Journal of Medical Entomology Advance Access published March 5, 2015

DIRECT INJURY,,FORENSICS Survey of the Genetic Diversity of Forensically Important (Diptera: ) from Egypt

1,2,3 2 1 ABEER M. SALEM, FATMA K. ADHAM, AND CHRISTINE J. PICARD

J. Med. Entomol. 1–9 (2015); DOI: 10.1093/jme/tjv013 ABSTRACT Minimum postmortem interval estimations of a corpse using blow larvae in medicolegal investigations require correct identification and the application of appropriate developmental data of the identified fly species. Species identification of forensically relevant blow could be very difficult and time consuming when specimens are damaged or in the event of morphologically indistinguishable immature stages, which are most common at crime scenes. In response to this, an alternative, accurate determination of species may depend on sequencing and molecular techniques for identification. Chrys- omyinae specimens (n ¼ 158) belonging to three forensically important species [ (Wiedemann), (F.), and Chrysomya marginalis (Wiedemann)] (Diptera: Calli- phoridae) were collected from four locations in Egypt (Giza, Dayrout, Minya, and North Sinai) and sequenced across the mitochondrial cytochrome oxidase subunit I (COI) gene. Phylogenetic analyses using neighbor–joining, maximum likelihood and maximum parsimony methods resulted in the same topological structure and confirmed DNA based identification of all specimens. Interspecific divergence between pairs of species was 5.3% (C. marginalis–C. megacephala), 7% (C. albiceps–C. megacephala), and 8% (C. albiceps–C. marginalis). These divergences are sufficient to confirm the utility of cytochrome oxidase subunit I gene in the molecular identification of these flies in Egypt. Importantly, the maximum intraspecific divergence among individuals within a species was <1% and the least nucleotide divergence between species used for phylogenetic analysis was 3.6%. This study highlights the need for thorough and diverse sampling to capture all of the possible genetic diversity if DNA barcoding is to be used for molecular identification.

KEY WORDS Blow fly, , COI, phylogenetic analysis

Insects of the genus Chrysomya are composed of a entomologists because some species within this genus group of blow flies that exhibit important roles in the are ectoparasitic, causing myiasis in (typically decomposition ecology of the environment. These flies, livestock) and humans (Zumpt 1965, Erzinclioglu often referred to as carrion flies, recycle dead organic 1989). Following death, these flies are able to detect materials from the environment. Because these and locate carrion sources rapidly, in some instances are among the first to arrive at carrion, they represent within minutes (Amendt et al. 2004), and as such, a large proportion of insects recovered from human Chrysomya are of great interest to ecologists and foren- corpses in cases of suspicious deaths or murders (Byrd sic entomologists (Kamal 1958, Norris 1965, Rognes and Castner 2000). Chrysomya are native to the Old 1991, Wells and Greenberg 1992, Gomez et al. 2003, World tropics, subtropics, and Australia (Zumpt 1965); Gabre et al. 2005, Singh et al. 2011). however, Chrysomya species have been successful at To use entomological evidence in criminal investiga- invading new geographic areas in the New World tions, correct identification of the specimen and accu- (Baumgartner and Greenberg 1984). Chrysomya spe- rate calculation of larval age are critical for estimating a cies also draw the attention of medical and veterinary precise minimum postmortem interval, as long as ovi- position or larvaposition followed death. Recently, there has been a shift in using molecular techniques (DNA sequencing) to ensure fast and reliable species identifi- 1 Department of Biology, Indiana University Purdue University India- cation of forensically important flies (Sperling et al. napolis (IUPUI), 723 W. Michigan Street, SL 306 Indianapolis, IN 1994, Benecke 1998, Harvey et al. 2003a,b, Zaidi et al. 46202. 2 2011). Two reasons for an alternative technique include Department of Entomology, Faculty of Science, Cairo University, the difficulty in identification for immature stage in- P.O. Box 12613, Giza, 12613, Egypt. 3 Corresponding author, e-mail: [email protected] or sects, and few or no comprehensive morphological keys abmsalem @iupui.edu. exist. A number of different techniques are used for

