Table Of Content2001. The Journal of Arachnology 29:388-395
MONOAMINES IN THE BRAIN OF TARANTULAS
(APHONOPELMA HENTZI) (ARANEAE, THERAPHOSIDAE):
DIFFERENCES ASSOCIATED WITH MALE
AGONISTIC INTERACTIONS
Fred Punzo: Dept, of Biology, Box 5F, University of Tampa, 401 W. Kennedy Blvd.,
Tampa, Florida 33606 USA
Thomas Punzo: 2782 Johnson Dr., Albuquerque, New Mexico 50052 USA
ABSTRACT. Experiments were conducted to determine the effects of male-male agonistic encounters
on changes in monoamine neurotransmitter concentrations in the supraesophageal ganglion (brain) ofthe
tarantula, Aphonopelma hentzi. Serotonin levels were significantly reduced 30 min after fighting in both
dominant (66.5 ±9.1 SE nmol/mg protein) and subordinate (42.8 ± 7.6) animals as compared to isolated
controls (89,7 ± 13,2), and these differences persisted for up to 24 h. A similar decrease was found for
octopamine concentrations in dominant (43.7 ± 7.7) and subordinate (31.2 ± 4.9) spiders when compared
to controls (56.9 ± 5.8). In addition, serotonin and octopamine levels were significantly lower in subor-
dinate vs. dominant spiders. Agonistic interactions had no effect on the concentrations of dopamine,
norepinephrine, and epinephrine. In isolated control spiders, serotonin (89.7 ± 13.2 SE nmol/mg protein)
was present in highestconcentration in thebrain, followedby octopamine (56.9 ±5.8 nmol/mg), dopamine
(22.4 ± 3.8 pmol/mg), norepinephrine (15.3 ± 4.7 pmol/mg), and epinephrine (0.57 ± 0.2 pmol/mg). The
results indicate that following agonistic encounters, monoamine concentrations in the brain decrease to
different levels in winners and losers. This is the first demonstration that the establishment ofsocial status
causes changes in brain monoamines in spiders.
Keywords: Agonistic interactions, Aphonopelma, CNS monoamines, males
Activation of monoaminergic systems in ganglion (SEG) of arthropods lies above the
the central nervous system (CNS) has been esophagus and consists of three major brain
implicated in the mediation of short-term and regions: the protocerebrum, which contains
chronic physiological stress responses as well the ‘higher brain centers' including the optic
as aggressive and social dominance relation- lobes and nerves; the central body, corpora
ships in numerous taxa (Eichelman 1987; pedunculata and a variable number of ganglia
Bicker & Menzel 1989; Haney et al. 1990; and associated neuropils; the deuterocere-
Summers et al. 1995). Most of this work has brum, which innervates the antennae (reduced
focused on social interactions in vertebrates in arachnids); and the tritocerebrum, which in-
exposed to staged encounters between con- nervates the mouthparts (Horridge 1965; Gup-
specifics under laboratory conditions. For ex- ta 1987). In spiders, the CNS is highly con-
ample, the social status ofsubordinate animals densed anteriorly, and the SEG consists
is associated with an increase in the utilization primarily of the cheliceral and optic nerves,
of the indolalkylamine neurotransmitter (NT) optic neuropils (masses of axonal fiber tracts)
serotonin (5-HT, 5-hydroxytryptamine) in var- for primary and secondary eyes (‘corpora pe-
ious brain regions from fish to mammals (Ha- dunculata’), and a loosely organized central
&
ney et al. 1990; Winberg et al. 1997; Punzo body (Babu 1985; Wegerhoff Breidbach
2000a). In addition, activation of brain cate- 1995).There have been few studies on neuro-
cholaminergic systems including dopamine chemical parameters associated with behavior
(DA), norepinephrine (NE), and epinephrine in general, and agonistic interactions specifi-
(Epi) in vertebrates is associated with in- cally, in arthropods and other invertebrates.
RNA
creased levels of aggression and dominance For example, increases in protocerebral
(Eichelman 1987; Matter et al. 1998). and protein synthesis have been shown to ac-
In general, the brain or supraesophageal company learning in molluscs (Kerkut et al.
