Swarming Squid Sperm: A Strategy in Sneakiness

Sneaky swarming squid sperm. Yeah, let’s talk about that. ‘Cause you hear that and you gotta know, right? But before all the sperm and the swarming is the amorous squid. Let’s start there.

As you may expect, squid have both a male and a female. Male squid produce spermatophores, packets of sperm that they can transfer to the females. Female squid carry around these sperm packets until they are ready to spawn. That can be quite some time in some species. When they are ready, they will use the stored sperm to fertilize and then release hundreds or thousands of eggs into the water as jelly-like strands. That’s about what we know about squid reproduction, the rest is relatively mysterious.

A newish study in Current Biology sheds some light on the mysterious nature of squid sperm. The study organism is Loligo bleekeri, one of the more common of the pencil squids (Loliginidae) in Japan and southern Korea. It is moderately large (40 cm) with very short arms. It is a polyandrous species, meaning that males only mate with one female, but females mate with many males. It is a good mating system for researchers interested in mate choice and sperm competition (oh yeah, there’s a whole subdiscipline of the science of sperm competition – rethinking your job now aren’t you?). These have been shown to drive sperm evolution (yes, that’s a thing) and morphology to optimize fertilization success. Because in this game, it’s all about how many babies you have.

One of the things that makes this squid species particularly interesting is the dimorphism among males. Large “consort” males do all the work. They compete with other males, court females with colorful body displays, and guard the female until she spawns his offspring. Smaller “sneaker” males are just that: sneaky. They rush in under the nose (or beak, as it were) of the consort male, attach their spermatophore and book it on outta there. The dimorphism in males is reflected in their mating as well as their size. Consort males place their spermatophores inside the female’s oviduct, while the sneaker males just stick it onto the external body surface near to the seminal receptacle near the mouth. It isn’t as close to the eggs, but it must be a successful otherwise why do it? What is it that makes this stick-and-ditch strategy so successful?

To find out, the researchers dissected consort and sneaker males to recover their spermatophores. Then sperm were released into test tubes, diluted and tagged with fluorescent labels (each type with a different label). They observed that when the sperm suspension was drawn into a capillary tube the sneaker, but not the consort, sperm aggregated (or “swarmed”) to form a regularly striped pattern along the tube. And, when sneaker and consort sperm were mixed, still only the sneaker sperm swarmed. The sperm weren’t slowing down or sticking together, so what was causing the swarming? It’s not like the sperm are problem solving. So the next thought was: Maybe it’s a chemical response. So a filter assay was designed where two chambers were separated by a filter so fine that only small molecules could get though. A sperm suspension was put into the lower chamber and then each type of sperm added to the top to see where it swam. Again, only sneaker sperm migrated toward the filter. Okay, so it must be some kind of chemical attractant, but what and how?

Again, labeled sperm suspensions were put into capillary tubes. Then bubbles of different gases were microinjected into the solution. This assay revealed that carbon dioxide (CO2) attracted sneaker, but not consort, sperm. This CO2 is likely generated by the sperm via the carbonate system. Not exactly a super-simple system. To tease apart the mechanism, they developed caged carbonate (you’re thinking Han Solo…me too, but not quite the same) to sculpture gradients of bicarbonate (a basic solution, pH-wise). This system allowed them to determine that swarming depends on acidic (CO2 and/or H+) gradients but not on a biocarbonate gradient. Next, they found that carbonic anhydrases (CAs) are involved in swarming as CO2 sensors in cells.

But let’s go back to the acid thing (as both CO2 and H+ increase acidity). The researchers used a pH-sensitive dye to look at the acid gradient during swarming. They observed that the middle of the swarm acidified first, producing a H+ gradient outwards. When they added a buffer, the swarming disappeared. When they put a pipette of acid (H+) into the suspension, both sneaker and consort sperm moved toward it. But remember that only CO2 attracted the sneaker sperm. Additionally, the pH at which these types of sperm responded was different. They found that only sneaker sperm lowered their intracellular pH with environmental pH. This means that only sneaker sperm have a H+ transport system that allows for the CO2 attraction. And finally, they showed that calcium (Ca2+) influx controls cause the sperm to turn around when they reach the end (weak part) of the gradient.

