Author Archives: Floyd A. Reed

Manipulating mosquito eggs

Now that we have stable caged populations of Culex mosquitoes and have worked out an egg laying on demand system the next incremental step is to separate the individual eggs from freshly laid egg rafts for microinjection.  (We have been advised that injecting them while together in an egg raft does not work well because of the pressure they put on each other during injections...)  Jolene worked out the timing to get fresh rafts, disassociated them, and lined them up on wet blotting paper (to keep them from drying out) on a microscope slide.

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To the immediate left of the eggs is an extra layer of paper to brace them for injection from the right.  They were very light in color at first but they got darker (and harden) quickly by the time the picture was taken.  She put this first set back into water to see if manipulating them affects survival.  (For more photos and news check out Jolene's research blog.)

In other related news, we have some of the plasmids we need cloned and I am working on cloning the last one this week (that contain the genetic modifications for injection), and my plasmid preps are working!  Fingers crossed, with any luck we can try serious microinjection to generate germ line Culex transformations three weeks from now during the next round of fresh egg production.  We are planning to generate the self docking system described (in Anopheles gambiae) by Meredith et al. 2013 in Culex.

We've been noticed!

It is no coincidence that my last post was in August.  Once classes started up I have had a very busy schedule and little time for anything else.

However, the thing that triggered this post is, I happened across an article that discussed one of our recent publications.  It is on the IGTRCN (insect genetic technology research coordination network) website in an article by Frank Crisione "Transgenic Underdominance: Pushing Transgenes into Populations".  It is good to know that people out there are reading our publications.

In a similar vein, a grad student here at UH in another lab (I am on his graduate committee) just published a nice article about his work for the general public, "Can a small fish answer the big question?" by Kirill Vinnikov.

Fluorescent Chromosomes

Kenton's project has progressed quickly.  We sorted out the issue with fluorescence (the light source was not aligned properly through the scope).

Here are some updated Drosophila chromosome images.  They have a stain that dyes the DNA and is causing it to glow so you can see the chromosomes.

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You can see the banding in the close up above.

Take a look at this!

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and the detail below.

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The spread isn't good but that's a clear banding pattern!

Now that this is working in Drosophila he has moved on to damselflies; his original interest.  He is trying different tissues at different life stages to see which ones give the best chromosomes.  The damsel fly chromosomes are smaller and there are many more of them so it is a bit challenging, but there are some more tricks up our sleeves to try out in order to increase resolution.  Kenton already has some promising images but I do not want to post them here yet because they are new and potentially publishable results.  However, expect an update on this topic in the future.

35 Egg Rafts!

The last year has been up and down in terms of establishing a Culex colony in the lab.  We have learned a lot though.  It is critical that they be able to reproduce in the lab in order to maintain colonies.  When we got our first egg raft (the females lay eggs on water in floating "rafts") it was cause for celebration for the lab.  However, we almost lost the colony twice over the summer.  We are trying to keep the descendants from the original ones that reproduce in the lab going and add in fresh wild individuals to prevent inbreeding depression until they are lab adapted.  Jolene has been working on increasing the numbers and we now have individuals in four separate cages in two incubators.  She is now working with ways to get eggs "on demand" because the microinjection procedure can only be done with very fresh eggs.  She arranged this over the last week and expected some eggs this morning.  There were 35 new egg rafts when she came in!  Each raft can easily have 100 eggs so we had well over 3,000 eggs this morning to work with!

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There are 25 rafts in this one tray alone!

Update: The next morning there were two smaller rafts.  That puts the number of Culex eggs in the colony this generation at approximately 3,650!

Boards full of equations

SympatricEquation
Above is a photo of Áki Láruson working on a model of sympatric speciation (photo used with permission from Áki).  Áki is studying speciation and evolution in sea urchins, which includes diving, collecting, dissecting, microscope, and wet lab work; however, another part of what many of us do is to work with and try to better understand mathematical models of evolution.  It can get messy, and often takes a lot of patience and time to think through the process.  I have filled up many boards like this in the past (and Áki has another board full on the other side of the room out of camera view).  Often starting from something fairly simple you end up at a point that is very messy, but sometimes, just past that point, things start to work out and simplify, and you discover something new and insightful---or weird and interesting---or both.

Polytene Chromosome Imaging

One undergraduate in the lab, Kenton Asao, is interested in chromosome evolution and has been troubleshooting protocols to prepare insect polytene chromosomes for imaging.  I worked with polytene chromosomes many years ago in grad school---but not since---so I am quite rusty and need to brush up on this myself, and this is something we would like to be able to routinely do in the lab.  Long story short, it wasn't working starting out so we sent some photos (to make sure we were dissecting out the right tissues) and asked for some advice from Kevin Cook (D. of Biology, Indiana U.) who kindly sent us his protocol.  We continued troubleshooting and we were getting quite frustrated trying different stains and imaging approaches but nothing seemed to be working (we also asked for advice from Gert de Couet in the department here at U.H. who loaned us some different dye and reagents to try out).  In the meantime we tried extracting DNA and doing some PCR for some damsel flies and that didn't even work...!  Then this morning Kenton found me and said that he needed to show me something.  After taking a look I went back to my office to get our microscope camera and set it up to record some images.

