INTRODUCTION

As lobsters grow, they shed their rigid exoskeleton by molting. Just prior to the molt, when they are hard-shelled, lobsters become highly aggressive. Just after the molt when they are soft-shelled, lobsters become timid and tend to flee (Scrivener, 1971; Tamm and Cobb, 1978; Atema and Cobb, 1980; Cromarty et al., 1991).

20-HE rises and falls over the molt-cycle (Snyder and Chang 1991a, b), together with the changes in aggressive behavior. Snyder and Chang, 1991a,b have also reported high concentrations of 20-HE, and the subsequent ecdysteroid metabolites in the urine of American lobsters.

Our previous experiments have shown that blood from premolt lobsters enhance excitatory junction potentials (EJPs) and depress inhibitory junction potentials (IJPs) at the claw-opener neuromuscular junction (Schwanke et al., 1983, 1990).

20-HE also alters the neuromuscular properties of the claw-opener and phasic flexor systems in intermolt animals (Cromarty and Kass-Simon, 1998) in a way that is consistent with molt-related behavioral differences (Tamm and Cobb, 1978; Cromarty et al., 1991).

When 20-HE is injected into the lobsters' hemolymph, aggressive behavior during agonistic encounters increases sharply (Bolingbroke and Kass-Simon, 2001). These results indicate that one of the factors contributing to the heightened aggressiveness of lobsters is the molting hormone itself.

In addition to affecting the internal aggressive state of an animal, 20-HE may also be acting as a chemical signal during aggressive encounters.

This study addresses whether:

Exposure to 20-HE alters aggressive, defensive and avoidance behaviors

The lobster olfactory neurons detect 20-HE

Membrane receptors for 20-HE are present on the sensilla (ORN dendrites)

PHYSIOLOGY METHODS The lateral antennular filament was excised and inserted into a Teflon tube attached to an

olfactometer in which artificial seawater (ASW) was flowing at 10 cm/s.

The proximal end of the antennule was inserted into a separate recording chamber containing Homarus saline. Several annuli were removed from the cut proximal end of the filament, thus exposing both the antennular nerve and artery.

The artery was then cannulated, and the preparation was perfused with oxygenated Homarus saline delivered directly into the artery through a glass cannula at a flow rate of 0.4–0.8 ml/min (see Derby, 1995).

Chemical stimuli were injected into the ASW flowing over the aesthetasc sensilla by activation of an electronic valve. The electronic valve allowed for a determination of the beginning of the stimulation period and response latency. Each odorant trial was followed by an ASW injection wash followed by a 2 minute inter-trial period.

Single-unit responses were recorded extracellularly from the exposed axons of ORNs in the saline dish by using fine-tipped glass suction electrodes (tip inner diameter ~ 2–5 µm), coupled to a differential AC amplifier.

Activity was monitored on-line and recorded on a microcomputer in digitized form using a Powerlab

® interface and 5.4.2 software (AD Instruments). Spikes were sorted according to their

waveforms using Spike Histogram Software of the Chart 5.4.2 package (AD Instruments).

For each stimulus and neuron, the numbers of spikes during 2000-ms periods following initiation of the response were counted as measures of the response intensity.

Dose dependence was evaluated with regression analysis (Excel).

“Best cells” are defined as those ORNs that have a maximum response to a particular odorant.

BEHAVIOR METHODS• Two female intermolt adult lobsters, one exposed to puffs of either artificial seawater (ASW), -ecdysone, or 20-HE and the other deprived of antennule olfaction and exposed only to (ASW) (5ml/5 min) were allowed to fight in a staged 30 minute interaction. Thirty fights are carried out ASW (n=10), -ecdysone (n=10), 20-HE (n=10).

• Only animals in perfect physical condition are used in a fight, and each animal was used only once. To bias the fight in favor of the larger lobster, each fight contains one small and one large combatant, with a minimum of 10% difference in carapace length (Scrivener, 1971). The larger lobster receives puffs of the treatment substance in ASW, while the smaller lobster receives puffs of ASW only. Each lobster’s nephropores are capped with a polyethylene plug to prevent urine release.