Whew! That’s a lot of compact information! So let’s put it together in a whole-organism, what-the-heck-is-going-on kind of way. Why does it matter that sneaker sperm like CO2? Remember back to the placement of the spermatophores by each of the males. When the female releases her eggs, the consort male’s sperm has first access because it is in the oviduct. They fertilize a lot of eggs but not all. Then the female holds her eggs in her arms while she swims to a good substrate to release them. Squid arms and mouth are not all that far away from each other. This is when the sneaker male sperm goes to work. The swarming allows the sperm to stay close to the site of egg deposition and may be sensing CO2 released from the eggs; both increase the chances of fertilization. And, in the end, that’s what it’s all about.



Hirohashi, N., Alvarez, L., Shiba, K., Fujiwara, E., Iwata, Y., Mohri, T., Inaba, K., Chiba, K., Ochi, H., Supuran, C., Kotzur, N., Kakiuchi, Y., Kaupp, U., & Baba, S. (2013). Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2 Current Biology, 23 (9), 775-781 DOI: 10.1016/j.cub.2013.03.040


And for a little more info, here's an earlier study on the same topic:


Iwata, Y., Shaw, P., Fujiwara, E., Shiba, K., Kakiuchi, Y., & Hirohashi, N. (2011). Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours BMC Evolutionary Biology, 11 (1) DOI: 10.1186/1471-2148-11-236



(image via MarineBio.org -- Note that this species is Loligo vulgaris, the European squid. It is weirdly difficult to find images of L. bleekeri, but this image gives you some of the characteristics of the genus.)

Moving at a Snail's Pace

This post is about slime. Well, it's about moving around in slime. *squish*

Mollusca, or mollusks, is a large and highly diverse phylum of invertebrate animals. Within this phylum is the Class Gastropoda, or gastropods, which include snails and slugs. Generally, gastropods, specifically snails, have an asymmetrically spiral (coiled on one side) shell that functions as a portable retreat. Slugs are almost identical to snails except they lack a shell. The snail and slug body consists of a head, foot, and visceral sac/hump, and mantle (pallium). The head includes a mouth surrounded by one or two pairs of tentacles which often carry eyes and a pharynx containing coarse or fine teeth on the radula (like a tongue). The foot is the main locomotive organ and is usually the part that is visible outside of the shell. On the sole of the foot are mucus glands that secrete the slime that the gastropods crawl on.The visceral sac contains most of the inner organs and the mantle is a tissue fold covering it.

Though snails and slugs have no external extremities they are quite capable of moving around in their environment. Understanding this movement has been of interest to scientists and engineers for some time, even inspiring new classes of robotic movement and adhesive locomotion. It is known that a series of pulses of muscle contraction and relaxation traveling along the central part of the foot's ventral surface allows the snail to move forward, and only forward in terrestrial gastropods. The pulses of muscles are called pedal waves, the regions of the foot between pedal waves are called interwaves, and the distance between the two is called the wavelength. When these waves interact with the mucus secreted by the gastropod propulsive forces are transmitted to the ground. In describing snail locomotion, the number of waves is classified according to their number and direction. They are classified as a single train of pedal waves (monotaxic) or as a two (ditaxic) or four (tetrataxic) series of waves. It is also known that the crawling speed is directly proportional to the speed and frequency of the pedal waves.

A 2010 study in the Journal of Experimental Biology takes a look at the mechanism by which the propulsive forces are generated during gastropod locomotion. To accomplish this the researchers used a newly developed force-cytometry method where they calculate the spatial and temporal distribution of pedal forces from measurements of the deformation produced by the snail when it contacts a surface of known elastic properties. This allows them to study the movement is great detail. They can measure the horizontal traction stresses to the surface underneath the snail/slug without any interference with the animal's body. Neat. The study also analyzes the kinematics (motion without reference to the forces causing it) of the pedal waves and its significance of the generation of traction force. They did this to find the relationship between speed/wavelength and velocity, to determine if the waves maintain a constant speed/wavelength, to find how the snail accelerates and decelerates, and to see if a change in speed is accomplished by increasing the number of waves or by varying the speed/wavelength.