Some background.  Generally chromosomes are very tiny and difficult to image with standard equipment.  However, some cells undergo endoreduplication where the chromosomes divide but the cells do not.  In some of our muscle and liver cells there are actually four (tetraploid) or eight (octoploid) copies of each chromosome instead of the normal two copies (diploid--one from each parent) that we are used to thinking about.  In older fly larvae that are about to pupate and metamorphose into adults the salivary gland chromosomes divide approximately nine times resulting in 512 copies of each of two starting chromosome copies or about 1,024 total copies per cell.  In addition they stay physically associated with each other so that they form large (gigantic on a cellular level) structures.  Each individual chromosome is made up of about half DNA (actually a single very long strand of DNA) and half proteins that organize the DNA into the structure of the chromsome.  The density and composition of proteins changes along the chromosome so that different regions have different staining levels that result in a banding pattern.  In polytene chromosomes all 1,000+ copies are organized together into these large structures.

This first image below is of some cells in larval salivary glands under the microscope.  Within the cells you can make out the nuclei and maybe the first hints of the darker chromosome structure.

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The next image is with higher magnification.  You can see the cell nucleus as spheres with darker chromosomes winding around.  In preparing the tissue for these images we apply a lot of pressure to purposely try to rupture the cells and spread the chromosomes (i.e., this is not necessarily what they would normally look like).

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In the image below one of the nuclei has been squashed and "spread" a bit more than the others.  The chromosomes look like darker squiggles.

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The image below is zoomed in to an even higher level.  You can start to make out the dark and light banding pattern along the chromosomes.

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Below are two nuclei.  The one on the left has been spread more than the one on the right, and the banding pattern is easier to make out---at least for the section that is spread.

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In the image below it looks like some of the strands are starting to shear.  However, the end of one chromosome is visible to the right so I wanted to focus on it and see if I could identify which chromosome it belonged to.

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Below is zoomed in some more on this area, and this is pushing what the optics in our system are capable of.  Just down from the end of the chromosome is a "puff" where it widens out.  These correspond to areas with active transcription of genes.

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There are cytological maps of the chromosomes available at flybase to compare to.  Here is the image at the end of the X-chromosome.

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Based on the similarity in the banding pattern and the presence of the puff I suspect this is the end of the X-chromosome.  The gene yellow (y) which affects body pigment is at the very end and white (w) which results in red eyes when functioning correctly (and happens to be the first genetic variation discovered in Drosophila melanogaster by T. H. Morgan's lab) is just down from the puff at the second dark band.

Ironically the best images today were not from the orcein stained slides but from Hoechst 33258 stain, but strangely the chromosomes did not appear to fluoresce under UV light.  Kenton wrote down all the steps he took today and the most important thing for now is to replicate the process tomorrow to make sure we can get good images of the chromosomes.  Then we will try to fine tune and troubleshoot some more.

Onward to Sydney and then back to Hawai'i

After Melbourne Jolene and I flew to Sydney to attend the GSA annual meeting where both of us gave presentations.  I talked about our plans with underdominance here in Hawai'i and Jolene talked about her work with immunity gene diversity in some island bird species.  After the meeting I returned to Hawai'i but Jolene is traveling on to New Zealand for an invited talk at Otago.

A great day in Melbourne!

We met with Prof. Scott O'Neill and his lab today.  We discussed what we are trying to do in Hawai'i with underdominance and possibilities with Wolbachia in mosquitoes in Hawai'i and Wolbachia strains from Hawaiian Drosophila species.  He is dean of the college and very busy but took time to talk with us and set up a tour and meetings with people working in his lab.  Their main focus is on modifying mosquitoes to block the spread and transmission of Dengue (the virus that causes Dengue fever) by using Wolbachia injections.  Long story short, certain Wolbachia (a type of symbiotic bacteria) strains can infect and be stably transmitted over generations in mosquitoes (and many other insect species).  If at a high enough starting frequency they increase in frequency in local populations and move toward 100% frequency.  Importantly, Wolbachia has also been shown to reduce the transmission of certain disease causing viruses and Plasmodium.  Originally they used a MelPop strain (from Drosophila melanogaster fruit flies) that reduced mosquito lifespan and virus transmission but it reduced fitness of the mosquito too much in the wild and would be lost.  They switched to a Mel strain (also from Drosophila melanogaster) that also (incompletely) blocks transmission and is able to become stably established in the wild.  They are conducting releases of Wolbachia infected mosquitoes in Australia and work in SE Asia.  We also discussed the regulatory aspects of their work both in Australia and internationally.  We got to see how they were raising and working with the mosquitoes, from cages to collecting and feeding to biosecurity setup (most of the work is at level 2 but they have a level 3 lab for the Dengue virus work).  We even got to see them do mosquito (Aedes aegypti) egg micro-injection and Jolene had a shot at arranging the eggs under a microscope and injecting them with a new experimental Wolbachia strain using a micromanipulator.  The people here were extremely helpful and friendly and we got to ask them questions for several hours---we were literally there all day and they took us out to lunch midday.  We have plenty of new pointers to help us with working with the mosquitoes in our lab.  The data they are getting back from the releases are impressive.  They are collecting mosquitoes from the release sites and areas around them weekly and plotting maps of the increase in allele frequency of the Wolbachia infected mosquitoes and the geographic spread from the release range.  On top of this you can see population fluctuations, drift, the effects of rainfall on the wild populations, etc.

A nature walk

I had some free time after the conference ended and before my flight back to Hawai'i---this is very rare for me.  So, I took advantage of the opportunity to go for a nature walk around the UMBC campus.  I saw lots of plants and animals including several deer, rabbits, squirrels, a groundhog, geese, barn swallows, etc.  I also brought along a camera and got photos!  This is a space holder post for now, because I have to get get ready to the airport soon, but I will update with some pictures and commentary as soon as I get a chance.