Methods for the recovery of mitochondrial DNA sequences from museum specimens of myiasis-causing flies

Authors

  • A. C. M. Junqueira,

    Corresponding author
    1. Laboratório de Genética Animal, Universidade Estadual de Campinas (UNICAMP), São Paulo, Brazil
      Ana Carolina Martins Junqueira, Laboratório de Genética Animal, Universidade Estadual de Campinas (UNICAMP), DGE/CBMEG, C.P. 6010, Campinas, São Paulo 13087-970, Brazil. E-mail: anacmj@obelix.unicamp.br
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  • A. C. Lessinger,

    1. Laboratório de Genética Animal, Universidade Estadual de Campinas (UNICAMP), São Paulo, Brazil
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  • A. M. L. Azeredo-Espin

    1. Laboratório de Genética Animal, Universidade Estadual de Campinas (UNICAMP), São Paulo, Brazil
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Ana Carolina Martins Junqueira, Laboratório de Genética Animal, Universidade Estadual de Campinas (UNICAMP), DGE/CBMEG, C.P. 6010, Campinas, São Paulo 13087-970, Brazil. E-mail: anacmj@obelix.unicamp.br

Abstract

Abstract Mitochondrial DNA (mtDNA) sequences from eight species of myiasis-causing flies, stored for up to 50 years, were amplified successfully. Universal primers were used to amplify six specific regions from total genomic DNA, including five mtDNA genes. The comparison of phenol/chloroform, DNAzol® and Chelex techniques for DNA extraction showed that the DNAzol® reagent was the most efficient in retrieving DNA from museum specimens, although the Chelex extraction procedure is currently the most frequently reported method. Comparison of the universal primer sequences with the homologous sequences of Cochliomyia hominivorax Coquerel and Chrysomya putoria Wiedemann (Diptera: Calliphoridae) revealed mismatches that could contribute to the low recovery of a short sequence from subunit II of cytochrome oxidase. The ability to characterize mtDNA markers from museum specimens should be useful in comparative studies of contemporary samples and should help in elucidating species introduction, colonization and dispersal.

Introduction

Until recently, morphological analysis was the only tool available for scientists to determine the relationships of museum specimens. However, the improvement in techniques of molecular biology over the past two decades has allowed the retrieval of DNA from well-preserved specimens. Studies with damaged DNA have shown that genetic information can be recovered from preserved specimens, but such samples are reduced to a few hundred base pairs because of oxidative and hydrolytic damage. Hydrolysis results in the deamination of nucleotide bases and cleavage of base-sugar bonds, creating baseless sites (Lindahl, 1993). When the base is lost, the chain is weakened and eventually cleaved (Cano, 1996). Oxidation leads to the chemical modification of nucleotide bases and eventual destruction of the ring structure of base and sugar residues in the DNA molecule (Pääbo, 1989; Lindahl, 1993).

The molecular approaches applied to museum material are based mainly on the enzymatic amplification of short, high copy number DNA sequences, such as those of mitochondrial DNA (mtDNA), using the polymerase chain reaction (PCR). The use of molecular markers for comparing the genetic information of live and preserved organisms could provide some idea of the population genetic changes that have occurred and of the mechanisms behind these processes (Cano, 1996; Roy et al., 1994).

In this work, we compared the efficiency of three techniques for DNA extraction based on the recovery of specific PCR products from preserved specimens of Calliphoridae and Oestridae (Diptera). These are the main families of myiasis-causing flies, which contain species of economic, medical, veterinary and forensic importance, including obligate or facultative livestock parasites. We also undertook an analysis of the universal primers used for amplifying mtDNA fragments and describe the difficulties of recovering and storing DNA under appropriate conditions.

Materials and methods

Specimens

Dried pinned specimens of Cochliomyia hominivorax Coquerel, C. macellaria Fabricius, Chrysomya albiceps Wiedemann, C. megacephala Fabricius, C. putoria Wiedemann, Lucilia eximia Wiedemann, Hemilucilia segmentaria Fabricius and Dermatobia hominis Linnaeus Jr., were provided by six entomological collections. Alcohol-preserved specimens (adults and larvae) of C. hominivorax, C. putoria and D. hominis were also obtained from three entomological collections. Fresh specimens were collected and maintained in the laboratory (Leal et al., 1982; Infante & Azeredo-Espin, 1995) or frozen at −70°C and used as extraction and PCR controls.

