Structural map of the polytene chromosomes from the salivary glands of South American fruit fly Anastrepha fraterculus Wied (Diptera, Tephritidae)

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1 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN: (Print) (Online) Journal homepage: Structural map of the polytene chromosomes from the salivary glands of South American fruit fly Anastrepha fraterculus Wied (Diptera, Tephritidae) Giardini M. Cecilia, Fabián Milla & Fanny C. Manso To cite this article: Giardini M. Cecilia, Fabián Milla & Fanny C. Manso (2009) Structural map of the polytene chromosomes from the salivary glands of South American fruit fly Anastrepha fraterculus Wied (Diptera, Tephritidae), Caryologia, 62:3, To link to this article: Published online: 10 Feb Submit your article to this journal Article views: 101 View related articles Full Terms & Conditions of access and use can be found at

2 CARYOLOGIA Vol. 62, no. 3: , 2009 Structural map of the polytene chromosomes from the salivary glands of South American fruit fly Anastrepha fraterculus Wied (Diptera, Tephritidae) Giardini* M. Cecilia, Fabián Milla, Fanny C. Manso Laboratorio de Genética de Insectos de Importancia Económica-Instituto de Genética Ewald A. Favret, INTA, (1712) Castelar, Buenos Aires, Argentina. Phonr/Fax: int Abstract This work presents a preliminary map showing the polytenized autosomic chromosomes from the salivary glands of South American fruit flies Anastrepha fraterculus (Wied) (Diptera, Tephritidae) sampled in Argentina, where it is a serious pest of commercial fruits. We base their identification on constant morphological structures (landmarks) and features of each autosome. The chromosome number is 2n= 10+XX/XY, but due to the intimate pairing between the two homologues, the number of observed elements is reduced to half of the autosome number, in polytenized tissues. Besides widening the cytological knowledge of the species, this map will allow the identification of chromosome rearrangements and would help to elucidate the taxonomic identity of A. fraterculus from other geographic origins. Key words: Anastrepha fraterculus, polytene chromosomes, salivary glands, spatial map. INTRODUCTION *Corresponding author: mgiardini@cnia.inta.gov.ar Polytene chromosomes are very important for the analysis of numerous aspects of the organization of the chromosomes at interphase and of the genome in general (Zhimulev 2001). In addition, they are a system in which the differential genetic activity and its control can be analysed directly at the level of the genes themselves (Ashb u r n e r 1970). Polytene chromosomes are also an excellent tool to study phylogenetic relationships among closely related species and are used to distinguish members of a complex species group (Ga r c í a-ma rt i n e z et al. 2009). The formation of a polytene chromosome is associated with the elimination of the whole mechanism of mitosis after each DNA duplication. As a result, the cell cycle consists only of two periods: synthesis and inter-synthesis, at the end of which, the sister chromatids do not segregate, but remain paired one with each other in different degrees (Koltzoff 1934; Bauer 1935). The nuclear membrane and the nucleolus remain intact during the consecutive cycles of DNA replication (Zh i m u l e v 2001). The banding and interbanding pattern of each polytene chromosome is not only speciesspecific but also characteristic of that chromosome in that tissue and/or in that developmental state (Su m n e r 2003). The South American fruit fly Anastrepha fraterculus (Wied) (Diptera, Tephritidae) is an important pest for fruit production in Argentina (St o n e 1942). This species is native to the Americas and is distributed throughout the tropical and subtropical regions (between the latitudes 27ºN and 35ºS) (St e c k 1999). The chromosome number of A. fraterculus is 2n= 10+XX/XY (Me n d e s 1958). Many population studies have been carried out on its mitotic chromosomes (So l f e r i n i and Mo r g a n t e 1987; Basso and Ma n s o 1998; Basso et al. 2003; Sel i v o n et al. 2005a; 2005b). They have proved very useful because they allow the comparison of the members of each pair of homologues and the identification of differences between them, due to the somatic pairing characteristic of Diptera. These observations are not possible in meiotic metaphase because of the small size of the bivalents. In order to validate the results of the studies

