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USE OF DEVELOPMENT DATA TO ESTIMATE COLONIZATION TIME OF THE -CAUSING , bezziana (DIPTERA: ) 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: , 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 -related material as evidence in myiasis-causing fly species commonly criminal investigations1. Entomological infesting human and farm 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 (Diptera) are the most common group highlights the lack of information to apply of 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 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 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 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 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 , the Indian laboratory conditions. This material served subcontinent and Southeast 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.

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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 2: Six lightly sclerotized (pale brown) Figure 1: Heavy bands of dark, robust, thorn-like spines papillae or branches in anterior spiracles

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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 (Table 01). Two live larvae were collected from a 65- year-old female diabetic patient on Case No. – 02 19/04/2016 at 13.00 h at the Teaching Hospital, Peradeniya. A larval infested Two live larvae were collected from a 30- wound was located under the 2nd left toe. year-old male patient on 18/03/2016 at The wound was first noticed by the patient 12.00 h at the Teaching Hospital, three weeks prior to being admitted to the Peradeniya. According to the clinical hospital. Collected larvae were in the examination, both legs of the patient had 3rdinstar stage (Table 01). severe wounds with the right heel infested with larvae.

Table 1: Background information of patients from which larvae were- collected. Case Sex Age Wound Life stage of the fly Number of larvae No. location collected collected 01 M 76 Right heel 3rd instar 4 02 M 30 Right heel 2nd instar 2 03 F 65 2nd toe, left foot 3rd instar 2

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Estimation of Colonization Time

Table 2: ADD values for the experimental development time of C. bezzianacalculatedusingdevelopment data given by Spradbery JP, 2001

Life Stage Threshold Developing Development Exp.DD ADD Minimum Temperature Time (h) (day= h/24) 10 °C 28 °C

Egg 20 h(0.83 days) 14.9 14.9

1st Instar 24 h (1day) 18 32.9

2nd Instar 24 h (1day) 18 50.9

3rd Instar 120 h (5days) 90 140.9

Pupa 168h(7days) 126 266.9 Total ADD 266.9

The lower threshold temperature for the fly species was considered 10°C 16. This is because in many calliphorid related studies this temperature was the standard lower threshold temperature of larval growth.

Table 3: Calculation of Degree Day (DH) and Accumulated Degree Day (ADD) for case study 01 with lower threshold minimum of 100C Date prior to Mean body DD ADD infestation Temperature 28/02/2016 37 27 27 27/02/2016 37 27 54 26/02/2016 37 27 81 25/02/2016 37 27 108 24/02/2016 37 27 135 *23/02/2016 37 27 162 22/02/2016 37 27 189 21/02/2016 37 27 216

*- Day attains the 3rdinstra larvae

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According to the above calculation, in case calculate the TOC associated with the study 01, eggs have been laid 6 days prior to PMI12.In Forensic entomology, two basic removal of larvae (23/02/2016). In case 02, methods have been used to calculate the eggs have been laid two days prior to PMI namely temperature related methods removal of larvae (16/03/2016). And, in and stage of succession12. ADD/ADH case 03, eggs have been laid 6 days prior to method and isomegalen/isomorphen removal (13/04/2016). diagram method are categorized under the temperature related methods which are the DISCUSSION major techniques of calculating the TOC. The use of temperature-related methods is Although this is not the first investigation to based on the hypothesized linear document C. bazziana infesting human relationship between the temperature and hosts in Sri Lanka, it is the first to utilize development of flies12. ADD/ADH method entomological techniques to estimate the was used here to identify the TOC of TOC of humans by this species. An wounds by larvae, as it is the standard and extensive study carried out by most sophisticated technique used in many Kumarasinghe et al18,in Colombo and forensic cases to estimate the development Kaluthara district over 18 months, which rate of flies over a period of time21. was initiated in 1997, recorded 16 myiasis cases with 14 involving C. bazziana. Prior In this study, several assumptions were to the current study, other three C. bazziana made. Typically with calculating ADD, infestations were only known from two ambient temperatures are used as the incidents of nasal myiasis19and one of function of the rate of development. But in cutaneous myiasis20. these cases, human body temperature was employed due to the host body serving as Risk factors leading to myiasis include poor the primary source of heat for larval personal hygienic practices, improper development. However, the impact of wound management and less concerning of ambient temperature in conjunction with good environmental hygiene19. The three body-associated heat on the development of patients in this study were from rural areas these insects is not known. associated with low socioeconomic development. A second issue that there was no data published on the development of C. Consequently, patients do not have proper bazziana at this temperature. Therefore, the access to health care professionals. The lack calculation was done using the available of resources possibly explains the poor temperature (28 °C) given by Spradbery17. wound care observed which could have led As the rate of larval development very with to ulceration and prolonged exposure to varying temperatures, this may have myiasis-causing flies. In this study, the affected the calculated ADD. Development infestation site associated with each patient studies done for blowflies (Diptera: was the foot. Kumarasinghe et al18 recorded Calliphoridae) show that the development 15 of 16 cases involving foot myiasis. They times within species vary for a variety of observed that this commonly occurred due species22, 23. to non-wearing of shoes by patients who came to hospitals from rural areas of the A third assumption was the data from country. Spradbery17 would be appropriate for application in the current study. The In this study, an attempt was made to variation could be the result of a number of calculate the TOC by C. bezziana using factors, such as experimental design, ADD. Historically, this method is used to environment, and genetic variation. Tarone

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