Sarcophaga peregrina (Robineau-Desvoidy, 1830), a synanthropic flesh fly species found in different parts of the world, is of medical and forensic importance. Traditional methods of inferring developmental age rely on the life stage of insects and morphological changes. However, once the larvae reach the pupal and adult stage, morphological changes would become barely visible, so that the classic method would be invalid. Here, we studied the cuticular hydrocarbon profile of S. peregrina of the whole life cycle from larval stage to adult stage by GC–MS. Sixty-three compounds with carbon chain length ranging from 8 to 36 were detected, which could be categorized into four classes: n-alkanes, branched alkanes, alkenes, and unknowns. As developmental increased, branched alkanes dominant, and the content of high-molecular-weight hydrocarbons is variable, especially for 2-methyl C19, DiMethyl C21, docosane (C22), and tricosane (C23). This study shows that the composition of CHC could be used to determine the developmental age of S. peregrina and aid in postmortem interval estimations in forensic science.
Postmortem interval (PMI) estimation is crucial for forensic investigations (DuPre 2013, Madea 2015). Insect evidence is a helpful evidence for PMI estimation when the victim is deceased for more than 72 h (Ren et al. 2018). Some members of the order Diptera are necrophagous arthropods abundant in cadaverous fauna, particularly the Sarcophagidae, Calliphoridae, and Muscidae families (Wang et al. 2008, Amendt et al. 2011). Therefore, the exact estimation of the developmental time of necrophagous flies is essential for determining the precise PMI (Al-Qahtni et al. 2021). The larval size, duration of development, and developmental accumulated temperature (Niederegger et al. 2010, Wang et al. 2016, Yang et al. 2017, Wang et al. 2018, Hu et al. 2019, Zhang et al. 2019) are merely suitable for the larvae stage. Once larvae reach the pupal and adult stage, morphological changes of the surface of the pupa would become barely visible, so that the classic method would be invalid (Amendt et al. 2011, Wang et al. 2017). Hence, new indicators need to be found for PMI estimation (Alotaibi et al. 2020).
Cuticular hydrocarbons (CHCs) are the main components of the wax layer covering insects' cuticles (Chung and Carroll 2015, Ginzel and Blomquist 2016, Botella-Cruz et al. 2021). Their function is mainly to prevent insects from environmental stress and serve as pheromones (Ginzel and Blomquist 2016, Blomquist and Ginzel 2021, Botella-Cruz et al. 2021). As Rutledge et al. (2014) reported, lacking dimethyl branched hydrocarbons in the Buprestidae family may be the reason why they were recognized as prey by female wasps. In addition, methyl alkanes could elicit various male-specific behaviors, leading to mating (Adams and Holt 1987). N-nonadecane (C19) was used to discriminate between female and male hosts by the egg parasitoid Trissolcus basalis (Wollaston) (Colazza et al. 2007). Insects can adjust the relative abundance of hydrocarbons to acclimatize the environment (Botella-Cruz et al. 2021). Moreover, measuring CHC composition could be used as a potential method to predict the age of flies, especially for the pupal developmental time (Zhu et al. 2006, Ye et al. 2007, Zhu et al. 2007, Alotaibi et al. 2020).
