NEUROBIOLOGY,PHYSIOLOGY,BIOCHEMISTRY Cuticular Hydrocarbon Composition in Pupal Exuviae for Taxonomic Differentiation of Six Necrophagous

1 GONGYIN YE, KAI LI, JIAYING ZHU, GUANGHUI ZHU, AND CUI HU

Institute of Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China

J. Med. Entomol. 44(3): 450Ð456 (2007) Downloaded from https://academic.oup.com/jme/article/44/3/450/854682 by guest on 27 September 2021 ABSTRACT Gas chromatography-mass spectrometry was used to analyze the cuticular hydrocar- bons extracted from the pupal exuviae of six necrophagous ßies: Aldrichina grahami (Aldrich), Chrysomya megacephala (F.), Lucilia sericata (Meigen), Achoetandrus rufifacies (Macquart), Boet- tcherisca peregrina (Robineau-Desvoidy), and Parasarcophaga crassipalpis (Macquart). A discriminant model including the variables of peak 1 (tricosane), peak 7 (9-,11-,13-methyl-pentacosane), peak 21 (11,12-;9,13-dimethyl-hexacosane), peak 24 (octocosane), peak 41 (7,11-dimethyl-nonacosane), peak 42 (3-methyl-nonacosane), peak 46 (2-methyl-hentriacontane), and peak 51 (unknown) was con- structed, which allowed a complete separation of the pupal exuviae of the six species. These results indicate that cuticular hydrocarbons as chemotaxonomic characters for of forensic importance are of high value and feasibility.

KEY WORDS forensic entomology, necrophagous ßy, cuticular hydrocarbon, pupal exuviae, gas chromatography-mass spectrometry

Forensic entomology may be used to resolve the tim- ysis, demonstrated the powerful potential forensic ap- ing of criminal events by studying the insects and plication of CHCs extracted from the adult ßies, in other associated with a body to estimate separating the Phormia regina (Meigen) according to the postmortem interval (PMI), the cause of death, both location and gender. and the site of death (Benecke 2001). Generally, pupal Up to the present, there has been a lack of research exuviae found at the scene are useful for estimating dealing with CHCs as taxonomic biochemical markers the PMI because their chemical degradation is slow; for pupal exuviae of forensic importance. There are sometimes, they can be found in the proximity of human potential advantages to using pattern recognition anal- remains even after many years. Accurate species iden- ysis of CHCs to achieve a satisfactory degree of res- tiÞcation is the Þrst step. However, species identiÞcation olution. In our previous study of the insect community of pupal exuviae by morphological methods is difÞcult succession on pig (Sus L.) carcasses as they decompose, because few characters are available, and they are easily the biology, ecology, and biochemistry of common car- destroyed by adult emergence. Even though some re- rion-breeding ßies was studied (Hu 2000, Ma et al. 2000). searchers can use molecular techniques to separate Here, a gas chromatography-mass spectrometry (GC- them, the major biochemical components, such as DNA MS) investigation has been undertaken into the use of and protein, are typically decomposed. Development of CHCs as chemotaxonomic characters for pupal exuviae a rapid and accurate identiÞcation method would be of six forensically important ßiesÑAldrichina grahami beneÞcial for practical Þeld use. (Aldrich), Chrysomya megacephala (F.), Lucilia sericata In some insect species, cuticular hydrocarbons (Meigen), Achoetandrus rufifacies (Macquart), Boettch- (CHCs), which provide a protective layer that limits erisca peregrina (Robineau-Desvoidy), and Para- moisture loss (Page et al. 1990), constitute Ͼ90% of the crassipalpis (Macquart)Ñin Hangzhou, surface lipids. They play a role as biological materials, Zhejiang province, China. similar to sex pheromones, acting as recognition sig- nals to produce behavioral responses in social and Materials and Methods gregarious insects such as mosquitoes, termites, and cockroaches, According to recent publications (Phil- Insects. Adult calliphorids (A. grahami, C. mega- lips et al. 1990, Thorne et al. 1994, Chapman et al. 1995, cephala, L. sericata, and A. rufifacies) and the sarcoph- Mahamat et al. 1998), CHCs have been extensively agids (B. peregrina and P. crassipalpis), identiÞed using investigated as cues for insect species and colony rec- keys of Fan (1992) and Xue and Zhao (1996), were ognition. Byrne et al. (1995), using discriminant anal- collected with fresh pig liver pitfall traps in a suburb of Hangzhou, Zhejiang province, China, in 2003. They 1 Corresponding author, e-mail: [email protected]. were maintained at 24ЊC and 75% RH under a12-h

