Comparative Biochemistry and Physiology, Part B 190 (2015) 27–36 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B j ourna l homepage: www.e lsev ie r .com/ locate /cbpb Analysis of lipid and fatty acid composition of three species of scorpions with relation to different organs Aldana Laino a, Camilo Mattoni b, Andrés Ojanguren-Affilastro c, Mónica Cunningham a, C. Fernando Garcia a,⁎ a Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), CCT-La Plata CONICET-UNLP, 60 y 120,1900 La Plata, Argentina b Laboratorio de Biología Reproductiva y Evolución, Instituto de Diversidad y Ecología Animal (CONICET-UNC), Av. Vélez Sarsfield 299, X5000JJC Córdoba, Argentina c Museo Argentino de Ciencias Naturales Bernardino Rivadavia , CABA, Argentina Abbreviations: C, Cholesterol; CE, Cholesteryl esters; Free fatty acids; HC, Hydrocarbons; MUFA, Mono Phospholipids; PtdCho, Phosphatidylcholine; PtdEtn, Pho Phosphatidylserine; PUFA, Polyunsaturated fatty acid; Triacylglycerols; UFA, Unsaturated fatty acid. ⁎ Corresponding author at: INIBIOLP, Fac. Cs. Médicas (1900), Argentina. Fax: +54 221 425 8988. E-mail address: cfgarcia1123@yahoo.com.ar (C. Fernan http://dx.doi.org/10.1016/j.cbpb.2015.08.005 1096-4959/© 2015 Elsevier Inc. All rights reserved. a b s t r a c t a r t i c l e i n f o Article history: Received 18 May 2015 Received in revised form 14 August 2015 Accepted 14 August 2015 Available online 21 August 2015 Keywords: Fatty acid Lipids Phospholipids Scorpion Triacylglycerides Within arthropods most of the information related to the type of mobilization and storage of lipids is found in insects and crustaceans. Literature is scarce with relation to scorpions. This order is a remarkably important model of the biochemistry, since it is characterized as an animal with very primitive traits which have varied minimally through time. In the present study we characterize and compare lipids and fatty acids present in three species of scorpion: Timogenes elegans, Timogenes dorbignyi, and Brachistosternus ferrugineus, focusing the study on the main organs/tissues involved in the dynamics of lipids. As found in the fat body of insects, hepato- pancreas of crustaceans and midgut diverticula of spiders, the hepatopancreas of the three species studied here turned out to be the organ of lipid storage (great quantity of triacylglycerides). With relation to the hemolymph andmuscles, a great quantity of phospholipidswas observed, which is possibly involved inmembrane formation. It is important to highlight that unlikewhat happens in insects, in scorpions themain circulating energetic lipid is the triacylglyceride. This lipid is found in greater proportion in the hepatopancreas of females, surely for repro- duction. The fatty acid of the different organs/tissues analyzed remained constant in the three species studied with certain characteristic patterns, thus observing saturated and unsaturated most abundant fatty acids of C16 and C18. Finally, it could be observed that in T. elegans, T. dorbignyi and B. ferrugineus scorpions, there is a lack of 20:4 that generates a special condition within fatty acids of arthropods. © 2015 Elsevier Inc. All rights reserved. 1. Introduction Lipid molecules are important in different organisms because they represent an effective energy storage, are main components of biological membranes and also have signaling function of remarkable importance. Within a given lipid class, the incorporation of a variety of fatty acids differing in chain length and unsaturation gives rise to a great variability of different lipid species. How this immense lipid variability is deployed in vivo, and to what extent it can be altered in response to exogenous factors (such as nutrition) without jeopardizing the lipid homeostasis at the organism level remains unclear (Shevchenko and Simons, 2010). Nowadays much effort is being made to improve the traditional methodology employed in the analysis of the lipids of insects, as for example the work recently performed by Tzompa-Sosa et al. (2014) in which the influence of the different CerPCho, Sphyngomielin; FFA, unsaturated fatty acid; PL, sphatidylethanolamine; PtdSer, SFA, Saturated fatty acid; TAG, , UNLP, Calle 60 y 120, La Plata do Garcia). methods of lipid extraction (aqueous versus organic solvent) in the lipid composition of 5 species of insects is analyzed, or that performed by Carvalho et al. (2012) where mass spectrometry is employed for building lipidomes of insects related to the different stages of development. Although it is known that lipids are stored in an organ named fat body in insects (Gilbert and Chino, 1974; Arrese and Soulages, 2010), and hepatopancreas in crustaceans (O'Connor, J.M., Gilbert, L.I., 1968; García et al., 2004), little is known about Class Arachnida with regard to the organ of lipid storage and the nature of stored lipids and their distribution to their respective places of utilization (Laino et al., 2009, 2011a). Class Arachnida presents a great diversity of species distributed in 11 orders, in which the information related to the lipids is in some cases disperse, in others scarce, and in a great majority nonexistent. It is unquestionable that Araneae is the Arachnid order containing the most information related to this subject. The first study performed where the uptake, storage, and mobilization of lipids were described, was recently reported in the spider Polybetes pythagoricus (Holmberg, 1875) (Laino et al., 2009); subsequently, in the same species, lipid trans- ference was observed between midgut-diverticula and lipoproteins (Laino et al., 2011a). Finally, an advance on the knowledge of the role of different lipids in the embryo development of Schizocosa malitiosa http://crossmark.crossref.org/dialog/?doi=10.1016/j.cbpb.2015.08.005&domain=pdf http://dx.doi.org/10.1016/j.cbpb.2015.08.005 mailto:cfgarcia1123@yahoo.com.ar http://dx.doi.org/10.1016/j.cbpb.2015.08.005 http://www.sciencedirect.com/science/journal/10964959 www.elsevier.com/locate/cbpb 28 A. Laino et al. / Comparative Biochemistry and Physiology, Part B 190 (2015) 27–36 (Tullgren, 1905) and P. pythagoricus was also recently achieved (Laino et al., 2011b, 2013). Scorpions (Class Arachnida, Order Scorpiones) are chelicerates with an ancient history that have changed little since the Silurian (450million years) (Millot and Vachon, 1968) and are considered as one of the oldest known terrestrial lineages (Zouari et al., 2006). Therefore, they have been chosen as models to perform biochemical studies as those carried out by Louati et al. (2011) and Zouari et al. (2005) where digestive en- zymes were studied, thus allowing for the interpretation of the digestive process in animals with primitive traits. Many studies have described scorpionmorphology, digestive system (Goyffon and Martoja, 1983; Warburg et al., 2002; Gefen, 2008; Warburg, 2012) and their venoms (Almaaytah and Albalas, 2014; Harrison et al., 2014), but very few have studied the lipids and fatty acids of these organisms. The only study related to lipids and fatty acids of TAG and PL in scorpions has been performed in the hemolymph and the hepatopancreas of the buthid Leiurus quinquestriatus (Ehrenberg, 1828) through the use of thin layer and gas–liquid chromatography (El-Salhy et al., 1981). Fatty acids of the body of other buthid: Centruroides vittatus (Say, 1821) were analyzed comparatively with other terrestrial arthropods (Uscian and Stanley-Samuelson, 1994). Lipoproteins of the scorpionid Pandinus imperator (Koch, 1842) were studied by Schenk et al. (2009), in which the authors described an un- usual quantity of phosphatidylserine (PtdSer) in the lipoprotein which is possibly related to the production or the efficiency of the venom. In the present study we characterize and compare lipids and fatty acids present in three neotropical species of the scorpion Family Bothriuridae, Timogenes elegans (Mello-Leitão, 1931), Timogenes dorbignyi (Guérin Méneville, 1843), and Brachistosternus ferrugineus (Thorell, 1876). We focused the study on the main organs/tissues involved in the dynamics of lipids (hepatopancreas, hemolymph, mus- cle and gonad). In addition, the possible differences between males and females of Brachistoternus ferrugineus were analyzed. This is the first time this kind of study is performed in Bothriuridae. This family presents a Gondwanic distribution, but most of its genera and species occur in the neotropics (Kovařík