388
—
PUNZO & PUNZO—TARANTULA BRAIN MONOAMINES 389
1970; Adamo & Chase 1991), decapod crus- trations of acetylcholine (ACh), norepineph-
taceans (Punzo 1985), insects (Lin & Roelofs rine (NE), and acetylcholinesterase (AChE) as
1992; Punzo 1996), and spiders (Punzo compared to younger instars (Punzo 1993).
1988a). Cycloheximide-induced inhibition of Theraphosid spiders exhibit a variety ofag-
brain protein synthesis impaired learning and gressive behaviors. Some of these involve
memory in insects (Jaffe 1980) and spiders male-male agonistic interactions (Baerg 1958;
(Punzo 1988a), as well as innate phototactic Minch 1977; Punzo & Henderson 1999; Pun-
behavior in tenebrionid and passalid beetles zo 2000b), while others involve males and fe-
&
(Punzo Jellies 1980). Changes in levels of males, especially during courtship and mating
brain monoamines have been implicated in a (Costa & Perez-Miles 1992; Shillington &
variety of ontogenetic shifts in behavior in Verrell 1997), and sometimes end in sexual
honeybee workers including the onset ofnest- cannibalism (Punzo & Henderson 1999). A
guarding behavior (Moore et al. 1987) and previous study showed that agonistic interac-
discrimination between olfactory cues (Mac- tions between paired conspecific males of
millan & Mercer 1987). Aphonopelma hentzi (Girard 1854) were ob-
With respect to aggression and agonistic in- served in 24 out of 27 (88.9%) staged en-
teractions, octopamine turnover rates in- counters in the laboratory (Punzo & Hender-
creased significantly in crickets after fighting son 1999). These encounters were initiated by
with conspecifics (Adamo et al. 1995). In- vigorous leg-fencing with each protagonist
creased foraging activities and nest defense pushing forcefully against its opponent. These
were correlated with higher concentrations of fencing bouts were interrupted from time to
octopamine (OA), dopamine (DA), and sero- time with at least one of the males exhibiting
tonin (5-HT) in the SEG ofworker honeybees a threat display (elevation of the anterior end
(Harris & Woodring 1992). Indeed, ithas been of the body, first pair of legs and pedipalps,
OA
suggested that is part of a general arousal and opening of the fangs). At least one male
system which prepares insects for a variety of exhibited a strike response toward his oppo-
vigorous skeletal-muscular activities, territo- nent in 11 out of 24 cases (45.8%). In eight
rial defense, and helps the animal deal with of these instances (33.3%), one of the males
stressful conditions (Corbet 1991; Orchard et was killed.
al. 1993). Increased 5-HT levels in the brain The purpose ofthis study was to investigate
(SEG) have been implicated in the onset of neurochemical parameters associated with ag-
flight behavior in weevils (Guerra et al. 1991). onistic interactions between males ofthe ther-
Lobsters exhibiting dominance over conspe- aphosid spider, Aphonopelma hentzi. Neuro-
cifics exhibited higher levels of CNS 5-HT transmitters and comodulators are important
when compared with subordinate animals regulatory molecules required for the trans-
(Kravitz 1988). mission ofinformation (nerve impulses) along
Changes in SEG amine concentrations as neural pathways involved in the control of
well as otherNTs were shown to be associated motor movements as well as 'mood’ and mo-
&
with ontogenetic shifts in behavior in solifug- tivational states (Ansell Bradley 1973).
ids (Punzo 1993, 1994). First nymphal instars Specifically, we were interested in whether or
(Nl) typically have poorly developed chelic- not there were any differences in the concen-
erae, and are gregarious, do nothuntprey, and trations of monoamines (OA, DA, NE, Epi,
remain in the nest with their siblings and ma- 5-HT) in the brains (SEG) ofdominant (‘win-
ternal parent. However, after molting, second- ners’) vs. subordinate (‘losers’) males follow-
instar nymphs (N2) possess functional chelic- ing agonistic encounters. To our knowledge,
erae and become aggressive (Punzo 1998a). this is the first study to address neurochemical
They will cannabilize one another if they do correlates of aggression in spiders.
