Reflection!

Over the whole year from the beginning to now, I have started from deciding my own topic to conducting and rounding up my own research on the sexual behaviors of pill bugs. Although the key factor in this experiment is time, I am glad that I was able to finish the four cycles and theoretically prove my hypothesis that the “male effect” does exists. In fact, the statement is already proven by purely observing the stages of growth the baby pill bugs are in right now. Currently, the biggest challenge is counting the pill bugs, for the purpose of statistical prove and analysis. My mentor, Mr. Calos, wish for me to publish a paper or report next year, so the statistical data is very important and will accurately support my hypothesis with real proof. When completing my poster, my report has been laid out with a format of a formal lab report, so the information that I have written will be useful for that. To reflect on my internship for this past year, I would like to present some of the challenges I have come across. First, it was gender determination. To determine the gender of pill bugs under the microscope can be tricky, and I have to be extra careful to not make a mistake, hence I have to observe each bug for at least 30 seconds with more than just a few looks at the hind legs to confirm its gender and minimize errors. I have improved in doing this in a less timely manner, which is satisfying. Second, it was managing the living environment for pill bugs. Pill bugs entertain moist areas; therefore the spraying of the tank has to be maintained on a regular basis, while balancing the risk of growing molds or fungi in the tanks. Even now, the growing of fungi occurs but the most helpful thing to do is to just remove them and re-feed the bugs. Third, it is challenging to run two separate trials at the same time, because the difference between the two outcomes makes it tough to determine whether the hypothesis is defeated or supported based on just the different outcomes. While the most recent challenge is to count bugs quickly and using bare eyes to spot them takes a very long time, I am enjoying the process of seeing little pill bugs climb up and out of the soil. Some successes I have experienced are the regular schedule that the cycles lie on, easily allowing the time control to be more accurate and fair to the other sets of pill bugs. Another is gaining the skills of sexing bugs as I become more effective. Time-management planning also plays a vital role in this experiment. I was able to keep the two interactions with males in the same timeframe. Although the four cycles of the experiment ended, I am still working in my final stage of counting the bugs, but an early thanks to Mr. Calos for his guidance and help all along.

Cycle 4!

Exciting news, it worked! New babies are born in Box #8, my second experiment trial (using Boxes #5-8) is actually closer to my predicted results than my first trial. The babies found in box #8 are newborn, they are so tiny compare to the ones in box #6, and they are still white, not transparent (as they develop transparent layers and a dark line in the middle of their body when they get older). The comparison mainly lies between box #7 to box #8, with box #8 having an additional encounter with a male for two more hours than the ones in box #7.  The first trial is also going well, with babies appearing in box #4, but interestingly enough, box #3 has no babies. This is the end of the experimental period, even though my results in my first trial and my second trail vary a little bit between each other, I have proven two things: prolonged presence does have an influence on the pregnancy period of the female, secondly, that female pill bugs can still reproduce with no male presence.

Here’s a picture of a baby in box #6 with the parent, compare the size:

The size of one baby is tiny compare to a grown pill bug.

Cycle 3!

Cycle 3 is the week when baby pill bugs are being born in large numbers. Box #6 shows the most positive results in terms of closeness towards the predicted outcome. What this means is that Box #6 is the box with the longest presence of male (because it has a male all along), and its babies are born the earliest since when I discover babies in Box #6, there aren’t babies in Boxes #5, 7 or 8 yet (they are in one set of experimental trial), which supports the “male effect” exists.  All throughout this week, I am observing the babies growing and I found an interesting factor that even in Box #1 and Box #5 (where it contains female x female, supposedly my negative control group) have babies. So I left it the way it is but still found out that the babies born in Box #6 grew faster and are in bigger size than the new born ones in other boxes, proving that prolonged male existence does have an impact. Next week, cycle 4, I am going to continue observing the appearance of babies and look for babies that will be hatched in Boxes #3, 4, 7 and 8. One more week till experiment is complete!

Cycle 2!

Start of cycle 2 on April 12th and 17th for #1,5 and #2-4,6-8 respectively. Following the plan of the experiment, this is also a week without any interferece on anything. As usual, continual spraying of 10-15 sprays per time to keep the area moist. One interesting thing is because of the moist, the left-over food has been covered with fungi, most likely molds. In #8, I found a mushroom on Tuesday; I was unable to take a picture of it but it looks a flower. It won't stay but I had to remove all the filter papers for the food area as well as under the "hosue" made by egg containers. Some of the containers actually have mold on them too so they were replaced. It is mostly maintence work this week and the interference of male presence will occur next week. Meanwhile, I had to control the amount of food fed to them so to prevent mold growing on the soil and I cannot spray on the food but the soil.

Here's a picture of that fungi mushroom that grew in #8:

I just learned about fungi in my AP Biology class so it was very cool to see one;

I brought it to class and we were all fascinated!

Cycle 1!

