Is the Culprit still on the Loose?

— Written by Vivian Lee

Through observing the presence of the 16s ribosomal unit from the bacterial samples that were cultured on various agar plates, the next step of identification was to get a closer look into the fingerprint. By using RFLP, which stands for Restriction Fragment Length Polymorphism, more distinctive banding patterns from the samples were differentiated through various enzymes. In RFLP, enzymes play an important role, as they break the DNA samples into small pieces at various restriction sites, they make each fragment distinguishable from one another on an agarose gel through gel electrophoresis. The enzymes that were used include HaeIII, DdeI, HinfI, HhaI, and RSAI. As the identification continued, the samples that underwent both a PCR and the RFLP were sent off for sequencing.


Figure 1: Through Gel electrophoresis, Polymerase Chain Reaction (PCR) results displayed the presence of the 16s ribosomal unit in multiple sea star bacterial samples. The presence of the 16s ribosomal unit is identifiable at around 540bp.

So what does sequencing my samples mean? Sequencing the bacterial DNA samples allowed me to identify which strain of bacteria matched the DNA of our sample. Through this recognition, results have shown that there is a presence of Vibrio, as well as other bacteria, including the Pseudoalteromonas. Through these results, what can be said about the role of Vibrio and its effect on Sea Stars is still being questioned.  The identification of Vibrio puts us one step closer to understanding that among the Sea Stars that were sampled, sick stars had Vibrio present. There are still numerous questions that need to be answered and various parameters that need to be taken into consideration. Are there are Sea Stars in intertidal that are currently alive and healthy, and if so, how are these survivors different amongst different species? Genetically, are these survivors going to advance to defend themselves in the future from another possible disease outbreak? The next approach to isolating other strains of bacteria, not only Vibrio may lead to another possible culprit that may be responsible for the deaths of these stars.


Figure 2: Gel results from a RFLP. Gel displays four different bacterial samples that experinced RFLP using five different enzymes (HaeIII, DdeI, HhaI, RsaI, HinfI, Sau3AI)

Fingerprinting the Culprit

— Written by Vivian Lee


Figure 1. Sea star tube feet sample in 100% ethanol

What are some things that come into mind when we think of DNA fingerprinting? Perhaps, an episode of CSI or Criminal Minds about identifying a murder suspect might be the first thought that crosses our minds. Similar to finding a murder suspect, bacteria DNA fingerprinting is being studied in my lab with Dr. Nguyen to catch a possible culprit, a bacterium known as Vibrio, which may be responsible for the sudden deaths of sea stars that occurred across the West Coast in 2014. In hopes of finding the presence of Vibrio on sea stars, we will culture the bacteria on plates in different agar environments, some that are rich in nutrients more than others to grasp how this bacterium behaves in the ocean, and the effects that it harbors on sea stars in the Rocky Intertidal.

Let’s take a step back and sink ourselves into an episode of a crime investigation scene where there is a case investigator and a crime scene personnel collecting sources of evidence such as hair or blood, swabbing around the perimeter to collect any other possible articles of DNA to then have it analyzed in a lab. Likewise, I play a similar role in which I collect DNA of sea stars out in the field, to then have the DNA extracted, whereupon, an amplification is done through Polymerase Chain Reaction (PCR). Once the fragments of DNA are separated by gel electrophoresis, I observe the fragment size to


Figure 2. Species Pisaster ochraceus found

analyze the presence of the 16s ribosomal subunit. This gene prompts the identification of a bacteria present in a bacterium swab sampled from a sea star. There are two methods in which DNA and the presence of multiple bacterial communities are collected from the stars. One, where I bargain with the sea stars as they give me a fair game of tug of war with their tube feet and when I win, in exchange I take a few tube feet back to the lab as seen in Figure 1. These organisms have the capability of regenerating their tube feet, as well as their arms, which allows the methodology of stealing their tissue quick and convenient. Because sea stars possess this trait of restoration to heal damaged tissues, questions arose when these Echinoderms began “melting”, one arm at a time when they became infected with the Sea Star Wasting Disease. Secondly, to observe which bacterial communities place sea stars under huge amounts of distress, areas where white lesions form on the body as seen in Figure 2 are swabbed, along with the exterior tissues that do not have white lesions, therefore to compare the presence of Vibrio in “healthy” and “non-healthy” sectors of the body.


