Leopard Sharks

Methods
Subjects
While it appears likely that different species of sharks would share similarities in their navigational strategies, there will likely be interspecies differences. Since leopard sharks (Triakis semifasciata) were selected in Andrew Nosal’s study, from which much of the experimental setup has been borrowed, it appears that these sharks are the best candidates for this study. While more would certainly be better for statistical analysis, 36 sharks were captured in Nosal’s study, and it can be expected that similar numbers of sharks can be captured for this study.

Location
Leopard sharks (Triakis semifasciata) inhabit the Pacific coast of the North America. In Andrew Nosal’s study, the coast of La Jolla, California was chosen for the study. The location has previously been documented for attracting large numbers of leopard sharks between June and December and will likely facilitate the capturing process (Nosal, Caillat, Kisfaludy, Royer, & Wegner, 2014). Moreover, for consistency, it appears best to conduct the experiment near the

original test location. Two sites, site A and site B, will be chosen as release points for these sharks. Site A will be approximately 10 kilometers away from the coast while site B will be approximately 20 kilometers away from the coast.

Figure 1: Location of site A, site B, and the capture site (drawn not to scale)

Experiment 1: Magnetoreception
In this experiment, sharks will be captured near the Californian shore with depths within 2 meters.

In the first group, approximately ten sharks will first have the magnets attached to the dorsal surface of their heads. These sharks will then be transported to point A, approximately 10 kilometers away from the shore. A second group of around ten sharks will undergo sham treatment and have non-magnetic objects of similar shape and weight as the magnets attached to the dorsal surface of their heads. These sharks, too, will be released at point A, approximately 10 kilometers away from the shore. In a third group of ten sharks, magnets will also be attached to the dorsal surface of their heads, but they will be released at point B, approximately 20 kilometers away from the shore as a comparison to the first group. All the sharks will have a GPS (Global Positioning System) tracking device attached to monitor their

movements.

Figure 2: Location of magnet attachment in leopard sharks.

Experiment 2: Olfaction
In this experiment, ten sharks will be captured off the Californian shore again with a depth within 2 meters. These sharks will not undergo any treatment and will be released at point A discussed above. Ten additional sharks without treatment will be released at point B. The water sample can be collected near the surface every 100 meters in the sharks’ trajectory towards the shore. All the sharks will have a GPS tracking device attached to monitor their movements. Next, liquid chromatography will be used to separate, identify, and quantize the chemical composition of the samples.

Experiment 3: Temperature
In this experiment, ten sharks will be captured off the California shore again with a depth within 2 meters. The sharks will not undergo any treatment; however, in addition to the GPS tracking device, a transmitter that can relay the surrounding oceanic temperature will be attached to the sharks. These sharks will then be released at point A. The same process will be repeated for an additional ten sharks that will be released at point B. The relationship between temperature and the sharks’ movements can then be analyzed through statistical tools such as regression analysis. Additionally, sharks’ trajectories can be analyzed in the context of sea surface temperature maps, which can have resolutions of 1 kilometer, generated by weather satellites.

Expected Results
Experiment 1: Magnetoreception
First, disrupting magnetoreception in sharks could potentially have little or no effect on the sharks’ navigational task. One possible explanation for such results will be that while sharks can perceive the Earth’s magnetic fields, it has little contribution to their navigational task. Another explanation could simply be that sharks possess other sensory modalities that could cover for the loss of magnetoreception. In contrast, it is possible that disrupting magnetoreception in sharks could potentially have a significant effect. Since magnetosensory input has been hypothesized to hold information regarding the direction in which the shark must swim towards the desired target, it is possible that the sharks could swim in wrong or even opposite direction away from the coast. While one explanation would certainly be that magnetoreception plays a role in shark navigation, it is important to note that such result could potentially arise from the fact that these sharks are exposed to unnatural magnetosensory stimuli. For instance, while the sharks normally do not utilize magnetoreception in navigation, the overwhelming sensory input from the magnets could potentially disorient and disrupt their navigation. While both extremes of the little to significant results are certainly possible, with the indications that other sensory modalities such as olfaction plays a role in navigation, it appears that the results would be something in between the two. For instance, despite the disruptions in magnetoreception, the sharks could recognize and follow certain odors that lead to coast. Thus, they might still head towards the coast but in a tortuous manner that deviates from their usual highly directed,
straight trajectories.

Figure 3: Potential shark trajectories towards the coast. On the left is the control group that demonstrates highly oriented behavior towards the coast. On the right is the experimental group that takes on a more tortuous path towards the coast.

