Author Archives: Floyd A. Reed

Our DNA Barcoding Project

The basic idea of DNA barcoding is that essentially every species has a unique DNA sequence that is diagnostic of that species.  It is inefficient to sequence different genes for comparison of different samples or even whole genomes, so it makes sense to pick a common place in the genome for comparison across species.  A great deal of effort has gone into determining the best gene sequence to use for barcoding.  It should be variable enough that essentially all species are unique, but not too variable (at least in the primer binding regions) so that the same set of primers can work in almost all species and that reasonable sequence alignments can be made to compare species.

Why?  Many species are described at only one stage of their life cycle.  In many insects the larvae look alike but the adults are completely different from each other (or in one famous case the opposite is true, link).  Similarly, many marine fish have tiny larvae in the plankton that look nothing like the adult forms, and there are cryptic look-alike fish species.  There is an organized effort to barcode as many fish species as possible (link).  And take the example of mushrooms.  The much more common mycelium fibers growing in soil look nothing like their occasional fruit---the mushrooms that we are used to.  Also, there is the problem of identifying what unobserved species are in an environment from the DNA they leave behind in the soil or water.  In many cases, like monitoring the species in the environment, and understanding which environments are important throughout a species' life-cycle, it would be helpful to be able to connect the dots and to know precisely which described species are present at different developmental stages or just in general.

Also, in a more forensic sense, it is handy to be able to test food and find out which species it comes from.  There are laws about which species can be harvested and how food ingredients have to be advertised.  DNA barcoding provides a useful and powerful way to verify the claims of a label (e.g. link).

Finally, there is a educationally valuable, direct, tangible connection for the students; a sample they find themselves and decide to select connects to modern molecular biology in the lab and then the results connect to the natural history of evolution on our plant that ultimately connects all living organisms.

However, DNA barcoding has been surprisingly polarizing; it has plenty of lovers and haters in biology.  Classical taxonomy is a skill that takes years to develop for a specific group of species of the taxonomist's specialization.  Some see DNA barcoding as a cheap and easy way to circumvent this process, resulting in a rapid naming of different species in a sample with no real deeper understanding of the important differences between them.  Like any new technological approach there will be an adjustment period as DNA barcoding finds and settles in on its role in biology.

(For more background information about DNA barcoding see the iBOL website, the introduction at CBOL, CSH's barcoding 101, and the DNA barcoding wikipedia page, which includes a discussion of criticisms that are not as forthcoming on the other websites.)

I am teaching a genetics course with a laboratory component (with the first lab last fall semester).  In my research lab we started using DNA barcoding to identify which species were present as mosquito larvae in standing water.  I expanded this to test some other species I came across and decided to include this as a project for the undergraduate students.   In many ways, Hawai'i is one of the most uniquely diverse places on the planet, so describing this diversity capitalizes on a natural strength of Hawai'i.

COI, cytochrome c oxidase I, found in the mitochondrial genome, has, for various reasons, become the standard barcoding locus for animals.  Both matk and rbcL have been promoted as barcoding regions in plants and ITS in fungi.  We have settled on rbcL for plants because the PCR amplification is more reliable (in our lab experience) over a diverse range of plants than matK.  We have not tried fungi samples yet but may include them soon.

Last semester I asked the students to bring in a (legally and safely obtained) tissue sample for DNA barcode testing.  We spent some time, effort, and resources on this and I don't want the results to simply disappear at the end of the semester, only existing as a student experience.  So, I have been curating the results and adding them to an online database so that researchers and students all over the world can access the results and use them in their own research---including a growing resources for students to compare results to in our teaching lab.  This has been somewhat on the back-burner as a free weekend project and I have just now finished submitting the animal samples.  There are even more plant samples, especially flowering plants, that I expect to finish curating this summer.  So, this post is to talk a bit about the initial results focusing on the animal samples.

Also, in another sense, I am also trying to learn more about the natural history of Hawai'i, and have my own gaps in understanding of species taxonomy---especially in the tropics.  Generating DNA barcodes from samples collected here provides a focus to help fill these gaps in.  Without this focus, on working out the relationships of the few species at hand, the bigger picture is too overwhelming.  This creates a backbone for us to connect new species to as we come across them in the future.

The BOLD Systems (Barcode of Life Data Systems) website is a publicly searchable repository of DNA barcode data.  In addition to the sequences you can also record the exact location a sample is collected from, when it was collected, an image of the sample, etc.  On their front page there is a map highlighting the geographic distribution of samples in the database.