VC The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: [email protected] 2JOURNAL OF MEDICAL ENTOMOLOGY molecular identification, which include direct sequenc- distributed in adjacent areas were included from pub- ing of DNA (Sperling et al. 1994), restriction fragment lished databases in our phylogenetic analyses. This length polymorphism (Ratcliffe et al. 2003) analysis, study is important because it intends primarily to estab- and amplified fragment length polymorphism (Picard lish baseline data for DNA sequencing of some abun- and Wells 2012) analysis. Though restriction fragment dantly encountered Chrysomyine in Egypt. length polymorphism and amplified fragment length polymorphism are useful techniques, sequencing vari- ous genetic loci has become the predominant method. Materials and Methods Various regions of nuclear and mitochondrial DNA have been proposed and used for molecular identifica- Samples. In total, 158 adult blow fly specimens tion of calliphorid species: 1) the nuclear internal tran- were collected from four locations in Egypt: Giza, scribed spacer locus (Zaidi et al. 2011); 2) an Dayrout, Minya, and North Sinai between March and alternative internal transcribed spacer locus (Nelson June 2013 (Table 1). Flies were collected using a sweep et al. 2008); 3) the nuclear protein coding gene EF-1a net in fish markets, or using fish-baited traps. Samples (Caterino et al. 2000); 4) mitochondrial 16S rRNA of flies were kept alive in a cage until they were killed gene (Li et al. 2010); the mitochondrial small rRNA in 70% ethanol. Specimens were identified to species (12S) gene, 5) the nonconserved control, or AT-rich, re- using morphological characters, according to published gion of the mtDNA (Stevens and Wall 1997); 6) the cy- taxonomic keys (Setyaningrum and Al Dhafer 2014). tochrome b gene (Gilarriortua et al. 2013); and 7) DNA Extraction. DNA was extracted from the mitochondrial coding genes as cytochrome oxidase sub- head of the flies using DNeasy Blood and Tissue Kit units I (COI; Harvey et al. 2008, Singh et al. 2011)and (Qiagen, Valencia, CA) following manufacturer’s II (COII; Sperling et al. 1994, Wallman and Donnellan instructions. The remaining tissue was retained as a 2001). Mitochondrial loci are useful because of high voucher specimen at 20C in ethanol at Indiana copy number (good for degraded samples), and the University–Purdue University Indianapolis. presence of conserved regions for the design of univer- Polymerase Chain Reaction (PCR) sal primers flanking the locus (Benecke and Wells Amplification and COI Sequencing. For every 2001). The identification of a universal DNA locus is extract, the COI fragment was amplified as two sepa- optimal, and most of the current research has used the rate overlapping fragments. Primer combinations COI, which includes the barcode region (648 bp; 1a þ 2wereusedforChrysomya albiceps Hebert et al. 2003). Proposed sequenced regions rang- (Wiedemann), whereas primer combinations 1b þ 2 ing from 229 bp to the entire COI gene has resulted in were used for Chrysomya megacephala (F.) and Chrys- various degrees of success for the molecular identifica- omya marginalis (Wiedemann) (Table 2). Each 20-ml tion of Calliphorid species (Wallman et al. 2005, Zhang PCR reaction was composed of 10 mlof2 Promega et al. 2007, Desmyter and Gosselin 2009, Park et al. Master Mix (Promega Corp., Madison, WI), 2 mlof 2009). Thus, the longer or more complete COI locus is each primer (10 pmol/ml; Integrated DNA Technologies more robust. However, COI sequencing for molecular Inc., Coralville, IA), 2 mloftemplateDNA,and6mlof identification has failed in discrimination between PCR water (Promega Corp.). Amplification was per- some closely related species within the genera Calli- formed using either Mastercycler (Eppendorf Inc., phora (Wallman et al. 2001), Lucilia (Wells et al. 2007, Hamburg, Germany) or Veriti 96-Well (Life Technolo- DeBry et al. 2013), and Chrysomya (Harvey et al. gies Inc., Carlsbad, CA) thermal cyclers using the fol- 2008). Multi-gene approaches are proposed alternatives lowing conditions: 94C for 2 min, 35 cycles of 94C to COI sequencing (Zaidi et al. 2011, Dai et al. 2012); (30 s), 47C(30s),72C (90 s), and a final extension at however, the resulting phylogenetic trees based on 72C for 7 min. The sizes of the amplified fragments COI fragments were similar to those based on three were verified using agarose gel electrophoresis. different gene fragments (Zaidi et al. 2011). PCR products were purified using ExoSAP-IT (USB Finding an appropriate locus for molecular identifi- Corp., Cleveland, OH) according to the manufacturer’s cation of species is crucial, but so is thorough sampling instructions. Sequencing, done in both directions, was of the potential genetic variation across populations of done in a total volume of 5 ml using BigDye Terminator each species in the location for which this method is to v3.1 Cycle Sequencing Kit (Life Technologies Inc.) be applied. In Egypt, few studies evaluated the use of according to Singh et al. (2010) using 3.2 pmol of each COI and COII fragments as genetic markers in the PCR primer and 1 ml of purified PCR product in a identification of local species of forensic impact (Aly Mastercycler thermal cycler (Eppendorf Inc.). Cycling and Wen 2013, Aly et al. 2013, Aly 2014). The results conditions for sequencing reactions were performed found that long COI fragment outperformed a shorter according to manufacturer recommendations but the one in species identifications, and recommended using annealing temperature was lowered to 48Cwhenpri- a larger sample size to confirm their findings. As a re- mers TY-J-1460a, C1-N-2191, and C1-N-2293 were sult, we aim to improve the genetic resources available used. The cycle sequencing products were purified for Egyptian carrion flies using a large population sam- using BigDye XTerminator Purification Kit (Life Tech- ple to determine that species can be reliably identified nologies Inc.) using manufacturer’s specifications. Auto- using long COI sequencing, and that the intraspecific mated sequencing was performed using Applied divergences do not vary across ecoregions in Egypt. Biosystems 3500 genetic analyzer (Life Technologies Additional sequences of carrion flies that are commonly Inc.) using default conditions. The resulting sequences 2015 SALEM ET AL.: IDENTIFICATION OF Chrysomya 3