and Ojanguren-Affilastro, 2013). The three studied species in this contribution where chosen be- cause they are well known species from the Chaco phytogeographic province as defined by Cabrera (1976), an area that possess the highest scorpion diversity of Argentina, in which we are performing several ethological and ecological studies (Nime et al., 2013, 2014). These three species are ground dwellers that construct their burrows in open soil, are active during spring and summer, spending the rest of the year in hibernation inside of their burrows. Brachistosternus ferrugineus is the smallest and most abundant of them;with an average size of 4 cm, is also the most abundant scorpion in meridional Chacoan environments (Nime et al., 2013, 2014). Timogenes species are less abundant and bigger; T. dorbignyi is a medium sized species (CA. 6 cm), and T. elegans is by far the biggest of them (and also of the family), reaching 12 cm (Kovarik and Ojanguren-Affilastro, 2013). All these scorpion species are active and generalist predators of the epigean arthropod fauna, their preys being limited mostly by their size and capability of manipulation. The main objectives of this work were to characterize and compare lipids and fatty acids present in three species of scorpion: Timogenes elegans, Timogenes dorbignyi, and Brachistosternus ferrugineus, focusing the study on themain organs/tissues involved in the dynamics of lipids. In one species, Brachistosternus ferrugineus, the abundance and avail- ability of materials allowed us to search for differences between sexes. 2. Materials and methods 2.1. Animal and organ/tissue isolation A total of 90 adult scorpions of Brachistosternus ferrugineus (45 females and 45 males), 20 adults of Timogenes elegans (males) and 10 adults of Timogenes dorbignyi (males) were analyzed. The individuals were collected using UV lamps at night in March 2014 in Reserva Natural Formosa and neighboring areas, in Formosa Province, Argentina [24°17′00″S61°48′00″W], after which they were moved to the laboratory and kept in glass cages at 25 °C with 16/8 h light/dark cycle for two days unfed, before being sacrificed. Animals were anesthetized with ethyl ether previous to treatment, and finally sacrificed. Hemolymph was obtained by severing their legs, and the scorpions were centrifuged in a tube at low speed, using the same technique as Cunningham et al., 1994 for spiders. A ventral inci- sionwasmade in themesosomal tegument hepatopancreas and gonads were carefully dissected out, similar to that performed in spiders with modifications (Laino et al., 2009). Legs, chelas, and telson were also dissected. Legs and chelas together were considered as muscle. 2.2. Lipid characterization Lipids from hepatopancreas, hemolymph, muscle, and whole body were extracted following the procedure by Folch et al. (1957). Quantita- tive determination of lipid classes was performed by thin layer chroma- tography coupled to a flame ionization detector in a latroscan apparatus model TH-10 (Iatron Laboratories, Tokyo, Japan), after separation on Chromarods type S-III (Ackman et al., 1990; García et al., 2002a). Lipid classes were quantified with monoacylglycerol as an internal standard. The total lipids were determined by gravimetry (Cunningham and Pollero, 1996). Due to the scarcity of T. dorbignyi hemolymph, we failed to perform lipid characterization, however wewere able to characterize fatty acids (see below). Lipids were separated by a sequence of three different solvent sys- tems. Firstly, by the use of hexane/benzene (70:30 v/v) chromarods were dried and partially scanned to determine apolar lipids. In order to determine neutral lipids, the development in benzene/chloroform/ formic acid (70:25:1 v/v) was performed. Finally, the determination of polar lipids was achieved by the development in chloroform/acetone/ methanol/acetic acid/ water (30:40:10:10:5 v/v). Quantification was performedwith calibration curves of standards run under the same con- ditions. Reference lipids used as standards were hydrocarbons of C24, cholesteryl oleate, tripalmitin, oleic acid, cholesterol, phosphatidyletha- nolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin. Three determinations of three independent pools were performed. 2.3. Fatty acid characterization Fatty acidmethyl esters from total lipid sampleswere preparedwith BF3–MeHOaccording to themethod byMorrison and Smith (1992). The analysis was performed by gas–liquid chromatography in a HP-6890 capillary chromatograph (Hewlett Packard, Palo Alto, CA) on an Omegawax 250 30 m × 0.25 mm fused silica column with a 0.25 μm phase (Supelco, Bellefonte, CA). The column temperature was pro- grammed for a linear increase of 3 °C per min from 175 to 230 °C. Peaks were identified by comparison with retention times of Supelco 37 component fatty acid methyl esters mix (Supelco). For the case of the different tissues, three pools independent from one another were analyzed. 2.4. Statistical analyses Statistical comparison of quantity of lipids and percentage of differ- ent lipid classes of whole body, hepatopancreas, muscle and hemo- lymph was done using a one-way ANOVA after checking for normality and homogeneity of variances. Lipids and fatty acid composition are shown as mean ± standard deviation (SD). Significant differences (p b 0.05 or p b 0.001) were compared using the Tukey's post hoc test and Student's t-test. Data were analyzed using GraphPad InStat 3.01 (GraphPad Software, San Diego California USA). Fig. 1.Quantity of lipids present in thewhole body, hepatopancreas,muscle and hemolymphofmales of Timogenes elegans, T. dorbignyi, and Brachistosternus ferrugineusmales and females. Values are expressed as total lipidmg/wetweight. Values express themeans±SDof three pools of tissue analyzed separately. Different letters (a, b, c, d) above the bar indicate statistically significant differences in quantity of lipids between the four differentmodels of study analyzingwhole body, hepatopancreas,muscle andhemolymphdeterminedby Tukey's post hoc test, same letter above are not significantly different; level of significance p b 0.05. 29A. Laino et al. / Comparative Biochemistry and Physiology, Part B 190 (2015) 27–36 3. Results After quantifying the lipids of the different samples, it could be de- termined that the quantity of lipid remained constant in the whole body of the four models analyzed, and ranged between 42.9 mg/g (wet weight) for males of B. ferrugineus and 59 mg/g in males of T. dorbignyi. In the hepatopancreas the quantity of lipids was higher, with values ranging from 62 to 118 mg/g. In hepatopancreas of females B. ferrugineus the value was significantly lower than in hepatopancreas of males (88.9 to 118 mg/g). In muscles the quantity varied between 22 mg/g in B. ferrugineus females to 58.4 mg/g in B. ferrugineus males. Fig. 2. Percentage of different lipid classes in whole body, hepatopancreas, muscle and hemol Values are the means ± SD of three pools, analyzed separately through chromatography co statistically significant differences between whole body, hepatopancreas, muscle and hemolym significantly different level of significance p b 0.05. HC (hydrocarbons), CE (cholesteryl esters), nolamine), PtdSer (phosphatidylserine), PtdCho (phosphatidylcholine), and CerPCho (sphingo Finally, the quantity of hemolymphatic lipids was about 3 mg/ml (Fig. 1). Principal lipids in whole body and hepatopancreas are the triacylglycerides (TAG), around 42%, both contain phosphatidylcholine (PtdCho) with 20% and 38% respectively. Also, 18% of hydrocarbons (HC) were found in whole body. Hemolymphatic lipids are represented by 57% of PtdCho. A great percentage of phosphatidylserine (PtdSer) was found (20%) (Fig. 2). In bothwhole body andmuscle of T. dorbignyi the predominant lipid was PtdCho (around 60%), whereas it TAG was in the hepatopancreas (75%). (Fig. 3). ymph of male of Timogenes elegans. Percentages are analyzed for each lipid in particular. upled to a flame ionization detector. Different letters (a, b, c, d) above the bar indicate ph for each class of lipid determined by Tukey's post hoc test, same letter above are not TAG (triacylglycerides), FFA (free fatty acids), C (cholesterol), PtdEtn (phosphatidyletha- myelin). Fig. 3. Percentage of different lipid classes in whole body, hepatopancreas and muscle of male of Timogenes dorbignyi. Percentages are analyzed for each lipid in particular. Values are the means ± SD of three pools, analyzed separately mediante chromatography coupled to a flame ionization detector. Statistical analyses and abbreviations of lipids are as in Fig. 2. 