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I was making my usual feeding and checking rounds at the marine lab last Wednesday, when I saw this:

Pugettia producta, molting.  Time 10:09:12

This crab is a kelp crab, Pugettia producta. It is one of the common crab species on the California coast; you can find them in the low intertidal clinging to algae. Many of them are this golden-brown color, coincidentally(?) the same color of the kelp Macrocystis pyrifera. Juveniles are often reddish or dark brown in color, again matching or blending in with the algae where you see them. This particular crab has always been this color, at least since it has been in my care.

Crustaceans, as all arthropods, periodically molt their entire exoskeleton in one fell swoop. The exoskeleton splits along the transverse seam between the carapace and the abdomen, then the crab sort of slithers out backward. The entire exterior of the body, including legs, antennae, and mouthparts, is left behind as a larger version of the crab scuttles away to hide out for a few days until its new shell hardens.

I've kept lots of crabs and seen lots of molts show up in their tanks, but have never caught one in the act before. From when I started watching, in the photo above, to the final wiggle out of the old exoskeleton took no longer than 5 minutes.  Here's the sequence of photos documenting the molt:

Pugettia producta molting. Time 10:09:41
Pugettia producta molting. Time 10:12:18
Pugettia producta, molting. Time 10:13:57

Pretty nifty, eh?

 

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.

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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?

 

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 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.

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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.

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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!

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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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