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An animal behavior seminar lectured by Fiona Cross detailed a few studies performed on the behavioral strategies of jumping spiders. While doing research an America, she mostly paid attention to Portia Africana, a tiny species of spider with incredible cognitive abilities when you consider its brain is smaller than a thumbtack.

She detailed the results from the studies done with Portia, revealing to the audience that the spider displays acts of specialized behavior with several strategies that allow it to be so versatile. For example, Cross described how throughout her studies, Portia paid attention to prey type and prey number with each consecutive trial using lures as bait. Portia would hesitate to approach prey if there were less of them, and would find no interest in the leaves used as the alternative lure in the trials it was subjected to.

I also learned some interesting facts about Portia compared to other species of spider: One of them was that compared to another jumping spider, Nephila Clavipes, Portia’s eyesight is superior. Another learned fact about Portia was about its aggressive behavior. Portia uses mimicry as a strategy to lure and attack other spiders. When it encounters other spider nests, Portia will act like prey stuck on its enemy’s web, by mimicking the vibrations of a struggling insect. When the other spider approaches closely enough, Portia will attack and ingest the spider. m

Because of how interesting this last piece of information was before she closed the seminar, a future proposal for an experiment involving Portia could be its frequency of aggressive mimicry with live spiders of a different species compared to its own cannibalistic species.

Morphology refers to the characters an organism that have developed over time throughout the course of evolution. A lot about an organism's lifestyle can be understood by looking at their morphology. For example, many organisms that live underwater have common traits such as the absence of hind limbs and the presence of fins and tails. Aside from habitat, morphology can also tell us what diet an organism might have. From presence of teeth to the different shapes and forms of teeth it is possible to discern what an organism eats. A more useful way to look at morphology is the classification of animals. Animals belonging to the same family often have a few shared characters which makes it easier for us to organize them. Characters of the skull and limbs are often the most useful in determining the group a certain animal belongs to. In mammals, a lot can be said about the number and placement of digits and characters of the skull such as postorbital processes, mandible shape, presence of horns or cones, and a great number of other things. The organization of animals using morphology is mostly accurate, but can be improved upon by using genetic techniques, which can more accurately place animals together in groups using genome rather than just looking at shared characters. Still, morphology is a good place to start.

Animals have been experimentally shown to navigate their environments using landmarks, beacons, and cognitive maps. A landmark an object or structure distinguishable from the background environment that gives information about the position of and direction to a goal, while a beacon directly locates the goal. A cognitive map is an image of the environment stored in the animal’s memory. The use of landmarks has been demonstrated in animals as different as wasps and birds. In wasps, a test was done where a circle of pinecones was placed around a wasp’s nest, and then moved once the wasp had left. When the wasp returned, it looked for its nest in the pinecone circle, evidencing it use of landmarks. In hummingbirds who were trained to locate a flower based on two differently coloured poles, it was found that moving the poles caused them to look for the flower at its absolute location from the poles. Additionally, Clark’s nutcrackers have been shown to use cognitive maps. In a test where seeds were buried halfway between two landmarks, the nutcrackers managed to consistently locate the seeds even as the landmarks’ absolute distance from each other changed. It was concluded that the nutcrackers had learnt the relationship between the landmarks and the seeds rather than judging distance and position of the seeds based on the landmarks. This has generally been accepted as evidence of a cognitive map in Clark’s nutcracker.

Housing the milkweed bugs

    We placed the milkweed bugs into a 30 x 16 x 21 cm arena at room temperature (~21℃) under a black roof. We gave the bugs shelled sunflower seeds in a dry petri dish as their source of food and water from a cotton stopper on a test tube filled with water as their source of water. We placed the bugs on a 8L:16D day-night cycle and allowed them vision of their surroundings through the plastic walls of the arena. 

