RESEARCH ARTICLE

Juvenile Development,
Ecdysteroids and Hemolymph Level
of Metabolites in the Spider

Brachypelma albopilosum

(Theraphosidae)

MARIE TRABALON

1

∗

AND

CATHERINE BLAIS

2

1

Department of Biology, Universit´e Rennes 1, UMR—6552 CNRS Ethologie, Rennes, France

2

Department of Biology, UPMC Univ Paris 06-INRA, UMR—1272 PISC, Paris, France

In the present work, juvenile development and physiological state of mygalomorph Brachypelma
albopilosum were investigated by means of individual rearing under controlled conditions. Males
required 4–5 years for development from first juvenile instar to adulthood, passing through 8 to
12 juvenile molts. Females developed to adults in 5–6 years with a variable juvenile molt number
from 9 to 13. The development and growth of males and females took place in a similar way
until the last juvenile molt leading to subadults. Ecdysteroids, total lipid, cholesterol, and protein
concentrations increased along with the different development instars in both males and females.
After the last juvenile molt, spiders presented morphological and biochemical sex differences.
Subadult and adulthood males were smaller in size and weight than females; hemolymph levels
of ecdysteroids, total lipids, cholesterol, and glucose were higher in males. These physiological
and biochemical differences can be correlated to the different sexual development between males
and females.

J. Exp. Zool. 00:1–12, 2012.

©

2012 Wiley Periodicals, Inc.

How to cite this article: Trabalon M, Blais C. 2012. Juvenile development, ecdysteroids and
hemolymph level of metabolites in the spider

Brachypelma albopilosum (Theraphosidae). J.

Exp. Zool. 00:1–12

ABSTRACT

J. Exp. Zool.
00:1–12, 2012

The spider subfamily Theraphosinae is a mygalomorph group
from the New World. The genus Brachypelma can be found
from Mexico to Central America (Smith, ’94; Y ´a ˜nez, ’99). The
destruction of its natural habitat and a high mortality (99%)
before sexual maturity (Baerg, ’58) are two factors that affect
the overall population, and combined with the illegal trade in-
volving the capture of juvenile and adult tarantulas could cause
extinction of these spiders. To regulate this trade and prevent
their endangerment, all the species of the genus Brachypelma
have been listed in appendix II of the Convention on Interna-
tional Trade in Endangered Species (CITES). The spiders of this
genus are long lived and, compared to other genera of the same
subfamily, they grow slowly (Smith, ’94). The males can reach
maturity in 7–8 years, living only 1 year or less after the last
molt, while the females reach maturity in 9–10 years, and then
live 10 years more than males (Locht et al., ’99). The extreme
scarcity of several species of Brachypelma, combined with po-
tential threats of habitat degradation and illegal trafficking, has

led to the need for captive breeding for future reintroduction
(Y ´a ˜nez et al., ’99). However, little is known of the development
and physiology of these spiders.

The ontogeny of spiders is divided into several different, main

stages (Vachon, ’57): an embryonic, a prelarval–larval, a juve-
nile, and an imaginal or adult stage. The last instar of the juve-
nile stage corresponds to the subadult instar, but a spider does
not become sexually mature until its makes the transition from
juvenile to imago (Foelix, ’96). After the imaginal molt, sex-
ual maturity is reached and the general rule is that they stop

∗

Correspondence to: Marie Trabalon, Universit´e Rennes 1, UMR 6552—

Ethologie animale et Humaine, Campus de Beaulieu, 35042 Rennes cedex,
France. E-mail: [e-mail]

Received 15 July 2011; Revised 18 November 2011; Accepted 2 December

2011

Published online xxxx in Wiley Online Library (wileyonlinelibrary.com).

DOI: 10.1002/jez.1717

©

2012 WILEY PERIODICALS, INC.

2

TRABALON AND BLAIS

molting, but the females of some nonaraneomorph species (see
Mygalomorpha) will continue to molt throughout their whole
life. The number of juvenile molts undergone depends on the
ultimate body size of the spider.

Body size is a key attribute of many organisms because it

affects directly their ability for survival and competition, their
fecundity, and other components of fitness. Because body size
is determined by patterns of growth throughout an organism’s
ontogeny, body size depends on both endogenous mechanisms
and environmental factors. The most commonly used method
of testing for body size regulation in Arthropods is to look for
a significant relation between premolt size and the amount of
growth during the subsequent molt cycle, a method referred
to here as “growth increment analysis” (Twombly and Tisch,
2000; 2002). Condition indices based on mass, size, or mass/size
relation (Kotiaho, ’99; Rolff and Joop, 2002) are often used as
shortcuts, but are not direct measures and sometimes may fail
to correlate with fitness (Rolff and Joop, 2002).

Ecdysteroids are considered as phylogenetically old chemical

signals (Karlson, ’83). In all arthropod taxa, they exert compa-
rable functions such as regulation of molting, development, re-
production, and differentiation. Knowledge of the function and
mode of action of ecdysteroids is only fragmentary in arachnid
groups in comparison to some other arthropod groups. In spi-
ders, the endocrine regulation of postembryonic development
is not known and the role of ecdysteroids is a much neglected
field of research. Ecdysteroids have been detected in only six
arachnid female species: Opilio ravennae (Romer and Gnatzy,
’81), Pisaura mirabilis (Bonaric and De Reggi, ’97), Coelotes ter-
restris (Trabalon et al., ’92), Tegenaria domestica, and T. atrica
(Trabalon et al., ’92, ’98, 2005; Pouri´e and Trabalon, 2003).
Bonaric (’87) showed that fluctuations of ecdysteroids during
the molting cycle in P. mirabilis are similar to those reported
for other arthropods. In female T. atrica, 20-hydroxyecdysone
inhibits cannibalism during sexual activity and changes sex
pheromone production (Trabalon et al., 2005). At present, there
is no study carried out with male spiders.