Table 1. List of species, collection dates, locations, and the number of individuals used in this study

Species Location No. of Haplotypes Date of (Latitude/Longitude) individuals collection (no. haplotypes)

C. albiceps Giza, Egypt 30 (4) AF083657, C. alb VI, IX, and XII 27 May 2013 (Wiedemann, 1819) (3000 N/31 10 E) Minya, Egypt 9 (3) C. alb I, V, and VI 12 Mar. 2013 (30 70 N/30 33 E) Dayrout, Egypt 23 (7) AF083657, C. alb I, III IV, 4 June 2013 (27 34 N/30 49 E) VII, VIII, X, and XI North Sinai, Egypt 8 (2) C. alb I and III 2 May 2013 (31 02 N/33 00 E) C. megacephala Giza, Egypt 24 (4) C. meg I, V, XI, and XII 27 May 2013 (F., 1794) (30 00 N/31 10 E) Minya, Egypt 1 (1) C. meg IX 12 Mar. 2013 (30 70 N/30 33 E) Dayrout, Egypt 25 (7) C. meg I, II, III, IV, VIII, X, and XI 4 June 2013 (27 34 N/30 49 E) North Sinai, Egypt 18 (5) C. meg I, VI, VII, VIII and V 2 May 2013 (31 02 N/33 00 E) C. marginalis Giza, Egypt 17 (5) C. mar I, II, III, IV and V 27 May 2013 (Weidemann, 1830) (30 00 N/31 10 E) Dayrout, Egypt (27 34 N/30 49 E) 3 (1) C. mar VI 2 May 2013 Also listed are the numbers of haplotypes found in each geographic location. Refer to Table 3 for GenBank accession numbers associated with each haplotype.

Table 2. List of combinations and primer sequences used in the amplification of the COI locus

