OPOLE SCIENTIFIC SOCIETY NATURE JOURNAL No 45 – 2012: 55-64 DETECTION AND IDENTIFICATION OF ALIEN DNA IN MUSEUM SPECIMENS OF HETEROPTERA USING MOLECULAR TECHNIQUES – A POSSIBILITY FOR APPLYING IN * FORENSIC ENTOMOLOGY ALEKSANDRA RAKOWIECKA, JERZY A. LIS1 Center for Biodiversity Studies, Department of Biosystematics, Opole University, Oleska 22, 45-052 Opole, Poland; 1 e-mail: cydnus@uni.opole.pl, http://www.cydnidae.uni.opole.pl ABSTRACT: Studies on nuclear DNA of museum specimens of pentatomoid bugs (Cydnidae, Dinidoridae, Thyreocoridae, and Tessaratomidae) are presented. Sequences of nuclear 28S rDNA subunit were analysed in the aspect of its usefulness in forensic entomology. Results of the study demonstrated that microorganisms and parasites detected by PCR methods can be useful in determining the geographical origin of the host-species with degraded DNA. KEY WORDS: forensic entomology, nuclear DNA, 28S rDNA, Heteroptera, Pentatomoidea, museum specimens, alien DNA. Introduction In last two decades, molecular techniques have been applied in many fields of science, such as phylogenetics, biogeography, medicine or forensic investigations. Nowadays, the analysis of DNA extracted from biological traces is widely used, especially in modern forensic investigations, where a molecular approach to identification of both, victims and criminals, are used to a large extent. Nuclear DNA in particular, seems to be a significant source for such analyses; however, also mitochondrial markers showed good feasibilities in human or animal species identification (Bellis et al. 2003, Caenazzo et al. 2008, Lis et al. 2011). ___________________________________________________________________________________________________________________________________________ * This paper presents partial results of the first author’s Master’s thesis (Detection and identification of microorganisms and parasites in museum specimens using molecular techniques – a possibility for applying in forensic biology), submitted to the Department of Biosystematics, Opole University, Opole, Poland, under the supervision of Prof. Jerzy A. Lis. 56 Application of DNA obtained from insects found at a crime scene can be regarded as the entirely new approach in forensic entomology. Since the late 19th century, insects are used to estimate PMI (post mortem interval) in homicide and other investigations. Most recently, many studies have demonstrated that DNA extracted from insect tissues can provide more informative evidences for investigations. In literature, there are numerous papers describing cases when a variety of blood feeding insects were used even to obtain human DNA (e.g., Wells et al. 2001, Spitaleri et al. 2006, Szalanski et al. 2006, Bucheli et al. 2010, Kester et al. 2010, Rasmy 2011). However, there are no papers which present the utility of alien DNA, obtained from the insect tissues, in forensic investigations. Therefore, in this paper the usefulness of alien DNA for such studies is considered. Material and methods 17 samples from museum specimens of four families of pentatomoid bugs (Cydnidae, Dinidoridae, Thyreocoridae, Tessaratomidae – Table 1) were selected for nuclear DNA analyses (one region of nDNA, i.e., 28S rDNA was analysed). All specimens come from the Heteroptera collection at the Department of Biosystematics (Opole University, Poland) (Lis et al. 2011). DNA extraction, purification and amplification were performed at the Centre for Biodiversity Studies (Department of Biosystematics, Opole University, Poland) using techniques described by Lis et al. (2011). Two primer sequences, i.e., 28Sa and 28Sb, were used for PCR amplification (Whiting et al. 1997, Edgecombe et al. 2002.). Sequencing was conducted at the Health Care Centre GENOMED (Warsaw, Poland). The trace files (electropherograms) produced by the automated DNA sequencers were edited by the Trace Data File Editor – Figs 1-8). For identification of alien DNA found in studied insects, obtained sequences were sent to the Web Browser for conducting BLAST searches (using blastn on NCBI). Results Readable sequences were obtained from all studied specimens. Unfortunately, no traces of original