In Crustacea, hemolymph metabolites, such as the levels of

glucose (Telford, ’68) and protein (Dall, ’74), undergo changes
in correlation with the molt stage while they are undoubtedly
related to metabolic changes associated with metamorphosis.
There is very little information available on metabolites (lipids,
proteins, and glucides) of hemolymph in spiders. Cohen (’80)
analyzed the chemical composition of the hemolymph of two
species of araneid spiders, and Punzo (’89) for six species of
lycosid spiders. All these analyses were conducted on pooled
samples of hemolymph. The changes in hemolymph proteome
of mygalomorph Brachypelma albopilosum females were exam-
ined for the first time in relation to their developmental stage
(subadult and adult period) by Trabalon et al. (2010). Recently,
Trabalon (2011) analyzed the lipid composition of hemolymph
from B. albopilosum adult females with respect to agonistic be-

havior. However, data are not yet available for the variations of
hemolymph levels of lipids, proteins, and glucides in relation to
juvenile development and sex in spiders.

Therefore, the first aim of the present study was to observe,

under environmentally controlled conditions, the development
of male and female B. albopilosum during the juvenile stage
up to the adult molt. The second aim was to analyze the basal
levels of ecdysteroids and metabolites (proteins, lipids, and glu-
cose) in hemolymph after each juvenile and the imaginal molts.
Because of the large size of B. albopilosum, it is possible to ob-
tain large individual volumes of hemolymph without killing the
animal. It is therefore ideally suited for use in studies of arthro-
pod physiology, as the problem of pooling hemolymph does not
exist with this species, and in addition, repeated samplings over
a period are possible. Observations and samplings were con-
ducted in the same spider, in order to obtain full knowledge of
juvenile–imaginal development of B. albopilosum.

MATERIALS AND METHODS

Animals

The curly haired tarantula, B. albopilosum (Valerio, ’80; until
2010 B. albopilosa), is a native of Costa Rica and Honduras
terricolous, living in the tropical rainforest, either around the
base of large trees, near rivers, or in patches of cleared rainforest.
All the B. albopilosum spiders used in the different tests reported
here came from laboratory stock (permit No. 540048—Pr´efecture
de Meurthe et Moselle, France).

The studied spiders (n

= 500) were from five egg sacs built

by spiders in the laboratory. Twenty days after their emergence
from the egg sac, the spiderlings were housed individually in
1-L plastic containers (16 cm

× 8 cm × 8 cm) during the larval

period. After the first juvenile molt, they were maintained in
8-L glass boxes (27 cm

× 18 cm × 16 cm), before being finally

transferred to 14-L plastic containers (32 cm

× 22 cm × 20 cm)

after the last juvenile molt (subadult instars). A 50:50 mixture
of potting soil and vermiculite was used for bedding.

Spiders were bred at 23

± 2

◦

C, with 60

± 10% relative

humidity under a 12:12 hr photoperiodic cycle. Animals were
fed ad libitum with a standardized diet of Tenebrionidae larvae
(Zophobas morio) and larvae or adult Blattidae (Blabera fusca,
Pleriplaneta americana). These preys were selected according to
the suitability of their body size for the experimental animals at
the different developmental stages.

For each individual spider, the number of juvenile molts until

the imaginal molt, the duration of individual instars, and the
time of death were recorded by checking the spiders at 1–2 days
interval. The sex of the animals could be checked only in adults.
The two characteristics of adult males, “mating spurs” on the
tibia of leg I and the “palpal organs” at the end of pedipalp tarsi,
were apparent only after the imaginal molt. A fully differentiated
epigyne appears in adult females.

J. Exp. Zool.

BIOCHEMICAL STATE IN TARANTULA

3

Weight and Body Size

Morphological measurement and hemolymph sampling were
performed after each molt in the same spiders, in the fourth
(A-4), the third (A-3), the second (A-2), and the first (A-1) in-
star before the final instar (A or adult stage). In order to avoid
any change in hemolymph composition related to the molt-
ing cycle (Stewart and Martin, ’70; Collatz and Mommsen, ’75),
groups were sampled in the intermolt instars (30

± 2 days after

each molt) and 24 hr after feeding. Spiders were anesthetized by
chilling at 4

◦

C for 1 hr. Weight measurements were taken with

a Sartorius electronic balance (Palaiseau, France) (

±0.01 g).

For determination of body volume, cephalothorax and

abdominal sizes were measured with callipers (

±0.01 mm,

Manostat Corp., Basel, Switzerland). For thorax volume, the
width at the widest part and the length from pedicel to the
region between the eyes were recorded. For abdomen volume
calculation, width at the widest part and length from the pedicel
to the region between the spinnerets were measured. The equa-
tion for a probate spheroid, V

= 4/3 π (ab

2

)/8, where a

= length

and b

= width of the cephalothorax or abdomen (in mm), was

used for the final volume.

