Author Archives: Floyd A. Reed

What is the best approach to address avian malaria in Hawai'i right now?

I started this lab blog in the interest of transparency, so that the public could see what we were doing. To that end I am putting down my recent thoughts about what we should/could be doing in the near future to address avian malaria and native bird conservation.

I have been to several meetings about Hawaiian forest bird conservation since I arrived in Hawai'i five years ago. I have talked to people that have been going to these same types of meetings for 25 years or longer. There is a frustration that has been communicated to me and that I am also sensing that at each meeting the urgency of doing something is discussed but that it doesn't translate into sufficient action in the field. (However, positive things have occurred such as establishing reserves, reforesting, and bringing some species into captive breeding programs, but these alone are not sufficient.) The birds have been going extinct for over a century and are continuing to be lost. In the next five to ten years, unless something changes dramatically, we will loose several additional species.

At this last meeting a person (I am not sure if they want to be identified here) spoke with emotion about the po'ouli (Melamprosops phaeosoma) which was lost in 2004. Part of the problems were egos, infighting, political maneuvering, and personalities that stood in the way of action. By the time it was decided that they should be brought into captivity it was too late.

I have been thinking critically about what I can do that would be most effective and most likely to succeed and to try to step back and ask, if as much ego, self interest, politics, and personalities were removed from the equation as possible, what approach suggests itself. I came here to Hawai'i with the idea of porting underdominance that we had engineered in fruit flies to (non-native introduced) Culex mosquitoes in order to transform the wild population to not pass on the parasite that causes avian malaria. I am still interested in doing this, but several things are making me question if this is the best course of action in the short term. Laven first proposed using cytoplasmic incompatibility (due to Wolbachia) to suppress wild populations in the 1960's and there have been successful applications of this. Other labs are working on using Wolbachia in insect pest management approaches in various ways. Because of this I have purposely steered clear of cytoplasmic incompatibility and tried to develop my own approach: both to create a new option that could be used and to not interfere with, crowd the field, or compete with other efforts.

However, it is now five years later and no one has (in the lab) a Wolbachia based option in Hawai'i. I have been trying repeatedly to raise funding from NSF and NIH in addition to other sources to conduct our underdomiannce work (as well as develop new sterile insect techniques), and have been unsuccessful (which is extremely personally frustrating). Writing these grant applications takes time and at this point they have only wasted time. I also have been working on several scientific publications but combined with my teaching load and committee work this also slows down what I could be doing in the lab. At the end of the day underdominance is a genetic modification, which may slow down regulatory approval and inhibit public acceptance and it is (probably) more technically challenging to engineer then a Wolbachia approach. Importantly, since I am having to pay for this out of pocket, with my own personal funds, it is also (probably) much cheaper to develop a Wolbachia based approach.

We have established a colony of Culex mosquitoes in the lab from local populations of Culex here in Hawai'i and have optimized our rearing technique. We have in-lab microinjection instruments and a technique optimized for Culex mosquitoes and have our injection survival rate up to 13% now. We also have Drosophila melanogaster, Drosophila simulans, and access to Hawaiian Drosophila; all as sources of various strains of Wolbachia. We can raise our Culex with tetracycline to remove their endogenous Wolbachia. Isolate Wolbachia from these other insects and inject it into our Culex. (Or just inject new Wolbachia---do we really need to clear the old Wolbachia first?) Then set up some crosses and test if the cytoplasmic incompatibility between the two types works and they do not have viable offspring. This is not a genetic modification in the sense of inserting new DNA sequences into the Culex nuclear genome, which may aide regulatory approval and public acceptance.

This could all proceed very quickly; especially if I take a temporary break from writing new grant applications and publications and stop all new projects (which sounds dangerously close to career suicide but I have tenure now so I might as well use it to get something done). I could then provide the mosquitoes to other labs here in Hawai'i for additional testing (I know Carter Atkinson is already interested in this approach and I asked him if he minded if I went ahead a tried to do this; he agreed and gave me the go ahead, which is great that we are already not conflicting with each other!) and ultimately this could be an option to use either alone or in combination with other techniques: releasing males with a different type of Wolbachia to mate with wild female Culex, so that there are no offspring, to suppress the population of mosquitoes in the next generation.

