Proceedings of 6th International Fruit Fly Symposium 6–10 May 2002, Stellenbosch, South Africa pp. 415–418
Studies on highly repetitive DNA in cryptic species of the Anastrepha fraterculus complex (Diptera: Tephritidae) L.S. Rocha & D. Selivon* Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, 05508-900 São Paulo, Brazil
Genomic DNA of Anastrepha sp. 1 aff. fraterculus and Anastrepha sp. 2 aff. fraterculus (from Brazil) and Anastrepha sp. 4 aff. fraterculus (from Ecuador) was cleaved with restriction endonucleases EcoRI and ClaI, and electrophoresed in agarose gels. All cleavages yielded bands of repetitive DNA sequences. Restriction patterns were similar in sp. 1 and sp. 2, while sp. 4 showed a distinct restriction pattern. DNA fragments from three bands were eluted, labelled and used as probes for hybridization in nylon membranes: S1C-2.1 (from a fragment of 2.1 kb yielded by ClaI in sp. 1); S1C-0.32 and S2C-0.32 (fragments of 320 bp yielded by ClaI, respectively, in sp. 1 and sp. 2). Southern blot with probe S1C-2.1 produced different hybridization patterns to sp. 1 and sp. 2 hybridization. In dot blot experiments, probes S1C-0.32 and S2C-0.32 hybridized to DNA from several Anastrepha species (sp. 1, sp. 2, sp. 3, sp. 4, A. obliqua, A. amita, A. sororcula, A. grandis, A. serpentina). Only probe S2C-0.32 hybridized to DNA from Ceratitis capitata. These results showed that sp. 1, sp. 2 and sp. 4 from the fraterculus complex can be distinguished by means of restriction endonuclease patterns of total genomic DNA, besides hybridization patterns of S1C-2.1, S1C-0.32 and S2C-0.32 probes. Remarkably, these results also show that some of these repetitive DNA sequences are broadly spread among the taxa, since they are shared by representatives of the genus Anastrepha and by C. capitata.
INTRODUCTION The fruit fly Anastrepha fraterculus, a remarkable pest in Brazilian fruit orchards, in fact comprises a complex of cryptic species, the so-called ‘fraterculus complex’. Two cryptic species belonging to this complex have been characterized in Brazil, Anastrepha sp. 1 aff. fraterculus and Anastrepha sp. 2 aff. fraterculus, through analysis of isoenzymes, external morphology of the eggs, morphometry of adult body structures, analysis of heterochromatin by the C-banding method, and interspecific crosses (Selivon 1996; Selivon & Perondini 1998; Selivon et al. 1999). Subsequent studies on a variant morphotype and on a sample from Guayaquil, Ecuador, suggested the existence of other entities in the fraterculus complex, which were named Anastrepha sp. 3 aff. fraterculus and Anastrepha sp. 4 aff. fraterculus, respectively (Selivon et al. 2004). Sequences of highly repetitive DNA are usually subjected to rapid evolutionary alterations. Since they are usually non-coding sequences, mutations and other alterations that occur in these sequences should not confer a deleterious effect. Thus, in several cases, natural selection does not impede the propagation of mutations acquired by these sequences. Owing to DNA’s high rate of change, even in sibling species, sequences of highly repetitive DNA tend to be unique (Hankeln et al. 1989). *To whom correspondence should be addressed. E-mail:
[email protected]
Previous studies showed that the entities of the fraterculus complex present karyotypic differences in relation to the size of sex chromosomes and their heterochromatic blocks shown by C-banding (Selivon 1996; Selivon et al. 2004). Since it is known that heterochromatic regions contain highly repetitive DNA, we decided to perform a detailed analysis of repetitive DNA in species of the fraterculus complex. Cleavage of genomic DNA by restriction endonucleases allows the analysis of highly repetitive DNA. Cleaved genomic DNA when electrophoresed in gels may exhibit prominent, highly repetitive DNA bands. The restriction pattern formed by the number and size of these bands normally differs between species, and can be studied on a comparative basis (Bachmann et al. 1993). We report here the first results of a restriction analysis of genomic DNA of specimens of the ‘fraterculus complex’, Anastrepha sp. 1, A. sp. 2 and A. sp. 4, using endonucleases ClaI and EcoRI, with a view to improving and complementing the characterization of cryptic species of the A. fraterculus complex. MATERIALS AND METHODS Samples of Anastrepha sp. 1, A. sp. 2 and A. sp. 4 were analysed. Genomic DNA was extracted from thoraxes of adults according to the ‘single fly DNA extraction method’ (Jowett, 1986). Each 36 mg of tissue yielded about 20 µg of DNA, which was totally cleaved with restriction endonuclea-
