Fingerprinting the Culprit

— Written by Vivian Lee

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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

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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.

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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.

Operation PURFECT (Purple Urchins Routinely Feeding in Extreme Climate Temperatures) Methodology

— Written by William Dejesus

I am measuring consumption of giant kelp by purple sea urchins, Strongylocentrotus purpuratus, living in experimental temperature waters. Twelve tanks were evenly divided into two different temperature seawater tables. The ambient temperature water table will stay consistent with current water temperatures outside, usually around 16-17°C. The warm water table is kept between 21-22°C. Drippers are used at the end of the inflow tubing to ensure consistent flow rate across tanks. The warm water table system contains a reservoir bucket that is used to pump the water to each tank ensuring consistent temperatures across the tanks.

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Warm water table with inflow tubing from reservoir

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Reservoir allowing incoming seawater to warm up

Urchins in this experiment were collected from various areas around the Channel Islands. The size of the urchins was measured upon entry to the lab, where almost all fell between 4 and 6 centimeters in diameter. All urchins were given a week of acclimation to the storage table. All relevant urchins are given a 3-day temperature and starvation acclimation period before being distributed to their individual tanks for trials. Using an acclimation period minimizes the affects of prior food availability and behavioral responses to increased temperatures. The twelve urchins subject to feeding trials are given 1 pre-weighed blade of giant kelp each per trial. Only blades of kelp are being used for feeding because of urchins’ prior known preference for this part of the plant. Each trial will last approximately 3 days, with deviations accounted for in rate calculations. The trial period was determined to be long enough for significant consumption but short enough that kelp degradation was minimal. There will be two trials each week with a 24 hour “No Food” break once a week. This allows for the trials to remain structured on a weekly basis. Statistically relevant deviations between trials with and without the “No Food” break before feeding will be acknowledged if found. Any experimental urchins that yield little to no kelp consumption (>1g) will be removed from experimentation after two consecutive trials with minimal consumption. These tanks will be replaced with acclimated urchins when the next trial begins.  Consumption of kelp is measured using wet weights of the kelp before and after each trial. Each blade is spun in a salad spinner before being weighed to reduce extra water weight in a consistent fashion.

Crabs Gone Wild: A Look at the Mysterious Foraging Behavior of Rock Crabs

— Written by Gabbie Baillargeon

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Two female brown rock crabs (Cancer antennarius)

Any research involving the study of living animals is simultaneously exciting and frustrating due to the fact that lab experimentation involves manipulation of individuals who have a brain of their own and don’t always want to cooperate nicely.  Although the general goal of my research project is to study the foraging behavior of brown rock crabs on California mussels, the specifics within addressing this research query have shifted in response to my preliminary observations of crab behavior.  Through this research project, I have come to find that doing science is a much more elaborate, drawn out, and unpredictable process of discovery than the simple flowchart of the scientific process in my high school biology book.

In order to start experimenting on the rock crabs, all of the crabs must undergo a set acclimation phase where they are allowed to get settled into their new, luxury tank habitat.  Following acclimation, the plan was to start all of the crabs in individual trials where both crab density, number of crabs per tank, and prey density, number of mussels per tank, would be systematically manipulated.  Simply, this experiment involves changing two variables: food availability and crab predator abundance, with the goal being to measure how the crabs respond to these changes by counting the number of mussels consumed and documenting behavior during feeding. All was going according to

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A gravid female yellow rock crab

plan for the first few weeks as crabs came into the lab they were placed in tanks, fed, and played nice with one another. However, due to little previous research published or documented observations available for reference, I had the privilege of witnessing first hand a wide array of surprising crab behaviors before the experiment even began.  For those of you who, like myself, were blissfully unaware of rock crabs’ night time job as a ninja, I am here to tell you that they are exceptionally stealthy little beasts who are capable of destroying every plastic tank barrier I repair. Without the tank barrier in place properly, crabs could move freely between the tanks which makes it impossible to accurately measure which crabs consumed which mussels – a vital aspect of data collection.  Along the way, a few crabs were lost in the battle for scientific understanding as some crab fights would end in death for the loser.  There was even an instance of crab suicide, where the crab simply found a way to escape its tank and met its unfortunate end drowning in the open air.   Additionally, many of the female crabs collected started to become gravid, or possessing eggs, during the acclimation period. To avoid any bias in the study, all females who were not gravid were going to be used in the experiment.