0022-2585/07/0450Ð0456$04.00/0 ᭧ 2007 Entomological Society of America May 2007 YEETAL.: CUTICULAR HYDROCARBON COMPOSITION 451 light/dark cycle to establish populations. Eggs ovipos- bons extracted from the pupal exuviae are listed in ited within 2Ð3 h were placed in fruit jars supplied with Table 1. Four classes of hydrocarbons were identi- fresh pork. Sawdust was added for pupation. Pupae Þed: 1) n-alkanes, 2) monomethylalkanes, 3) dimethyl- were transferred to petri dishes for emergence. As alkanes, and 4) n-alkenes. Representative GC spectra adults emerged, pupal exuviae were collected and of CHCs of six necrophagous ßies are shown in Fig. frozen at Ϫ70ЊC. 1. In all six species, the n-alkanes constituted the CHC Extraction and GC-MS Analysis. The extrac- greatest percentage (Ͼ55%, on average) of the CHC tion procedure of Byrne et al. (1995) was slightly components, followed by monomethylalkanes modiÞed. One pupal exuviae of each species was (16.85ϳ52.31%), dimethylalkanes (1.13ϳ4.78%), and n- immersed in 2 ml of redistilled hexane in 10- by alkenes (2.07ϳ3.76%). Among different species, the 35-mm glass tubes, at room temperature for 15 min. CHC abundance in A. grahami was much greater than in The pupal exuviae were then removed from the the others. The CHC proÞles were highly similar be-