This week is the first week of cycle 1, which is planned for everything to settle and stay in the initial setting, with no inferference except continual spraying for a moist environment and feeding. To continue on my last blog record, I sex-ed bugs to complete my set up to have 14 females and 2 males in #1-8. All of the bugs in #2-4 and #6-8 are placed in on April 10, 2012, which means cycle 2 starts on April 17th for them. With #1 and #5, they are kept the same since the start of the experiment, so despite the fact that their cycles started on April 5th, it is all right for them to stay for the four cycles. As for all other bugs, I placed the other sex-ed males in a colony tank and some of the ones are left unsex-ed in another colony because they hid under soil. However, I found some babies in that tank, they are tiny in size compare to grown pill bugs, about 2mm long and 1mm wide. Their bodies are transparent but their structures are already developed.

Here's a picture of the baby pill bug that I isolated and placed into a petri dish:

I have seen a couple more babies in the un-sexed colony tank, exciting!

Official Start of Cycles!

The bugs came in on yesterday, I placed them all in a "colony tank" and sorted them out today. Unlike last time, there are a lot more males than females, which is a big CHALLENGE because I need more females than males. I ended up only being able to start #1 and #5 with four females. For all #1-8, I sprayed 30 times because they seem to be drying out faster than I expected. Throughout this and the coming week, I will be sexing more and continue taking care of the rest of the sex-ed bugs.

Here's me looking into the microscope and waiting for them to expose their hind legs:

Fun observation: pill bugs get "impatient" under the light as they struggle to escape from it.

Sexing them takes less time now, even though it still requires about 1-2 minutes per bug.

Final Preparation!

After confirming that the experimental trial worked, it is time to start. I plan to start off with two experimental sets, hence I will need 8 boxes, 4 boxes for each set. Each box is set up like the pre-experimental trial box, with approximately 280g of soil, 200ml soaked water, a little shelter made by the egg container and a food area shaped by a piece of filter paper (that way, there won't be any molds). I came in late this week to spray 15 times in #1-8 to moist the area in preparation when pill bugs come in next week. I will briefly explain again how #1-8 is used. In #1 and #5, there is the negative control group, female + female. In #2 and #6, there is the positive control group, male + female, which we can expect a second generation. Then #3 and #7 are the treatment 1 group, female + female with periodic interaction with males for once. Last but not least, #4 and #8 are the treatment 2 group, female + female with periodic interaction with males for twice. Once they all come in next week, I will sex them and then the cycle starts!

This is how my experimental set-up is...(cleaner than it usually is)

See the one experimental set on the left - the four piled up boxes.

Found Babies!

It's after break - that means it's the time to start off the real thing! Well, before then, I of course have to take care of what was left off and be prepared to start everything the week after. Over the two weeks of vacation, I was researching about pill bug pregnancy. Here's what I found. Pill bugs eggs are stored in the brood poutch of the mother for a period of time (approximately 1 month) before being hatched. To know if the female pill bug is pregnant or not, you will see white patches near the hind legs area.

Here's a picture of what a pregnant pill bug looks like:

This is just the early stage of pregnancy...

and this is definitely what is called "a later stage of pregnancy".

Now that I returned with more knowledge on pill bug's reproduction cycle, I shall proceed. One great news for me (or even maybe you) when I check my pre-experimental tanks...There are egg shells lying all over the box! Which means...the mommy-pill bug gave birth! The parents are placed in the box for about one month, exactly about the time the offspring would be born. How great! This perfectly works out as how I wanted it to, postive sign to move forward and get the real experiment going.

Spring Break!

Due to a two-week spring vacation from March 3rd to 19th, I cannot start my experiment. However, Ms. Mroczka helped with keeping the soil moist and feeding the bugs while I was away from campus. The bugs are kept alive and I am thankful for her assistance with my project. Once I return on campus, I will start my experiment on the second week. During spring break, I am to research and learn about pill bug pregnancy. More information will be posted once I finished my research. Have a good break everyone!

Setting Up for the Official Start!

Time to officially start the experiment. During this meeting, the trial box was more dry so I was able to complete my set up for the trial run. I placed one male and female inside the box. They are the grown up type (and possibly not virgins anymore). I used filter paper to serve as their food area and the poked holes on the box lid will provide them with enough air. After setting up my trial experiment, I moved onto setting up boxes for the actual experiment. For those boxes, the set up has to be the same so that the environment will act as a control instead of variable. I put in about 382.3g of dirt in every box, with each box weighing approximately 67g (without lid) and 95g (with lid) and poured in 200mL of distilled water. I also prepared labels and data tables for each of these boxes to keep track. If all goes well (especially the trial box), I will just have to put in the different experimental groups of pill bugs in the boxes and then the experiment will begin!

The trial box looks like:

The pill bug (male) on the filter paper is trying to get food!

Pre-experimental Trial!

After planning the experiment, I prepared a trial set up to test if my setting would work for the actual experiment. I used a small food container half filled with dirt, along with a pair of egg container for a shadowed area. In order for the dirt to be moist, I poured 300mL of water but after a while, the egg container softens and is soaked with water. Too wet, opps! For next week, Mr. Calos will bring in more of the same materials so I can set up for my real experiment. Meanwhile, I have to wait until the test box dries so I can put in two bugs from the colony tank. Also, my meeting time changed to Thursday due to a different spring semester schedule. I will work independently on the experiment every Tuesday.

