Conservation Genetics 5: 103–104, 2004.© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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DNA sequence from butterfly frass and exuviae

Julie FeinsteinAmbrose Monell Collection for Molecular and Microbial Research, Division of Invertebrate Zoology, AmericanMuseum of Natural History, 79th Street at Central Park West, New York, New York 10024, USA(Phone: 212-769-5663; Fax: 212-496-3380; E-mail: jfstein@amnh.org)

Received 2 January 2003; accepted 19 February 2003

Key words: butterfly larvae, non-destructive sampling, Pieris rapae, Vanessa cardui, Vanessa virginianus

The early instar larvae of many butterflies are crypticand minute. Identification often requires microscopy.One cannot simultaneously prepare samples for micro-scopy (Peterson 1967) and optimal DNA preservation(Reiss et al. 1995). Insect tissue storage in DNAisolation buffer, in fact, works best when the insectmaterial is homogenized (Reiss et al. 1995). Theseissues become critical in the case of tropical insects,where, for many species, male and female forms havenot yet been linked, where larval and adult forms havenot yet been associated, and where new species arelikely to be found. Field collectors face a dilemma ofmutually exclusive options. A non-destructive methodof DNA isolation that would leave specimens intactfor collection or alive for observation and rearingwould be useful. Most caterpillars consume high bulkplant material throughout their larval stages, growingrapidly and producing large amounts of frass relativeto their size. Molecular scatology may prove fruitfulin their study. Additionally, butterflies are holometa-bolic, undergoing periodic ecdyses. Their shed skins(exuviae) may be viewed as another reservoir of DNA.

Two species of butterfly were investigated in thisstudy: Vanessa cardui L., the “Painted Lady”, andPieris rapae L., the “Cabbage Butterfly”. A matingpair of P. rapae plus two gravid females, (desig-nated females f1, f2 and f3) were caught wild inPalisades Park, Bergen Co., New Jersey, and caged,individually, with the wild host plant, “garlic mustard”(Alliaria officinalis). The females oviposited on andunder the leaves during three days of captivity. Eggswere removed and reared on cabbage leaves followingstandard protocols of butterfly culture (Pyle 1984).P. rapae exuviae were not found in the cages of thegrowing larvae. Presumably, they were eaten, as iscommonly the case (Aiello 1993). Five P. rapae cater-

Table 1. Primers for PCR and sequencing of P. rapae ND5(pieND5n) and V. cardui ND1 (vanND1n) designed for this study

pieND5L1 5’-CGGGGATTTCTGCTGTTTATG-3’

pieND5H1 5’-CTTAAACCTCCCATATAACG-3’

pieND5H2 5’-GAAACTCCCTGAAACTACTC-3’

pieND5H3 5’-TCCCCCCTAAAAAATTTCTC-3’

vanND1L1 5’-GGGGTTTTAATTGGGGTTGC-3’

vanND1H1 5’-AGAAGATCAACCCGCAATC-3’

pillars were reared (see Table 2). Individuals were isol-ated on clean leaves for frass collection. The sampleconsisted of three to five 5 mm diameter pellets. Frasssamples were stored in DMSO buffer (0.25M EDTA,40% dimethylsulfoxide, saturated with NaCl, pH 8.0).The caterpillars were allowed to develop through thepupal stage and collected in the adult form after emer-gence. Adults were euthanized with ethyl acetate andstored in DMSO buffer. V. cardui larvae were obtainedfrom a commercial supplier (Insect Lore, P.O. Box1535, Shafter Calif. 93263). Late larval instars areshipped, alive, in artificial medium. V. cardui larvalhabit differs from that of P. rapae. The larvae formwebs of silk and do not consume cast off exuviae.Shed skin is left attached to the silk networks and theshed skin of the ultimate instar remains attached to thepupal case. The last larval skin was collected fromfour individuals at pupation. In addition to exuviae,a combined frass sample for the cohort was collectedfrom the communal cage to test the possibility of PCRamplification of DNA from frass of this species. Uponemergence, each adult was collected and stored inDMSO buffer.