solvent. After extraction, CHCs were concentrated tween C. megacephala and L. sericata. Downloaded from https://academic.oup.com/jme/article/44/3/450/854682 by guest on 27 September 2021 to dryness under vacuum for 30 min and then re- The chain lengths of n-alkanes in all species ranged suspended in 10 ␮l of redistilled hexane before from C23 to C30. Pentacosane occurred in all species GC-MS injection. except A. grahami. Odd-numbered alkanes were the Aliquots (3 ␮l) were analyzed by gas chromatogra- predominant compounds and accounted for 52.4%, on phy (GC) using a HewlettÐPackard 6890A (Agilent average, in all species except C. megacephala where Technologies, Palo Alto, CA) with splitless injection they represented 75.6%. The most abundant odd-num- mode. The GC was linked to a HewlettÐPackard 6890B bered alkanes were heptacosane (18.56%) and non- mass (Agilent Technologies) selective detector inter- acosane (21.11%), on average. Except in A. grahami, faced with a HewlettÐPackard 3393A computing inte- hentriacontane was abundant and reached the highest grator (Agilent Technologies) and equipped with a hy- level (15.44%) in B. peregrine and the lowest (4.44%) drogen ßame ionization detector and a 30-m fused Þlm in C. megacephala. silica capillary column (0.25 mm i.d., 0.25 mm Þlm thick- Thirty-four monomethylalkanes were identiÞed. ness). The program started at an initial temperature of Variations were mostly in odd-numbered chain lengths. 50ЊC, increased gradually to 200ЊCat25ЊC/min, and then The peaks of 9-, 11-, 13-, and 15-methylalkanes with equal to 270ЊCat3ЊC/min with a Þnal hold of 20 min. Electron main chain lengths usually overlapped. The variations impact mode was performed at 70 eV. The inlet tem- and abundances (all Ͼ1%) of monomethylalkanes were perature was set at 250ЊC. The column, with a head the largest in A. grahami and included: 9-,11-,13-methyl- pressure of 11.3 psi and a temperature of 300ЊC, used pentacosane, 3-emthyl-pentacosane, 2-methyl-hexade- ultrapure helium as the carrier gas and was run at a cane, 9-,11-,13-methyl-heptacosane, 3-methyl-heptaco- constant ßow of 40 ml/min. The temperature of the sane, 5-methyl-heptacosane, 2-methyl-octocosane, GC-MS interface was 280ЊC. Components were charac- 9-,11-,13-,15-methyl-nonacosane, 3-methyl-nonacosane, terized by their individual mass spectra, which were 7-methyl-nonacosane, 2-methyl-triacontane and 9-,11-, compared with those of standards, and matched by En- 13-,15-methyl-hentriacontane. hance ChemStation (Agilent Technologies) with NIST Five alkenes, including two pentacotenes (0.29 and 98.L library. Ten analyses were conducted for each 0.63%), two heptacotenes (0.94ϳ1.15%), and one non- species. acotene (0.75%) were identiÞed. The unsaturated bonds Data Analysis. Generally, the carbon chain lengths of in the carbon chains were not determined because of CHCs in insects are greater than C23, which results in limitations of the methodology. retention times Ͼ18 min. Therefore, only peaks exceed- Small variations and low abundances of dimethyl- ing an 18-min retention time were selected for analysis. alkanes were observed in all six species, and included The hydrocarbon peaks of each replicate were num- 3,7-dimethyl-pentacosane (0.3%), 2,4-dimethyl-hexade- bered according to their retention times. Percentages of cane (0.21%), 3,4-dimethyl-heptacosane (0.87%), 11,15-; each compound were calculated by computing the area 9,13-dimethyl-heptacosane (2.85%), 2,4-dimethyl-octo- under each peak, which produced a relative abundance cosane (0.51ϳ0.81%), 9,17-dimethyl-nonacosane (0.53ϳ value for each hydrocarbon component. Taxonomic 2.02%), 7,11-dimethyl-nonacosane (0.79%), and 11,15-; analysis was carried out by stepwise discrimination 11,17-dimethyl-nonacosane (0.34ϳ1.96%). (Byrne et al. 1995). Discriminant analysis was conducted Discriminant Analysis. Using a stepwise multiple with SPSS 11.0 statistical software (SPSS 11.0 for Win- regression analysis by the Fisher discriminant method, dows, SPSS Inc., Chicago, IL). Discriminant functions eight spectral peaks were determined as characteristic were constructed by applying WilkÕs lambda method variables among the pupal exuviae. They included with Fratios as variables. The spectral data were taken up peak 1 (tricosane), peak 7 (9-,11-,13-methyl-pentaco- for use when F Ͻ 8 and F Ͼ 5. Otherwise, the data were sane), peak 21 (11,12-;9,13-dimethyl-heptacosane), rejected. These peaks represented Ͻ1% of the sums of peak 24 (octocosane), peak 41 (7,11-dimethyl-nona- the areas for all the peaks in each species. cosane), peak 42 (3-methyl-nonacosane), peak 46 (2- methyl-hentriacontane), and peak 51 (unidentiÞed). Peaks 1, 7, and 24 occurred in all species. Peak 21 was Results present only in P. crassipalpis. Peaks 41 and 51 were CHC Composition. The names, retention times, present only in A. grahami. Peaks 42 and 46 were and percent compositions of principal hydrocar- absent in both C. megacephala and P. crassipalpis. All 452 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 3

Table 1. CHC composition of pupal exuviae of six necrophagous flies

Retention % composition (means Ϯ SD) Peak time Hydrocarbon no. A. C. Lucilia A. B. P. (min) grahami megacephala sericata rufifacies peregrina crassipalpis 1 21.30 Tricosane 0.54 Ϯ 0.33 2.86 Ϯ 0.59 0.14 Ϯ 0.08 6.44 Ϯ 1.43 0.39 Ϯ 0.30 0.12 Ϯ 0.05 2 23.54 Quadracosane 0.22 Ϯ 0.11 0.29 Ϯ 0.09 0.12 Ϯ 0.06 0.50 Ϯ 0.21 0.36 Ϯ 0.24 0.11 Ϯ 0.06 3 24.99 2-Methyl-quadracosane 0.47 Ϯ 0.25 4 25.31 Pentacontene a 0.29 Ϯ 0.13 5 25.47 Pentacontene b 0.63 Ϯ 0.25 6 25.87 Pentacosane 6.08 Ϯ 1.46 3.78 Ϯ 0.62 3.29 Ϯ 0.70 6.33 Ϯ 1.36 0.77 Ϯ 0.31 1.74 Ϯ 0.78 7 26.67 9-,11-,13-Methyl-pentacosane 1.08 Ϯ 0.40 1.05 Ϯ 0.31 0.19 Ϯ 0.09 1.41 Ϯ 0.65 0.20 Ϯ 0.07 1.92 Ϯ 2.75 8 26.84 7-Methyl-pentacosane 0.49 Ϯ 0.31 1.21 Ϯ 1.28 9 27.59 3-Methyl-pentacosane 2.21 Ϯ 1.05 0.34 Ϯ 0.12 0.27 Ϯ 0.16 0.65 Ϯ 0.31 0.62 Ϯ 0.26

