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I've shown you how sea urchin eggs are fertilized in the lab, and you've watched the fertilization membrane develop in real-time.

One day a few years ago, my colleague, Betsy, and I set up shop to spawn urchins.  We do this just about every year because it is super fun and we both enjoy watching larval development; plus, if all goes well we end up with a cohort of urchins whose genetic lineage is known to do growth experiments.

Anyway, after we shot up the urchins and they began spawning we took a sample of eggs to check on their shape.  They should be uniformly round and about 80 microns in diameter.  The first slide that we set up looked like this:

How did this egg get fertilized?

See that egg in the center, with the fertilization membrane?  Somehow that egg got fertilized.  This sample of eggs had not been in contact with sperm or any tools that might have been in contact with sperm, so how did this single egg get fertilized?  None of the other eggs on the slide had been fertilized, nor was there any visible sperm swimming around.

Betsy and I never did figure out what was going on here.  We decided it was one of the Mysteries of Life, and continue to marvel at all the complexities of life that we don't understand.  That's what makes being a biologist so cool--it wouldn't be nearly as much fun if we already understood everything.

In my next post I'll show you pictures of sea urchin larval development.

1

One of the all-around coolest things I do with my students is spawn sea urchins to show them fertilization.  We can actually watch fertilization occur under the microscope.  And since the early stages of development are the same in sea urchins and humans the students get to see how their own lives started--not in dishes of seawater and probably not on a microscope slide, but you get the drift.  I've probably spawned and fertilized sea urchins dozens of times, and I never get tired of it.

Part of the reason we can spawn urchins on demand (sort of) is that they are broadcast spawners.  In nature, urchins of both sexes shed their gametes to the outside and fertilization and all ensuing development occur in the water column.  This is convenient for us because it means we can culture the larvae and observe them at various stages of development.

Gametogenesis is seasonal in urchins, with the local purple urchin (Strongylocentrotus purpuratus) generally ripe from December through March-ish.  In the lab we can manipulate the timing of gametogenesis by subjecting the urchins to artificial photoperiod, tricking them into "thinking" that they are experiencing winter when the calendar says otherwise.

Fertilization is a complex series of events, some of which happen very quickly and some of which are a bit slower.  Here's a brief rundown:

  1. Sperm fuses with the outer layer (called the vitelline layer) of the egg.
  2. Sperm nucleus begins to enter the cytoplasm of the egg.  This causes the egg membrane to become impenetrable to other sperm and is called the fast block to polyspermy.  The egg is impenetrable about 1 second after the sperm and egg membranes begin to fuse.
  3. Once an egg has been penetrated by a sperm, vesicles in the outer layer of the egg fuse with the egg's plasma membrane and release cortical granules into the space between the plasma membrane and the vitelline layer.
  4. The granules trigger a cortical reaction that results in the lifting of the vitelline layer off the egg surface.  The vitelline layer hardens and is now called a fertilization envelope.  The hardened envelope keeps other sperm from penetrating the egg and is referred to as the slow block to polyspermy.

Why are there two blocks to polyspermy?  Everyone knows that it takes only one sperm to fertilize an egg.  It turns out that if multiple sperm enter an egg at the same time, development goes awry.  I've had cultures that I fertilized with too high a concentration of sperm that get through the early stages fine but crash soon afterwards.  So polyspermy is bad and it definitely makes sense that natural selection would come up with redundant ways to prevent it.

All this is to set up the following video clip.  These eggs were spawned at the end of February 2012 for my zoology class, and after all these years I was finally to record fertilization as it occurred in real time.  Actually, I can't take credit for the recording; Sid and Moriah were the ones who figured out how to make the camera play nice with the microscope and actually record video to a computer.

What you will see at the beginning is several large dark eggs on a yellow background, with lots of little sperms wiggling around.  There are way too many sperm for this particular set of eggs NOT to be dealing with polyspermy, by the way.  A few seconds into the video you will see what looks like a bubble forming around some of the eggs. The bubble is the fertilization envelope rising off the egg surface.  There's one egg that seems to be holding out, but by the end of the 1-minute-long video all the eggs will be fertilized.

