Metamorphosis-related changes in the free fatty acid profiles of Sarcophaga (Liopygia) argyrostoma (Robineau-Desvoidy, 1830)

The flies of the Sarcophagidae, widespread throughout the temperate zone, are of great significance in Medicine, Veterinary science, Forensics and Entomotoxicology. Lipids are important elements of cell and organelle membranes and a source of energy for embryogenesis, metamorphosis and flight. Cuticular lipids protect from desiccation and act as recognition cues for species, nest mates and castes, and are a source of various pheromones. The free fatty acid (FFA) profile of cuticular and internal extracts of Sarcophaga (Liopygia) argyrostoma (Robineau-Desvoidy, 1830) larvae, pupae and adults was determined by gas chromatography–mass spectrometry (GC–MS). The larvae, pupae and adults contained FFAs from C5:0 to C28:0. The extracts differed quantitatively and qualitatively from each other: C18:1 > C16:1 > C16:0 > C18:0 predominated in the cuticular and internal extracts from the larvae and adults, while 18:1 > C16:0 > C16:1 > C18:0 predominated in the pupae. The FFA profile of the cuticle varies considerably between each development stage: C23:0 and C25:0 are only present in larvae, C28:0 in the pupal cuticle, and C12:1 and C18:3 in internal extracts from adults. The mechanisms underlying this diversity are discussed herein.


Results
The present work characterises the chemical composition of cuticular and internal FFAs of S. argyrostoma. Three types of extraction were performed for the larval, pupal and adult material. The cuticular lipids were found in the petroleum ether (extract I) and dichloromethane (extract II) extracts, and the internal lipids in extract III. The total masses of the extracts are shown in Table 1. Cuticular extracts of larvae amounted to 0.002 mg in the cuticular extracts (0.00017 mg per larva) and 0.900 mg in the internal extracts (0.075 mg per larva). Greater quantities of cuticular extracts were obtained from pupae 1.530 mg (0.077 mg per pupa) than larvae; however, lower quantities of internal extracts were obtained from pupae (0.520 mg; 0.026 mg per individual). The most efficient extraction was observed for adults: the cuticular extract yielded 5.630 mg (0.469 mg per insect), and the internal extract yielded 7.660 mg (0.638 mg per insect). The masses of the extracts are shown in Table 1.
These extracts were further analysed by GC-MS. A comparison of the FFA profiles of the cuticle surface (sum of extracts I and II) and the internal structures of the insect is given in Table 2; the raw data is appended in Table S1. The highest total FFA content was observed in adults, in both the cuticular (1865.278 ± 19.580 µg/g of insect body) and internal extracts (3811.660 ± 9.217 µg/g of insect body), while the lowest was observed for pupae: 70.821 ± 2.381 µg/g insect body mass in the cuticular extract, and 63.654 ± 1.167 µg/g insect body mass in the internal extract. In larvae, the total FFA content equalled 186.576 ± 7.550 μg/g insect body mass in the cuticular extracts, and 190.665 ± 8.849 μg/g insect body mass in the internal extract. Only the extracts from the adults demonstrated a statistically significant difference in total FFA content between cuticular and internal fractions (p < 0.001).
The internal lipids had a similar fatty acid profile, with the exception that C5:0 was absent. Most FFAs were present at similar levels; however, C9:0, C17:0, C18:0, C22:0, C23:0, C24:0, C25:0 and C26:0 were significantly higher in the cuticle extract. The two extracts were found to have similar total amounts of FFAs. The total ion current (TIC) chromatogram of fatty acids (TMS esters) of the ether extract (Extract I) from the larvae is given in Fig. 2.