Sarcophaga peregrina (Robineau-Desvoidy, 1830), a synanthropic flesh fly species found in different parts of the world (Ren et al. 2021), is of medical and forensic importance. It is a vector of diseases and has a necrophagous habit (Sukontason et al. 2014). Furthermore, Lee et al. (2011) reported that S. peregrina could cause nasal myiasis. To further reveal the mechanisms underlying these characteristics, Ren et al. (2021) provided the valuable chromosome-level de novo genome assembly of S. peregrina. However, there is little known about the hydrocarbon profile of S. peregrina. Many researchers have focused on the compound profile of insects as they relate to developmental age and hydrocarbon composition (Zhu et al. 2006, Ye et al. 2007, Roux et al. 2008, Moore et al. 2016, Moore et al. 2017, Sharma et al. 2021), including in Chrysomya rufifacies (Macquart, 1843) (Zhu et al. 2006, Pechal et al. 2014, Sharma et al. 2021), Calliphora vomitoria (Linnaeus, 1758) (Roux et al. 2008, Moore et al. 2016), Calliphora vicina (Robineau-Desvoidy, 1830) (Roux et al. 2008, Moore et al. 2016), Protophormia terraenovae (Robineau-Desvoidy, 1830) (Roux et al. 2008), Lucilia sericata (Meigen, 1826) (Moore et al. 2013, Moore et al. 2017), Cochliomyia macellaria (Fabricius, 1775) (Pechal et al. 2014), Aldrichina grahami (Aldrich, 1930) (Xu et al. 2014). However, all the above insects belong to the Calliphoridae family. Yet, the Sarcophagidae family is also important in forensic entomology (Ren et al. 2018), and the chemical profiles applied to aging of the different life stages of forensically relevant sarcophagidae are still limited (Frere et al. 2014). Thus, to determine the cuticle hydrocarbons profile of S. peregrina, we collected S. peregrina from larvae to adults fed with pig lung at 25°C, and their age-dependent changes of CHCs were investigated using gas chromatography–mass spectrometry (GC–MS). This study provides a new age estimation method of S. peregrina and improves this insect's value for PMI estimation in forensic investigations.
Materials and Methods
Insect Materials
Sarcophaga peregrina was obtained from a laboratory colony (Guo's Laboratory, Changsha, China) initiated from pig carcasses placed in Changsha city (28°12′N, 112°58′E), Hunan province, China. Species identification was performed according to the method of Fan et al. (Fan 1992). Morphological pictures of S. peregrina are presented in Supp Fig. 1 (tjab172_suppl_supplementary_figure_s1.pdf) (online only). The color of adults is grayish-black. The postabdomen of male adults has fringes that curl up at the end. The pre-paramere is elongated, and its terminal is flat and thin. The post-paramere is obviously shorted than the pre-paramere. Surstylus is of a blunt round triangular shape. Adult flies were raised in a nylon cage (35 × 35 × 35 cm3), which was placed in the artificial climate box (LRH-250-GSI, Taihong Co., Ltd, Shaoguan, China) set at temperature 25.0 ± 1.0°C, 75% RH, and photoperiod of 12:12 (L:D) h cycle. The adults were fed with freshwater and milk powder mixed sugar with a ratio of 1:1 in a dish (12 cm diameter). Fifteen grams of fresh pig lung was placed in the cage in a culture dish to induce larviposition. Larvae that larviposited within 2 h were collected and reared in a plastic bowl (18 cm diameter, 5 cm height) with a moderate amount of pork lung. The bowl was put into a box (25 × 25 × 12 cm3) with 2 cm of silver sand covering the bottom of the box until pupation. After eclosion, the adults were reared as mentioned before. The above feeding process was carried out in the artificial climate box set at temperature 25.0 ± 1.0°C, 75% RH, and photoperiod of 12:12 (L:D) h cycle. We collected about 5–10 samples at 3:00 pm during the life cycle. Ten larvae were collected every day until pupation, and then 10 pupae were gathered every other day. Finally, five adults were collected every other day in the first 10 d. In total, we collected 70 larvae, 50 pupas, and 30 adults. The larval instar was determined by the number of posterior spiracle slits (Kotzé et al. 2015). All samples were stored separately in 1.5-ml cryovials and immediately frozen in liquid nitrogen and stored at –80°C for the subsequent CHCs extraction. Each sample was extracted separately and detected three times to avoid the technical error.
