clean glass slide and minced in a drop of 60% acetic acid. These slides were dried on a hot plate at a surface temperature of 45°C for 15 min. The dried slides were stained with 2% Giemsa followed by thorough washing in distilled water.
C-banding The method of Sumner et al. (1972) was adopted with modification in duration and tempera- ture of incubation in 2 SSC for induction of C-bands. The fresh unstained dried slides were warmed over a hot plate at surface temperature of 50°C. These slides were treated in 0.2 N HCl at room temperature for 20 min. Slides were thoroughly washed in distilled water. The washed slides were incubated in couplin jar containing 5% Barium 1 Hydroxide, (Ba(OH)2), kept in water bath at constant temperature of 60°C for 4 to 4 /2 min. These incubated slides were washed in distilled water and rinsed in 0.1 N HCl followed by one more wash in distilled water. These slides were further washed thoroughly for few minutes in tap water fol- lowed by another wash in distilled water. The slides were incubated in 2 SSC (Double Standard Saline Citrate—0.88 g sodium citrate and 1.75 g of sodium chloride dissolved in 100 ml of distilled water), for 90 to 120 min at 60°C. After this treatment, the slides were rinsed twice in distilled water and stained in diluted 2% Giemsa for 30 min.
Quinacrine staining Quinacrine (Q-banding) staining was carried out using a similar method to that of Caspersson et al. (1969a). The dried slides were stained for 15 to 20 min in a 0.5% quinacrine mustard. After staining, the slides were washed 3 times in MacIlvaine’s phosphate-citrate buffer, pH 4.1, and then mounted in the same buffer under a sealed coverslip for fluroscence observation.
Observations, photography and morphometric measurements Observations were made with “LEITZ” Research Microscope. Microphotographs were made in ‘WILD’ and ‘LEITZ’ Research Microscope with ‘WILD’ camera regulated by MPS-45 Photoau- tomat. All prints were made following the conventional methods. Well spread chromosomal pho- tomicrographs were enlarged and measurements were made using the dial calipers. The LR values (Relative length) were calculated following the method of Laven et al. (1964) and the ideograms were constructed by the conventional method.
Results Metaphase chromosomes The result of the cytological studies includes analysis of metaphase chromosomes and C- and Q-banding techniques in B. zebina are presented here. Metaphase plates prepared from somatic and meiotic cells from male and female of B. zebina revealed diploidy 2n 10. All the 5 pairs of chromosomes were submetacentric and were arranged in a graded series length wise. There was no sign of heterogamety in the males of B. zebina because all the 5 chromosomal pairs were homomorphic in nature. The total length of chromosomal complements ranged from 67.54 to 86.36 mm with an average 75.56 mm. The karyotype has been illustrated in Figs. 1–3. The details of analysis are shown in Table 1, Fig. 4. Chromosomal pair I was shorter than the other chromosomal pairs but the arm ratio of 1.50 was found to be comparatively higher than the other chromosomal pairs. Chromosomal pair II was longer than pair I, but the arm ratio of 1.36 was found to be comparatively lower than the pair I. Pair III and IV had lower arm ratio of 1.29 and 1.22 respectively compared to other pairs. These chromosomal arms showed a slight overlap between them. Since they differed in length, no 192 K. N. Venkatachalapathy and H. P. Puttaraju Cytologia 71(2)
Table 1. Average chromosome analysis of Blepharipa zebina
Chromosome Total Length of each Average % of total Chromo measurement length Arm ratio Centromeric chromosome length of complemental somes on drawing of pair (SD) index (SD) pair (mm) length (SD) mm (100 10) (mm)
I 2.74 0.81 1.81 5.36 0.260 10.75 5.37 14.54 0.166 1.50 0.612 34.10 2.75 0.79 1.85 5.39 0.269 II 3.35 0.78 2.49 6.62 0.591 13.27 6.63 17.95 0.721 1.36 0.318 37.48 3.40 0.77 2.48 6.65 0.572 III 3.83 0.81 2.71 7.35 0.571 14.86 7.43 20.10 0.260 1.34 0.423 37.20 3.77 0.81 2.93 7.51 0.382 IV 4.40 0.79 3.04 8.23 0.160 16.46 8.23 22.26 0.710 1.43 0.172 37.18 4.38 0.77 3.08 8.23 0.272 V 5.01 0.81 3.47 9.29 0.392 18.58 9.29 25.13 0.561 1.44 0.731 37.41 5.03 0.80 3.50 9.33 0.671
III V I V IV V II III II IV I IV III I IV II I II V III 12 56 25
20 I I II III 15 II II IV IV
% of TCL % of III 10 V III 5 V IV I V 34IIIIII IV V 78 Figs. 1–4. Male meiotic metaphase-I. (1), Female meiot- Figs. 5–8. C-banded chromosomal complement from ic metaphase-I. (2), Somatic metaphase. (3), testes. (5), C-banded chromosomal comple- Idiogram. (4). ment from ovary. (6), Q-banded chromosomal complement from testes. (7) Q-banded chro- mosomal complement from testes (8).
