The hybrids are winning!

Although at this stage it’s a close race. Two and a half weeks ago I spawned sea urchins in the lab, setting up several purple urchin crosses with the hope of re-doing the feeding experiment that I lost this past summer when I was on the DL (that’s Disabled List, for those of you who don’t speak baseball). I was also fortunate enough to set up a hybrid cross, fertilizing purple urchin (Strongylocentrotus purpuratus, or “Purp”) eggs with red urchin (Mesocentrotus franciscanus, or “Red”) sperm. I would have done the reciprocal hybrid cross (red eggs by purp sperm) as well if I’d gotten any of female red urchins to spawn. However it wasn’t really spawning season for the reds, and I consider myself lucky to have persuaded that one male to release some sperm for me.

This is the first time that I’ve tried to raise the hybrid larvae, although I know it can be done because my colleagues Betsy and John did it many years ago, before I came to the marine lab. All of my larvae are the exact same age and are being raised side-by-side, so I can make direct comparisons between the Purp by Purp crosses and the Purp by Red hybrids. Incidentally, when speaking or writing about a hybrid cross the convention I’ve adopted is to reference the female parent first, so when I say Purp by Red I mean a Purple eggs fertilized by Red sperm. A Red by Purp hybrid would logically result from red urchin eggs fertilized by purple urchin sperm.

My experience raising sea urchin larvae is that things almost always go well for the first 48 hours or so; most (but not all) of the fertilized eggs develop into embryos and undergo the crucial processes of gastrulation and hatching. In some cultures the hatching rate is close to 100%. After that there’s a window of 3-4 days when cultures can crash for no apparent reason, although food availability or quality may be a factor. If the larvae make it past their first week of post-hatching life they generally cruise along until the next danger period which occurs at about 24 days. I change the water in the culture jars and observe the larvae under the microscope twice a week.

Today the larvae are 18 days old. It’s a little early for that second mortality period, but some of the Purp by Purp cultures never really took off. The larvae don’t seem to be growing or developing as quickly as I’m used to. Perhaps this has to do with lower water temperatures, especially after the prolonged period of high temps in 2014-2015. In any case, two of the four Purp by Purp crosses are doing well and the other two are just hanging in there.

There are two things I can see with the naked eye that give me a heads-up when cultures are crashing: the first sign is an accumulation of debris at the bottom of the jar and the second is an absence of larvae in the water column. The debris can be due to excess food, a build-up of fecal matter (not usually the case, as I’m pretty good at doing the water changes on time), the disintegration of larval bodies, or some combination thereof. If the water column is clear then the culture has already crashed and everybody is dead.

Today one of my jars had crashed. The water column was very clear and there was a lot of fluff at the bottom of the jar. I’d been wondering if I could figure out what the fluff was made of, so I sucked up a bit in a pipet and examined it under the microscope. I thought I’d see dead algal cells or pieces that look like defecated algal cells. This is what I saw:

18 January 2017
© Allison J. Gong

Silly me. I had forgotten that the corpses of pluteus larvae would disintegrate pretty quickly, leaving behind only the skeletal rods. The rods get all tangled together and trap the organic stuff, which is probably a mixture of uneaten and defecated algal cells and the soft tissues of the larval bodies. This explains the clear water column in the jar.

While the Purp by Purp larvae have had mixed success so far, the Purp by Red hybrids have been doing well. From the outset they appeared to be more robust than the Purps, and even though the fertilization rate was only about 50% the post-hatching mortality seems low. The hybrid larvae are also larger than the Purps, and are developing more quickly. In the two photos below the scale bar indicates 100 µm.

Pluteus larva of Strongylocentrotus purpuratus, age 18 days.
17 January 2017
© Allison J. Gong

Pluteus larva of a hybrid cross between S. purpuratus and Mesocentrotus franciscanus, age 18 days.
17 January 2017
© Allison J. Gong

The hybrid larva is about 10% larger than the Purp larva. Other than that they look similar, but to me the hybrid larva seems farther along the developmental process: its arms are proportionally longer and have a more mature look (although I don’t have any way to describe that to a naive observer). There’s something about the gestalt of the animal that makes me think it’s more robust than the Purp individual.

