Vol.:(0123456789)1 3 J Comp Physiol B DOI 10.1007/s00360-017-1120-7 ORIGINAL PAPER Embryonic and post-embryonic development inside wolf spiders’ egg sac with special emphasis on the vitellus M. Trabalon1 · F. Ruhland1 · A. Laino2 · M. Cunningham2 · F. Garcia2 Received: 30 May 2017 / Revised: 18 July 2017 / Accepted: 25 July 2017 © Springer-Verlag GmbH Germany 2017 “juveniles 2” was 38% of the initial calorie stocks in the eggs. Keywords Lipovitellin · Biochemical status · Energetic state · Developmental period · Arachnids Introduction Embryogenesis in oviparous animals proceeds indepen- dently through the direct influence of maternal activity. In many animal species, a large quantity of nutritive mate- rial or yolk (vitellus) is deposited in the egg, the amount depending on the length of time before the young animal can feed itself. The yolk provides the developing embryo, be it a worm, a tadpole or a chicken, with the nutrients essential for growth within its birthplace, the egg (Byrne et al. 1989). The term yolk does not refer to any particular substance but in fact includes proteins and lipids with a rel- atively small amount of carbohydrates, vitamins and min- erals, all of which substances occur in various proportions in the eggs of different vertebrate and invertebrate animals (Byrne et al. 1989). These compounds are usually associ- ated leading to the formation of lipovitellins (LV) that are a major nutrient source for oviparous embryos (Kunkel and Nordin 1985; Wallace 1985). Most arthropods lay centrol- ecithal eggs and LV have been purified and characterized from several species: crustaceans (Chen et al. 2004; García et al. 2006; Kawazoe et al. 2000; Lubzens et al. 1997) and insects (Chino 1997; Dhadialla and Raikhel 1990; Tufail and Takeda 2008) but data concerning arachnids, except three Acari (Boctor and Kamel 1976; Chinzei et al. 1983; Tatchell 1971), and two spider species (Laino et al. 2011, 2013) are poor. Abstract The development of Pardosa saltans wolf spi- ders inside an egg sac includes two periods: an embryonic period and a post-embryonic period after hatching. We investigated spiderlings’ energy expenditure to assess ener- getic costs during the different embryonic and post-embry- onic developmental stages during which they are confined within their egg sac. We focused on the following devel- opmental stages: egg, embryonic stages 1 and 2, and two stages, separated by a moult, during post-embryogenesis inside the egg sac: “juvenile instars 1 and 2” until emer- gence of 2 instar juveniles from their egg sac. We present the first biochemical characterization of the vitellus of wolf spiders’ eggs, embryos and juveniles. Lipovitellins (LV) are composed of four apolipoproteins of 116, 87, 70 and 42 kDa, respectively, and LV represent 35–45% of total protein during development. The principal LV lipids are triglycerides, phospholipids, free fatty acids and sterols. Egg caloric content averaged 127 cal/g (proteins: 91 cal/g, lipids: 33 cal/g, carbohydrates: 3 cal/g). During develop- ment from undivided egg to emerged “juvenile 2”, 67% of proteins, 51% of carbohydrates and 49% of triglycerides stocks were depleted. At the end of the post-embryonic period, at emergence from egg sac, body energy stock of Communicated by G. Heldmaier. * M. Trabalon marie.trabalon@univ-rennes1.fr 1 Université de Rennes 1, UMR-6552 CNRS EthoS, Campus de Beaulieu, 263 avenue du Général Leclerc, CS 74205, 35042 Rennes Cédex, France 2 Instituto de Investigaciones Bioquímicas de la Plata “Prof. Dr. Rodolfo R. Brenner” (INIBIOLP), CCT-La Plata CONICET-UNLP, La Plata, Argentina http://crossmark.crossref.org/dialog/?doi=10.1007/s00360-017-1120-7&domain=pdf J Comp Physiol B 1 3 In the process of embryogenesis, utilization of reserved nutrients is auto-regulated related to the program of embry- ogenesis and differentiation (Masetti and Giorgi 1989; Raikhel and Dhadialla 1992). Hatching of the egg usu- ally occurs when histogenesis is complete and newly born arthropods have diverse forms (Minelli et al. 2013). The time required to complete embryonic development var- ies widely among different arthropod taxa, ranging from 30 h to more than 3 months (Hinton 1981). Studies on the role of yolk during embryogenesis have been restricted mainly to changes in the yolk protein content of insect eggs (Yamashita and Indrasith 1988; Izumi et al. 1994). During insect embryonic development the carbohydrate/ protein and protein/fat ratios decline with age and there is a succession of sources of energy, carbohydrate preceding protein and protein preceding fat (Dovner 2012). Little is known about embryonic development and yolk utilization in spider. Spider lays centrolecithal eggs and most spiders enclose their eggs in some form of silken egg sac (Foelix 2011). Embryonic development of eggs occurs within this struc- ture and is often divided into two parts by the incident of birth or hatching: the prenatal part and the postnatal part. The prenatal part (or embryonic period) encompasses development from the time eggs are fertilized until the typi- cal shape of the spider’s body is established. Descriptions of spiders’ embryonic development are confusing because authors use different terms (e.g. pre-larva, larva, pullus, pre-nymph or embryo) for the different embryonic stages (Canard 1987; Canard and Stockmann 1993; Downes 1987; Mittmann and Wolff 2012; Wolff and Hilbrant 2011). After hatching, development is qualified as postnatal (or post- embryonic). Spiders hatch in the cocoon and emergence can sometimes last a few hours or days before the first instar juveniles are released (Canard 1987; Vachon 1957). Emer- gence from the egg sac takes place after the moult which releases a juvenile equipped with all its organs (Vachon 1958; Wurdak and Ramousse 1984). As the development of spiders inside egg sacs includes several stages, embryos have to rely on energy supplied by