BOLD-world-map

We are contributing to the purple dot over O'ahu in Hawai'i.  Zooming in, here is a map of our samples in O'ahu.

BOLD-map-oahu

And even closer showing the Honolulu/Manoa region around campus.

BOLD-map-manoa

Currently we only have 60 samples in the online database, but I am expecting that to quickly grow as we optimize the lab project.  Here is a summary by taxonomic class.

BOLD-data-summary

40% of the samples are from flowering plants (Magnoliopsida), while 30% are, not surprisingly, from insects.  There is also a project image browser.

BOLD-image-library

And summaries of the data like this accumulation curve.

BOLD-accumulation-curve

This shows the slightly diminishing returns as our sample collection progresses.  For 60 samples we have (at least) 37 unique species represented (however, several samples could not be identified to the species level).  Some species are beginning to be sampled more than once.

I organized the samples taxonomically, again currently only the animal ones, on this page (link) (the page is currently a draft and will need some rounds of cleanup and revision) with links to aid learning more about each taxon and links to the BOLD entry for each sample.  I also indicate if the species is native, endemic (only found in Hawai'i), brought here by the ancient Polynesians, threatened or endangered (students are given clear guidelines about what they can and cannot legally collect (no coral, no vertebrates except under special conditions, no threatened or endangered species, etc.) the endangered samples in the database were obtained legally (students are also given common sense safety guidelines for collecting)).

Many of the samples could be identified down to a species level, but some could not, which can raise a flag of interest to check out further as a possible new species.  I suspect the mantis sample is a Chinese mantis (Tenodera sinensis) but I am not certain and there are some look alikes among this group so I left it as unknown at the genus and species level.  On the other hand, one of the students brought in an insect that seems to fit into the cockroach family (Blattodea) but is not turning up any obvious close genetic relatives on the databases (BOLD and GenBank).  A similar story goes for a Xanthid crab and for a hover fly.  However, as more samples are collected and the database grows we might get some matches in the future from positively ID'd specimens that will help resolve this.

Finally, I couldn't resist generating a phylogenetic tree using our data and BOLD's online tools.  DNA barcoding is not designed to behave well in terms of reconstructing more ancient relationships among species.  It is aimed at resolving species identifications.  So I was surprised at how well the data preformed in clustering the insect species (which contain our largest number of animal samples).

barcode-insect-tree-labeled

The bar at the upper right represents a distance in the tree of 2% sequence divergence.  The tree is rooted with a crustacean (an arthropod but not an insect) sequence (not shown).  The flies (Diptera) were grouped together and within this we get mosquito, fruitfly, and hover fly groups.  Within the fruitflies we also get a Hawaiian Drosophila clade.  Finally we see that one Drosophila heteroneura sample is distant from Drosophila silvestris while another D. heteroneura is quite close to D. silvestris.  This is not as surprising as it might first appear because these two species are found to hybridize quite frequently in the wild.

A Hawaiian Octopus and Cowrie

This is a rare find.  In another video that I made with my underwater fishing pole camera (see earlier post here), I (unknowingly) went right by an octopus (he'e in Hawaiian, also commonly called by the Japanese name tako here in Hawai'i) in a shallow Mokule'ia reef on O'ahu.  He/she blends in well but the suction cups on the upturned arm give it away.  This might be a day octopus (Octopus cyanea) but this is really just a guess.  Clicking on the image below will link you to the original video.

oct

Even better, the octopus has caught a cowrie!  It is hard to see but as the camera passes over you can catch the spotted pattern of (probably) a reticulated cowrie (Mauritia maculifera) just behind and under the octopus. Here in Hawai'i, octopi love to hunt cowries (leho in Hawaiian) and the cowries grow to some of the largest sizes in the world!  However, large ones are now rare in the waters around O'ahu.  In the image below that octopus is out of focus in the foreground and the mottled pattern of the cowrie shell is visible just behind it.  (There is also a sea urchin which gives a size comparison.)

cow

He'e love leho so much that they are used as a lure for catching tako.  You can read more about that in the links from here, here and here.