Pretty cool, eh?

 

5

Yesterday I went in for my allergy shots. I've been doing this immunotherapy for several years now, after innumerable yearly bouts of debilitating bronchitis that lasts for 6-8 weeks. Silly me. If I had done the allergy shots back in my 20s, I wouldn't have had to suffer all these years.

My allergy scratch test was. . . interesting, shall we say. The nurse drew a grid on my back and started pricking me with antigens. By the time she got to the end of the first row the pricks on the left had left welts bigger than the box they were in. By the end of the test my back was one big itchy welt. The allergist was impressed. "You are a very allergic young lady!" he pronounced.

The upshot is that I get four shots to cover the environmental allergens--trees, weeds, pollen, dust mites, cats and dogs, and molds. For the past year or so I've also gotten an injection of honeybee venom, since I am a beekeeper and will get stung more frequently than the average person. My progress has been slow because of my overactive immune system, but back in May I reached my maintenance dosages of all five shots.

Yesterday I went in for my shots as usual and felt fine immediately afterwards. By law I have to wait 30 minutes after the shots before leaving so I was just sitting there knitting. Twenty-five minutes into my wait I started feeling flushed in my face and neck, and weird all along my GI tract. The nurse took me back into a room and took my vitals. My blood pressure was low-ish but my O2 sat was fine and my breathing unaffected. Just to be safe they called in the doctor to check on me. He gave me a dose of Benadryl and prednisone.

That must have been about the time my blood pressure started tanking. I remember feeling vaguely woozy and unhappy about the state of affairs. My guts were still griping and I was feeling hot on my face and cold everywhere else. They gave me an IM shot of epinephrine to stop the allergic reaction. The doc said, "This will stop the allergy but make you feel lousy." Boy, he wasn't kidding. My heart was pounding and I was still shivering.

By this time I was lying down feeling sorry for myself. I never lost consciousness but probably would have had I been sitting upright. My blood pressure didn't come back much and I got another shot of epinephrine and they started an IV to get some fluids into me. At this time they called 911 and were starting to look really worried. My blood pressure was about 60/30. That's pretty damn low, even for someone like me whose BP is on the low end of normal anyways. Apparently by the time the EMTs came to get me I was really pale. At least I was able to get onto the gurney myself.

This was my first time inside an ambulance. The EMT, a very nice man named Jorge, tried to start another IV in my other hand but couldn't get it going because my veins had collapsed due to lack of pressure. I was strangely unworried when he told me that. It took about 2 minutes to drive from the allergy doc's office to the hospital, where they set me up in the ER for observation. Since I had been given all the appropriate meds at the allergist's office they didn't give me anything else after I got to the hospital. By that time my BP had risen to 100/70, which is close to normal for me.

"Observation" in the clinical sense means just that. I was left alone for the most part, with a nurse coming in to check my vitals every half-hour or so at the beginning. The ER doc came in at the beginning and I didn't see her again until hours later. She told me they needed to keep me until the effects of the epinephrine wore off, to make sure the allergic reaction didn't start up again. Poor Alex had to take the day off work and sit with me. What a guy! He let me read the Time magazine he had scrounged from somewhere and found me a sandwich to eat. The hospital discharged me at about 4:30 p.m., almost six hours after I had been dropped off.

What does a blood pressure of 60/30 feel like, you ask? It's strange. I could hear my heart thumping because of the epinephrine, but my head was empty feeling and slow. I think I was talking coherently but don't know if I was actually making any sense. My thought process was very slow and I remember having to think about words before I could say them. All in all, I don't recommend the experience.