not disperse from the nest. This pronounced METHODS
increase in aggression is associated with sig-
nificant changes in brain 5-HT and DA levels, Animals. Males were collected during
although OA levels remained relatively con- July and August of 1997 at a site 3.5 km S of
stant throughout postembryonic development Elgin, Texas (30°32^N, 97°29'W; Bastrop
(Punzo 1—994). In addition, later nymphal in- County). This site consisted ofa dry wash and
stars (N5 N8) exhibited higherbrainconcen- surrounding flood plain consisting of sand.
390 THE JOURNAL OF ARACHNOLOGY
gravel and adobe soils, with numerous rocks, attempt to flee after making initial contact
rock crevices, and burrows. The dominant with its opponent. These trials were not used
vegetation included prickly pear cactus in data analysis. In all other cases {n = 60
(Opuntia), catclaw {Mimosa), mesquite {Pro- trials), following initial contact, one ofthe spi-
sopis), broom weed {Xauthocephalum), and ders would begin to push with its front pair
mesquite grass {Bouteloua), Adult males rang- of legs against its opponent. The other spider
ing in size from 4.2-6.7 g were abundant dur- rapidly responded in a similar fashion (leg-
ing this period and were easily found moving fencing). In other cases, after initial contact,
about the surface between 2000-0300 h (Cen- one or both spiders would exhibit the threat
tral Standard Time). Spiders were collected display, followed by another bout of leg-fenc-
and weighed to the nearest 0.1 g using a ing. In a few instances the fighting escalated
Ohaus Model 87 portable electronic balance. until one or both spiders attempted to bite the
Spiders were transported to the laboratory other. An encounter trial was terminated if at
and housed individually in plastic cages (20 any time during the encounter one of the spi-
X 16 X 8 cm). They were provided with water ders backed away and rapidly fled from the
ad libitum and fed three times per week to vicinity of the other spider and attempted to
satiation on a mixed diet of crickets {Gryllus crawl out ofthe chamber. The spider that held
sp,), mealworms {Tenebrio molitor), and its ground was recorded as the 'winner’ (dom-
grasshoppers {Schistocerca sp.). They were inant animal), and the spider that fled, the
maintained at 22 °C ± 1°, 65% RH, and a Toser’ (subordinate). Each pair of contestants
photoperid regime of 12L:12D in a Percival were subjected to only one encounter trial as
Model 805 environmental chamber (Boone, described by Summers & Greenberg (1995) in
Iowa). Adult males were kept in these condi- their study ofmale-male aggression in lizards.
tions for two weeks and then re-weighed on We conducted a total of 60 encounter trials
the day before the initiation of encounter tri- comprising 60 pairs of contestants {n = 120).
als. Since previous studies on arachnids have Ne—urochemical analyses of brain tis-
indicated that differences in body size can in- sues* Immediately following their designa-
fluence the outcome of aggressive encounters tion as dominant or subordinate (based on the
(Faber & Bayliss 1993; Punzo 1998c, 2000b), outcome of encounter bouts), paired contes-
only males ofapproximately similar size (6.2- tants were randomly assigned to one of three
6.7 g) were used for encounter trials and sub- groups; each group consisted of40 spiders (20
sequent neurochemical analyses. Voucher pairs). Spiders in group 1 (Gl) were anaes-
specimens have been deposited in the Inver- thetized with CO thirty min after encounter
2
tebrate Collection at —the University ofTampa. trials, and their brains (SEG) removed in a
Encounter trials. We used a rectilinear cold room, weighed to the nearest 0.1 g on an
glass arena (26 X 16 X 12 cm) divided into electronic analytical balance, and frozen at
halves by an opaque divider to stage conspe- -80°C as described by Punzo (1988b) for
cific male encounters as described by Punzo subsequent neurochemical analyses. Group 2
(1998c). To summarize, the floor of the arena (G2) and group 3 (G3) spiders were anaesthe-
was provided with a layer of loose sand to a tized and their brains frozen at 24 hr and 48
depth of 2 cm. All observations were con- h, respectively, after encounter trials. In this
ducted under Black lighting (BioQuip One., way, we were not only able to determine what
Model 2804, Gardena, California). We used a neurochemical changes, if any, followed
Panasonic PS 150 tape recorder to record ver- male-male aggression, but also how rapid the
bal descriptions of each encounter. response might be, and how long these chang-
Before each encounter trial, a male spider es might persist. The brains from another
(chosen at random) was placed at each end of group of 20 spiders (G4) maintained in iso-
the arena, separated by the opaque divider. A lation and notexposed to encountertrials were
trial was initiated by removing the divider and used as controls.
