DOI: 10.2478/s11686-014-0242-2 © W. Stefański Institute of Parasitology, PAS Acta Parasitologica, 2014, 59(2), 301–304; ISSN 1230-2821 Confirming (Diptera: Oestridae) ophthalmomyiasis by larval DNA barcoding

Bjørn Arne Rukke1*, Symira Cholidis2, Arild Johnsen3 and Preben Ottesen1 1Department of Pest Control, Norwegian Institute of Public Health, Lovisenberggata 8, 0456 Oslo, Norway; 2Department of Ophthalmology, Oslo University Hospital, Kirkeveien 166, 0450 Oslo, Norway; 3Natural History Museum, University of Oslo, Sars gate 1, 0562 Oslo, Norway

Abstract DNA barcoding is a practical tool for species identification, when morphological classification of an organism is difficult. Herein we describe the utilisation of this technique in a case of ophthalmomyiasis interna. A 12-year-old boy was infested during a summer holiday in northern Norway, while visiting an area populated with . Following medical examination, a Diptera larva was surgically removed from the boy’s and tentatively identified from its morphological traits as Hypoderma tarandi (L.) (Diptera: Oestridae). Ultimately, DNA barcoding confirmed this impression. The larval cytochrome c oxidase subunit 1 (COI) DNA sequence was matched with both profiles of five adult H. tarandi from the same region where the boy was infested, and other established profiles of H. tarandi in the Barcode of Life Data Systems (BOLD) identification engine.

Keywords Ophthalmomyiasis, species identification, DNA barcoding, Hypoderma tarandi

Introduction polar geographical range (Zumpt 1965). Eggs are laid directly on guard hairs of these . When the larvae hatch, they Ophthalmomyiasis is a condition where larvae of some penetrate the skin, migrating subcutaneously to dorsal areas of Diptera invade the human eye or its coverings. In ophthal- the bodies. At this point, they become encased, forming granu- momyiasis externa, the organisms are found in the conjuncti- lomatous cysts (so-called warbles), and they cut holes for val sac, or rarely, in a warble on the eyelid. In ophthalmo- breathing. After 9–11 months of further development, the larvae interna, the larvae actually enter the globe of the eye exit the host to pupate in the ground (Kearney et al. 1991). (Kearney et al. 1991) which may damage the retina, lens, or Identification of parasitic from larvae usually requires iris, ultimately leading to retinal detachment, lens displace- an intact specimen, so that the cephalopharyngeal skeleton and ment, uveitis, and glaucoma (Entezari et al. 2004, Francesconi posterior spiracles can be studied (Kearney et al. 1991). In and Lupi 2012). ophthalmomyiasis, this may be difficult to achieve, and alter- Most cases of ophthalmomyiasis are caused by two native methods of identification are often sought. Time-con- species: (L. Jr) (human bot ) and suming identification of H. tarandi through histological Oestrus ovis L. ( bot fly) (Diptera: Oestridae). The first sections has been done (Kearney et al. 1991). Serological test- is found throughout Central and South America (Anderson ing (Western blot) for H. tarandi has also been used to confirm 2006), while the latter has nearly world-wide distribution infestations (Lagacé-Wiens et al. 2008, Kan et al. 2012). but is not known to inhabit Norway (Kearney et al. 1991). DNA barcoding is a relatively new approach that may have The two species most often cause ophthalmomyiasis externa value in this setting. It provides rapid and accurate species (Anderson 2006). In northern Scandinavia and Canada, the identification (with potential for automation) by using short, reindeer warbler fly, Hypoderma tarandi (L.) (Diptera: Oestri- standardized gene regions as internal species tags (Hebert et dae), is an occasional cause of ophthalmomyiasis (Kearney al. 2003a). Among other things, it may be used to determine et al. 1991, Syrdalen et al. 1982, Lagacé-Wiens et al. 2008, taxonomic placement if an organism is damaged, if only frag- Kan et al. 2012, 2013). It is a non-biting fly whose obligate en- ments remain, or if morphology is insufficient to discriminate doparasitic larvae typically affect caribou/reindeer in circum- between species (Frézal and Leblois 2008). For animals, a part

*Corresponding author: [email protected] 302 Bjørn Arne Rukke et al.