• Puffs of treatment solution were delivered via a system of tubes glued as a backpack to the carapace of each lobster. Test substances were delivered directly onto the area immediately surrounding each lobster’s antennules.

• All fights were videotaped by a Sony camcorder suspended above the tank. Behaviors are then analyzed at a later time, according to a previously established ethogram (Mello et al., 1999; Bolingbroke and Kass-Simon, 2001).

• Behaviors were ranked based on the published ethogram according to their aggressive content and classified as aggressive, defensive, or avoidance. The more aggressive a behavior is, the higher its rank.

• Parameters measured for each animal, for each pair of combatants, and for each of the three types of treatments were:

Frequency - Total number of behaviors per 30 minute fight.

Average Rank (AR) – Calculated by multiplying the frequency of each behavior by its rank on the ethogram and dividing by the total number of observed behaviors.

SUMMARY AND CONCLUSIONSBehavior:

Exposure to 20-HE increases the overall activity level of the exposed animal such that the average rank of aggressive behaviors, the frequency of defensive and avoidance behaviors are increased.

The opponents of the exposed animals become more aggressive, performing more aggressive behaviors towards the exposed animal and more redirected wall behaviors.

Physiology:

• Our experiments indicate that perfusion of 20-HE over lateral antennule filaments causes large-scale excitation of individual olfactory receptor neurons (ORNs).

• ORNs respond to 20-HE in a dose-dependent manner. These ORNs are broadly tuned and respond to amino acids common in their diet. Other steroids produce significantly lower levels of excitation and are not dose-dependent.

Biochemistry:

Sufficient total protein could be extracted from the following tissues: walking legs (WL), eyestalks (EY), intact antennules (IA), shaved antennule stalks (ST), aesthetascs (IA) and guard setae (GS).

SDS-PAGE gels indicate that the extracted protein has a MW that approximates that of the drosophila ecdysone receptor.

Western spot blots demonstrate that the following tissues (walking legs, eyestalk, intact antennules and antennule stalks) label with the antibody against the drosophila ecdysone receptor (EcR).

Biochemical data suggest that intact antennules and antennule shaved stalks have the EcR–further experiments will determine whether the membrane of aesthetascs and guard setae express the ecdysone receptor.

Our studies indicate that 20-HE is able to be perceived, affects social interactions, modulates internal aggressive states and neuromuscular transmission in Homarus americanus. This suggests that 20-HE acts both as a lobster hormone and a pheromone.

ABSTRACTDuring fighting, lobsters urinate on each other. Blocking urine excretion obliterates established relationships (Breithaupt and Atema 1993, Breithaupt et al., 1999). Over the molt cycle blood and urine titers of the molting hormone 20-hydroxyecdysone (20-HE) vary. Concomitant with changes in hormone levels are changes in aggressive behavior. We have previously shown that 20-HE increases the internal aggressive state and modifies potentials at neuromuscular junctions (Cromarty and Kass-Simon 1998, Bolingbroke and Kass-Simon 2001).

Here we present evidence that 20-HE acting as a pheromone, reduces the inherent aggressiveness in intermolt animals. Agonistic encounters were staged in which the nephropores were blocked and artificial seawater (ASW) puffed onto the antennules of the smaller combatants whose antennules had also been desensitized. The antennules of the larger combatants were puffed with 20-HE, alpha ecdysone, or ASW. Animals exposed to 20-HE became significantly more defensive and more timid than animals exposed to alpha ecdysone or ASW. In experiments in which isolated antennule lateral filaments were perfused with 20-HE, alpha ecdysone and other steroid metabolites, only 20-HE increased olfactory receptor neuron excitation to levels comparable to those of food odorants, in a dose-dependent manner.

