— Written by Carlos Estrada

Step 1: Collecting Gametes

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!