Fig. 1. from the paper showing the ventral surface of the banana slug

That all sounds very...complicated. So how do you measure the pedal waves of a snail's foot? If you know any biomechanists then you know they like to do two things - put animals on treadmills and put animals on transparent surfaces. In this case they tested banana slugs (Ariolimax californicus and A. buttoni), grey field slugs (Deroceras reticulatum), and garden snails (Helix aspersa) by placing them on transparent surfaces, illuminating them, and then recording them crawling with digital cameras. Turns out that if you illuminate the body in different ways you can get different information about movement. Add all of those ways together and you get a 3D reconstruction of the snail's foot as it moves.
The researchers found that when a snail/slug moves forward there are alternating pedal wave and interwave regions propagating from the tail forward to the head, but the interwaves remain stationary with respect to the ground. That result wasn't all that surprising, and agreed with previous studies. When they looked a little closer at the high-resolution images they found that the organization of the waves was not symmetrical and did not move at a constant speed. They observed steady wave acceleration followed by abrupt deceleration, a variable speed of pedal waves which modulated the magnitude of stresses under each wave. This was unexpected and observed in more than one of their test species, suggesting that the pattern is mechanically relevant to locomotion. They also found that the net forward force was generated beneath each stationary interwave. This is where the animal is pressing the foot against the ground and then pulling it backwards, propelling the body forwards. The foot is actually lifted during the pedal waves. Another result showed that that the crawling speed increased with pedal wave frequency. Not all that surprising, have more waves then move faster. And the mucus, we can't forget the mucus. This study's experiments showed that the slugs were able to move themselves over very thin threads of mucus without changing the pedal wave pattern or frequency. This suggests that the amount of pressure applied by the foot doesn't really matter for propulsion. Considering the rugged surfaces that snails and slugs move on that is not all that hard to believe.

The take home message? Mucus is helpful but it is the muscle movements that allow snails/slugs to crawl.

Read the study here, and there are videos in the supplemental materials:
Lai, J. H., J. C. del Alamo, J. Rodriguez-Rodriguez, J. C. Lasheras (2010) The mechanics of the adhesive locomotion of terrestrial gastropods. Journal of Experimental Biology: 213(22), 3920. (DOI: 10.1242/jeb.046706)

Cone of Silence

Genus Conus LINNAEUS, 1758. Not really a taxa that many people give much though to, but cone shells (or cone snails) are ubber cool. There are about 500 extant species of Conus, that's the largest genus of marine invertebrates. These mollusks are found between latitude 40° North and the 40° South parallel. That means you can find them in tropical and subtropical oceans including the Indo-Pacific, Panamic, Caribbean, West African, South African, Peruvian, Patagonic, and Mediterranean Seas. You can find a few other species outside of this region but they tend to be localized in South Africa, Southern Australia, and Southern Japan. Cone snails live in the intertidal muds and sandflats, areas where the high and low tides alternate, but you can also find some offshore or in deep waters.

When picturing the structure of a cone shell, think of something like an underwater snail. They have a strong, muscular foot with a flat sole that is truncated or widely rounded at the front and pointed at the back. The foot can be striped or pimpled, but the coloring is really variable, not just due to genetics but environmental factors as well. On each side of the head they have an eye on a stalk, stalks that are wide at the bottom and narrow at the end. The mouth of this animal is very elastic and includes sharp and often hooked teeth, allowing the cone shell to swallow large prey. Being a cone shell, they are covered by a shell. This shell is spiral shaped and can have interesting patterns, and they are very desirable to shell collectors.

Most people, including me, find the cone snails' venom to be its most interesting feature. We're talking venom that is often fatal, or at the very least causes temporary paralysis, respiratory trouble, or swelling and inflammation (depending on the species). The composition of this venom varies depending on the species, the individual, or even between injections by the same individual. The active components are small, disulfide-rich peptides called conotoxins or conopeptides, and they cause paralysis in the victim. The specific paralytic components include alpha-, omega- and mu-conotoxins which all prevent neuronal communication, each targeting a different aspect of the process. Alpha-conotoxins target the nicotinic ligand gated channels, omega-conotoxins target the voltage-gated calcium channels, and the mu-conotoxins target the voltage-gated sodium channels. These toxins are particularly interesting to scientists, especially neurobiologists and medical researchers, because they can be used to identify specific ion channels.

