Author Archives: Ruth Mauntz

uniprot SPLAT

We spent most of this morning working on our annotated star transcriptome in IPython, and I have to say it’s been a humbling experience. I could list off a series of unfortunate events between me and my Mac this morning, but I clearly had no idea what I was doing this morning in the midst of a flurried scripting extravaganza.

Some hard lessons learned from working on transcriptomics in computer lab today:

1) ASK/Google if you get stuck in the scripting. There are so many resources available (explainshell.com, wiki, forums, ROSALIND). Keeping this class blog and going over scripts that have been tweeted, screenshot, and posted has a huge help in retracking my steps to fix potential issues and definitely sells me on open network science.

2) Keep practicing!

3) Save your IPython notebook (which I thought I did on Day 1 but can’t find anywhere).

3) Get a good night’s sleep.

4) Keep a written list of commands until you know them. There are also some other tricks to helping keep track of your code such as ‘#’ for when you want to make a note in the middle of your script, and ‘!’ which always precedes a command (and you definitely don’t want to include a space).

Learning new languages is guaranteed to be difficult but I am amazed at how quickly it is to work with the thousands of contigs Steven assembled and can’t wait to start dipping our hands into looking at differential expression between our sea star samples. I’ve linked to a pdf to a 2012 Nature paper describing the use of software such as Cufflinks and TopHat (which you can use in Galaxy!) to assemble and analyze RNA-seq reads.

http://www.nature.com/nprot/journal/v7/n3/pdf/nprot.2012.016.pdf

we be all night

Special thanks to Queen B for helping us give voice to our passion for science. More substantial posts to come once I figure out how to save my iPython scripts!

“Stuck in Lab” (feat. Sarah, Ruth, and Reyn)

I’ve been thinking, I’ve been thinking
I get filthy when that vector get into me
I’ve been thinking, I’ve been thinking
Why can’t I keep these slides in focus?
Laby I want you, na na
Why can’t I keep these slides in focus?
Laby I want you, na na

Primers on ice, primers on ice
Feeling like an animal with these cameras all in my grill
Flashing lights, flashing lights
These bands are faded, faded, faded
Laby, I want you, na na

Can’t keep my eyes off that UV
Laby I want you, na na
Stuck in lab, I want you

We woke up in the morning saying,
“How the hell did this shit happen?”
Oh laby, stuck in lab we be all night
Last thing I remember is your beautiful cultures spinning up in that flask
Stuck in lab

We be all night, in laaaab lab
We be all night, in laaaaab lab

We be all night,
And everything alright
No complaint, your FISH is so fluorescent under these lights
Laby, I’m thinking,
Park it in the rig 7 to 11
I’m sterilizing, st-sterilizing, if you scared, call that sales rep
I’m thinking, get my brain right
Filter water, oyster life
Kim wipes, we wipe it out like wash rags we wear it out
Laby, I’m thinking, I’m yelling, on the phone with the sales rep
Then I lay the transect halfway then write it on my clipboardt, clipboardt, clipboardt
Riding on that boat, riding, riding on that boat
I’m culling on that, culling, culling on that eel grass
Been spinning all this, spinning, spinning all in this good, good

We woke up in the morning saying,
“How the hell did this shit happen?”
Oh laby, stuck in lab we be all night
Last thing I remember is your beautiful cultures spinning up in that flask
Stuck in lab

We be all night, in laaaab lab
We be all night, in laaaaab lab

Hold up
That Lisa is the shit if I do say so myself
If I do say so myself, if I do say so myself
Hold up,
Intertidal got doused time to back up all of that mouth
That you had all in the car, talking ’bout you the baddest bitch thus far
Talking ’bout you be counting that turd, I wanna see all the laby that I heard
You know I can quantitate, hope you got a standard curve, Uh
PCR in the gel rig, fucked up my control
Put the product right to the side aint got the time to put gloves on, on site
Catch a charge I might, beat the rig up like Mike
In ’97 I bite, I’m Drew, Harvell Harvell
Laby no I don’t play, now bake the cake, DNA
Said, “bake the cake, DNA!”
I’m nice, for y’all to reach these heights you gonna need 3 genes
4, 5, 6 types, sleep tight
We count again in the morning, your hemocytes is my breakfast
We going in, we be all night

We be all night, in laaaab lab
We be all night, in laaaaab lab

Never tired, never tired
I been sipping, that’s the only thing that’s keeping me on fire, me on fire
Didn’t mean to spill that coffee all on my attire
I’ve been eating watermelon
I want your lesions right now, laby I want you, right now
Can’t keep my eyes off that UV
Laby I want you

We be all night, in laaaab lab
We be all night, in laaaaab lab

laby in the lab

A shout out to all the scientists that have spent hours painstakingly dissecting the sea stars from Ruby Beach. We are on track to start some really exciting histological and genetic analyses on the sick stars and the water filtrates that is going to be very helpful in supplementing the transcriptomic data.