Our new sepia allele is now listed in flybase

Fruit flies, like humans, carry a lot of recessive deleterious alleles.  When wild chromosomes are made homozygous there is a dramatic loss of fitness.  We have been curious about the effects of this and Louis Boell aggressively inbred wild caught Drosophila melanogaster for a few generations by reestablishing the line from single females each generation.  Not only did we see a change in behavior of the flies, several mutant phenotypes appeared, more than I expected.  (We also carefully measured changes in wing morphology during several generations of inbreeding, to compare to Louis' results comparing wild and lab mice jaw morphology, but saw no significant effect.)  Myles Tabios, a biology major and former student of my BIOL375 Genetics class, investigated one of these mutants in the lab and discovered it was a new allele of a gene called sepia (which encodes a PDA synthase enzyme) by a classical genetic complementation test.  Then he designed his own PCR primers,  sequenced the gene and found the mutation results from a 40 base pair deletion that completely disrupts the gene sequence.  I checked the Bloomington Stock Center (that maintains mutant Drosophila stocks as a resource for genetics researchers) and they only have one sepia allele: sepia[1], the first allele discovered in Morgan's lab in the 1920's.  Until now sepia[1] was also the only allele that was described on a molecular level.  I contacted them and said that we likely have an amorph (a completely non-functional allele) and they are interested in adding it to the collections!  They try to maintain at least two amorphs of each gene for testing...  I sent them the stock and the allele, sepia[Kiel] (the allele originated from a wild population in Kiel, which incidentally is where Drosophila melanogaster was first described by Meigen---but that is a different story), has now showed up in the newest version of flybase (link).

flybase_se_Kiel

It's nice to have a study that initially found no positive result (no change in morphology with inbreeding) to end up resulting in a lasting impact no matter how small it might be.

We have egg rafts!!!

For the last six months we have been trying to get Culex mosquitoes to breed in the lab so we can establish stable lab populations isolated from wild Hawaiian Culex.  As mosquitoes go, Culex are notoriously difficult to rise in the lab.  (In contrast, Aedes albopictus, the "tiger mosquito," are comparatively easy, almost trivial, to raise.)  We tried lots of approaches the past six months.  We caught wild larvae and brought them into the lab to raise.  We were quickly able to get the larvae to live but we could not get the adults to feed.   Then we figured that out but they would die quickly, after a couple days.  It took us a long time to troubleshoot that but we seem to have finally hit on the trick.  ...then we could not get them to mate and lay eggs.  We brought in egg rafts from the wild (Culex lay their eggs in a floating cluster called an egg raft) to drive the numbers in the cage up but nothing seemed to work...  A large part of my recent trip to NCSU last month was to ask for advice on how to raise Culex.  We incorporated this with some new things we were able to come up with and it looks like it is finally paying off!  I am not putting all the details here (although that is my first impulse) because we are planning to publish the new tricks we discovered as a methods paper.  Part of the frustration along the way is that we want to ultimately be able to genetically modify the mosquitoes; but this is useless if we can't maintain the modification for more than a single generation.  Now that we have complete lab generation cycles we can allow the mosquitoes to adapt to the lab for a few generations to optimize everything before starting genetic modifications with microinjections of plasmids.

Long story short, we have our first egg rafts from lab reared mosquitoes today!  This morning it was one and now we have two!  They are normal size like wild rafts.  They should hatch in the next 24 hours...

...then on to Sweden! Conservation Genomics

After NCSU in Raleigh (which by-the-way I should say it felt strange to be back in the east coast in March with near freezing temperatures, etc.) I was off on an overnight flight to London Heathrow then to Stockholm to a conservation genomics workshop (link) organized by Aaron Shafer and Jochen Wolf of Uppsala University.  A man was waiting for me at the airport with my name on a card as soon as I walked out.  He drove me out into the country to the place the workshop would be held for the next few days.  The location was amazing and the most surreal aspect for me in the rapid transition from Hawai'i.  It was a medieval mansion/castle (Wiks slott) in a rural Scandinavian farm and forest landscape.  After arriving it started snowing and several inches accumulated.  We all stayed on site so there was no travel back and forth to the nearest town.  We ate meals in a lower floor with long tables, small barred windows set deep into thick walls, and arched walls and ceilings.  I soon discovered the castle had hidden passage ways, suits of armor, etc!  I also had jet lag so in the middle of the night I couldn't sleep so I went for a walk, and to top it off there was a full moon hanging over the castle!

MeAtVampireCastle

The point of the workshop was to discuss if there was a general reason to include new genome level technologies (versus classical molecular genetics of a few loci) in species conservation genetic applications.  The discussions and presentations were a lot of fun and I'm hopeful some impact will come of it. It was also nice to be back in Europe after being away for 2 1/2 years.  There are lots of things I like about Europe and I was a bit homesick.  It was also fun to listen to and read Swedish and to try to decipher the meanings.  I was surprised that I could recognize some words based on their similarity to German and/or English.