nutriments in the vitel- lus provided by mothers. Notably, the yolk of an egg cell must contain all the energy needed for embryonic develop- ment, hatching and moulting, as well as for all the activi- ties of a young spiderling before it actually catches its first prey (Anderson 1978; Schaefer 1976). This raises questions concerning use of vitellus. Therefore, we investigated spi- derlings’ energy expenditure to assess their energetic costs during the embryonic and post-embryonic development periods while they were confined within their egg sac. We selected the free-moving wolf spider, Pardosa sal‑ tans, as our model lycosid species. These spiders live in forests, woodlands and copses and sometimes nearby grass- lands and hedgerows. The breeding season of P. saltans extends from April to September when adult spiders cop- ulate and females’ maternal behaviour develops. Their reproduction usually presents two peaks in France: one in late spring–early summer, and a second in autumn (Ruh- land et al. 2016a, b). P. saltans’ maternal behaviour can be divided into the following stages: construction, trans- port, care, perforation of the egg sac, and care of spider- lings after they emerge from the egg sac. P. saltans females carry their egg sacs for about 25–30 days and mothers open their egg sac to facilitate the emergence of their young, as do other spiders (Ruhland et al. 2016a, b; Whitcomb et al. 1966). Our aim was threefold: first, to identify two embryos’ late developmental stages and two juveniles’ developmen- tal stages inside the egg sac laid by female P. saltans and to evaluate the contribution of protein, carbohydrates and lipids to the development of spiderlings; second, to identify the LV and the lipids present during spiderlings’ develop- ment; and third, to evaluate the contributions of protein, carbohydrates and triglycerides to the maintenance of juveniles’ energy. We hypothesized that: (1) trophic eggs inside the egg sacs allow juveniles to reconstitute their energy reserves after hatching and before emergence; (2) triglycerides are used as a priority source of energy. The recently proposed embryonic and post-embryonic develop- ment stages for Cupiennius salei (Wolff and Hilbrant 2011) and Parasteatoda tepidariorum (Mittmann and Wolff 2012) provided us a basis for describing the developmental stages of P. saltans. We applied this system with the aim to estab- lish a standardized description of spiders’ developmental stages. Materials and methods Ethics statement Our research conformed to legal requirements and guide- lines established for the treatment of animals in research using invertebrate species and for their care using accepted ethical laboratory standards. The species used for the experiments is not endangered or protected in Europa. Spider collection and rearing Our subjects were young adult females and were cap- tured during the copulation period in a forest near the Paimpont biological station (property of the University of Rennes 1; France; 48°00′05.67″North, 2°13′46.65″West) in April–June 2015. Females were housed individually in circular terrariums (10 cm diameter × 5 cm high) with- out any substrate on bottom and were kept at 20 ± 1 °C, with 57 ± 1% relative humidity under a L:D, 14:10 h J Comp Physiol B 1 3 photoperiodic cycle. Spiders were fed every other day ad libitum, with either juvenile cricket (Acheta domestica and Nemobius sylvestris; 5 days after egg emergence) or adult flies (Drosophila melanogaster). All females were checked four times a day (8–9h, 11–12h, 14–15h, 17–18h) to record oviposition. Experimental groups The spiders studied came from 100 egg sacs built by spiders in the laboratory. They were divided into six experimental groups related to embryonic and post-embryonic stages inside the egg sac, before emergence of young (Fig. 1). The embryonic period was divided into three groups: 1–2 h (egg stage, n = 20), 5 days (“embryo 1” instar, n = 20), and 10 days (“embryo 2” instar; n = 20) after oviposition and before hatching. The post-embryonic period included two groups after hatching: juveniles after the 1st moult, i.e. 15 days after oviposition (“juvenile 1” instars; n = 20) and after the 2nd moult, i.e. 20 days after oviposition (“juvenile 2” instars; n = 20); and one group at the moment of emer- gence from egg sac, i.e. 30 days after oviposition (“juve- nile 2E” instars; n = 20). All egg sacs were dissected on micrographs and egg, embryo, and juvenile developmental instars were investigated. Individuals from each develop- mental instar were placed in an Eppendorf tube (1.5 mL) then weighed using a Sartorius electronic balance (Palai- seau, France) (±0.01 g). The entire mass was divided by the number of individual inside tube. Development period and energetic state After weighing, individuals in the Eppendorf tubes were freeze-killed at −18 °C. Each individual was mixed in 50 µL of water, homogenized (5 min) and vortexed for 30 min and centrifuged at 10,000 rpm, for 10 min at 5 °C (Ruhland et al. 2016b). Three aliquots (10 µL/aliquot) of supernatant were taken to assay carbohydrates and pro- teins. Lipids from the homogenate sample were extracted with 600 µL of methanol–dichloromethane (1v/2v) (Sigma, Saint-Quentin Fallavier, France), vortexed for 30 min and centrifuged at 10,000 rpm, for 10 min at 4 °C. The pellets were discarded and the supernatant was aliquoted for fur- ther analyses. We triplicated the analysis of each sample and then calculated the ratio of lipids, proteins, and carbo- hydrates related to wet mass. Energetic state was assessed from 20 extracts for each developmental stage. Fig. 1 Biological cycle of the embryonic and post-embryonic development of P. saltans (Lycosidae) in the egg sac until emergence, under labo- ratory conditions (20 ± 1 °C, 57 ± 1% relative humidity) J Comp Physiol B 1 3 Quantification of carbohydrates and free glucose Total carbohydrate concentration