Category Archives: Estrada, Carlos

Was the heat too hot for L. pictus to handle?

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— Written by Carlos Estrada

After a temperate and not-so-long summer, the Lytechinus pictus project has come to an end. In that time, I’ve unfortunately caused the deaths of a few of my beloved urchin, but in the process raised hundreds of thousands of larvae. All in the name of science! And have I got science to report! We’ll start with what was surprisingly the most difficult part of project, taking pictures.

Morphometrics

Some quick analysis of variance (ANOVA) showed that there was no significance between the lengths of the larvae and the temperatures in which they were reared at any stage. The gastrula stage held the strongest relationship between rearing temperature and length, but very little relationship was seen with the prism and pluteus stages. This conclusion corresponds with that of Hammond and Hoffman (2013), which found that increased temperatures had no significant effect on larval skeletal length. Developmental timelines, however, differed as predicted with larvae reared in the 20°C buckets reaching all three stages much quicker than those reared in 17°C buckets.

Thermal Tolerance

Preliminary data shows significance between rearing temperature and thermal tolerance at both gastrula and pluteus stages. The prism stage, however, shows no significant difference between the two rearing temperatures.  All life stages showed little to no mortality until they reached higher temperatures, in which mortality increased dramatically. Although the beginning in mortality can differ, 31°C seems to be the temperature at which mortality reaches 90-100%.

Heat Shock: hsp70 Expression

Unfortunately, my first attempt at attaining any data on the expression of the heat shock protein hsp70 through gel electrophoresis did not go well. No bands appeared on my gel, but don’t panic! After a few tweaks to our formula, we’re ready to make another stab at it first thing in the morning. My partner-in-crime, Erin DeLeon Sanchez, has run her own gel in the past with pluteus DNA that showed bands. She also went to the trouble of conducting qPCR and found that hsp70 expression was stable throughout all both rearing temperatures and heat shock temperatures.

Discussion

Very little ecological research has been done on Lytechinus pictus and I can’t express how proud I am to have done my part. But more studies are needed to better understand the relationship between this urchin and it’s changing environment. Such studies could help us predict not only this species current and potential future biogeography, but that of its prey and predators as well.

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

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— Written by Carlos Estrada

Step 1: Collecting Gametes

Carlos_Urchin

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!

 

Turning up the heat on the painted sea urchin

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— Written by Carlos Estrada

With global ocean temperatures and acidification on the rise, it is becoming increasingly important to study in what ways marine life may be affected. Slow-moving echinoderms, such as sea urchins, may be especially vulnerable due to their inability to migrate far distances in a short time. Ocean acidification has been shown to stunt the skeletal growth of purple urchin larvae whereas elevated temperatures increases their developmental rate (Padilla-Gamino 2013). If these increases in acidity and temperature continue, urchin larvae size may decrease, leading to further predation and low survivability. These effects on urchins are not only threatening to this species, but to the entire ecological system in which they belong. Sea urchins are important in keeping kelp forests in check and also serve as food to a variety of organisms, including the California sheephead and otters. Any detriment to urchin development may prove disastrous to this delicate balance, which is why this summer I will be studying an urchin species not previously studied in the Hofmann lab known as Lytechinus pictus, commonly referred to as the painted sea urchin.

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Lytechinus pictus. Picture taken in the Hofmann Lab’s seawater room

Unlike the purple urchin, which is found as far north as Vancouver and as far south as Baja California, L. pictus is found from central California all the way down to the Ecuador. This makes L. pictus particularly interesting to study due to its tropical habitat and wide-ranging temperatures that fall anywhere between 9°C and 23°C. My project will attempt to answer a few questions regarding the effects of increased rearing temperatures on L. pictus larval development. The focus of this project will be to find the thermal tolerance of L. pictus and whether thermal tolerance varies across developmental stages by observing four different stages. This will allow me to see if there are any developmental stages that are most vulnerable to increased water temperatures. Morphometrics will also be involved by taking pictures of these different stages to check for differences in size or abnormalities. Lastly, but certainly not leastly, my project will have me look at whether thermal tolerance reflects expression of the heat shock protein hsp70. What makes this project so exciting (other than learning about invertebrate development, as well as new molecular skills!) is that very few studies have been conducted on L. pictus, making it feel as though I’m traveling directly into uncharted waters. So, hopefully, by the end of this summer we will have a better understanding of how echinoderms in warmer climates might fare when faced with increasing ocean temperatures.