RESEARCH ARTICLE
Juvenile Development,
Ecdysteroids and Hemolymph Level
of Metabolites in the Spider
Brachypelma albopilosum
(Theraphosidae)
MARIE TRABALON1" AND CATHERINE BLAIS2
1
Department of Biology, Universit� 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
ABSTRACT
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.
J. Exp. Zool.
Exp. Zool. 00:1 12
00:1 12, 2012
The spider subfamily Theraphosinae is a mygalomorph group led to the need for captive breeding for future reintroduction
from the New World. The genus Brachypelma can be found (Y�ńez et al.,  99). However, little is known of the development
from Mexico to Central America (Smith,  94; Y�ńez,  99). The and physiology of these spiders.
destruction of its natural habitat and a high mortality (99%) The ontogeny of spiders is divided into several different, main
before sexual maturity (Baerg,  58) are two factors that affect stages (Vachon,  57): an embryonic, a prelarval larval, a juve-
the overall population, and combined with the illegal trade in- nile, and an imaginal or adult stage. The last instar of the juve-
volving the capture of juvenile and adult tarantulas could cause nile stage corresponds to the subadult instar, but a spider does
extinction of these spiders. To regulate this trade and prevent not become sexually mature until its makes the transition from
their endangerment, all the species of the genus Brachypelma juvenile to imago (Foelix,  96). After the imaginal molt, sex-
have been listed in appendix II of the Convention on Interna- ual maturity is reached and the general rule is that they stop
tional Trade in Endangered Species (CITES). The spiders of this
genus are long lived and, compared to other genera of the same
"
Correspondence to: Marie Trabalon, Universit� Rennes 1, UMR 6552
subfamily, they grow slowly (Smith,  94). The males can reach
Ethologie animale et Humaine, Campus de Beaulieu, 35042 Rennes cedex,
maturity in 7 8 years, living only 1 year or less after the last
France. E-mail: [e-mail]
molt, while the females reach maturity in 9 10 years, and then
Received 15 July 2011; Revised 18 November 2011; Accepted 2 December
live 10 years more than males (Locht et al.,  99). The extreme 2011
Published online xxxx in Wiley Online Library (wileyonlinelibrary.com).
scarcity of several species of Brachypelma, combined with po-
DOI: 10.1002/jez.1717
tential threats of habitat degradation and illegal trafficking, has
� 2012 WILEY PERIODICALS, INC.
2 TRABALON AND BLAIS
molting, but the females of some nonaraneomorph species (see havior. However, data are not yet available for the variations of
Mygalomorpha) will continue to molt throughout their whole hemolymph levels of lipids, proteins, and glucides in relation to
life. The number of juvenile molts undergone depends on the juvenile development and sex in spiders.
ultimate body size of the spider. Therefore, the first aim of the present study was to observe,
Body size is a key attribute of many organisms because it under environmentally controlled conditions, the development
affects directly their ability for survival and competition, their of male and female B. albopilosum during the juvenile stage
fecundity, and other components of fitness. Because body size up to the adult molt. The second aim was to analyze the basal
is determined by patterns of growth throughout an organism s levels of ecdysteroids and metabolites (proteins, lipids, and glu-
ontogeny, body size depends on both endogenous mechanisms cose) in hemolymph after each juvenile and the imaginal molts.
and environmental factors. The most commonly used method Because of the large size of B. albopilosum, it is possible to ob-
of testing for body size regulation in Arthropods is to look for tain large individual volumes of hemolymph without killing the
a significant relation between premolt size and the amount of animal. It is therefore ideally suited for use in studies of arthro-
growth during the subsequent molt cycle, a method referred pod physiology, as the problem of pooling hemolymph does not
to here as  growth increment analysis (Twombly and Tisch, exist with this species, and in addition, repeated samplings over
2000; 2002). Condition indices based on mass, size, or mass/size a period are possible. Observations and samplings were con-
relation (Kotiaho,  99; Rolff and Joop, 2002) are often used as ducted in the same spider, in order to obtain full knowledge of
shortcuts, but are not direct measures and sometimes may fail juvenile imaginal development of B. albopilosum.
to correlate with fitness (Rolff and Joop, 2002).