in a 10-µL homogenate sample was assessed using a colorimetric method (Total Carbohydrate Assay kit, Sigma-Aldrich, St Louis, USA) based on the phenol–sulfuric acid method in which poly- saccharides are hydrolyzed and then converted to fur- fural or hydroxylfurfural with a yellow-orange colour as a result of the interaction between the carbohydrates and the phenol. We divided the carbohydrate level data by fresh weight to obtain concentrations in µg/mg. Free glucose concentrations in a 10-µL homogenate sample were determined using a colorimetric method after enzymatic oxidation in the presence of glucose oxi- dase (Glucose-test, Randox, Crumlin, Co, Antrim, UK). The hydrogen peroxide formed reacted in the presence of peroxidase with phenol and 4-aminophenazone to form a red-violet quinoneimine dye as the indicator. We divided the free glucose level data by fresh weight to obtain con- centrations in µg/mg. Identification and quantification of proteins and lipovitellin Quantification of proteins Total protein content in a 10-µL biological sample was determined according to Bradford’s method (1976) with a Coomassie protein assay kit (Thermo Scientific, Cergy Pontoise, France) using bovine serum albumin as the standard. We divided the protein level data by fresh weight to obtain concentrations in µg/mg. Anti‑lipovitellin rabbit serum preparation Antibodies directed against purified lipovitellin of Schizo‑ cosa malitiosa (SmLV) were prepared in rats (Laino et al. 2013). Rats were given subcutaneous injections of about 1 mg of SmLV emulsified in Freund’s complete adjuvant (Sigma Chemical Co., St. Louis, MO). A booster injec- tion containing about 1 mg of antigen with Freund’s incomplete adjuvant was administered after 4 weeks, and then 1 mg of antigen without adjuvant was administered 2 weeks later. One week later, rats were bled through car- diac puncture. The blood collected was allowed to clot for 30 min at room temperature and then overnight at 4 °C. After centrifugation, the serum obtained was stored at −70 °C until use. The specificity of the antiserum was verified by immunoblotting against different proteins. The antiserum reacted only with the proteins of the LV fraction. Gel electrophoresis and western blot analysis Eggs (freshly laid) were analysed by dissociating electro- phoresis (SDS-PAGE) using a 12% acrylamide (Laemmli 1970) and stained with Coomasie Brilliant Blue R-250 (Sigma Chemical Co., St. Louis, MO). Molecular weights (MW) were calculated as previously described (Laino et al. 2011). Proteins were separated by SDS-PAGE elec- trophoresis and electroblotted for 1 h at 100 V (Trans- Blot SD Semi Dry Transfer Cell, Bio-Rad Laboratories, Hercules, CA, USA) from the unstained gel to nitrocellu- lose membranes using 48 mM Gly, 39 mM Tris, pH 9.2, and 20% MeOH buffer. After being blocked overnight at 4 °C with 3% (w/v) non-fat dry milk in 0.15 M NaCl, 10 mM Tris–HCl, pH 7.4, the membranes were incubated with the anti-LV serum (1:5000) for 2 h. Specific antigens were detected by goat anti-rat. IgG horseradish peroxidase conjugate (1:1000) immunoreactivity was visualized by enhanced electrochemiluminescence (ECL). Enzyme‑linked immunosorbent assay (ELISA) development A standardized enzyme-linked immunosorbent assay (ELISA) was developed to quantify LV at different embry- onic and post-embryonic instars. The procedure was based on Engwall and Perlmann’s (1972) assay. The standard curve was obtained using purified SmLV. Nunc-Immuno- plate Polisorp microtiter plates were loaded with 50 μL/ well of the LV standard (0–400 ng) dissolved in a buffer containing 35 mM sodium bicarbonate and 15 mM sodium carbonate at pH 9.6 (coating buffer). Samples of eggs, embryos or juveniles were diluted with the coating buffer. Aliquots of 50 μL were incubated at 37 °C for 90 min. The antigen was then taken out, and each well was filled with 300 μL of PBS, pH 7.4, containing 3% (w/v) non-fat dry milk. The plates were incubated at room temperature for 2 h and subsequently washed three times with 0.05% (v/v) Tween in PBS. The anti-LV rat serum diluted in PBS- Tween (1:1000) containing 3% non-fat dry milk was poured into each well. Plates were incubated overnight at 4 °C and washed three times. Goat anti-rabbit IgG horseradish per- oxidase conjugate (Thermo Scientific) diluted 1:1000 in PBS–0.05% Tween-3% non-fat dry milk was added (50 μL) and incubated 2 h at 37 °C. After four washes as before, 50-μL aliquots of substrate solution, ABTS (2, 2′-Azino- bis [3-ethylbenzothiazoline-6-sulfonic acid]–diammonium salt), and H2O2 (Bio-Rad) were added to each well and were incubated at room temperature for 15 min. The reac- tion was stopped with 2% oxalic acid (50 μL). The absorb- ance was read at 405 nm in a Beckman Coulter, Inc. Instru- ments (DTX 880). Non-fat dry milk in PBS was used in all the assays as blank and negative control. All analyses of samples were triplicate and the data were averaged. J Comp Physiol B 1 3 Identification and quantification of lipids Characterization of lipids and fatty acids Lipids were extracted from freshly laid eggs following Folch et al.’s (1957) procedure. Lipids were evaluated quan- titatively by thin-layer chromatography (TLC) coupled to a flame ionization detector in an Iatroscan apparatus model TH-10 (Iatron Laboratories, Tokyo, Japan), after separation on Chromarods type S-III (Ackman et al. 1990; Laino et al. 2011). The general procedure for separation and identifica- tion of lipids has been described previously by Cunningham and Pollero (1996). Fatty acid methyl esters (FAME) from total lipids of eggs were prepared with BF3–MeOH accord- ing to Morrison and Smith’s (1992) method. Analyses were performed by gas–liquid chromatography (GLC-FID) using an HP-6890 capillary GLC (Hewlett-Packard, Palo Alto, CA), fitted with an Omegawax 250 fused silica column, 30 m × 0.25 mm with 0.25 μm phase (Supelco, Bellefonte, CA). The