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Measuring and labelling a new batch of rock crabs to prepare for running foraging trials

A series of small pre-experiment foraging trials were conducted to get a sense of how the crabs interact with each other, how many mussels they consume, and how they utilize tank space.  The most surprising find of this small study was that groups of female crabs consumed many more mussels than groups of male crabs, indicating that there is a difference in their foraging behavior.  Given this new information, along with the predicament of females become pregnant, the experiment was reconsidered to answer a slightly different question within the same frame of investigating the interface of rock crab foraging and human impact on their populations.  My experimental design shifted to reflect this new path of investigation, as the study now tested only males. To accommodate a shortened timeline and restricted tank availability, the number of prey and predator densities was altered as well.   All in all, the crabs provide a good laugh, a frustrated yell, and sometimes a proud smile along the journey of designing and running foraging trials to better understand their feeding preferences and behaviors.

 

 

Guide on How to Spawn, and Experiment on, Urchin Larvae

— Written by Carlos Estrada

Step 1: Collecting Gametes

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Left: Male L. pictus expelling sperm. Right: Female L. pictus dispensing eggs

To begin raising urchin larvae, we first have to “convince” the parents to reproduce. But instead of dimming the lights and lighting a few candles in hopes that the urchins will hit it off, we take matters into our own hands. We start by injecting 1-2mL of KCl around the mouth of the urchin, which causes them to stiffen and release their gametes. Since male and female urchins are morphologically similar, their gametes become their only differentiator. If sperm begins to flow out, the urchin is obviously male and is placed on ice to keep cool. If eggs begin to ooze, the urchin is female and is left inverted on an overfilled 50mL conical tube, where the eggs will begin to drizzle down and gather at the bottom. This process is repeated with as many urchins needed to gather one male and at least five females.

Step 2: Fertilization

Before mixing the sperm with eggs, we make sure that the sperm is healthy by checking for motility. Once motility is confirmed, we move onto the eggs by homogenizing them and check for a coefficient of variation under 10%. Doing this allows us to have a rough estimate of how many eggs we’ve collected from each female and helps calculate the volume needed to have an equal amount of eggs between all females. Next, we test fertilization using a dilute amount of sperm and eggs, looking for a >95% fertilization rate. If successful, we fertilize the entire batch and transfer the fertilized eggs into our rearing buckets at either 15°C or 20°C.

Step 3: Developmental Timeline

Checking for the developmental stage is done by taking a small, concentrated sample from the bucket every 30mins-1hr, and placing them under the microscope. Urchin developmental stages are so variable from one another, that confirming the stage can be quite easy!

Step 4: Sampling

Sampling is done through a seemingly simple, yet complex, system involving buckets, mesh, beakers, and soft-line tubing. Once the larvae are siphoned into a collection beaker and then concentrated into a 15mL conical tube, which is homogenized for an egg count.

Step 5: Thermal Tolerance, Heat Shocking, and Morphometrics

Thermal tolerance trials are conducted by transferring larvae into a heat block with a gradient of temperatures that will help determine the temperature at which L. pictus is no longer able to function. The larvae are left in the heat block for an hour and subsequently checked for mortality. Heat shock trials will be done by placing larvae into four different temperatures, allowing for one-hour recovery, and then flash freezing for future extraction of the hsp70 heat shock protein. Morphometrics will be used to take pictures of the larvae to measure sizes and to determine whether increased temperatures influenced development. Once the trials are finished, down the drain they go!

 

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.

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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.

Ara’s Fishy Adventures

— Written by Ara Yazaryan

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Counting dorsal rays of a rockfish

As a research assistant in the Caselle laboratory, my entire experience revolves around fish. My day starts at 9 o’clock, when I report to the lab and march over to the microscope for duty. One of the ongoing projects in the Caselle lab which I have the good fortune to partake in is the SMURF monitoring program at Santa Cruz island. The SMURF discussed here, contrary to the little blue people that may come to mind, is a large black “cushion” of intertwined black fencing material. SMURF stands for Standard Monitoring Unity for the Recruitment of Fishes. This large object is meant to mimic kelp structure, and gives planktonic fish larvae a place to settle and grow. Every two weeks, these SMURFs are netted and the fish are collected and frozen for later study. And this is where my abilities with a microscope come in! One of my primary laboratory responsibilities is to count and sort the SMURF fishes into each respective species. Though some species, such as Cabezon (Scorpaenichthys marmoratus) are instantly recognizable, other families of fish, such as juvenile rockfish (Sebastes) and the Clinids require a closer look. In order to separate them into the respective species, all rockfish must be observed under a dissecting microscope in order to count their fin rays. Each species of rockfish has a unique combination of dorsal and anal fin rays, serving to identify these individuals when they are young and have not yet developed their respective mature morphological features. I examine these fish one-by-one and sort them, and then place them into a final freezer storage.

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Otoliths – note the concentric ring structure

When my eyes need a break from the microscope, I move on to fish dissections. Another of my laboratory responsibilities is extracting the otoliths from fish heads. After opening the brain case, these “ear-stones” are extracted and saved for storage. Eventually, the otoliths will be polished and observed under a microscope. As a fish mature, information about what it eats, rates of growth, and potential environmental stressors is stored inside their otoliths. All this important information can be gleaned by extracting and observing the otoliths.