PCR and sequencing primers were designed fromthe GenBank entry (Accession #AB033594) for the

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Table 2. PCR and sequence results for adults, frass and exuviae ofP. rapae and V. cardui

P. rapae V. cardui

Larva Adult pcrs Frass pcrs Larva Adult Exuviae Frass

(c, d & e) (c) (d) (e)

1 f1f √a √ √Xb 1

√ √—

2 f1√ √ √

X 2√ √

—

3 f2√ √ √ √

3√ √

—

4 f3√ √ √

X 4√ √

—

5 f3√ √ √

X cohort — —√

a√ indicates a successful PCR and sequencing reaction with blanknegative control. bX indicates that no PCR product was amplified.ND5 PCR products and sequences from P. rapae were obtained with3 H strand primers paired with pieND5L1, to produce products ofc200 base pairs (pieND5H1), d475 base pairs (pieND5H2) and e650base pairs (pieND5H3). fF1, 2 or 3 indicates the female parent of theP. rapae caterpillar. ND1 PCR products and sequences of V. carduifrass and exuviae were obtained with the primer pair vanND1L1 andvanND1H1.

partial sequence of the mitochondrial gene dehydro-genase subunit 5 (ND5) of P. rapae (pieND5 primers,Table 1), to amplify fragments of 200, 475 and 650base pairs. V. cardui ND5 sequences were not avail-able, so primers (vanND1 primers, Table 1) weredesigned from a partial sequence of dehydrogenasesubunit 1 (ND1) of Vanessa virginianus (Accession#AF412752), to amplify a fragment of about 300 basepairs. DNA was extracted with the Qiagen DNEasyTissue Kit, (Qiagen, Inc., Valencia, CA) with themodification that a final elution volume of 100 µLwas used to concentrate the extract. Adult DNA wasextracted from sections of abdomen. PCR reactionswere performed using the Qiagen HotStart Kit. Each(40 µL) reaction contained 1 mM of each primer, 1U taq polymerase, 25 µM dNTPs, 1X PCR buffer(1.5 mM MgCl2), and 5 µL of DNA extract. Reagentblanks were prepared as negative controls for eachDNA/primer pair combination. PCR cycle profilesconsisted of initial enzyme activation at 95 ◦C for 15minutes, then 40 cycles of 94 ◦C, for 30 seconds,55 ◦C for 30 seconds and 72 ◦C for 30 seconds. PCRproducts were separated on 2% agarose gels, stainedwith ethidium bromide and visualized with UV light.PCR products were sequenced on an Applied Biosys-tems (Foster City, CA) 377 fluorescent sequencer,following standard protocols.

Good quality sequences were obtained fromextracts of P. rapae adults and frass and from V. carduiadults, frass, and exuviae as indicated in Table 2. Allfrass and exuviae sequences were exactly consistentwith that of the adult form of the individual that gener-ated the sample. ND5 fragments up to 475 base pairswere successfully amplified and sequenced from allP. rapae adult and frass samples. Only one of the fiveP. rapae frass samples allowed the amplification of aPCR product of 650 bases. The variable success ofthis amplification suggests that long target fragmentsof DNA exist in variable amounts in frass extracts. Asingle haplotype was found for ND5 of P. rapae, andwas deposited in GenBank (Accession #AY327133).V. cardui adult, exuviae and frass sample sequences allshowed a single unambiguous haplotype (Accession#AY327134).

These results confirm the utility of caterpillarfrass and exuviae as sources of DNA. Larvae maybe collected for microscopy or rearing experimentswith the simultaneous collection of frass or exuviaefor genetic analysis. This is the first report of DNAsequences obtained from insect frass and exuviae. Theuse of these novel sources need not be limited tobutterflies. The method is suitable for all larval insectsand may find wider applicability among invertebrates.

References

Aiello A (1993) How to prepare publishable reports of lepidopteranlife histories. News of the Lepidopterists’ Society, 1, 6–10.

Peterson A (1967) Larvae of Insects: An Introduction to NearcticSpecies. Part 1. Lepidoptera and Plant Infesting Hymenoptera,6th edn. Ohio State University.

Pyle RM (1984) Handbook for Butterfly Watchers. HoughtonMifflin Co., Boston/New York.

Reiss RA, Schwert DP, Ashworth AC (1995) Field Preservation ofColeoptera for Molecular Genetic Analyses. Environ Entomol,24(3), 716–719.