Twelve classes of chemical compounds were identified from the empty puparial cases of the three fly species at retention time 18.78 to 35.03. The percentages and the types of the extracted compounds were listed in Table 1. The profiles for Chrysomya albiceps and Lucilia sericata were very similar. Alcohol represented the highest percentage of compounds with 28.6% in L. sericata. However, in the three fly species L. sericata, Ch. albiceps and Chrysomya marginalis, alkanes (n-alkanes, branched alkanes and cycloalkanes) constitute the major component of cuticular hydrocarbons with 28.5, 50 and 89.4%, respectively. The chromatographs in Fig. 1 showed the characteristic peaks for each fly. Among studied species, the CHCs abundance in Ch. albiceps was lower than that in the others (18 compounds) as shown in Table 2a and 2b.
The retention times, names, and the frequency of each hydrocarbon in the three flies were listed (Table 2a & 2b). Forty-two compounds were identified with chain lengths ranging from C12 to C45. Heptacosane (7.6%) is the n-alkane dominated the chemical profile of Ch. marginalis, while Dodecane is the major one found in L. sericata (2.36%) and Ch. albiceps (1.88%). The predominant methyl branched alkane is 2-Methyltetracosane in L. sericata and C. albiceps and accounted for 5.53 and 4.92%, respectively. While the most abundant methyl branched alkane in C. marginalis is 2,6,10,14-Tetramethylhexadecane, (2.65%). The three species shared one compound in common which is the cycloalkane, 1-(2-Octyldecyl)octahydropentalene. Halogen branched hydrocarbons were detected in the chemical profiles of L. sericata and C. albiceps, but none was found in C. marginalis profile. Also, alkenes with different function groups as acid anhydride, alcohol and ester were detected in L. sericata and/or C. albiceps as illustrated in Table 2a & 2b peaks number 29, (26, 18) and (11, 7), respectively.
Alkadienes were represented in L. sericata with peak 30 (aldehyde) and in L. sericata and C. albiceps profiles with peak 35 (alcohol). The only cycloalkadienes observed in the chromatogram is the ketone compound, 3-(Dodecenyl)dihydro-2,5-furandione (peak 36) in the profiles of L. sericata and C. marginalis. The later species revealed several specific compounds demonstrated by peaks 34, 33, 31, 28, 27, 25, 23, 20, 17, 15, 13, 9, 8, 6, 5, 4 and 2. The chromatogram of C. marginalis shows more alkanes than those found in other species. According to test equality of group means, most peaks differed significantly (P < 0.01) among all species.
Discriminant analysis
According to multiple regression analyses using Fisher discriminant method, twelve spectral peaks identified as characteristic variables among the three species of flies. They include peak 1 (Hydroxymethylcyclododecane), peak 2 (Heneicosane), peak 3 (Dodecane), peak 4 (Triacontane), peak 5 (Tetracosamethyl-cyclododecasiloxane), peak 6 (Tricosane), peak 7 (Oxalic acid, allyl pentadecyl ester), peak 10 (9-t-Butyl-4-iodo-2,2-dimethyladamantane), peak 11 (Oxalic acid, allyl octadecyl ester), peak 12 (2-Ethyl-1-decanol), peak 14 (2-Butyl-1-octanol), and peak 18 (Phytol).
Peaks 1, 3, 11, 12, 14 and 18 were found in Lucilia sericata. Peaks 1, 3, 7, 10, 14, and 18 were identified in Chrysomya albiceps. However, in Chrysomya marginalis, peaks 2, 4, 5 and 6 were only presented. According to one-way ANOVA, all the peaks showed significant differences among the studied species.
Two canonical standardized functions were obtained by discriminant analysis (Table 3). Function 1 explained 83.8% of the variations in the dependent variables (Fly species) and function 2 interpreted 16.2% of variable rate. To validate this result, species were plotted according to their scores on these two functions. Each individual was correctly assigned to its species as observed in Fig. 2 permitting establishment of a confident identification.
In Table 4, the unstandardized coefficients of the canonical discriminant function are displayed. The higher the value of the coefficient, the higher the ability to predict the change in the dependent variable. The canonical discriminant function for the three species is discernible.