species DNA were recovered (all obtained sequences represented alien DNA). Sequences length varies from 60 bp to 338 bp (see Table 2, and Figures 1-8), and, usually, are characterized by a high level of noises. In five cases (see Table 2) obtained sequences suggested similarities of studied DNA to DNA of Saccoglossus kowalevskii (Hemichordata), seven sequences suggested similarities to fungi, two – to species of Cryptosporidiidae (Apicomplexa), another two – to species of Drosophila (Diptera), and one – to Nematostella vectensis (Cnidaria). No similarity of the recovered sequence to any species from GenBank was found only for Strombosoma impictum (Thyreocoridae), most probably because of its sequence length (60 bp) and high level of degradation. 57 Discussion Fungi. Though seven obtained sequences of alien DNA suggested close similarities to fungi species (Table 2), in four cases more than a single result were received from GenBank for each entry, i.e., for Ochetostethus balcanicus, Coridius laosanus, Megymenum paralleum, and Galgupha difficilis. Such situation may be caused by unclear systematic position of many fungi species, e.g., resulted Lodderomyces elongisporus can be a sexual stage of Candida parapsilosis, which is closely related to C. tropicalis (James et al. 1994). All fungi species, for which DNA was sequenced, are distributed worldwide (according to the Global Biodiversity Information Facility [GBIF] website). It seems possible that in all cases this alien DNA originated from a contact with man (i.e., Malassezia globosa, Ajellomyces dermatidis, Penicillium marneffei – suggesting human skin diseases; Candida tropicalis – usually occurring on human hands), or, was acquired from host plants during feeding process (Phaeosphaeria nodorum, Ashbya gossypii, Mycosphaerella punctiformis, Botryosphaeria dothidea – all are plant pathogens) (Smith and Stanosz 2001, Verkley 2004, Phillips et al. 2005, Hospenthal and Rinaldi 2008, Kirk et al. 2008, Blixt 2009). Apicomplexa. Two pathogenic protists, i.e., Cryptosporidium hominis and C. parvum were detected in Peltoxys sataranus and Coridius laosanus (Table 2). Both protist species are human parasites; moreover, C. parvum, is also pathogenic to cattle (Hashim et al. 2006). Most probably this alien DNA originated from a contact with man, because studied specimens of both heteropterans come from India and China, where both pathogens are injurious to people (Hashim et al. 2006). Cnidaria. In one case (i.e., Coridius nepalensis), results of our DNA study show similarities to DNA of Nematostella vectensis (Cnidaria). According to the Global Biodiversity Information Facility [GBIF] website, this cnidarian species of the family Edwardsiidae, is distributed from eastern coasts of the United States to the southern coasts of Great Britain. Because, the similarity of DNA sequences is relatively low (84%) and concerns only 195 bp (out of the total 288 bp) we assume this result as totally unbelievable, but we can’t give a sound explanation for it (see also: Putnam et al. 2007). Diptera. Also, in Tessarotoma quadrata, results of the DNA study was ambiguous (see Table 2), i.e., 100% similarity, but only on the length of 79 bp (out of the total 325 bp). Most probably, the presence of DNA of other (parasitic) dipteran species, which DNA was never before sequenced (and therefore not present in GenBank), is the only explanation. Hemichordata. In five cases (Table 2) obtained results indicated a similarity of alien DNA to DNA of Saccoglossus kowalevskii (class Enteropneusta). Because, it is impossible to find this species inside the insect body, we assume this fact as a result of a contamination by human DNA (see also: Acorn Worm Genome Project, www.hgsc.bcm.edu/content/acorn-worm-genome-project). 