We returned to the allergy doc to show them I was still alive and to ask if we could leave my car there. They were all glad to see me standing upright. The doc said that anaphylaxis manifests in several ways: hives, difficulty breathing due to swelling in the airway, and a sudden drop in blood pressure. I never had the first two, but had the third in spades. And I didn't have just an anaphylactic reaction, but a severe anaphylactic reaction. Until then I hadn't realized just how bad it was. I am very grateful for the mandatory 30-minute wait after allergy shots. The waiting period was extended from 20 minutes to 30 minutes a while back, and if I had waited only 20 minutes I would have been on the road to the marine lab when the reaction occurred.

Today I am more or less back to normal, except for the Benadryl hangover. It is amazing how quickly the body recovers from such a severe shock like anaphylaxis. I think I'll wait until tomorrow before driving, though. And it remains to be seen what we'll do about continuing the immunotherapy injections. I had been rather cavalier about the whole thing but now will definitely be more conservative and cautious.

As I suspected, the little Dendronotus veligers didn't last very long.  On Wednesday the very last survivors had kicked the proverbial bucket.  All that was left in the jar was some debris and scum from leftover food.  They lasted nine days post-hatching, which is about the norm for me when I've tried to raise nudibranch larvae.  Something just happens (or doesn't happen) around Day 10 and they all crash after a week or so of apparently vigorous life.  Someday I may figure out what's going on.  In the meantime, RIP, little guys.

On the more fun side of marine biology, there's a new exhibit at the Seymour Center that is extremely cool.  Someone brought in a buoy that had been out in the ocean for a long time.  It's a perfect example of a fouling community.

People who have boats or just spend time in marinas know about fouling communities.  They're all the stuff that gets scraped off the bottoms of boats.  It's also the same stuff that grows on pilings and the underside of floating docks.  In this case the term "fouling" refers to early recruiting animals and algae that grow quickly to monopolize space.  Many of the fouling species seen in harbors are invasive non-natives.

A few years ago I hung a box of slides off one of the docks at the Santa Cruz Yacht Harbor and left them there for several months to see what would grow.  Here's what recruited and grew on a single slide measuring about 5x7.5 cm:

Fouling community of invertebrates and algae on a glass slide.
© Allison J. Gong

As you can see, it's a very colorful world down there!  The brightest red curly stuff is an introduced species of bryozoan called Watersipora.  It is a fast grower and can overtake the other stuff and form large clumps.  It grows as an encrusting sheet over surfaces, but when two sheets make contact they grow up each other and form those curly upright bits.  To model how this works, hold your hands in front of you, palms down, with the fingers facing each other.  Push your hands together until your fingertips meet, then continue to move them towards each other.  What happens is that your hands flex and your finger tips get moved upwards until your palms come together in a praying position.  If your hands were encrusting sheets of bryozoan colonies, that's how you'd get those curly pieces.

Anyway, the buoy on display at the Seymour Center has a lot of large barnacles.  The barnacles have been actively feeding and molting since they arrived last week.  They are definitely the most animated critters growing on the buoy, as shown here:

 

Barnacles are crustaceans that lie on their backs entirely encased in hard shells glued to other surfaces.  They feed by extending their thoracic appendages and sweeping them through the water to capture detritus and plankton.  It's a strange way to make a living, but it does work.

 

 

 

The best thing about where we live is that all we have to do is walk to the edge of the back deck and we're looking down into wild-ish nature.  I say "wild-ish" because while it is one of the natural arroyos common on the central California coast, there is a utilities access road at the bottom of it that is used by lots of pedestrians, cyclists, dogs, and the occasional municipal employees in a city truck.  But in the early mornings I feel that I have the entire canyon to myself, since most sensible people aren't awake at the crack of dawn on a regular basis.

This morning I was playing with some little birds when I remembered that the first spring we lived here I was able to "catch" a chickadee and a juvenile finch.  By "catch" I mean "persuade to come feed from my hand," not actually put into a cage or anything.  Chestnut-backed chickadees and both purple and house finches are year-round residents that readily come to our seed feeders.  We have a core group of 3-5 chickadees that visit us daily; the number varies from season to season.  Chickadees are vocal and friendly.  For little birds, they're surprisingly tame.