Ecdysteroids are considered as phylogenetically old chemical
MATERIALS AND METHODS
signals (Karlson,  83). In all arthropod taxa, they exert compa-
rable functions such as regulation of molting, development, re- Animals
production, and differentiation. Knowledge of the function and The curly haired tarantula, B. albopilosum (Valerio,  80; until
mode of action of ecdysteroids is only fragmentary in arachnid 2010 B. albopilosa), is a native of Costa Rica and Honduras
groups in comparison to some other arthropod groups. In spi- terricolous, living in the tropical rainforest, either around the
ders, the endocrine regulation of postembryonic development base of large trees, near rivers, or in patches of cleared rainforest.
is not known and the role of ecdysteroids is a much neglected All the B. albopilosum spiders used in the different tests reported
field of research. Ecdysteroids have been detected in only six here came from laboratory stock (permit No. 540048 Pr�fecture
arachnid female species: Opilio ravennae (Romer and Gnatzy, de Meurthe et Moselle, France).
 81), Pisaura mirabilis (Bonaric and De Reggi,  97), Coelotes ter- The studied spiders (n = 500) were from five egg sacs built
restris (Trabalon et al.,  92), Tegenaria domestica, and T. atrica by spiders in the laboratory. Twenty days after their emergence
(Trabalon et al.,  92,  98, 2005; Pouri� and Trabalon, 2003). from the egg sac, the spiderlings were housed individually in
Bonaric ( 87) showed that fluctuations of ecdysteroids during 1-L plastic containers (16 cm � 8 cm� 8 cm) during the larval
the molting cycle in P. mirabilis are similar to those reported period. After the first juvenile molt, they were maintained in
for other arthropods. In female T. atrica, 20-hydroxyecdysone 8-L glass boxes (27 cm � 18 cm � 16 cm), before being finally
inhibits cannibalism during sexual activity and changes sex transferred to 14-L plastic containers (32 cm � 22 cm � 20 cm)
pheromone production (Trabalon et al., 2005). At present, there after the last juvenile molt (subadult instars). A 50:50 mixture
is no study carried out with male spiders. of potting soil and vermiculite was used for bedding.
In Crustacea, hemolymph metabolites, such as the levels of Spiders were bred at 23 ą 2ć%C, with 60 ą 10% relative
glucose (Telford,  68) and protein (Dall,  74), undergo changes humidity under a 12:12 hr photoperiodic cycle. Animals were
in correlation with the molt stage while they are undoubtedly fed ad libitum with a standardized diet of Tenebrionidae larvae
related to metabolic changes associated with metamorphosis. (Zophobas morio) and larvae or adult Blattidae (Blabera fusca,
There is very little information available on metabolites (lipids, Pleriplaneta americana). These preys were selected according to
proteins, and glucides) of hemolymph in spiders. Cohen ( 80) the suitability of their body size for the experimental animals at
analyzed the chemical composition of the hemolymph of two the different developmental stages.
species of araneid spiders, and Punzo ( 89) for six species of For each individual spider, the number of juvenile molts until
lycosid spiders. All these analyses were conducted on pooled the imaginal molt, the duration of individual instars, and the
samples of hemolymph. The changes in hemolymph proteome time of death were recorded by checking the spiders at 1 2 days
of mygalomorph Brachypelma albopilosum females were exam- interval. The sex of the animals could be checked only in adults.
ined for the first time in relation to their developmental stage The two characteristics of adult males,  mating spurs on the
(subadult and adult period) by Trabalon et al. (2010). Recently, tibia of leg I and the  palpal organs at the end of pedipalp tarsi,
Trabalon (2011) analyzed the lipid composition of hemolymph were apparent only after the imaginal molt. A fully differentiated
from B. albopilosum adult females with respect to agonistic be- epigyne appears in adult females.