10 28.21 Hexacosane 0.92 Ϯ 0.24 1.10 Ϯ 0.21 1.47 Ϯ 0.20 0.56 Ϯ 0.25 0.45 Ϯ 0.17 0.63 Ϯ 0.50 Downloaded from https://academic.oup.com/jme/article/44/3/450/854682 by guest on 27 September 2021 11 28.40 3,7-Dimethyl-pentacosane 0.30 Ϯ 0.09 12 29.69 2-Methyl-hexacosane 2.88 Ϯ 1.06 0.25 Ϯ 0.09 0.16 Ϯ 0.06 1.22 Ϯ 1.36 13 30.01 Heptacontene a 0.94 Ϯ 0.45 0.97 Ϯ 1.08 14 30.19 Heptacontene b 1.51 Ϯ 0.94 1.10 Ϯ 0.84 15 30.58 Heptacosane 15.21 Ϯ 4.08 34.83 Ϯ 5.23 39.85 Ϯ 4.23 5.22 Ϯ 0.72 3.65 Ϯ 0.99 12.58 Ϯ 2.90 16 31.11 2,4-Dimethyl-hexacosane 0.21 Ϯ 0.11 17 31.31 9-,11-,13-Methyl-heptacosane 2.82 Ϯ 1.25 0.93 Ϯ 0.17 0.90 Ϯ 0.32 1.30 Ϯ 1.54 0.42 Ϯ 0.13 8.08 Ϯ 1.40 18 31.44 7-Methyl-heptacosane 0.42 Ϯ 0.11 0.26 Ϯ 0.09 0.19 Ϯ 0.06 0.43 Ϯ 0.17 19 31.74 5-Methyl-heptacosane 1.05 Ϯ 0.20 0.14 Ϯ 0.03 0.33 Ϯ 0.10 1.47 Ϯ 0.27 20 32.03 3,4-Dimethyl-heptacosane 0.87 Ϯ 0.27 21 32.06 11,15Ð9,13-Dimethyl-heptacosane 2.85 Ϯ 0.51 22 32.31 3-Methyl-heptacosane 4.69 Ϯ 1.98 4.13 Ϯ 0.50 3.89 Ϯ 0.96 1.13 Ϯ 0.62 0.92 Ϯ 0.25 10.54 Ϯ 2.40 23 32.52 Unknown (C29) 0.67 Ϯ 0.30 0.88 Ϯ 0.32 1.01 Ϯ 0.20 24 32.98 Octocosane 1.42 Ϯ 0.36 3.52 Ϯ 0.29 2.15 Ϯ 0.19 0.72 Ϯ 0.19 0.87 Ϯ 0.19 1.60 Ϯ 0.32 25 33.24 Unknown (C29) 1.54 Ϯ 0.92 2.07 Ϯ 0.65 26 33.80 Unknown (C29) 0.67 Ϯ 0.34 0.57 Ϯ 0.53 0.53 Ϯ 0.27 0.34 Ϯ 0.09 1.68 Ϯ 1.00 27 34.21 6-Methyl-octocosane 0.30 Ϯ 0.10 0.52 Ϯ 0.14 28 34.57 4-Methyl-octocosane 1.23 Ϯ 0.71 29 34.71 2-Methyl-octocosane 4.63 Ϯ 0.87 5.05 Ϯ 1.95 1.16 Ϯ 0.43 1.95 Ϯ 1.12 30 35.14 3-Methyl-octocosane 0.70 Ϯ 0.38 0.51 Ϯ 0.13 0.38 Ϯ 0.16 31 35.41 Nonecontene 0.75 Ϯ 0.38 32 35.70 Unknown 1.59 Ϯ 0.79 4.20 Ϯ 3.93 33 35.87 Nonacosane 11.58 Ϯ 3.23 29.64 Ϯ 3.34 25.99 Ϯ 5.95 16.76 Ϯ 8.37 21.44 Ϯ 4.73 21.25 Ϯ 4.42 34 36.10 2,4-Dimethyl-octocosane 0.51 Ϯ 0.09 0.79 Ϯ 0.34 0.81 Ϯ 0.69 35 36.49 Unknown (C30) 0.49 Ϯ 0.29 36 36.87 9-,11-,13-,15-Methyl-nonacosane 5.96 Ϯ 1.99 2.30 Ϯ 0.80 7.06 Ϯ 2.54 6.97 Ϯ 4.03 8.06 Ϯ 1.75 37 37.16 7-Methyl-nonacosane 2.02 Ϯ 0.74 0.75 Ϯ 0.27 1.27 Ϯ 0.36 0.71 Ϯ 0.26 1.44 Ϯ 0.24 38 37.49 5-Methyl-nonacosane 0.92 Ϯ 0.32 1.07 Ϯ 0.33 0.24 Ϯ 0.13 0.99 Ϯ 0.52 1.14 Ϯ 0.34 1.26 Ϯ 0.28 39 37.74 11,15-;11,17-Dimethyl-nonacosane 