Back to our first spring here.  The chickadees are easy to watch.  They aren't afraid of people and come right up to us as they flit between feeders and bushes.  I started hanging out on the deck with some seeds in my hand and, sure enough, soon one little guy was brave enough to trust me:

My little chickadee!

Yep, that's a wild bird perched on my hand.  This is the only picture we managed to catch, although he was a repeat visitor through the summer.

Sometimes we are lucky enough to see a new (to us) bird at a feeder.  Our neighbor also has seed feeders and a hummingbird feeder, and two years ago we saw this handsome fellow:

A very studly male rose-breasted grosbeak

Plus, we get to look down on birds in flight.  How cool is that?  More on our avian neighbors in another post.

The Dendronotus veligers are still alive.  I've been running into the same difficulties I've always had when trying to rear nudibranch larvae:  hydrophobic shells that tend to get stuck in the surface tension of the water.  Larvae that are trapped at the surface can neither swim nor feed.

We can pretty easily rear sea urchin larvae in culture by stirring jars on a paddle table.  The stirring keeps the larvae and their food in suspension; without stirring the larvae would settle on the bottom and die.  Nudibranch veligers are stronger and faster swimmers than sea urchin larvae and I thought I could get away with not stirring them, as I worried that the paddles might break the larval shells.

Two jars are being stirred on the paddle table.

Two jars of larvae are being stirred on the paddle table, along with several jars of sea urchin juveniles that resulted from a spawning I did back in late February.  The paddles move back and forth and keep the water moving, ensuring that the larvae have pretty consistent access to food.

It's a little early to tell, but it seems that there may be fewer larvae trapped at the surface in these jars.  And I didn't see more smashed or broken larvae in these jars compared to the others.  I'll look at them again tomorrow to reassess.

One jar being bubbled and two beakers with no water movement

One jar of larvae is being gently bubbled, to see if this helps break the surface tension.  I started with bubbling that was too gentle, and the other day upped the airflow a bit.  There is a slow circular current in the jar that might be helping.

The two beakers in the front of this table have no agitation at all.  These larvae are dependent on oxygen dissolving into the water from the surface, and I'm a little worried that they might be a little oxygen-stressed.  They are definitely getting stuck at the surface, so I doubt this will be a long-term solution to that particular problem.

Tomorrow I will change the water in all the jars and beakers, and try to assess the amount of stuckage in each.  Hopefully either stirring or bubbling will be the way to maximize survival of my larvae.

3

Today a lot of my Dendronotus eggs had hatched on their own, swimming through the water as bona fide veliger larvae.  Nudibranch larval culture has officially started!

These bad boys are much more spherical now--whew!-- which makes me think that pointy-shell thing I saw last week was an artifact of their premature hatching.  Now they look like little swimming bubbles.  Interestingly, their shells are mostly empty.  My invertebrate larval culture guide says that planktotrophic larvae (those that feed while in the plankton) such as these hatch with relatively tiny bodies that grow as the larvae feed. We'll see if that holds for these guys.

I captured some video of the little veligers zooming around.  Here they are at 10X magnification:

Here's another short video clip of some veligers that were conveniently squished under the coverslip.  This kept them from swimming away and I was able to film them at higher magnification.  You can see the little velum whirling away and then being retracted.  See also how most of the shell space is empty?

So, now that these guys have hatched and have all that empty space inside their shells to fill up, they need to eat.  What do I feed them, you ask?  Well, because I was in a hurry to get them something, anything, to eat this morning, I fed them a bit of Isochrysis galbana, which is a haptophyte.  Algal taxonomy is not well established yet, and there are many ways of classifying both micro- and macroalgae.  I hesitate to wade into those murky waters, so suffice it to say that Isochrysis is a unicellular alga, golden-brown in color but neither a diatom nor a dinoflagellate.

This is what Isochrysis galbana looks like in culture.  We grow it in 1000-mL flasks of sterilized seawater and nutrients.