Hemolymph Sampling

For hemolymph chemical composition, 40 spiders per sex were
studied: for males, 5 spiders that molted 8 times, 5 that molted 9
times, 25 that molted 10 times, and 5 that molted 11 times; for
females, 5 spiders that molted 9 times, 25 that molted 10 times,
5 that molted 11 times, and 5 that molted 12 times.

After weight and body measurements, the pericardium of

each spider was punctured with a needle, and 200

μL of

hemolymph was withdrawn in the presence of sodium citrate
buffer pH 4.6, using a positive displacement pipette. The whole
sampling procedure lasted less than 20 sec. This brief period of
handling precluded induction of an endocrine-dependent stress
response that would influence levels of hemolymph metabolites
during the time of sampling. All spiders survived the anesthesia
and hemolymph sampling procedures. Hemocytes were removed
by centrifugation at 10,000 rpm for 10 min at 4

◦

C and the result-

ing plasma was aliquoted for further analyses of ecdysteroids,
total lipids, cholesterol, glucose, and proteins.

Quantification of Ecdysteroid Levels

Ecdysteroids from 50

μL of plasma were extracted twice with

1 mL of methanol. After centrifugation, the pellets were dis-
carded and the combined supernatants were evaporated to dry-
ness. Ecdysteroids were detected with an enzyme immunoassay
(EIA) adapted from the method described by Porcheron et al.
(’89), by using goat anti-rabbit IgG (Jackson Immunoresearch
Lab) and 2-succinyl-20-hydroxyecdysone coupled to peroxidase
as the enzymatic tracer. Enzymatic activity was measured us-
ing ortho-phenylene-diamine (Sigma, Saint-Quentin Fallavier,
France) as substrate. The polyclonal anti-20-hydroxyecdysone

antiserum AS4919 was used (Porcheron et al., ’89). It presents
approximately an equal affinity for 20-hydroxyecdysone and
ecdysone. In routine experiments, calibration curves were gen-
erated with 20-hydroxyecdysone (4,000–31.2 fmol/tube). Dried
samples were resuspended in EIA sample buffer and quantified
in duplicate.

Quantification of Total Lipids and Cholesterol

Total lipids in plasma (50

μL) were measured with a colorimetric

method (Atlas Medical, Cambridge, UK). This method converts
lipids, but not saturated fatty acids, to a pink-colored complex
in the presence of sulfuric acid, phosphoric acid, and vanillin.
Results were expressed as an “index of total lipids.”

Cholesterol in 10

μL of plasma was determined using choles-

terol/cholesteryl ester detection kit (Abcam, Cambridge, UK) us-
ing a colorimetric method. In the assay, free cholesterol is oxi-
dized by cholesterol dehydrogenase to generate NADH that re-
acts with a sensitive probe resulting in strong absorbance at
450 nm.

Quantification of Glucose

Concentration of glucose in a 10-

μL plasma sample was deter-

mined using a colorimetric method after enzymatic oxidation in
the presence of glucose oxidase (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 indicator.

Quantification of Proteins

Protein content in a 10-

μL plasma sample was determined

according to the method of Bradford (’76) with a Coomassie
(Bradford) protein assay kit (Thermo Scientific, Cergy Pontoise,
France) using bovine serum albumin as the standard.

Statistical Analysis

Chi-square analyses were used to compare the maturation rates
of the different stages and the sex ratios. Since all data were
normally distributed, group differences were determined using
a two-way ANOVA (factor sex

× factor instar). Specific mean

comparisons were then made using t-test with Bonferroni cor-
rection. Statistical level of significance was P

≤ 0.05. Results

were expressed as means

± SE.

RESULTS

Sex ratio, Mortality, and Number of Juvenile Molts

Of the 500 young spiders studied until adult instars, 37% (n

=

186) were female, 25% (n

= 123) were male, and 38% (n = 191)

died before reaching the imaginal molt and sex determination.
Total mortality was 60% per egg sac and half of the deaths were
associated with exuviations. The spiders remained sometimes
attached to their old cuticle and died 1–2 days later. Mortality
was the greatest at the 3rd juvenile molt and was greatly reduced
after the 5th juvenile molt (

<2% of total mortality per egg sac).

J. Exp. Zool.

4

TRABALON AND BLAIS

Table 1.

Relative frequency of Brachypelma albopilosum male

(n

= 123) and female (n = 186) reaching the adult stage, scored af-

ter the 5th to 13th juvenile molts. The female and male frequencies
were compared using

χ

2

test.

Adult males

Adult females

Molts

Number

Frequency (%)

Number

Frequency (%)

5th

0

0

0

0

6th

0

0

0

0

7th

0

0

0

0

8th

33

26.83*

0

0

9th

41

33.33

NS

40

21.50

10th

25

20.33*

68

36.56

11th

15

12.20*

64

34.41

12th

9

7.31

NS

10

5.38

13th

0

0

NS

4

2.15

*P

< 0.05; NS, not significant.

The number of juvenile molts before the adult molt varied

along with sex and individual spiders (Table 1). The frequency
of adult molt after the 8th, 10th, 11th, and 13th juvenile molts
was different between males and females (

χ

2

= 3.64–33.64, P =

0.003). The adult molt occurred with the highest frequency after
the 10th and 11th juvenile molt in females (71% of females, n

=

132/186), and after the 8th to 9th juvenile molt in males (60%
of males, n

= 74/123).