Primer combination Primer sequences (50-30) References

1a (TY-J-1460a þ C1-N-2191) TACAATTTATCGCCTAAACTTCAGCC Silva-Branda˜o et al. (2005) CCCGGTAAAATTAAAATATAAACTTC Simon et al. (1994) 1b (TY-J-1460a þ C1-N-2293) TACAATTTATCGCCTAAACTTCAGCC Silva-Branda˜o et al. (2005) AGTAAACCAATTGCTAGTATAGC Wells and Sperling (1999) 2 (C1-J-2183a þ TL2-N-3014) CAACATTTATTTTGATTTTTTGG Simon et al. (1994) TCCAATGCACTAATCTGCCATATTA Simon et al. (1994) were quality trimmed and manually aligned using the X03240.1) mitochondrial DNA sequence. From 158 BioEdit v7.2.5 sequence alignment editor (Hall 1999). successfully sequenced individuals, 97 have 1,509 bp of All new haplotype sequences were deposited into Gen- COI gene and the rest have sequences ranging from Bank with the accession numbers KM407601and 1,400 to 1,491 bp. The observed haplotypes for each KM434339–KM434366 (Table 3). species from each locality is listed in Table 1.Foreach Phylogenetic Analysis. An additional 14 sequences species, many individuals from the four locations of other carrion feeding calliphorids, including Chryso- shared a single haplotype; however, these data also mya species that are highly probable to be found on demonstrated the number of unique haplotypes corpses in Egypt, were obtained from GenBank (Table observed. For example, 60% (n ¼ 42) of the specimens 3) to include other flies for comparison. MEGA 6.0 of C. albiceps,68%(n ¼ 46) of C. megacephala,and (Tamura et al. 2013) was used to calculate pairwise dis- 55% (n ¼ 11) of C. marginalis individuals had unique tances, nucleotide compositions, and transition/trans- haplotypes (Table 3). No insertion or deletion (indels) version ratios and to construct neighbor-joining (NJ) was observed within the aligned sequences over this and maximum likelihood (ML) trees using Kimura’s region. Of the 403 variable sites identified, only 259 two-parameter model of substitution and 500 bootstrap were parsimony-informative characters. replicates. PAUP v4.0b10 (Swofford 2002) was used for Intra- and Interspecific Variation. Intraspecific maximum parsimony (MP) analysis with 500 bootstrap variation accounted for <1% for all specimens included replicates of unweighted heuristic searches. The MP in this study (Table 4). The exception was the previous tree was viewed and edited by MrEnt v2.5 (Zuccon inclusion of published Egyptian-derived sequences, and Zuccon 2013). KC249675.1 and KC249676.1 (Aly 2014), that had a sequence divergence from all other C. megacephala Results sequences between 1.6 and 2.6%. Because of this sur- prising result, we translated all of the new sequences Sequencing Data. The sequences obtained were and the published sequences, and it appears that the aligned corresponding to positions 1,475–2,983 of the KC249675.1 and KC249676.1 sequences has nine Drosophila yakuba Burla (GenBank accession number amino acids changes and thus is likely the result of 4JOURNAL OF MEDICAL ENTOMOLOGY

Table 3. Species, collection location, number of specimens, haplotypes details, and GenBank accession numbers for individuals used in the phylogenetic analyses

Species Location No. of Haplotypes No. of individu- Reference and GenBank specimens als sharing accession number haplotype

Chrysomya albiceps Egypt (various locations, 70 C. alb I 28 KM407601a (Wiedemann, 1819) see Table 1) AF083657.1 19 (Wells and Sperling, 1999) C. alb III 2 KM434339a C. alb IV 1 KM434340a C. alb V 1 KM434341a C. alb VI 2 KM434342a C. alb VII 1 KM434343a C. alb VIII 1 KM434344a C. alb IX 1 KM434345a C. alb X 1 KM434346a C. alb XI 1 KM434347a C. alb XII 12 KM434348a Ismailia, Egypt 1 KC249681.1 1 (Aly, 2014) Allier, France 1 KF919014.1 1 (Sonet et al. 2013) Chrysomya megacephala Egypt (various locations, 68 C. meg I 22 KM434355a (F., 1794) see Table 1) C. meg II 1 KM434356a C. meg III 1 KM434357a C. meg IV 2 KM434358a C. meg V 1 KM434359a C. meg VI 1 KM434360a C. meg VII 2 KM434361a C. meg VIII 18 KM434362a C. meg IX 1 KM434363a C. meg X 1 KM434364a C. meg XI 4 KM434365a C. meg XII 14 KM434366a Ismailia, Egypt 2 KC249676.1 2 (Aly, 2014)b KC249675.1 Calicut, India 1 AJ426041.2 1 (Stevens et al. 2008) Chrysomya marginalis Egypt (various locations, 20 C. mar I 1 KM434349a (Weidemann, 1830) see Table 1) C. mar II 2 KM434350a C. mar III 4 KM434351a C. mar IV 9 KM434352a C. mar V 1 KM434353a C. mar VI 3 KM434354a Karoo, South Africa 1 AB112862.1 1 Harvey et al. 2003b Chrysomya putoria Near Chilbre, Panama 1 AF295554.1 1 (Wells and Sperling, 2001) (Weidemann, 1830) Kitwe, Zambia 1 AB112831.1 1 (Harvey et al. 2003b)