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Large Male Sheephead (Semicossyphus pulcher) collected at Anacapa Island

The third project that I have just begun to work with is the monitoring of California Sheephead (Semicossyphus pulcher) stomach contents between sites at Anacapa Island. Fish were captured by spear or hook-and-line (much to my inner angler’s delight) and their stomach contents will be examined to determine how MPAs affect their feeding habits and population levels. The overarching goal of these three projects, and the information I aspire most to learn about, is how California fish communities are responding to Global Change. Fishing, climate-change, Ocean warming and acidification, and pollution all have the potential to degrade our rich fish stocks. By studying and monitoring these fish, I hope to gain a better understanding of California’s complex marine ecosystems. In particular, I want to more thoroughly understand how MPAs conserve fish populations, and correspondingly alter dietary structures. I will use the Sheephead (As Dr. Caselle has made very clear, there is no “s” in the middle) as a model organism for this experimentation. I seek to answer the question: Does dietary composition of Sheephead change inside and outside of Marine Protected Areas? Using observation of stomach contents coupled with statistical quantification, I set out to answer this question over the course of my REU program. Though only two weeks have gone by, I feel as if I have gained a year’s worth of scientific immersion. Between lab work, field research, and peer camaraderie, I have gained a much clearer understanding of what research in the marine science field consists of. I look forward to what is to come, and am excited to examine many more fish in the upcoming weeks at UCSB.

What does a changing climate mean for Phytoplankton and what does this mean for our planet?

— Written by Ean Eberhard

The microscopic marine algae known as Phytoplankton are incredibly important to our planet. Although minuet in size these organisms have an immense amount of responsibility within both aquatic and terrestrial ecosystems.

Phytoplankton are primary producers, the foundation of the marine food web, feeding everything from zooplankton to massive whales. As a primary producer these phytoplankton create the foundation of the aquatic food web and thus bare the weight of the rest of the aquatic food web. In other words, the fluctuation in phytoplankton abundance could mean a collapse in the web.  Phytoplankton are not only a source of food but are also a large contributor to the biological carbon pump, responsible for most of the carbon dioxide transfer from the atmosphere to the ocean. For this reason, changes in the growth of phytoplankton could have a great effect on atmospheric concentrations of carbon dioxide. Phytoplankton can also be detrimental. When nutrients levels are high, phytoplankton can grow out of control, creating algal blooms which produce extremely toxic compounds with many harmful effects.   There is no doubt that the balance in phytoplankton abundance is crucial to all life. Unfortunately, phytoplankton are experiencing more stress due to a changing climate.

For the next seven weeks I will be studying the photochemistry of phytoplankton in response to three different stressors. This experiment will be a multifactorial design that will give a more realistic look into how the stress of a changing climate is much more complex than rising temperatures. These three stressors include variances in light intensity, temperature, and CO2 levels. Each of these stressors correspond to variances in the phytoplankton’s environment due to the change in climate. I will dive deeper into the response of these phytoplankton by measuring cell abundance, cell efficiency, Dissolved Inorganic Carbon and pH. This experiment will give us an insight into just how exactly phytoplankton may respond to a changing world and the findings may provide an insight into how the effects on phytoplankton could have a ripple effect on all other life.

Will one of Japan’s most famous delicacies survive a changing climate?

— Written by Will Dejesus

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Purple urchin feeding on giant kelp

Sea urchin is a famous Japanese delicacy used in sushi, called uni. The gonads, or sex organs, is the part of the urchin that is actually eaten. It is high in protein and contains omega-3 fatty acids, which can help lower blood pressure. Sea Urchins can be found along the West coast from Baja to Seattle. Sea Urchin harvesting along the coast of California has become one of the highest valued fisheries in the area. Almost 75% of the urchins caught along the California coast are sent to Japan, where they import over 250 million dollars worth every year.

Not only are sea urchins a relevant part of the food and fishing industry, but they also play a critical role in the kelp forest communities, as their preferred food source is giant kelp. With few native predators, sea urchins have the power to decimate kelp forests, which are already struggling with changes in ocean chemistry. Areas where sea urchin population has sky rocketed, kelp density has been nearly wiped out in a short period of time. From ocean to table, sea urchins play an important role in coastal communities on land and below the surface.

I am interested in taking a closer look at the feeding habits of these invertebrates given future predicted changes in ocean temperature. Their reactions to temperature changes in general and especially in feeding behavior is all of interest in this study. This will begin to paint a clearer picture of what we can expect our marine ecosystems and seafood industry to look like in the future.