CHC Extraction
After frozen samples were thawed to ambient temperature, they were cleaned in ultrapure water with a small degreasing brush and blotted dry with filter paper. Then, each individual was immersed in 1 ml redistilled hexane with the internal standard tetracosane, C24, 1 µg/ml in hexane in a 2-ml glass vial at room temperature for 30 min. Meanwhile, an n-alkanes mix from heptane to tetracontane, C7–C40 (1 µg/ml) resolved in 1-ml redistilled hexane was used as an external standard. Next, the immersed liquid was transferred into a new 2-ml glass vial filtered by a syringe filter with a 0.45-µm aperture nylon membrane. After that, the sample was concentrated to dryness under vacuum and then dissolved in 200-µl redistilled hexane before GC–MS analysis.
GC–MS Analysis
CHCs analysis was carried out by GC–MS (Agilent Technologies, 7890B-5977A GC/MSD), with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 µm). Samples of 1 µl were injected splitless at 250°C. The oven temperature program was initiated at 40°C for 2 min then ramped to 200°C at 20°C/min, then to 320°C at 3°C/ min and finally, it was held for 8 min. The temperature of the GC–MS interface was 280°C. Ultrapure helium was used as the carrier gas with a pressure of 11.3 psi. Electron impact mode was set at 70 eV, and ion source temperature was set at 230°C. EI mass spectra and related literature comparing retention index data were used to identify CHCs (Kováts and Weisz 1965, Espelie and Bernays 1989, Bernier et al. 1998, Carlson et al. 1998, Pang et al. 2007, Krkosová et al. 2008, Park et al. 2020). The recognition of homologous peaks (retention index) was more important than chemical identification (Roux et al. 2008). Each stage has one original chromatogram, which is shown in Supp Fig. 2 (tjab172_suppl_supplementary_figure_s2.pdf) (online only).
Statistical Analysis
With the focus on hydrocarbons, the peak areas were integrated using the MSD ChemStation Data Analysis F.01.03, and only compounds with a consistent peak area percentage above 0.5% were included. Hydrocarbons were identified using a library search (NIST14), Kovats Index based on external standards, and literature (Kováts and Weisz 1965, Espelie and Bernays 1989, Bernier et al. 1998, Carlson et al. 1998, Pang et al. 2007, Krkosová et al. 2008, Park et al. 2020). Each compound's peak area was then divided by the peak area of the internal standard to correct for any variation on the final volume of the extract and finally divided by the total peak area to give the percentage value. After data filtering, the Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) models were used to visualize the data obtained from ChemStation in a graphic form. SIMCA 14.1 software was used to do cross-validation as well. Selecting hydrocarbons with variable importance of projection (VIP) parameter > 1 according to the OPLS-DA model, the fitting equation was constructed and visualized by OriginPro version 8.6 (OriginLab Corporation, Northampton, MA; SCR: 015636) and GraphPad Prism 6 (GraphPad Inc., La Jolla, CA; SCR: 002798).
Results
The Hydrocarbons Profile of S. peregrina
All CHCs of S. peregrina detected from the first day (larvae, 0 d) to the 26th day (adults, 25 d) are shown in Table 1. Sixty-three CHCs were identified by GC–MS analysis, including 23 n-alkanes, 30 branched alkanes, 4 alkenes, and 6 unknown compounds with the carbon chain length between C8 and C36. Two conclusions could be deduced from Fig. 1. The first is that most compounds were distributed at the adult stage, followed by the first-instar stage, and the other stages were similar. The other is that n-alkanes and branched alkanes were the two primary compounds of all detected hydrocarbons, while branched alkanes dominated with development time. Dimethyl-C21 alkane had the most proportion of CHCs in larvae, including first instar, second instar, third instar, and wandering, while its abundance showed a downward trend with larval growth. When S. peregrina developed into the pupal stage, tricosane (C23) and dimethyl-C21 alkane were the two main hydrocarbons. We found that nonane (C9) prevails in the first 2 d after S. peregrina eclosion. Then, pentacosane (C25) and heptacosane (C27) became the two most dominant compounds after becoming adults 2 d later.