30 A. Laino et al. / Comparative Biochemistry and Physiology, Part B 190 (2015) 27–36 Most abundant lipids in males of B. ferrrugineus were as follows: in whole body TAG and PtdCho (40%), in hepatopancreas TAG (82%), in muscle PtdCho (68%) and in the hemolymph PtdCho (52%) and PtdSer and HC (around 15%) (Fig. 4). In Fig. 5 the lipid composition of females of B. ferrugineus is shown. In whole body and hepatopancreas, TAG turned out to be the main component with around 60%, followed by PtdCho with 30% and 14% respectively. In muscle and hemolymph PtdCho was found to make up around 70%, TAG 10%. Again, a high percentage of PtdSer (15%) was found in hemolymph. With respect to T. elegansmales the most abundant fatty acids were 18:1 in hepatopancreas, gonads, whole body and hemolymph (50%, 46%, 40%, and 28% respectively); in muscle they were 18:2, 18:0, 18:1 with around 26% and in telson they were 18:0 with 36% (Table 1). Fig. 4. Percentage of different lipid classes in whole body, hepatopancreas, muscle and hemo particular. Values are the means ± SD of three pools, analyzed separately through chromatogra are as in Fig. 2. With relation to T. dorbignyimales, the most abundant fatty acids of whole body, hepatopancreas, gonad and hemolymph were 18:1 with 35% for the first two and 43% for the last two ones), in muscle 18:2 (31%) and in telson it is 48% of 18:0 (Table 2). Table 3 represents the percentages of fatty acids of B. ferrugineus males. In whole body, muscle and telson the predominant tones were 18:1 and 18:2 (between 32% and 40%), in hepatopancreas 18:0 with 36%, in gonads 18:1 with 45% and in the hemolymph 16:0 and 18:1 (around 30%). Finally, the most abundant fatty acids found in females of B. ferrugineuswere 43% and 37% of 18:0 in the whole body and hepa- topancreas, respectively. In muscle the values found were around 32% 18:2 and 18:0. In both telson and gonads the most abundant FA were 18:1 with 31% and 42%, respectively, .Meanwhile in hemolymph was found to have 42% of 16:0 (Table 4). From the comparative analysis of lymph of males of Brachistosternus ferrugineus. Percentages are analyzed for each lipid in phy coupled to a flame ionization detector. Statistical analyses and abbreviations of lipids Fig. 5. Percentage of different lipid classes in whole body, hepatopancreas, muscle and hemolymph of Brachistosternus ferrugineus females. Percentages are analyzed for each lipid in particular. Values are the means ± SD of three pools, analyzed separately through chromatography coupled to a flame ionization detector. Statistical analyses and abbreviations of lipids are as in Fig. 2. 31A. Laino et al. / Comparative Biochemistry and Physiology, Part B 190 (2015) 27–36 fatty acids of the main saponifiable lipids, differences between TAG of hepatopancreas and phospholipids (PL) of the same tissue were ob- served in T. elegans (Table 5). Here, an increase of 16:0, 18:1, and a de- crease in 18:2 was observed. This pattern is repeated in T. dorbignyi and B. ferrugineus (Table 6, 7, and 8). For the case of the muscle of T. elegans, T. dorbignyi, andmales of B. ferrugineus, there is a great differ- ence at the level of the sum of saturated where in TAG is between 78% and 87% and in PL only from 35% to 48%. This difference is given by an increase in 16:0 and 18:0, and a decrease in mono unsaturated and poly unsaturated (Table 5, 6 and 7). In females of B. ferrugineus an in- crease in TAG of muscle is also observed, but with a pattern of fatty acids different from the aforementioned (Table 8). 4. Discussion As expected, in the three species studied here, it was observed that the organ with greater lipid content was the hepatopancreas (60 to 118 mg/g). It is possibly themost important storage organ of scorpions, Table 1 Fatty acid composition ofwhole body, hepatopancreas, muscle, telson, gonad and hemolymph o and quantified by GLC-FID. Values are themeans± SD of three independent analyses of three p whole body, hepatopancreas,muscle, telson, gonad and hemolymph for each percentage of fatty levels of significance, p b 0.05. SFA (saturated fatty acid), MUFA (mono unsaturated fatty acid) T. elegans male Whole body Hepatopancreas Muscl 14:0 1.11 ± 1a 1.59 ± 1a 0.50 15:0 0.40 ± 0.3a 0.47 ± 0.4a 0.20 16:0 15.90 ± 6a 20.11 ± 3a 11.20 16:1 0.90 ± 0.6a 1.33 ± 1.1a 0.90 17:0 0.90 ± 0.7a 0.80 ± 0.9a 0.60 17:1 1.70 ± 1.2a 0.55 ± 0.5a 0.90 18:0 16.50 ± 3a 8.38 ± 2a 26.50 18:1 40.80 ± 7a 50.74 ± 7.5a 24.20 18:2 19.40 ± 3a 13.83 ± 1a 30.80 18:3 0.50 ± 0.4a 1.85 ± 1a 1.60 20:0 0.90 ± 0.9a ND 0.30 20:3 ND ND 1.50 22:0 0.60 ± 0.5a 0.26 ± 0.2a 0.30 ∑SFAs 36.31 ± 12.4a 31.62 ± 7.9a 39.60 ∑MUFAs 43.40 ± 8.8a 52.62 ± 9.1a 26.00 ∑PUFAs 19.90 ± 3.4a 15.68 ± 2ab 33.90 ∑UFAs 63.30 ± 12.2a 68.30 ± 11.1a 59.90 not only for the quantity of lipids stored in it, but also for the presence of TAG asmajority lipid (see further). This organ is described in other scor- pions because of its great importance for nutrient storage and metabo- lism (Ramamurthi et al., 1975; Warburg et al., 2002) and has likewise high lipid concentrations (Raghavaiah and Ramamurthi, 1977). It is analogous to theM-diverticula of spiders (Laino et al., 2009), the insect's fat body (Seifert and Rosenberg, 1977; Warburg et al., 2002) and the hepatopancreas of crustaceans (O'Connor, J.M., Gilbert, L.I., 1968; Al-Mohanna and Nott, 1986; García et al., 2002b). In the three species of scorpions studied, a majority content of TAG was observed in the hepatopancreas ranging from 42% to 82%, coincid- ing with that found in the hepatopancreas of the scorpion Leiurus quinquestriatus (El-Salhy et al., 1981). In the M-diverticula of the spider Polybetes pythagoricus TAG constitutes the main lipid storage form, representing about 74% of the total lipid (Laino et al., 2009). In insects they represent around 70%–90% in the fat body (Bailey et al., 1975; Sobotnik et al., 2006) and in crustaceans' hepatopancreas around 68% (González Baró and Pollero, 1988). f males of Timogenes elegans.Data on fatty acid classes are expressed asweight percentage ools. Different letters (a, b, c, d and e) indicate statistically significant differences between acid determined by Tukey's post hoc test; same letters indicate non-significantly different , PUFA (poly unsaturated fatty acid), and UFA (unsaturated fatty acid). e Telson Gonad Hemolymph ± 0.4a 1.11 ± 0.5a 1.06 ± 1a 1.80 ± 0.7a ± 0.2a 0.41 ± 0.3a 0.24 ± 0.3a 2.60 ± 0.1b ± 4ab 14.23 ± 3ab 15.33 ± 4a 25.90 ± 0.3ac ± 0.7a 2.14 ± 0.3a 0.54 ± 0.4a 4.40 ± 0.1b ± 0.5a 1.03 ± 0.7a 0.21 ± 0.3a ND ± 0.8a ND 0.19 ± 0.2a ND ± 5b 36.42 ± 5c 14.52 ± 3a 14.80 ± 1.4a ± 4b 18.92 ± 2b 46.6 ± 6.5a 28.40 ± 1ab ± 4.1b 20.42 ± 2.1ac 16.5 ± 1.5a 11.00 ± 0.8ad ± 1.2a 0.46 ± 0.3a 0.85 ± 0.7a 0.20 ± 1.5a ± 0.4a 0.14 ± 0.3a ND 4.20 ± 0.5b ± 1.3a 2.80 ± 0.7a 3.19 ± 0.5a ND ± 0.2a 1.70 ± 1.1a 0.26 ± 0.4a 6.30 ± 0.8b ± 10.7a 55.0 ± 10.9a 31.6 ± 8.7a 55.60 ± 3.8a ± 5.6ab 21.06 ± 2.3b 47.4 ± 7.1a 32.80 ± 1.1ab ± 6.6ac 23.68 ± 9.4a 20.5 ± 2.7a 11.20 ± 2.3ab ± 12.2a 44.7 ± 11.7a 67.9 ± 9.8a 44.00 ± 3.4a Table 2 Fatty acid composition of whole body, hepatopancreas, muscle, telson, gonad and hemolymph of males of Timogenes dorbignyi. Data on fatty acid classes are expressed asweight percent- age and quantified by GLC-FID. Values are the means ± SD of three independent analyses of three pools. Different letters (a, b, c, d and e) indicate statistically significant differences be- tween whole body, hepatopancreas, muscle, telson, gonad and hemolymph for each percentage of fatty acid determined by Tukey's post hoc test; same letters indicate non-significantly different levels of significance, p b 0.05. SFA (saturated fatty acid), MUFA (mono unsaturated fatty acid), PUFA (poly unsaturated fatty acid), and UFA (unsaturated fatty acid). T. dorbygnyi male Whole body Hepatopancreas Muscle Telson Gonad Hemolymph 14:0 0.90 ± 0.7a 0.90 ± 0.1a 0.43 ± 0.2a 0.66 ± 0.4a 1.49 ± 0.2a 4.10 ± 1.8b 15:0 0.40 ± 0.5a 0.50 ± 0.3a 0.22 ± 0.1a 0.23 ± 0.1a 0.60 ± 0.3a 1.50 ± 1a 16:0 17.30 ± 2.1a 21.97 ± 1ac 11.13 ± 1.5ad 22.60 ± 2.5ac 20.48 ± 2.1ac 25.00 ± 1.5bc 16:1 2.10 ± 0.9a 1.40 ± 0.5a 0.43 ± 0.2a 0.80 ± 0.4a 0.88 ± 0.6a 4.30 ± 1b 17:0 0.20 ± 0.1a 1.30 ± 0.2b 0.60 ± 0.3ac 0.50 ± 0.2a 1.06 ± 0.2bc ND 17:1 0.76 ± 0.6a ND 1.24 ± 0.6a ND ND ND 18:0 23.40 ± 3.1a 12.04 ± 1.5b 