Duration of Development Instars

There was no significant intrasex difference for the duration of
the juvenile period whatever the number of molts in males (F

=

2.30, P

= 0.06) and in females (F = 1.49, P = 0.21) (Table 2).

However, the duration of the juvenile instars and the time until
adulthood (Fig. 1 and Table 2) were different in females and
males and significantly longer in females (F

= 7.89, P < 0.0001).

Males required between 4.78 to 4.99 years for development

with 8–12 juvenile molts (Fig. 1A and Table 2). Adult molts
generally occurred in the laboratory from August to October.

Females required between 5.51 to 5.90 years until the adult

molt (Fig. 1B and Table 2). The number of juvenile molts ranged
from 9 to 13, and adult molts occurred during July to October.

Mass/Size

The body size increased with each instar until the adult molt
in males and in females. Body weight and the volume of
cephalothorax and abdomen increased with the same pattern
in juveniles from both sexes (Figs. 2 and 3). The weight of fe-
males that reached adulthood after 9, 10, 11, 12, or 13 instars
was not different, i.e., 17.4

± 0.7 g (F = 1.32; P = 0.12). Males

that reached adult stage after 11 or 12 instars were heavier

Table 2.

Duration of development (expressed in days and years,

mean

± SE) of B. albopilosum from first juvenile molt until the

imaginal molt. The durations of the stages were compared between
males and females using ANOVA test.

Males (

n

= 123)

Females (

n

= 186)

Duration of stages

Days

Years

Days

Years

Juvenile stage (first to last juvenile molt) with
8 molts

1,474

± 24

4.04

-

-

9 molts

1,598

± 22

4.38

1,814

± 33*

4.97

10 molts

1,523

± 24

4.17

1,818

± 22*

4.98

11 molts

1,583

± 26

4.34

1,878

± 31*

5.14

12 molts

1,567

± 22

4.29

1,758

± 24*

4.82

13 molts

-

-

1,804

± 20

4.94

Mean

1,548

± 24 4.24 1,814 ± 26

4.97

Subadult stage (last juvenile molt to imaginal molt) reached after
8 molts

272

± 28

0.74

-

-

9 molts

195

± 22

0.53

338

± 33*

0.93

10 molts

269

± 21

0.74

216

± 25*

0.59

11 molts

167

± 39

0.46

232

± 33*

0.64

12 molts

253

± 22

0.69

357

± 22*

0.98

13 molts

-

-

209

± 25

0.57

Mean

231

± 22

0.63

314

± 28

0.86

Total juvenile–imaginal stage with
8 molts

1,746

± 19

4.78

-

-

9 molts

1,793

± 19

4.91

2,152

± 33*

5.90

10 molts

1,792

± 22

4.91

2,034

± 29*

5.57

11 molts

1,750

± 18

4.79

2,110

± 35*

5.78

12 molts

1,820

± 22

4.99

2,115

± 21*

5.79

13 molts

-

-

2,013

± 31

5.51

Mean

1,792

± 22 4.91 2,084 ± 29

5.71

Bold values represented duration mean of development whatever the num-
ber of molt.
*Significantly different between sex at P

< 0.0001.

(14.4

± 0.2 g) than those that reached it after 8, 9, or 10 in-

stars: 13.1

± 0.4 g (F = 2.25, P = 0.05). After the last juvenile

molt, leading to subadult instars (A-1), the body size (weight and
volumes) was significantly different between males and females
(F

= 28.93, P < 0.0001). Subadult and adult females were thus

significantly heavier than males (F

= 4.90, P < 0.001), i.e.,

14.5

± 0.5 and 17.5 ± 0.7 g for females compared to 12.6 ±

0.4 and 13.7

± 0.6 g for males (Fig. 2). Concerning the size of

cephalothorax and abdomen, there was also a significant sex
difference after the A-2 molt (Fig. 3). In subadult (A-1) and
adult instars (A), females were significantly larger than males
(F

= 2.89–3.98, P = 0.05–0.01). The volume of cephalothorax

and abdomen of adult females and males did not vary whatever
the number of juvenile instars.

J. Exp. Zool.

BIOCHEMICAL STATE IN TARANTULA

5

Figure 1.

Duration of development (days) in Brachypelma albopilosum males (A) and females (B) from the first juvenile molt (A-12 before

the adult instar) until the adult instar (A). Males, M8–M12, and females, F9–F13, were numbered according to the number of the molts to
become adults: M8: males that molted 8 times (n

= 33); M9: 9 times (n = 41); M10: 10 times (n = 25); M11: 11 times (n = 15); M12:

12 times (n

= 9); F9: females that molted 9 times (n = 40); F10: 10 times (n = 68); F11: 11 times (n = 64); F12: 12 times (n = 10), and

F13: 13 times (n

= 4).

The durations of the stages were compared between the different groups of males and the different groups of females using ANOVA test:
*Significantly different with females at P

< 0.0001.

Ecdysteroid Titers in Hemolymph

The hemolymph ecdysteroid titers in females and in males that
reached adulthood after a different number of instars were not
different (F

= 1.30 and 1.49, P = 0.17 and 0.21).

Figure 4 shows that the hemolymph ecdysteroid titers, mea-

sured 30 days after each molt, varied significantly with the sex

of spiders (F

= 4.91, P = 0.001). In females, ecdysteroids levels

were constant about 7.44

± 1.01 ng/mL in all instars checked

about. In juvenile males, ecdysteroid levels were rather constant
until A-2 instar: 5.68

± 0.87 ng/mL. They increased signifi-

cantly afterwards to 12.38

± 1.84 ng/mL in A-1 (subadults), and

remained stable in A (adults) at 11.22

± 1.41 ng/mL.

