Friday, August 28, 2015

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.



ResearchBlogging.orgHirohashi, 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:


ResearchBlogging.orgIwata, 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.)

Wednesday, August 26, 2015

Chemists Know

An epic chemistry parody of Frozen's "Let It Go"

So good.




(the moles never bothered me anyway...)

Monday, August 24, 2015

The Secret Anatomy of Toys

Jason Freeny is an artist and toy designer. He creates interesting anatomy illustrations and sculptures of toys. They are a mixture of detailed anatomy, advanced graphics, and pop iconography. Here are a few of my favorites:


Digitals:

"Kitty Anatomy"
"Pneumatic Anatomica"


"Goldfish Cracker"


Sculptures:
"Dissected Sully"
"Yoshi Anatomical Sculpt"
"Cutaway 8" Anatomical My Little Pony"

You can see lots more over at the Moist Production website.

Thursday, August 20, 2015

Falling with Style: Controlled Gliding in Spiders


Sometimes I read a paper because the methods catch my eye. I can just imagine some scientists sitting around a table with a beer and saying, “I wonder what would happen if we just dropped a bunch of spiders from the tops of trees.” An article published online yesterday did just that.

Barro Colorado Island is a man-made island is located in Gatun Lake, created by filling of the Panama Canal. It is covered in tropical rainforests, and its inhabitants have been studied extensively. It would be a mistake to look at a forest as only ground habitat. The canopy supports a tremendous abundance of life, particularly arthropods. These critters are particularly tasty to predators and must find a way to escape within their decidedly hazardous habitat that is located over 90 feet (30 meters) off the ground. Falling is a bit of a risk. But what if you do fall? You could land in the understory or on the ground which, if it doesn’t kill you, is both unfamiliar and full of predators. To avoid this potentially lethal scenario, many wingless arthropods have developed the ability to orient their bodies (via visual cues, appendages, and other structures) such that they are more likely to fall towards tree trunks.

The genus Selenops is a large and common group of nocturnal spiders. They are easy to find and collect, often hiding under bark or in crevices. The researchers went out into the forests and collected a bunch of these spiders. The spiders were then weighed and photographed. The images were then analyzed for the horizontally projected areas of different segments and appendages. Then these data were put together to give “effective wing loading.” Here’s where we get to the fun part. The spiders were put into individual plastic cups and taken up into the canopy. The cups were held at a known distance from a tree trunk, inverted and tapped to release the spider. Geronimo! These drop tests were filmed at 60 frames per second so that glide index (ratio of horizontal distance from the tree trunk to the total distance traveled) could be calculated. The videos also allowed for the measurement of how the legs were being used to maneuver. The spiders were also scored in terms of their performance, either directly reaching the tree, indirectly or irregularly gliding towards the trunk, or failing completely and landing elsewhere.



Most of the spiders had a successful, directed decent without the aid of draglines or balloons. They fall several meters and then glide to a trunk. During this fall, they were observed to adopt body postures that orient their bodies to descend head first with the forelegs out to the side and slightly forward and the rest of the legs out and back. This foreleg asymmetry was shown to significantly change body heading meaning that they are using their legs to control their glide trajectory. Also, glide index was shown to decrease with increasing body mass. They hypothesize that this negative relationship means that larger spiders must accelerate under gravity to airspeeds where aerodynamic lift becomes significant relative to body weight.

This is an interesting result because other arachnids do not show it. These spiders have developed the ability to control their glide trajectory. This means that they have evolved novel mechanisms of body righting and maneuvering. Gliding spiders….cool.


ResearchBlogging.orgStephen P. Yanoviak, Yonatan Munk, & Robert Dudley (2015). Arachnid aloft: directed aerial descent in neotropical canopy spiders J. R. Soc. Interface, 12 : 10.1098/rsif.2015.0534

(image via Toy Story screencap)

Monday, August 17, 2015

Tuesday, August 11, 2015

Mutualism a.k.a Caterpillars Drugging Ants To Do Their Bidding

From the study - Figure 1. Attendant Workers of
Pristomyrmex punctatus standing on or around
Narathura japonica caterpillars
The manuscript is done! Submitted! Summer interns are finished. Boot up Normal Life Mode, please. Recommence blogging. So many good papers have come out during my hiatus. Where to start…where to start…

If you have read this blog for any amount of time then you will come across my fascination with ant manipulation, particularly zombification. This is why my cursor stopped over a new paper in Current Biology about caterpillars manipulating ants to do their bidding.