The Orthogonal Partial Least Squares Discriminant Analysis
The OPLS-DA models were used to visualize the hydrocarbon distribution of S. peregrina at different developmental stages. Adults, pupa, and larvae can be obviously differentiable, as shown in Fig. 2A, with R2X 71.6%, R2Y 86.3%, and Q2Y 84.2%. To ascertain CHCs differences in the larvae stage, the OPLS-DA model was conducted again, excluding adults and pupa, and the result is shown in Fig. 2B with R2X 84.6%, R2Y 74.0%, and Q2Y 62.1%, indicating significant differences between compounds of the first-instar, second-instar, third-instar, and wandering stage. Figure 3 shows that our models achieved good resolution within different stages. Because of the short duration of the first instar, we did not show it in the picture, while the second, third instar, and the wandering stage met the desired impact and could be distinguished by hydrocarbons with developmental time (Fig. 3A–C). The first 5 d (7–11 D) cluster together in the pupa stage, but 13–15 D can be distinguished from each other, shown in Fig. 3D. For the adult (Fig. 3E), the variety of the hydrocarbons profile was also plainly discernable, with clear differentiation among days, while 16–17 D and 19–25 D could be discriminated more strongly. R2X, R2Y, and Q2Y were presented in Fig. 3.
Fig. 1.
The distribution of four compound classes in all life stages of S. peregrina. Four compound classes: branched alkanes, n-alkanes, alkenes, unknown. Life stages: first instar, second instar, third instar, wandering, pupa, adult. The number in parentheses after the life stage is the total kind of hydrocarbons detected by GC–MS in this stage.
The Variation Tendency of Hydrocarbons With VIP Parameter > 1
There are 36 hydrocarbons selected according to the variable importance of projection (VIP) parameter > 1 from the OPLS-DA ( Supp Fig. 3 (tjab172_suppl_supplementary_figure_s3.pdf) [online only]). Four of them (2-methyl C19, DiMethyl C21, C22, and C23) with R2 > 0.8 and exiting the whole life cycle are shown in Fig. 4. Cubic, parabola, and allometric equations were used to fit the hydrocarbons variety. The y-axis of the tendency chart was represented as the percentage of related composition, while the x-axis was regarded as development time.
Discussion
This study aims to explore whether the different stages of S. peregrina can be differentiable by CHCs analysis. Our results showed the potential for utilizing the CHCs profile to estimate insect age. Other studies have reported that utilizing hydrocarbons to distinguish the life stages of insects is possible (Zhu et al. 2007, Roux et al. 2008, Moore et al. 2013, Zhu et al. 2013, Frere et al. 2014, Pechal et al. 2014, Xu et al. 2014, Sharma et al. 2021). For example, Zhu et al. reported CHCs as a potential indicator in indicating the age of A. graham, which might be further used to determine the PMI (Xu et al. 2014). Sharma et al. (2021) extracted hydrocarbons from the pre-pupa stage of C. rufifacies, and Moore et al. (2013) did a similar experiment with L. sericata. Both of them demonstrated that CHCs profiles could establish the developmental stages, allowing for fly age estimation.
Hydrocarbons with carbon chain length between C8 and C36 were detected in S. peregrina by GC–MS. As studies had shown before, high-molecular-weight hydrocarbons of C20–C40 were the most significant compounds of insect cuticles (Ye et al. 2007, Roux et al. 2008). Similar results were found in this study, while we found that some low-molecular-weight hydrocarbons, especially C8, C9, and 4-Methyl-C10, also had stage specificity. Sharma et al. manifested C9 as representative of low-molecular-weight hydrocarbons in C. rufifacies, showing stage specificity in larvae (Sharma et al. 2021). The liner alkanes were dominant compounds from the third instar to the immature adult stage, but branched alkanes had the highest content than other compounds in the first-instar, second-instar, and mature adult stage. There was a certain similarity with Sharma's report (Sharma et al. 2021), as it showed liner alkanes have the dominant abundance in cuticular components of preadult of C. rufifacies. We found that the first instar and adult delivered the broadest range of hydrocarbons in S. peregrina. The flies that belonged to Calliphoridae had the broadest range of hydrocarbons in egg and adult, as Roux et al. (2008) reported. The varieties observed in hydrocarbon patterns depended on these flies' maturation and development stages.