column temperature was programmed for a lin- ear increase of 3 °C/min from 175 to 230 °C. Peaks were identified by comparison with retention times of Supelco 37 component fatty acid methyl ester mix (Supelco). Quantification of triglycerides and cholesterol Total triglycerides in a 10-µL biological sample were assessed using a colorimetric method (Triglycerides test, Randox, Crumlin, Co. Antrim, UK). The triglycerides were determined after enzymatic hydrolysis with lipases. The indicator was a quinoneimine formed from hydrogen perox- ide, 4-aminophenazone and 4-chlorophenol under the cata- lytic influence of peroxidase. We divided triglyceride level data by fresh weight to obtain concentrations in µg/mg. Cholesterol in a 10-µL biological sample was deter- mined using cholesterol/cholesteryl ester detection kit (Abcam, Cambridge, UK) using a colorimetric method. In this assay, free cholesterol was oxidized by cholesterol dehydrogenase to generate NADH that reacts with a sen- sitive probe resulting in strong absorbance at 450 nm. We divided the cholesterol level data by fresh weight to obtain concentrations in µg/mg. Energetic equivalent of proteins, carbohydrates and lipids We estimated the total calories provided by different energy substrates (carbohydrates, proteins and lipids) using Beni- ngher and Lucas’s (1984) conversion factors applied to arthropods (Garcia-Guerrero et al. 2003; Heras et al. 2000; Laino et al. 2013). The conversion factors were: 4.3 kcal/g for protein, 4.1 kcal/g for carbohydrate and 7.9 kcal/g for lipid. Statistical analyses Statistical analyses were performed using STATISTICA 6.0 for WINDOWS (Statsoft Inc.). As all our data satisfied the requirements for parametric statistics, we analysed ours using one-way ANOVAs. When differences between means were significant at the p < 0.05 level, post hoc Tukey’s (HSD) tests were applied. Linear regressions assessed vari- ations of biochemical variables over time during embryonic and post-embryonic development. Results are expressed as means ± SE. Results Development and instars Females lay 35 ± 10 eggs and these eggs are initially coated with a sticky substance that holds them together (Table 1). Eggs are spherical and contain a large quantity of homog- enous yolk (Fig. 1a). P. saltans’ eggs are always envel- oped in two layers, an inner vitelline membrane and an outer chorion. Egg incubation, or embryonic development, lasts 14–15 days and post-embryonic development lasts 14–15 days. Embryonic development After 5–10 days of incubation, “embryo 1” stage is char- acterized by the closure of the opisthosomal region (Fig. 1b). A frontal view shows that the chelicerae partly covered the labrum. The walking legs are segmented into seven podomeres. Yolk is shifted from the prosoma to the opisthosomal parts of the embryo. 10 days after oviposi- tion, “embryo 2” stage is characterized by the fact that the labium starts to protrude and together with the labrum forms a beak-like structure (Fig. 1c). The eyes can be detected in their position underneath the prosomal cuticle. The walking legs show prominent endites and legs still bend ventrally. Stage 2 embryos hatch from the eggs after 14–15 days of embryonic development. Post‑embryonic development The stage after hatching is called the juvenile instar. This instar is divided into two. Juveniles remain in the cocoon’s outer chamber where they undergo one moult after hatch- ing (“juvenile 1” instar, Fig. 1d), then a second moult 5–6 days later (“juvenile 2” instar, Fig. 1e). First instar juveniles’ walking legs are arranged laterally around the prosoma. The opisthosoma is oval and can be up to twice the size of the prosoma and it still contains yolk reserves. The first instar juveniles do not move and their cuticle is J Comp Physiol B 1 3 transparent and unpigmented. No sensory hairs are visible on the cuticle. After the second moult, the opisthosoma of instar “juvenile 2” increases in volume; their walking legs become slightly longer (Fig. 1e). The cuticle is pigmented showing brown dots and patches. Second instar juveniles are relatively active (frequent leg movements). The mother opens the cocoon 25–30 days after oviposition, and “Juve- niles 2E” emerge from the egg sac and climb onto their mother’s back (Fig. 1f). Developing embryos were visible in the majority of the eggs (91%) after 5 days of incubation and only 9% of the eggs were sterile (Table 1). During the post-embry- onic period, 18% of the juveniles died inside the egg sac. Weights of embryos and juveniles varied significantly dur- ing development (F(5,108) = 11.83, p < 0.0001; Table 1): after hatching and their first moult, instar “juveniles 1” were significantly less heavy than instar “embryos 2” (Tukey test: p < 0.01); after the second moult and at emer- gence instar “juveniles 2” were significantly heavier than instar “juveniles 1” (Tukey test: p < 0.02). Carbohydrates and free glucose levels Levels of carbohydrates in the eggs averaged 0.83 ± 0.19 µg/ mg wet mass after oviposition. Their levels varied signifi- cantly during embryonic and post-embryonic development (F(5,108) = 4.06, p = 0.002; Fig. 2a). They decreased signifi- cantly from egg to “embryo 2”: 0.63 ± 0.08 µg/mg (Tukey test: “embryo 2” vs “egg”: p = 0.05) then again during post- embryonic development until emergence: 0.41 ± 0.08 µg/ mg (Tukey test: “embryo 2” vs “juvenile 2E”: p = 0.01). A linear regression indicated a significant correlation between the values of calories provided by carbohydrates and the variations of total calories during the embryonic and the post-embryonic periods (F(5,94) = 6.19, p = 0.0003; Y = −0.0813x + 0.8863, R2 = 0.99). Free glucose levels in the eggs averaged 0.18 ± 0.01 µg/ mg wet mass after oviposition. Free glucose levels varied significantly during embryonic and post-embryonic devel- opment (F(5,108) = 4.63, p = 0.01; Fig. 2b). They increased Table 1 Development