Category Archives: 2-Research Methods

Materials and Methods of Ara’s Fishy Project

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— Written by Ara Yazaryan

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Me and my prized catch, a nice sized male Sheephead (Semicossyphus pulcher)

My experiment, now finalized, is very exciting: I get to study what the Sheephead of Anacapa are eating! My project is a quantitative assessment of Sheephead (Semicossyphus pulcher) stomach contents across several different sites and populations at Anacapa, part of the Channel Island chain. Anacapa has a unique conservation history, as the MPAs established here are of varying ages. One site is 40+ years old, and is one of the oldest Marine Protected Areas in the state of California. Another was established relatively recently, and the remaining areas of the island remain open for public use. Across these three study sites, I seek to determine the diets of Sheephead across different ages and areas.

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A fully dissected and processed Sheephead, complete with gut contents in tray

First, Sheephead were collected from these three sites (with appropriate research licensing of course) via SCUBA spear-fisherman or – much to my angling delight – with hook-and-line. Next, the fish were dissected in lab. Various parts of the fish were saved for future study: the organs, otoliths, eyes, muscle tissue, gonads, and gills. But for my efforts, the stomach intestine was squeezed to release the slurry of partially digested fish food. These stomach contents were then weighed, placed into a Falcon Tube, and preserved in ethanol to prevent decomposition (and the horrendous smell that accompanies it). Upon preservation, I place these stomach contents into an examining tray to view under a dissecting microscope, under a snorkel vent to alleviate the less-than-stellar odour. Using a low-power scope to help me view the small pieces, I correspondingly separate the contents into categories. Based on my findings thus far, the majority of the sheephead gut contents consist of urchin spines and shell fragments, kelp encrusted in bryozoan colonies, small crabs, gastropods, and bivalves. However, a few unique finds have been unveiled: such as unidentified fish spines and scales (Sheephead

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A sample of Sheephead stomach contents under a microscope: a hermit crab, urchin spines and tests, as well as mussel shell fragments can be seen.

are not normally known to be piscivorous), octopi, pieces of gravel and sand, and even a plastic nurdle (a small bead of resin plastic such as polyethylene used in the production of larger plastic objects). Such variable stomach contents, once separated, will correspondingly be assessed to determine the composition by percent volume. Data will then be pooled from many fish from all three sites at Anacapa to determine dietary trends.

Though identifying, separating, and sorting each shell fragment may seem tedious at times, I am nonetheless driven by curiosity and the pursuit of scientific knowledge. Much like the Californian gold miners of old descending into deep, dark mines to unearth unknown treasures; I plunge my tweezers with nervous anticipation into the mushy lumps of partially digested Sheephead guts, facing constant perils and danger. Okay, maybe I am being a tad hyperbolic, but hopefully my point comes across. My stomach content examinations are part of a larger effort to understand fish populations, and the effects MPAs have on fish conservation. So I carry on with my unwavering efforts, to explore the vast unknown wilderness that is the world of fish guts!

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In the midst of gory dissection, complete with gloves, photo courtesy of Dr. Jenn Caselle

Method to the Madness

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— Written by Ean Eberhard

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The experiment takes place in a cold room where the two multicultivators are shown side by side. Both are connected to the CO2 tank to the right which supplies a constant amount of CO2 to the 16 tubes.

The experimental design begins with the creation of artificial sea water. To this I add three different nutrient solutions; main stock, trace metal stock, and vitamin stock. This combination of nutrients and artificial sea water makes up F/2 media used for growing the culture of Phytoplankton that is specifically studied in this project. The name of this species is Thalassiosira pseudonana. Once the culture is healthy and acclimated the experiment can proceed. The experiment is split into two weeks, each week being split into two phases. The first phase is a three day long acclimating phase for the cultures to be monitored in their tubes. Monitoring the culture at this time assures that each tube is growing healthy before entering the second phase of 4 days, the experimental phase. The first week consist of two multicultivators (seen below), one set to maintain 15°C and the other 25°C. Each multicultivator contains eight tubes that are each inoculated with the same culture of Thalassiosira pseudonana. In each multicultivator there is a set of three tubes at low light intensities of 100µmol(photon)/m2/s, 200µmol(photon)/m2/s, and 300µmol(photon)/m2/s, a set of two tubes at medium light intensities of 400µmol(photon)/m2/s and 500µmol(photon)/m2/s, and a final a set of