To be effective the venom must be delivered from the cone shell to the prey. The cone shell itself is relatively slow and unable to swim, and yet it hunts other, faster marine organisms such as fish. The venom is synthesized in the epithelial cells of a long, convoluted venom gland and stored in the gland's lumen. When the cone snail zeros in on its prey it extends it's proboscis which is loaded with venom and tipped with a specialized radula tooth that functions as both a harpoon and hypodermic needle. The snail then shoots it (by a ballistic mechanism, we're talking around 400 miles per hour) into the prey to deliver the venom. It is known that the distal end of the venom gland dilates into an oval structure called the venom bulb and it has been suggested the this bulb functions in venom transport, perhaps like a peristaltic pump. If you look at other animals that use jet propulsion, like scallops and squid, you see that the closing of their valves requires a burst contraction of the adductor muscle. This muscle shows high levels of glycolytic enzymes as well as arginine kinase (a type of phosphagen kinase).



Figure 1 showing the venom apparatus of cone snails.
Also, Figure 1A is probably the best figure I've ever seen in a peer reviewed paper.

 A study in the Journal of Proteome Research takes a closer look at the Australian cone species Conus novaehollandiae and Conus victoriae in order to shed some light on the role of the venom bulb, or pump. Specifically they look at the levels of enzymes and kinases integral to pump function. In terms of methods they did a protein extraction and 2-dimensional gel electrophoresis, a one dimensional gel electrophoresis of the venom gland and bulb proteins, a cDNA (complementary DNA) isolation and identification of arginine kinase and BIP (immunoglobulin binding protein), and an in situ hybridization of the venom bulb using an arginine kinase specific probe.

After lots of tables and graphs, some colorful and pretty and some not-so-much, they found that the venom bulbs contain high concentrations of arginine kinase. The presence of this kinase enables the venom bulb to contract very rapidly and repeatedly. That means that the cone snail can quickly force the venom through the venom duct and out through the proboscis and into the harpooned prey. In addition to the kinase, morphological examination of the bulb showed the organ to be highly muscularized. Three distinct muscle layers are separated by a tunic-like collagen sheet and the outer muscle layer, in particular, contains radially, spirally organized collagen fibers. Ok, cool. Layered muscle. What does that matter? Well, if we go back to the squid comparison you see that squids have inner and outer surfaces of muscle lined with collagen tunics. These tunics are stronger than the muscles and prevent the muscle from stretching longitudinally during contraction. This restriction and contraction allows the squid to propel water through it's jet at very high speeds. Now, the cone's venom bulb is less complex but it is likely that the function is similar. So rather than just holding the venom, these researchers found that the venom bulb is an active participant in the injection event itself. Previous studies have shown that the venom is pressurized before injection. This study shows that repeated burst contractions of the venom bulb in combination with the relaxation of the proboscis leads to a sudden ballistic discharge of the radula tooth, where it is shot into the prey and the pressurized venom pumped in by ongoing, repeated burst contractions of the venom bulb (you got an image of that in your head right? Wow!).

Read more in the article:
Safavi-Hemami, Helena , Neil D. Young, Nicholas A. Williamson, Anthony W. Purcell (2010) Proteomic Interrogation of Venom Delivery in Marine Cone Snails: Novel Insights into the Role of the Venom Bulb. Journal of Proteome Research: 9(11), 5610–5619. (DOI: 10.1021/pr100431x)

Learn more about cone shells at these links:
http://www.coneshell.net/pages/pa_genus_conus.htm
http://www.venomdoc.com/conotoxins.html
http://grimwade.biochem.unimelb.edu.au/cone/

Snails on Speed

This little blurb on ScienceNOW sums up nicely a new article published in The Journal of Experimental Biology:

