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

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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 

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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)



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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.



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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 



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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



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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. The work was partly supported by funding from the project 
ECOS-MINCyt (France-Argentine) No. A16B03.

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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