26.61 ± 4.4a 48.00 ± 6.1c 13.08 ± 2.7b 10.40 ± 1b 18:1 32.16 ± 4.7a 38.00 ± 5.1ab 24.17 ± 2.4ac 14.43 ± 1.5c 45 ± 1b 42.00 ± 1.5ab 18:2 20.18 ± 2.9a 17.45 ± 3ab 31.56 ± 5.7d 11.63 ± 2bc 13.82 ± 2ac 9.40 ± 0.5b 18:3 0.60 ± 0.3a 5.23 ± 0.6b 0.77 ± 0.3a 0.35 ± 0.2a 1.02 ± 0.3a ND 20:0 0.70 ± 0.1a 0.30 ± 0.2b 0.29 ± 0.1b 0.15 ± 0.1b ND ND 20:3 0.70 ± 0.2a 0.60 ± 0.1a 1.40 ± 2.1a 0.50 ± 0.3a 2.15 ± 0.3a 1.60 ± 1 22:0 0.32 ± 0.3a 0.29 ± 0.1a 0.83 ± 0.4a 0.17 ± 0.1a 0.52 ± 0.4ab 1.10 ± 0.5ac ∑SFAs 43.22 ± 6.9a 37.29 ± 3.5a 40.11 ± 7a 72.31 ± 9.5b 37.24 ± 5.7a 42.1 ± 5.8a ∑MUFAs 35.02 ± 6.2a 39.40 ± 5.6a 25.84 ± 3.2ac 15.23 ± 1.9bc 45.88 ± 1.6a 46.3 ± 2.5ad ∑PUFAs 21.48 ± 3.2a 23.27 ± 3.6ab 33.73 ± 8.1b 12.48 ± 2.5a 16.99 ± 2.6a 11 ± 1.5a ∑UFAs 56.50 ± 9.4a 62.67 ± 9.2a 59.57 ± 11.3a 27.71 ± 4.4b 62.87 ± 4.2a 57.3 ± 4a 32 A. Laino et al. / Comparative Biochemistry and Physiology, Part B 190 (2015) 27–36 With regard to the composition of fatty acids of the hepatopancreas of the three species studied, the chromatographic patterns match those described for fatty acids of PL and TAG of the hepatopancreas of the scorpions L. quinquestriatus and Centruroides vittatus, where the main fatty acids were 18:1, 18:2, 18:0 and 16:0 (El-Salhy et al., 1981; Uscian and Stanley-Samuelson, 1994). This same pattern was also observed in whole body preparations and M- diverticula of four labidognath spider species (Uscian and Stanley-Samuelson, 1994; Laino et al., 2009), as well as in one millipede species (Order Polydesmida) (Uscian and Stanley-Samuelson, 1994). It is known that in insects, lipids have a rel- atively high quantity of unsaturated fatty acids C18 (Uscian and Stanley- Samuelson, 1994). Also, in general, the profile found in thepresentwork matches with that recently reported for five species of insects (Tzompa- Sosa et al., 2014). In our case it is important to highlight that although in the three species studied here, 18 carbonswere themost abundant fatty acids found, there is a difference in the percentage of 18:0 between the two species of T. elegans andmales and females of B. ferrugineus, the dif- ference being more than double in this last species. This difference is possibly due to the alimentary habit of the different species as deter- mined by Schartau and Leidescher (1983) when they compared fatty acids of tarantula Aphonopelma hentzi (Girard 1852) with those of the scorpion Leiurus quinquestriatus. Table 3 Fatty acid composition of whole body, hepatopancreas, muscle, telson, gonad and hemolymph percentage and quantified byGLC-FID. Values are themeans±SDof three independent analyse betweenwhole body, hepatopancreas, muscle, telson, gonad and hemolymph for each percenta different levels of significance, p b 0.05. SFA (saturated fatty acid), MUFA (mono unsaturated f B. ferrugineus Male Whole body Hepatopancreas Musc 14:0 0.61 ± 0.5a 0.90 ± 0.7a 0.90 ± 15:0 0.39 ± 0.3a 0.30 ± 0.1a 0.40 ± 16:0 12.81 ± 3.1a 20.80 ± 4.2ac 11.60 16:1 0.59 ± 0.4a 1.80 ± 0.9a 0.90 ± 17:0 0.92 ± 0.7a 0.60 ± 0.5a ND 17:1 1.07 ± 0.5a ND 0.80 ± 18:0 14.79 ± 4.1a 36.40 ± 7.2b 12.40 18:1 34.43 ± 4.5a 25.05 ± 3.5ab 34.90 18:2 31.16 ± 6.8a 11.38 ± 2.3b 33.99 18:3 1.40 ± 0.8a 0.87 ± 0.5a 1.00 ± 20:0 0.72 ± 0.7a 0.57 ± 0.4a 0.56 ± 20:3 0.42 ± 0.2a 0.53 ± 0.3ab 1.90 ± 22:0 0.64 ± 0.3a 0.36 ± 0.4a 0.27 ± ∑SFAs 30.88 ± 9.7a 59.93 ± 13.2b 26.13 ∑MUFAs 36.10 ± 5.4a 26.85 ± 4.4ab 36.60 ∑PUFAs 32.98 ± 7.6a 12.78 ± 3.1b 36.89 ∑UFAs 69.08 ± 13a 39.63 ± 7.5bc 73.5 ± The information related to lipids present in muscle in Class Arachnida is very scarce. The best representation perhaps stems from studies performed on spiders, where the transference of radiolabelled lipids from hemolymphatic proteins to muscle was observed (Laino et al., 2009).With relation to the quantity of lipids present in themuscle of the three species studied, values ranging from25 to 58mg/gwere ob- served, which correspond to 50% of lipids found in the hepatopancreas; this seems to be reasonable because it is not a storage tissue. PL are the majority, surely due to their importance in membrane formation. It is worth mentioning that the so-called “muscle” of the present work is represented by legs and chelas. The analysis of those appendages was performed separately since they possess different muscle/cuticle rela- tionship. A similar composition of lipids and fatty acids was observed for both appendages (results not shown). Majority fatty acids found in the muscle of T. elegans, T. dorbignyi, and B. ferrugineus are 18:2, 18:1, and 18:0. This coincides with the only information reported by scorpions, where Uscian and collaborators analyzed fatty acids of TAG and PL of the scorpion Centruroides vittatus, though unfortunately legs, head and thorax were analyzed as a whole (Uscian and Stanley-Samuelson, 1994). From these experimental re- sults we can conclude that the lipid composition of fatty acids seems to be quite stable among the different species of scorpions (even of males of Brachistosternus ferrugineus. Data on fatty acid classes are expressed as weight s of three pools. Different letters (a, b, c, d and e) indicate statistically significant differences ge of fatty acid determined by Tukey's post hoc test; same letters indicate non-significantly atty acid), PUFA (poly unsaturated fatty acid), and UFA (unsaturated fatty acid). le Telson Gonad Hemolymph 0.7a 1.48 ± 0.7a 1.00 ± 0.6a 4.46 ± 1.4b 0.3a ND ND 3.96 ± 1.3b ± 3.7ad 19.85 ± 2.7a 18.40 ± 2.2a 32.38 ± 2.8b 0.7a ND 1.20 ± 0.7a 7.00 ± 2b ND 0.70 ± 0.3a ND 0.5a ND ND 1.15 ± 0.2a ± 3.2a 13.12 ± 2.9a 11.50 ± 2.1a 11.47 ± 0.9a ± 4.7a 40.99 ± 5.2ac 45.50 ± 5.1ac 27.68 ± 2.8a ± 4.8a 22.40 ± 3.7ac 15.28 ± 2.1bc 7.88 ± 0.2b 0.2a ND 1.70 ± 0.3a 0.72 ± 0.1a 0.9a ND 1.90 ± 0.2a 1.77 ± 0.1a 0.3bd 1.36 ± 0.5be 0.50 ± 0.4ab ND 0.1a 0.80 ± 0.5a 1.96 ± 0.7b 1.00 ± 0.4a ± 8.9ac 35.25 ± 6.8a 35.46 ± 6.1a 55.03 ± 6.8ad ± 5.9a 40.99 ± 5.2a 46.70 ± 5.8ac 35.82 ± 4.8a ± 5.3ac 23.76 ± 4.2ad 17.48 ± 2.8b 8.60 ± 0.3b 11.2a 64.75 ± 9.4ac 64.18 ± 8.6ac 44.42 ± 5.1ac Table 4 Fatty acid composition of whole body, hepatopancreas, muscle, telson, gonad and hemolymph of females of Brachistosternus ferrugineus. Data on fatty acid classes are expressed asweight percentage and quantified byGLC-FID. Values are themeans±SDof three independent analyses of three pools. Different letters (a, b, c, d and e) indicate statistically significant differences betweenwhole body, hepatopancreas, muscle, telson, gonad and hemolymph for each percentage of fatty acid determined by Tukey's post hoc test; same letters indicate non-significantly different levels of significance, p b 0.05. SFA (saturated fatty acid), MUFA (mono unsaturated fatty acid), PUFA (poly unsaturated fatty acid), and UFA (unsaturated fatty acid). B. ferrugineus Female Whole body Hepatopancreas Muscle Telson Gonad Hemolymph 14:0 0.90 ± 0.7a 0.90 ± 0.4a 0.52 ± 0.1a 1.19 ± 0.4a 2.00 ± 0.9a 2.40 ± 2a 15:0 0.39 ± 0.3a 0.8 ± 0.4a 0.19 ± 0.1a 0.30 ± 0.2a 0.90 ± 0.7ac 2.90 ± 2bc 16:0 18.73 ± 3.2a 20.51 ± 3.1a 13.1 ± 1.9a 20.38 ± 2.7a 21.8 ± 3.1a 41.70 ± 10a 16:1 1.01 ± 0.1a 1.80 ± 0.4a 0.99 ± 0.7a 2.15 ± 1.3a 1.50 ± 0.7a ND 17:0 0.83 ± 0.7a 0.92 ± 0.1a 0.77 ± 0.5a 0.82 ± 0.4a ND 1.20 ± 0.4a 17:1 0.10 ± 0.1a 0.14 ± 0.1a ND ND 1.30 ± 0.2b ND 18:0 43.85 ± 5.2a 37.70 ± 4.7a 34.1 ± 3.9a 19.14 ± 3.7b 10.9 ± 1.9b 17.90 ± 9b 18:1 10.74 ± 2.1a 17.60 ± 2.1a 9.57 ± 2.2a 31.14 ± 4.9b 42.8 ± 5.1c 9.10 ± 2.1a 18:2 18.89 ± 2.1a 14.39 ± 2.1a 35.6 ± 4.1b 19.36 ± 2.1a 13.9 ± 1.5a 17.60 ± 0.2a 18:3 3.00 ± 0.7a 4.76 ± 0.7b 2.04 ± 0.7ab 0.73 ± 0.4bc 2.15 ± 0.7ab 0.70 ± 0.4a 20:0 0.82 ± 0.4a 0.58 ± 0.3a 0.11 ± 0.1ab 0.91 ± 0.7a 1.5 ± 0.3ac 1.40 ± 0.7a 20:3 ND 0.36 ± 0.2a 2.20 ± 0.7a 2.32 ± 2.1a 0.69 ± 0.2a 2.70 ± 1a 22:0 0.38 ± 0.2a 0.26 ± 0.2a 0.71 ± 0.5ab 1.53 ± 0.7bc 0.44 ± 0.4ab 2.43 ± 0.1c ∑SFAs 65.91 ± 10.7a 62,47 ± 9.6a 49.5 ± 7.1a 44.29 ± 8.8a 37.5 ± 7.3a 69.93 ± 24.2a ∑MUFAs 11.85 ± 2.3a 19.54 ± 2.6a 10.5 ± 2.9a 33.30 ± 6.2b 45.6 ± 6c 9.10 ± 2.1a ∑PUFAs 21.89 ± 2.8a 19.50 ± 3a 39.8 ± 5.5b 22.41 ± 4.6a 16.7 ± 2.4a 21.00 ± 1.6a ∑UFAs 33.74 ± 5.1a 39.05 ± 5.6ab 50.4 ± 8.4ac 55.71 ± 10.8bc 62.3 ± 8.4c 30.10 ± 3.7ac 33A. Laino et al. / Comparative Biochemistry and Physiology, Part B 190 (2015) 27–36 comparingdifferent families), since in themuscle a higher percentage of 18:2was foundwith relation to all the tissues/organs studied. There are severalworks in insects determining thatmembranes can be affected by the type of diet of the different species (Carvalho