Setting up the experiment

    Twelve hours prior to the experimental setup we starved the milkweed bugs by removing the sunflower seeds from their arena. We took a 34 x 29 x 24 cm cardboard box and lined it with a black rubbish bag. We covered the bottom of the box with a layer of ice chunks. We placed a 30 x 16 x 21 cm arena with a blue roof into the box and packed the cleft between the box and the arena with ice. We lined a 30 x 16 x 21 cm arena with a black rubbish bag and clipped a 60W heat lamp onto the top lip of the arena. We roofed the arena with clear plastic clingfilm. We allowed the cold arena to reach 4.6℃ and the hot arena to reach 26.0℃.

Temperature switching and measurements

    We took 20 milkweed bugs from the housing arena and placed 10 of them in the cold arena and 10 of them in the hot arena. We let them habituate for 30 minutes. We simultaneously switched the hot and cold arenas. One lab member removed the rubbish bag wrapping, the clingfilm roof, and unclipped the heat lamp of the hot arena while another removed the cold arena from the ice-packed box and removed its roof. We placed the hot arena into the ice-packed box and put shelled sunflower seeds in a petri dish and a cotton water source in the arena. At the same time, we clipped the heat lamp onto the cold arena, wrapped it with the rubbish bag, and roofed it with clear plastic clingfilm. 

    We recorded temperature and bug activity as defined by movement in both arenas simultaneously: in 15 second intervals for the first 120 seconds, in 30 second intervals until the 720 second mark, and in 60 second intervals until the 1500 second mark. 

    After the experiment had been run we placed the three arenas under the same conditions as the housing arena.

Today’s seminar, lectured by Fiona Cross, involved insight from studies done on behavioral strategies of jumping spiders. While doing research here, she mostly paid attention to Portia Africana, a tiny species of spider with incredible cognitive abilities when you consider its brain is smaller than a thumbtack. Results from the studies done with Portia revealed that it displays acts of specialized behavior with several strategies that allow it to be so versatile. For example, Fiona described how throughout her studies, Portia paid attention to prey type and prey number with each consecutive trial using lures. Portia would hesitate to approach prey if there were less of them, and would find no interest in leaves used as the alternative lure in the trials it was subjected to. I also learned some interesting facts about Portia compared to other species of spider: One of them was that compared to Nephila Clavipes, Portia’s eyesight is superior. An even better, more interesting fact about Portia I learned was its aggressive behavior using mimicry as a strategy to lure and attack other spiders. Portia, when encountering other spider nests, will pretend to act like a helpless insect stuck on the web by mimicking the vibrations of a struggling bug. When the other spider approaches closely enough, Portia will attack and eat the spider. Because of how interesting this last piece of information was before she closed the seminar, a future proposal for an experiment involving Portia could be its frequency of aggressive mimicry with live spiders of a different species compared to its own species (because Portia is also known to display acts of cannibalism).

The seed coat is designed to allow a seed to survive in conditions that would have been intolerable to the adult plant. The seed is able to wait until suitable environmental conditions occur before germination (Sanford, 1963). In most cases, seeds can be germinated in moist substrate that is stored in dim light or even dark. The temperature can be 15-30℃.Germination rates can occur between 16 hours and up to 10 days in most cases (Sanford, 1963).

In order to conclusively identify the organelle tagged with the mCherry fluorophore, we used immunofluorescence to visualize the hypothesized organelle. The mCherry tag was hypothesized to be on the nuclear lamina, so we stained the nuclear lamina with a GFP antibody. This allowed us to perform microscopy on fixed cells and see the colocalization of the mCherry tag and GFP stain. Because the antibody used was a known nuclear lamina antibody, its colocalization with the mCherry tagged molecules proves that the tag is on the same molecule as the antibody. Therefore the mCherry tag is in fact on the nuclear lamina. 