of the cytochrome c oxidase subunit 1 (COI) gene has been identified by a taxonomic expert (Arne C. Nilssen, University chosen as the official DNA barcoding region in the Interna- of Tromsø, Norway), based on morphological traits. Later they tional Barcode of Life initiative (Hebert et al. 2003b). DNA were DNA barcoded at the Natural History Museum, Univer- barcoding has earlier been used in identification of Hypo- sity of Oslo. derma larvae, including H. tarandi (Otranto et al. 2003, 2004, Kan et al. 2012), but the region of the COI gene then used is DNA barcoding different from the standard DNA barcoding region, making comparisons difficult. DNA was extracted from the midsection of the larva and from In the present study, we report a case of ophthalmomyia- one leg from each of five adult flies, using the E.Z.N.A. tissue sis in a boy who was infested while visiting northern Nor- extraction kit (Omega Bio-Tek Inc., Norcross, USA). The bar- way. DNA barcoding was used to confirm the initial suspicion coding region of the COI gene was amplified using LepF1 of H. tarandi, based on morphological traits, by comparing (ATTCAACCAATCATAAAGATATTGG (Hajibabaei et al. the larva’s sequence with profiles of H. tarandi in the Bar- 2006)) and LepR1 (TAAACTTCTGGATGTCCAAAAAATCA code of Life Data Systems (BOLD) identification engine. (Hajibabaei et al. 2006)) primers. A 10-uL PCR reaction was Additionally, to enhance the barcode library of H. tarandi, performed by standard protocol (50 ng template DNA, Ampli- we barcoded five adult specimens of this fly from the north- Taq DNA polymerase [Applied Biosystems, Foster City, USA], ern part of Norway. 55ºC annealing temperature). The PCR product was purified using ExoSap-It (United States Biochemical, Cleveland, USA), cycle-sequenced in both directions with BigDye 3.1 (Applied Materials and Methods Biosystems, Foster City, USA) and run on an ABI 3130xl Ge- netic Analyser (Applied Biosystems, Foster City, USA). For the The fly larva five adult specimens, sequencing in both direction produced high-quality, unambiguous COI sequences, 658 bp long. These In October, 2011, a 12-year-old boy was referred by a private sequences were added to the Barcode of Life Data Systems ophthalmologist to the Department of Ophthalmology at Oslo (BOLD), under the project Hypoderma Diptera (HYPDI), and University Hospital. He had been vacationing in Måsøy Mu- submitted to Genbank (Genbank accession numbers KF147191- nicipality, Finnmark County, in the northern part of Norway in KF147195). For the larvae, only the forward primer yielded late July, 2011. A herd of reindeer was in the vicinity. a high-quality sequence (636bp long), with no ambiguous nu- Since September, 2011, the boy had suffered a left unilat- cleotide positions. This sequence (Genbank accession number eral panuveitis. Visual acuity of the affected eye was 0.1 JX961601) was entered into the BOLD identification engine (20/200), compared with 1.0 (20/20) in the other healthy eye. (http://www.boldsystems.org/index.php/IDS_OpenIdEngine), Clinical examination revealed a moderate anterior uveitis and filtering for a species-level match by barcode. A neighbour-join- severe vitritis, but a clear view of the retina was not possible. ing gene tree was created in Mega 5.1, with the Kimura-2- He was otherwise healthy, claiming no history of trauma to parameter substitution model and 1000 bootstrap iterations, the eye. The uveitis was treated with topical and systemic using all publicly available Hypoderma COI sequences from steroids but failed to appreciably improve. Genbank and/or BOLD. On his third visit to Oslo University Hospital, a parasitic larva was discovered in the anterior chamber of the affected eye. Surgery was performed that day, and the larva was ex- Results tracted in its entirety. At follow up, peripheral retinal detach- ment was found, as well as subluxation of the crystalline lens. Microscopy Consequently, retinal surgery with vitrectomy was done, and cataract surgery was later performed, incorporating a Cionni The intact larva was subjected to microscopic study. It was ring. After surgery, best corrected visual acuity was 0.8 less than 5 mm in length. The body shape and patterning of (20/25), but the patient still suffered mild anterior uveitis and spines at its rear resembled a first-stage larva of H. tarandi, as glaucoma. described by Ferrar (1987). Rather than pursuing more elab- The larva was sent to Norwegian Institute of Public Health orate morphological analysis, we opted for DNA barcoding as for routine morphological analysis. Later, DNA barcoding was a time-saving measure. carried out at the Natural History Museum, University of Oslo. DNA barcoding Adult H. tarandi The BOLD identification engine returned a 98.26–99.05 Five adult H. tarandi had been sampled in July 2009 from the sequence similarity between the larva and the five adult northern part of Norway (Finnmark County), the same region H. tarandi flies from Northern Norway (Table I). Also, there where the 12 year old boy was infested. Initially the flies were was a 98.58–98.74% sequence similarity between our larva Confirming ophthalmomyiasis by barcoding 303