J. Exp. Zool.
BIOCHEMICAL STATE IN TARANTULA 3
Weight and Body Size antiserum AS4919 was used (Porcheron et al.,  89). It presents
Morphological measurement and hemolymph sampling were approximately an equal affinity for 20-hydroxyecdysone and
performed after each molt in the same spiders, in the fourth ecdysone. In routine experiments, calibration curves were gen-
(A-4), the third (A-3), the second (A-2), and the first (A-1) in- erated with 20-hydroxyecdysone (4,000 31.2 fmol/tube). Dried
star before the final instar (A or adult stage). In order to avoid samples were resuspended in EIA sample buffer and quantified
any change in hemolymph composition related to the molt- in duplicate.
ing cycle (Stewart and Martin,  70; Collatz and Mommsen,  75),
Quantification of Total Lipids and Cholesterol
groups were sampled in the intermolt instars (30 ą 2 days after
Total lipids in plasma (50 �L) were measured with a colorimetric
each molt) and 24 hr after feeding. Spiders were anesthetized by
method (Atlas Medical, Cambridge, UK). This method converts
chilling at 4ć%C for 1 hr. Weight measurements were taken with
lipids, but not saturated fatty acids, to a pink-colored complex
a Sartorius electronic balance (Palaiseau, France) (ą0.01 g).
in the presence of sulfuric acid, phosphoric acid, and vanillin.
For determination of body volume, cephalothorax and
Results were expressed as an  index of total lipids.
abdominal sizes were measured with callipers (ą0.01 mm,
Cholesterol in 10 �L of plasma was determined using choles-
Manostat Corp., Basel, Switzerland). For thorax volume, the
terol/cholesteryl ester detection kit (Abcam, Cambridge, UK) us-
width at the widest part and the length from pedicel to the
ing a colorimetric method. In the assay, free cholesterol is oxi-
region between the eyes were recorded. For abdomen volume
dized by cholesterol dehydrogenase to generate NADH that re-
calculation, width at the widest part and length from the pedicel
acts with a sensitive probe resulting in strong absorbance at
to the region between the spinnerets were measured. The equa-
450 nm.
tion for a probate spheroid, V = 4/3 Ą (ab2)/8, where a = length
and b = width of the cephalothorax or abdomen (in mm), was
Quantification of Glucose
used for the final volume.
Concentration of glucose in a 10-�L plasma sample was deter-
mined using a colorimetric method after enzymatic oxidation in
Hemolymph Sampling
the presence of glucose oxidase (Glucose-test, Randox, Crumlin,
For hemolymph chemical composition, 40 spiders per sex were
Co. Antrim, UK). The hydrogen peroxide formed reacted in the
studied: for males, 5 spiders that molted 8 times, 5 that molted 9
presence of peroxidase with phenol and 4-aminophenazone to
times, 25 that molted 10 times, and 5 that molted 11 times; for
form a red violet quinoneimine dye as indicator.
females, 5 spiders that molted 9 times, 25 that molted 10 times,
5 that molted 11 times, and 5 that molted 12 times.
Quantification of Proteins
After weight and body measurements, the pericardium of
Protein content in a 10-�L plasma sample was determined
each spider was punctured with a needle, and 200 �L of
according to the method of Bradford ( 76) with a Coomassie
hemolymph was withdrawn in the presence of sodium citrate
(Bradford) protein assay kit (Thermo Scientific, Cergy Pontoise,
buffer pH 4.6, using a positive displacement pipette. The whole
France) using bovine serum albumin as the standard.
sampling procedure lasted less than 20 sec. This brief period of
handling precluded induction of an endocrine-dependent stress Statistical Analysis
response that would influence levels of hemolymph metabolites Chi-square analyses were used to compare the maturation rates
during the time of sampling. All spiders survived the anesthesia of the different stages and the sex ratios. Since all data were
and hemolymph sampling procedures. Hemocytes were removed normally distributed, group differences were determined using
by centrifugation at 10,000 rpm for 10 min at 4ć%C and the result- a two-way ANOVA (factor sex � factor instar). Specific mean
ing plasma was aliquoted for further analyses of ecdysteroids, comparisons were then made using t-test with Bonferroni cor-
total lipids, cholesterol, glucose, and proteins. rection. Statistical level of significance was P d" 0.05. Results
were expressed as means ą SE.
