Author Archives: Collin Closek

Week 3: Data Fun

This week we’re moving from the field & lab to computational research…doing 21st century science! (joking…)

We started with the compiled contigs from a Sea Star Transcriptome. Prior to this RNA was extracted – mRNA – 6 libraries were made – compiled seq reads were clustered into contigs using CLC.

Today we got our bearings of the data set using iPython, which allowed us to run commands in the terminal, while simultaneously collecting notes on the commands ran. In addition we were introduced to SQLShare from UW, which allows to compile/join datasets and share datasets as well.

It was a great introduction to the tools and resources out there on the web that we can tool around with (e.g. iPlant). We also learned a few new commands to add to the tool belt:

!fgrep  #faster, but less capable than grep

!awk   #lets you have reports of your data and do data extraction

!sed   #replaces content you don’t want to a new content

 

Human Health & Coral Health

Scientists have begun to consider an organism as a sum of it’s parts, that is its associated microbes. Rather than the old view of all microbes being foes and definitively separate from the organism (host), it’s becoming more clear that most, if not close to all microbes, under ambient conditions are beneficial symbionts of the animal that can also be host- or tissue-specific. The human microbiome is a popular example of this, where associated bacterial community differences are correlated with location in or on the body (e.g. gut vs. skin on hand, even left vs. right hand) as well as ethnicity (e.g. specific enzymes and microbes help people of Japanese-origin digest seaweed).

There has been strong support for the human microbiome project and in parallel coral scientists have also focused more on the microbiome associated with corals, termed the coral holobiont. This emphasis has in large part been a response to better understand coral health and result of increasing disease occurrences. Much like in human health studies, research has highlighted that the coral holobiont is a complex metaorganism, which contains many more facets: archaea, bacteria, fungi, protists, viruses. The functions these players and their interactions with each are poorly understood. Today and yesterday’s lectures on coral health and immunity outlined some of the many victories, as well as large gaps that still remain. Over the last 70 years progress has been made in understanding the coral-animal as well as its health, however new sequencing tools are making it clear that we’ve only begun to scrape the surface of our understanding of these simple, yet complex and microbially-diverse organisms.

More on these advancements and gaps soon.

 

Sea Star: Round 2

Our Sea Star experiment began on 07/31/2014.

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We set-up 15 tanks in a cold room (set to 20C) with filtered/autoclaved sea water within our experimental sea table (3×5). Our objective is to test what is in the water that the stars circulated after 20hrs. The conditions tested were asymptomatic, exposed without lesions, and 1 or more lesions (4 of each). We also had 1 of the follow in the remaining 3 tanks: control initial (T0), temp logger, and control finish (T1) in the center (see Fig. Exp. Set-up below).

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All star samples were collected well into the night last night (after 20 hrs). We meticulously collected the samples in order to not expose stars to one another, and subsequently   water from each will be filtered.

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Sea Stars: Round 1

48 stars were collected from Ruby Beach in Olympic National Park (6 asymptomatic, twisting, lesioned)
~6 asymptomatic
Could not find disease from this site 2 weeks ago
Mo was there 3-4 days ago and saw diseased -> fairly wavy – so likely washed away
Set up tanks: 3 conditions x 4 specimens, plus 3 tanks – 2 control (to be sampled before and after)

test: DNA extraction quality and concentration, new primers

To Do:
Time-lapse Pisaster footage
DNA extraction troubleshoot
Biochemical
New primers-PCR
Histology
——
qPCR
Transcript
Proteomics

Many pathogens, but temperature tips the scale

Today we heard about many pathogen infections from around the globe that included different domains and kingdoms. One fungal infection, one polychaete worm, and two bacterial infections. Three of these involved different species of abalone as a primary host and one involved the infamous Pacific oyster, C. gigas.

While they occurred on different continents, temperature was the key in triggering the spread in of these diseases. In each case, the pathogen was either detectable and/or caused obvious malformities.

The most intriguing case was the Sabellid Polychaete, which infests various gastropods. The example focused on was farmed abalone (Haliotis midae), however it does also infests Tegula and less so limpets. Some facts that make this case cool was that it had been recently brought in from South Africa to the West Coast of the US. Infestation signs were noticeable, as the Sabellid causes the shell to build up and deform.

Kuris&Culver1999Fig4

Sabellid worms build, secrete a mucus tube, and modify the edge of the shell. Another cool fact: they’re functional hermaphrodites. It takes 1 to cause an outbreak, Kuris & Culver (1999) – YIKES!

TerebrHet
Also, reproduction increases above 16C, cooler temps also allow infestation to occur, but less so. (It can still reproduce 11C, but much less.)
Eventually, the Sabellids were established in the wild in Tegula funebralis, due to ignorant practices of one hatchery. This oversight allowed 17,000 viable worms/day escape through the effluent and into the open ocean.

Kuris&culver1999Fig3b

Luckily this worm doesn’t move far, and the efforts of Carolynn Culver’s work at UCSB removed 1.6 million viable gastropod hosts. The team continued to remove viable hosts and eventually continuous monitoring showed that the Sabellid was irradiated.
Image credits: Kuris & Culver (1999), Invertebrate Biology

Case Studies: Murder, She Wrote…in the Ocean

With every classic whodunit, there is the crime, the perpetrator, and the victim. While Angela Landsbury always seemed to solve the case, the reality is gathering the evidence, examining the appropriate areas, and fitting all the pieces together is challenging; not to mention that it rarely happens in an hour segment.