J. Exp. Zool.

6

TRABALON AND BLAIS

Figure 2.

Body size (weight) of B. albopilosum males (A) and females (B) in juveniles (A-4, A-3, A-2), subadults (A-1), and adults (A).

Males, M8–M12, and females, F9–F13, were numbered according to the number of the molts to become adults. Same abbreviations and
sample size as in Figure 1.
The weights for each instar in one sex were compared using the ANOVA test: *Significantly different with age at P

< 0.05; NS, not

significant.

Total Lipids and Cholesterol Levels in Hemolymph

The level of lipids and cholesterol in females and males that
reached adulthood after a different number of instars was not
different (F

= 0.63–1.59, P = 0.60–0.17).

Figure 5A, B shows that the index of total lipids (F

= 37.68,

P

< 0.0001) and the level of cholesterol (F = 8.16, P = 0.001)

in the hemolymph increased significantly after each instar. The
index of total lipids became significantly higher in males than in
females in the A-2 instars (402.6

± 46.5 vs. 260.3 ± 28.8 mg/L;

P

< 0.05) and until the final instar. The level of cholesterol was

significantly higher in males than in females only in the final
instars (9.45

± 0.35 vs. 4.65 ± 0.49 mg/L; P < 0.0001).

J. Exp. Zool.

BIOCHEMICAL STATE IN TARANTULA

7

Figure 3.

Body size (volumes) of B. albopilosum males (A and B) and females (C and D) in juveniles (A-4, A-3, A-2), subadults (A-1),

and adults (A). Males, M8–M12, and females, F9–F13, were numbered according to the number of the molts to become adults. Same
abbreviations and sample size as in Figure 1.
The body sizes for each instar by sex were compared using the ANOVA test; NS, not significant.

Glucose and Protein Levels in Hemolymph

The levels of glucose and proteins in females and males that
reached adulthood after a different number of instars were not
significantly different (F

= 0.73–1.44, P = 0.26 and 0.0.57).

The glucose level in hemolymph increased significantly after

each molt in males (Fig. 6A; F

= 9.11, P = 0.01). Males had

a significantly higher level of glucose than females during the
last juvenile, subadult, and adult instars: from 0.09 to 0.13 g/L

J. Exp. Zool.

8

TRABALON AND BLAIS

Figure 4.

Hemolymph ecdysteroid titers in B. albopilosum males (dotted line, n

= 40) and females (solid line, n = 40) n juveniles (A-4,

A-3, A-2), subadults (A-1), and adults (A). Ecdysteroid concentrations are expressed in nanogram of 20-hydroxyecdysone equivalents per
milliliter hemolymph.
Averages for each instar were compared using the Student’s t-test: *Significant difference at P

< 0.05, **Significant difference at P <

0.01; NS, not significant.

in adults. In female spiders, the level of glucose did not vary
significantly during development (0.07–0.09 g/L; P

= 0.09) and

was significantly lower than in males.

The level of proteins (Fig. 6B) increased significantly un-

til the A-1 instar (subadult instars) in both sexes (F

= 14.05,

P

= 0.001): 16–44 g/L in females and 13–46 g/L in males. The

protein level was not significantly different between males and
females at adulthood (F

= 1.42, P = 0.34).

DISCUSSION-CONCLUSION

Sex ratio, Mortality, and Development

Our results have shown that only 40% of the young spiders’
B. albopilosum survived and developed until the adult stage un-
der environmentally controlled conditions (319 adults/500 larval
spiders). Males represented only 40% of the surviving adults. The
proximal physiological causes of this mortality are unknown at
the present time. Death occurred either just before molting or
during ecdysis. Approximately one-half of the animals died be-
fore any external sign of ecdysis initiation had appeared; the
remainder died stuck in their exoskeleton or once emerged from
ecdysis with extremely distorted legs.

Brachypelma albopilosum presented a slow growth and de-

velopment as shown in other spiders, Nephila clavipes (Higgins,
2000). Males should have normally one or two fewer molts than
females, but this was not an absolute rule. The duration of each
juvenile instar was negatively correlated with the rate of increas-

ing mass, and the number of juvenile instars was dependent on
the sex of the spider. Thus, B. albopilosum reached adulthood
after 8–12 instars for the males and 9–13 instars for the females,
and needed approximately 5–6 years to mature from the first lar-
val instars. A low growth rate in juvenile arthropods increases
the duration of each juvenile instar, and is associated with an
increased risk of prereproductive mortality (Higgins and Rankin,
2001).

During juvenile development of B. albopilosum, there is an

expansion of biomass and of all its constituents. The growth
curves of males and females were similar until the last juve-
nile instar (subadult instars). The size of adults after the imag-
inal molt was not correlated to the number of juvenile molts
in a large part in B. albopilosum, and slowly growing females
passed through several instars and reached maturity at a larger
size than rapidly growing males. Thus, adult females may reach
5–6 cm in cephalothorax–abdomen length and weigh 14–17
g, the males being somewhat smaller (4–5 cm in length and
13–14 g in weight).