Table I. Output from the Barcode of Life Data Systems (BOLD) identification engine, showing sequence similarity of the suspected Hypoderma tarandi larva with the ten highest specimen hits

Specimen Phylum Class Order Family Species Location similarity (%) Arthropoda Insecta Diptera Oestridae Hypoderma tarandi Norway 99.05 Arthropoda Insecta Diptera Oestridae Hypoderma tarandi Norway 98.74 Arthropoda Insecta Diptera Oestridae Hypoderma tarandi Norway 98.74 Arthropoda Insecta Diptera Oestridae Hypoderma tarandi Canada 98.74 Arthropoda Insecta Diptera Oestridae Hypoderma tarandi Canada 98.74 Arthropoda Insecta Diptera Oestridae Hypoderma tarandi Canada 98.58 Arthropoda Insecta Diptera Oestridae Hypoderma tarandi Canada 98.58 Arthropoda Insecta Diptera Oestridae Hypoderma tarandi Norway 98.26 Arthropoda Insecta Diptera Oestridae Hypoderma tarandi Norway 98.26 Arthropoda Insecta Diptera Calliphoridae Lucilia sericata – 85.38 and four other H. tarandi specimens from Canada which were Lucilia sericata (Meigen) (Diptera: Calliphoridae). Note that already present in BOLD. Otherwise, a considerable gap the BOLD database contains sequences from Hypoderma existed for second-best matched species (85% similarity with bovis (L.), Hypoderma lineatum (de Villers), and Hypoderma

Fig. 1. Tree of CO1 sequences from unknown larva and five adult Hypoderma tarandi sampled from same part of Norway together with other specimens of Hypoderma sp. from BOLD 304 Bjørn Arne Rukke et al.