et al., 2012), similar to what can occur in the hepatopancreas, but at muscle level. The results based on analyzingmuscle in thepresentwork do not showa significant difference among species. Based on the results of the chromatography analysis of hemolymphatic lipids, PL were found to be the majority lipids in the circulation of T. elegans, as well as in males and females of B. ferrugineus, possibly transporting these PL to the different organs to cover the structural requirements of plasmaticmembranes. This finding is in agreement with the one described in the scorpion Leiurus quinquestriatus (El-Salhy et al., 1981), in spiders (Cunningham et al., 2007) and crustaceans (Lee and Puppione, 1978; García et al., 2004). On the other hand, it does not coincide with the results described by Schenk and collaborators in the scorpion Pandinus imperator, where Table 5 Fatty acid composition of TAG and PL of hepatopancreas andmuscle ofmales of Timogenes elegans. Data on fatty acid classes are expressed as weight percentage and quantified by GLC-FID. Values are the means ± SD of three independent analyses of three pools. Student's t-test was used to compare the significance of the differences fatty acid compo- sition of PL with respect to TAG in hepatopancreas and PL with respect to TAG in muscle. SFA (saturated fatty acid), MUFA (mono unsaturated fatty acid), PUFA (poly unsaturated fatty acid), and UFA (unsaturated fatty acid). T. elegans Male PL TAG Hepatopancrease Muscle Hepatopancrease Muscle 14:0 3.47 ± 0.9⁎ 1.84 ± 0.7⁎⁎ 1.93 ± 0.2 9.20 ± 1.1 15:0 1.42 ± 0.5 1.34 ± 0.3⁎⁎ 0.69 ± 0.5 3.50 ± 0.4 16:0 16.91 ± 2.7⁎ 16.5 ± 1.2⁎⁎ 24.36 ± 2.7 39.80 ± 4.2 16:1 ND ND 1.48 ± 0.7 4.20 ± 0.5 17:1 0.21 ± 0.1 ND ND ND 18:0 13.31 ± 2.7 15.65 ± 1.7⁎ 9.62 ± 0.7 30.00 ± 3.5 18:1 31.99 ± 4.5⁎ 28.97 ± 2.7⁎⁎ 54.19 ± 7.1 9.80 ± 1.3 18:2 25.55 ± 2.9⁎⁎ 28.05 ± 2.1⁎⁎ 7.73 ± 0.4 2.80 ± 0.3 18:3 2.55 ± 0.9⁎ ND 0.10 ± 0.1 0.30 ± 0.4 20:0 0.71 ± 0.5 ND ND ND 20:3 2.83 ± 0.7 7.57 ± 0.3 ND ND 22:0 1.06 ± 0.6 ND ND ND ∑SFAs 36.88 ± 7.9 35.33 ± 3.9⁎⁎ 36.60 ± 4.1 82.50 ± 9.2 ∑MUFAs 32.2 ± 4.6⁎ 28.97 ± 2.7⁎⁎ 55.67 ± 7.8 14.00 ± 1.8 ∑PUFAs 30.93 ± 4.5⁎⁎ 35.62 ± 2.4⁎⁎ 7.83 ± 0.5 3.10 ± 0.7 ∑UFAs 63.13 ± 9.1 64.59 ± 5.1⁎⁎ 63.50 ± 8.3 17.10 ± 2.5 ⁎ p b 0.05. ⁎⁎ p b 0.001. only 30% of PL were found in hemolymphatic lipoproteins (Schenk et al., 2009). Asmentioned previously, PL are structural lipids per excellence, con- taining a PL in particular which is characterized by a highly negative charge and pI 1.2 (Abramson et al., 1964): phosphatidylserine. This PL is found at an unusually high concentration in the scorpion Pandinus imperator. It seems that the negative charge of the PL may be related to the efficiency or quantity of venom generated (Schenk et al., 2009). This fact could be certain since in an early study of venom of the scorpion Leiurus quinquestriatus a great quantity of PtdSer was ob- served by thin-layer chromatography (Marie and Ibrahim, 1976). In T. elegans, as well as in males of B. ferrugineus and females of B. ferrugineus the quantity of PtdSer was very high, similar to that found in P. imperator. The pattern of fatty acids of telson – a structure with venom gland as majority component – did not show great differences with those of the other tissues, leading to the conclusion that at least in telson there are no lipids with qualitatively and/or Table 6 Fatty acid composition of TAG and PL of hepatopancreas andmuscle, of male of Timogenes dorbignyi. Data on fatty acid classes are expressed as weight percentage and quantified by GLC-FID. Values are the means ± SD of three independent analyses of three pools. Student's t-test was used to compare the significance of the differences fatty acid compo- sition of PL with respect to TAG in hepatopancreas and PL with respect to TAG in muscle. SFA (saturated fatty acid), MUFA (mono unsaturated fatty acid), PUFA (poly unsaturated fatty acid), and UFA (unsaturated fatty acid). T. dorbygnyi Male PL TAG Hepatopancreas Muscle Hepatopancreas Muscle 14:0 3.19 ± 0.7⁎ 3.43 ± 0.4⁎ 1.70 ± 0.4 5.17 ± 0.4 15:0 1.29 ± 0.6 1.57 ± 0.2⁎⁎ 0.79 ± 0.5 6.74 ± 0.7 16:0 18.82 ± 2.1⁎ 24.35 ± 2.8⁎ 27.80 ± 2.9 44.83 ± 4.9 16:1 1.39 ± 0.2 1.57 ± 0.7 1.66 ± 0.9 ND 17:1 1.00 ± 0.7 1.43 ± 0.9 ND ND 18:0 13.65 ± 3.1 17.77 ± 2.1⁎ 10.04 ± 1.5 30.65 ± 3.1 18:1 28 ± 0.9⁎ 24.16 ± 2.9⁎⁎ 30 ± 1 9.98 ± 0.7 18:2 29.28 ± 1.1⁎ 17.87 ± 3.2⁎⁎ 27.02 ± 1 2.63 ± 0.7 18:3 0.70 ± 0.4 ND 0.80 ± 0.7 ND 20:0 ND 0.65 ± 0.5 ND ND 20:3 2.29 ± 0.4 6.20 ± 0.9 ND ND 22:0 ND 1.00 ± 0.8 ND ND ∑SFAs 36.95 ± 6.5 48.70 ± 6.8⁎ 40.33 ± 5.3 87.39 ± 9.1 ∑MUFAs 30.39 ± 1.8 27.10 ± 4.5⁎ 31.66 ± 1.9 9.98 ± 0.7 ∑PUFAs 32.27 ± 1.9 24.00 ± 4.1⁎⁎ 27.82 ± 1.7 2.63 ± 0.7 ∑UFAs 62.66 ± 3.7 51.20 ± 8.6⁎⁎ 59.28 ± 3.6 12.61 ± 1.4 ⁎ p b 0.05. ⁎⁎ p b 0.001. Table 7 Fatty acid composition of TAG and PL of hepatopancreas and muscle of males of Brachistosternus ferrugineus. Data on fatty acid classes are expressed as weight percentage and quantified by GLC-FID. Values are the means ± SD of three independent analyses of three pools. Student's t-test was used to compare the significance of the differences fatty acid composition of PL with respect to TAG in hepatopancreas and PL with respect to TAG in muscle. SFA (saturated fatty acid), MUFA (mono unsaturated fatty acid), PUFA (poly unsaturated fatty acid), and UFA (unsaturated fatty acid). B. ferrugineus Male PL TAG Hepatopancreas Muscle Hepatopancreas Muscle 14:0 5.26 ± 0.7⁎⁎ 2.6 ± 0.5 0.70 ± 0.6 2.26 ± 0.7 15:0 2.37 ± 0.4⁎ 1.3 ± 0.5⁎⁎ 0.90 ± 0.5 4.50 ± 0.5 16:0 24.20 ± 0.9⁎ 15.9 ± 1.7⁎⁎ 26.39 ± 0.6 41.00 ± 5.4 16:1 1.47 ± 0.7 1.1 ± 0.6⁎⁎ 2.45 ± 0.3 5.40 ± 0.8 17:1 0.61 ± 0.5 1.1 ± 0.2 1.01 ± 0.1 ND 18:0 12.97 ± 2.1⁎ 14 ± 2.3⁎ 7.76 ± 0.3 30.20 ± 5.2 18:1 30.84 ± 2.7⁎ 25.4 ± 4.5⁎ 39.46 ± 3 10.00 ± 3.4 18:2 19.33 ± 2.1⁎ 28.3 ± 3.1⁎ 10.22 ± 1.5 6.33 ± 0.8 18:3 1.47 ± 0.1⁎ 1.51 ± 0.7⁎ 0.62 ± 0.5 0.10 ± 0.1 20:0 ND ND 3.13 ± 0.3 ND 20:3 1.60 ± 0.8⁎ 5.90 ± 0.7 3.75 ± 0.4 ND 22:0 ND 2.00 ± 0.7 3.03 ± 0.5 ND ∑SFAs 44.80 ± 4.1 35.8 ± 5.7⁎ 41.91 ± 6.7 77.96 ± 11.8 ∑MUFAs 32.90 ± 3.9 27.6 ± 5.3⁎ 42.90 ± 3.4 15.4 ± 2.8 ∑PUFAs 22.40 ± 3⁎ 35.71 ± 4.5⁎⁎ 14.50 ± 2.4 6.43 ± 0.9 ∑UFAs 55.32 ± 6.9 63.31 ± 9.8⁎ 57.40 ± 5.8 21.83 ± 5.1 ⁎ p b 0.05. ⁎⁎ p b 0.001. 34 A. Laino et al. / Comparative Biochemistry and Physiology, Part B 190 (2015) 27–36 quantitatively different fatty acids. The relation of PtdSer with the venom is not clear enough at present and needs to be studied further. Although data related to the circulation of energetic lipids in scorpions are restricted only to the species Leiurus quinquestriatus (El- Salhy et al., 1981), currently, and including the present work, it seems that in these arthropods the majority energetic lipid is the TAG. This fact coincides with what we described in two araneomorph spiders (Cunningham et al., 1994, 2000, 2007) but differs significantly from whatwas described for groupswith diacylglycerides as themajority en- ergetic lipid, as is the case in two mygalomorph spiders (Haunerland and Bowers, 1987; Laino et al., 2015) and in insects (Thomas and Gilbert, 1968; Peled and Tietx, 1975; Ryan et al., 1988; Rodenburg and Van der Horst, 2005; Weers and Ryan, 2006). Table 8 Fatty acid composition of TAG and PL of hepatopancreas and muscle of females of Brachistosternus ferrugineus. Data on fatty acid classes are expressed as weight percentage and quantified by GLC-FID. Values are the means ± SD of three independent analyses of three pools. Student's t-test was used to compare the significance of the differences fatty acid composition of PL with respect to TAG in hepatopancreas and PL with respect to TAG in muscle. SFA (saturated fatty acid), MUFA (mono unsaturated fatty acid), PUFA (poly unsaturated fatty acid), and UFA (unsaturated fatty acid). B. ferrugineus Female PL TAG Hepatopancreas Muscle Hepatopancreas Muscle 14:0 2.94 ± 0.4 2.0 ± 0.4 3.21 ± 0.4 2.70 ± 0.7 15:0 1.22 ± 0.2 1.4 ± 0.2⁎ 1.29 ± 0.3 2.89 ± 0.4 16:0 23.72 ± 3.1 19.9 ± 2.2⁎ 31.00 ± 4.2 34.40 ± 3.5 16:1 1.35 ± 0.3 1.14 ± 0.2⁎ 1.31 ± 0.2 3.25 ± 0.5 17:1 1.29 ± 0.2 1.15 ± 0.2 0.95 ± 0.7 ND 18:0 11.25 ± 1.3⁎ 16.3 ± 1.7⁎ 8.16 ± 0.5 22.00 ± 2.5 18:1 31.10 ± 4.1 23.1 ± 2.5 38.00 ± 3.9 28.08 ± 3.1 18:2 18.56 ± 2.1⁎ 24.3 ± 3.1⁎⁎ 12.00 ± 1.9 6.30 ± 0.7 18:3 6.02 ± 0.7⁎ 2.50 ± 0.3⁎⁎ 2.96 ± 0.3 0.10 ± 0.1 20:0 ND ND 0.34 ± 0.1 ND 20:3 1.72 ± 0.4⁎ 6.91 ± 0.9 0.39 ± 0.2 ND 22:0 0.82 ± 0.5 1.06 ± 0.4 ND ND ∑SFAs 39.9 ± 5.5 40.8 ± 4.9⁎ 44 ± 5.5 61.99 ± 7.1 ∑MUFAs 33.7 ± 4.6 25.4 ± 