appreciable difficulty was encountered in distinguishing them. Pair V was found to be longer in length relatively to other pairs. Its arm ratio of 1.36 was found to be similar to that of pair II. The average arm ratio of pair I to V was 1.41 both in male and female. The centromeric index (Ic) was found more or less of same in all the chromosome pairs from I to V and averaged 36.67. The present study on the karyotype of B. zebina further revealed that all the chromosomes were biarmed with submetacentric in nature. Unlike in other Cyclorraphan insects, with an excep- tion of few species of Muscidae, the characteristic feature of absence of heterogamity in male poses problem in identification/differentiation between X and Y chromosome. Therefore C-banding tech- nique was applied to know the morphological characterisation of these sex chromosomes and the results are presented here under.
C-banding pattern in metaphase chromosomes Application of C-banding to study the chromosomal complements in population of B. zebina collected from Bastar and Nowrangpur revealed indistinct C-banding in both male and female. All the 5 pairs of chromosomes in male revealed small paracentric C-bands and in the short arm of V 2006 An Analysis of Chromosomes in the Uzi Fly 193
Table 2. Comparison of the present somatic chromosomes complement of Blepharipa zebina with those of other members of the family tachinidae studied earlier by boyes and wilkes (1953)
Sex chromosomes
YI (X) Si Avg. Avg. TCL Species No. arm ratio (mm) I–VI % TCL Arm ratio % TCL Arm ratio
1 Aplomya caesar 3.5 1.00 11.3 1.68 1.70 57.7 2 Aplomya mitis 4.0 1.25 9.1 1.51 1.72 45.5 3 Blepharipa zebina*———— 1.41 37.0 4 Ceracia dentata 5.6 1.25 6.2 2.61 1.53 52.7 5 Ceromasia auricaudata 4.4 1.22 6.0 1.67 1.23 54.8 6 Drino bohemica 3.8 1.21 7.4 2.09 1.22 63.7 7 Eumea wesermanni ——5.9 2.17 1.28 50.8 8 Exorista bombycis** 3.7 2.11 3.1 — 1.43 68.5 9 Lydella grisescens 18.4 1.24 19.8 1.25 1.22 69.8 10 Madremyia saundersii 4.3 1.00 12.9 1.43 1.22 52.8 11 Mericia 3.6 — 7.0 1.12 1.35 42.7 12 Nemorilla pyste ——13.0 1.18 1.21 55.4 13 Neophorocera hamata 3.4 1.10 4.8 1.41 1.60 68.3 14 Omotoma fumiferance 8.7 2.25 10.9 3.97 1.35 64.3 15 Phryxe pecosensis 4.1 1.13 6.6 2.60 1.17 48.9 16 Spathimeigenia sp. 4.4 1.17 6.0 2.80 1.61 53.8 17 Winthemia datanae ——31.0 1.13 1.34 54.0 18 Winthemia occidentis 6.0 2.88 16.2 7.53 1.25 64.0
TCL, Total complement length. * Present study. ** Manjunatha and Puttaraju (1992b).
chromosome the constitutive heterochromatin is placed interstecially (Fig. 5). Howevewr, these bands were not constant in all the chromosomal pairs of both male and female individuals. In case of female, all chromosomal pairs has not displayed discrete C-positive bands. The C-bands were not distinguishable between 2 homologues of any pair. Hence, here also the distinction between X and Y chromosome was found difficult. Further there was no chromosomal pair found completely hete- rochromatic in nature as in other cyclorraphan insects.
Q-banding Q-banding studies were made only in male meiotic cells of both the populations (Figs. 7, 8) es- pecially to trace out the variation between X and Y-chromosomes. Q-banding revealed fluorescence in all the 5 pairs of chromosomes without any prominent bands. Further, there were no brighter bands throughout on any of the chromosomal complements which otherwise would have con- tributed for distinction of X and Y-chromosomes.