Planning the Mating Experiment!

To test the male-effect on pill bugs, the experimental setup requires different sexes of pill bugs to be separated into different groups. During the month of February to March, it is the time of the year that female pill bugs are predicted to be very probably entering the pre-parturial intermoult (179). The experimental design will involve the following groups of pill bugs:

                  1) Negative control group = female + female
                  2) Positive control group = male + female*
                  3) Treatment group 1 = female + female, each will be further mated once
                  4) Treatment group 2 = female + female, each will be further mated twice
*The purpose of positive control group is to be compared with the other three groups.

 All of these groups of pill bugs should be placed into identical tanks/boxes, filled with moistened dirt and enough food for several months and maintained in the same conditions. After two or three weeks, females from the treatment groups should be placed into petri dishes to individually confront a male for a limited amount of time (e.g. one hour). Encounters should be repeated on a regular basis until “a male initiated a mating sequence (typically consisiting of mounting attempts and two successive mating postures to inseminate the right and left female genital ducts)” (179). Towards the end of the experiment, females in the negative control group would have to be dissected to verify their virginity as well as to confirm the presence of sperm in the genital ducts of all other females in the positive control and the two other treatment groups.


For the full text of the published research article, please visit:
http://www.tandfonline.com/doi/abs/10.1080/07924259.2010.9652331

FRANÇOIS LEFEBVRE & YVES CAUBET (2010): Female-extended control over their reproductive investment: the role of early mating interactions on oocyte maturation in the terrestrial crustacean Armadillidium vulgare (Latreille, 1804), Invertebrate Reproduction & Development, 54:4, 177-186.

Slight Break for Service Day!

In honor of Dr. Martin Luther King Jr., today is community service day for all Emma students. My service was the Boys & Girls Club of Troy Canvas Painting Project, held on campus. I have to miss my internship today but I still regularly took care of the pill bugs that I sorted out last week. Every couple of days, I will have to water the dirt, so that the environment get moisty. I feed the pill bugs with fish food as well. Ready to start the experiment soon!

Sorting Bugs Out!

Mr. Calos orderd pill bugs during the meeting on Wednesday. They came in today so I have to sort them out, or else they would die in their white foam container.

These are the procedures that I performed when sorting the bugs by gender:
#1 Transfer all bugs into a large plastic transparent container
#2 Gently use paint brush to remove the bugs one by one onto petri dish
#3 Flip the bug upside down on petri dish
#4 Place petri dish under microscope
#5 Observe carefully under bright light
#6 Wait until bug is still on the dish
#7 Focus on the hing legs part of the bug
#8 Place the bug into the tank (either male or female) that I have prepared earlier
#9 Mark down number of bug(s) in each tank
#10 Repeat procedure 1-10 until all bugs are sorted

The process took more than two hours but it was rewarding. In the end, I sorted 11 males and 34 females. It is definitely a great start to the project!

This is the setup...notice the bug in the petri dish!

Research and Finish Setup!

From last week’s assignment to look for a reliable published reading, I came across pill bug's “male effect”. The “male-effect” is characterized by a significant shortening period of the pre-parturial intermoult (PPI) during which oocyte maturation spontaneously takes place. The prolonged presence of male along with females is known to boost female reproductive physiology. From the paper, the study resulted in the ideas that the “continuous presence of a male can speed up vitellogenesis and the moulting cycle, so to reduce female PPI by 15-20 day” (177). Results also indicate that the earlier and longer the copulations, the stronger the “male effect” can be. From the published data, a conclusion of “females are adjusting their reproductive physiology according to the presence/absence and the intensity of male mating stimuli” (177) was proven. During this meeting, Mr. Calos ordered pill bugs. I also prepared two tanks (for the convenience after sorting out males and females) in preparation for the bugs to arrive on Friday.


For the full text of the published research article, please visit:

http://www.tandfonline.com/doi/abs/10.1080/07924259.2010.9652331

FRANÇOIS LEFEBVRE & YVES CAUBET (2010): Female-extended control over their reproductive investment: the role of early mating interactions on oocyte maturation in the terrestrial crustacean Armadillidium vulgare (Latreille, 1804), Invertebrate Reproduction & Development, 54:4, 177-186.

Project Begins!

This week, I am actually starting the project - Mr. Calos and I set up my experiment station downstairs of Weaver. I then started researching about the determination of gender for pill bugs. I interestingly found out that male pill bug has a pair of long blade-like appendages near its hind legs while female does not. The way to know that is to hold the bug gently and turn it upside down to check. Here is the link as to where I got the information: http://snailstales.blogspot.com/2008/02/is-it-boy-or-girl.html. I then moved on to observe some of the pill bugs; I was not able to get one by just using my hands, so I will have to use some forceps to help me. Before my next meeting, I will have to look for some published books that discuss about pill bug’s sexual behaviors, and learn more.

Here is a picture of what a male pill bug will look like: