-19- USE OF DEVELOPMENT DATA TO ESTIMATE COLONIZATION TIME OF THE MYIASIS-CAUSING FLY, chrysomya bezziana (DIPTERA: CALLIPHORIDAE) COLLECTED FROM HUMAN WOUNDS Bambaradeniya Y.T.B.1, Karunarathne W.A.I.P.*1, Goonerathne I.2, Kotakadeniya R.B.3, Tomberlin J.K.4 1Department of Zoology, Faculty of Science, 2Department of Forensic Medicine, 3Department of Surgery, Faculty of Medicine, University of Peradeniya, Sri Lanka & 4Department of Entomology, Texas A & M University, College Station, Texas, USA ABSTRACT Forensic entomological techniques are environment, based on the time scale highly accepted for forensic investigations calculated from above method. all over the world, especially to estimate the time of colonization (TOC) as related to the Key words: Forensic entomology, time of postmortem interval (PMI) of human or colonization, accumulated degree day, other vertebrate remains as well as with myiasis, Chrysomya bezziana cases of neglect or abuse. Here, the accumulated degree days (ADD) method Corresponding author: [email protected] was used to calculate the TOC as related to three cases of myiasis associated with individuals admitted to the teaching hospital, Peradeniya during 2016. INTRODUCTION Chrysomya bezziana was recorded as the responsible species for all three cases. Forensic entomology is the application of Chrysomya bezziana is an obligatory arthropod-related material as evidence in myiasis-causing fly species commonly criminal investigations1. Entomological infesting human and farm animals mainly in evidence can be used to resolve three major tropical and subtropical countries. questions. Such material can be used to According to the present study, time of determine when, how and where a particular colonization of wounds by C. bezziana in crime scene was committed2. these three cases ranged from two to six days. Accordingly, the present paper Flies (Diptera) are the most common group highlights the lack of information to apply of insects used as evidence in forensic such forensic entomology investigations and investigations3. In some cases, immature the future steps to be taken to improve this flies are present in wounds of living field in Sri Lanka. In the future, forensic vertebrates. These events are referred to as medical and judiciary authorities in the myiasis4. Myiasis is considered the world’s country can incorporate this technique to fourth most common travel-associated skin pursue legal cases on verification of the disease after cutaneous larva migrans, malpractices of caregivers of wound pyodermas and pruritic arthropod-reactive patients in the professional and personal dermatitis5. Even though myiasis has been Sri Lanka Journal of Forensic Medicine, Science & Law-May 2016-Vol.7 No.1 -20- reported globally, such events most amount provided by surrounding thermal commonly occur in tropical and subtropical conditions. Therefore theoretically, the countries5. Typically myiasis associated development of a fly from egg to adult can larvae feed on the host’s living or dead be predicted based on the temperature tissue, liquid body substance, or on ingested conditions experienced. Lower and upper- foods6. Based on the host-larval temperature thresholds also bind insect relationship, myiasis is classified into three development. Immature insects exposed to major groups, 1) obligatory, 2) facultative, temperatures outside of this range and 3) accidental7. Larvae causing experience retarded growth or death13. obligatory myiasis infest live tissues where Based on data generated through as facultative larvae consume dead tissue. development studies of these insects, Accidental myiasis occurs when chance entomologists can estimate the TOC of ingestion of fly eggs or larvae that survive human or animals by these insects. The in the gastro intestinal tract6. Emphasizing present paper discusses the application of the infestation location, myiasis can be ADD/ADH to estimate TOC of myiasis- cutaneous, oral, nasal or gastrointestinal. causing larvae using already available Cutaneous myiasis is the most commonly development data set for the myiasis- encountered clinical form of the disorder causing fly species. and further can be classified into three groups: (1) furuncular, (2) creeping METHODOLOGY (migratory), and (3) wound (traumatic)8. Three patients (Table 01) diagnosed with Diptera is one of the largest insect orders, cutaneous myiasis were admitted to the with approximately 150,000 species in 150 surgery ward, teaching hospital, Peradeniya families, and 10,000 genera9. Several fly from February to April 2016. Larval species cause myiasis in humans. Examples samples were collected from each patient on include, but are not limited to, the primary the day of their admission. The larvae were screwworm Cochliomyia