DNA extraction

The three techniques used in this work were based in different principles for DNA extraction. The DNAzol® reagent (Gibco BRL/Life Technologies, Gaithersburg, MD, U.S.A.) contains the chaotropic agent guanidine thiocyanate and a detergent mixture in a lysing solution which permits selective precipitation of DNA from a cell lysate (Chomczynski et al., 1997). The Chelex® 100 (Bio-Rad, Hercules, CA, U.S.A.) is a chelating resin that protects the DNA during the boiling step and sequesters divalent heavy metals that could introduce DNA damages (Walsh et al., 1991). The phenol/chloroform procedure is based in two different organic solvents for deproteinization, including several separation and purification steps (Sambrook et al., 1989).

Fresh, pinned and alcohol-preserved adult specimens were washed in sterile distilled water and the wings were removed with sterile pincers. The specimens were transferred to sterile, screw-capped 1.5 mL microcentrifuge tubes.

For extractions using the DNAzol® procedure, each sample was ground in 500 µL of DNAzol® with a sterile pestle. A further 500 µL of DNAzol® was added and the contents were mixed twice by inversion. After centrifugation at 16 000 g for 10 min, 500 µL of ice-cold absolute ethanol were added to the supernatants for DNA precipitation. After 5 min at room temperature, the samples were again centrifuged at 16 000 g for 5 min. The DNA pellets were washed once in 95% ethanol. After air drying for 10 min, the DNA was resuspended in 50 µL of sterile 1 × TE (1 mm Tris-HCl, 0.1 mm EDTA, pH 7.0). For DNA extractions of thorax, abdomen and legs, the initial volume of DNAzol® was reduced to 250 µL, and the volumes of absolute and 95% ethanol were also reduced proportionally.

DNA extraction with Chelex 100 consisted of grinding the samples in 100 µL of 10% Chelex 100 and incubating at 56°C overnight. Then the samples were mixed with vortex at high speed for 10 s, incubated in boiling water for 5 min and mixed again in vortex for 10 s. The tubes were centrifuged at 15 000 g and the supernatants were transferred to new screw-capped tubes (Walsh et al., 1991; Cano et al., 1992).

The phenol/chloroform DNA extraction was done as reported in Infante & Azeredo-Espin (1995). The specimens were homogenized in 100 µL of grinding buffer (2 m Tris, 5 m NaCl, 50% sucrose, 0.5% EDTA) pH 7.5 using a sterile pestle. A further 100 µL of lysis buffer (2 m Tris, 10% SDS, 0.5% EDTA, 2% DEPC) pH 9.0 were added. The lysate was incubated in ice for 15 min, after which an equal volume of phenol pH 7.6 was added, mixed with vortex and incubated in ice for 2 min. The tubes were centrifuged at 2600 g for 5 min and the supernatant was transferred to a new tube. An equal volume of phenol/chloroform: isoamyl alcohol 24 : 1 were added, mixed with vortex, incubated for 2 min in ice and centrifuged at 2600 g for 5 min. The supernatant was transferred to a new tube and an equal volume of chloroform : isoamyl alcohol 24 : 1 was added, mixed in vortex, incubated in ice for 2 min and centrifuged at 2600 g for 5 min. After the last centrifugation, 100 µL of 1 × TE, 0.05 volumes of 3M sodium acetate (∼20 µL) and 2.5 volumes (∼900 µL) of ice-cold absolute ethanol were added to the supernatant, mixed by inversion and incubated at −20°C for 2 h to precipitate the DNA. After a centrifugation at 15 000 g for 30 min, the supernatant was discarded and the pellet resuspended in 50 µL of 1 × TE. All the DNA extractions were stored at −20°C.