Quantification of Ecdysteroid Levels
RESULTS
Ecdysteroids from 50 �L of plasma were extracted twice with
1 mL of methanol. After centrifugation, the pellets were dis- Sex ratio, Mortality, and Number of Juvenile Molts
carded and the combined supernatants were evaporated to dry- Of the 500 young spiders studied until adult instars, 37% (n =
ness. Ecdysteroids were detected with an enzyme immunoassay 186) were female, 25% (n = 123) were male, and 38% (n = 191)
(EIA) adapted from the method described by Porcheron et al. died before reaching the imaginal molt and sex determination.
( 89), by using goat anti-rabbit IgG (Jackson Immunoresearch Total mortality was 60% per egg sac and half of the deaths were
Lab) and 2-succinyl-20-hydroxyecdysone coupled to peroxidase associated with exuviations. The spiders remained sometimes
as the enzymatic tracer. Enzymatic activity was measured us- attached to their old cuticle and died 1 2 days later. Mortality
ing ortho-phenylene-diamine (Sigma, Saint-Quentin Fallavier, was the greatest at the 3rd juvenile molt and was greatly reduced
France) as substrate. The polyclonal anti-20-hydroxyecdysone 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 Table 2. Duration of development (expressed in days and years,
(n = 123) and female (n = 186) reaching the adult stage, scored af- mean ą SE) of B. albopilosum from first juvenile molt until the
ter the 5th to 13th juvenile molts. The female and male frequencies imaginal molt. The durations of the stages were compared between
were compared using �2 test. males and females using ANOVA test.
Adult males Adult females Males (n = 123) Females (n = 186)
Molts Number Frequency (%) Number Frequency (%) Duration of stages Days Years Days Years
5th 0 0 0 0 Juvenile stage (first to last juvenile molt) with
6th 0 0 0 0 8 molts 1,474 ą 24 4.04 - -
7th 0 0 0 0 9 molts 1,598 ą 22 4.38 1,814 ą 33* 4.97
8th 33 26.83* 0 0 10 molts 1,523 ą 24 4.17 1,818 ą 22* 4.98
9th 41 33.33NS 40 21.50 11 molts 1,583 ą 26 4.34 1,878 ą 31* 5.14
10th 25 20.33* 68 36.56 12 molts 1,567 ą 22 4.29 1,758 ą 24* 4.82
11th 15 12.20* 64 34.41 13 molts - - 1,804 ą 20 4.94
12th 9 7.31NS 10 5.38 Mean 1,548 ą 24 4.24 1,814 ą 26 4.97
13th 0 0NS 42.15
Subadult stage (last juvenile molt to imaginal molt) reached after
8 molts 272 ą 28 0.74 - -
*P < 0.05; NS, not significant.
9 molts 195 ą 22 0.53 338 ą 33* 0.93
10 molts 269 ą 21 0.74 216 ą 25* 0.59
11 molts 167 ą 39 0.46 232 ą 33* 0.64
12 molts 253 ą 22 0.69 357 ą 22* 0.98
The number of juvenile molts before the adult molt varied
13 molts - - 209 ą 25 0.57
along with sex and individual spiders (Table 1). The frequency
Mean 231 ą 22 0.63 314 ą 28 0.86
of adult molt after the 8th, 10th, 11th, and 13th juvenile molts
was different between males and females (�2 = 3.64 33.64, P =
Total juvenile imaginal stage with
0.003). The adult molt occurred with the highest frequency after
8 molts 1,746 ą 19 4.78 - -
the 10th and 11th juvenile molt in females (71% of females, n =
9 molts 1,793 ą 19 4.91 2,152 ą 33* 5.90
132/186), and after the 8th to 9th juvenile molt in males (60%
10 molts 1,792 ą 22 4.91 2,034 ą 29* 5.57
of males, n = 74/123).