In biochemical experiments an antibody (DDA2.7) made against the ecdysone receptor (Talbot et al., 1993) was used to label tissue extracts from whole antennules, aesthetasc sensilla, guard setae, eyestalk and walking-leg muscle. Biotin-labeled pan-mouse secondary antibody and strepavidin-FITC was used to visualize antibody binding. Western and spot blots revealed significant labeling in eyestalks, walking-leg muscles, whole antennules, and aesthetasc sensilla.

These findings suggest that 20-HE is perceived by the aesthetasc sensilla, likely at a membrane bound receptor, which ultimately results in a significant modification of aggressive behavior.

PERCEPTION OF THE STEROID MOLTING HORMONE (20-HE) MODIFIES AGGRESSIVE BEHAVIOR IN THE AMERICAN LOBSTER, Homarus americanus.

Stuart Cromarty1, Debra Coglianese2 Lenore Martin3 and G. Kass-Simon2

1Department of Natural Sciences, Assumption College, Worcester, MA. 2Department of Biological Sciences, University of Rhode Island, Kingston, RI. 3Cell and Molecular Biology, University of Rhode Island, Kingston, RI.

Rank of Aggression Ethogram

140 Meral Spread

Behaviors are divided into three categories – aggressive, defensive, or avoidance. Behaviors are ranked according to the degree of aggressiveness, with the most intensely aggressive behaviors given the highest rank.

Location of the Sensilla (Aesthetascs)

Antennules

Lateralfilament

Antenna

Close up of Sensilla (Aesthetascs)

Guard hair

Sensilla(Aesthetascs)

LateralAntennule

(Derby, 1995)

Experimental Design and Setup

(See Cromarty and Derby 1995 for complete technique and protocol)

SCHEMATIC OF EXPERIMENTAL PROTOCOL

s e a w a t e r

- E c d y s o n e

2 0 - H E

L o b s t e r BL o b s t e r A

s e a w a t e r

s e a w a t e r

s e a w a t e r

s e a w a t e r

- E c d y s o n e

2 0 - H E

L o b s t e r BL o b s t e r A

s e a w a t e r

s e a w a t e r

s e a w a t e r

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ASW

Small Lobster OlfactionBlocked Receives ASW only

Large Lobster Olfaction Not Blocked 20- , Receives HE-, ecdysone or ASW

ASW

ASW -ecdysone

ASW 20-HE

R2 = 0.9355

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60

70

20HE (10-4) 20HE (10-5) 20HE (10-6) 20HE (10-7) 20HE (10-8) 20HE (10-9)

ORN SPIKE NUMBER

20-HE

Linear(20-HE)

R2 = 0.0114

0

10

20

30

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50

60

ECDY (10-4) ECDY (10-5) ECDY (10-6) ECDY (10-7) ECDY (10-8) ECDY (10-9)

ORN SPIKE NUMBER

ECDYSONE

Linear(ECDYSONE)

R2 = 0.1023

0

10

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ECDY (10-4) ECDY (10-5) ECDY (10-6) ECDY (10-7) ECDY (10-8)

ODORANT CONCENTRATION (100uM to 100nM)

ORN SPIKE NUMBER

PONASTERONEA

Linear(PONASTERONEA)

R2 = 0.0026

0

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45

50

CHOL (10-4) CHOL (10-5) CHOL (10-6) CHOL (10-7) CHOL(10-8)

ODORANT CONCENTRATION (100uM to 100nM)

ORN SPIKE NUMBER

CHOLESTEROL

Linear(CHOLESTEROL)

Single search odorants commonly found in lobster prey items are dose-dependent

Mixture search odorants are dose-

dependent

18% of the ORNs recorded are 20-HE best cells

Olfactory receptor neurons respond to all steroids

but only to 20-HE in a dose-dependent manner

Lobster Extract Total Protein MWs by 7.5% SDS-PAGE. Eggs= Drosophila eggs, ES=Eyestalks, AM= Abdominal muscle, AG= Abdominal ganglion, TG= Thoracic ganglion.Standards are Kaleidoscope MW stds 212 kDa, 121 kDa, and 96 kDa.

ESST1 Eggs AGAM TGST1

212

121

96

MW(kDa)

SDS-PAGE gels demonstrate a band at approximately 85 to 98kDA

which is comparable to the known MW of the ecdysone receptor

Lobster Extract Total Protein MWs by 7.5% SDS-PAGE. AE=Aesthetasc sensilla,GS=Guard setae, ST=Antennule Stalks shaved, IA=Intact antennules,AM=Abdominal muscle, TG=Thoracic ganglia. Standards are Kaleidoscope MWstds 203.9 kDa, 125.5 kDa, and 82.2 kDa.

AE1GS1ST1 IA2 Std AM TG AE+GSmixIA1ST2

203.9

125.5

82.2

MW(kDa)

• Nerve Cord Extracts Total Protein by 10% SDS-PAGE. ANC=Abdominal nervechord, TNC=Thoracic nerve chord. Std 1 has bands at 212 kDa, 121 kDa, 96 kDaand 48 kDa. The main lobster extract bands are greater than 96 kDa by thismethod.

212

121

96

48

ANCTNC Std 1Std 2

203.9

125.5

82.2

MW (kDa)MW (kDa)

REFERENCESAtema J, Cobb JS (1980) Social Behavior. In: The biology and management of lobsters. Vol 1. Cobb JS, Phillips BF (eds), pp 409-450. New York: Academic Press.Bolingbroke MB, Kass-Simon G (2001) 20-Hydroxyecdysone causes increased aggressiveness in female American lobsters, Homarus americanus. Horm and Behav 39:144-156.Breithaupt,T, Atema, J (1993) Evidence for the use of urine signals in agonistic encounters of the American lobster. Bio. Bull. 185:318.Breithaupt,T, Lindstrom, DP, Atema, J (1999). Urine release in freely moving catheterised lobsters (Homarus americanus) with reference to feeding and social activities. J exp Biol 202:837-844.Cromarty SI, Cobb JS, Kass-Simon G (1991) Behavioral analysis of the escape response in the juvenile lobster Homarus americanus over the molt cycle. J exp Biol 158:565-581.Cromarty SI and Kass-Simon G (1998) Differential effects of a molting hormone, 20-Hydroxyecdysone, on the neuromuscular junctions of the claw opener and abdominal flexor muscles of the American lobster. Comp Biochem Physiol 120(2):289-300.Derby CD (1995) Single unit electrophysiological recording techniques from crustacean chemoreceptor neurons. In: Spielman AI, Brand JG (eds) CRC Handbook on experimental cell biology of taste and olfaction: Current techniques and protocols. CRC, Boca Raton, FL, pp 241-250. Schwanke ML, Cobb JS, Kass-Simon G (1983) Physiological evidence for a blood-borne factor affecting aggressive behaviors in lobsters. Am Zool 23:947A.Schwanke ML, Cobb JS, Kass-Simon G (1990) Synaptic plasticity and humoral modulation of neuromuscular transmission in the lobster claw opener during the molt cycle. Comp Biochem Physiol 97C:143-149.Scrivener JCE (1971) Agonistic behavior of the American lobster, Homarus americanus (Milne-Edwards) Fish Res. Bd Can Tech Rep 235:1-128.Snyder MJ, Chang ES (1991a) Ecdysteroids in relation to the molt cycle of the American lobster, Homarus americanus. I. Hemolymph Titers and Metabolites. Gen Comp Endocrin 81:133-145.Snyder MJ, Chang ES (1991b) Ecdysteroids in relation to the molt cycle of the American lobster, Homarus americanus. II. Excretion of Metabolites. Gen Comp Endocrin 83:118-131.Talbot WS, Swyryd, EA, Hogness, DS (1993). Drosophila tissues with different metamorphic responses to ecdysone express different receptor isoforms. Cell 73:1323-1337.Tamm GR, Cobb JS (1978) Behavior and the crustacean molt cycle: Changes in aggression of Homarus americanus. Science 200:79-81.