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When taking a closer look at an individual multicultivator, one can see the aerators placed into each of the eight tubes. Behind the tubes are a set of LED lights which will be set to different intensities when running.

three tubes with high light intensities of 600µmol(photon)/m2/s, 700µmol(photon)/m2/s, and 800µmol(photon)/m2/s. Each tube is supplied with a continues flow of CO2 at 1000ppm with a pressure of 2psi. The multicultivators are set to begin a light cycle at 5am and go into a dark cycle at 5pm. These two multicultivators are designed to give us a total of 16 different environments for the phytoplankton to grow in, varying initially by temperature and light.

 

With the experiment started, the sampling begins. Each day, from each tube, I sample for, Non-photochemical quenching, light curve, chlorophyll, particulate organic carbon, nutrients, and pH. For the beginning, middle, and end of each experiment I sample for dissolved inorganic carbon, biogenic silica, and flow cytometry.

Sampling for Non-photochemical quenching and Light Curve

When sampling for NPQ and LC3, I remove the tubes and invert three times then pipette 3ml of the cultures from each tube into a cuvette, giving me a total of 16 cuvettes for NPQ measurements and 16 cuvettes for LC3 measurements. The cuvettes are placed in complete darkness for 30 minutes for a dark adaptation. After the 30 minutes the cuvettes are individually placed into an AquaPen, which will give a value for NPQ and LC3.

Sampling for Chlorophyll, Particulate Organic Carbon, Biogenic Silica, and Nutrients

Using a filtering apparatus and filter papers, I collect samples for Chlorophyll, POC, and Bsi by filtering the NPQ and LC3 samples. Each tube gets its own filter for Chlorophyll, POC, and Bsi. The Chlorophyll and Bsi samples are stored in the freezer for a later analysis while the POC filters are incubated and left to dry. The filtrate from these tubes are pooled together by light intensity, the low light intensities (100, 200, 300µmol(photon)/m2/s) are combined for a nutrient sample while the medium light intensities are pooled together for another nutrient sample and finally a third nutrient sample is taken from the high light intensities of 600, 700, and 800µmol(photon)/m2/s. These nutrient samples are also stored in the freezer for later analysis.

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The filtering apparatus uses a pump to create suction. A filtering paper specific to the material being collected is placed on a surface above the vacuum. The samples of NPQ and LC3 are filtered through the papers. The filtrate is collected for nutrients below and the material collected rest on the filter paper.

Sampling for Flow Cytometry

For flow cytometry measurements I pipette 1ml of the culture from each of the 16 tubes. I then fix these samples with 50µl of CH2O to kill the cell while preserving its structure. These samples are then stored in the freezer until processed and counted with the flow cytometer.

Sampling for Dissolved Inorganic Carbon

To sample for dissolved inorganic carbon (DIC) I fill a serum bottle full of the culture from each tube and add 50µl of mercury chloride. This gives me a total of 16 samples. The samples are then crimped shut to exclude gas exchange and stored in freezer for later analysis.

Sampling for pH

Sampling for pH is done for every tube. I pipette 3ml of the growing cultures from each tube into their own glass cuvette. These cuvettes are then placed in a dry bath for 5 minutes or until they reach room temperature. An initial reading is then taken by placing the cuvettes into a spectrophotometer where each cuvette is given three values at three different wavelengths. The cuvettes are then removed and treated with 50µl of m-cresol and then left for 5 minutes longer. The cuvettes are placed back into the spectrophotometer for another set of readings. Both the initial and treated values for each tube are entered into an excel spreadsheet with a preexisting equation that gives the value of pH. I should end with a total of 16 pH readings.

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To the left is the spectrophotometer that gives the values that are then used to calculate the pH for each tube. Above are 5 cuvettes demonstrating the visual grade of variance amongst pH when treated with m-cresol.

 

From Beach to Beach

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— Written by Emma Saas

Rising sea levels and beach erosion create problems for important intertidal species, such as beach hoppers, normally found on sandy beaches. Their sandy environment is shrinking, and scientists are wondering how well they will find new real estate. The distribution and abundance of different each hopper species may vary among southern California shores, but one can find beach hoppers, such as Megalorchestia corniculata, on many beaches. The dispersal of beach hoppers from beach to beach may be an important factor in the distribution of the four common species.