Finally a small world note.  I already knew Jochen Wolf, one of the organizers, because we worked at the same institute in Germany before he moved to Sweden and I moved to Hawai'i.  Also, the other organizer Aaron Shafer was at the same institute in Canada with Jolene Sutton who is working here on the mosquito/avian malaria project.

...and I'm back. First a note about NCSU.

That was a whirlwind trip.  It was very surreal to be on a tropical island one day and in a castle in Sweden set in a snowy landscape on another day.  First of all I want to post the slides for my presentation at NCSU in Raleigh, my first stop.  Here is a link to my presentations page.  At NCSU I was asked to include some background on the social landscape here in Hawai'i as it pertains to genetic pest management.  This is something I am trying to get my head around so I presented my understanding at this point---how GMO crops and classical biocontrol (which genetic pest management tends to be initially compared to) are connected to issues of sovereignty and self determination in Hawai'i.  I also wanted to make the point---and was able to discuss this point to a great extent---that we tend to only think of regulatory and research funding agencies and labs doing research as the major players and tend to forget about the public until a technology is ready for deployment (then it is too late to make major changes and we tend to try to convince the public to accept what we have already decided).  I met with a lot of people and got a lot of good feedback, from techniques to raise and work with Culex mosquitoes in the lab to embedding non-biologists in the group for feedback on social issues to shape the ongoing research program.

NCSU has an NSF IGERT program to focus on cross disciplinary genetic pest management (link).  I was able to meet with a lot of people in this program during the visit.  At the moment the current cohort of students is focused on invasive mice on islands and ways to suppress the population.  They are doing some pretty exciting stuff.

Traveling

Sorry, I've been really bad about making posts over the last few months.  Mostly this is because I am teaching two new classes this semester and it has taken a lot of my time.  However, this week I am traveling!  First to visit NCSCU and then to a Conservation Genomics workshop in Uppsala.  I will post updates about each in the next few days.

Student Genetics Book Reading Recomendations

Towards the end of the semester I asked my students for their recommendation on extra credit reading assignments next year.  Here is a plot of their response with books that were suggested by two or more students (single entries, which were slightly more than a quarter of the total response, are grouped together).

BookReccomend

The large number of students that recommended The Selfish Gene by Dawkins surprised me.  Actually it makes me a bit suspicious that they are already reading this for another class...  The four next most popular books are ones that were options this year, so I suspect they are a bit inflated, but are still good books that are relevant to genetics.  Controlling Human Heredity by Paul is also one that was among the options this year but it was not recommended as highly--it was dry and less enjoyable to read, but I still think it is an important book for them to be exposed to (the history of eugenics programs in the United States).  Still, the students have helped me identify several books that I can take a closer look at for reading options in class next year.

Here are some of the single entry student recommendations that caught my attention (this is not an endorsement for or against):

Lords Of The Harvest: Biotech, Big Money, And The Future Of Food by Daniel Charles

Genetically Modified Athletes: Biomedical Ethics, Gene Doping and Sport : by Andy Miah

Genetic Witness: Science, Law, and Controversy in the Making of DNA Profiling  by Jay Aronson

Evolution: Making Sense of Life by Carl Zimmer and Douglas Emlen

Everyone Here Spoke Sign Language: Hereditary Deafness on Martha's Vineyard by Nora Ellen Groce

Deep Ancestry: Inside the Genographic Project by T. Spencer Wells

Deep Ancestry: Inside the Genographic Project by Spencer Wells

Banana: The Fate of the Fruit That Changed the World by Dan Koeppel

The Red Queen: Sex and the Evolution of Human Nature by Matt Ridley

Power, Sex, Suicide: Mitochondria and the Meaning of Life by Nick Lane

Why We Get Sick: The New Science of Darwinian Medicine by Randolph M. Nesse and George C. Williams

Incognito: The Secret Lives of the Brain by David Eagleman

The Demon-Haunted World: Science as a Candle in the Dark by Carl Sagan and Ann Druyan

Mapping Human History: Genes, Race, and Our Common Origins by Steve Olson

Mutants: On Genetic Variety and the Human Body by Armand Marie Leroi

Crying Hands: Eugenics and Deaf People in Nazi Germany by Horst Biesold and Henry Friedlander

Survival of the Sickest: The Surprising Connections Between Disease and Longevity by Sharon Moalem and Jonathan Prince

Invasion of the Genes: Genetic Heritage of India by B. S. Ahloowalia

The Violinist's Thumb: And Other Lost Tales of Love, War, and Genius, as Written by Our Genetic Code by Sam Kean