Amplification

Extracted DNA was diluted 1 : 5, 1 : 10 and 1 : 20 in sterile 1 × TE and 5 µL were used as a template in PCR reactions. The PCR amplifications were done with 1 × PCR buffer (200 mm Tris-HCl pH 8.4, 500 mm KCl), 1.25 mm MgCl2, 200 µm of each deoxynucleoside triphosphate (dNTP), 0.5 µm of each primer, and 1.25 U of Taq DNA polymerase (Gibco-BRL/Life Technologies), in a final volume of 25 µL. These PCR conditions have been optimized for fresh and frozen samples and then used for dried and alcohol 80% preserved specimens, guaranteeing that the reactions were not suboptimal for different techniques used during the DNA extraction. The amplification reactions from thorax and legs DNA were conducted with 5 µL of the extract, without dilutions.

The primers used were mainly from the UBC Insect mtDNA oligonucleotide set (Simon et al., 1994). Five regions of the mtDNA were analysed: subunit I of cytochrome oxidase (COI) with the primers C1-J-2195 and C1-N-2329, subunit II of cytochrome oxidase (COII) with the primers C2-J-3400, C2-N-3494 and C2-N-3661, cytochrome b (Cyt. b) with the primers CB-J-10612 and CB-N-10920, a region including the partial sequence of ribosomal RNA 16S and subunit I of NADH genes (16S/ND1) amplified with the primers N1-J-12585 and LR-N-12866, and the control region using the primers TI-N-24 and CR-J-433. This last primer was designed based on the C. megacephala sequence (Lessinger & Azeredo-Espin, 2000). These regions were selected based on the total length of the sequences to be amplified and were limited to a few hundred base pairs to provide more reliable PCR products as the DNA of these samples was highly degraded and fragmented.

The PCR reactions were run on a model 9600 Thermocycler (Perkin Elmer, Foster City, CA, U.S.A.). During the first cycle, the DNA templates were denatured at 94°C for 3 min, followed by 40 cycles consisting of a 1-min denaturation at 94°C, annealing for 1 min 15 s at 42–50°C, depending on the primer pair used, and an elongation step at 72°C for 2 min. The final cycle involved an extended elongation at 72°C for 7 min. The amplification of the control region fragment required a modification of the elongation temperature to 60°C. In all amplifications, a control reaction with no template was included to ensure that contaminating DNA would not be amplified instead of the museum specimen target molecules. The products of the PCR reactions were evaluated by electrophoresis in 2.0% agarose gels in 1 × TAE (40 mm Tris – Acetate, 1 mm EDTA) buffer.

Cloning and sequencing

An aliquot of each amplification product was purified by dialysis in 1 × TE buffer, pH 7.4 on a VM 0.05 µm filter (Millipore, Bedford, MA, U.S.A.) for 20 min. The products were then cloned into the vector pUC 18 SmaI/BAP using a SureClone ligation kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ, U.S.A.), according to the manufacturer's instructions. After preparation of plasmid DNA by alkaline lysis (Sambrook et al., 1989), the recombinant clones were confirmed and used as templates for automatic sequencing with the Big Dye™ terminator kit (Perkin Elmer) according to the manufacturer's instructions. A total of 45 cycles were performed during the PCR reaction for sequencing. The sequences obtained were analysed in BLASTN using the GenBank (Altschul et al., 1997) database and aligned with ClustalW (Thompson et al., 1994).

Results

DNA extraction

Of the three methods for DNA extraction evaluated, the DNAzol and Chelex techniques provided consistent and well-resolved PCR products from museum specimens. Dried and alcohol-preserved samples yielded no PCR products when extracted with phenol/chloroform. For this reason, this technique was not used for subsequent DNA extractions from museum specimens. Fresh and frozen specimens yielded PCR products with all three methods.

To establish which technique was the most efficient in recovering DNA from museum specimens, three alcohol-preserved larvae of D. hominis were sectioned longitudinally into two identical parts. One half was extracted with DNAzol and the another with Chelex. The results showed that DNAzol provided the best recovery of DNA from preserved specimens (Table 1).