Yachts are a little bit pricey for our tiny beach hoppers, so how can they move from one beach to the next? The answer may be drift kelp, an important resource for marine environments that is produced by off-shore kelp forests. Beach hoppers can cling to drifting kelp and perhaps use this mechanisms as a lift to new beaches. How well they can cling, however, is an important question. Ideally, beach hoppers may be able to survive rising sea levels by clinging to drift kelp after it washes onto the shore and then leave with it as it floats to its next destination. An added bonus? Kelp is also food for these traveling beach hoppers! They’re able to sustain themselves for free while traveling (personally, Delta charges me excessive amounts for the same thing), which could enhance their survival on longer trips to a new beach.

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Beach hopper climbing in Macrocystis pyrifera, otherwise known as giant
kelp, commonly found on California beaches washing in from the kelp forests

Sea level rise will lead to habitat changes and a requirement for flexibility. According to recent studies, 25% of beaches are eroding worldwide and up to 67% of beaches in southern California could disappear by the end of the century. Creatures who made their homes on sandy beaches will see a significant narrowing of their stomping ground. Their ability to disperse to and from different beaches on kelp may reflect how well they can move around once sea levels are consistently encroaching on their living space.

Finding needles in a hay stack

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— Written by Maria Rivera

Collecting hundreds of kilograms of sediment samples is challenging but finding microscopic shark scales in the midst of hundreds of sand grains is another thing entirely. In the process of extracting denticles from large sediment samples, several steps such as lab processing, many gallons of acetic acid, and hundreds of hours of microscope time are required. First of all, we need to collect the samples (bulk of sediments), from a region with high and low shark abundance to be able to make comparisons. For example, with my project on a small island in the Caribbean called Curaçao, there are samples from two sites. One site has high human impacts while the other has less human impacts. A potential hypothesis might be that the more impacted site might have less sharks and therefore less denticles.

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Fig.1 shows the microscopes used in the lab when we are searching for denticles in a sample.

Once the samples are collected, we then weigh each sample in the lab. These samples are then separated into different sized fragments using sieves with various mesh sizes (2 mm, 500µm (micrometers), 250µm, 106µm, and 63µm). The denticles, which are the size of several strands of hair, are found in the 106, 250, and 500 µm fractions. The bulkiness of these samples would make it nearly impossible to pick through, like finding a needle in a hay stack, thus, we add 10% acetic acid to reduce the size of the mass significantly. This gets rid of all the carbonate (coral and shell) material, which compromises over 90% of the samples’ mass. We also have to rinse the sample with hydrogen peroxide to get rid of any excess organic material. All that is left is the non-organic and non-carbonate residue that the denticles are found in. Now it is feasible to pick through the different sized grains and identify our denticles under a microscope. Once we identify our denticles in a sample, we measure them and look at the ridges and how they are shaped. This helps us determine the type of shark form which it was shed. When we identify the denticles, we can plot our data and analyze/make inferences about how shark communities vary across different human interference sites. Going back to Curaçao, once we have all the data, we would be able to analyze what types of sharks roam each site and their relative abundance, as well as how that has changed over time. This will shed a light to how humans have impacted the coral reef shark communities in both areas.

It is important, however, to realize that a number of factors can impact the number of denticles we find in a sample. For example, denticle abundance can be affected by denticle shedding rates, transportation, and coral reef growth and sedimentation rates. In regards to shedding rates, we do not know how often sharks shed denticles or whether shedding rates differ between pelagic and demersal species, and this can impact how many denticles we find in a sample. When it comes to transportation, denticles can be transported horizontally by currents as they sink. However, our collection sites are located on very sheltered, low energy reefs, and modeling exercises have shown that sinking denticles do not travel very far. With coral growth, if a coral reef is growing at a fast rate, denticles would be deposited and then buried at a quicker rate. As a result, denticle densities per amount sediment would be lower at faster accumulating reefs. To correct for this, we date corals to determine how much time is encapsulated in each sediment sample. These are just some of the few things that we need to consider when we are analyzing denticle abundance and trying to relate that to shark abundance.

Fingerprinting the Culprit

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— 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

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— 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

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— 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

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— 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!