Chrysomya bezziana Malaysia 1 JX187377.1 1 (unpublished) (Villeneuve, 1914) Bogor, Indonesia 1 AF295548.1 1 (Wells and Sperling 2001)

Chrysomya rufifacies Miami, USA 1 AF083658.1 1 (Wells and Sperling 1999) (Macquart, 1842) Petaling Jaya, Malaysia 1 AY909054.1 1 (Tan et al. 2009) Phormia regina Hopland, United States 1 AF295550.1 1 (Wells and Sperling 2001) (Meigen, 1826) Lucilia sericata Seoul, South Korea 1 EU880209.1 1 (Park et al. 2009) (Meigen, 1826) Calliphora vicina Brussels, Belgium 1 KF918981.1 1 (Sonet et al. 2013) (L., 1758) a New sequence. b Specimen is not included. some sequencing errors; therefore, it was excluded trees, the calliphorid species were assigned correctly to from all phylogenetic analyses. the subfamilies Chrysomyiinae, Luciliinae, and Calli- The interspecific divergence between the examined phorinae with >82% bootstrap support for Chrysomya calliphorids varied from 3.6% for the closely related group and support of 98% for the relation between C. albiceps– (Macquart) group to the sister groups Calliphorinae and Luciliinae. Haplo- 11.7% for the species pair C. rufifacies–Calliphora vicina types of each Chrysomya species were monophyletic (L.). Within the genus Chrysomya,themaximum and correctly assigned to their genera with bootstrap nucleotide divergence was 8.6 % for C. rufifacies– support of 96%, as shown in Figures 1–3.Atthespe- (Villeneuve) (Table 4). cies level, tree topology reflects a strong relation Phylogenetic Results. The topology of the NJ tree between C. albiceps and C. rufifacies, which clustered differed from the ML and MP trees at a single node together with 100% support. Also, C. marginalis (as indicated by “*” in Fig. 1), though this node is formed a distinct group then joined the group of weakly supported (41% bootstrap support). From all Chrysomya putoria (Weidemann) with >71% support, 2015 SALEM ET AL.: IDENTIFICATION OF Chrysomya 5 and in turn both of them are located in the same clus- number of published papers reporting the molecular ter that includes C. megacephala with a support of 89 identification of forensically relevant flies from Egypt and 80%, respectively, as shown in ML and MP trees using COI or COII fragments was limited to few publi- (Figs. 2 and 3, respectively). cations (Aly and Wen 2013, Aly et al. 2012, 2013, Aly 2014). This is the first study about the molecular identi- fication of Egyptian C. marginalis that was recorded Discussion from Egypt and established itself in different regions across the country (Schumann 1986, Shaumaretal. This study has updated the currently available data 1989, Morsy et al. 1991, Abd El-bar and Sawaby 2011). on Egyptian Chrysomya with longer COI sequence The current study is a preliminary survey of com- and the inclusion of a greater number of flies to assess monly found Chrysomya blow flies from Egypt and the the degree of population structure. To date, the inclusion of other sequences of Chrysomya species that Table 4. Intra- and interspecific divergences expressed as per- are highly probable to be found in Egypt is due to simi- centage of the analyzed COI gene using NJ approach with Kimu- lar and favorable environmental conditions, the absence ra’s two-parameter of geographical barriers and the export–import and movement of animals between Egypt and the surround- Species v 12345678 ing areas (Sudan, Libya, and Saudi Arabia) from which those flies are recorded and observed (Idris 1985, Setya- 1 C. albiceps 0–0.8 ningrum and Al Dhafer 2014). Our results revealed 2 C. marginalis 0–0.3 8.0 3 C. megacephalaA 0–0.6 7.0 5.3 numerous haplotypes among all specimens, thus more 4 C. bezziana – 7.9 5.6 5.0 genetic variation than previously observed. None, how- 5 C. putoria – 7.6 4.4 4.4 5.1 ever, appeared to be population specific or belonging to 6 C. rufifacies – 3.6 7.9 8.4 8.6 8.2 different ecological zones. This deviates from published 7 Phormia regina – 8.5 7.8 7.9 7.8 7.5 10.1 8 Lucilia sericata – 10.7 9.1 9.2 10.0 9.8 11.2 9.4 works that observed little to no genetic variation on a 9 Ca. vicina – 10.9 10.3 10.9 10.2 9.9 11.7 10.1 8.5 global scale; however, these studies suffered from small v, intraspecific divergence. samples (Harvey et al. 2003a) and a thoroughly and a Specimens with the accession numbers (KC249676.1 and exhaustive collection of specimens is the only way to KC249675.1) are not included. sample the true population genetic variation.