Washington Post article on gene drive

https://www.washingtonpost.com/news/energy-environment/wp/2016/09/07/this-new-gene-technology-could-wipe-out-entire-species-to-save-others/

There are several big news items recently, a lot of it corresponding to the IUCN conference here in Honolulu, that I need to post updates about. (And I have fallen behind in posting in general as the classes have started up this semester.) I just returned from a "mosquito meeting" on the Big Island, more about that soon. One big item in the lab is that I am going to change our lab strategy temporarily from underdominance to Wolbachia with the mosquitoes (and more about that soon). For the moment here is a link to a Washington Post article by Chelsea Harvey who called, interviewed me by phone late last week, and quoted me in the article.

The Kākāpō Speciome: the first 40 completed

The kākāpō is a large flightless New Zealand parrot and there are only 125 individuals left.  The Kākāpō 125 project aims to sequence the genomes of all the individuals in the species.  This will be the first time that an entire species genomes (a speciome?) has been sequenced.  This could be very useful for evolutionary genomics as all future individuals will be a result of an already sequenced ancestor. The projects stated objective:

"To sequence the genomes of all remaining 125 kākāpō. This will allow us to better understand the genetic variation between individuals which in turn allows us to breed pairs even more selectively. It also serves as a foundation for potential future genetic intervention in order to more quickly and successfully breed kākāpō."

The project recently made an announcement that the sequencing of 40 individuals has been completed and the sequence data will be freely shared with interested labs.

Other species such as the ʻalalā may soon follow.

National Geographic on Gene Drive, Avian Malaria, and Hawai'i

National Geographic just posted an article that includes CRISPR technology, gene drive, and mentions the situation of avian malaria in Hawai'i (link).

In related news, we just resubmitted our application to NIH, again, for funding to to use CRISPR and gene drive technology in genetically modifying Culex mosquitoes to address both avian malaria in Hawai'i and human diseases.

Vox: The Seven Biggest Problems in Science

Vox posted an article, "The 7 biggest problems facing science, according to 270 scientists". I would have cast some of it differently and there are some alternative issues I might have focused on but it is worth reading over.  It closely mirrors a conversation I had with a grad student last week about struggling to deal with a feeling of cynicism and pessimism in science versus trying to stay positive and focused on research. The section headings are copied below to give you a feeling of what is discussed.

  1. "Academia has a huge money problem"
  2. "Too many studies are poorly designed"
  3. "Replicating results is crucial — and rare"
  4. "Peer review is broken"
  5. "Too much science is locked behind paywalls"
  6. "Science is poorly communicated"
  7. "Life as a young academic is incredibly stressful"

Can bacteria or parasites take advantage of RNAi against host genomes?

This is an idea I've had floating around in the back of my mind for a few years now.  I keep some ideas secret in the short-run because I hope to be able to investigate them some day. However, I also have to face reality, with my teaching load and limited research funding there are a wide range of projects that I simply won't be able to follow up on.  I figured it would be better to just put the idea out there so someone can check into this if they want.

RNA interference is a widely used and very useful genetics tool where gene expression can be reduced by double stranded RNA that matches the mRNA of the target gene. There is a great deal of literature on this and I am not going to attempt a summary or review of this technology and its natural role here. Instead I want to focus on a curious fact. For a wide range of species you can simply feed bacteria to the organism that have been modified to express double-stranded RNA and this can create phenotypes by silencing certain genes.

Okay, if this is so easy and straightforward (and quite short sequences of RNA can accomplish this) have any bacteria evolved to take advantage of inactivating genes of a host Eukaryote? Perhaps as a pathogen to block immunity and promote infection, or in a more symbiotic role to tweak host phenotypes? For that matter have any Eukaryotic parasites evolved this ability to excrete dsRNA or antisense RNA to alter host phenotypes?  Part of this is a comparative genomics question and if candidate sites are found the follow-up study of activating or inactivating the gene sequence can be done to see if there is a response across species.

Culex Egg Rafts

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Culex mosquitoes eggs stick together and are laid in "rafts" that float on the surface of water. In the picture above are four freshly laid egg rafts from our lab mosquito colony, each laid by a single female. They are on the end of a paintbrush that we used to lift them out of the water. They get darker as they age. The two lower rafts are almost the same age; the dark one above them is older and the very light one at the top is less than 1/2 an hour old. To prepare them for microinjection you want to use the very youngest one like the raft at the top. They are seperated from each other and arranged on a glass slide and injected using a microscope and micromanipulator for fine detailed control. They also harden as they age and the light ones can be injected without breaking the thin glass needle that is used. Once they harden you have to use a brief treatment with bleach to remove the out egg surface (chorion) which lowers the rate of survival.