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Table 1. Approximate sizes of highly repetitive DNA bands obtained after cleavage of DNA from Anastrepha sp. 1, sp. 2 and sp. 4 with endonucleases EcoRI and ClaI. Sizes of bands Agarose 1000 (bp)
Enzyme
Sample
Standard agarose (kb)
EcoRI
sp. 1 sp. 2 sp. 4
7.8–3.2–2.0 7.8–3.2–2.0 7.8–3.2–1.7
850 850 850
ClaI
sp. 1 sp. 2 sp. 4
9.0–5.2–2.1 9.0–5.2–2.1 9.0–5.2–2.1
1050–850–490–450–320 1050–850–490–450–320 950–900–850–600
ses ClaI and EcoRI according to the manufacturer’s recommendations (Invitrogen-Life Technologies, U.S.A.). Samples of cleaved DNA were electrophoresed on agarose gels with running buffer TAE 1X, according to Sambrook et al. (1989). DNA fragments over 1000 bp were analysed in 0.8% standard agarose gels, whereas fragments under 1000 bp were analysed in 3% agarose 1000 (Invitrogen-Life Technologies, U.S.A.). DNA from bands containing highly repetitive DNA were eluted from gels through the Concert Gel Extraction system (Invitrogen-Life Technologies), and the eluted DNA was biotin-labelled with a Bioprime kit (Invitrogen-Life Technologies, U.S.A.). Southern and dot blots were performed using nylon membranes as described in Sambrook et al. (1989). In Southern blots, 20 µg of endonucleasedigested DNA was applied to each sample. In dot blots, each blot contained 1 µg of integer DNA. For hybridization, biotin-labelled probes were used against target DNA fixed in membranes as suggested in the Photogene kit protocol (Invitrogen-Life Technologies), with the following modification in post-hybridization washes (for 100% similarity): firstand second washes, 1 × SSC, 1% SDS (5 min at 65°C); third wash, 0,1 × SSC, 1% SDS (30 min at 55°C); fourth wash, 2 × SSC (5 min at room temperature). Detection of the chemiluminescent hybridization signal was carried out according to the Photogene kit (Invitrogen-Life Technologies). Signals were registered using Kodak X41 X-ray films, and exposure times ranged from 0.5 to 15 min. RESULTS AND DISCUSSION Total cleavage of DNA samples from Anastrepha sp. 1, Anastrepha sp. 2 and Anastrepha sp. 4 with restriction endonucleases EcoRI and ClaI yielded visible bands, whose sizes ranged from about 9 kb to 300 bp. The electrophoretic patterns of these
fragments of highly repetitive DNA were identical to sp. 1 and sp. 2 (Table 1). This restriction analysis suggests that the arrangement of highly repetitive DNA is quite similar in the two species, being distinct in sp. 4 (Figs 1 & 2). Southern blot, dot blot and hybridization analyses under highly stringent conditions (100% similarity) were done with biotin-labelled probes obtained from three highly repetitive DNA restriction fragments: 2.1 kb fragment from sp. 1 cleaved by ClaI (S1C-2.1), 320 bp fragment from sp. 1 cleaved by ClaI (S1C-0.32) and 320 bp fragment from sp. 2 cleaved by ClaI (S2C-0.32) (see Table 1). In Southern blots, probe S1C-2.1 hybridized to the fragments of 2.1 kb from sp. 1, sp. 2 and sp. 4 cleaved by ClaI (Fig. 3), showing a high degree of homology among these highly repetitive
Fig. 1. Agarose gels (0.8%) of DNA from Anastrepha sp. 1, sp. 2 and sp. 4, cleaved with EcoRI. Molecular marker 8HindIII, in kb at left. Size of highly repetitive DNA bands are indicated.
Rocha & Selivon: Highly repetitive DNA in cryptic species of the Anastrepha fraterculus complex
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Fig. 2. Agarose 1000 gel (3%). Lane ‘lad’, molecular marker ladder 100 bp, with sizes at left. Other lanes, DNA from sp. 1, sp. 2 and sp. 4 cleaved with ClaI. Sizes of bands in Table I.
Fig. 3. Southern blot and hybridization of probe S1C-2.1 against DNA from Anastrepha sp. 1, sp. 2 and Guayaquil (Gua) sp. 4 cleaved with ClaI. Molecular marker 8HindIII, at right. Fragments size in kb.