Fig. 1. NJ tree of the Egyptian Chrysomya based on 1,509 bp of COI gene with branch length using Kimura’s two- parameter model of substitution and 500 bootstrap supports. Node not shared with other trees is indicated by *. C.alb, C. albics; C. meg, C. megecephala; C. mar, C. marginalis. 6JOURNAL OF MEDICAL ENTOMOLOGY

The present study showed no overlap between inter- Interestingly, when the previously published sequences specific and intraspecific nucleotide divergences: the of C. megacephala (GenBank accession nos. KC249675 distance between species was >3%.Thisisimportant and KC249676) from another Egyptian location (Ismai- as all species included here (and ones that could be) lia) were included within our analysis, the intraspecific are well differentiated using COI sequencing. The dis- divergence (1.6 to 2.6%) was found within C. megace- cussion on the percentage threshold for species separa- phala individuals from the same country where the dis- tion is still ambiguous: Wells et al. (2001) stated that a tance was surprising given the limited distance percent of 1% divergence for COI þ II sequences between the two locations (Ismailia and Minya about within carrion fly species and 3% divergence between 300 km) and thus elicited greater attention. Further species is adequate threshold and this was supported examination of the sequence data revealed what is by Harvey et al. (2003a). For DNA barcoding to be an likely the result of sequencing errors, and thus caution accurate method of identification, the intraspecific should be exercised if using this data for molecular divergence should >3% and the interspecific distance identification (Aly 2014). Harvey et al. (2008) illustrated must exceed this percent (Hebert et al. 2003). The that the discovery of an outlying sample must address awareness of establishing group-specific thresholds was the question of whether it is an extreme sample or if it supported by the findings of Hebert et al. (2004), Har- represents an unsampled population. The high intra- vey et al. (2008), Boehme et al. (2012) and Nelson specific divergence within C. megacephala individuals et al. (2007), who illustrated that the interspecific varia- have also been noticed in a study by Jordaens et al. tion between Chrysomya species is <0.5%, while the (2013) , who concluded the high divergence (4.3%) of closely related Conicera species showed interspecific this species may be due to hybridization or introgres- variation of only 1.97%. The 3% threshold standard sion, incomplete lineage sorting, and to was satisfactorily used by Meiklejohn et al. (2011) and misidentifications. also to explain our results where no overlap was A larger data set provides valuable and reliable infor- observed within and among species in this study. mation concerning the population structure and

Fig. 2. ML tree of the Egyptian Chrysomya based on 1,509 bp of COI gene with branch length using Kimura’s two- parameter model of substitution and 500 bootstrap supports. C. alb, C. albics; C. meg, C. megecephala; C. mar, C. marginalis. 2015 SALEM ET AL.: IDENTIFICATION OF Chrysomya 7

Ullerich and Schottke (2006), Harvey et al. (2008), Uller- ich and Schottke (2006) and Singh et al. (2010). In conclusion, the sufficient bootstrap support and the phylogenetic analysis using three methods (NJ, ML, and MP) used in this study revealed similar and consistent data to support the reliability of the 1,509 bp of the COI gene to differentiate between subfamilies, genera, and species of blow flies, and also assured the phenotypic classifications that are commonly found in taxonomic reviews. As far as we know, this study exam- ines for the first time molecular identification of C. albiceps and C. megacephala from Egypt using a greater proportion of COI gene (1,509 bp), since Aly used COI with some species, also the first incidence to molecular analysis of the Egyptian C. marginalis.The blow flies of the Egyptian fauna require further investi- gation and analysis before use in forensic entomology cases.

Acknowledgments This work was funded by the Egyptian Cultural Affairs and Mission Sector (Cairo, Egypt) in the form a Scholarship to A.M.S. as a part of her joint supervision program. Addi- tional funds were provided by the School of Science (IUPUI) in the form of start-up funds awarded to C.J.P. Thanks also are extended to my colleagues in C.J.P’s laboratory, whom were of great help and support.

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