11 molts 1,750 ą 18 4.79 2,110 ą 35* 5.78
12 molts 1,820 ą 22 4.99 2,115 ą 21* 5.79
Duration of Development Instars
13 molts - - 2,013 ą 31 5.51
There was no significant intrasex difference for the duration of
Mean 1,792 ą 22 4.91 2,084 ą 29 5.71
the juvenile period whatever the number of molts in males (F =
Bold values represented duration mean of development whatever the num-
2.30, P = 0.06) and in females (F = 1.49, P = 0.21) (Table 2).
ber of molt.
However, the duration of the juvenile instars and the time until
*Significantly different between sex at P < 0.0001.
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
(14.4 ą 0.2 g) than those that reached it after 8, 9, or 10 in-
with 8 12 juvenile molts (Fig. 1A and Table 2). Adult molts
stars: 13.1 ą 0.4 g (F = 2.25, P = 0.05). After the last juvenile
generally occurred in the laboratory from August to October.
molt, leading to subadult instars (A-1), the body size (weight and
Females required between 5.51 to 5.90 years until the adult
volumes) was significantly different between males and females
molt (Fig. 1B and Table 2). The number of juvenile molts ranged
(F = 28.93, P < 0.0001). Subadult and adult females were thus
from 9 to 13, and adult molts occurred during July to October.
significantly heavier than males (F = 4.90, P < 0.001), i.e.,
14.5 ą 0.5 and 17.5 ą 0.7 g for females compared to 12.6 ą
Mass/Size
0.4 and 13.7 ą 0.6 g for males (Fig. 2). Concerning the size of
The body size increased with each instar until the adult molt
cephalothorax and abdomen, there was also a significant sex
in males and in females. Body weight and the volume of
difference after the A-2 molt (Fig. 3). In subadult (A-1) and
cephalothorax and abdomen increased with the same pattern
in juveniles from both sexes (Figs. 2 and 3). The weight of fe- adult instars (A), females were significantly larger than males
(F = 2.89 3.98, P = 0.05 0.01). The volume of cephalothorax
males that reached adulthood after 9, 10, 11, 12, or 13 instars
and abdomen of adult females and males did not vary whatever
was not different, i.e., 17.4 ą 0.7 g (F = 1.32; P = 0.12). Males
the number of juvenile instars.
that reached adult stage after 11 or 12 instars were heavier
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 of spiders (F = 4.91, P = 0.001). In females, ecdysteroids levels
The hemolymph ecdysteroid titers in females and in males that were constant about 7.44 ą 1.01 ng/mL in all instars checked
reached adulthood after a different number of instars were not about. In juvenile males, ecdysteroid levels were rather constant
different (F = 1.30 and 1.49, P = 0.17 and 0.21). until A-2 instar: 5.68 ą 0.87 ng/mL. They increased signifi-
Figure 4 shows that the hemolymph ecdysteroid titers, mea- cantly afterwards to 12.38 ą 1.84 ng/mL in A-1 (subadults), and
sured 30 days after each molt, varied significantly with the sex remained stable in A (adults) at 11.22 ą 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 in the hemolymph increased significantly after each instar. The
The level of lipids and cholesterol in females and males that index of total lipids became significantly higher in males than in
reached adulthood after a different number of instars was not females in the A-2 instars (402.6 ą 46.5 vs. 260.3 ą 28.8 mg/L;
different (F = 0.63 1.59, P = 0.60 0.17). P < 0.05) and until the final instar. The level of cholesterol was
Figure 5A, B shows that the index of total lipids (F = 37.68, significantly higher in males than in females only in the final
P < 0.0001) and the level of cholesterol (F = 8.16, P = 0.001) instars (9.45 ą 0.35 vs. 4.65 ą 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 glucose level in hemolymph increased significantly after
The levels of glucose and proteins in females and males that each molt in males (Fig. 6A; F = 9.11, P = 0.01). Males had
reached adulthood after a different number of instars were not a significantly higher level of glucose than females during the
significantly different (F = 0.73 1.44, P = 0.26 and 0.0.57). 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 ing mass, and the number of juvenile instars was dependent on
significantly during development (0.07 0.09 g/L; P = 0.09) and the sex of the spider. Thus, B. albopilosum reached adulthood
was significantly lower than in males. after 8 12 instars for the males and 9 13 instars for the females,
The level of proteins (Fig. 6B) increased significantly un- and needed approximately 5 6 years to mature from the first lar-
til the A-1 instar (subadult instars) in both sexes (F = 14.05, val instars. A low growth rate in juvenile arthropods increases
P = 0.001): 16 44 g/L in females and 13 46 g/L in males. The the duration of each juvenile instar, and is associated with an
protein level was not significantly different between males and increased risk of prereproductive mortality (Higgins and Rankin,
females at adulthood (F = 1.42, P = 0.34). 2001).