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Agatha Christie and other murder-mystery genre novelists have also made the pieces come together seamlessly, however researching disease is normally less clearcut. Today, Carolyn provided 3 case studies which are exemplary marine disease cases.

Case Study #1: Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD) in salmonids. Chinook salmon are particularly susceptible. In the 1960’s Chinook salmon were moved to Lake Michigan! The population grew too large, resulting in reduced prey population, and they kept restocking the lake. So the population kept increasing, but eventually fell and continued to be low (regardless of continuing to restock). Eventually the Lake Michigan population did start to re-establish. So what was killing the salmon? Answer: Renibacterium salmoninarum. When tested against the initial stock from WA, high mortality resulted, while the stock in MI was less impacted. Essentially, during this 20+ year period the introduction of the salmon to Lake Michigan was a large selection experiment. Where the natural population is more susceptible to infection, but resistance traits were selected for within the natural genetic distribution, resulting in a more R. salmoninarum-resistant salmon genotypes.

Coho_36432_7

Case Study #2: Candidatus Xenohaliotis californiensis (bacterium) in Black Abalone (BA) cause Withering Syndrome (WS). The syndrome was magnified by warm water events in 1980’s
BA were densely populated, as sea otters were hunted to almost extinction at the beginning of the 20th century, and the pathogen had a densely susceptible host. During 1985-1990 99% of BA were wiped out. Except on San Nicolas Island (the most distant Channel Island), where 95-98% mortality occurred. Rickettsia (RLO) arrived in 1992 and the pop dropped from 25,000 to 200 in 2001. The population began to increase from 2001. Other locations, e.g. Carmel had naive or non-selected because there are lesions, but not active disease as on San Nicolas. Carolyn & Lisa performed a 490 day exposure trial (CRAZY!) with these two populations. Using a ISH probe they visualized WS RLO and a new (much larger) RLO. It took ~700 days til mortality set in, longer than previous studies. TEM shows phage-like hyperparasites. The pathogen was infected with the phage, causing reduced pathogenicity. Essentially, reduced metaplasia resulted, which decreased the parasite in the digestive gland, then a decrease in pedal catabolism, and ultimately an increase in length of time til mortality. So the phage reduces pathogenicity of WS-RLO.

Interestingly, green abalone was less susceptible to WS, while white abalone had almost 100% mortality. Another interesting trend occurred with temperature and susceptibility in pinto, pink, and red abalone…temperature determined whether the species was susceptible.

Abalone-map-copy

Case Study #3: Selective Breeding of the Eastern Oyster reduces pathogenicity of MSX (Multinucleiated sphere X) or Delaware Bay Disease from Haplosporidium nelsoni. In 1959 MSX was found in Chesapeake Bay. H nelsoni infects cells of the digestive tubule as spores and causes mass mortality. Selection for MSX-resistant C. virginica has helped to reduce mass mortality. Then, DERMO- Perkinsus marinus - thought to be a fungus at first – related to dinoflagellates was introduced in 1992 and spread to warm conditions. This secondary pathogen became the main disease and caused further mass mortality, however climate conditions allow you to predict outbreaks. The question now is whether selection for dual-resistance is possible.

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Selection for these fitness traits were a common theme throughout the day and a clear loophole in natural populations to allow disease to reduce populations (when perhaps they’ve increased beyond sustainable numbers), but also a way for the populations to reseed and be less susceptible in the future. These same types of impacts may also be taking place under ocean acidification (OA) conditions. Carolyn presented data today showing that populations under high OA have lagged and often reduced sporulation. One more player to all of these marine health mysteries!

Link

Today we dove headfirst into the “primitive” world of invertebrate immunity and host response. Molluscan & crustacean models were the focus. While primitive, marine invertebrate immune systems have evolved/persisted for millions of years, and they seem to have been quite effective so far. However, most marine invertebrates have not been studied to the extent that their terrestrial counterparts have. Unlike vertebrates, marine invertebrates have been thought to only have an innate immune response and no long-term immune “memory” (i.e. adaptive immune response). These large differences and lack of knowledge make studying invertebrate immune systems challenging.

Innate vs. Adaptive Immunity - Various innate responses and the physical location of where those responses take place in the host.

Innate vs. Adaptive Immunity – Various innate responses and the physical location of where those responses take place in the host.

However, evidence is pointing to potential adaptive immune response(s) in invertebrate hosts. It is clear that immune response is still in many ways a large black box, but a recent review by Joseph Sun and colleagues (2014) point to the evidence and missing links in this enigma.

Vertebrates vs. Invertebrates and the black hole of knowledge regarding invertebrate immune responses. http://emboj.embopress.org/content/early/2014/03/27/embj.201387651

Vertebrates vs. Invertebrates and the black hole of knowledge regarding invertebrate immune responses. http://emboj.embopress.org/content/early/2014/03/27/embj.201387651

Additionally, there are some recent advances that have helped us understand the invertebrate-host response, as well as connect it to environmental variation and stressors. qPCR and gene expression profiles are allowing us to target genes and get a more systemic understanding of the host response. Carolyn and Steven showed us multiple examples from their labs as well as other colleagues, where these studies have shed light on poorly understood invertebrate immune responses and signaling cascades. We’ll be continuing to examine these topics further and I’m thoroughly interested in this topic, so expect more posts on these and similar topics in the upcoming weeks!