allowing the animals to interact. Within a pe- After thawing, all glandular and peripheral
riod of time ranging from 0.5-8.5 min over fatty tissue was carefully removed from the
all trials, the contestants made contact with surface of the SEG (Murdock & Omar 1981).
one another (usually with a front leg). In afew The SEG were then weighed to the nearest
trials, one of the spiders would immediately 0,01 g on a Sartorius Model 54C electronic
PUNZO & PUNZO—TARANTULA BRAIN MONOAMINES 391
analytical balance. Brain protein determina- weights and brain protein values between
tions were conducted using the standard pro- dominant, subordinate or isolated control spi-
cedure described by Lowry et aL (1951) and ders {P < 0.5), Serotonin was the monoamine
expressed as percent (brain protein/brain found in the highest concentration in the
weight) (Meyer et al. 1984). The SEG from brains of isolated control of A. hentzi (Table
the dominant and subordinate spiders were an- 1), This was followed in decreasing order by
alyzed to determine the concentrations of the OA, DA, NE, and Epi.
monoamine eeurotransmitters, 5-HT, OA, DA, The effects of agonistic interactions be-
and NE, using high performance liquid chro- tween coespecific males on SEG monoamine
matography with electrical detection (HPLC- concentrations at various time intervals fol-
ED, Beckman Model 47A) as described by lowing encounter trials are shown in Table 1.
Brandes et al. (1990). To summarize, each Serotonin (5-HT) levels were significantly re-
brain tissue sample was placed in a 750 pi duced in spiders losing aggressive encounters
glass vial and homogenized in 50 pi of a 200 (subordinates) for up to 24 h as compared to
mM perchloric acid (PA) solution. Following dominant animals (t = 9.4, P < 0,01). This
homogenization, an additional 50 pi of PA difference persisted for at least 24 h, with lev-
were added to each vial. Samples were then els returning to normal after 48 h (F = 3.36,
centrifuged at 10,000 g and 4 °C for 3 min in P < 0.05). In addition, the brains ofdominant
a Sorvall Model 100A high speed refrigerated spiders contained significantly lower levels of
centrifuge. Twenty pi of supernatant were in- 5-HT than those of the isolated controls (t —
jected directly into the HPLC column (40 cm 6.8, P < 0.05) for up to 24 h. These changes
in length, with a 0.2 p pore diameter) packed in 5-HT and OA levels associated with fight-
with Hypersil and provided with a Hewlett- ing occurred quite rapidly since changes were
Packard 760E detector (0.40 V). The mobile detected after only 30 min following an en-
phase (flow rate, 3000 psi) used to elute the counter.
monoamines consisted of 12% acetonitrile, 20 A similar pattern was found for OA levels
mM mM
sodium acetate, 100 sodium dihy- which were also significantly reduced in sub-
drogen orthophosphate, 2.5 mM octane sul- ordinate vs. dominant animals (t = 6,2, P <
fonic acid, and 0.3 mM EDTA disodium salt 0.02). However, the reduced levels of OA as-
adjuested to pH 4.2 and filtered through a 0.45 sociated with agoeisitic interactions returned
pm filter. Each sample was compared to 5-HT to control levels within 24 hr. With respect to
and DA standards tested at the beginning of DA, NE, and Epi, no differences were found
each assay run and retested at 30 min inter- between spiders exposed to agonistic encoun-
vals. Monoamine concentrations were ex- ters and controls at any time interval {P <
pressed as nmol or pmol/mg protein as des- 0.5).
ribed by Meyer et al. (1984).