58 Conclusions In forensic entomology, when a crime has been committed, most often a murder, insects around and on the victim‘s body become the very important evidence. The precise DNA sequence of insects that appear on a corpse has been carefully documented by forensic entomologists. Such data are usually utilized for estimation of post-mortem interval (PMI), to locate scenes of murder crimes, and for toxicological analyses in the absence of human tissues and fluids normally taken for such purposes. Results of the present study have additionally demonstrated the potential utility of insects as good samples of alien DNA, which can be utilized for forensic purposes. The presence of alien DNA (e.g., DNA of fungi, or parasite DNA) in insect tissues may provide helpful information for resolving the problem of geographic origin of forensically important samples; as the most important this can be used to locate scenes of murder crimes. Bibliography Bellis C., Ashton K.J., Freney L., Blair B., Griffiths L.R. 2003. A molecular genetic approach for forensic animal species identification. Forens. Sci. Int. 134: 99-108. Blixt E. 2009. On Phaeosphaeria nodorum in wheat. Acta Uni. Agric. Sueciae 2009, 1, 55 pp. Bucheli S.R., Batheway J.A., Gangitano D.A. 2010. Necrophagous caterpillars provide human mtDNA evidence. J. Forens. Sci. 55: 1130-1132. Caenazzo L., Ceola F., Ponzano E., Novelli E. 2008. Human identification analysis to forensic purposes with two mitochondrial markers in polyacrilamide mini gel. Forens. Sci. Int.: Gen. 1: 266-268. Edgecombe G.D., Giribet G., Wheeler W.C. 2002. Phylogeny of Henicopidae (Chilopoda: Lithobiomorpha): a combined analysis of morphology and five molecular loci. Syst. Entomol. 27: 31-64. Hashim A., Mulcahy G., Bourke B., Clyne M. 2006. Interaction of Cryptosporidium hominis and Cryptosporidium parvum with primary human and bovine intestinal cells. Infect. Immun. 74: 99-107. Hospenthal D.R., Rinaldi M.G. (eds.) 2008. Diagnosis and Treatment of Human Mycoses. Series: Infectious Disease. Humana Press, XVI, 428 pp. James S.A., Collins M.D., Roberts I.N. 1994. The genetic relationship of Lodderomyces elongisporus to other ascomycete yeast species as revealed by small-subunit rRNA gene sequences. Lett. Appl. Microbiol. 19: 308-311. Kester K. M., Toothman M.H., Brown B. L., Street W.S., Cruz T.D. 2010. Recovery of environmental human DNA by insects. J. Forens. Sci. 55: 1543-1551. 59 Kirk P.M., Cannon P.F., Minter D.W., Stalpers J.A. 2008. Dictionary of the Fungi (10th ed.). Wallingford: CABI, 221 pp. Lis J.A., Ziaja D.J., Lis P. 2011. Recovery of mitochondrial DNA for systematic studies of Pentatomoidea (Hemiptera: Heteroptera): successful PCR on early 20th century dry museum specimens. Zootaxa 2748: 18-28. Phillips A.J.L., Rumbos I.C., Alves A., Correia A. 2005. Morphology and phylogeny of Botryosphaeria dothidea causing fruit rot of olives. Mycopathologia 159: 433–439. Putnam N., Srivastava M., Hellsten U., Dirks B., Chapman J., Salamov A., Terry A., Shapiro H., Lindquist E., Kapitonov V., Jurka J., Genikhovich G., Grigoriev I., Lucas S., Steele R., Finnerty J., Technau U., Martindale M., Rokhsar D. 2007. Sea Anemone genome reveals ancestral Eumetazoan gene repertoire and genomic organization. Science 317 (5834): 86-94. Rasmy A.H. 2011. The human lie but spiders do not lie: An overview on forensic acarology. Egypt. J. Forens. Sci. 1: 109-110. Smith D.R., Stanosz G.R. 2001. Molecular and morphological differentiation of Botryosphaeria dothidea (anamorph Fusicoccum aesculi) from some other fungi with Fusicoccum anamorphs. Mycologia 93: 505-515. Spitaleri S., Romano C., Di Luise E., Ginestra E., Saravo L. 2006. Genotyping of human DNA recovered from mosquitoes found on a crime scene. Int. Congr. Ser. 1288: 574-576. Szalanski A. L., Austin J.W., McKern J.A., McCoy T., Dayton Steelman C., Miller D.M. 2006. Time course analysis of bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), blood meals with the use of polymerase chain reaction. J. Agric. Urban Entomol. 23: 237-241. Verkley G.J., Crous P.W., Groenewald J.Z., Braun U., Aptroot A. 2004. Mycosphaerella punctiformis revisited: morphology, phylogeny, and epitypification of the type species of the genus Mycosphaerella (Dothideales, Ascomycota). Mycol. Res. 108: 1271-1282. Wells J.D., Introna F., Di Vella G., Campobasso C.P., Hayes J., Sperling F. 2001. Human and insect mitochondrial DNA analysis from maggots. J. Forens. Sci. 46: 685-687. Whiting M.F., Carpenter J.C., Wheeler Q.D., Wheeler W.C. 1997. The Strepsiptera problem: phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Syst. Biol. 46: 1-68. 