Second entry.

These past few weeks, I’ve really felt that I have been able to narrow in on my project, and the accumulation of completed protocols, samples, and successful data has contributed to my current feeling of satisfaction and efficiency. In my project, I have become very comfortable and familiar with the RNA Extraction and qPCR protocols as I have completed both procedures numerous times for all of my samples. My samples consist of the head, gut, abdomen, and thorax of several nurse and forager bees which have undergone one of two different treatments. By using these samples, we are able to examine the expression of specific heat shock genes from four different tissues that we commonly look at. By specifically targeting nurses and foragers, we wish to determine which type of bee experiences a lower expression of these heat shock genes, and thus which type of bee has a lower overall heat shock response. Nurses and foragers are at different stages of their life cycles. Nurses are younger bees which care for the larvae, and foragers are older. By looking at the expression of these heat shock genes in both ages of bees, we wish to understand whether or not heat shock is more prevalent in older or younger bees. The primers used for the qPCR for this aspect of the project are actin, which acts as the control, and Hsc 70-4 (Hsp 68). The latter is a gene involved in the heat shock response. Working on this aspect of my project has made me very comfortable with the RNA Extraction and qPCR procedures, and my results have been very pleasing and encouraging. I used to be very tentative, and as the data improved every time I completed these protocols, I began to feel increasingly comfortable and confident in what I was doing. I feel excited to continue doing this to go through all of my samples and create a cohesive spreadsheet with all my data.
 
Another aspect of my project is creating double stranded RNA to perturb these heat shock response genes in the honey bee. I am learning how to set up gel electrophoresis, and how to isolate the bands in the dark room in order to eventually create double stranded RNA. Working in this lab, especially working with the double stranded RNA, has caused me to realize something about myself. Before SRI, I always thought of myself as a conscientious, organized, and careful person, however I am now realizing the degree of meticulousness, dexterity, and care that science requires. I can feel myself becoming a better and more efficient scientist everyday, as I am remembering to be even more conscious and focused on every little thing that I do in the lab. These qualities that I already thought I had are being improved every day.
 
We have been experimenting with newborn bees lately. The initial intention we had in using newborn bees was to potentially run longer term experiments with them since a newborn bee would be able to last longer than an older bee. It is also easier to handle newborn bees because they are too weak and young to fly or sting. We were able to easily manipulate them into cages by hand, and conduct our tests on them. Some problems arose in using newborns, and we needed to brainstorm and come up with innovations to our technique in using the newborns. One of these problems was that the newborns would eat little to none of the food that we provided them, because in that stage of life it is not really necessary for them to eat too much. This was a problem for us because in order to conduct our tests on the newborns, we use the food as the means to infect them with microsporidia or treat them with a certain drug, like Halofuginone or Dithiothreitol​. We found a method that we could use to make sure that the newborns would eat. Because newborns can neither fly nor sting, we decided we could literally hand feed each and every one of them using a micropipette. While this did work and we were successful in feeding them with the treatments, it was incredibly time consuming. Another problem that arose was that a very large percentage of them would die very quickly. They wouldn’t live long enough for us to be able to observe the effects of our experiments. We thought that the reason for their high death rate could be because we were handling them too much, and that in their young and fragile state, picking them up by hand to both put them in their cage and try to feed them individually could have been too stressful for them. In order to try and resolve this problem, we decided against the hand feeding and to also try and simply pour them out of a box that held them into their test cages so we do as little manhandling with them as possible. We are currently seeing if making these tweaks to our methodology will be beneficial to achieve what we want. This process taught me how science is all about trial and error. It is common that something does not go as planned or work out in your favor, and when this happens you need to be able to ask the important questions and find creative ways to resolve any problems that may arise.
 
I have been doing a lot of spore counting with the samples we collect regularly from bees that we dissect. The purpose of spore counting is to record which bee midgut samples are infected and with how many spores. We do this using a hemacytometer. With the information gathered from spore counting, we make food for the bees we are testing on. Professor Snow taught us how to make dilutions with a positive tube containing the spores and sugar water to use to infect our bees. It has been really helpful to understand how to do this because it is central to many of the tests that we run in the lab. The same approach is used to incorporate the drugs and double stranded RNA to the food for the bees.
 
I have found the process of completely finishing an experiment all the way through to collecting and analyzing my data very rewarding and satisfying. Professor Snow has taught me how to input my data on a spreadsheet and to look at the numbers and try to analyze and figure out exactly what it could mean. I have learned that so much can be derived from numbers, such as which procedure along the way may have skewed certain data points in one way or another, or what the relationship between related data points may mean in the large scheme of things. Being part of this lab and having my own project really makes me feel that I am learning about all parts of science, including the actual lab work as well as the analysis and understanding of the results. My goal for the upcoming weeks is to continue with my project and to finish up all of my samples to analyze the data as a whole, as well as to get started on the poster describing our projects.

Setting up for a qPCR plate for my nurse and forager samples.

Doing RNA extraction from my nurse and forager samples.