Total protein could be extracted from WL, AE, GS, ST, IA,EY but not from cuticle

WL AE GS ST IA EY CUStd Std Std Std

Total Protein Stain (Amido Black) of Lobster Tissue on Nitrocellulose1uL protein sample spotted per square, all 12 top and bottom squares (24 total) labeled with Brailleindentations to facilitate identification. Top row left (#1) and right (#12) spots are identical 1 uL x 5mg/mL BSA std. Spots #2-#11 are serial dilutions of 1 uL x 5 mg/mL BSA x 100uL previousstd/100uL TBS. Bottom row from left show the Lobster Tissue Extracts (1uL each) : WL=Walkinglegs, AE=Aesthetasc sensilla, GS=Guard setae, ST=Antennule Stalks shaved, IA=Intact antennules,EY=Eyestalks, CU=Cuticle. Bottom standards on right are Biotin-labeled MW and primary antibodiesDDA2.7 (first two) and Sheep anti-chironomous EcR (78-92) (last two).

WL AEStd GS ST IA EY CU Std StdWL AEStd

[BSA stds]

StdStdGS ST IA EY CU Std StdWL AEStd

WL AE GS ST IA EY CUStd Std Std Std Std

Std WL IAGSAEST AE+GS

DDA2.7 positive western spot blots indicate the presence of the ecdysone receptor in WL, EY, IA and ST

DDA2.7 Positive Western Blots on Lobster Tissue Extracts (1uL each).Spot Blots on Nitrocellulose paper, blocked with 5%BSA-TBS, Primary Ab was DDA2.7, secondary Ab was polyclonal goat antimouse1IgGs conjugated with biotin. Visualization was with HRP-SA and TMB. 1uL protein sample was spotted per square, all 12 top and bottom squares (24 total) labeled with Braille indentations to facilitate identification. Top row left (#1) and right (#12) spots are identical 1 uL x 5 mg/mL BSA std. Spots #2-#11 are serial dilutions of 1 uL x 5 mg/mL BSA x 100uL previous std/100uL TBS. WL=Walking legs, AE=Aesthetasc sensilla, GS=Guard setae mechanoreceptors, ST= Antennule shaved Stalks, IA=Intact antennules, EY=Eyestalks, CU=Cuticle.

ACKNOWLEDGEMENTS

Grant# IBN0234569 (GKS)Grant# IBN0234555 (SC)

The monoclonal antibody DDA2.7 was developed by Carl Thummel and was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.

EXPERIMENTAL SETUP

Fighting area with lobsters receiving puffed treatments

Tube system glued to carapace delivers treatment to antennule area

Catheter blocks normal urine release from each lobster during experiment

351.6351.6

The average rank of the aggressive behaviors of animalsexposed to 20-HE is significantly higher than that of

-ecdysone-exposed or ASW-exposed animals

ASW Alpha 20-HE114

116

118

120

122

124

126

128

130

*

The frequency of avoidance behaviors of animalsexposed to 20-HE is significantly higher than thatof -ecdysone-exposed or ASW-exposed animals

A S W A l p h a 2 0 - H E

0

1 0

2 0

3 0

4 0

5 0

6 0

*

The frequency of defensive behaviors of animals exposedto 20-HE is significantly higher than that of -ecdysone-

exposed or ASW-exposed animals.

A S W A l p h a 2 0 - H E

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

*

The frequency of aggressive behaviors of opponents ofanimals exposed to 20-HE is significantly higher than

that of opponents of -ecdysone-exposed orASW-exposed animals

A S W A l p h a 2 0 - H E

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

*

The frequency of aggressive wall behaviors (redirectedbehaviors) was significantly higher in opponents of 20-

HE exposed lobsters than that of opponents of -ecdysone-exposed or ASW-exposed animals

A S W A l p h a 2 0 - H E

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

1 6 0

*

(R2 =

(R2 =

(R2 =

(R2 =

0

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(10-4M) (10-5M) (10-6M) (10-7M) (10-8M)