0.74 Ϯ 0.36 0.34 Ϯ 0.19 1.96 Ϯ 1.14 1.93 Ϯ 0.49 40 37.95 9,17-Dimethyl-nonacosane 0.53 Ϯ 0.16 1.31 Ϯ 0.44 2.02 Ϯ 1.40 41 38.21 7,11-Dimethyl-nonacosane 0.79 Ϯ 0.37 42 38.32 3-Methyl-nonacosane 1.29 Ϯ 0.41 0.66 Ϯ 0.14 3.79 Ϯ 2.35 28.50 Ϯ 3.02 6.67 Ϯ 1.25 43 38.54 Unknown (C31) 0.40 Ϯ 0.22 0.22 Ϯ 0.11 44 39.26 Triacontane 0.89 Ϯ 0.25 0.62 Ϯ 0.27 1.58 Ϯ 0.73 2.03 Ϯ 0.49 0.99 Ϯ 0.30 45 40.50 12-,14-,15-Methyl-triacontane 0.71 Ϯ 0.21 0.27 Ϯ 0.11 0.89 Ϯ 0.54 0.28 Ϯ 0.09 46 41.78 2-Methyl-triacontane 2.87 Ϯ 0.45 0.58 Ϯ 0.38 0.43 Ϯ 0.21 8.07 Ϯ 0.83 0.58 Ϯ 0.28 47 43.00 Unknown 0.90 Ϯ 0.58 3.17 Ϯ 1.26 48 43.14 Unknown 3.85 Ϯ 0.95 49 43.48 Hentriacontane 4.44 Ϯ 1.09 5.65 Ϯ 3.25 12.05 Ϯ 1.61 15.44 Ϯ 4.05 6.42 Ϯ 3.81 50 43.63 Unknown 3.56 Ϯ 1.51 51 44.48 Unknown 2.28 Ϯ 0.34 52 44.87 9-,11-,13-,15-Methyl-hentriacontane 3.55 Ϯ 0.73 0.92 Ϯ 0.52 13.19 Ϯ 5.78 5.99 Ϯ 3.71 1.21 Ϯ 0.33 53 45.44 7-Methyl-hentriacontane 3.43 Ϯ 0.76 54 46.14 Unknown 1.06 Ϯ 0.39 1.29 Ϯ 0.85 55 46.57 Unknown 1.07 Ϯ 0.35 56 46.76 Unknown 0.38 Ϯ 0.13 57 47.21 3-Methyl-hentriacontane 1.18 Ϯ 0.55 1.53 Ϯ 0.37 peaks differed signiÞcantly (P Ͻ 0.01) among the an- rate determinations using functions 1 and 2 are alyzed species. Through discriminant analysis, Þve un- shown in Fig. 2, where species were plotted accord- standardized discriminant functions were obtained ing to their scores on these two functions. A. gra- (Table 2). hami, B. peregrine, A. rufifacies, and P. crassipalpis Functions 1 and 2 were interpreted with a total can be clearly distinguished from C. megacephala or success variable rate of 70.82 and 98.65% for func- L. sericata. The difference between C. megacephala tions 1, 2, 3, and 4. A statistical analysis (one-way and L. sericata is not clear, but they could be dis- analysis of variance (ANOVA) showed signiÞcant tinguished using functions 2 and 3. Complete sep- differences (P Ͻ 0.01) among all functions. Accu- aration of all species was achieved by applying all May 2007 YEETAL.: CUTICULAR HYDROCARBON COMPOSITION 453 Downloaded from https://academic.oup.com/jme/article/44/3/450/854682 by guest on 27 September 2021