3 polytene chromosomes in anastrepha fraterculus 205 with mitotic chromosomes, other studies should be carried out. In polytenized tissues the number of observed elements is reduced to half due to the intimate pairing between the two homologues. In the Mediterranean fruit fly Ceratitis capitata (Wied) (Diptera, Tephritidae) five polytene elements are found, corresponding to the five autosomes; both the X and Y chromosomes are heterochromatic and do not form banded polytene elements (Be d o 1986; 1987; Za c h a r o p o u l o u 1987; Gariou-Papalexiou et al. 2002). The same situation has been observed in many other tephritids such as the Mexican fruit fly Anastrepha ludens (Loew) (Ga r c í a-ma rt i n e z et al. 2009), the olive fruit fly Bactrocera oleae (Rossi) (Za m b e ta k i et al. 1999; Mav r a g a n i-tsipidou 2002), the melon fly Bactrocera cucurbitae (Coquillett) (Sh a j a h a n et al. 2002) and the Queensland fruit fly Bactrocera tryoni (Froggatt) (Zh a o et al. 1998). The karyotypic variability observed in populations of A. fraterculus (Wied) (Diptera, Tephritidae) has generated some controversy about its systematics. Some authors, evaluating different features such as karyotype (Basso and Ma n s o 1998; Basso et al. 2003), sexual behaviour and mating incompatibility (Pe t i t-ma rt y et al. 2004), highly repetitive DNA (Al b e rt i et al. 2002) and mitochondrial DNA (Alberti et al. 2008), consider any A. fraterculus having origin in Argentina, to be only one highly polymorphic species. Others researchers, studying populations from Brazil, define it as a complex formed by at least four species (Zu c c h i et al. 1998; Se l i v o n et al. 2005a; Go d ay et al. 2006). We present here a preliminary spatial map showing the polytenized autosomic chromosomes of A. fraterculus from Argentina. Spatial maps (Sh a r a k h o v et al. 2001) allow the identification of each of the five autosomes present in this species by means of useful and practical characteristic morphological markers. In addition to that, and based on this structural map, we made an approximation to the linear map of polytene chromosomes following the Br i d g e s (1935) labelling system. Materials and Methods Stocks - Larvae from two laboratory strains were used in approximately equivalent amounts to study polytene chromosomes: 294 and Lab-Tuc. Strain 294 comes from wild guavas collected in a private orchard of Ituzaingó, Buenos Aires, Argentina (34º 40 S, 58º 40 W) and it was maintained since 1996 in our laboratory. The individuals of Lab-Tuc line are originally a strain reared since 1997 in Estación Experimental Provincial Obispo Colombres, Tucumán, Argentina (26º 78 S, 65º 38 W) and held in our laboratory since The used larvae were late third-stage about to complete the cycle. Preparation and observation of polytene chromosomes - Salivary glands from A. fraterculus thirdstage larvae were removed and two procedures were followed in order to obtain slides. 1) As described by Yoon et al. (1973), the larvae were dissected in Ringer solution; the glands were hydrolyzed in acetic acid 45% for 1 min., and hydrolyzed in HCl for 30 sec. Then they were stained in warm lacto acetic orcein for 1 min. Excess of stain was removed before squashing by washing the glands twice in a drop of lactic acid: acetic acid 60% 1:1. The tissue was mounted and dispersed with a needle, sealed and examined with optic microscope. 2) As described by Ashburner (1989), the larvae were dissected in Ringer solution; the glands were hydrolyzed in acetic acid 45% for 1 min. and stained in lacto acetic orcein for min. Then the tissue was mounted and dispersed with a needle, sealed and examined with optic microscope. Image capture and analysis - Out of the 168 slides, 97 were further studied due to their better quality. Slides were observed with x100 magnification. Nuclei where chromosomes were well expanded were photographed and analysed without taking into consideration the position of the cell in the gland. Between 10 and 20 nuclei per slide were analyzed. The chromosome markers found were recorded and counted. Preparation of the spatial and linear maps - Once each polytenized autosomic chromosomes was identified, images were digitalized and then assembled using image processing software (PhotoShop CS). These were used to prepare the spatial map and the linear map later. The chromosomes were measured from the spatial map with Micro Measure 3.3 software. Results and Discussion No significant differences were observed between the two procedures used to obtain the slides. In general, the chromosomes length is not variable, unlike the thickness and the puffing pattern, which suffers modifications, since it is a dynamic system associated to the transcriptional activity of each chromosome segment. In A. fraterculus these