60 Table 1. Details of examined specimens. Species and family Geographic origin Year collected Adrisa flavomarginata (Voll.) Cydnidae New Caledonia 2008 Adrisa flavomarginata (Voll.) Cydnidae New Caledonia 2008 Chilocoris assmuthi Bredd. Cydnidae Pakistan 1972 Ochetostethus balcanicus Wagn. Cydnidae Croatia 1957 Peltoxys sataranus Lis & Lis Cydnidae India 2002 Pseudoscoparipes nilgiricus Lis Cydnidae India 1976 Coridius laosanus (Dist.) Dinidoridae China 1915 Coridius nepalensis (Westw.) Dinidoridae Thailand 1995 Coridius prolixus (Leth.) Dinidoridae Togo 1954 Cyclopelta obscura (Leth. & Serv.) Dinidoridae Thailand 1995 Cyclopelta siccifolia (Westw.) Dinidoridae Nepal 1986 Cyclopelta siccifolia (Westw.) Dinidoridae Sri Lanka 1894 Megymenum brevicorne (F.) Dinidoridae Thailand 1995 Megymenum paralleum Voll. Dinidoridae Thailand 1995 Tessarotoma quadrata Dist. Tessaratomidae Vietnam 1990 Galgupha difficilis (Bredd.) Thyreocoridae Brasil 1975 Strombosoma impictum (Stål) Thyreocoridae Zaire 1932 61 Table 2. Similarities of obtained sequences to those available from GenBank (using BLAST); (length of the sequence and similarity percentage for each species are shown in square brackets). Studied species Family [length of sequence in bp] Highest similarity to species from GenBank Adrisa flavomarginata Cydnidae [338] Saccoglossus kowalevskii [93% (311)] Harrimaniidae (Hemichordata) Adrisa flavomarginata Cydnidae [338] Saccoglossus kowalevskii [93% (311)] Harrimaniidae (Hemichordata) Chilocoris assmuthi Cydnidae [335] Saccoglossus kowalevskii [92% (305)] Harrimaniidae (Hemichordata) Ochetostethus balcanicus Cydnidae [295] Phaeosphaeria nodorum [92% (411)] Phaeosphaeriaceae (Fungi: Dothideomycetes) Lodderomyces elongisporus [92% (411)] Debaryomycetaceae (Fungi: Saccharomycetes) Peltoxys sataranus Cydnidae [292] Cryptosporidium hominis [95% (279)] Cryptosporidiidae (Apicomplexa: Eucoccidiorida) Pseudoscoparipes nilgiricus Cydnidae [291] Malassezia globosa [89% (357)] Malasseziaceae (Fungi: Exobasidiomycetes) Coridius laosanus Dinidoridae [287] Cryptosporidium hominis [88% (183)] Cryptosporidiidae (Apicomplexa: Eucoccidiorida) Cryptosporidium parvum [88% (183)] Cryptosporidiidae (Apicomplexa: Eucoccidiorida) Coridius nepalensis Dinidoridae [288] Nematostella vectensis [84% (195)] Edwardsiidae (Cnidaria: Actiniaria) Coridius prolixus Dinidoridae [288] Malassezia globosa [81% (237)] Malasseziaceae (Fungi: Exobasidiomycetes) Cyclopelta obscura Dinidoridae [330] Saccoglossus kowalevskii [92% (305)] Harrimaniidae (Hemichordata) Cyclopelta siccifolia Dinidoridae [290] Mycosphaerella punctiformis [87% (327)] Mycosphaerellaceae (Fungi: Dothideomycetes) Cyclopelta siccifolia Dinidoridae [293] Botryosphaeria dothidea [85% (313)] Botryosphaeriaceae (Fungi: Dothideomycetes) Megymenum brevicorne Dinidoridae [337] Saccoglossus kowalevskii [91% (302)] Harrimaniidae (Hemichordata) 62 Megymenum paralleum Dinidoridae [326] Talaromyces stipitatus [88% (361)] Trichocomaceae (Fungi: Eurotiomycetes) Penicillium marneffei [88% (361)] Trichocomaceae (Fungi: Eurotiomycetes) Ajellomyces dermatitidis [88% (361)] Ajellomycetaceae (Fungi: Eurotiomycetes) Galgupha difficilis Thyreocoridae [290] Candida tropicalis [84% (315)] Incertae sedis (Fungi: Saccharomycetes) Penicillium marneffei [84% (315)] Trichocomaceae (Fungi: Eurotiomycetes) Ashbya gossypii [84% (315)] Saccharomycetaceae (Fungi: Saccharomycetes) Strombosoma impictum Thyreocoridae [60] No similarity found Tessarotoma quadrata Tessaratomidae [325] Drosophila simulans [100% (79)] Drosophilidae (Diptera) Drosophila melanogaster [100% (79)] Drosophilidae (Diptera) 63 Figures 1-4. Electropherograms showing the same fragment of selected sequences. 1 – Adrisa flavomarginata (Cydnidae), 2 – Ochetostethus balcanicus (Cydnidae), 3 – Pseudoscoparipes nilgiricus (Cydnidae), 4 – Coridius laosanus (Dinidoridae). 64 Figures 5-8. Electropherograms showing the fragment of selected sequences. 5 – Coridius nepalensis (Dinidoridae), 6 – Megymenum paralleum (Dinidoridae), 7 – Galgupha difficilis (Thyreocoridae), 8 – Strombosoma impictum (Thyreocoridae).