We’ll see how the pure Purps and the hybrids do from here on. I actually have the Purp larvae divided up into different feeding treatments, which I may discuss in a future blog post. In the meantime I’m trying to baby the hybrid larvae as much as possible, to maximize their probability of successful metamorphosis in six weeks or so.

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You can’t push a string

Northern California is currently being pummeled by a meteorological phenomenon called an atmospheric river. The storms produced by these “rivers” tend to be warm and can be very wet, such as the Pineapple Express storms that carry atmospheric moisture from Hawai’i to California. The weather station on the roof of our house has recorded 4.26 inches of rain over the past three days, and more will come in the next few days. In addition to the rain, the atmospheric river has brought very strong winds, gusting to 40+ mph. Combined with the saturated ground, winds like this can uproot trees and utility poles. So far we haven’t lost power, but are prepared with candles, firewood, and extra water. . . just in case.

Data from our weather station in Santa Cruz, CA.
8 January 2017

I am not a meteorologist, and this is not a blog about weather. I mention all the rain because it brought out the worms. Earthworms, to be more precise.

Earthworms are oligochaete worms in the phylum Annelida, which also contains the polychaetes (marine segmented worms) and hirudineans (leeches). The body plan for annelids is based on segmentation, or metamerism. Let me explain what that means.

Imagine a round water balloon. Now imagine two sets of rubber bands encircling the balloon along perpendicular axes. You’d have something like this:

where the green and red lines indicate different muscle types. Remember that we’re discussing a three-dimensional object here. We’ll call the red lines circular muscles, and the green lines longitudinal muscles. Now picture in your mind what happens when the circular muscles contract; how does this change the shape of the water balloon? What happens when the longitudinal muscles contract?

An annelid’s body consists of many fluid-filled segments, each with its own set of circular and longitudinal muscles. The segments are arranged along the anterior-posterior axis, with the head being located at the anterior end.

Adjacent segments are separated by a layer of tissue called a septum (anatomically speaking, a septum is any tissue that divides a cavity into two or more smaller spaces; think of the septum that divides your nasal cavity into left and right nostrils). An incidental amount of fluid may escape from one segment into the next, but for the most part they function as separate water balloons. Water isn’t compressible but is deformable, so contracting muscles around one part of the water balloon simply displaces that water to another part, and the balloon’s shape changes. Because each segment in our worm has its own complement of body wall musculature, its shape can be modified independently from that of its neighbors.

Rather than draw up another pedagogical worm, I’ll show you a real one. As I mentioned earlier all the recent rains have brought the earthworms out from their burrows. I was out and about myself this afternoon, and took pictures. This is the anterior (front) end of a worm. Not much of a head, is there? Earthworms are poorly cephalized, which makes sense when you consider that they live underground: an animal that spends almost all of its time in complete darkness has no need for eyes, and having sensory organs hanging off the body would impede its burrowing activities.

Earthworm on wet pavement.
8 January 2017
© Allison J. Gong

That pale pink apparently unsegmented bit of worm is the clitellum, a glandular structure used in reproduction. Another feature you can see is the difference in size among all the segments. Some of them are much wider than others. These are the localized deformations. The anterior-most segments are the widest; which type of muscle is contracted in this part of the worm? Which muscles are contracted in the segments immediately in front of the clitellum?

Earthworm on wet pavement.
8 January 2017
© Allison J. Gong

The annelid body plan originally evolved to facilitate burrowing through soft substrates. The fluid in each segment provides a stiffness against which the body wall muscles can contract, and the separation of adjacent segments allows the aforementioned localized deformations. An earthworm burrows by making its front end long and pointy (by contracting the circular muscles), jabbing it into the soil, swelling those anteriormost segments (by relaxing circular muscles and contracting longitudinal muscles), and pulling the rest of the body along. Next time you have a live earthworm at your disposal, watch how it moves either on top of or through the ground.

As you may imagine, while an earthworm’s body volume remains constant, its shape varies greatly. This has consequences for internal anatomy as well. For example, an earthworm’s gut is essentially a straight tube within a tube; it doesn’t have distinct compartments or side chambers as ours does. But it can’t really be straight, can it? If the overall body shape of an individual worm it follows that the internal morphology must be equally plastic. This means that the blood vessels and major nerve cord remain functional whether the worm is stretched out or scrunched up. Kinda hard to imagine that in the body of any vertebrates.