4 206 giardini, milla, manso patterns are influenced not only by the age of the individual but also by its maturation stage. In this species, we face the inconvenient of the lack of synchronization in the development, since it is possible to obtain flies emerging with approximately 10 days of difference from eggs collected at the same moment (Ma n s o 1998). As a way to overcome this problem we analysed larvae at the same developmental stage regardless of its age, obtaining a constant banding and puff patterns, which allowed the preparation and analysis of the maps presented in this work. We did not observe a typical chromocentre resulting this in the separation of the individual chromosomes (Figure 1). Moreover, the chromocentre has not been observed in trycogene cells (Be d o 1986) or salivary gland tissues (Za c h a r o p o u l o u 1987; Gariou-Papalexiou et al. 2002) of C. capitata, Culex pipiens (L.) (Diptera, Culicidae) (Za m- b e ta k i et al. 1998), A. ludens (Ga r c í a-ma rt i n e z et al. 2009), and many other dipteran species. We observed the presence of the nucleolus in the analysed cells. The nucleolus presented an oval shape with a dark circle in its centre (Fig- Fig. 1 A larval gland polytene nucleus showing the five polytene elements Ta b l e 1 Morphological markers (Landmarks) Chromosome II III IV V VI % of the total polytenized kariotype Landmarks Straight initial end (zone 1), banded zone and puff (zone18) Loop (zone 39) and discontinuity (limit zone 24 and zone 25) Ends (zone 41 and 60) and coiled zone (zones 51-56) Ends (zones 61 and 80) and curved zone (69-72) Loop (zones 87-98), fan (zone 81) and discontinuity (zone 98) Frequency (%) n=97

5 polytene chromosomes in anastrepha fraterculus 207 Fig. 2 Nucleolus. ure 2) in agreement with what was described by Zhimulev Description of chromosomes - In mitosis, autosomic chromosomes of A. fraterculus are acrocentric, although some submetacentric variants have also been found (Basso and Ma n s o 1998). The Polytene chromosomes were characterized following the Bridges labelling system (Br i d g e s 1935). Chromosomes were labelled from II to VI according to their size but, so far, it has not been possible to establish the equivalence between mitotic and polytene chromosomes. Figure 3 shows the spatial map, Figure 4 the linear map, and Table 1 summarises the morphological markers (landmarks) proposed to identify each chromosome. Chromosome II - This chromosome represents a 25.6% of the total polytenized kariotype. 1. Straight end with eight bands. 2. Four bands. The first one with an interruption in the middle. 3. Four dark and irregular bands. 4. Puff with five curved bands. 5. Puff with non-defined bands and two darker ones. 6. Small puff with two large, dark bands. Towards the end, a dotted band, followed by a weak band. 7. Dark band with an interruption in the middle, followed by another dark band that continues with a dotted band, and a thin, weak band at the end. 8. Five bands. 9. Two dotted bands. 10. Three dark bands. 11. Prominent puff displaying three bands. 12. Small region with three bands. 13. Narrow chromosomal segment with seven thin clear bands. 14. Zone without a defined banding, which displays a peak at one side. 15. Puff with bands only at the ends. 16. Two dotted bands. 17. Eight bands. 18. Large puff towards one side. 19. Seven bands. 20. End with four dotted bands. Marker - The markers that allow the identification of this chromosome are: the initial straight end, the large banded segment that comprises several zones and the puff in zone 18.

6 208 giardini, milla, manso Fig. 3 Spatial map. Chromosome III - This chromosome represents a 24.2% of the total polytenized kariotype. 21. Tip with nine thin bands. 22. Four dark bands. 23. Zone without bands. 24. Curving of the chromosome that adopts the form of a hook, followed by discontinuity or interruption of the chromosome. 25. Coiled fan. 26. Six curved bands. 27. Chromosomal segment displaying five bands. 28. Thin band. 29. Two merged band. 30. Three curved bands. 31. Two defined bands. 32. Curved zone without defined bands. 33. Rhomboid-shaped segment with nine bands. 34. Zone with diffuse and curved bands.

7 polytene chromosomes in anastrepha fraterculus 209 Fig. 4 Linearized spatial map. 35. Thick and dotted band. 36. Three similar bands. 37. One irregular band. 38. Ten bands. Towards one end, the portion of the chromosome which would correspond to the loop of the following zone is not aligned. 39. Loop, possibly caused by non-homologous zones that are located towards one end of the chromosome. 40. Five bands at one end of the chromosome. Marker - The loop that corresponds to zone 39, together with the discontinuity displayed in the limit between zones 24 and 25, help in the identification of this chromosome. Chromosome IV - This chromosome represents a 17.7% of the total polytenized kariotype. 41. Triangular end with three small bands. 42. Large and dark band accompanied by a clearer band. 43. Clear zone that ends with a dark band. 44. Part of the puff without band. 45. Two narrow bands at the end of the puff. 46. Two bands.