Isochrysis galbana in culture

According to the literature, veligers of Dendronotus frondosus can be raised on a mixture of Isochrysis galbana and a red alga called Rhodomonas salina.  And it just so happens that we also have R. salina in culture, so starting tomorrow the veligers will get a mixture of algae for their breakfast.

2

Every spring the barn swallows return to the marine lab, not exactly on the first day of spring as in San Juan Capistrano, but I always know it's really spring when they arrive.  They build their mud nests against the eaves of the buildings, and spend time chattering at us from the fences.

However, the swallows don't always choose the best location for their nests.  About two weeks ago a pair of swallows were determined to build their nest here:

Not a good place to build a nest.

The poor birds would build up a small pile of mud, only to get all twitterpated and bent out of shape whenever anybody walked out the door, which is every few minutes.  I'm not sure if the proto-nests fell down by themselves or were hosed off, but it took the birds about a week to take the hint.

Then they decided to build the nest here, which makes a lot more sense:

A much better site for a nest.

Doesn't the little guy (or gal) look pretty satisfied up there?  This site is farther away from any doors and is on a building that people don't go into or out of nearly as frequently, so the swallows should be able to raise and fledge their young successfully.

3

The marine gastropods and bivalves go through a larval stage called a veliger.  This larva gets its name from the ciliated structure, called a velum, that the animal uses for swimming.  Veligers have shells--1 for gastropods and 2 for bivalves--and can withdraw the velum into the shell.  Even gastropods that lack shells as adults, such as nudibranchs, have shells as larvae.

The egg mass from Dendronotus is still intact and the embryos are developing nicely.  This morning when I looked at it through the microscope I could see the little larvae swimming around inside their egg capsules.  I wanted to take a closer look under the compound scope, and when I teased apart the egg mass some of the larvae were forced to "hatch" prematurely.  They're not yet ready for life on their own but now they're out in the real world swimming, for better or for worse.

Not being one to let an opportunity like this go to waste, I took some video of the almost-veligers.

You can see the cilia on their little velums whirling around.  The larvae aren't as spherical as I had expected, based on what I've seen in other nudibranchs, and I think it'll be fun seeing how they develop.  More as things unfold!

1

What better way to start a new blog than to talk about sex?

This morning at the Seymour Center I noticed a blob of what looked like nudibranch eggs on the wall of one of the tanks. Looking around for the likely culprit I saw three big nudibranchs on the tank. Ooh, cool!

One of two slugs of this species in this tank.

This is Dendronotus iris, a large nudibranch, or sea slug. This bad boy/girl had a foot (the flat white bit that you see reflected in the aquarium glass) that was about 15 cm long. The brownish branched structures on the slug's back are its cerata, which function as gills. These animals do not have the ctenidium, or gill, that is typical of marine snails. Other nudibranchs carry their gills in a single plume that surrounds the anus.

This species is distinguished from D. iris by its coloration and some details of its anatomy.

There is one other big slug in this tank. It has a paler body color and cerata that are banded with orange and tipped with white.

Nudibranchs are among the rock stars of marine invertebrates--they are flamboyantly colored, have short adult lives with lots of sex, and leave beautiful corpses when they die. After a planktonic larval life of a few weeks, adult nudibranchs spend their time eating, copulating, and laying eggs. Each slug is a simultaneous hermaphrodite, capable of functioning as both male and female, and mating involves an exchange of sperm. In some other species of nudibranch the act of love can be followed by an act of cannibalism.

Nudibranchs lay egg masses in ribbons or strings that are characteristic of the species. It turns out that Dendronotus egg masses look like Top Ramen noodles:

Egg mass of Dendronotus.

Each of those individual little white blobs is an egg capsule that contains 10-30 developing embryos. These eggs were deposited yesterday (3 June) and the embryos have been developing but are not yet at any distinct stage. With water temperature at about 13C, I think they'll develop pretty quickly.

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