Chapter 1: Functional Specialization and Arm Length in Octopus bimaculoides<br/>Although studies are limited, there is some evidence that octopuses use their arms for specialized functions. For example, in Octopus maya and O. vulgaris, the anterior arms are utilized more frequently for grasping and exploring (Lee, 1992; Byrne et al., 2006a), while posterior arms are more frequently utilized for crawling in O. vulgaris (Levy et al., 2015). In addition, O. vulgaris uses favored arms when retrieving food and making contact with a T-maze as dictated by their lateralized vision (Byrne, 2006b). O. vulgaris also demonstrates a preference for anterior arms when retrieving food from a Y-maze (Gutnick et. al. 2020). In Octopus bimaculoides bending and elongation were more frequent in anterior arms than posterior arms during reaching and grasping tasks, and right arms displayed deformation more frequently than left arms, with the exception of the hectocotylus (R3) in males (Kennedy et. al. 2020). Given these observed functional differences, the goal of this study was to determine if morphological differences exist between different octopus arm identities, coded as L1-L4 and R1-R4. In particular, the relationship between arm length and arm identity was analyzed statistically. The dataset included 111 intact arms from 22 wild-caught specimens of O. bimaculoides (11 male and 11 female). Simple linear regressions and an analysis of covariance were performed to test the relationship between arm length and a number of factors, including body mass, sex, anterior versus posterior location, and left versus the right side. Mass had a significant linear relationship with arm length and a one-way ANOVA demonstrated that arm identity is significantly correlated with arm length. Moreover, an analysis of covariance demonstrated that independent of mass, arm identity has a significant linear relationship with arm length. Despite an overall appearance of bilateral symmetry, arms of different identities do not have statistically equivalent lengths in O. bimaculoides. Furthermore, differences in arm length do not appear to be related to sex, anterior versus posterior location, or left or right side. These results call into question the existing practice of treating all arms as equivalent by either using a single-arm measurement as representative of all eight or calculating an average length and suggest that morphological analyses of specific arm identities may be more informative.<br/><br/>Chapter 2: Predicting and Analyzing Octopus bimaculoides Sensitivity to Global Anesthetic<br/>Although global anesthetic is widely used in human and veterinary medicine the mechanism and impact of global anesthetic is relatively poorly comprehended, even in well-studied mammalian models. Invertebrate anesthetic is even less understood. In order to evaluate factors that impact anesthetic effectiveness analyses were conducted on 22 wild-caught specimens of Octopus bimaculoides during 72 anesthetic events.Three machine learning models: regression tree, random forest, and generalized additive model were utilized to make predictions of the concentration of anesthetic (percent ethanol by volume) from 11 features and to determine feature importance in making those predictions. The fit of each model was analyzed on three criteria: correlation coefficient, mean squared error, and relative error. Feature importance was determined in a model-specific manner. Predictions from the best performing model, random forest, have a .82 correlation coefficient with experimental values. Feature importance suggests that temperature on arrival and cohabitation factors strongly influence predictions for anesthesia concentration. This likely indicates the transportation process was incurring stress on the animals and that cohabitation was also stressful for the typically solitary O. bimaculoides. This long-term stress could lead to a decline in the animal’s well-being and a lower necessary ethanol concentration (Horvath et al., 2013). This analysis provides information to improve the care of octopus in laboratory settings and furthers the understanding of the effects of global anesthetic in invertebrates, particularly one with a distributed nervous system.

Neuromodulation is an emerging field of research that has a proven therapeutic benefit on a number of neurological disorders, including epilepsy and stroke. It is characterized by using exogenous stimulation to modify neural activity. Prior studies have shown the positive effect of non-invasive trigeminal nerve stimulation (TNS) on motor learning. However, few studies have explored the effect of this specific neuromodulatory method on the underlying physiological processes, including heart rate variability (HRV), facial skin temperatures, skin conductance level, and respiratory rate. Here we present preliminary results of the effects of 3kHz supraorbital TNS on HRV using non-linear (Poincaré plot descriptors) and time-domain (SDNN) measures of analysis. Twenty-one (21) healthy adult subjects were randomly assigned to 2 groups: 3kHz Active stimulation (n=11) and Sham (n=10). Participants’ physiological markers were monitored continuously across three blocks: one ten-minute baseline block, one twenty-minute treatment block, and one ten-minute recovery block. TNS targeting the ophthalmic branches of the trigeminal nerve was delivered during the treatment block for twenty minutes in 30 sec. ON/OFF cycles. The active stimulation group exhibited larger values of all Poincaré descriptors and SDNN during blocks two and three, signifying increased HRV and autonomic nervous system activity.

Migration allows animals to track favorable environments and avoid harmful conditions but is energetically costly. There are different types of migration, such as tidal/daily, seasonal, and lifetime. Locust migratory swarms are one such famous phenomena that can have dramatic effects on human livelihoods. During long-distance flight, locusts rely on lipid oxidation from fat stores, while initial flight is fueled by carbohydrates. However, limited studies have tested how dietary macronutrients affect insect flight performance. Therefore, we asked: How do different dietary macronutrient ratios affect prolonged flight migration? We predicted that high carbohydrate diets would lead to high body lipid synthesis which would increase flight performance. We reared locusts in three crowded cages from 5th instar to adulthood on artificial diet varying in p:c ratio, supplemented with lettuce and water tubes, ad libitum. We used 7-14-day old adult males for flight performance assays where each day we used new individuals for tethered flight for 12 h in wind tunnels (~12 km·h-1) and video recorded their flight. We found that locust flight duration and quality increased with a decrease of dietary p:c ratio. Using control groups of locusts, we estimated that across 1 day of flight (up to 12 h), locusts lost on average in all treatments ~25 or ~30% of their total body lipid content. We concluded that long distance flight is improved by a high carbohydrate and low protein diet for L. migratoria by increasing their fuel sources. This work was supported by NSF # 1942054.