Fig. 1. Representative gas chromatographs of CHCs in pupal exuviae of six necrophagous ßies. 454 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 3

Table 2. Cuticular hydrocarbos coefficients of discriminant It is widely accepted that CHC analysis provides a functions of six necrophagous flies (unstandardized) wealth of information for distinguishing species (spe- cies complex or populations), including cockroaches Peak Function no. (Everaerts et al. 1997, Carlson and Brenner 1988), 12345mosquitoes (Milligan et al. 1986, Phillips et al. 1990, 1 Ϫ77.28 35.71 Ϫ25.16 20.64 82.42 Kruger 1991), P. regina (Byrne et al. 1995), a tabanid 7 48.87 11.61 9.51 40.69 54.91 (Sakolsky et al. 1999), and termites (Howard et al. 21 231.24 87.16 110.35 482.96 91.05 24 318.47 Ϫ40.75 Ϫ220.68 Ϫ39.12 293.41 1988, Thorne et al. 1994, Takematsu and Yamaoka 41 664.54 839.33 Ϫ468.32 Ϫ267.18 179.94 1999), even though the differences in CHCs may be 42 1.56 17.81 53.90 Ϫ8.81 28.04 small. The identiÞcation of pupal exuviae of forensi- Ϫ Ϫ 46 215.59 59.81 18.70 62.83 15.27 cally important species is a major problem, because 51 Ϫ366.30 Ϫ1,268.50 433.32 226.93 142.97 Constant Ϫ0.76 0.80 Ϫ0.71 Ϫ3.60 Ϫ10.45 there are no easy methods for species identiÞcation.

This study demonstrates that CHCs of pupal exuviae Downloaded from https://academic.oup.com/jme/article/44/3/450/854682 by guest on 27 September 2021 are chemotaxonomically diagnostic for A. grahami, C. Þve functions. This result also was validated by megacephala, L. sericata, A. rufifacies, B. peregrine, and cross-regression analysis. P. crassipalpis, with 100% correct identiÞcation by Besides the aforementioned typical functions, we cross-regression analysis based on discriminant func- adopted a Bayesian discriminant function, where only tions. To our knowledge, this is the Þrst report of a the values of each pupal exuviae needed to be com- separation methodology for pupal exuviae of forensic puted. It is easy to see, that the highest scores repre- interest. It suggests directions for future taxonomic sent the sampled species. The Bayesian discriminant research in forensic entomology, especially for pupae functions for the six necrophagous ßies were clearly and pupal exuviae. They are difÞcult to identify for discernible (Table 3). two reasons: 1) there are few morphological charac- ters available and 2) the useful chemical components (e.g., proteins, enzymes, DNA) of pupal exuviae nat- Discussion urally decompose. Amorim and Ribeiro (2001) inves- The results showed that the six necrophagous ßies tigated the possibility of identifying the pupae of C. had distinct CHC proÞles. Their CHCs are similar to megacephala, Chrysomyia putoria (Wiedemann), and those of other insects and consisted of alkanes, Cochliomyia macellaria (F.). They discovered only monomethylakanes, and dimethylalkanes (Kruger et three useful morphological characters for identiÞca- al. 1991, Estrada-Pena et al. 1994, Byrne et al. 1995). In tion: circum-spiraculum obvious or not, the diameter addition, alkenes, minor components of CHCs in many of postspiraculum, and the lubricity of pupal exuviae. dipterans, were detected. Heptacosane and nonaco- Lack of morphological characters restricts their use- sane were the most abundant compounds found in fulness. Although some entomologists in China (Xue these six species, similar to what is found in other et al. 1984; 1985) distinguish common species of ßy insects (Lockey 1988, Louloudes et al. 1962, Byrne et pupae by morphological characters, this is difÞcult for al. 1995). forensic scientists. Furthermore, adult emergence de-