All day yesterday and today I spent mostly in the molecular lab trouble-shooting the star PCR using primer sets for our candidate sea star microbe. Sarah and I used the forward primer from set A with the reverse primer from set B in an attempt to bypass issues we’ve been having with primer-dimers and non-specific binding, but unfortunately the 2 kb amplicon is not showing up on the gel (which really should max out at 1.5 kb anyways). Not to fear! We are able to redesign new primers and hopefully will be on our way to validating them ASAP. These can be used to detect presence of pathogen (cPCR), probing for viable cells in various tissue (ISH), and for detecting the presence the pathogenic DNA that might have sloughed off the deteriorating stars into tank water.

As the sea star experiment moves forward full throttle, the oyster filtration experiment is coming to an end. We should have one more day of eel grass incubation and can move forward with histology and cPCR, which Casey and I started DNA extractions for today.

 

Day 2 of oyster filtration

After finishing the seawater sampling at 0hr, 1hr, 2hr, 4hr, 8hr, and 24hr, we split into teams to measure, weigh, and dissect the various tissues from our oysters. We had 10 oysters in triplicate for each condition: sea water+penstrep+cultured laby treated (experimental) and sea water+penstrep+SSA treated (negative control). This came out to 60 little gills and digestive glands that had to carefully be sampled. It takes time to shuck and dissect 60 oysters but its oddly very relaxing.

Crassotrea gigus ready for shucking

Crassotrea gigus ready for shucking

Cross-sections of gill and digestive gland tissue from 10 laby-treated oysters

Cross-sections of gill and digestive gland tissue from 10 laby-treated oysters

The cross sections are fixed for 24 hours and will be used for histological analysis to look for any sign of viable laby cells they might filter through their gills from the water column. We also took tissues samples for DNA extraction and cPCR analysis to determine whether we can find laby DNA in the gills or digestive gland.

Eel grass incubating with laby-treated, oyster-filtered seawater

Eel grass incubating with laby-treated, oyster-filtered seawater

Finally we added eel grass to the beakers for a 3 day incubation to determine whether the oysters might filter out this eel grass pathogen and prevent infection and/or lesioning.

 

 

More to disease than cells and slides

After another long day in the lab troubleshooting primer sets and working with cultured laby cells that just would not cooperate, I have to take a step back and reflect on the bigger picture. We finished our day by unexpectedly pulling out histology slides of sick and healthy sea star tissues prepared earlier by Colleen and going through disease diagnosis 101.

Somewhere in the maze of coelomocytes there may be a pathogenic culprit, but the vastly unstudied physiology of the sea star leaves much to be understood. Thankfully, Carolyn talked at length today on several case studies of fungal, metazoan, and bacterial infections in shellfish, and while these epidemics may be vastly different from sea star wasting disease, there is something to be learned in each case.

In New Zealand, the Haliotis iris abalone underwent an infection via fungal hyphae, which caused lesions to form on the inner surface of the prized iridescent shell and cause eventual loss of shell integrity (obviously a problem for the intertidal creature). X-ray of the shell reveals the large lesions, and cross-sectional histology of the cell reveals hyphae structures.

Another abalone pathogen, the polychaeted sabellid, arose in abalone farms in California after being imported from South Africa on farmed abalone, and also infect the shell. This metazoan instead forms tubes on the outer shell perpendicular to the leading edge and extends a branchial crown outward for feeding. Amazingly, these polychaetes are functional hermaphrodites and can spawn an epidemic with just one worm! It can easily be visualized under a microscope but requires vigorous abalone husbandry and water control to manage an outbreak.

The both of these infectious agents,coupled with the various protists and bacteria already studied that infect the digestive epithelia of shellfish, leave me with a profound respect for invertebrate pathologists. Where do we start looking for disease in echinoderms? What tissues might look suspect under histology and what pathogens might be at play in neurological or inflammatory responses? I am intrigued and look forward to learning more about the art of disease diagnosis.

Hopefully the sea stars on the West Coast will soon be another case study of a disease diagnosed and contained.

Primed for PCR

Today was spent in much speculation troubleshooting our sea star PCR experiment. The laby gel seemed to show clear bands for two out of three primer sets, but the sea star gel was problematic in that there was no clear band patterning between our sick and healthy sea stars, and even our negative controls (which contain only water and hopefully no template strand microbial DNA).

Some issues discussed were that the GC% content and self-complementarity of our primer sets seem too high. This self-annealing could lead to primer-dimer formation, outcompeting our template viral DNA strand for amplification. After a quick BLAST, it might be necessary for primer redesign. It might also be helpful for us to increase the annealing temperature to 57C, but the experiment is on its way to being repeated because results are always more reassuring when you see them twice.