of P. saltans’ eggs and spiderlings in the egg sacs Number and weights in relation to embryonic and post-embryonic developmental periods under laboratory conditions (20 ± 1 °C, 57 ± 1% relative humidity; n = 100 egg sacs). Mean levels were compared between stages using ANOVA and post hoc Tukey (HSD) tests *Significant difference among other instars at p < 0.05 Developmental stage Days after oviposi- tion Number/egg sac Weight (mg/egg or mg/instar) Freshly laid eggs 0 (1–2 H) 35.1 ± 1.7 0.481 ± 0.028* Embryonic period Embryo 1/undeveloped egg 5–6 31.1 ± 2.5/4.2 ± 0.5 0.589 ± 0.063 Embryo 2/undeveloped egg 10–11 28.2 ± 3.3/5.0 ± 0.8 0.602 ± 0.026 Post-embryonic period Juvenile 1/undeveloped egg 15–16 26.6 ± 2.3/4.8 ± 0.4 0.465 ± 0.025* Juvenile 2/undeveloped egg 20–21 26.1 ± 2.7/4.2 ± 0.5 0.597 ± 0.032 Juvenile 2 at emergence Living/undeveloped egg 30 24.7 ± 1.7/5.2 ± 0.6 0.623 ± 0.036 Fig. 2 Carbohydrates (a) and glucose (b) concentrations in P. sal‑ tans’ freshly laid eggs, embryos and juveniles in relation to embry- onic and post-embryonic developmental stages (n = 20/instar). The average levels for each instar were compared using ANOVA and post hoc Tukey (HSD) tests. **Significant difference among other instars at p < 0.01 J Comp Physiol B 1 3 significantly from egg to “embryo 2”: 0.29 ± 0.04 µg/ mg (Tukey test: “embryo 2” vs “egg”: p = 0.01; “embryo 2” vs “embryo 1”: p = 0.01) then decreased significantly after the first post-embryonic moult (Tukey test: “embryo 2” vs “juvenile 1”: p = 0.01). At emergence (stage “juve- nile 2E”), free glucose levels had decreased significantly: 0.11 ± 0.01 µg/mg (Tukey test: “juvenile 2” vs “juvenile 2E”: p = 0.01). A linear regression did not indicate any significant correlation between values of calories provided by free glucose and variations of total calories during the embryonic and the post-embryonic periods (F(5,94) = 1.81, p = 0.147; Y = −0.014x + 0.2429, R2 = 0.11). Protein levels and lipovitellin Protein levels in the eggs averaged 22.21 ± 0.78 µg/mg wet mass (Fig. 3) and decreased significantly during the two periods of development in the egg sac (embryo and juvenile instars) until emergence from egg sac, i.e. 6.97 ± 0.70 µg/ mg in instar “juvenile 2E” (F(5,108) = 43.76, p < 0.0001). A linear regression revealed a significant correlation between the values of calories provided by proteins and variations of total calories during the embryonic and the post-embryonic periods (F(5,95) = 12.61, p < 0.001; Y = −2.699x + 24.259, R2 = 0.965). Analyses of image of dissociating electrophoresis and immunoblot analyses of proteins from eggs, embryos, juve- niles and SmLV revealed the presence of different bands in 1-D gels visualized by blue staining (Fig. 4a). No signifi- cant differences could be evidenced concerning the values of bands between duplicate gels showing the reproducibil- ity of the experiments. Four protein bands were identified during the different developmental instars: protein bands 116, 87, 70, and 42 kDa (Fig. 4a) and corresponded to LV (Fig. 3b). A standardized enzyme-linked immunosorbent assay (ELISA) was developed to quantify LV at different embryonic and post-embryonic stages (Fig. 4c, d). LV rep- resented 35% of total proteins in the eggs. Lipovitellin lev- els did not vary significantly during development (35–45%) in relation to total protein. Lipids and fatty acids levels TLC and GLC-FID from the egg extracts were composed of a mixture of lipids (Table 2). Triglycerides, phosphati- dylcholine, lysophosphatidylcholine and free fatty acids accounted for the main lipid content; the levels of other lipids were lower. Oleic (C 18:1) and linoleic (C 18:2) acids were the dominant unsaturated fatty acids; palmitic (C 16:0) and stearic (C 18:0) acids were the dominant satu- rated fatty acids (Table 3). Triglycerides and cholesterol levels Triglyceride levels in eggs averaged 4.15 ± 0.38 µg/mg wet mass (Fig. 5a) and did not vary significantly dur- ing embryonic development and post-embryonic periods but at emergence, triglyceride levels stage “juvenile 2E” (2.06 ± 0.19 µg/mg) were significantly lower than those of the other instars (F(5,108) = 6.02, p = 0.0005). A linear regression showed a significant correlation between the values of calories provided by triglycerides and variations of total calories during the embryonic and post-embryonic periods (F(5,96) = 3.961, p = 0.008; Y = −0.133x + 4.225*X, R2 = 0.904). Cholesterol levels in eggs averaged 0.63 ± 0.06 µg/ mg wet mass (Fig. 5b). During the “embryo 1” stage cho- lesterol levels were significantly lower than in the eggs 0.26 µg/mg (Tukey test: “egg” vs “embryo 1”: p = 0.0007), then increased significantly during the juvenile post-embry- onic period and at the emergence (stage “juvenile 2E”) cho- lesterol levels were significantly higher: 0.74 ± 0.05 µg/mg. (F(5,108) = 43.76, p < 0.0001). Energetic equivalence Just after oviposition (Fig. 6), the total caloric content of eggs was 127 ± 5 cal/g wet mass, the energy equivalent of which was represented mainly by proteins (71%) supple- mented by lipids (26%), and carbohydrates (3%). During the embryonic and post-embryonic periods, total caloric contents decreased significantly between each devel- opmental instar (ANOVA: F(5,108) = 22.62, p < 0.0001; Y = −14.715*X + 146.05; R2 = 0.939; Fig. 6a). At emer- gence from the egg sac, the energetic state of “juvenile 2E” (47.93 ± 5.34 cal/g wet mass) was significantly lower than those of the other developmental instars (Tukey test: “egg” Fig. 3 Protein concentrations in P. saltans’ freshly laid eggs, embryos and juveniles in relation to embryonic and post-embryonic developmental stages (n = 20/instar) J Comp Physiol B 1 3 vs “juvenile 2E”, p < 0.0001), and its energy equivalent was mainly due to proteins (62%) and lipids (34%) (Fig. 6b). Discussion Our results show that the development of P. saltans in the egg sac can be divided into