Does the title of this post make any sense now?

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Fine distinctions

Sea urchins have long been among my favorite animals. From a purely aesthetic perspective I love them for their spiky exterior that hides a soft squishy interior. I also admire their uncanny and exasperating knack for getting into trouble despite the absence of a brain or centralized nervous system. Have you ever been outsmarted by an animal without a brain? I have. It’s rather humbling.

Red sea urchins (Mesocentrotus franciscanus) and purple sea urchins (Strongylocentrotus purpuratus) share a common geographic range along the northeastern Pacific but generally live in different habitats. S. purpuratus is the common urchin in tidepools, while reds are almost always subtidal (although I have seen them in the intertidal on very low minus tides). The two species’ habitats do overlap a bit, as the purple urchin can live in subtidal kelp forests alongside the reds. There is a commercial fishery for the gonads of red urchins, which are prized as uni by sushi aficionados. I’ve tried uni once, and it tasted exactly the way I imagined the gonads of a sea urchin would taste. Not a fan. I’d much rather make a different use of urchin gonads.

The other week I collected some urchins from the field, hoping that they’d have nice full gonads. Gametogenesis in many marine invertebrates, including sea urchins, is governed at least partly by annual light cycles. Provided they have sufficient food, purple urchins have ripe gonads and spawn in the winter, from December through March. Reds spawn in the spring, from March through June. In my experience the best time to induce spawning of purps in the lab is December or January, when the urchins have developed gonads but likely haven’t spawned yet. There is no way of knowing the sex of any given urchin or the condition of its gonads, so this exercise is somewhat of a crap shoot even with the best of planning.

Ready to induce spawning!
30 December 2016
© Allison J. Gong

Today I shot up my eight field-collected purps, hoping to get at least one male and one female out of the deal. I got lucky with the timing, as one of the smallest urchins was a female and began spewing out eggs. This little female gave a lot of eggs! She was followed by three males and two more females. So out of my eight purps I ended up with three of each sex, and a spawning rate of 75% ain’t bad.

I set up some mating crosses and fertilized all of the eggs. I divided the little female’s eggs into two batches and fertilized them with the sperm of two different males (M1 and M2). Each of the other females’ eggs was fertilized by M1, who gave huge amounts of sperm. When I checked on the eggs about two hours post-fertilization most of them had gone through the first cleavage division and seemed to be developing normally and on schedule.

2-cell embryos of Strongylocentrotus purpuratus
30 December 2016
© Allison J. Gong

Just for the hell of it I decided to shoot up some of the red urchins we have in the lab. I didn’t really think they’d spawn, as it’s not the season for them to be gravid. Red urchins are large, heavy animals with long and sharp spines and they are much more difficult to handle. Four of the five that I shot up did nothing, as expected. It took a long time, but just as I was about to give up on them the biggest red began dribbling out a couple thin streams of sperm. I examined the sperm under the microscope and they were very active and healthy. Fortunately I hadn’t returned the purps to their tanks, and two of the female were still putting out some eggs. I rinsed the purp eggs into a clean beaker, pipetted up some of the red sperm, and added it to the eggs.

Sea urchin eggs are covered by a thick jelly coat. In the video you can see many of the red urchin sperm embedded in the jelly coat of the egg. Despite the frantic activity of the sperm, fertilization (as evidenced by the rising of the fertilization envelope off the surface of the egg) took much longer than it does when eggs and sperm come from the same species.

Egg of a purple sea urchin (Strongylocentrotus purpuratus) fertilized by sperm from a red urchin (Mesocentrotus franciscanus)
30 December 2016
© Allison J. Gong

Look at that beautiful zygote! Fertilization success in this hybrid cross was low, only about 50%. The eggs that did get fertilized went through the first cleavage division after about two hours later, which is right on time.

Eggs of a purple sea urchin (Strongylocentrotus purpuratus) fertilized by sperm from a red urchin (Mesocentrotus franciscanus)
30 December 2016
© Allison J. Gong

It remains to be seen whether or not the few hybrid embryos I have continue to develop. I have a colleague who has hybridized red and purple urchins successfully in the past, and has raised the offspring to adulthood. I don’t have any expectations of great success with this little experiment, but it would be very informative to raise known hybrid urchins. I’ve seen animals in the field that look like hybrids and there’s no reason to assume that hybridization between these two free-spawning species never occurs. The adults can be found living side-by-side subtidally, and there’s enough overlap in their reproductive seasons that some individuals of each species could very well spawn at the same time. On the other hand, hybridization that can be forced in the lab doesn’t necessarily occur in the field. I dumped a lot of red urchin sperm on those purple urchin eggs, and such high sperm concentration may overcome any mechanisms of reproductive isolation that exist under real-life conditions.