2.9 40.26 ± 4.8 31.3 ± 3.6 ∑PUFAs 26.3 ± 3.2⁎ 33.7 ± 4.3⁎⁎ 15.35 ± 2.4 6.4 ± 0.8 ∑UFAs 60.04 ± 7.8 59 ± 7.2⁎ 55.61 ± 7.2 37.73 ± 4.4 ⁎ p b 0.05. ⁎⁎ p b 0.001. The composition of fatty acids of the hemolymph showed a similar pattern in the three species studied, most of them being fatty acids of 16 and 18 carbons, similar to those described by El-Salhy et al. (1981) for L. quinquestriatus, in insects and crustaceans (Gilbert and O'Connor, J.M., 1970; Gilbert et al., 1977; Pattnaik et al., 1979). Another constant characteristic related to the hemolymph of scorpions is that, similarly to what was observed in L. quinquestriatus, in T. elegans, T. dorbignyi and B. ferrugineus the C20 fatty acids do not exceed 4%. This differs from the percentages of fatty acids of 20 carbons previously reported for insects and crustaceans, found to be higher than the ones reported here (Gilbert and Chino, 1974). Assays using radiolabelled acetate rein- force the hypothesis of the absence of arachidonic acid (20 carbons) in scorpions (Centuroides sculpturatus (Wood, 1863)), and confirm their presence in mygalomorphae spiders (Aphonepelma sp.) (Ross and Monroe, 1970). Subsequently, Schartau and Leidescher (1983) while working with another mygalomorph spider, Aphonopelma hentzi, confirmed these observations. In a later work (Uscian and Stanley- Samuelson, 1994) they observed almost twice the 20:4 PL of that found for the scorpion Centruroides vittatus, when comparing the PL of the tarantula Grammostola sp. It is important to highlight that with regard to the composition of fatty acids of hemolymph and hepatopancreas in the males of the threemodels, the hepatopancreas is enriched in 18:2 and impoverished in 16:0, coinciding with what was reported in L. quinquestriatus (El Salhy et al., 1981). As determined by El Salhy et al. (1981), it is clear that there ismore than one pool of fatty acids in the hepatopancre- as with different degrees of interchange with the hemolymph. This hy- pothesis can be extended to the different organs analyzed; for example whatwas found in gonads,where not only the gonad is enriched in 18:2 but also in 18:1, and impoverished in 16:0. No great differences be- tween patterns of fatty acids or lipids were observed between males and females. Variations are possibly found at the level of quantity of lipids, which are surely accompanied by increasing concentrations of water and glycogen as described for scorpions in general (Warbug, 2012). This fact may be reflected by a lower concentration of lipids found in the hepatopancreas and by a greater percentage of TAG in the whole body of females of B. ferrugineus. In 2011 Lease andWolf observed in females of terrestrial arthropods (several representatives of Class Arachnida and Class Insecta) the lipid content (dry weight) was higher than that found in males. However, significant differences were not observed for the specific case of scorpions, because their study seems to be incomplete with relation to males n = 1. Further comparative studies with other species are necessary in order to clarify this issue. A differential pattern of fatty acids was observed when the majority saponifiable lipids (PL and TAG) found in the hepatopancreas and mus- cle of the males of the three species were compared. More specifically, when TAG and PL of the hepatopancreas are compared, it can be noted that TAG have an enrichment of 18:1, 16:0 and an impoverish- ment of 18:2 with regard to PL, coinciding with the results described for the hepatopancreas of L. quinquestriatus (El-Salhy et al., 1981) and complete abdomen of C. vittatus (Uscian and Stanley-Samuelson, 1994). For the case of PL and TAG of muscle, a great increase in the per- centage of saturated fatty acids of over 75% mainly given by 16:0 and 18:0 was observed in TAG, at the expense of a decrease of over 80% of poly unsaturated and over 35% mono unsaturated. This difference may be in the different studies, disguised by a great quantity of PtdCho in the muscular tissue. Although it is certain that the physiological requirements and envi- ronmental constraints argue against a characteristic fatty acid pattern in the arthropods as discussed for insects earlier (Stanley-Samuelson et al., 1988), it is evident that there are patterns of lipids and fatty acids that are shared by the three studied species (Family Bothriuridae) and that match with what was described for unrelated scorpions, as L. quinquestriatus, C. vittatus (Family Buthidae) and P. imperator (Family Scorpionidae). Further studies are necessary to discover new 35A. Laino et al. / Comparative Biochemistry and Physiology, Part B 190 (2015) 27–36 information which may lead to new interpretations of the lipidic characteristics of these primitive animals. Acknowledgments This work was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-PIP no. 075) and Agencia Nacional de Promoción Científica y Tecnológica (PICT no. 2010-1270 and PICT no. 2010-1764), Argentina. All authors are mem- bers of CONICET, Argentina. The authors are grateful to Rosana del Cid and Sara Ceccarelli for the revision of the English, Mario Ramos the fig- ure design, Eva Illara de Bozzolo for their excellent technical and Sergio Mijailovsky for mass specrometry. We are also indebted to Renzo Adilardi for his help in the field work, to Administración de Parques Nacionales for providing the necessary permits to conduct our collec- tion campaign, and to all the staff working in Reserva Nacional Formosa, whose collaboration was substantial for our field work. References Abramson,M.B., Katzman, R.,Gregor,H.P., 1964. Aqueous dispersions of phosphatidylserine. Ionic properties. J. Biol. Chem. 239, 70–76. Ackman, R.G., McLeod, C., Banerjee, A.K., 1990. An overview of analyses by chromarod- iatroscan TLC-FID. J. Planar Chromatogr. 3, 450–490. Almaaytah, A., Albalas, Q., 2014. Scorpion venom peptides with no disulfide bridges: a review. Peptides 51, 35–45. Al-Mohanna, S.Y., Nott, J.A., 1986. B-Cells and digestion in the hepatopancreas of Penaeus semisulcatus (Crustacea: Decapoda). J. Mar. Biol. Assoc. UK 66, 403–414. Arrese, E.L., Soulages, J.L., 2010. Insect fat body: energy, metabolism, and regulation. An. Rev. Entomol. 55, 207–225. Bailey, E., Horne, J.A., Izatt, M.E., 1975. The effects of allatectomy on the lipid composition of the fat body and haemolymph of adult Locusta. Comp. Biochem. Physiol. B 52, 525–528. Cabrera, A.L., 1976. Regiones fitogeográficas argentinas. Enciclop. Arg. Agric. Jard. Tomo II Fs. 1. Ed. ACME. Bs. As, Argentina (1-85 pp). Carvalho, M., Sampaio, J.L., Palm, W., Brankatschk, M., Eaton, S., Shevchenko, A., 2012. Effects of diet and development on the Drosophila lipidome. Mol. Syst. Biol. 8, 600. Cunningham, M., Pollero, R.J., 1996. Characterization of lipoprotein fractions with high content of hemocyanin in the hemolymphatic plasma of Polybetes pythagoricus. J. Exp. Zool. 274, 275–280. Cunningham, M., Pollero, R.J., Gonzalez, A., 1994. Lipid circulation in spiders. Transport of phospholipids, free acids and triacylglycerols as the major lipid classes by a high- density lipoprotein fraction isolated from plasma of Polybetes pythagoricus. Comp. Biochem. Physiol. B 109, 333–338. Cunningham, M.L., Gonzalez, A., Pollero, R.J., 2000. Characterization of lipoproteins isolated from the hemolymph of the spider Latrodectus mirabilis. J. Arachnol. 28, 49–55. Cunningham, M., García, F., Pollero, R.J., 2007. Arachnid lipoproteins: comparative aspects. Comp. Biochem. Physiol. C 146, 79–87. El-Salhy, M., Gustafsson, I., Grimelius, L., Vessby, B., 1981. The lipid composition of the haemolymph and hepatopancreas of the scorpion (Buthus quinquestriatus). Comp. Biochem. Physiol. B 69, 873–876. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. García, F., Cunningham, M.L., González Baró, M.R., Garda, H.A., Pollero, R.J., 2002a. Ef- fect of fenitrothion on the physical properties of crustacean lipoproteins. Lipids 37, 673–679. García, F., González Baró, M.R., Pollero, R.J., 2002b. Transfer of lipids between hemolymph and hepatopancreas in the shrimp Macrobrachium borellii. Lipids 37, 581–585. García, F., González Baró, M.R., Garda, H., Cunningham, M., Pollero, R.J., 2004. Fenitrothion-induced structural and functional perturbations in the yolk lipoproteins of the shrimp Macrobrachium borellii. Lipids 39, 389–396. Gefen, E., 2008. Sexual dimorphism in desiccation responses of the sand scorpion Smeringurus mesaensis (Vaejovidae). J. Insect Physiol. 