Region A (82bp amplicon)

Length Melt temp GC% Self-complementarity
5’ → 3’ 20 57.3C 50% 4
3’ → 5’ 20 57.5C 45% 4

Region B (94bp amplicon)

Length Melt temp GC% Self-complementarity
5’ → 3’ 20 55.6C 42% 6
3’ → 5’ 19 55.4C 53% 6

 

We also had a really fun tour of the ocean acidification lab, where various ongoing experiments require sterile seawater (pumped from the ocean and sent through a series of filters decreasing in pore size to 0.2 microns), and vigorous temperature and pH control. Carolyn is working on an oyster hybrid vigor experiment with a local WA hatchery to outcross and possibly develop strains of oyster which are more resilient to changes in CO2 and temperature in the water.

Another really cool upcoming oyster experiment involves oysters filtering pathogenic laby from the seawater and slowing or preventing infection of eel grass. More to come on that in the next few days!

 

17 days later

Carolyn and Steven took us on a journey through invertebrate immunology, which is important for wound healing and tissue repair, pathogen response (viral, bacterial, and fungal), and stress response (i.e. CO2, heat).

The invertebrate immune response recognizes pathogenic motifs through Toll-like receptors and pattern recognition proteins (PRPs), which are an Ig-super family domain containing FREPs. I’ve included a Venn diagram which simplifies the cellular and humoral response, but don’t let the oversimplification fool you; there is cross-talk between both responses via chemokines and other immunological signaling events.

Screenshot 2014-07-28 23.39.01

Another interesting and confounding “tidbit” from lecture today is the hypothetical quasi-adaptive immune system. The dogma that invertebrates do not possess an adaptive immune response, activated by multivariate recombination events, may actually be oversimplifying the invertebrate immune system. For example, oysters have an experimentally determined anticipatory response to infection, not a trait expected from a static immune response (Roberts et al 2012). Also, shrimp injected with Vibrio harveyi were shown to have heightened levels of circulating PRPs at least 17 days later and retained some recognition of bacterium and showed evidence of immune “priming” (Rowley et al 2007).

However, the plot thickens! Pathogens can find ways to evade host immune response. Parasites such as bonamia can survive quite well in the hemocytes of the oyster cellular response by secreting substances which essentially distracting the hemocyte from the pathogen and prevent the ultimate phagocytic end that this pathogen warrants. We ran two DNA gels today, one with primers against cultured laby and one with primers against a candidate microbe that might be infecting the sea stars (results pending). Determining the presence of the pathogen is just the first step in solving the puzzle of wasting disease; we have a lot of work ahead of us!

Collin hopes that his pathogenic DNA bands visualize after gel electrophoresis

Collin hopes that DNA electrophoresis can identify the presence of virulent pathogen in the sea stars

marinus maximus

“lots of eel grass”

Okay, so maybe it means something closer to “giant eel grass”, but it comes close to describing the amount of Zostera marinus surveyed today at Indian Cove.

We spent another day conducting eel grass surveys to ascertain the affects of culling lesioned eel grass on plant viability, this time at Indian Cove on nearby Shaw Island.

blue arrow is Indian Cove, red arrow is Friday Harbor

blue arrow is Indian Cove, red arrow is Friday Harbor

This time the winds were quiet and tide was low enough to confidently survey the site. We tackled 12 square meter areas, harvesting every eelgrass from the rhizome up that had either been culled or left as a control a month ago (3 quadrats, 2 controls and 2 culls in each). We want to determine whether there are any positive benefits to culling infected blades from the environment, so we brought our blades back to lab in baggies to count lesions, measure blade lengths, and ultimately determine the percent prevalence of disease.

Once back in lab and all cleaned up from another day in the field, we spent hours recording shoot length and counting lesions (which are dead areas of blade surrounded by darker bands of tissue). The most extreme lesions Sarah and I clipped, washed in sterilized sea water and ethanol, and plated on a plate of SSA with penicillin+streptomycin @21C.

Zostera marina in the field

Zostera marina in the field

Zostera marina in the lab

Zostera marina in the lab with lesions

 

TGIF!

Knee deep in False Bay

Today began very rainy yet again as we set out for an eel grass wasting disease survey at False Bay on the SE side of the island. Originally we intended to survey transects that were previously culled to assess a possible preventative measure against eel grass wasting, but high water levels during low tide made for some creative improvisation.

We chose to collect fresh eel grass samples from more shallow waters to culture in the lab (p.s. 10C water doesn’t really get to you hurt after you become numb to it!). We collected 20 of the second-longest shoots from each 10m increment, and brought them to Friday Harbor in baggies with fresh seawater. The shoots were measured by length and width, scored for any lesions (areas of necrotic tissues surrounded by definitive dark bands), and any lesions measured by length and width as well (results pending).

Each group plated clippings from a green, healthy shoot and a lesioned shoot onto sterile SSA broth+penicillian+streptomycin and left to incubate overnight at 21C. We had some free time this afternoon so I went through Culturing 101 with Carolyn and Amanda to make 10-fold serial dilutions of previously cultured laby and plated those at 21C to optimize culturing conditions. I got to see live spindle-shaped laby under the microscope for the first time! Very cool :)

cultured Labyrinthula zostera colonies

cultured Labyrinthula zostera colonies