Shark abundance shown with dermal denticles

— Written by Maria Rivera

How can we know how many sharks there are in a specific area? We cannot track or observe them as easily as other animals, especially land animals. This is a major issue because we do not have sufficient data that tells us about shark communities in the past nor present. Without this knowledge, we cannot analyze how humans have impacted shark communities near reefs. Fortunately, there are new techniques being developed that help us measure the relative abundance of sharks near coral reefs. One of these new techniques involves analyzing dermal denticles. Dermal denticles are the microscopic tooth-like scales that cover sharks’ bodies. Sharks shed these denticles, and they are deposited and preserved in coral reef sediments. Hundreds of denticles can be preserved in the sand, recording a timeline of what shark communities where like in the past and how they have changed.

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Fig. 1 shows different types of denticles that belong to different kinds of sharks. Denticle A has high ridges for swimming, while denticle B is smooth and thick for protection. Photographs courtesy of Natalie Minouei taken on July 6, 2018.

How do denticles help us estimate/determine the relative abundance of sharks? By comparing the denticles we find in sediments from different regions and time periods, we can qualitatively estimate the abundance of sharks. Not only do the number of denticles help us interpret the abundance of sharks near reefs, but we can also use the wide array of denticle shapes and sizes to learn more about the ecology of sharks. Variation in denticle morphology serves different purposes. For example, thin denticles with high ridges are for drag reduction and allow the shark to swim faster (Fig. 1). These denticle characteristics are common with pelagic sharks that spend a majority of the time in open waters and are fast swimmers, like great whites for example. In contrast, there are denticles that can be smooth and thick for abrasion strength, this provides protection to the shark from the ocean floor (Fig. 1). Denticles like these would be found on sharks that live near reefs, such as nurse sharks. Variations of denticles for different sharks gives us an insight into the structure and composition of the shark communities roaming that reef.

We can also look at denticle distributions in areas across gradients of human impacts to investigate how shark communities are affected by human activities. For example, if there is a lot of fishing in an area, there might be lower numbers of sharks and thus lower number of denticles. If we compare that to a place that is less impacted by humans, we would expect to see more sharks and more denticles. This can help us understand how humans have impacted the marine ecosystem through time and in a context of local baselines. This is a great way to analyze the relative abundance of sharks near coral reefs when there are limited tools and scientists cannot go physically count sharks one by one in the ocean.

Studying Santa Barbara Fisheries: Spotlight on Rock Crabs

— Written by Gabbie Baillargeon

 

Walking along Stern’s Wharf, accompanied by the bustling crowd of downtown Santa Barbara tourists and locals alike, signs proudly boasting the sale of delicious rock crabs seem to line the boardwalk.  Similar to their more famous and tasty cousin, the Dungeness crab, rock crabs are a group of crab that comprise a productive and lucrative fishery along the coast of Southern California and the Northwestern Pacific states.  Three distinct species of rock crab are found off the shores of California: Yellow (Cancer anthonyi), Red (Cancer producutus), and Brown (Cancer antennarius) rock crabs.  All three species are available to purchase as seafood, though red rock crabs have the highest demand due to their larger size and sweeter meat.  For the amount of recreational and commercial fishing that surrounds Rock Crabs, shockingly little research has been dedicated to studying them and even less information is available to shed light on how these crabs operate in the unique environmental conditions of Santa Barbara.

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Two yellow rock cabs inside a cinderblock, a favorite resting spot.

My research project investigates how changing fishing pressures on rock crab populations alters their foraging behavior.  In order to mimic fishery pressure, different densities of rock crabs will be put in controlled environments and fed different densities of California mussels (Mytilus californianus) to measure their predation rates in each case. The goal of this project is to determine if changing the number of rock crab competitors of a single species will affect how they forage.  In an effort to better understand how fishing plays a role in shaping the nearshore marine environment, the potential consequences of varied foraging behavior in response to population shifts on the larger ecosystem will be analyzed.

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A Brown rock crab being measured and tagged after collection from Stern’s Wharf.

In the early stages of the project, plenty of challenges quickly multiplied when working with the rock crabs or “little monsters” as we fondly like to think of them.  The rock crabs have earned this nickname as they seem to have a bit of a angst-ridden destructive streak in them, especially when it comes to following some basic tank rules.  After only 24 hours, the crabs had deftly removed their identifying leg bands – I’m sure they planned this operation carefully and helped each other out.  Then, instead of being happy with their buffet of mussels and clean tank to play in, some crabs decided that they simply must explore what was on the other side of the tank barrier and crawl into their neighbor’s tank.  The mussels are bluer on the other side, right? Lastly, although the names red, yellow, and brown rock crab seem to indicate that it would be simple to distinguish between the three species, I painfully came to find out that could not be farther from the truth.  Despite the roadblocks, learning to care for the crabs and become excited over little things such as a crab finally deciding to eat mussels, has all been a part of the learning curve and given me invaluable skills of patience, problem-solving, and a lightning-fast reaction to a feisty crab claw.