Table 1.   Comparison of DNA recovery with DNAzol and Chelex.
Samples Extraction
method
Size of amplified mtDNA regions
COII
137bp
COII
155bp
COI
180bp
COII
305bp
16S/ND1
315bp
Cyt b
357bp
  1. += successful amplification; –= unsuccessful amplification.

D. hominis(A 155)Chelex
(A 156)DNAzol++++
D. hominis(A 157)Chelex
(A 158)DNAzol+++
D. hominis(A 159)Chelex
(A 160)DNAzol

Amplification

Low (1 : 5) dilutions of DNA extracts from museum specimens provided well-resolved PCR products when compared to dilutions of 1 : 10 and 1 : 20. The size of the amplified products varied from 137 to 404 bp, depending on the primers and regions used (Fig. 1, Table 2). DNAzol extraction recovered specific PCR products from specimens stored for up to 50 years (Table 2), indicating that the genetic information of well-preserved organisms can be accessed and used for comparative studies.

Figure 1.

 Agarose gel (2.0%) with mtDNA PCR products. Lane 1 = 137 bp fragment of the COII gene from a C. putoria leg extracted with DNAzol; lane 3 = 155 bp fragment of the COII gene from an alcohol-preserved larva of D. hominis extracted with DNAzol; lane 5 = 180 bp fragment of the COI gene from a pinned adult of C. hominivorax extracted with DNAzol; lane 7 = 305 bp fragment of the COII gene from a pinned adult D. hominis extracted with DNAzol; lane 9 = 320 bp of the A + T region of a pinned adult of C. putoria extracted with DNAzol; lane 11 = 315 bp fragment including sequences of the 16S and ND1 genes from a C. macellaria thorax extracted with Chelex; lane 13 = 357 bp fragment of the cytochrome b gene from a pinned adult of C. putoria extracted with DNAzol; lane 15 = 404 bp fragment of the A + T region from a pinned adult of C. putoria extracted with DNAzol. Lanes 2, 4, 6, 8, 10, 12, 14 and 16 are PCR controls and Lane S contains the molecular size markers. Small bands (below 118 bp) correspond to primer dimers.

Table 2.   Museum specimens and size of mitochondrial DNA regions used in this study.
MuseumSpeciesSexDateLocalityExtraction methodSize of amplified mtDNA regions
COII 137bpCOII 155bpCOI 180bpCOII 305bp16S/ND1 315bpCyt b 357bp
  • Dried thorax;

  • ‡dried adult;

  • §

    alcohol 80%;

  • ¶frozen;

  • ††

    ††fresh.

  • += successful amplification, –= unsuccessful amplification, *= sex not identified.

  • 1

    Zoology Museum, University of São Paulo,

  • 2

    2 National Museum, Rio de Janeiro,

  • 3

    3 Emilio Goeldi Paraense Museum,

  • 4

    4 Entomological Collection, Federal University of Amazonas,

  • 5

    5 Entomological Collection, State University of Campinas,

  • 6

    Dr Carlos G. Malbrán National Institute of Microbiology – Buenos Aires, Argentina,

  • 7

    The Natural History Museum of London. AM – Amazonas, Brazil, BA – Bahia, Brazil, RJ – Rio de Janeiro, Brazil, SP – São Paulo, Brazil, RS – Rio Grande do Sul, Brazil, MG – Minas Gerais, Brazil, AR – Argentina, FG – French Guiana.