"Talk about an oxymoron: A snail on speed. No, researchers weren't trying to make the gastropods slide faster—they were trying to improve their memories. When the great pond snail (Lymnaea stagnalis) wades into water low in oxygen, it extends a special breathing tube to the surface. A team of researchers trained snails not to do this by repeatedly poking at their breathing tubes when the snails tried to extend them. Two days later, the team again placed the snails in low-oxygen water. The snails trained in normal water had already forgotten their training, and they extended their breathing tubes twice as often as snails trained in methamphetamine-laced water, the researchers report tomorrow in The Journal of Experimental Biology. The results suggest that meth improves memory, something that has been previously observed in creatures with large, complex brains like rats and humans. But since the snails store their memories in a simple, three-neuron network, the team hopes that studying the meth effect in these gastropods will help pinpoint how the drug's memory magnification powers work."

The original report:
Knight, Kathryn. (2010) Meth(amphetamine) may stop snails from forgetting. The Journal of Experimental Biology: published online. (DOI: 10.1242/jeb.046664)

The ScienceNow story:
http://news.sciencemag.org/sciencenow/2010/05/scienceshot-snails-on-speed.html

Tree Octopus

Save The Pacific Northwest Tree Octopus!

http://zapatopi.net/treeoctopus/

This is a website that I've known about for a while, but it is fun to check back with on occasion. If you've never seen it before, its entertaining.

Its a parody site but on the left-hand side of the screen there is some cephalopod news (which includes the last story I blogged) and a link to the Cephaloblog.

Enjoy!

Octo HD

Class Cephalopoda are a group of mollusks that include octopuses (or octopi), squid, cuttlefish and nautiluses and can be found in all the oceans of the world. They are known for their high intelligence, ability to change color and texture rapidly, advanced eyes/eyesight, defensive ink clouds and jet powered locomotion. One of the characteristic traits of mollusks is a hard, protective external shell, and although the nautilus has an external shell, most other cephalopods do not (they are either greatly reduced or internalized). In the past, cephalopods were one of the dominant forms of ocean life; today, however, we see approximately 800 species.

Octopuses, in particular, are known for their intelligence. Several studies have shown that octopuses also have a large capacity for learning. A classic experiment illustrating this is one octopus learning from another how to open a jar to get the crustacean reward inside. And aquarium owners are well aware of the great lengths that octopuses go to in order to rearrange their tanks or even escape!

A recent study published in the Journal of Experimental Biology tested octopus behavior in a new way. Since many cephalopods use visual signals to interact with conspecifics, predators and prey, images can be used to gain insight into behavior. The researchers (Pronk, Wilson, and Harcourt) were working with the Gloomy Octopus (for real), Octopus tetricus, a native of the Sydney Harbor. They set up high definition televisions next to octopus tanks and showed them images of crabs and other octopuses. They then evaluated the octopuses response (excitable, aggressive, etc).

It isn't that this TV method hasn't been tried in the past, it has and it failed. The cause is likely the fault of the TV and not the octopus. Remember my mention of the sophisticated eyesight characteristic of cephalopods? Well, those sophisticated eyes looked at that old 26 frames per second TV and made no sense out of it. But high def TVs that run at 50 frames per second? Oh yeah!

When the octopuses were shown images of their favorite food (and one of mine), crabs, they rushed towards the TV and tried to attack it. When shown images of other octopuses they retreated to the back of their aquarium to hide. After several weeks of repeating the experiment the researchers got a good picture of the octopuses' "mood swings." Individual octopus tended to react in a consistent way to these stimuli within the same day, but within a week their behavior could be very different. The octopuses were also given images of items they had never seen before, such as a jar. Sometimes they would be bothered by the jar and other times they would be curious and go take a look at it. Overall, the authors concluded that the "gloomy octopus show temporal discontinuities, and hence display episodic personality." Basically, they are moody little things.

So now there is an excuse to buy fancy new tech -- to play with octopuses! I mean...ahem...to test octopus behavior and intelligence.

The paper abstract: http://jeb.biologists.org/cgi/content/abstract/213/7/1035

(image from scuba-equipment-usa.com)