The predominant FFA in all developmental stages was C18:1 (77 µg/g insect body mass in larvae, 30-31 µg/g insect body mass in pupae, and 891-1873 µg/g insect body mass in adults, respectively). High concentrations of C16:1 was measured only in larvae (42-56 µg/g of insect body) and adults (476-917 µg/g of insect body). The third most dominant acid was C16:0 (33-34 µg/g insect body mass in larvae, 12-15 µg/g insect body mass in pupae, and 311-640 µg/g insect body mass in adults). Table 2. Fatty acid contents in the cuticular and internal lipids extracted from Sarcophaga argyrostoma [µg/g of body mass ± SD]. FFA free fatty acids, SD standard deviation, ND not detected; statistically significant differences are marked with the same letters (ANOVA, Test HSD Tukey, p < 0.05), see Table S1 for raw data.    www.nature.com/scientificreports/ Significant differences regarding the presence of individual FFAs were observed regarding the between developmental stages. C23:0 and C25:0 were observed only in larvae, whereas C28:0 was detected only in the cuticle of pupae. In turn, C12:1 and C18:3 were present only in internal extracts from adults. Interestingly, several FFAs present in larvae and adults were absent from pupae: C11:0, C19:1, and C19:0 absent from both extracts; C20:4 and C20:5 absent from the cuticle; C13:0, C20:3, C20:1, and C26:0 absent from the internal extract.
Glycerol and cholesterol were observed in all extracts. However, glycerol content was higher in the extract from the adults (23.366 ± 0.632 μg/g insect body mass in the cuticular extract and 93.437 ± 2.506 μg/g insect body mass in the internal extract) and larvae (3.064 ± 0.071 μg/g insect body mass in the cuticular extract and 30.370 ± 0.517 μg/g insect body mass in the internal extract) than from the pupae (0.316 ± 0.021 μg/g insect body mass in the cuticular extract and 0.3202 ± 0.014 μg/g insect body mass in the internal extract). In larvae and adults, the glycerol concentrations were ten-times and four-times higher in the internal extracts than the cuticle, while in pupae, both concentrations were nearly equal. The results have been showed in Table 3.
The highest concentration of cholesterol was observed in the extracts from adult cuticular (33.256 ± 0.514 μg/g insect body mass) and internal extracts (92.327 ± 0.509 μg/g insect body mass); the highest content was observed inside the insect body. In contrast, in the larval and pupal extracts, a higher content of cholesterol was observed in the cuticular fractions of larval (4.527 ± 0.000 μg/g of insect body) and pupal extracts (7.604 ± 0.174 μg/g of insect body) than in their corresponding internal fractions: 2.958 ± 0.015 μg/g insect body mass in larvae and 5.676 ± 0.196 μg/g insect body mass in pupae. The results have been showed in Table 3.

Discussion
The FFAs comprise a huge and diverse group of lipids. Our present findings demonstrate high quantitative and qualitative FFA content diversity between the three developmental stages: larvae, pupae and imago. They also confirm the occurrence of metamorphosis-related changes in the FFA profiles in S. argyrostoma as observed in other insect species [37][38][39][40][41][42][43][44] .
The differences in distribution of fatty acids in the body of the insect are summarised in Table 2. The agedependent differences in cuticular lipid content have been observed in S. bullata imagoes: FFAs were found to constitute 26% of all cuticular lipids in new-borns and 45% in 7-day-old flies, suggesting that lipid synthesis or transport to the cuticle surface may be incomplete in newly-emerged adults. The author also proposes that lipid transport occurs primarily through the unhardened cuticle of newly-emerged adults, and that transport is essentially complete by the time the adult cuticle is fully hardened 56 . FFAs are also used as precursors of cuticular hydrocarbons. Oenocyte-directed RNAi knock-down of D. melanogaster CYP4G1 or NADPH-cytochrome P450 reductase results not only in flies deficient in cuticular hydrocarbons, but also in the accumulation of midchain fatty acids, which might suggest that they play a role in hydrocarbon synthesis 57 .
The results of the GC-MS analysis indicate that the extracts from adults possess higher FFA content than in preimaginal stages of flies, which is in accordance with previous findings 26 in Sarcophaga carnaria. Sun and Brookes also report a lower level of FFA in the fat body of Sarcophaga bullata larvae (from 0.93 to 2.92%, depending on age) than in adults 58 ; they also note that C18:1 > C16:0 > C18:0 > C16:1 FFAs predominated in three-day-old larvae, and C18:1 > C16:0 > C16:1:C18:2 in nine-day-old larvae, and that the amount of C18:2 and C16:1 increased, and C18:0 decreased, during six days of rearing 58 .