Figure 3a. Coal Oil Point Rocky Intertidal. b. Emma Saas and Vivian Lee making observations at Hazards Canyon intertidal. c. Species Pisaster ochraceus affected by SSWD

Sampling the species of Pisaster ochraceus non-local sites such as Hazards Canyon in comparison to the local intertidal at Coal Oil Point, where it has become barren of sea stars raises questions concerning to what is holding one species over another back from making a comeback to recovery. By surfacing one of many hypotheses proposed about the cause of the Sea Star Wasting Disease through observing past bacterium samples that were collected during the Sea Star Wasting Disease is currently preparing me to compare the presence of the 16s ribosomal subunit in current stars that may have survived the disease.

The Attack of Vibrio and the Plummeting Deaths of Sea Stars

— Written by Vivian Lee

A keystone species, one that is largely depended upon by other species, acts as a fundamental instrument for balancing an ecosystem at a constant equilibrium. Thus, when a keystone species population declines or has an absence in an ecosystem, the consequences are detrimental. Recently, the Rocky Intertidal Community of the West Coast has been facing consequences such as overpopulation since the sea star wasting epidemic. In 2013, a mysterious disease spread from Washington State to as far North as Alaska and as far South as  Mexico, sweeping across the coast and taking away many sea stars species in its path.

As white lesions form on the body, the disease takes away each arm at a time, and clinging onto what becomes the last of themselves, the sea stars waste away until only a sheer outline of ossicles remain ghostly on the rocks of the intertidal. The wasting disease leaves sea stars hopeless as the progression to death can take less than a few days, leaving them unable to salvage themselves; causing populations to plummet to the verge of local extinction. Amongst the many species, Pisaster ochraceus was among those attacked the hardest by the disease,producing questions and possible hypothesis not only for the Pisaster ochraceus but the rest of the sea stars. Being one of the biggest marine epizootic outbreaks that occurred, questions are still on the rise, parameters have been continuously observed as the cause of the disease are countless.


Figure 1: Photos above display three different species of sea stars. 1a displays  two Pisaster ochraceus sea stars (orange and brown) and Pisaster brevispinus (pink). 1b is a closer view of the brown Pisaster ochraceus. 1c. Sea star species known as Pisaster giganteus

Is it the rise of seawater temperature along the west coast? Could the El Nino event have played a part in intensifying the pathology in infected stars from certain sites compared to others? Perhaps the salinity of the water? Has something changed in these Echinoderms’ diets? Finding a concluding answer has been as difficult as finding a needle in a hay stack; however, an explanation of the disease has been monitored. Vibrio, a bacteria found in sea water and some marine organisms such as crab and eel can affect both humans and other marine organisms. Vibrio was found on many sea stars that were collected for observation. This bacterium may have opened the doors that weaken the sea star, causing the white lesions to form and eventually leading to death. Site by site along the Southern California Coast, multiple bacterial swab samples of sea stars were collected during the time span during and after the outbreak. Whether the stars showed phenotypic/ physical attributes of sickness or not, samples were taken to observe the presence of the bacteria.

Previously, a collaborative team from Dr.Hofmann’s lab at UC Santa Barbara collected swab samples of Pisaster ochraceus from sites in Southern California ranging from Lompoc to San Diego, including the Channel Islands. This summer, a continuation of detecting the presence of the bacteria will be studied. Through this, we anticipate grasping a better understanding of the biodiversity of the bacterial community associated with sea stars from  samples collected during and after the outbreak. We hope to find current sea stars in sites locally around Santa Barbara as well to understand and monitor the differences compared to when the outbreak occurred.