Prior to the development of all experiments, l established access to my target species: Squilla mantis via local fisherman in Malaga, Spain. I also designed and built a field laboratory where I maintained animals, conducted electrophysiology experiments, and filled neurons for fluorescent microscopy in a local home. The majority of experiments with S. mantis were carried out during their available season in the fishing industry from November to March in both year 1 and year 2. During these field seasons, experiments were conducted in Spain (3-5 trips per year) and in Cambridge. The specific work achieved for each primary objective are as follows:
1.DEFINE STRIKE BEHAVIOR: Restrained animals were induced to strike via mechanical stimulation during HSV recordings. Strikes were induced both in air and in water to measure the effects of drag on the kinematics of the strike. Strike speeds were calculated using a custom code in Matlab and R. The results of this work suggests that mantis shrimp modulate the speed of their strikes (not all-or-nothing).
2.CURRENT INJECTION: different regions of the nervous system were stimulated to identify the location of strike control circuits. Strikes could only be induced by stimulating the circumesophogeal connectives (CEC). Lesion experiments demonstrate strikes may be induced independent of brain input. HSV analysis of current stimulated strikes demonstrate that only strike acceleration is engaged and not braking mechanism.
3. ANATOMY: Light microscopy of CECs revealed population of giant axons (>40um) in S. mantis. CEC and strike motor nerve axons were labelled with multiple fluorescent dyes to trace connections between the two nerves. Over 30 tissue samples were prepared for subsequent 3D imaged using 2 photon microscopy. CT scans generated high-resolution 3D scans of the striking appendages. These data served as a guide for muscular electrode placements and for a publication on the biomechanics the strike latch.
4. ELECTROPHYSIOLOGY: A stable, partially restrained extracellular recording preparation was designed Squilla mantis. The animal is restrained such that only the rotating appendage is free to move during a strike. Custom electrodes were used to record from the CEC. To monitor strike activity, EMG silver electrodes simultaneously record from the strike-driving muscles. Initial analyses identified descending neuronal units that respond to specific moving target visual stimuli. These same units also respond to mechanical stimulation of the antennae. While these data demonstrate presence of descending neurons with multi-sensory input, their targets remain unknown.
Figure 1. Diagram and unpublished data from S. mantis electrophysiological preparation. A Diagrams of experimental preparation viewed from top and the side (grey inset). (B) Light micrograph CEC crossection. * denote large diameter axons (>40um). D, dorsal; L, lateral. (C) Spontaneous spiking from CEC recording. D Spiking from same recording in response to moving target stimuli. Inset shows stimulus screen and region where most visual responses occurred (dashed ellipse, dorsal visual field). Green line, stimulus start; red line, stimulus stop. E Coactivity in CEC and lateral extensor muscle recordings during a spontaneous strike (not in response to stimuli). Strike movement occurs at approximately 2 s. Top, orange trace is CEC recording and bottom, red trace is EMG recording for C-E.
Figure 2. Angular velocity of strikes stimulated in a single animal via mechanical stimulation or current injection. A. Maximum angular velocity of mechanically stimulated strikes are greater than strikes forced by current stimulation to the CEC. B. Though the two stimuli produce different strike speeds, both current injection and mechanical stimulation accelerate the appendage at the same rate. Deceleration of the appendage is highly variable in current elicited strikes, suggesting that the braking mechanism of the strike is not recruited b