hominivorax removed from the wounds of each patient Coquerel (Diptera: Calliphoridae) in the and transferred into separate vials (7ml New World, Chrysomya bezziana bijou bottles) containing 3ml saline as a Villeneuve (Diptera:Calliphoridae), means to keep them alive. Larvae were Chrysomya megacephala Fabricius brought to the Entomology laboratory, the (Diptera: Calliphoridae), Wohlfahrtia University of Peradeniya for assessment. magnifica Schiner (Diptera: Sarcophagidae) Two to three larvae were preserved in 70% in the Old World10.Chrysomya bezziana is ethanol and the remaining were placed on a an obligate parasite, which causes myiasis 50g of piece of fish inside a petri dish, in a wide range of vertebrates including which was then stored in an uncontaminated livestock, domestic pets, and humans7. This insect-rearing jar (1L) containing saw dust species occurs throughout much of tropical at a depth of 1.25cm and stored under and subtropical Africa, the Indian laboratory conditions. This material served subcontinent and Southeast Asia from as pupation site for the post-feeding larvae southern China to New Guinea11. under room conditions. The mouth of the jar was covered with cloth mesh to prevent Accumulated Degree Days/Hours adult flies from escaping and jars were (ADD/ADH) are used to calculate TOC of placed on a shelf under ambient decomposing remains by insects12. temperature. Emerged flies were killed Poikilothermic creatures, such as insects, using ethyl acetate and curated using need a certain amount of energy to develop standard entomological techniques14. from one point of their life cycle to another. Preserved larvae and emergent adults were This growth is proportionate to the energy identified using identification keys 15. Sri Lanka Journal of Forensic Medicine, Science & Law-May 2016-Vol.7 No.1 -21- Stage of development was recorded for each RESULTS larva preserved from each case. ADD values for each identified life stage were calculated Species Identification using the following equation16: Larval samples were collected from three ADD (Accumulated Degree Days) = Time is patients admitted to the Peradeniya taken to attain each life stage (days) X Teaching Hospital during a period of three (surrounding temperature* – lower months in 2016. Larvae in all samples were threshold temperature of the species) identified as C.bazziana .C.bazziana larvae (*This can be environment/ experimental are easily distinguished from related species temperature or human body temperature) by the presence of several prominent characters such as pinkish coloration of the To estimate the colonization time for each body, heavy bands of dark, robust, thornlike sample, initially, ADD values were spines (Fig. 01), 4-6 lightly sclerotized (pale calculated for development data obtained brown) papillae or branches in anterior from a previously published laboratory spiracles (Fig. 02), heavily sclerotized study (Spradbery, 2002)17.For this study, the incomplete peritreme in dark brown to larval development temperature was blackish posterior spiracle and with 3 slit- considered 37°C as they were collected like spiracular openings at approximately from human wounds. The lower threshold 45º to the horizontal plane15 (Fig. 03). The temperature for the fly species was adult flies of C. bezziana were identified considered 10°C16. These calculated ADD using characters such as metallic blue, values for each development stage bluish purple or blue/green body color with corresponded to the standard energy amount predominantly orange colored heads and needed to attain each stage respectively. burgundy-coloured eyes15 (Fig. 04). 0.3mm 0.9 mm Figure 1: Heavy bands of dark, robust, Figure 2: Six lightly sclerotized (pale brown) thorn-like spines papillae or branches in anterior spiracles Sri Lanka Journal of Forensic Medicine, Science & Law-May 2016-Vol.7 No.1 -22- 0.6 mm 7.8 mm Figure 3: Heavily sclerotized incomplete Figure 4: Blue/green body color of adult C. peritreme in dark brown to blackish posterior bezziana fly spiracle and with 3 slit-like spiracular openings CASE DETAILS Case No. – 01 Four live larvae were collected from a 76- Patient sensed a burning type irritation from year-old male diabetic patient on 29/02/ the wound five days prior to admission to 2016 at 09.00 h at the Teaching Hospital, the hospital. One larva was preserved while Peradeniya. Clinical examination revealed the others were reared to the adult stage. the presence of the larvae infesting a wound The preserved larva was in the 2nd instar in the right heel of the patient. In the stage (Table 01). laboratory, two larvae from the total were preserved and the remaining two were Case No. – 03 reared to get the adults. The larvae were 3rd instarat the time of collection