DISCUSSION
All statistical procedures followed those de-
scribed by Sokal & Rohlf (1995). Compari- Although the profile for monoamine con-
sons between mean concentrations of mono- centrations in the SEG ofA. hentzi (5-HT >
amine NTs for the various groups were OA > DA > NE < Epi) is in general agree-
conducted using an analysis of variance (AN- ment with what little information is available
OVA), followed post-hoc by a Duncan’s mul- on the neurochemistry of spiders, differences
tiple range test at a significance level of0.05, in the NT profiles for spiders from different
Significant differences between dominant and families have been reported (Florey 1967;
subordinate males following aggressive en- Meyer et al. 1984; Meyer 1991) Similar con-
counters were determined using an indepen- centrations were reported for NE and DA
dent-samples t test {P < 0.05), from the brain ofthe theraphosid, Aphonopeh
ma eutylenum Chamberlin 1918, although no
RESULTS data were presented for 5-HT and OA (Meyer
Brain weights for all spiders ranged from et al. 1984). In contrast, the brain ofPardosa
8.98-9.32 mg (mean: 9.11 ± 0.56 SE). Brain amentata (Clerck 1932) contained much high-
protein/brain weight (%) ranged from 7.2-7.6. er concentrations of NE (174.9 pmol/mg ±
An analysis of variance (ANOVA) indicated 5.0 SE), a condition most likely associated
that there were no differences in mean brain with the noradrenergic system of the optical
392 THE JOURNAL OF ARACHNOLOGY
—
Table 1. Concentrations of various monoamines (in nmol or pmol/mg protein) in the supraesophageal
ganglia (SEG) ofAphonopelma hentzi following agonistic interactions between conspecific males. Brain
analyses were conducted from tissue extracted from isolated control spiders, as well as from the brains
of dominant and subordinant spiders removed 5 min (20 pairs; N = 40) 24 h {n = 20 pairs), and 48 h {n
— 20 pairs) after an encounter trial. Data expressed as means; values in parentheses represent (± SE).
Values followed by asterisks are significantly different than controls: ** (P < 0.01); * (P < 0.05). See
text for details.
Time after encounter
Neurotransmitter Controls 5 min 24 h 48 h
Serotonin (5-HT) (nmol/mg) 89.7 (13.2)
Subordinate 42.8** (7.6) 51.3** (8.3) 92.2 (12.6)
Dominant 66.5* (9.1) 73.9* (10.4) 86.3 (9.5)
Octopamine (nmoi/mg) 56.9 (5.8)
Subordinate 31.2** (4.9) 54.8 (8.1) 60.1 (10.6)
Dominant 43.7* (7.7) 55.3 (5.2) 57.8 (8.2)
Dopamine (pmol/mg) 22.4 (3.8)
Subordinate 19.6 (5.1) 23.6 (7.1) 21.9 (5.5)
Dominant 22.2 (7.8) 20.6 (3.5) 24.4 (6.2)
Norepinephrine (pmol/mg) 15.3 (4.7)
Subordinate 18.1 (5.8) 14.3 (2.9) 17.3 (6.6)
Dominant 16.6 (4.4) 18.5 (3.1) 14.9 (4.1)
Epinephrine (pmol/mg) 0.57 (0.2)
Subordinate 0.61 (0.1) 0.55 (0.2) 0.58 (0.1)
Dominant 0.52 (0.2) 0.63 (0.3) 0.56 (0.2)
brain centers which are more highly devel- activate endocrine stress mechanisms in ver-
oped in salticids (Meyer & Jehnen 1980). Ly- tebrates, which are thought to be mediated by
cosids and agelenids also contained higher changes in CNS neurotransmitters brought
levels of DA and NE as compared to thera- about primarily via activation of monoamin-
&
phosids, although not as high as salticids ergic systems (Ansell Bradley 1973; Ei-
(Meyer 1991). chelman 1987). Eor example. Summers &
The most pronounced changes in mono- Greenberg (1995) showed that 5-HT levels
amine levels involved 5-HT. They occurred decreased significantly after one h and one
among subordinate males 30 min after fight- day in the brains (diencephalon and non-optic
ing, although 5-HT levels were reduced in lobe midbrain) oflizards (Anolis carolinensis)
dominant males as well. Thus, fighting be- losing aggressive interactions. Similarly, no
tween male spiders resulted in a decrease in changes were detected for NE and DA levels
SEG serotonin levels. To our knowledge, this over this time interval. However, subordinate