Let’s start with mutualism. This is a topic that I have visited in the past, and in ants for that matter. It’s a nice little relationship between species that involves an exchange of goods and/or services. In the natural world, this often means food and protection.

In this study, the researchers chose the Japanese oakblue butterfly (Narathura japonica), a lycaenid belonging to the Theclinae subfamily of butterflies. Many in this group are myrmecophilic, meaning they associate (often mutualistically) with ants in some way. The Japanese oakblue caterpillar has a specialized exocrine gland, the “dorsal nectary organ (DNO),” that is located on the seventh abdominal segment and is flanked by tentacle organs (TO). The DNO secretes sugar- and amino acid-rich honeydew while the TO secretes scents to “talk” to the ants. A “Come on down!” or “Danger, Will Robinson!” type thing. The ants tend to the caterpillars and keep them safe for a nice, sugary food reward. But is that all to the story? Obviously not or this post would end here.

To do this experiment, butterfly eggs and their associated ants (Pristomyrmex punctatus) were collected and reared separately. Then three test situations were set up with 50 ants per treatment:

  1. “Experienced” ants – had free access to the caterpillars and their DNO secretions
  2. “Inexperienced” ants – no caterpillar access, just some sugar soaked cotton balls
  3. “Unrewarded” ants – had access only to caterpillars that had their DNO’s blocked (a little bit of clear nail polish goes a long way)

After 3 days in their test situation, 10 ants from each treatment were moved to Petri dishes that were set on pieces of white paper with a line on it to divide the dishes into 2 halves. After the ants acclimated to their new little plastic arenas, they were observed to see how many times they crossed the center line (“locomotory activity”). Also at the 3 day time point, ants and caterpillars were frozen in liquid nitrogen until their brains could be dissected out, specifically removing the optic lobes. Now, I’ve done some pretty small dissections, but those come nowhere close to ant brain removal! Wow, just wow. Once those itty bitty brains were out, they were processed for liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for serotonin, dopamine, octopamine, and tyramine. Very simply, that means making an ant-brain-aerosol that is then separated and identified by component.

They found that experienced ants had significantly less locomotory activity than the other two groups. So what does an ant walking, or in this case not walking, across a line even mean? Well, the fact that the ants are staying put signals that they are “standing guard” for the caterpillars. Okay, let’s say that standing means guarding, how do we know that this is really caterpillar-related and not just standing there? Well, first of all, it was only the experienced ants that did this. Second, the researchers observed that the caterpillars often “everted their TOs,” meaning that they turned them outward. This is typically a response the caterpillar makes when it is attacked by a predator – “Raise shields!” Experienced ants responded differently than the other two when they saw this caterpillar behavior in that they responded aggressively. This aggression is a response to the caterpillars’ alarm, one that has the ants defending against the predator. The fact that only experienced ants had these responses suggests that something in the DNO secretions is eliciting these defense behaviors.

So what is it about these secretions? That’s where the LC-MS/MS comes in. Biogenic amines are known function as neurotransmitters, neuromodulators, and/or neurohormones. This means that they can modify behavior in insects. DNO secretions contain biogenic amines. This analysis showed that experienced ant brains had low dopamine levels. Now, that’s important because dopamine has been shown to be involved in both locomotory activity and aggression in well studied organisms like fruit flies. Starting to see some links here, yes? To confirm the linkage, ants from each treatment were given reserpine, a small-molecule inhibitor that depletes dopamine but not serotonin in the brain. This test resulted the same behaviors, but the LC-MS/MS showed increased dopamine and serotonin in the ant brains. So same but different.

There is another aspect to consider: Who loses if the mutualism goes away? The honeydew is not the sole source of nourishment for the ants. They can leave and be still be fine. The caterpillar has much more to lose than the ant (its life via predation). So the caterpillars must be doing something besides sugar-loading their ants.  This is where the caterpillar gets sneaky - finding a way to make their ants to both stick around and defend against predators. As the authors put it, they they insert “manipulative drugs [into the honeydew] that could function to enforce cooperative behavior…from attendant ants.” Put that way, I’m okay calling it “ant mind control.”


ResearchBlogging.orgHojo, M., Pierce, N., & Tsuji, K. (2015). Lycaenid Caterpillar Secretions Manipulate Attendant Ant Behavior Current Biology DOI: 10.1016/j.cub.2015.07.016

p.s. The supplementary materials have a nice little video of ants in lined Petri dishes.

(image is Figure 1 from the above paper)
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