sequences in the three species. This probe also hybridized to a 5.2 kb fragment yielded by ClaI in sp. 1; however, it did not hybridize to the similarsized fragments of 5.2 kb yielded by ClaI in sp. 2 and sp. 4. This indicates that the sequence of S1C-2.1 is shared by the three species but it is differentially arranged in the sp. 1 genome. In dot blots (Fig. 4),probes S1C-0.32 and S2C-0.32 hybridized to DNA from sp. 1, sp. 2, sp. 4 and Anastrepha sororcula, A. obliqua, A. amita, A. grandis and A. serpentina, but not to DNA of Drosophila melanogaster (Diptera: Drosophilidae) or Sciara ocellaris (Diptera: Sciaridae) (data not shown). Although sp. 4 does not exhibit a 320 bp band after cleavage by ClaI, sequences very similar to S1C-0.32 and S2C-0.32 are present in its genome. Despite their similar size, S1C-0.32 and S2C-0.32 showed arguable qualitative differences. The present data show that sp. 1, sp. 2 and sp. 4, despite being isomorphic species, can be distinguished through analysis of highly repetitive DNA, either by restriction or hybridization analyses, in addition to other molecular characteristics (Selivon et al. 2004). However, the DNA fragments obtained in this study are somewhat distinct from those usually described as highly repetitive DNA.
In contrast to the features of satellite-DNA, the gel bands obtained here are not assembled in ladder-like fashion, and some of them are long fragments (over 2 kb). Moreover, it is uncommon to find the presence of highly repetitive sequences shared by several species within a genus or a family, as shown by hybridization of S1C-0.32 and S2C-0.32. This could indicate that S1C-0.32 and S2C-0.32 are exceptionally-conserved, highly repetitive DNA. Similar cases were described by Brutlag (1980) and Beridze (1982). Another possibility is that these sequences are rDNA or mtDNA, which are more likely to be evolutionarily conserved. However, rDNA and mtDNA are moderately repeated, and do not usually form bands in agarose gels after restriction analyses. Following this hypothesis, one would have to consider that these sequences are present in exceptionally higher numbers of copies in Anastrepha. However, tests using probes of 26S rDNA from the midge Chironomus led to this hypothesis being rejected, at least for ribosomal sequences. This analysis in fact revealed the presence of atypical repetitive DNA, and it is not yet clear whether or not they are coding sequences. Prelimi-
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entities in this complex of species is a necessary step for the successful development and application of programmes intended to control these fruit fly pests. ACKNOWLEDGEMENTS This study was supported by FAPESP (processes 98/10701-4 and 99/06164-6). D.S. is a research fellow of CNPq.
Fig. 4. Dot blot and hybridization of probes S1C-0.32 andS2C-0.32 against DNA from Anastrepha sp. 1, sp. 2, and Guayaquil (Gua) sp. 4, Anastrepha sororcula, A. obliqua, A. amita, A. grandis, A. serpentina and Drosophila melanogaster.
nary data on sequencing fragments of 500 bp suggest that this fragment really is representative of ‘junk DNA’. However, additional studies are necessary to elucidate the nature of these sequences. In spite of its unknown nature, they may be employed as taxonomic markers for cryptic species of the fraterculus complex. The correct recognition of the different biological
REFERENCES BACHMANN, L., SCHIBEL, J.M., RAAB, M. & SPERLICH, D. 1993. Satellite DNA as a taxonomic marker. Biochemical Systematics and Ecology 21: 3–11. BERIDZE, T. 1982. Satellite DNA. Springer-Verlag, Berlin. BRUTLAG, D.L. 1980. Molecular arrangement and evolution of heterochromatic DNA. Annual Review of Genetics 14: 121–144. HANKELN, T., KEYL, H.G. & SCHMIDT, E.R. 1989. DNAprobes for the investigation of chromosome evolution in Chironomus. Part II: Repetitive sequences. Acta Biolica Debr Oecologica Hungarica 2: 219–227. JOWETT, T. 1986. Preparation of nucleic acids. In: Roberts, D.B (Ed.) Drosophila, a Practical Approach. Oxford, U.K. SAMBROOK, J., FRITSCH, E.F. & MANIATIS, T. 1989. Molecular Cloning: A Laboratory Manual (2nd Edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. SELIVON, D. 1996. Estudo sobre a diferenciação populacional em Anastrepha fraterculus ( Wiedemann) (Diptera: Tephritidae). Doctoral thesis, Depto de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo. SELIVON, D. & PERONDINI, A.L.P. 1998. Eggshell morphology in two cryptic species of the Anastrepha fraterculus complex (Diptera: Tephritidae). Annals of the Entomological Society of America 91: 473–478. SELIVON, D., PERONDINI, A.L.P. & MORGANTE, J.S. 1999. Haldane’s rule and other aspects of reproductive isolation observed in the Anastrepha fraterculus complex (Diptera: Tephritidae). Genetics and Molecular Biology 22: 507–510. SELIVON, D., VRETOS, C., FONTES, L. & PERONDINI, A.L.P. 2004. New variant forms in the Anastrepha fraterculus complex (Diptera: Tephritidae). In: Barnes, B.N. (Ed.) Proceedings of the 6th International Symposium on Fruit Flies of Economic Importance. 253–258. 6–10 May 2002, Stellenbosch, South Africa.