54, 798–805. Gilbert, L.I., Chino, H., 1974. Transport of lipids in insects. J. Lipid Res. 15, 439–456. Gilbert, L.I., O'Connor, J.M., 1970. Lipid metabolism and transport in arthropods. In: Florkin, M., Scheer, B.T. (Eds.), Chemical Zoology. Academic Press, New York, pp. 229–252. Gilbert, L.I., Goodman, W., Bollenbacher, W.E., 1977. Biochemistry of regulatory lipids and sterols in insects. In: Goodwin, T.W. (Ed.), Biochemistry of lipids. University Park Press, Baltimore.pp, pp. 1–50. González Baró, M.R., Pollero, R.J., 1988. Lipid characterization and distribution among tissues of the freshwater crustacean Macrobrachium borellii during an annual cycle. Comp. Biochem. Physiol. B 91, 711–715. Goyffon, M., Martoja, R., 1983. Cytophysiological aspects of digestion and storage in the liver of a scorpion, Androctonus australis (Arachnida). Cell Tissue Res. 228, 675. Harrison, P.L., Abdel-Rahman, M.A., Miller, K., Strong, P.N., 2014. Antimicrobial peptides from scorpion venoms. Toxicon 88, 115–137. Haunerland, N.H., Bowers, W.S., 1987. Lipoproteins in the hemolymph of the tarantula, Eurypelma californicum. Comp. Biochem. Physiol. B 86, 571–574. Kovařík, F., Ojanguren Affilastro, A.A., 2013. Illustrated Catalog of Scorpions. Part II. Bothriuridae; Chaerilidae; Buthidae I., Genera Compsobuthus, Hottentotta, Isometrus, Lychas, and Sassanidotus. Jakub Rolčík Publisher. Clairon Production, Czech Republic (400 pp). Laino, A., Cunningham, M., García, F., Heras, H., 2009. First insight into the lipid uptake, storage and mobilization in arachnids: role of midgut diverticula and lipoproteins. J. Insect Physiol. 55, 1118–1124. Laino, A., Cunningham, M., Heras, H., García, F., 2011a. In vitro lipid transfer between lipoproteins and midgut-diverticula in the spider Polybetes pythagoricus. Comp. Biochem. Physiol. B 160, 181–186. Laino, A., Cunningham, M., Heras, H., García, F., 2011b. Isolation and characterization of two vitellins from eggs of the spider Polybetes pythagoricus (Araneae: Sparassidae). Comp. Biochem. Physiol. B 158, 142–148. Laino, A., Cunningham, M., Costa, F.G., Garcia, C.F., 2013. Energy sources from the eggs of the wolf spider Schizocosa malitiosa: isolation and characterization of lipovitellins. Comp. Biochem. Physiol. B 165, 172–180. Laino, A., Cunningham, M., Suárez, G., Garcia, C.F., 2015. Identification and character- ization of the lipid transport system in the tarantula Grammostola rosea. OJAS 5, 9–20. Lee, R.F., Puppione, D.L., 1978. Serum lipoproteins in the spiny lobster, Panulirus interruptus. Comp. Biochem. Physiol. B 59, 239–243. Louati, H., Zouari, N., Miled, N., Gargouri, Y., 2011. A new chymotrypsin-like serine protease involved in dietary protein digestion in a primitive animal, Scorpio maurus: purification and biochemical characterization. Lipids Health Dis. 10, 121. Marie, Z.A., Ibrahim, S.A., 1976. Lipid content of scorpion (Leiurus quinquestriatus, H and E) venom. Toxicon 14, 93–96. Millot, J., Vachon, M., 1968. Ordre des scorpions. In: Grassé, P.P. (Ed.), Traité de zoologie anatomie, systématique, biologie, tome VI. Masson et compagnie, Paris, pp. 386–436. Morrison, W.R., Smith, L.M., 1992. Preparation of fatty acid methyl esters and dimethylacetals from lipid with boron fluoride-methanol. J. Lipid Res. 5, 600–608. Nime, M.F., Casanoves, F., Vrech, D., Mattoni, C.I., 2013. Relationship between environ- mental variables and the surface activity of the scorpions in a reserve of Arid Chaco. Argen. Invert. Biol. 132 (2), 145–155. Nime, M.F., Casanoves, F., Mattoni, C.I., 2014. Surface activity, sex ratio and diversity of scorpions in two different habitats in an Arid Chaco reserve. Argen. J. Insect Conserv. 18 (3), 373–384. O´Connor, J.M., Gilbert, L.I., 1968. Aspects of lipid metabolism in crustaceans. Am. Zool. 8, 529–539. Pattnaik, N.M., Mundall, E.C., Trambusti, B.G., Law, J.H., Kezdy, F.J., 1979. Isolation and characterization of a larval lipoprotein from the hemolymph of Manduca sexta. Comp. Biochem. Physiol. B 63, 469–476. Peled, Y., Tietx, A., 1975. Isolation and properties of a lipoprotein from the haemolymph of the locust, Locusta migratoria. Insect Biochem. 51, 61–72. Raghavaiah, K., Ramamurthi, R., 1977. Quantitative distribution of lipid and utilization of 1-14C-acetato in the scorpion Heterometrus fulvipes (Koch). Comp. Physiol. Ecol. 2, 62–66. Ramamurthi, R., Satyanarayana, K., Selvarajan, V.R., Swami, K.S., 1975. Distribution & synthesis of glycogen in the scorpion Heterometrus fulvipes (C. L. Koch). Indian J. Exp. Biol. 13, 446–447. Rodenburg, K.W., Van der Horst, D.J., 2005. Lipoprotein-mediated lipid transport in insects: analogy to the mammalian lipid carrier system and novel concepts for the functioning of LDL receptor family members. Biochim. Biophys. Acta 1736, 10–29. Ross, R.H., Monroe, R.E., 1970. Utilization of acetate 1-14C by the tarantula, Aphonepelma sp., and the scorpion, Centruroides sculpturatus, in lipid synthesis. Comp. Biochem. Physiol. 36, 765–773. Ryan, R.O., Senthilathipan, K.R., Wells, M.A., Law, J.H., 1988. Facilitated diacylglycerol exchange between insect hemolymph lipophorins. Properties of Manduca sexta lipid transfer particle. J. Biol. Chem. 263, 14140–14145. Schartau, W., Leidescher, T., 1983. Composition of the hemolymph of the tarantula Eurypelma californicum. J. Comp. Physiol. 152, 73–77. Schenk, S., Gras, H., Marksteiner, D., Patasic, L., Prommnitz, B., Hoeger, U., 2009. The Pandinus imperator haemolymph lipoprotein, an unusual phosphatidylserine carrying lipoprotein. Insect Biochem. Mol. Biol. 39, 735–744. Seifert, G., Rosenberg, J., 1977. Feinstruktur der Leberzellen von Oxidus gracilis. Zoomorphologie 88, 145–162. Shevchenko, A., Simons, K., 2010. Lipidomics: coming to grips with lipid diversity. Nat. Rev. Mol. Cell Biol. 11, 593–598. Sobotnik, J., Weyda, F., Hanus, R., Cvacka, J., Nebesarova, J., 2006. Fat body of Prorhinotermes simplex (Isoptera: Rhinotermitidae): ultrastructure, inter-caste differences and lipid composition. Micron 37, 648–656. Stanley-Samuelson, D.W., Jurenka, R.A., Cripps, C., Blomquist, G.J., Renobales, M., 1988. Fatty acids in insects: composition, metabolism and biological significance. Arch. Insect Biochem. 9, 1–33. Thomas, K.K., Gilbert, L.I., 1968. Isolation and characterization of the hemolymph lipopro- teins of the American silkmoth, Hyalophora cecropia. Arch. Biochem. Biophys. 127, 512–521. Tzompa-Sosa, D.A., Yi, L., van Valenberg, H.J.F., van Boekel, M.A.J.S., Lakemond, C.M.M., 2014. Insect lipid profile: aqueous versus organic solvent-based extraction methods. Food Res. Int. 62, 1087–1094. Uscian, J.M., Stanley-Samuelson, D.W., 1994. Fatty acid compositions of phospholipids and triacylglycerols from selected terrestrial arthropods. Comp. Biochem. Physiol. B 107, 371–379. http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0005 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0005 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0010 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0010 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0015 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0015 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0020 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0020 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0025 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0025 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0030 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0030 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0030 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0305 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0305 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0035 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0045 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0045 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0045 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0050 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0050 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0050 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0050 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0055 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0055 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0055 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0040 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0040 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0070 