Hemolymph Chemical Composition and Ecdysteroids

Hemolymph chemical composition from spiders that reached the
adult stage after a different number of molts (8th, 9th, 10th, 11th,
12th, or 13th) was similar. The number of molts did not influence
the chemical composition.

Our results showed that the concentration of lipids increased

in the plasma after each instar and during the development of

J. Exp. Zool.

BIOCHEMICAL STATE IN TARANTULA

9

Figure 5.

Hemolymph concentrations of lipids (A) and cholesterol (B) in B. albopilosum males (dotted line, n

= 40) and females (solid line,

n

= 40) in juveniles (A-4, A-3, A-2), subadults (A-1), and adults (A).

The average levels for each instar were compared using the Student’s t-test: *Significant difference at P

< 0.05, **Significant difference

at P

< 0.01; NS, not significant.

B. albopilosum juveniles. However, our results showed a sexual
difference as the level of lipids was higher in male spiders af-
ter the A-3 instar: 730 mg/L of lipids in adult males compared
to 600 mg/L in females. Males have not been studied previ-
ously but comparable values have been obtained in adult female
spiders (Araneus gemma and Argiope trifasciata: 580 and 410
mg/L, respectively; Cohen, ’80). Lipids are a compact form of
energy storage and thus the storage molecules of choice. They
play key roles in insect biochemistry as sources of energy, struc-

tural components, and as hormones (Stanley-Samuelson et al.,
’88). Lipids present in the hemolymph of adult B. albopilosum
females have been identified (Trabalon, 2011) as hydrocarbons,
four free fatty acids (palmitic, linoleic, oleic, and stearic acids),
and cholesterol. Lipid levels are hypothesized to have evolved
as a regulatory factor of predation and agonistic behaviors in
tarantula females. Trabalon (2011) showed that the female was
able to modulate her aggressive behavior according to the lev-
els of circulating lipids. Indeed, females with high levels of

J. Exp. Zool.

10

TRABALON AND BLAIS

Figure 6.

Hemolymph concentrations of glucose (A) and proteins (B) in B. albopilosum males (dotted line, n

= 40) and females (solid line,

n

= 40) in juveniles (A-4, A-3, A-2), subadults (A-1), and adults (A).

The average levels for each instar were compared using the Student’s t-test: *Significant difference at P

< 0.05, **Significant difference

at P

< 0.01; NS, not significant.

circulating lipids presented no aggressive interactions with their
congeners. Tarantula males were less aggressive than females.
Our results showed that all males presented levels of lipids signif-
icantly higher than the females. We showed here that cholesterol
levels varied as did total lipid levels with the sex of the spider
after the imaginal molt: they were higher in males (9 mg/L) than
in adult females (5 mg/L). Cholesterol plays an important role in
arthropod physiology as component of cuticular surface waxes,

as a constituent of cell membranes, and as a precursor to steroid
hormones such as ecdysteroids.

In our study, we report for the first time the variation of

hemolymph ecdysteroids in a batch of male and female spiders
during juvenile development. Ecdysteroid concentrations in fe-
males were constant during the successive stages of development
(7 ng/mL). In juvenile males, ecdysteroids were at the same level
as in females but increased after the last juvenile molt to reach a

J. Exp. Zool.

BIOCHEMICAL STATE IN TARANTULA

11

peak at 12 ng/mL during the transition from subadults to adults.
During these instars, our results showed that males possessed
also higher levels of lipids and cholesterol than females. These
differences can be bound to the fact that B. albopilosum males
do not molt any more in adulthood, while in adult females, a
molt occurs once a year throughout the survival period, as in
other mygalomorph spiders (Stradling, ’78; Miyashita, ’92). But
these differences can also be connected to the fact that after
the imaginal molt, males display spermatogenesis, then transfer,
and store the sperm in their pedipalp organs, before searching
for a female mate. In B. albopilosum, we observed that males
loaded their pedipalps with sperm 30 days after the imaginal
molt; so spermatogenesis should have begun before, some days
after this molt, when high levels of ecdysteroids were present.
Ecdysteroids could promote spermatogenesis in male spiders, as
has been shown in many species of insects (Hagedorn, ’85) and in
ticks (Zhang et al., ’95). Ecdysteroids should have a stimulatory
role on early spermatogenesis involving mitoses and meioses.
After sperm storage, male spiders are involved in an intense
mate-searching activity under natural conditions. In this polyg-
ynous mating system, costs of locomotion are high and adult
males have higher energy life styles than females, as has been
shown in other tarantulas (P´erez-Miles et al., 2005). Glucose is
the primary energy source for arthropod and vertebrate tissues.
Its concentration in subadult and adult males was higher (0.10–
0.12 g/L) than in juvenile males and females. These differences
could be explained by a higher requirement of energy in males
linked to a higher metabolism in connection with sexual activity.

In B. albopilosum females, our results showed a low level

of lipids, cholesterol, and ecdysteroids in comparison to postju-
venile males. They were unmated and we observed that their
ovaries were in the previtellogenic phase of oocyte develop-
ment (M. Trabalon, unpublished data). In female spiders of other
species such as C. terrestris and Tegenaria sp., the highest lev-
els of total circulating ecdysteroids were detected during the
vitellogenic phases of oocyte development (Trabalon et al., ’92,
’98). In T. atrica, copulation stimulated the synthesis/liberation
of ecdysteroids and vitellogenesis (Pouri´e and Trabalon, 2003).
We can thus presume that after mating, ecdysteroid titers in B.
albopilosum females would quickly increase. Studies of the vari-
ation of ecdysteroid levels in females before and after mating
would allow verification of this hypothesis.