two periods: an embryonic period lasting 14–15 days and a post-embryonic period last- ing for 15 more days before spiderlings emerge. The mor- tality rate was low, only 11% of the embryos/juveniles died during this developmental period. Some authors divided spiders’ embryonic period into an early and a late devel- opment period (Canard 1987; Foelix 2011; Vachon 1957). Our study considered only the late development period that we divided into two stages: “embryo 1” and “embryo 2”. These two stages are morphologically comparable to stages 13 and 14 of Parasteatoda tepidariorum (Mittmann and Wolff 2012) and stages 19 and 21 of Cupiennius salei (Wolff and Hilbrant 2011). By their morphological descrip- tions, our “juvenile 1” and “juvenile 2” instars are also comparable to the two “post-embryo” stages of P. tepi‑ dariorum (Mittmann and Wolff 2012) and C. salei (Wolff and Hilbrant 2011). This pattern of post-embryonic devel- opment is similar to that found in some solitary (Downes 1987) and social spiders (Viera and Ghione 2007). Moreo- ver, our observations show that only 9% of the P. saltans eggs were sterile and not eaten by the juveniles inside their egg sac, in contradiction to our hypothesis and Canard’s report (1987), as we found them in the egg sacs after the emergence of the juveniles. These eggs are, therefore, not trophic eggs and the only energy resource of juveniles dur- ing their development inside egg sacs is their yolk reserve. Chemical identification of vitellus in eggs Eggs of P. saltans consist largely of vitellus which is dis- tributed in fine homogeneous granules and composed of proteins, lipids and carbohydrates. Carbohydrates are a minor constituent (3%) of P. saltans eggs, as for eggs of other arthropods (i.e. crustaceans: Heras et al. 2000; Hol- land 1978). Our results show that LV is the predominant lipoprotein in newly laid eggs as for crustaceans (Chaffoy Fig. 4 Soluble proteins of P. saltans’ freshly laid egg in relation to developmental stage, in percentage of LV in relation to the total pro- teins (mean ± SD) (n = 3). Electrophoresis (SDS-PAGE) of 20 µg of soluble protein for each well (a) and immunoblot of 5 µg of soluble protein for each well (b). SmLV lipovitellin of S. malitiosa, Emb 1 embryo 1, Emb 2 embryo 2, MW standard, Juv 1 juvenile 1, Juv 2 juvenile 2. c Changes of LV levels during P. saltans’ development. LV was quantified by ELISA. Polyclonal antibody against egg LV was used to immunoblot and ELISA. d Inset dose–response titration of LV ▸ J Comp Physiol B 1 3 de Courcelles and Kondo 1980; Telfer and Kulakosky 1984) and is involved in lipid storage. Protein extracts The levels of total egg proteins were similar to those in the eggs of other arthropods (Kunkel and Nordin 1985; Wal- lace 1985; Yamashita and Indrasith 1988). The results of ELISA and western blot analyses show that 35% of the total proteins present in P. saltans eggs were LV as in eggs of Schizocosa malitiosa (Laino et al. 2013) and Polybe‑ tes pythagoricus (Laino et al. 2011). LV is composed of four apoproteins of 116, 87, 70 and 42 kDa, respectively, and was present during embryonic and post-embryonic development. During arthropods embryogenesis, proteins present in the vitellus and those forming LVs are degraded by hydrolytic enzymes for embryo nutrition and energy supply (González Baró et al. 2000; Subramoniam 1991). Lipid extracts Our results show that the composition (triglycerides, phos- pholipids, sterols and hydrocarbons) of the lipid extracts of P. saltans eggs was similar to that reported for other arthro- pods (i.e. Chen et al. 2004; Garcia et al. 2006; Salerno et al. 2002; Tufail and Takeda 2008; Walker et al. 2006). Triglycerides and phospholipids are the most abundant constituents of egg extracts as for S. malitiosa (Laino et al. 2013), but proportions differed between these two species. This difference could be caused by different food habits (Pardosa ate flies in the laboratory during tests whereas S. malitiosa were fed mealworm larvae), similar to that observed for the hemolymph of Eurypelma californicum (Schartau and Leidescher 1983) and Brachypelma albopi‑ losum (Trabalon 2011) spiders. Triglycerides and phospho- lipids are lipids necessary for organogenesis, as for instance Table 2 Lipid composition of P. saltans’ freshly laid eggs, in per- centage (mean ± SD) of total weight and quantified by TLC-FID N = three independent analyses of three pools from ten egg sacs each Lipids Percentage/egg Diacylglycerols 2.1 ± 0.9 Free fatty acids 5.6 ± 1.7 Hydrocarbons 3.8 ± 0.6 Phospholipids 38.6 Phosphatidylcholine 28.4 ± 0.3 Lysophosphatidylcholine 5.5 ± 2.3 Phosphatidylethanolamine 3.0 ± 0.9 Sphingomyelin 1.7 ± 0.5 Sterols 3.5 Cholesterol 2.8 ± 0.7 Esterified sterols 0.7 ± 0.4 Triglycerides 45.9 ± 6.4 Table 3 Fatty acid composition of P. saltans’ freshly laid eggs, in percentage (mean ± SD) of total weight and quantified by TLC-FID N = three independent analyses of three pools from ten egg sacs each Free fatty acids Percentage/Egg Saturated 45.5 Myristic a. (C 14: 0) 2.4 ± 1.1 Pentadecanoic a. (C15:0) 2.1 ± 0.1 Palmitic a. (16:0) 24.0 ± 0.6 Stearic a. (C18:0) 15.2 ± 1.9 Arachidic a. (C20:0) 1.6 ± 0.4 Unsaturated 52.8 Palmitoleic a. (C16:1) 4.2 ± 2.1 Oleic a. (C18:1) 24.1 ± 1.2 Linoleic a. (C18:2) 19.9 ± 1.1 Linolenic a. (C18:3) 2.4 ± 0.3 Arachidonic a. (C20:4) 2.2 ± 0.1 Fig. 5 Triglycerides (a) and cholesterol (b) concentrations in P. sal‑ tans’ freshly laid eggs or bodies in relation to embryonic and post- embryonic developmental stage (n = 20/instar). Mean levels were compared between stages using ANOVA and post hoc Tukey (HSD) tests. *Significant difference among other instars at p < 0.05; **sig- nificant difference among other instars at p < 0.01 J Comp Physiol B 1 3 biomembrane formation, and are main energetic resources (Heras et al. 2000). The phospholipids present in egg extracts are