Fig. 2. CHCs of six necrophagous ßies distributed in the space of discriminant functions 1 and 2 (Ⅺ, A. grahami; ϩ, C. megacephala; ϫ, L. sericata; ‚, A. rufifacies; ƒ, B. peregrina; and छ, P. crassipalpis). May 2007 YEETAL.: CUTICULAR HYDROCARBON COMPOSITION 455

Table 3. Bayesian discriminant functions of cuticular hydrocarbons of six necrophagous flies

Peak A. grahami C. megacephala L. sericata A. rufifacies B. peregrina P. crassipalpis no. 1 79.99 237.60 Ϫ250.64 2,386.59 140.24 Ϫ258.12 7 378.05 1,092.35 626.81 Ϫ10.74 820.25 1,831.53 21 873.53 2,323.46 1,326.74 70.44 2335.94 11,094.36 24 2,418.76 9,244.17 5,713.57 Ϫ2,265.42 1,573.75 6,462.28 41 Ϫ20,985.89 12,192.93 7,290.99 Ϫ4,767.00 2,227.06 8,124.54 42 93.76 Ϫ30.93 Ϫ6.64 246.46 1,180.16 470.08 46 665.12 Ϫ921.88 Ϫ648.44 5,727.93 202.57 Ϫ1,097.19 51 31,556.74 Ϫ5,180.46 Ϫ3,022.51 Ϫ1,113.81 Ϫ787.52 Ϫ3,138.65 Constant Ϫ308.55 Ϫ171.11 Ϫ62.20 Ϫ306.15 Ϫ178.48 Ϫ244.62 Downloaded from https://academic.oup.com/jme/article/44/3/450/854682 by guest on 27 September 2021 stroys the pupal exuviae, making their identiÞcation study, Fig. 1 shows distinct GC patterns in CHC com- even more difÞcult. Using CHCs as a tool for identi- positions for each species, and discriminate analysis using Þcation has some major advantages: 1) broken exuviae CHC compositions of pupal exuviae results in accurate can be used for analysis; 2) CHCs are relatively stable identiÞcations. molecules that can be used for older specimens; 3) pinned museum specimens can be analyzed; 4) single specimens provide a useful GC-MS pattern; and 5) this Acknowledgments method is simple, feasible, and cost-effective when compared with traditional identiÞcation using mor- This study was supported by National Natural Science Foundation of China grant 39870681. phological characters, especially for forensic special- ists with few entomological knowledge. Some workers have reported that CHC composition References Cited varied with geographic populations, diet, and envi- ronmental temperature (Byrne et al. 1995; Liang and Amorim, J. A., and O. B. Ribeiro. 2001. Distinction among Silverman 2000; Desena et al. 1999a, 1999b). Page et al. the puparia of three blowßy species (Diptera: Callipho- (1990) postulated that CHCs are relatively stable met- ridae) frequently found on unburied corpses. Mem. Inst. abolic end products that seem to be genetically con- Oswaldo Cruz 96: 781Ð784. Benecke, M. 2001. A brief history of forensic entomology. trolled and are only slightly affected by environmental Forensic Sci. Int. 120: 2Ð14. factors. Stoffolano et al. (1997) reported that there Brown, W. V., R. Morton, M. J. Lacey, J. P. Spradbery, and were no diet-, age-, or sex-speciÞc differences in CHCs R. J. Mahon. 1998. IdentiÞcation of the geographical observed for adult P. regina. Cuvillier-Hot et al. (2001) source of adults of the old world screw-worm ßy, Chry- determined that the CHCs of ceylonense somya bezziana Villeneuve (Diptera: Calliphoridae), by Emery vary in their proportions according to age and multivariate analysis of cuticular hydrocarbons. 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Likewise, in this and geographic relationships in allopatric populations of 456 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 3

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