Discussion Karyotype analysis of 30 well spread metaphase plates from mitotic and meiotic cells of B. ze- bina revealed that, the diploid chromosomal number was 2n 10. Among the Calyptrate, B. zebina is one of the few species that does not confirm to the general rule of 2n 12. All the 5 pairs of chro- mosomes were submetacentric. There was no sign of heterogamity in the males of B. zebina, be- cause all the 5 chromosomal pairs were homomorphic in nature. The arrangement of chromosomes in the present study was in an ascending order in number, with the objective of comparing the kary- otypes of this taxon with those of other higher Dipterans including Tachinids (Manjunatha and Put- 194 K. N. Venkatachalapathy and H. P. Puttaraju Cytologia 71(2) taraju 1992, Boyes and Wilkes 1953), Muscids (Boyes et al. 1964), Sarcophagids (Boyes 1953) and Calliphorids (Boyes 1961). The comparison was possible with reference to the nature of the auto- somes and sex chromosomes. Interestingly in all the cytologically known species of the aforesaid families, the usual diploid number is 12 with a deviation in some species of Muscids, wherein the chromosome number was reduced to 10. In 1 species of Sarcophagid, Pseudosarcophag affinis, the somatic chromosome number is stated to be 19 or 20. P. a f finis is the only species having the highest number of chromo- somes and all being acrocentric except 1 pair which is submetacentric i.e. 9th pair (Boyes 1953). A karyotype with all acrocentric chromosomes is generally considered as a karyotype of an ancestral form. Based on this, the karyotype of P. a f finis can be considered as an ancestral one though there is a submetacentric pair, which may be a secondary feature (i.e. pericentric inversion). Since the diploid number is 19 or 20 in P. a f finis, a more generalised hypothetical karyotype may be con- ceived. So this hypothetical form may be having 20 acrocentric chromosomes, which may be the progenitor of other species with chromosomal structural changes. The nature of the autosomes in most of the cases supports this view, as they are either metacentric or submetacentric or a combina- tion of both. Such chromosomes can only be derived by translocations. Even the sex chromosomes in general are acrocentric in nature with larger X and smaller Y in tachinids. As an exception to this “general rule”, 2 species of Tachinidae showed some variations (Manjunatha and Puttaraju 1992, Boyes and Wilkes 1953). Further, Boyes and Wilkes (1953) re- ported that the X-chromosomes of Winthemia sp. is larger than the largest autosome and therefore suggested to categories it under a separate group. Manjunatha and Puttaraju (1992) demonstrated that the Y-chromosome of Exorista bombycis was larger than the X-chromosome, which is unique in this respect. But in the present study, B. zebina has no heteromorphic pair in any of the chromo- somal complement of male analysed and no clear variation in the number of chromosomal pairs (Table 2). Therefore, B. zebina is one of the few Calyptrate Dipteran, that the sex chromosomes ‘XY’ became so useless that they were lost and been inferred that they left sex determinations capa- bility under the control of autosomal pairs as suggested by Boyes (1961) and Avancini and Weinzierl (1994). This loss of sex determination role of XY resulted perhaps in a karyotype as 2n 10 and making B. zebina as an exception with a few other allied species, to the general rule of 2n 12 in all most all other Calypterates. Therefore, its inclusions in the family Tachinidae have been engimatic. The variations in the chromosomal number to that of other Calypterate are of great signifi- cance in the evolutionary cytogenetics. Further, absence of heteromorphic chromosomal pair of any complement in male seems to indicate that they have an indepedent evolutionary history.
C-banding C-banding is widely believed to stain constitutive heterochromatin in both plants and animals. But Kaul et al. (1978, 1989) in P. misera does not showed discrete C-banding in autosomes. Similar observations were recorded in the present study and revealed no discrete blocks of stained constitu- tive heterochromatin at the centromeric regions in the chromosomal pairs of all the observed metaphase complements. In this context characterization of C-banding in B. zebina may be species specific. This notion is suggested by Greilhuber and Speta (1976) in case of the Scilla hohenekeri group. However, the present findings of C-banding in B. zebina is in contrary to the C-banding studies of other allied species like E. sorbillans (Venkatachalapathy and Puttaraju 2000), Muscina stabu- lans (Parise et al. 1996), Parasarcophaga albiceps, P. orchidea, P. argyostoma, P. ruficornis and P. knabi (Kaul et al. 1978, 1989). The sex chromosomes of several members of the allied species viz., Chrysomia bezziana (Ul- lerich 1976), Lucilia cuprina (Bedo 1980), species of Parasarcophaga (Kaul et al. 1978, 1989), 2006 An Analysis of Chromosomes in the Uzi Fly 195
Musca domestica (EL Agose et al. 1992) and E. sorbillans (Venkatachalapathy and Puttaraju 2000) were found to be completely heterochromatic or one of its counter part showed completely or par- tially C-possitive in nature. However, in present study we could not identify the sex chromosomes by applying C-banding. Addendum to this no chromosomal pair or homologue was found with C- banding positive in B. zebina in contrary with the above and reinforces the observations of Parise et al. (1996) in case of Muscina stabulans, which does not possess a completely heterochromatic chromosomal pair or a homologue or part of it. According to them (l.c) in M. stabulans the loss of at least part of sexual chromosomal material, that is the constitutive heterochromatin, which could not be identified in the chromosomal, set anymore. Therefore, independent of the mechanisms in- volved in the diminishing of 1 chromosomal pair seems appropriate to conclude that, it is accompa- nied by loss of most of C-banded material present in the sexual chromosomes.
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