phosphati- dylcholine, lysophosphatidylcholine, phosphatidylethan- olamine and sphingomyelin. Phosphatidylcholine, accounts for about 95% of the total choline in tissues (Ueland 2011) and is a major plasma lipid constituent of vertebrates (Li and Vance 2008) and invertebrates (Laino et al. 2013), and is a major component of biological membranes, espe- cially cellular membranes. Fatty acids and cholesterol are transported in the plasma by lipoproteins. Phosphatidyl- choline synthesis is required for the transport of fat and cholesterol (Noga et al. 2002; Noga and Vance 2003). This phospholipid can be synthesized from choline via the cyti- dine diphosphocholine pathway or through methylation of another phospholipid, phosphatidylethanolamine (Gibellini and Smith 2010) present in extracts of P. saltans eggs. Sphingomyelin (or ceramide phosphorylcholine), another phospholipid present in extract of P. saltans eggs, is a type of sphingosine containing phospholipid that is synthesized by the transfer of a phosphocho- line residue from a phosphatidylcholine to a ceramide. Sphingomyelins constitute an important class of phospho- lipids present in the membranes of most eukarotic cells and lipoproteins (Slotte 2013; Slotte and Ramstedt 2007). This compound plays an important role in the expres- sion of specific cellular functions, such as transmission of intracellular information transmission and mainte- nance of membrane structure. Sphingomyelin interacts favourably with cholesterol and other sterols and sphin- gomyelins and cholesterol are co-located on the surface of lipoprotein particles in P. saltans as in other species (Slotte and Ramstedt 2007). Sphingomyelin seems to reg- ulate the distribution of cholesterol within membranes. Concerning yolk-derived sphingomyelin, palmitic acid accounts for about 80% of the fatty acid chains bound by amide bonds. Phosphatidylcholine and sphingomyelin are precursors for intracellular messenger molecules such as diacylglycerol that are also present in the extracts of P. saltans eggs. Diacylglycerol is released by degradation of phosphatidylcholine by phospholipases. Sterols (cho- lesterol and esterified sterols) in eggs represented 3.5% of the lipids in eggs. Cholesterol plays an important role in arthropod physiology as a cuticular surface wax, as a constituent of membranes in the lipid bilayer, and as a precursor for the synthesis of steroid hormones involved in moulting (Andersen 1979; Martin-Creuzburg et al. 2007; Merzendorfer and Zimoch 2003). Furthermore, the fatty acid composition, dominated by palmitic, oleic and linoleic acids, of P. saltans eggs is similar to that of another wolf spider, S. malitiosa (Laino et al. 2013) and its pattern is also comparable to that of P. pythagoricus LV (Laino et al. 2011). A major charac- teristic of fatty acids in general is that they function as a structural component of membranes to maintain proper fluidity and permeability (Trabalon 2011). We found that the energy content of P. saltans’ eggs was equivalent to 127 cal/g, a value similar to that for Schizocosa malitiosa eggs (Laino et al. 2013), but lower than those for other spiders (Anderson 1978; Laino et al. 2013), crustaceans (Amsler and George 1985; Petersen and Anger 1997; Clarke et al. 1990; Heras et al. 2000), or beetles (Sloggett and Lorenz 2008) (Table 4). For example, values for 3 species of spiders (Anderson 1978) ranged from 26 to 29 J/mg ash-free dry mass (7 cal/mg). These differences could be related to differences between indirect methods (respirometer and size egg) used by Anderson to measure the level of energy in eggs and probable values were overestimated by indirect methods. It thus remains difficult to make clear generalizations about the ecological and evolutionary implications of egg compositional differences in spiders as well as other arthropods. To facilitate cross-species comparisons, clear and comparable methods are required. Fig. 6 Calories provided by proteins, triglycerides and carbohydrates in P. saltans’ freshly laid eggs, embryos and juveniles during the embryonic and post-embryonic periods (n = 20/instars). a Total calo- ries/mg wet mass. b Total calories/mg wet mass in percent J Comp Physiol B 1 3 Chemical variations during embryonic and post-embryonic development As embryonic development proceeds, maternal reserves of vitellus are sequentially degraded. Our results show that carbohydrates (total sugars and free glucose), protein and lipid (triglycerides and cholesterol) levels varied during embryonic and post-embryonic development. During development inside egg sacs, only 51% of the initial carbohydrate stock was used. During embryonic development, carbohydrate levels decreased gradually and free glucose levels increased in instar “embryo 2” just before they hatched, probably due to glyconeogenesis or hydrolysis of glycogen. We hypothesized that this increase was related to the embryos’ need to tear the chorion apart, an activity that requires rapid mobilization of energetic components. Thus, during this period, 24% of the initial carbohydrate stock was used by embryos. Total carbo- hydrate and free glucose levels decreased after hatching, maybe as a consequence of chitin synthesis during cuti- cle formation, as in Zaluska vincula (Sloggett and Lorenz 2008). During post-embryonic development, 27% of the initial carbohydrate stock was used by juveniles and at the emergence from egg sacs, 49% of the initial carbohydrate stock was still present in instar “juvenile 2E” but the levels of free glucose level were lower. Levels of total proteins decreased gradually dur- ing embryonic and post-embryonic development. This decrease was not linked to the decrease of LV. Indeed, our results showed that P. saltans consumed LV proportionally to the other proteins as no significant vari- ations of the LV/protein ratio could be evidenced at any stage. This may be because spiderlings are born with an important reserve