Concerning hemolymph proteins, our results showed simi-

lar levels in B. albopilosum females and males, thus protein
concentrations were not influenced by sex. During juvenile de-
velopment, the levels of proteins increased between each instar,
but in subadults and adults the concentrations were compara-
ble, thus showing that they are not influenced by the imaginal
molt. In addition, protein concentrations measured in adults of
B. albopilosum were equivalent to those found in other spider
species (Schartau and Leidescher, ’83; Punzo, ’89; Pouri´e and
Trabalon, 2003; Zachariah et al., 2007). Male and female adults

show different exploratory behaviors: females are sedentary and
males wander. The hemolymph proteome of B. albopilosum has
been previously analyzed only in females and consisted mainly
of hemocyanin, actin, and another protein of unknown function
(Trabalon et al., 2010). The putative corresponding structures of
these proteins are the coagulogen protein and/or lipoproteins for
which quantitative differences between adult and subadult spi-
ders could be related to the molting process and protein compo-
sition according to the developmental stage. Within the frame-
work of future studies, it would be thus interesting to analyze the
distribution of these various protein constituents (lipoproteins,
hemocyanin, and actin) according to the sex and the exploratory
behavior of the adult spider.

CONCLUSION

Our results showed that males had a higher metabolic reserve
than females as observed in other spiders (Tanaka and Ito, ’82;
Watson and Lighton, ’94; Shillington, 2005). Some of the sexual
dimorphism can be explained by size differences, between males
and females, and behavior. Brachypelma albopilosum spiders
are an excellent model for addressing questions in evolution-
ary physiology because of variations in performance (locomotor
activity) and life history of the adult animals. The higher levels
of energy-giving compounds (lipids and glucose) could be an
adaptive strategy to support higher energy demands for males
during their active, locomotor search for females. Additional
work is needed to attest a correlation between behavior and
variation of ecdysteroids and metabolic constituents.

ACKNOWLEDGMENTS

We would like to thank Prof. Simon N. Thornton and Franc¸oise
Joubaud for reading the manuscript; Annick Maria (UPMC, Paris)
for technical assistance in ecdysteroid determinations; Jo¨elle
Couturier and Jean Charles Olry for animal maintenance (Uni-
versity of Nancy I).

LITERATURE CITED

Baerg WJ. 1958. The tarantula. Lawrence, Kansas: University of Kansas

Press.

Bonaric JC. 1987. Moulting hormones. In: Nentwig W, edidor. Eco-

physiology of spiders. Berlin: Springer Verlag. p 111–118.

Bonaric JC, De Reggi M. 1997. Changes in ecdysone levels in the

spider Pisaura mirabilis nymphs (Araneae, Pisauridae). Experientia
33:1664–1665.

Bradford MM. 1976. A rapid and sensitive method for the quantifi-

cation of microgram quantities of protein utilizing the principle of
protein-dye binding. Annal Biochem 72:248–254.

Cohen AC. 1980. Hemolymph chemistry of two species of araneid

spiders. Comp Biochem Physiol A 66:715–717.

Collatz KG, Mommsen T. 1975. Physiological conditions and variations

of body constituents during molting cycle of spider Tegenaria atrica
C.L. Koch (Agelenidae). Comp Biochem Physiol A 52:465–476.

J. Exp. Zool.

12

TRABALON AND BLAIS

Dall W. 1974. Indices of nutritional state in the western lobster,

Panulirus longipes (Milne Edwards). I. Blood and tissue constituents
and water content. J Exp Mar Biol Ecol 16:167–180.

Foelix RF. 1996. Biology of spiders. New York: Oxford University Press.
Hagedorn HH. 1985. The role of ecdysteroids in reproduction. In:

Kerkut GA, Gilbert LI, editors. Comprehensive insect physiology
biochemistry and pharmacology, Vol. 8. Oxford: Pergamon Press.
p 205–262.

Higgins L. 2000. The interaction of season length and development

time alters size at maturity. Oecologia 122:51–59.

Higgins LE, Rankin MA. 2001. Mortality risk of rapid growth in the

spider Nephila clavipes. Functional Ecol 15:24–28.

Karlson P. 1983. Why are so many hormones steroids? Hoppe-Seyler’s

Z. Physiol Chem 364:1067–1087.

Kotiaho JS. 1999. Estimating fitness, comparison of body condition

indices revisited. Oikos 87:399–400.

Locht A, Y´a ˜nez M, V´azquez I. 1999. Distribution and natural history

of Mexican species of Brachypelma and Brachypelmides (Thera-
phosidae, Theraphosinae) with morphological evidence for their
synonymy. J Arachnol 27:196–200.

Miyashita K. 1992. Postembryonic development and life cycle of Aty-

pus karschi D ¨onitz (Araneae: Atypidae). Acta Arachnol 41:177-186.

P´erez-Miles F, Costa FG, Toscazo-Gadea C, Mignone A. 2005. Ecology

and behaviour of the “road tarantulas” Eupalaestrus weijenberghi
and Acanthoscurria suina (Araneae, Theraphosidae) from Uruguay.
J Nat Hist 39:483–489.

Porcheron P, Morini`ere M, Grassi J, Pradelles P. 1989. Develop-

ment of an enzyme immunoassay for ecdysteroids using acetyl-
cholinesterase as label. Insect Biochem 19:117–122.