I’ll know more when I check on things tomorrow.

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Seashore to forest

I am fortunate to live in a place of great natural beauty. While the Pacific Ocean dominates much of the landscape, we are also partially surrounded by mountains. I grew up in the flatness of the San Joaquin Valley, a couple hours’ drive from both the sea and the Sierra Nevada but not near enough for either to have any appreciable effect on daily life. When I first moved here from the Sacramento area to start graduate school, I felt claustrophobic because I had been used to looking out in any direction and being able to see for miles around. I’ve long since grown accustomed to the fact that the only miles-long vistas we get are over the ocean and have come to appreciate the proximity of the mountains.

Here we are ideally situated so that ocean and mountain forest are close enough that both can be explored in a single day. And in fact, I did just that the other day, on Boxing Day. The elephant seal (Mirounga angustirostris) breeding season has started, and I wanted to go up to Año Nuevo State Park to see them. Alas, this idea didn’t occur to me soon enough to purchase tickets for the docent-led tour to the elephant seal reserve area, so we didn’t get close to the seals. But it was a gorgeously clear day and the scenery was every bit as spectacular as you’d expect from this part of the coast.

Año Nuevo Island lies a short distance to the southwest off Año Nuevo Point and is reachable only by kayak. The island is a marine wildlife refuge closed to the public, uninhabited by any humans except scientists. Elephant seals, northern fur seals (a type of otariid, or eared seal), rhinoceros auklets, western gulls, and Brandt’s cormorants all breed on the island. California sea lions don’t breed on the island, but several thousand use it as a haul-out site throughout the year. During the elephant seal pupping season white sharks come to the waters around the island to feed on pups as they learn how to swim.

Año Nuevo Island, viewed from Cove Beach atAño Nuevo State Park.
26 December 2016
© Allison J. Gong

It is not common for the air to be so clear. Usually there is fog or haze that obscures the buildings. There used to be a lighthouse on the island; the dilapidated tower was pulled down in the early 2000s to safeguard the wildlife. Some of the other buildings–a 19th century residence and foghorn station–are currently used as research facilities.

View to the west from Cove Beach.
26 December 2016
© Allison J. Gong

Even without a ticket for docent-led tour of the elephant seal reserve area, you can hike to the staging area from where the tours depart. The trail passes a freshwater pond that is home to two endangered California herps: The red-legged frog (Rana draytonii) and the San Francisco garter snake (Thamnophis sirtalis tetrataenia). Years ago I had a colleague in graduate school who studied the elephant seals up at Año Nuevo. I went in the field with him one day and got to wear the special blue research windbreaker. He told me that before being allowed to drive into the reserve area all of the researchers have to take a driving test that involves not running over plastic snakes that are placed in the road. This is to make sure that the endangered snakes won’t be inadvertently killed.

Freshwater pond at Año Nuevo State Park.
26 December 2016
© Allison J. Gong

We ate lunch at a lookout point of the tour staging area. Because the air was so clear we could see quite a way down the coast. Highway 1 as it passes under the cliffs immediately north of the Waddell Beach is visible at the far right edge of the photograph.

View towards Waddell Beach from Año Nuevo.
26 December 2016
© Allison J. Gong

After lunch we headed away from the coast and drove up Gazos Creek Road a few miles into the forest. It took all of about 15 minutes to go from beach to redwood forest. How cool is that? Two completely different ecosystems to explore easily within a day. Even the weather was different: sunny and warm at the beach, much cooler and damper among the trees.

Although we were up in the redwoods, this day I was fascinated by all of the moss growing on the trees. We’ve had a decent amount of rain so far, and the forests are satisfyingly wet and squishy. The creek we followed had washed out a bit of the road in a couple of places, and was closed to all traffic about 5 miles from the highway.