sinense Pleske (Diptera: Oestridae), all around 80% similar to Entezari S.M., Key L., Athari A., Ramin S. 2004. Ophthalmomyia- our test specimen (Fig. 1). sis interna posterior: Report of a case with lens subluxation and multiple retinal breaks. Annals of Ophthalmology, 36, 129–131. Ferrar P. 1987. A guide to the breeding habits and immature stages of Discussion Diptera Cyclorrhapha. E. J. Brill, Leiden, Netherlands, 907 pp. Francesconi F., Lupi O. 2012. Myiasis. Clinical Microbiology Re- Confirmation of our larva study specimen as H. tarandi is views, 25, 79–105. DOI: 10.1128/CMR.00010-11. Frézal L., Leblois R. 2008. Four years of DNA barcoding: Current valid, unless the larva belongs to a Hypoderma species not advances and prospects. Infection, Genetics and Evolution: currently represented in the BOLD database. Given that only Journal of Molecular Epidemiology and Evolutionary Genet- three species of Hypoderma are known to occur in Norway ics in Infectious Diseases, 8, 727–736. DOI: 10.1016/j.meegid. (H. tarandi, H. bovis and H. lineatum) (Ottesen 1993), this 2008.05.005. seems highly unlikely. Additionally, a considerable number of Hajibabaei M., Janzen D.H., Burns J.M., Hallwachs W., Hebert P.D.N. 2006. DNA barcodes distinguish species of tropical human myiasis cases due to H. tarandi infestation has been Lepidoptera. Proceedings of the National Academy of Sci- identified in Northern Norway recent years (Kan et al. 2012, ences of the United States of America, 103, 968–971. DOI: 2013), revealing that this is not an uncommon phenomenon. 10.1073/pnas.0510466103. The sequence discrepancy between our larval test specimen Hebert P.D.N, Cywinska A., Ball S.L., deWaard J.R. 2003a. Biolog- and both our barcoded adult flies from the same region and ical identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B, 270, 313–321. DOI: those from North America, illustrate a relatively high intra- 10.1098/rspb.2002.2218. specific sequence variation (mean sequence variation among Hebert P.D.N., Ratnasingham S., deWaard J.R. 2003b. Barcoding an- the five Norwegian specimens: 1%), which is in the same imal life: cytochrome c oxidase subunit 1 divergences among order of magnitude both on a small and large spatial scale. closely related species. Proceedings of the Royal Society of Similarly, Otranto et al. (2003) found a 1.59% sequence vari- London. Series B (Supplement), 270, S96–99 (2003). Kan B., Otranto D., Fossen K., Åsbakk K. 2012. Dermal swellings ation within H. tarandi using another part of the COI gene. and ocular injury after exposure to reindeer. The New Eng- Our study demonstrates the utility of DNA barcoding in land Journal of Medicine, 367, 2456–2457. DOI: 10.1056/ species identification of an early-life-stage parasitic fly. By NEJMc1201434. standard morphological inspection, this process is difficult and Kan B., Åsbakk K., Fossen K., Nilssen A., Panadero R., Otranto D. time-consuming even when facing an intact larva. Further- 2013. Reindeer -associated human myiasis, Scan- dinavia. Emerging Infectious Diseases, 19, 830–832. DOI: more, morphological inspection will not be possible in cases 10.3201/eid1905.130145. where the posterior part of the eye (retina and corpus vitreum) Kearney M.S., Nilssen A.C., Lyslo A., Syrdalen P., Dannevig, L. is affected. In order to remove the larva in such cases, vitrec- 1991. Ophthalmomyiasis caused by the reindeer warble fly tomy is performed. Such a procedure will damage the larva larva. Journal of Clinical Pathology, 44, 276–284. ruling out morphological inspection of an intact specimen. Lagacé-Wiens P.R.S., Dookeran R., Skinner S., Leicht R., Colwell D.D., Galloway T. D. 2008. Human ophthalmomyiasis interna This leaves DNA barcoding as a suitable method to detect lar- caused by Hypoderma tarandi, Northern Canada. Emerging val DNA to confirm clinical diagnosis when ophthalmomya- Infectious Diseases, 14, 64–66. DOI: 10.3201/eid1401.07 sis interna is suspected. 0163. Further development of a barcoding library, as we have Otranto D., Colwell D.D., Traversa D., Stevens J.R. 2003. Species done here, seems highly justified. A tool of this nature would identification of Hypoderma affecting domestic and wild ru- minants by morphological and molecular characterization. complement traditional identification methods, contributing Medical and Veterinary Entomology, 17, 316–325. DOI: to improved public health through enhanced diagnostics. 10.1046/j.1365–2915.2003.00446.x. Otranto D., Traversa D., Colwell D.D., Guan G., Giangaspero A., Acknowledgements. Professor Arne C. Nilssen kindly donated five Boulard C., Yin H. 2004. A third species of Hypoderma adult Hypoderma tarandi flies. We thank Kjersti S. Kvie and Gunnhild (Diptera: Oestridae) affecting and yaks in China: mo- lecular and morphological evidence. Journal of Parasitology, M. Martinsen for laboratory assistance and help with analyses. 90, 958–965. Ottesen P.S. 1993. Norwegian families and their species num- ber. Norwegian Institute for Nature Research. Report No.: References NINA Utredning 55, Trondheim, Norway, 40 pp. Syrdalen P., Nitter T., Mehl R. 1982. Ophthalmomyiasis interna pos- Anderson J.R. 2006. Oestrid myiasis in . In: (Eds. D.D. terior: Report of case caused by the reindeer warble fly larva Coldwell, M.J.R. Hall and P.J. Scholl) The oestrid flies – and review of previous reported cases. The British Journal of biology, host-parasite relationships, impact and management. Ophthalmology, 66, 589–593. CABI Publishing, Oxford, UK, 201–209. Zumpt F. 1965. Myiasis in man and animals in The Old World: a text- book for physicians, veterinarians and zoologists. Butter- worths, London, UK, 276 pp.

Received: August 7 ,2013 Revised: March 7, 2014 Accepted for publication: March 13, 2014