MZ-USP1C. macellaria (A85)1978São Paulo-SPDNAzol+++
C. macellaria (A88)1978São Paulo-SPDNAzol++
L. eximia (A65)1965Boca do Tucano-AMDNAzol++
D. hominis (A58)*1945Itatiaia-RJDNAzol+++
D. hominis (A69)*1950BoracÅa-SPDNAzol++
MNRJ2C. macellaria (A76)1993Rio de Janeiro-RJChelex++
C. macellaria (A87)1976Rio de Janeiro-RJDNAzol++++
C. macellaria (A106)*1976Rio de Janeiro-RJChelex+
MPEG3C. macellaria (A128)1995Breves-PADNAzol++
C. macellaria (A130)1983Serra Norte-PADNAzol+++
C. putoria (A132)1982Belém-PADNAzol++
C. megacephala (A134)1987Belém-PADNAzol+
C. putoria (A203)††2001Belém-PADNAzol++++++
C. putoria (A207)††2001Belérn-PAPhenol++++++
C. macellaria (A45)1987Manaus-AMDNAzol+
C. albiceps (A55)1988Manaus-AMDNAzol++
UFA4C. megacephala (A52)1987Manaus-AMDNAzol+++++
C. putoria (A53)1987Manaus-AMDNAzol+++++
L. eximia (A50)1988Manaus-AMDNAzol+++
C. hominivorax (A03)1992Morro do Chapéu-BADNAzol++++++
C. hominivorax (A07)1992Morro do Chapéu-BADNAzol+++
C. hominivorax (A17)1992Alfenas-MGPhenol++++++
C. hominivorax (A35)1987Porto Alegre-RSDNAzol++++
C. hominivorax (A36)1987Alfenas-MGDNAzol++++
C. hominivorax (A39)1987Porto Alegre-RSDNAzol++++++
UNICAMP5C. hominivorax (A43)1987Porto Alegre-RSDNAzol+++
C. hominivorax (A44)1987Porto Alegre-RSDNAzol+++
C. hominivorax (A49)1987Caraguatatuba-SPDNAzol+++
C. macellaria (A78)1993Rio de Janeiro-RJChelex++++++
C. hominivorax (A79)1992Morro do Chapéu-BAChelex++++++
C. hominivorax (A99)*1987Caraguatatuba-SPDNAzol+++++
INM6C. hominivorax (A33)1987Buenos Aires-ARDNAzol++++++
D. hominis (A156)§*1994Petit Saut-FGDNAzol++++
NHM7D. hominis (A158)§*1994Petit Saut-FGDNAzol+++
D. hominis (A163)§*1994Petit Saut-FGDNAzol++++

Although most of the amplifiable DNA was derived from the entire specimen, DNA extractions from specific parts of the dried samples were also tried in order to reduce the destruction of specimens and preserve important morphological traits. Thoracic DNA extractions with DNAzol and Chelex yielded PCR products, but from legs DNA resulting in a detectable PCR product was obtained only with the DNAzol technique. Extracts from the abdominal region yielded no amplification products.

The comparative analysis between sequences of the universal primers used in this work and its homologous regions of C. hominivorax and C. putoria (Fig. 2) allowed the evaluation of these primers for recovering the genetic information of museum specimens, considering the mutational events on the annealing sites and the total length to be amplified. As discussed below, this analysis could provide the most adequate primers pair to be used in initial attempts on amplifying DNA from preserved samples.

Figure 2.

Sequences of the universal primers from Drosophila yakuba, compared with the homologous regions of Cochliomyia hominivorax and Chrysomya putoria.

Discussion

Extractions from museum specimens usually contain sheared and interstrand cross-linked DNA, compromising the continuity and the denaturation of DNA strands during the PCR amplification (Dean & Ballard, 2001). Hydrolytic and oxidative damages also have major effects on DNA recovery by PCR, reducing the number of intact DNA molecules. For these reasons, short PCR products from museum specimens were expected to be recovered more efficiently than larger fragments. Generally, the smaller the region, the better the retrieval of DNA from damaged specimens (Pääbo et al., 1989; Lindahl, 1993; Cano, 1996). However, amplification of the 305 bp fragment of the COII region was much more reliable than that of the 155 bp fragment of the same gene (Table 2). The low recovery of this shorter product may reflect the occurrence of mismatches between the primers and the target sequence, which would in turn compromise the annealing step of the PCR reaction.