During juvenile development of B. albopilosum, there is an
expansion of biomass and of all its constituents. The growth
DISCUSSION-CONCLUSION
curves of males and females were similar until the last juve-
Sex ratio, Mortality, and Development
nile instar (subadult instars). The size of adults after the imag-
Our results have shown that only 40% of the young spiders
inal molt was not correlated to the number of juvenile molts
B. albopilosum survived and developed until the adult stage un-
in a large part in B. albopilosum, and slowly growing females
der environmentally controlled conditions (319 adults/500 larval
passed through several instars and reached maturity at a larger
spiders). Males represented only 40% of the surviving adults. The
size than rapidly growing males. Thus, adult females may reach
proximal physiological causes of this mortality are unknown at
5 6 cm in cephalothorax abdomen length and weigh 14 17
the present time. Death occurred either just before molting or
g, the males being somewhat smaller (4 5 cm in length and
during ecdysis. Approximately one-half of the animals died be-
13 14 g in weight).
fore any external sign of ecdysis initiation had appeared; the
remainder died stuck in their exoskeleton or once emerged from Hemolymph Chemical Composition and Ecdysteroids
ecdysis with extremely distorted legs. Hemolymph chemical composition from spiders that reached the
Brachypelma albopilosum presented a slow growth and de- adult stage after a different number of molts (8th, 9th, 10th, 11th,
velopment as shown in other spiders, Nephila clavipes (Higgins, 12th, or 13th) was similar. The number of molts did not influence
2000). Males should have normally one or two fewer molts than the chemical composition.
females, but this was not an absolute rule. The duration of each Our results showed that the concentration of lipids increased
juvenile instar was negatively correlated with the rate of increas- 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 tural components, and as hormones (Stanley-Samuelson et al.,
difference as the level of lipids was higher in male spiders af-  88). Lipids present in the hemolymph of adult B. albopilosum
ter the A-3 instar: 730 mg/L of lipids in adult males compared females have been identified (Trabalon, 2011) as hydrocarbons,
to 600 mg/L in females. Males have not been studied previ- four free fatty acids (palmitic, linoleic, oleic, and stearic acids),
ously but comparable values have been obtained in adult female and cholesterol. Lipid levels are hypothesized to have evolved
spiders (Araneus gemma and Argiope trifasciata: 580 and 410 as a regulatory factor of predation and agonistic behaviors in
mg/L, respectively; Cohen,  80). Lipids are a compact form of tarantula females. Trabalon (2011) showed that the female was
energy storage and thus the storage molecules of choice. They able to modulate her aggressive behavior according to the lev-
play key roles in insect biochemistry as sources of energy, struc- 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 as a constituent of cell membranes, and as a precursor to steroid
congeners. Tarantula males were less aggressive than females. hormones such as ecdysteroids.