Table 1
The frequency and percentage of each hydrocarbon classes in the three flies
Peak No. | Hydrocarbon class | Frequency (% of total abundance) |
Lucilia sericata | Chrysomya albiceps | Chrysomya marginalis |
1 | Cyclic Alkane | 2 (9.5%) | 2 (11.1%) | 2 (10.5%) |
2 | Alkane | 2 (9.5%) | 2 (11.1%) | 15 (78.9%) |
3 | Alcohol | 6 (28.6%) | 3 (16.7%) | --- |
4 | Ester | 2 (9.5%) | 2 (11.1%) | --- |
5 | Ketone | 3 (14.3%) | 2 (11.1%) | 1 (5.3%) |
6 | Halogenated alkane | 2 (9.5%) | 2 (11.1%) | --- |
7 | Ether | 1 (4.8%) | 2 (11.1%) | --- |
8 | Halogenated Cycloalkane | --- | 1 (5.6%) | --- |
9 | Acid | --- | --- | 1 (5.3%) |
10 | Acid anhydride | 1 (4.8%) | 1 (5.6%) | --- |
11 | Aldehyde | 1 (4.8%) | --- | --- |
12 | Epoxide | 1 (4.8%) | 1 (5.6%) | --- |
Table 2
a. Classes and percent composition of the compounds isolated from the cuticle of the three flies
Peak No. | Hydrocarbon class | Compound | RT | Percent composition | p-Value |
Lucilia sericata | Chrysomya albiceps | Chrysomya marginalis |
1 | Cyclic Alkane | Hydroxymethylcyclododecane | 18.78 | 8.13 ± 0.70 | 7.12 ± 0.22 | --- | 0.000 |
2 | Alkane | heneicosane | 18.87 | --- | --- | 1.04 ± 0.18ab | 0.000 |
3 | Alkane | Dodecane | 19.65 | 2.36 ± 0.32 | 1.88 ± 0.66 | --- | 0.000 |
4 | Alkane | Triacontane | 19.75 | --- | --- | 1.85 ± 0.38ab | 0.000 |
5 | Cyclic Alkane | Tetracosamethyl-cyclododecasiloxane | 19.83 | --- | --- | 0.83 ± 0.11ab | 0.000 |
6 | Alkane | Tricosane | 20.60 | --- | --- | 2.43 ± 0.36ab | 0.000 |
7 | Ester | Oxalic acid, allyl pentadecyl ester | 20.98 | --- | 0.99 ± 0.21a | --- | 0.000 |
8 | Alkane | Tetracosane | 21.41 | --- | --- | 4.12 ± 0.64ab | 0.000 |
9 | Alkane | Heptacosane | 22.21 | --- | --- | 7.60 ± 2.17ab | 0.000 |
10 | Halogenated Cycloalkane | 9-t-Butyl-4-iodo-2,2-dimethyladamantane | 22.41 | --- | 1.04 ± 0.14a | --- | 0.000 |
11 | Ester | Oxalic acid, allyl octadecyl ester | 22.50 | 2.50 ± 0.93 | --- | --- | 0.000 |
12 | Alcohol | 2-Ethyl-1-decanol | 22.52 | 3.06 ± 1.10 | --- | --- | 0.000 |
13 | Alkane | Pentatriacontane | 22.94 | --- | --- | 5.94 ± 0.57ab | 0.000 |
14 | Alcohol | 2-Butyl-1-octanol | 23.53 | 4.42 ± 0.17 | 2.69 ± 0.41 | --- | 0.000 |
15 | Alkane | Octacosane | 23.67 | --- | --- | 6.75 ± 0.77ab | 0.000 |
16 | Cycloalkane | 1-(2-Octyldecyl)octahydropentalene | 25.33 | 5.68 ± 0.58 | 0.86 ± 0.04a | 0.88 ± 0.25a | 0.000 |
17 | Alkane | Hexatriacontane | 24.36 | --- | --- | 5.91 ± 0.73ab | 0.000 |
18 | Alcohol | 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, (2E,7R,11R)- Phytol | 24.53 | 3.25 ± 0.35 | 2.35 ± 0.46a | --- | 0.000 |
19 | Ketone | N-[4-Bromo-n-butyl]-2-piperidinone | 24.61 | 4.07 ± 0.27 | 3.37 ± 0.25a | --- | 0.000 |
20 | Alkane | Nonacosane | 25.03 | --- | --- | 5.57 ± 0.32ab | 0.000 |
21 | Alkane | 2-Methyltetracosane | 25.05 | 5.53 ± 0.99 | 4.92 ± 0.33 | --- | 0.000 |
According to one-way ANOVA test, P < 0.000, represent significant effect of the studied factor. According to post-hoc least significant difference (LSD) test a, b represent significant differences (P < 0.05) as compared to Lucilia sericata and Chrysomya albiceps, respectively. |
