Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Gabriele Kass-Simon

Abstract

20-hydroxyecdysone (20E), the active principle of the molting hormone in the American lobster has significant effects on the animals’ agonistic behavior and has been shown to influence the outcome of agonistic interactions. Animals injected with 20E are significantly more aggressive than saline-injected control animals, and premolt animals, which have high circulating levels of 20E in their hemolymph, are more successful than intermolt animals in agonistic interactions. 20E has been shown to act as an internal modulator of neuromuscular physiology: there is an increase in the amplitude of excitatory post-synaptic potentials in the claw opener muscle and a decrease of them in the abdomen when 20E is perfused across the neurons. In addition to its humoral action, 20E appears to be an important signaling molecule sensed by the animal’s antennules, since the behavior of animals change when they are exposed to 20E. The purpose of this study was twofold: to reassess the internal hormonal effects of 20E on agonistic behavior in lobsters, and to provide biochemical evidence for the presence of 20E receptors on the antennules.

Fights were conducted between small lobsters injected with 20E and large lobsters injected with saline. The nephropores of each lobster were blocked to eliminate urine signals between the combatants. Using an ethogram, the frequency and intensity of aggressive, defensive and avoidance behaviors of animals in experimental fights were compared to those in control fights (large and small lobsters injected with saline). A significant difference was found in the aggressive content in the behavior of animals engaged in experimental fights and that of the animals engaged in control fights, such that the difference in aggressive content of defensive behaviors between 20E injected animals and their opponents was less than its difference between saline-injected animals and their opponents. These results suggest the aggressiveness of the defensive behavior of smaller treated animals was closer to that of their larger opponents than the behavior of smaller control animals was to their opponents. A post-hoc analysis comparing the control animals in this study to control animals in a similar experiment in which lobsters were injected with 20E and allowed to urinate freely showed that blocking urine release changes the dynamics of an agonistic interaction between lobsters.

Since 20E was previously shown to affect the neuromuscular properties of the claw opener muscle, force experiments were performed to test the effect of ecdysteroids on the claw closer muscle. A customized force transducer was constructed to measure the force and duration generated by the closer muscle of male and female lobsters after injection with alpha-ecdysone or 20E. The differences in force and duration before and after injection of 20E or alpha-ecdysone was compared to their differences after injection of saline. Alpha-ecdysone significantly increased the force generated by female crusher and cutter claws, and 20E potentially increased the force in female crusher claws. The results suggest that circulating ecdysteroids influence the claw closer muscle of females and could be a factor influencing agonistic interactions.

Because previous behavioral experiments indicated that 20E could be perceived by lobsters and could alter their behavior, experiments were performed to determine whether a 20E receptor (EcR) existed on the antennules of lobsters. In order to visualize the presence of an EcR, various tissues from lobsters were dissected, soluble and insoluble fractions extracted, and spot blots and Western blots performed. Spot blots indicate the presence of a 20E receptor in both the soluble (cytoplasmic/nuclear) and insoluble (membrane-associated) fractions of walking legs and eyestalks, but only in the membrane-associated fraction of the guard setae and aesthetasc sensilla. Western blots and Mass Spectrometry returned several different molecular weights for the EcR (75 kDa, 50 kDa, 40 kDa). The presence of an EcR in the membrane-associated fraction confirms that 20E can be perceived by the antennules of lobsters, while the various molecular weights suggest different isoforms may exist, which is consistent with various insect and crustacean species.

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