Moss-covered tree along Gazos Creek.
26 December 2016
© Allison J. Gong

We didn’t have a lot of time to poke around in the forest, but since we were in the area we stopped at Rancho del Oso on our way home to visit my favorite tree. Rancho del Oso is at the bottom of Big Basin Redwoods State Park. I take my ecology students there for the first field trip of the semester, because there I can introduce them to two of the ecosystems that define the natural history of Santa Cruz.

My favorite tree is a coast live oak (Quercus agrifolia) that lives just off the trail at Rancho del Oso. I love its gnarled branches that grow horizontally at ground level. It is an old, wise tree. Looking through its branches you see into the redwood forest of Big Basin. I normally photograph this tree at a different angle, looking into the forest away from the trail. This day I decided to shoot it from an angle parallel to the trail. I don’t think it’s quite as dramatic from this angle but there’s no denying the magnificence of the tree.

Coast live oak (Quercus agrifolia) at Rancho del Oso.
26 December 2016
© Allison J. Gong

Rancho del Oso is also the downhill terminus of the Skyline-to-the-Sea trail. The entire trail is about 30 miles, and most hikers take two or three days to hike the whole thing. I’m not much of a backpacker but one of the things I’d like to do this spring is the day hike from Big Basin down to Rancho del Oso. Doesn’t that sound like great fun?

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Return of the natives

When the most recent epidemic of seastar wasting syndrome (SSWS) began back in 2013, the forcipulate stars were the first to succumb. This group includes conspicuous members of intertidal and subtidal habitats, such as:

  • Pisaster ochraceus — the intertidal ochre star
  • Pisaster giganteus — the giant spined star, which lives in the low intertidal and subtidal
  • Pycnopodia helianthoides — the sunflower star, a huge monster of the low intertidal and subtidal.

In the past year or so, I’ve noticed P. ochraceus making a comeback at local intertidal sites. At first I was seeing stars in the 2-3 cm size range, and now I’m regularly seeing hand-sized ones clinging to the rocks.

4 mm juvenile Pisaster ochraceus star at Pescadero State Beach.
11 May 2016
© Allison J. Gong

You read that right. 4 mm in diameter. This is the tiniest forcipulate star that I’ve ever been able to ID in the field with any certainty.

Pair of Pisaster ochraceus stars in the low-mid intertidal at Natural Bridges.
22 July 2016
© Allison J. Gong

A hand-sized (dark orange) and much smaller (dark purple, tucked far back in the little cave) Pisaster ochraceus at Mitchell’s Cove.
28 November 2016
© Allison J. Gong

It seems pretty clear that the ochre stars, at least, are making a comeback. It’s likely that the larger ones are survivors of the SSWS plague. That little tiny one, though, may well be a post-SSWS recruit. Unfortunately we don’t know how fast they grow once they recruit to the benthos. We do know that when they recruit they’re about 500 µm in diameter, so even that little guy has grown a lot in however long it has been since it settled.

The really exciting news is that yesterday I saw my first P. giganteus since the SSWS outbreak began! I was up at Davenport Landing collecting sea urchins and saw this star in an urchin hole. The rock around here is a soft mudstone that is easily eroded. Urchins excavate holes by twisting their spines against the rock, and then live in them. Holes that are urchinless, for whatever reason, are quickly colonized by other organisms (including baby urchins).

A not-so-gigantic Pisaster giganteus star in an urchin hole at Davenport Landing.
13 December 2016
© Allison J. Gong

For a sense of size, this urchin hole is about 8 cm in diameter. The star is sharing it with a small anemone, most likely Anthopleura elegantissima.

Pisaster giganteus generally occurs lower in the intertidal than P. ochraceus, and I wouldn’t expect to see it on a tide that isn’t at least as low as -0.8 ft. It isn’t as closely associated with mussel beds as P. ochraceus, either, because it lives lower in the intertidal. Fortunately, this week’s low tide series includes a few days with tides below -1.0 ft, and I’m going back out today. I’ll be keeping my eyes open for not only Pisaster stars, but also the Pycnopodia that disappeared a few years ago. Although Pycnopodia gets very large, I don’t expect to see any really big ones running across the intertidal. However, Pycnopodia juveniles would indicate at least the beginning of a possible population recovery  from the SSWS plague.

So, wish me luck and keep your fingers crossed!

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