Endless Forms Most Beautiful: The New Science of Evo Devo by Sean B. Carroll

War Against the Weak: Eugenics and America's Campaign to Create a Master Race by Edwin Black

The Family that Couldn't Sleep: D.T. Max

The Making of the Fittest: DNA and the Ultimate Forensic Record of Evolution by Sean B. Carroll

The Genetics Revolution: History, Fears, and Future of a Life-Altering Science by Rose Morgan

The Philadelphia Chromosome: A Mutant Gene and the Quest to Cure Cancer at the Genetic Level by Jessica Wapner and Robert A. Weinberg

The Emperor of All Maladies: A Biography of Cancer by Siddhartha Mukherjee

Denialism: How Irrational Thinking Hinders Scientific Progress, Harms the Planet, and Threatens Our Lives by Michael Specter

The Ethics of Genetic Engineering by Roberta M. Berry

Is It in Your Genes?: The Influence of Genes on Common Disorders and Diseases that Affect You and Your Family... by Philip R. Reilly

The Mismeasure of Man by Stephen Jay Gould

Abraham Lincoln's DNA and Other Adventures in Genetics by Philip R. Reilly

 

 

 

A Few Common Tide Pool Fish Species

Final grades for the semester were due on Christmas Eve the 24th, and I had students calling me on my office phone and emailing me to ask about their grades up until the last minute on the afternoon of the 24th.  Then I was looking forward to a much needed break and went fishing with a video camera.  I'll try adding one of the videos here. It is down-sampled a bit to reduce the file size online.  This is a video of a Mokuleia tidepool on O'ahu.

You can clearly see three (?) fish species.  I am not a marine biologist (this is also part of a personal project on my part to learn more about marine natural history here in Hawai'i), so be suspicious of my attempts at identification.  I am fairly certain that two of the species are sharnosed mullets (Neomyxus leuciscus, uouoa in Hawaiian)--the larger long ones--and (juvenile)

SNMullet

blackspot sergeants (Abudefduf sordidus, Kūpīpī)--the ones with false "eye spots".

BSpotS

I suspect that flagtails are also present (Kuhlia spp., ʻāholehole) and the most common in this video, but I am less confident about their identification (see the update below, these are actually reticulated flagtails, Kuhlia sandvicensis).  There are also two brief glimpses of a third species that I suspect is a "rockskipper" zebra blenny.

If this works well then I will try uploading some more videos.

-----

I sent a picture of the flagtail from the video to some of my coworkers to ask about its identification.

HIflagtail

Dr. Kathleen Cole wrote this in reply:

Hi Floyd.
This looks more like the reticulated flagtail, Kuhlia sandvicensis (also referred to as aholehole). It has a broader distribution than K. xenura, the latter of which is an endemic. Distinguishing features include a pale gray rather than dark grey tail, sometimes with a pale margin (which shows up in your picture), and a flattish, rather than slightly concave, head profile. The eye tends to be smaller in sandvicensis, but that doesn't help much unless you have the two species side by side.
Juveniles of both species are found in shallow coastal waters and tide pools so are easy to encounter.
Kassi

I also came across an article about the flagtails in Hawai'i that discusses some of the difficulties and confusion in distinguishing between them.

Keeping up with email

This is something that I have struggled with for a long time and that has puzzled me.  How do people keep up with email?  I have tried all sorts of strategies.  I have over 200 students in my class this semester so I ask them to put the class code in the subject line to filter those to one folder.  I use completely different email addresses for work and family and typically do not use my work email when at home and vice versa.  I have a filter for people within the department that goes to another folder.  However, I am still way behind even just sifting through all the messages and keep missing important ones.  Here is a cropped screenshot of the times emails have come in between 10:00am and 10:30am this morning, and this is only for filtered emails within the department.

email-times

In this example, I am getting an important email every five minutes; these are from different people about different things.  Also, I did not cherry pick this; I'm sure if I searched I could find a denser set of messages.  It feels like as soon as I reply to one another one comes in.  And, this is not including the several phone calls and several people that stopped by my office this morning as well.  If replying to people was my only job I could keep up with it but it is not.  I have to prepare for class, grade exams, apply for grants, not to mention work in the lab, etc.

I know I am just complaining but I honestly don't see how people can keep up and still get everything else done.

-----

Update: A busy morning (Dec. 3).  I couldn't resist a screenshot of my inbox times.  (I can't wait for the semester to be done.)  ... I was interrupted by six people coming by my office before I finished posting this.

inbox-times