Our results showed that all males presented levels of lipids signif- In our study, we report for the first time the variation of
icantly higher than the females. We showed here that cholesterol hemolymph ecdysteroids in a batch of male and female spiders
levels varied as did total lipid levels with the sex of the spider during juvenile development. Ecdysteroid concentrations in fe-
after the imaginal molt: they were higher in males (9 mg/L) than males were constant during the successive stages of development
in adult females (5 mg/L). Cholesterol plays an important role in (7 ng/mL). In juvenile males, ecdysteroids were at the same level
arthropod physiology as component of cuticular surface waxes, 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. show different exploratory behaviors: females are sedentary and
During these instars, our results showed that males possessed males wander. The hemolymph proteome of B. albopilosum has
also higher levels of lipids and cholesterol than females. These been previously analyzed only in females and consisted mainly
differences can be bound to the fact that B. albopilosum males of hemocyanin, actin, and another protein of unknown function
do not molt any more in adulthood, while in adult females, a (Trabalon et al., 2010). The putative corresponding structures of
molt occurs once a year throughout the survival period, as in these proteins are the coagulogen protein and/or lipoproteins for
other mygalomorph spiders (Stradling,  78; Miyashita,  92). But which quantitative differences between adult and subadult spi-
these differences can also be connected to the fact that after ders could be related to the molting process and protein compo-
the imaginal molt, males display spermatogenesis, then transfer, sition according to the developmental stage. Within the frame-
and store the sperm in their pedipalp organs, before searching work of future studies, it would be thus interesting to analyze the
for a female mate. In B. albopilosum, we observed that males distribution of these various protein constituents (lipoproteins,
loaded their pedipalps with sperm 30 days after the imaginal hemocyanin, and actin) according to the sex and the exploratory
molt; so spermatogenesis should have begun before, some days behavior of the adult spider.
after this molt, when high levels of ecdysteroids were present.
Ecdysteroids could promote spermatogenesis in male spiders, as CONCLUSION
has been shown in many species of insects (Hagedorn,  85) and in Our results showed that males had a higher metabolic reserve
ticks (Zhang et al.,  95). Ecdysteroids should have a stimulatory than females as observed in other spiders (Tanaka and Ito,  82;
role on early spermatogenesis involving mitoses and meioses. Watson and Lighton,  94; Shillington, 2005). Some of the sexual
After sperm storage, male spiders are involved in an intense dimorphism can be explained by size differences, between males
mate-searching activity under natural conditions. In this polyg- and females, and behavior. Brachypelma albopilosum spiders
ynous mating system, costs of locomotion are high and adult are an excellent model for addressing questions in evolution-
males have higher energy life styles than females, as has been ary physiology because of variations in performance (locomotor
shown in other tarantulas (P�rez-Miles et al., 2005). Glucose is activity) and life history of the adult animals. The higher levels
the primary energy source for arthropod and vertebrate tissues. of energy-giving compounds (lipids and glucose) could be an
Its concentration in subadult and adult males was higher (0.10 adaptive strategy to support higher energy demands for males
0.12 g/L) than in juvenile males and females. These differences during their active, locomotor search for females. Additional
could be explained by a higher requirement of energy in males work is needed to attest a correlation between behavior and
linked to a higher metabolism in connection with sexual activity. variation of ecdysteroids and metabolic constituents.
In B. albopilosum females, our results showed a low level
of lipids, cholesterol, and ecdysteroids in comparison to postju- ACKNOWLEDGMENTS
venile males. They were unmated and we observed that their We would like to thank Prof. Simon N. Thornton and Fran�oise
ovaries were in the previtellogenic phase of oocyte develop- Joubaud for reading the manuscript; Annick Maria (UPMC, Paris)
ment (M. Trabalon, unpublished data). In female spiders of other for technical assistance in ecdysteroid determinations; Jo�lle
species such as C. terrestris and Tegenaria sp., the highest lev- Couturier and Jean Charles Olry for animal maintenance (Uni-
els of total circulating ecdysteroids were detected during the versity of Nancy I).
vitellogenic phases of oocyte development (Trabalon et al.,  92,
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