In the present study, C18:1 > C16:1 > C16:0 > C18:0 predominate in both cuticular and internal extracts from larvae and adults, while 18:1 > C16:0 > C16:1 > C18:0 predominate in pupae; those FFAs have also found to be characteristic of Diptera 59,60 . The dominant FFA is C18:1 in all extracts, with the highest proportions being found in the adult extracts (47.78% in cuticular and 49.14% in internal) and the internal extracts of pupae (47.85%). In previous studies, higher proportions of C18:1 have been described in the internal extracts from larvae (55.9%) and pupae (58.9%) of S. carnaria 26 . In S. bullata, lower levels of C18:1 were found in the cuticular extracts of adults; in addition, the C18:1 levels in the cuticular extract increased during adulthood from 29.0% in new-born and 34.3% in seven-day-old adults, while C16:1 decreased from 32.5 to 21.6% 56 .
In all extracts, the predominant FFA was found to be C18:1. One example of a C18:1 FFA is oleic acid. It has many biological properties, including the ability to provide a wide temperature window for growth. It provides the best environment for critical membrane proteins such as membrane ATPases, which function at optimum levels when oleic acid is present in the cell membrane 61 ; therefore, an increase in oleic acid level in response to, or in preparation for low temperatures, may maintain correct fluidity of the membrane without sacrificing the delicate balance needed to optimize the function of sensitive membrane proteins. Higher proportions of C18:1 have been found as an adaptation to low temperatures in Eurosta solidaginis 62 , Dolycoris baccarum and Piezodorus lituratus 63 and S. similis 64 . As oleic acid is energetically more favourable to manufacture than linoleic acid, due to it having one less double bond, insects that upregulate oleic acid rather than linoleic acid for low temperature use may be preserving finite energy reserves while still gaining the benefit of a wide window of fluidity 65 . The high amount of C18:1 observed in S. argyrostoma might be an example of adaptation to cold.
Polyunsaturated fatty acids (PUFA) are usually associated with biomembranes as phospholipid fatty acids. The proportion and composition of 20:5, 20:4 and 20:3 in the membranes, cuticle and so on vary according to life stage and tissue type 66 . Higher concentrations of C20:5 (1.59% of FFA content) and C20:4 (1.12%) were observed in the internal extracts in the present study; they are thought to be precursors of prostaglandins, leukotrienes and thromboxanes [67][68][69][70] . Various metabolites of C20:4 (arachidonic acid), known as eicosanoids, stimulate oviposition in crickets, regulate the function of Malpighian tubules in mosquitoes or ants, and control thermoregulation in cicadas 71 . They also play crucial roles in the mediation of insect cellular and humoral immunity 72,73 ; for example, in S. argyrostoma, they were found to mediate and coordinate the biosynthesis of NO and lysozyme in response to bacterial challenge 27 , and to participate in the LPS-dependent activation of the IMD pathway in Sarcophaga peregrina 74 . In a study of the internal extract of S. carnaria, Gołębiowski and co-workers found C20:5 to be present at 90-times higher concentrations in females than males; they propose that the compounds may play an important role in vitellogenesis 26 . Clements and co-workers 75 propose that arachidonic acid may play a role in the resistance of the Colorado potato beetle, Leptinotarsa decemlineata to neonicotinoid insecticide, and suggest that this may be associated with its regulatory role in cytochrome P450-dependent insecticide detoxification pathways. In the present study, a high concentration of C20:4 was observed in the extracts from the adults, which might suggest that S. argyrostoma is resistant to chemical insecticides; however, more detailed research is needed to confirm this. Table 3. Glycerol and cholesterol contents in the cuticular and internal lipids extracted from Sarcophaga argyrostoma [µg/g of body mass ± SD]. FFA free fatty acids, SD standard deviation, ND not detected; statistically significant differences are marked with the same letters (ANOVA, Test HSD Tukey, p < 0.05), see Table S1 for raw data. www.nature.com/scientificreports/ The FFAs in the insect cuticle have also been identified as resistance factors against fungal infection; for example, Gołębiowski and co-workers report that cuticular FFAs play a role in resistance to fungal infection by the flies Calliphora vicina, Lucilia sericata, C. vomitoria and S. carnaria 26,[39][40][41]76,77 . Also, literature data postulate that the chemical composition of cuticular FFAs may influence the susceptibility of cockroaches (Blatella germanica, Blatta orientalis) to infection by the fungus Metarhizium anisopliae 78 and by Conidiobolus coronatus 79 . Additionally, Smith and Grula indicate that cuticular FFAs in corn earworm larvae (Heliothis zea) can inhibit the germination and growth of Beauveria bassiana 80 . These examples illustrate the significant role played by cuticular FFAs in resistance or susceptibility to entomopathogens.