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0070 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0070 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0075 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0075 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0080 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0080 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0080 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0090 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0090 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0085 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0085 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0095 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0095 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0100 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0105 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0105 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0105 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0110 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0110 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0110 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0115 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0115 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0115 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0120 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0120 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0120 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0125 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0125 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0130 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0130 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0310 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0310 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0310 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0310 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0140 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0140 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0140 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0145 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0145 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0145 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0150 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0150 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0150 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0135 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0135 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0135 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0155 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0155 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0155 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0160 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0160 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0170 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0170 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0170 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0175 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0175 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0180 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0180 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0180 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0185 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0185 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0190 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0190 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0190 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0195 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0195 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0195 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0200 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0200 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0205 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0205 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0205 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0210 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0210 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0215 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0215 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0215 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0220 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0220 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0220 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0225 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0225 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0225 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0225 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0230 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0230 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0230 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0235 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0235 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0235 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0240 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0240 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0245 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0245 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0245 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0250 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0250 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0255 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0255 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0260 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0260 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0260 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0265 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0265 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0315 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0315 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0315 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0270 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0270 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0275 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0275 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0275 36 A. Laino et al. / Comparative Biochemistry and Physiology, Part B 190 (2015) 27–36 Warburg, M.R., 2012. Reviewing the structure and function of the scorpion's hepatopan- creas. Arthropods 1, 79–93. Warburg, M.R., Rivka, E., Rosenberg, M., 2002. The hepatopancreas of Scorpio maurus fuscus; seasonal changes in mass and water content as related to gender and oogen- esis. J. Zool. 256, 479–488. Weers, P.M., Ryan, R.O., 2006. Apolipophorin III: role model apolipoprotein. Insect Biochem. Mol. Biol. 36, 231–240. Zouari, N., Miled, N., Cherif, S., Mejdoub, H., Gargouri, Y., 2005. Purification and character- ization of a novel lipase from the digestive glands of a primitive animal: the scorpion. Biochim. Biophys. Acta 1726, 67–74. Zouari, N., Bernadac, A., Miled, N., Rebai, T., De Caro, A., Rouis, S., Carriere, F., Gargouri, Y., 2006. Immunocytochemical localization of scorpion digestive lipase. Biochim. Biophys. Acta 1760, 13865-1392. http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0280 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0280 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0285 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0285 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0285 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0290 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0290 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0300 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0300 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0300 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0295 http://refhub.elsevier.com/S1096-4959(15)00142-6/rf0295 Analysis of lipid and fatty acid composition of three species of scorpions with relation to different organs 1. Introduction 2. Materials and methods 2.1. Animal and organ/tissue isolation 2.2. Lipid characterization 2.3. Fatty acid characterization 2.4. Statistical analyses 3. Results 4. Discussion Acknowledgments References