Fig. 2.
OPLS-DA of the various compositions of cuticular hydrocarbons based on stages of S. peregrina. (A) Discriminated among larvae, pupa, and adults of S. peregrina with R2X 71.6%, R2Y 86.3%, and Q2Y 84.2%. (B) Discriminated among first instar, second instar, third instar, and wandering of S. peregrina with R2X 84.6%, R2Y 74.0%, and Q2Y 62.1%.
Fig. 3.
OPLS-DA of the various compositions of cuticular hydrocarbons based on different days of S. peregrina. (A) Discriminated in the second instar. (B) Discriminated in the third instar. (C) Discriminated in wandering. (D) Discriminated in the pupa. (E) Discriminated in adults. The time unit of D represents days old. R2X, R2Y, and Q2Y of each model are shown in the table of the picture.
Fig. 4.
The variation tendency of four hydrocarbons with VIP parameter > 1. The y-axis of the tendency chart is represented as the percentage of related composition, while the x-axis is regarded as development time. The table under the figure shows the parameters of the equation.
The age estimation of wandering and pupa developmental stages is difficult as there is little variety in size (body length, width, or weight; Niederegger et al. 2010, Wang et al. 2016, Yang et al. 2017, Wang et al. 2018, Hu et al. 2019, Zhang et al. 2019) and intrapupa varieties (Amendt et al. 2011, Wang et al. 2017). Nevertheless, CHCs profiles maybe aid to estimate the wandering and pupa developmental time. Different developmental times of wandering could be distinguished significantly (Fig. 3C). The pupal stage can be divided into three clusters (Fig. 3D). Intrapuparial morphological changes can indirectly attest to this phenomenon. After the emergence of the antennal outlines stage (about 114 h after pupariation), the body surface changes become obvious (Yang et al. 2017), influencing the puparium's hydrocarbons to some extent. Thus, the difference of CHCs of late pupa was more obvious than early pupa. Using CHCs of the family Calliphoridae has been reported to be a potential method to predict pupal age accurately (Roux et al. 2008).
Odd alkanes are always the domain hydrocarbons of flies (Roux et al. 2008, Sharma et al. 2021). The same result was found in this study. However, the mechanism and function of why odd alkanes showed a great preponderance are not understood. As Demkovich et al. studied, CHCs, especially odd alkanes, could help Amyelois transitella get pyrethroid resistance via reduced pesticide penetrance (Demkovich et al. 2021). According to Blomquist et al. (1987) whether even or odd carbon chains are synthesized depends on the substrate used as a precursor. Adding a unit of two carbons brings elongation of the carbon chain so that the precursor for odd alkanes is a 2-carbon unit (malonyl) and that for even alkanes is a 3-carbon unit (Blomquist et al. 1987). Even and odd branched carbon chains biosynthesis relies on valine and carbon skeletons of isoleucine (Ginzel and Blomquist 2016, Holze et al. 2021).
In summary, this study has shown the promising potential of CHC analysis to calculate PMI. Further research is required to investigate the reason for observing in some cases a pretty significant variation in relative abundance in a particular instar. Current data were obtained under standard laboratory conditions using a single species. Further work needs to examine the hydrocarbon composition and its stability under fluctuating temperatures and the effect of dealing with unknown species. Contrasting with relatively thorough studies of Calliphoridae, more studies need to focus on the cuticle hydrocarbon profile of the Sarcophagidae family to enrich our comprehension of these necrophilic flies.
Acknowledgments
We are grateful to Prof. Lushi Chen (Guizhou Police Officer College) for species identification. This study was supported by the National Natural Science Foundation of China (82072114 and 81772026), the Natural Science Foundation of Hunan Province (2020JJ4763), and the Fundamental Research Funds for the Central Universities of Central South University (2021zzts0940).