Table 2
Peak No. | Hydrocarbon class | Compound | RT | Percent composition | p-Value |
Lucilia sericata | Chrysomya albiceps | Chrysomya marginalis |
22 | Halogenated alkane | 1-Bromohexadecane | 25.18 | 4.87 ± 0.05 | 2.89 ± 0.29a | --- | 0.000 |
23 | Alkane | Dotriacontane | 25.68 | --- | --- | 3.53 ± 0.35ab | 0.000 |
24 | Ketone | 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione | 25.98 | 3.85 ± 0.09 | 2.99 ± 0.24a | --- | 0.000 |
25 | Alkane | 2,6,10,14-Tetramethylhexadecane (phytan) | 26.31 | --- | --- | 2.65 ± 0.18ab | 0.000 |
26 | Alcohol | 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]- (T-phytol) | 26.59 | 5.46 ± 1.10 | --- | --- | 0.000 |
27 | Alkane | 4-Methyldocosane | 26.91 | --- | --- | 2.21 ± 0.17ab | 0.000 |
28 | Acid | 3,5-Di-tert-butyl-4-hydroxy hydrocinnamic acid | 27.01 | --- | --- | 3.03 ± 0.46ab | 0.000 |
29 | Acid anhydride | 2-Dodecen-1-yl(-)succinic anhydride | 27.11 | 3.84 ± 0.91 | 1.38 ± 0.13a | --- | 0.003 |
30 | Aldehyde | 7,11-Hexadecadienal | 27.16 | 4.00 ± 2.82 | --- | --- | 0.000 |
31 | Alkene | Squalene | 27.50 | --- | --- | 1.57 ± 0.23ab | 0.000 |
32 | Halogenated alkane | 1,2-Dibromododecane | 27.66 | 3.21 ± 0.51 | 1.74 ± 0.28 | --- | 0.000 |
33 | Alkane | Tritetracontane | 28.07 | --- | --- | 1.13 ± 0.18ab | 0.000 |
34 | Alkane | Tetratetracontane | 28.63 | --- | --- | 0.89 ± 0.08ab | 0.000 |
35 | Alcohol | 12-Methyl-E,E-2,13-octadecadien-1-ol | 29.08 | 1.88 ± 0.27 | 4.73 ± 0.18a | --- | 0.003 |
36 | Ketone | 3-(Dodecenyl)dihydro-2,5-furandione | 23.73 | 1.06 ± 0.21 | --- | 5.73 ± 1.48ab | 0.000 |
37 | Alcohol | 1-Eicosanol | 29.88 | 5.19 ± 1.79 | --- | --- | 0.000 |
38 | Ester | Undec-10-ynoic acid, dodecyl ester | 30.23 | 2.77 ± 0.02 | 1.90 ± 0.44a | --- | 0.000 |
39 | Epoxide | 1,2–15,16-Diepoxyhexadecane | 31.15 | 2.90 ± 0.30 | 1.38 ± 0.18a | --- | 0.000 |
40 | Ether | 1-(Ethenyloxy)octadecane | 33.50 | 1.57 ± 0.34 | 3.74 ± 0.02 | --- | 0.001 |
41 | Ether | Oxirane, [(hexadecyloxy)methyl]- | 35.03 | --- | 1.58 ± 0.24a | --- | 0.000 |
According to one-way ANOVA test, P < 0.000, represent significant effect of the studied factor. According to post-hoc least significant difference (LSD) test a, b represent significant differences (P < 0.05) as compared to Lucilia sericata and Chrysomya albiceps, respectively.
Table 3
Summary of Canonical Discriminant Functions
Function | Eigenvalue | % of Variance | Cumulative % | Canonical Correlation |
1 | 18355.136a | 83.8 | 83.8 | 1.000 |
2 | 3537.382a | 16.2 | 100.0 | 1.000 |
Table 4
Canonical Discriminant Function Coefficients (unstandardized)
Characteristic peaks | Function |
1 | 2 |
C1 | − .867 | 3.551 |
C2 | 17.835 | 74.332 |
C3 | -37.899 | 29.267 |
C4 | 1.052 | 4.382 |
C5 | -3.858 | -16.078 |
C6 | -10.439 | -43.508 |
C7 | 112.916 | -75.184 |
C10 | 135.229 | 20.004 |
C11 | − .093 | 1.607 |
C12 | -7.051 | -3.120 |
C14 | -12.785 | 10.309 |
C18 | 46.312 | -9.549 |
(Constant) | -74.299 | -33.121 |