In the present work, higher levels of C9:0, C18:0 and C22:0 were found in the cuticular fractions of larvae, pupae and adults than the internal fractions. Of these, C9:0 and C18:0 have been found to inhibit the germination of C. coronatus spores 81 . Wrońska and co-workers 43 report a correlation between the concentration of C9:0 and C18:0 in the cuticle of Galleria mellonella larvae, pupae and imagoes, and the activity of C. coronatus enzyme cocktail. In addition, Boguś and co-workers 82 report a correlation between the concentration of C9:0, C18:0 and C22:0 in the cuticle of four medical and veterinary important flies: C. vomitoria, C. vicina, L. sericata and Musca domestica, and the enzymatic activity of C. coronatus.
Lipid accumulation and mobilisation is particularly important for the radical reconstruction of body structure and its biochemistry in holometabolous insects such as the Sarcophagae. However, these flies can also be larviparous, meaning that the egg develops internally, and females then give birth to first-instar larvae 83 .
The lipid content of holometabolous insects increases steadily during larval development, reflecting not only the metabolic requirements of the larva, but also the need to accumulate reserves for maintenance during metamorphosis 84 . However, in the present study, a higher concentration of particular FFAs was observed in the cuticular fraction; the levels of C9:0, C17:0, C18:0 and C23:0 were twice as high, C22:0, C24:0 and C26:0 were three times as high and C25:0 was five times as high. It is unusual to find odd-numbered fatty acids in insects, and as such, the presence C25:0 and C23:0 in the larval extracts merits further discussion. Pentacosanoic acid methyl ester is used as a pheromone by the European paper wasp Polistes dominulus for nest discrimination 85 . The C25:0 fatty acid has been found in both larval-larval (0.3% of FFA content) and larval-pupal (0.1% of FFA content) cuticular extracts from Dendrolimus pini exuviae; in contrast, C23:0 has only been found in in larvallarval extracts (0.1% of FFA content) 86 . C23:0 has also been described in the whole-body extracts of Allomyrina dichotoma 87 , Protaetia brevitarsis 88 , Tenebrio molitor 89 and in Cirina forda 90 larvae and Teleogryllus emma adults 89 , which are used as food in Asia.
Cholesterol was also found in higher levels in the cuticular extracts of the tested larvae. This contrasts with C. vicina, M. domestica and S. carnaria, where higher levels have been recorded in the internal extract. In addition, in contrast to the present study on S. argyrostoma, it has previously been found to be absent from cuticular extracts of S. carnaria larvae 38 . In the present study, glycerol was found to be present at higher concentrations in the internal extract, which is similar the distribution of glycerol in M. domestica larvae 38 .
During metamorphosis, most larval tissues decompose and adult structures are synthesized de novo from imaginal discs [91][92][93] . This process is dependent upon energy reserves, lipids for example, and anabolic precursors accumulate during larval growth 84 . Our present findings indicate a wide diversity of FFAs in the pupal stage; however, lower total FFA content was observed in both the internal and cuticular extracts of the pupal stage, compared with other developmental stages. A number of FFAs were found to be present at higher concentrations in the cuticular extract than the internal one, particularly C9:0, C12:0, C14:0, C18:0 and C22:0, each of which was present at twice the level in the cuticle. Wrońska and co-workers 43 report a high positive correlation between the concentration of C12:0 in the pupal cuticle of G. mellonella and the efficiency of entomopathogenic fungus C. coronatus chitinases and lipases in degrading it. In addition, similar to the present findings, S. carnaria pupae demonstrated a lack of C11:0, C19:1 and C20:4 in cuticular extracts and C20:1 in internal extracts; however, all FFAs were present in extracts from the larvae 26 .