Pouri´e G, Trabalon M. 2003. The role of 20-hydroxyecdysone on the

control of spider vitellogenesis. Gen Comp Endo 131:250–257.

Punzo F. 1989. Composition of the hemolymph of mygalomorph spi-

ders (Orthognatha). Comp Biochem Physiol A 93:757–760.

Rolff J, Joop G. 2002. Estimating condition: pitfalls of using weight

as a fitness surrogate. Evol Ecol Res 4:931–935.

Romer F, Gnatzy W. 1981. Arachnid oenocytes: ecdysone synthesis

in the legs of harvestmen (Opilionidae). Cell Tissue Res 216:449–
453.

Schartau W, Leidescher T. 1983. Composition of the hemolymph of

the tarantula Eurypelma californicum. J Comp Physiol 152:73–77.

Shillington C. 2005. Inter-sexual differences in resting metabolic

rates in the Texas tarantula, Aphonopelma anax. Comp Biochem
Physiol 142:439–445.

Smith A. 1994. Tarantula spiders: tarantula of the USA and Mexico.

London: Fitzgerald Publishing.

Stanley-Samuelson DW, Jurenka C, Crips GJ, Blomquist GJ, de

Renobales M. 1988. Fatty acids in insects: composition, metabolism
and biological significance. Arch Insect Biochem Physiol 9:1–33.

Stewart DM, Martin AW. 1970. Blood and fluid balance of common

tarantula, Dugesiella hentzi. Z Vergl Physiol 70:223–246.

Stradling DJ. 1978. The growth and maturation of the “tarantula”

Avicularia avicularia L. Zool J Linn Soc 62:291–303.

Tanaka K, Ito Y. 1982. Decrease in respiratory rate in a wolf spi-

der, Pardosa astrigera (L. Koch), under starvation. Resp Pop Ecol
214:360–374.

Telford M. 1968. The identification of sugars in the blood of three

species of atlantic crabs. Biol Bull 135:674-684.

Trabalon M. 2011. Agonistic interactions, cuticular and hemolym-

phatic lipid variations during the foraging period in spider females
Brachypelma albopilosa (Theraphosidae). J Insect Physiol 57:735–
743.

Trabalon M, Bautz AM, Morini`ere M, Porcheron P. 1992. Ovarian de-

velopment and correlated changes in hemolymphatic ecdysteroids
levels in two spiders, Coelotes terrestris and Tegenaria domestica
(Araneae, Agelenidae). Gen Comp Endo 88:128–136.

Trabalon M, Pouri´e G, Hartmann N. 1998. Relationships among canni-

balism, contact signals, ovarian development and ecdysteroid levels
in Tegenaria atrica (Araneae, Agelenidae). Insect Biochem Mol Biol
28:751–758.

Trabalon M, Niogret J, Legrand-Frossi C. 2005. Effect of 20-

hydroxyecdysone on cannibalism, sexual behaviour, and contact
sex pheromone in the solitary female spider, Tegenaria atrica. Gen
Comp Endo 144:60–66.

Trabalon M, Carapito C, Voinot F, Martrette JM, Van Dorsselar A,

Glibert C, Bertile F. 2010. Differences in Brachypelma albopilosa
(Theraphosidae) hemolymph proteome between subadult and adult
females. J Exp Zool A 313:651–659.

Twombly S, Tisch N. 2000. Body size regulation in copepod crus-

taceans. Oecologia 122:318–326.

Twombly S, Tisch N. 2002. Fitness consequences of the timing of

metamorphosis in freshwater crustacean. Oikos 97:213–222.

Vachon M. 1957. Contribution `a l’´etude du d´eveloppement post-

embryonnaire des araign´ees. Premi`ere note: G´en´eralit´es et nomen-
clature des stades. Bull Soc Zool Fr 82:337–354.

Valerio CE. 1980. Theraphosid spiders of Costa-Rica (Araneae,

Theraphosidae). 3. Sphaerobothria, Aphonopelma, Pterinopelma,
Citharacanthus, Crypsidromus and Stichoplastus. Rev Biol Trop
28:271–296.

Watson PJ, Lighton JRB. 1994. Sexual selection and the energetics of

copulatory courtship in the Sierra dome spider, Linyphia litigiosa.
Anim Behav 48:615–626.

Y´a ˜nez M. 1999. Las tar´antulas de M´exico, un mito que es benefice.

Especies: Revista Sobre Conservacion y Biodiversidad. Naturalia AC
M´exico DF 8:4–8.

Y´a ˜nez M, Locht A, Macias-Ordo ˜nez R. 1999. Courtship and mat-

ing behaviour of Brachypelma klaasi (Schmidt and Krause, 1994)
(Araneae: Theraphosidae). J Arachnol 27:165–170.

Zachariah TT, Mitchell MA, Guichard CM, Singh RS. 2007. Hemolymph

biochemistry reference ranges for wild-caught goliath birdeater
spiders (Theraphosa blondi) and Chilean rose spiders (Grammostola
rosea). J Zoo Wildlife Med 38:245–251.

Zhang WY, Zhu XX, Oliver Jr JH. 1995. Ecdysteroids and spermatoge-

nesis in the fourth instar nymph of the tick Ornithodoros parkeri.
Invert Reprod Develop 27:1–9.

J. Exp. Zool.