Experiment 2: Olfaction
The fact that the ocean water contains a mixture of many chemicals and that there are ecosystem differences in the coastal and offshore environments will likely lead to the identification of quite a few chemicals that are in gradient between site A or site B and the final position of the sharks near shore. As the coastal regions are likely to be richer in the sharks’ preferred prey, certain amino acids or nutrients that strongly corresponds to these animals can be predicted increase as closer to the shore. Moreover, as previous studies have indicated higher levels of chlorophyll a concentration near the coast of California, it appears highly likely that chlorophyll a concentration will increase closer to the coast. In contrast, certain chemicals previously found in the deeper waters of Gulf of Lower California such as fatty esters, free fatty acids, fatty alcohols, and hydrocarbons (Garrett, 1967) can be expected to decrease closer to the coast. In identifying the candidate chemicals, chemicals that exists in quantities below the threshold for the sharks’ olfactory senses or constant through the sharks’ trajectory can be removed. It is, however, important to realize that such technique observes the correlation between the sharks’ trajectories and chemical gradient. In fact, many of the candidate chemicals likely do not contribute the shark navigation. Moreover, it is possible that certain chemicals also utilized by the sharks were not present or constant in concentration in the particular trajectories undertaken by the sharks. While these chemicals would be absent in the list for potential chemical cues in this study, it is important to note that they do exist. Finally, while the experiment has adopted the hypothesis that chemicals cues exist in gradient, it is possible that sharks process the odors in a more complex manner beyond a simple increase or decrease. Nevertheless, the goal of the experiment is to identify potential chemicals that could potentially play a role in gradient chemical cue processing, and guide future experiments on whether sharks do respond to these chemical cues.

Experiment 3: Temperature
One simple integration of temperature cues would be simply following an increase or decrease in temperature towards the shore. It has previously been suggested that due to processes such as coastal upwelling, the temperature is generally lower closer to the shore (Nosal et al., 2016). Thus, the sharks could potentially follow such temperature gradient towards the coast. As sharks are ectoderms, another plausible finding would be a homeostatic movement pattern, where the sharks would move towards lower temperature when the surrounding water temperature is greater than its optimal temperature or move towards higher temperature when the surrounding water temperature is lower than its optimal temperature.

Figure 4: Possible relationship between temperature and sharks’ movements if the sharks were to move towards their optimal body temperatures
Such correlation would reveal that temperature during the sharks’ trajectory towards the coast would fluctuate within their optimal temperature range of around 13 °C to 16 °C; however, such results could simply be a byproduct of relatively stable oceanic temperature. Thus, it would be important to analyze the sharks’ movements in the larger context of the surrounding sea water temperature given by weather satellites. Now, when the sharks are faced with two paths, one with a cooler 10 °C and another with more optimal 14 °C, it would be interesting to observe how the sharks would respond.

Figure 5: It is possible that when given the choice between 10°C and 14°C pathways, more sharks would select the 14°C pathway more suitable to their body temperature.
It is important to note that this is a correlational study. Even if a correlation between temperature and the sharks’ movements is indeed found, there could potentially be a third variable such as chlorophyll a, mediated by temperature, that is actually guiding the sharks towards the shore. Nevertheless, in the case that the results indicate no correlation, while it is possible that other sensory inputs that guide the shark is concealing the correlations, the study would still lend support to the idea that temperature may not play a role in shark navigation. In fact, it would also potentially lend support to the idea that third variables that relates to temperature such as chlorophyll a, might not play a role in the navigation process.

Future Directions
If experiment 2 is able to identify candidate odorants, then it would be important to examine whether the sharks can actually perceive the chemicals. Neural physiological methods with the goal of identifying neurons that specifically respond to the chemical can be important in understanding the neural circuits that underlie the olfaction system and determining if the shark is able to perceive the chemical. Moreover, while only the chemical gradients in the sharks’ returning trajectories were observed in experiment 2, future studies could carry out more intensive examination of the oceanic chemical composition of the surrounding waters. Similarly, even if there was a correlation between temperature and shark trajectories, experiment 3 does not reveal whether temperature cues are indeed used in shark navigation. Thus, it would important to examine thermoreceptors and its underlying circuitry in shark. Different temperature can be applied to the shark, and the responses at both the neuronal and circuit level can be examined to gain a better understanding of the system. Finally, while all the experiments here are proposed to be conducted in leopard sharks, there are likely interspecies differences in navigational strategies. Thus, similar experiments can be conducted in other shark species