In addition, the concentration of cholesterol was higher in the cuticular extracts in the present study, in contrast to extracts from M. domestica, S. carnaria and C. vicina pupae 38 . Glycerol was found to be present at very similar concentrations in the cuticular and internal extracts of the tested pupae, as previously observed in extracts from C. vicina pupae 38 . Lower amounts of internal FFAs were recorded, which might be due to disintegration of larval fat body in the pupal stage 94 .
An interesting finding was the presence of the long-chain FFA C28:0 on the surface of the cuticle of pupae, which is quite unique for insects. It is an aliphatic primary acid which has been shown to be an antibiofilm and anti-adherence agent against Streptococcus mutans 95 ; it has so far only been detected in the cuticular wax of the honey bee Apis mellifera 96 and in the cuticular fraction from larvae and pupae of D. pini 86 . It is important to note that this FFA is absent in extracts from species which are also consider as significant tools in forensics, such as C. vicina 97 , C. vomitoria 40 and S. carnaria 26 . However, C28:0 has been found in chloroform extracts from C. vicina (C. erythrocephala) pupae by electron diffraction 98 .
The advantage of holometabolous development is the specialisation of stages: larvae for feeding and growth, and adults for reproduction. However, there are some examples of holometabolous insects, such as blood-feeding mosquitos, which require blood meal to obtain protein or lipids to achieve, or enhance, reproductive success. Research on S. crassipalpis has shown that lipids derived from adult dietary components constitute half of the storage materials of eggs, and these are used as an energy supply for the developing embryos 99 . The balance between lipogenesis and lipolysis is tightly regulated in insects, to match energy needs that vary in response to the changes in the environment [100][101][102] . FFAs are main source of energy for muscles during flight.
In the adult fly extracts, higher concentrations of particular short-FFA were observed in the cuticle than in the internal extracts; for example, the concentration of C5:0 was four-times higher in the cuticle. However, the opposite was observed in case of middle and long-chain FFAs: higher amounts of particular FFA was detected inside the body, particularly C20:4 and C20:5. Also, the cholesterol and glycerol concentrations were higher in www.nature.com/scientificreports/ the internal extracts; a similar situation was observed in extracts from M. domestica, S. carnaria and C. vicina flies; however, higher concentrations were observed in the cuticular fraction for male C. vicina 38 . The internal extract also included two unsaturated FFAs: C12:1 (0.02% of all FFAs) and C18:3 (0.37% of all FFAs). C12:1 has been found in extracts from the larvae, pupae and adults (both female and male) from two species: Dermestes ater and Dermestes maculatus, which are highly resistant to infection by the entomopathogen fungus C. coronatus 42 , as well as in cuticular extracts from Schistocerca gregaria 103 . The presence of C18:3 was detected in phospholipids in Sarcophaga similis 64 and in extracts from Culex pipiens mosquito, where it allowed adults to stand or hop on the medium surface 104 and support flight at emergence; however, it was less effective than arachidonic acid (C20:4) 105 . C18:3 is required by Lepidoptera and Hymenoptera to achieve complete metamorphosis 106 and has been used as a precursor of female moth sex pheromones 107 .
The fatty acid contents of insects can vary according to growth stage, temperature and dietary regime. It is well know that in the environment, flesh flies feed on nectar, fruit juice, decomposing matter such as excrement, and carrion as sources of protein [108][109][110] . Valverde-Castro and co-workers report the presence of high population densities of Sarcopagiae flies in places with decomposing fish, whose flesh is associated with high fat and protein content; both are needed by female flies for developing eggs, and for the growth and development of first instar larvae 5 . Insects from fly families like the Muscidae, Calliphoridae, Drosophilidae, and Stratiomyidae can develop on media containing human faeces and fruits; however, the authors note that the nutrient levels from that source are insufficient for the larval development of the Sarcophaginae 5 . In the present work, all adults had access to both meat, i.e. beef, and sugar. Studies have shown that more than 30% of the fatty acid content in beef is composed of oleic acid [111][112][113] , which might explain the high level of C18:1 in the extracts from adult flies. In addition, beef is a popular meat used for feeding Diptera flies, and high levels of C18:1 have also been observed in extracts from adult L. sericata 39 or C. vomitoria 40 . The higher levels of FFA in the extracts from adults might be explained also by the conversion of saccharose (sugar) to lipids 114 . Literature data indicates that female Sarcophaga start to feed on sugar after emergence and begin to feed on meat after three days, and that meat feeding is cyclic 115 . The insects are also able to take up dietary C20:5, which can alter the overall FFA profile of tissue phospholipids in lengthy feeding experiments [116][117][118] ; in addition, dietary administration of C20:5 might reverse the inhibitory effect of dexamethasone (an inhibitor of eicosanoid formation) during viral infection in the larvae of the parasitic wasp Pimpla turionellae 119 .