Hox genes are a family of genes that are vital to an animal’s embryonic development. The HOX genes belong to the homeobox gene family, which is the second-largest gene family. The Homeobox genes codes for the homeobox domain which are 61 amino acid that forms a helix turn helix structure. Homeobox domain is present in transcription factors that are important in development. Mutations in either the homeobox domain or in the HOX gene can cause serious developmental diseases. The expression pattern of HOX genes dictates the body plans of animals. In vertebrates, HOX genes are organized into clusters that are on different chromosomes named abcd which each contain 9 to 11 hox genes. In this proposal, we are focusing on HOXC genes. There are 9 HOXC genes. They are named HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, and HOXC13. All of the HOXC genes reside on chromosome 12.

The second experiment established that in the N15S, one glycosylation site is missing, which caused a change of molecular weight, allowing it to have a lower molecular weight than normal. After PNGase F treatment, however, the protein appears more similar to the wild type indicating that it is more like the wildtype compared to when it is not treated. In the P23H, the mutation that the group is interested in, there was also a lower molecular weight, which indicates that there is degradation. Compared to the control, both of the dimers were had a heavier molecular weight compared to that of the wild type which indicates that aggregation is more severe in the mutants, with the P23H being more severe compared to N15S(figure 3)

In the 3rd experiment, the total fluorescence was lower than the wild type in both of the mutants. The researchers hypothesized that this may be due to the aggregation of the mutant proteins. There was a reduction in the stability in N15S, compared to WT, and P23H was much less stable compared to both. However, because there was a total decrease of fluorescence signal, which indicates that the properties of that are expressed in this experiment causes some of the mechanism of pathogenesis of the mutants. 

In the fourth experiment, the experiment indicated that in N15S, there was a decrease in alpha helix, an increase in beta-sheet, random coils and turns compared to the wild type. In the P23H, there were even more differences compared to that of wild type with an increase in random coil and decrease in alpha-helix, beta-sheet and turns. This indicates that when the P23H mutation happens, there is a larger structural change compared to the N15S mutation. (figure 4, table1)

In the fifth experiment, the rescue experiment was conducted. The proteins were denatured, in the presence and absence of chlorin e6.  The proteins were then put through circular dichroism spectroscopy. In the resulting graph, the % helix content was lower in P23H with chlorin e6, but not in N15S. This indicates that the compound restored some of the stability to the protein with a mutation of P23H.  This implies that compound chlorin e6 may be a viable treatment for patients with a mutation of P23H but not with N15S.

The goal of the research is to compare the two mutants of the rhodopsin mutation, P23H, and S15N. Specifically, the researchers were interested in how the mutations in the N terminus domain affect their invitro biochemical properties, their UV visible absorption, Fourier transform infrared, circular dichroism and metarhodopsin II fluorescence spectroscopy properties.  While this was not a formal goal of the research, the study also found that chlorin e6have the ability to rescue mutated rhodopsin protein that had its stability affected.

In the experiment, the proteins were first prepared through PCR mutagenesis technique. The genes were then placed into HEK293S cell lines and the P23H and V15S opsins were established. The cells were harvested and the proteins were purified using immunoaffinity chromatography. 

In the first experiment, the proteins were subjected to UV spectrophotometry, which would establish the degree of misfolding of the mutated protein. For the second experiment, both of the mutant proteins had their oligosaccharide chain from rhodopsin glycosylation sites cleaved using PNGase F. The resulting immunoblot was used for densitometric analysis of the immunoblot was done using Image J, then the Box plot was used to summarize the result of the analysis. This method would establish the amount of aggregation that may occur in cells, which would disrupt their protein function. For the third experiment, MetaII fluorescence was measured for three proteins to measure the amount of aggregation that may occur in the cell. For the fourth experiment, Fourier transforms infrared spectra were collected for COS-1 cell rhodopsin to gain structural insight into the degree of misfolding in the two mutants. For the fourth experiment, circular dichroism spectra were measured with the samples of rhodopsin and Ce6 at 2.5 and 100 micro M this would indicate the stability of the presence and absence of Chlorin e6.