Comparison of the COII sequence of C. hominivorax (Lessinger et al., 2000; GenBank accession number AF260826), C. putoria (Junqueira et al. unpublished; GenBank accession number AF352790) and the homologous sequences of the universal primers (Simon et al., 1994) used for amplification of the 155 bp fragment revealed five mutational events involving three transversions and two transitions in the C2-J-3279 primer (Fig. 2), originally derived from the Drosophila yakuba mtDNA sequence (Clary & Wolstenholme, 1985). The low recovery of the Cyt b 357 bp fragment may also have been caused by mismatches between the primers and the homologous sequences of the species analysed, as the primer CB-J-10612 was the most variable (Fig. 2). Based on sequence analysis of the primer pairs, the pairs C2-J-3400/C2-N-3494 (137 bp) and C2-J-3400/C2-N-3661 (305 bp) showed few nucleotide substitutions (Fig. 2) and yielded reliable PCR products from different species and samples preserved over a wide period of time (Table 2). These results suggest that the 137 bp region of CO II should be used in initial attempts at recovering DNA from insect museum specimens, as the universal primers for this region are conserved and amplify a short sequence. In addition, the CO II gene may be very useful for comparative analysis because it has been sequenced for a wide variety of insects, thus providing direct comparisons among them (Caterino et al., 2000).

As reported previously (Pääbo, 1989; Post et al., 1993; Handt et al., 1994; Hauswirth, 1994; Phillips & Simon, 1995; Cano, 1996; Höss et al., 1996; Kelman & Moran, 1996), and based on the data in Table 2, the extent of the reduction in the size and genetic information of the retrieved fragments did not correlate with the age of the specimen, but was related to the environmental conditions under which the specimen was preserved. Other extrinsic factors, such as the type of fixation and its method of application, also have considerable effects, but vary according to the curator's collecting habits (Thomas, 1994).

Rapid desiccation after death, the chelation of copper and other metal ions, protection against exposure to UV light and storage at low temperatures in an alkaline environment favour the preservation of DNA (Eglinton & Logan, 1991). The method by which specimens are killed can also affect the yield and quality of DNA. Dillon et al. (1996) showed that specimens killed in ethyl acetate vapour gave very low yields of DNA, which could not be amplified by PCR.

Although speculative, the climate could be a major problem accelerating the DNA degradation and fragmentation when collections and specimens are not stored appropriately in dried environments with a stable temperature. Of the 55 dried specimens from Rio de Janeiro, Manaus and Belém used for DNA extraction, only 12 have been successfully amplified. These cities are located in coastal and/or rain forest areas, where the high humidity and temperatures could accelerate the degradation of DNA.

The sequenced fragments showed high nucleotide sequence identity with homologous regions of mtDNA from other Calliphoridae species in GenBank, thus indicating that contamination had been effectively avoided. Contamination is a particular problem in the analysis of DNA from museum specimens, as DNA from other organisms may be preferentially amplified.

In preliminary studies with fresh samples, the DNAzol technique also yielded suitable DNA for restriction fragment length polymorphism, random amplified polymorphism DNA and microsatellite analysis. DNA obtained by DNAzol can be stored for long periods of time and amplified after more than 3 years when stored at −20°C. This finding is particularly important because DNA extracted by Chelex shows a rapid degradation (Cano & Poinar, 1993).

The isolation and analysis of DNA from dried and alcohol-preserved specimens adds a direct temporal dimension to genetic and evolutionary studies (Wayne et al., 1999), as the genetic variability over time and the geographical distribution of inaccessible populations can be studied. Access to temporal changes in living organisms allows the reconstruction of their genetic past and provides important insights into a species' pattern of introduction, distribution and colonization.

Acknowledgements

The authors thank Drs M.C.F. do Val, D. Pamplona, C.J.B. Carvalho, N.D. Parallupi, M.C. Esposito, A. Harada and M.J.R. Hall for kindly providing specimens from entomological collections. M.C. Arias provided helpful comments on the Chelex procedure, and R.A. Rodrigues, M.S. Couto and M. Constantino Filho gave valuable technical assistance. We are grateful to Dr F.A.H. Sperling for constructive comments on the manuscript and to two anonymous referees for their critical review. This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 99/07355-0) and by a PADCT/CNPq grant 620097/95-7 to A.M.L.A.E. A.C.L. was supported by a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil). A.C.M.J. was supported by a fellowship from FAPESP.

Accepted 17 September 2001

Ancillary