Although sterols are essential substrates for insect steroid hormone (ecdysteroid) synthesis 120 , insects do not have the ability to synthesize sterols de novo due to a deficiency of the necessary enzymes 121,122 ; therefore, their diet is their main source. Research has indicated that some insects demonstrate a preference toward some FFAs in the diet. For example, the adult mosquitoes Aedes aegypti 123 and Anopheles gambi 124 , and the nymphal bug Triatoma infestans 125 are attracted by specific FFAs (mixed with L-lactic acid), and that some FFAs might discourage insects, like flies 126 or mosquitoes 127 . However, this preference for some FFAs could change over the course of development; for example, Drosophila melanogaster larvae prefer unsaturated FFAs whereas adults prefer saturated FFAs 128 .
Although diet generally affects the fatty acid profiles of insects, exceptional cases can occur. The biosynthesis of saturated palmitic (C16:0) and stearic acids (C18:0) and monounsaturated oleic acid (C18:1) seems to be widespread among insects, and accordingly, these fatty acids are the most abundant in their bodies 40,46 . Some insects from the Hymenoptera (N. vitripennis, for example) can synthesize the C18:2 FFA linoleic acid from oleic acid, thanks to presence of D12-desaturase, an enzyme, which is responsible for inserting a double bound at the D12-position 129 .
One of the important roles of health officials, particularly in tropical and subtropical regions, is the control of fly populations in urban and rural communities. Parasitoid wasps in the Order Hymenoptera (for example N. vitripennis) are regarded as biological control agents of flies 8,[130][131][132][133] . N. vitripennis wasps have been found to demonstrate a strong preference for Sarcophaga pupae, including S. argyrostoma, due to the greater production and rapid development of wasp pathogeny during infection by 6,134,135 .
The host preference might be connected with lipid metabolism: as parasitoid wasps are supposed to have lost their potential for lipogenesis during evolution due to environmental compensation, host lipids may well be essential and limiting factors for developing larvae 136,137 . In addition, Thompson and Barlow propose that the level of FFAs in parasitic Hymenoptera is determined by the FFA levels in their host lipid profile 138 . Similarly, research on parasitized S. bullata or S. crassipalpis pupae have shown increasing expression of genes involved in lipid biosynthesis and higher lipid content after venom injection 134,[139][140][141][142] . Hyperlipidaemia is also observed after administration of venom of Euplectrus separate to lepidopteran larva Pseudaletia separate 143 .
Our present findings illustrate the great diversity of FFAs between insect development stages: C23:0 and C25:0 are only present in larvae, C28:0 in the pupal cuticle, and C12:1 and C18:3 in the internal extracts from adults. This variation, occurring as a result of the remodelling processes of holometabolic insects and their adaptation to environment conditions might be a useful tool for the distinction of sarcophoids. Our findings regarding the chemical composition of the fatty acids of S. argyrostoma may play an important role in further studies on other significant lipid components of flies intended to improve our understanding of the taxonomy and physiology of insects, and thus may have a great impact on medical, veterinary, toxicology and forensic science.

Methods
Insect. S. argyrostoma were reared at 25 °C with 70% relative humidity and a 15:9 h photoperiod. The larvae were fed on beef ad libitum. The flies formed a puparium 14 days after hatching, and the adults emerged 14 days later. The species was confirmed by prof. Krzysztof Szpila from the Chair of Ecology and Biogeography (Nicolaus Copernicus University in Toruń, Poland). Post-feeding third instar larvae, freshly emerged pupae and 6-day-old sexually mature adults were used for lipid extraction. The sixth generation of insects were used in the study.