of LV that should satisfy their energy needs until they are able to feed themselves. Acari Boo‑ philus microplus present a similar strategy as their lar- vae consumed only 35% of the LV deposited in oocytes (Campos et al. 2008). The variations observed of protein levels could be linked to glyconeogenesis that enables embryos and juveniles to develop. Thus, during these periods 67% of their total protein stock was used. Triglyceride levels, contrary to proteins, did not vary significantly during development until the “juvenile 2” instar. Degradation of these triglycerides was lower (16%) during embryonic and post-embryonic develop- ment than in many other invertebrate embryos that nor- mally used 40–60% of the initial stock (e.g. Amsler and George 1985; Needham 1950; Petersen and Anger 1997). Contrary to our hypothesis, triglycerides appear not rep- resenting the major fuel for embryonic development for P. saltans as for other invertebrates (Clarke et al. 1990; Sasaki et al. 1986). The greatest degradation of triglyc- erides was caused by instar “juvenile 2” after their sec- ond moult and before emergence: 33% of the triglyceride stock was used during this last period and when instar “juvenile 2E” emerged they still had 49% of the initial stock. This conservation strategy of carbohydrate and tri- glyceride stocks enables young P. saltans to survive with- out eating for 6–7 days after emergence and to moult. A previous study showed that the spiderlings start foraging Table 4 Comparison of relative lipid, protein and calories in arthropod species with different taxonomic position habitat (carbohydrates were considered constant during embryogenesis by authors) Arthropod species Egg (mg) Lipid/protein in freshly laid eggs (%) Calories/mg of egg Lipid/protein in larvae or juvenile freshly hatched (%) Aquatic crustacean Euphausia superba (Amsler and George 1985) 30 31/57 – 51/49 Hyas araneus (Petersen & Anger 1997) 69 31/34 0.38 66/34 Macrobrachium rosenbergii (Clarke et al. 1990) 51 28/61 – 100/0 Macrobrachium borellii (Heras et al. 2000) – 29/29 1.31–0.6 – Terrestrial spiders Pardosa saltans (present study) 0.481 26/71 0.13 34/62 Schizocosa malitiosa (Laino et al. 2013) – 20/60 0.10 – Filistata hibernalis (Anderson 1978) 1.28–1.56 – 6.1–6.3 – Nuctenea cornuta (Anderson 1978) 0.44–0.55 – 6.1–6.4 – Peucetia viridans (Anderson 1978) 1.67–1.85 – 6.6–6.8 – Terrestrial insects Adalia bipunctata (Sloggett and Lorenz 2008) 0.13–0.14 42/50 1.43 27/72 Adalia decempunctata (Sloggett and Lorenz 2008) 0.11 40/53 1.67 – Anissosticta novemdecimpunctata (Sloggett and Lorenz 2008) 0.10 44/49 1.79 – J Comp Physiol B 1 3 7–8 days after emergence, after their first post-emergence moult (Ruhland et al. 2016b). Our results show that cholesterol levels increased gradu- ally during development. We hypothesize that these vari- ations correspond to embryos’ and juveniles’ needs for moulting, as arthropods moulting is controlled by steroid hormones (Cheong et al. 2015; De Almeida et al. 2003; Trabalon and Blais 2012). Indeed, our results show that cholesterol levels were high just before juveniles moulted. These compounds would be appropriate to satisfy embryos’ sterol requirements but they have not yet been investigated. In conclusion, contrary to our hypothesis, P. saltans do not lay trophic eggs and juveniles only develop from the vitellus. Variations of carbohydrate, lipid (particularly tri- glycerides) and protein levels induce energetic modifica- tions. Intriguingly only 62% of the total initial energetic stock was used to form the embryos and the juveniles. The remaining vitellus was used by embryos. This suggests that part of the vitellus content is used for embryonic differen- tiation and another part for juvenile differentiation, in and out of the egg. Vitellus is used during post-embryonic life during juvenile differentiation. Decrease of energetic stocks is linked mainly to the use of proteins (67% of the initial stock), carbohydrates (51%) and triglycerides (49%) stocks. Contrary to our hypothesis, triglycerides are not the main energy source for embryonic development. The energetic stock provided by P. saltans mothers to their eggs is impor- tant and enables young to develop and to emerge with suffi- cient energetic reserves (38% of the initial energy stock) to last until they can forage by themselves, i.e. 7–8 days after emergence. It will be interesting to study at how the vitellus changes after emergence until vitellus stock is depleted. Acknowledgements The authors would like to thank Dr Ann Cloarec and Pr Simon N. Thornton for reading and correcting the English. 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Dev Growth Differ 30(4):337–346 http://dx.doi.org/10.1186/1742-9994-8-15 Embryonic and post-embryonic development inside wolf spiders’ egg sac with special emphasis on the vitellus Abstract Introduction Materials and methods Ethics statement Spider collection and rearing Experimental groups Development period and energetic state Quantification of carbohydrates and free glucose Identification and quantification of proteins and lipovitellin Quantification of proteins Anti-lipovitellin rabbit serum preparation Gel electrophoresis and western blot analysis Enzyme-linked immunosorbent assay (ELISA) development Identification and quantification of lipids Characterization of lipids and fatty acids Quantification of triglycerides and cholesterol Energetic equivalent of proteins, carbohydrates and lipids Statistical analyses Results Development and instars Embryonic development Post-embryonic development Carbohydrates and free glucose levels Protein levels and lipovitellin Lipids and fatty acids levels Triglycerides and cholesterol levels Energetic equivalence Discussion Chemical identification of vitellus in eggs Protein extracts Lipid extracts Chemical variations during embryonic and post-embryonic development Acknowledgements References