DA
is the first demonstration ofan association be- males exhibited significantly lower levels
tween monoaminergic activity and aggression after one week than did subordinates after one
in spiders. Serotonin has been identified with h. Changes in the serotonergic content and
aggressive behavior in other arthropods as turnover between individuals of different so-
well. Injection of5-HT into lobsters and cray- cial status were found in the telencephalon
fish caused them to elevate and flex their tails, and diencephalon of territorial vs. satellite
which represent behavioral acts associated males in the lizard, Sceloporusjarrovi (Matter
with the expression of dominance (Yeh et al. et al. 1998). In addition, the levels of5-HT in
1996). the telencephalon and diencephalon were
These observations are interesting since a found to decrease significantly following
similar reduction in brain serotonin levels ac- male-male aggression in rodents (Haney et al.
companying fighting and territorial defense 1990; White et al. 1991) and fish (Winberg et
has been reported for a number ofvertebrates. al. 1997).
Indeed, it has been well established that a va- Immediately following aggressive defense
riety of stimuli, including social interactions, of territories, territorial male lizards {S. jar-
PUNZO & PUNZO—TARANTULA BRAIN MONOAMINES 393
rovi) exhibited higher Epi levels as compared with vertebrates is not possible since OA has
to males that did not experience aggressive not been identified as a NT in this group.
encounters (Matter et ah 1998). In contrast, no In conclusion, significant changes in brain
comparable changes in Epi levels in the SEG concentrations of 5-HT and OA result from
ofA. hentzi were observed after fighting. male-male agonistic encounters in tarantulas.
Octopamine levels also decreased signifi- It has been well established that 5-HT is a
cantly in males of A. hentzi exposed to ag- CNS monoamine involved in the expression
gressive interactions. This suggests that an ac- of dominance and aggression in vertebrates,
tivation of the octopaminergic system, in and the results of this study suggest that the
addition to serotonergic activation, follows establishment ofsocial status in spiders causes
aggression in spiders. This is not surprising changes in brain monoamine levels and may
since OA appears to be central in eliciting the play a role in the elicitation ofcommunicative
overall arousal response of arthropods (Krav- displays as well. Future studies should focus
itz 1988; Corbet 1991), and elevated OA ac- on other species of spiders as well as other
tivity has been shown to accompany stress arachnids in order to determine if similar
(Downer & Hiripi 1993; Harris & Woodring changes in monoamine profiles are associated
1992), increased locomotor activity (Orchard with aggression in these groups.
et al. 1993; Adamo et al. 1995), courtship ACKNOWLEDGMENTS
(Downer & Hiripi 1993), and a wide range of
systemic physiological responses including We are indebted to C. Bradford, R. Khatibi,
respiration, gastrointestinal peristalsis, cardio- M. Harvey, and R. B. Suter for commenting
acceleration, Malpighian tubule filtration, gly- on an earlier draft of the manuscript, B. Gar-
cogenolysis, and pheromone production (Cor- man for consultation on statistical analyses,
bet 1991) in insects. It has been further and J. Bottrell for assistance in the collection
suggested that certain behaviorpatterns canbe of specimens. A University of Tampa Faculty
triggered by the activation of specific octo- Development Grant and a Delo Foundation
paminergic pathways in arthropods, an idea Research Grant (DFRG-10-204) to FP made
known as the ‘orchestration hypothesis’ (Som- much of this work possible.
&
bati Hoyle 1984). For example, administra-
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