8 210 giardini, milla, manso 47. Three diffuse bands. 48. Clear zone adjacent to two dark zones. 49. Three small bands. 50. Four bands. The first one is the darkest ban 51. Extended region displaying six bands. 52. Puff with longitudinal bands. 53. Puff with irregular bands. 54. Extended puff with a dark irregular banding. 55. Puff with two pairs of small and clear bands. 56. Five darker bands in the other end of the puff. 57. Puff with longitudinal banding. 58. Two small and defined bands. 59. Three bands. 60. Small, dark, pointed ending. Marker - Both ends of the chromosome are characteristic and allow its identification. The coil or loop, which comprises the zones 51-56, can also be used as a marker. Chromosome V - This chromosome represents a 16.8% of the total polytenized kariotype. 61. Fan with two dark bands. 62. Two dotted bands. 63. Five bands. 64. Three defined bands of different thickness. 65. Zone with eight bands. 66. Region without a defined banding. 67. Part of the puff with three weak bands at one. 68. Two defined bands at the other end of the puff. 69. Two bands with different thickness. 70. A circular band and a dark band at the vertex of the chromosome. 71. Three defined bands. 72. A circular band. 73. Two bands. The second is joined in the middle forming a peak. 74. Three bands. 75. Non-defined banding. 76. Four bands. 77. Zone without defined banding. 78. Fifteen thin bands of similar intensity. 79. Four bands. 80. Final segment with four bands. Marker - Both ends of this chromosome, together with the curved zone that comprises zone 69-72, are characteristic and allow its identification. Chromosome VI - This chromosome represents a 15.3% of the total polytenized kariotype. 81. Fan with a defined band in the centre. 82. Dark band occupying the entire region 83. Clear zone 84. Not-defined banding. 85. Incomplete band which does not extend from side to side. 86. Irregular bands. 87. Dotted region. 88. Clear region. 89. Puff possessing a small weak band followed by a band joined at the centre and forking towards the sides. A small dot-like band and a dark band are observed as well. 90. Weak band followed by two dotted bands that are adjacent to a darker and irregular band. 91. Dotted band and a curved band which does not cover all the width of the chromosome. 92. Region with a thick and dark band followed by another smaller band that is adjacent to three curved bands that do not cover all the width of the chromosome. 93. Two bands joined by an intermediate bridge, followed by a band that is adjacent to a clear region and a dark band. 94. Dark region that is clearer in one side. 95. Thick band followed by two clearer bands. 96. Three dark bands 97. Two dotted bands. 98. Small dark band followed by two prominent bands that display a discontinuity between them, followed by two small, clear bands. 99. Two interrupted bands, followed by two other bands Clear zone and a thick band towards the end. Marker - The loop or coil that extends from the beginning of zone 87 to the discontinuity in zone 98. In addition, the initial fan and the discontinuity present in zone 98 are also markers of this chromosome. The characteristic zones that allowed us to individualize the chromosomes are fairly constant appearing in more than 78% of the analysed pictures (Table 1). When we carried out a simultaneous analysis of mitotic (in order to know the sex of the larva) and polytene nuclei, we found that neither the number of polytene chromosomes nor the banding pattern showed significant differences between males and females of A. fraterculus, thus confirming that sexual chromosomes do not polytenize. This result is in agreement with previous reports for A. fraterculus (Cá c e r e s et al. 2009), C. capitata (Be d o 1986; 1987; Za c h a r o p o u l o u 1987; Ga r i o u-pa pa l e x i o u et al. 2002), A. ludens (García-Martinez et al. 2009), B. olivae (Zambe -

9 polytene chromosomes in anastrepha fraterculus 211 ta k i et al. 1999; Mav r a g a n i-tsipidou 2002), B. cucurbitae (Re z a et al. 2002) and B. tryoni (Zh a o et al. 1998). The results presented in this work are promising since they represent a new field of study that will allow researchers to clarify and solve the complexity displayed by this species. The data provided by the use of polytene maps, are useful for the correct identification of A. fraterculus, and the methodology applied here could be helpful for the description of other dipteran species in order to broaden the knowledge of the family Tephritidae. Acknowledgements This work was financed with funds from the contract with the International Commission of Atomic Energy (Austria) n R2, SECyT and INTA. We are grateful to Daniel Díaz and Leonela Carabajal for proof-reading the manuscript and for their constant collaboration. We would like to thank Roberto Civitillo for his help with the processing of the images. 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