1. I.INTRODUCTION Huntingtin associated protein 1 (HAP1) is believed to play a role in organelle trafficking and to interact with inositol-(1,4,5) triphosphate receptor 1 which implies that it may be involved in calcium signaling. A human HAP1 homolog (T27A3.1) is found in Caenorhabditis elegans (C. elegans) and is expressed in a subset of neurons. A study of the knockout HAP1 homolog T27A3.1 in C. elegans was performed in order to examine its function. Based on our previous studies, we hypothesized that T27A3.1 might be involved in mechanosensation, growth, chemosensation and/or sensitivity to osmolarity. In this study, we have performed several behavioral assays including the body bend, growth assays, nose touch, ammonium acetate chemotaxis assay and osmotic avoidance. Assays were performed on T27A3.1, wildtype and mutant lines with documented behavioral phenotypes to confirm the effectiveness of the assays. In the body bend assay, worms are observed as they move freely on an agar plate. The worms move by generating sinusoidal waves along the length of their body. The number of forward locomotion waves are counted and averaged. With the growth assay, the worms were bleached and three days later an adult worm was transferred onto a clean plate. In the nose touch assay, a worm pick is placed perpendicular to the origin of movement of the organism. Once the worm comes in contact with the pick, sensory neurons (ASH, OLQ, and FLP) transmit a signal which causes the C. elegans to reverse their direction of movement (backwards). The percent of retraction was recorded. In the chemotaxis assay, either ammonium acetate (attractant) or sterile water were placed at diametrically opposed locations on an agar plate. Worms were placed in the center of the plate and allowed to move around for 60 minutes. Number of worms at each location was recorded. In the osmotic avoidance assay, the percentage of worms escaping from a 4M NaCl osmotic ring was examined. In all cases, there were no statistical differences between the wildtype and T27A3.1 knockout worms but known control lines were different from both groups. II. METHOD AND PROCEDURES Growth and Preparation of C. elegans  Worms were placed on a standard 6ml NGM agar that had a drop of an OP50.  To collect worms for the study, the plates were washed with 2 ml of S. Basal. The worms were transferred to a 1.5ml eppendorf tube. The worms were treated with bleach to isolate the eggs. After the worms have settled in the tube, 100 ml was transferred onto a new plate. These plates were used for the growth or nose touch assays. For the growth assay, the number of worms were counted after 3 days. Nose Touch Assay  We used a worm pick that was sterilized in 70% ethanol.  Placed the worm pick directly in front of a worm to allow the organism to run into at a 90° angle.  We simply tapped the side of the agar plate to stimulate movement and categorized their movement.  If the worm responded instantly (by reversing direction or recoiling), that individual was given a score of a “1”. If the worm was unresponsive to the worm pick (by crawling over, under, or alongside the worm pick), that worm was given a score of a “0”.  Body Bend Assay III. RESULTS V.DISCUSSION Various behavioral studies were done in order to understand the effects of the HAP1 homolog on C. elegans. Based on the results obtained from the body bend, growth, nose touch, chemotaxis, and osmolarity assays, there was not a statistically significant difference between the responses from the wild type worms (the N2 line) and the knockout worms (X4-1, X4-2, and X4-3). However, worms that were known to be deficient in these responses were used as controls and they all responded as expected confirming that the assays were performed appropriately. It is possible that the effect of T27A3.1 on behavior may not be very strong and hence, these assays may not be sensitive enough to detect differences. It is also possible that another gene may compensate for the lack of the T27A3.1. There are various genes that bind to the Hap1 protein and it is possible that some of these genes may compensate for some of the functions usually performed by T27A3.1. Thank you to the Caenorhabditis elegans center for supplying all of the control worms The knockout worms were kindly provided by Dr. Shohei Mitani, Tokyo Women’s Medical University, Japan This work was funded by the department of Natural Sciences at Clayton State University, an NIH grant number 1R03NS058376, and a fellowship from the Minorities Affair Committee of the American Society of Cell Biology The effects of a HAP1 homolog knockout on behavior in Caenorhabditis elegans 1 Clayton State University Department of Natural Sciences and 2 Emory University Department of Neurology Fran Norflus 1, Jingnan Bu 2, Evon Guyton 1, Miranda Holmes 1, Elliot Aronson 1, Caitlyn Van Heest 2, LaQuinte Brinson 1 and Claire-Anne Gutekunst 2 Chemotaxis Assay Agar plates were prepared as shown in Figure 1. We placed 10µL of ammonium acetate directly on the plate where indicated by the dot, and 10µL of sterile water on the other dot. An additional 10µL of sodium azide were placed on each dot. Worms were washed with 1mL of M9 solution and collected into a centrifuge tube. They were then centrifuged for one minute on lowest setting, refilled with de-ionized water, centrifuged once more. The supernatant was drawn without disturbing the pellet of worms. Approximately 10µL on worms were in the center of the plate, ensuring that at least 150 worms were present. The solution suspending the worms were allowed to dry. An hour after, plates were placed in cold room. Differences between strains were analyzed by One-way ANOVA. Osmolarity Assay The end a test tube was dipped in 4M NaCl solution with added bromophenol blue dye to form a ring on the center of small agar plate. Five L4 worms were individually picked and rinsed with M9 solution and placed in the center of the osmotic ring. After the M9 solution dried, the worms were allowed to move about for 30 minutes, and the number of worms in and out of the ring were recorded Osmolarity Assay Chemotaxis Assay Nose Touch Assay NH 4 Ac H2OH2O Figure 1 Body Bend Assay Growth Assay