ORN SPIKE NUMBER

TAU

AMMONIA

GLU

OH-PROLINE

Linear (TAU)

Linear(AMMONIA)Linear (GLU)

Linear (OH-PROLINE)

(R2 =

(R2 =

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(10-4M) (10-5M) (10-6M) (10-7M) (10-8M)

ODORANT CONCENTRATIONS (100uM to 10nM)

ORN SPIKE NUMBER

TNG

GMX

GLU BEST CELL

ONLY AT 10- 4M

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80

100

120

TNG (10-4) TNG (10-5) TNG (10-6) TNG (10-7) TAU (10-4) TAU (10-5) TAU (10-6) TAU (10-7)NH4CL (10-4) NH4CL (10-5) NH4CL (10-6) NH4CL (10-7) NH4CL (10-8) LGLU (10-4) LGLU (10-5) LGLU (10-6) LGLU (10-7) LGLU (10-8)

OHPRO (10-4) OHPRO (10-5) OHPRO (10-6) OHPRO (10-7) OHPRO (10-8)20HE (10-4) 20HE (10-5) 20HE (10-6) 20HE (10-7) 20HE (10-8) 20HE (10-9)

ORN SPIKE NUMBER

20-HE BEST CELL

AT 10 -5 AND 10-6M

0

20

40

60

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120

TNG (10-4) TNG (10-5) TNG (10-6) TNG (10-7) TAU (10-4) TAU (10-5) TAU (10-6) TAU (10-7)NH4CL (10-4) NH4CL (10-5) NH4CL (10-6) NH4CL (10-7) NH4CL (10-8) LGLU (10-4) LGLU (10-5) LGLU (10-6) LGLU (10-7) LGLU (10-8)

OHPRO (10-4) OHPRO (10-5) OHPRO (10-6) OHPRO (10-7) OHPRO (10-8)20HE (10-4) 20HE (10-5) 20HE (10-6) 20HE (10-7) 20HE (10-8) 20HE (10-9)

ORN SPIKE NUMBER

20-HE BEST CELL

TAU BEST CELLFOLLOWED BY GLU

THEN 20-HE

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TNG (10-4)TNG (10-5)TNG (10-6)TNG (10-7)TAU (10-4)TAU (10-5)TAU (10-6)TAU (10-7)

NH4CL (10-4)NH4CL (10-5)NH4CL (10-6)NH4CL (10-7)NH4CL (10-8)LGLU (10-4)LGLU (10-5)LGLU (10-6)LGLU (10-7)LGLU (10-8)

OHPRO (10-4)OHPRO (10-5)OHPRO (10-6)OHPRO (10-7)OHPRO (10-8)20HE (10-4)20HE (10-5)20HE (10-6)20HE (10-7)20HE (10-8)20HE (10-9)

ODORANTS PRESENTED

ORN SPIKE NUMBER

R2 = 0.9355

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20HE (10-4) 20HE (10-5) 20HE (10-6) 20HE (10-7) 20HE (10-8) 20HE (10-9)

ORN SPIKE NUMBER

20-HE

Linear(20-HE)

R2 = 0.0114

0

10

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30

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ECDY (10-4) ECDY (10-5) ECDY (10-6) ECDY (10-7) ECDY (10-8) ECDY (10-9)

ORN SPIKE NUMBER

ECDYSONE

Linear(ECDYSONE)

R2 = 0.1023

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30

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50

60

ECDY (10-4) ECDY (10-5) ECDY (10-6) ECDY (10-7) ECDY (10-8)

ODORANT CONCENTRATION (100uM to 100nM)

ORN SPIKE NUMBER

PONASTERONEA

Linear(PONASTERONEA)

R2 = 0.0026

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CHOL (10-4) CHOL (10-5) CHOL (10-6) CHOL (10-7) CHOL(10-8)

ODORANT CONCENTRATION (100uM to 100nM)

ORN SPIKE NUMBER

CHOLESTEROL

Linear(CHOLESTEROL)