Cephalove

Cephalopods have a lot to offer – tentacles, beaks, and big scary (and perhaps cute) eyeballs. Today, though, let’s look at a part of the cephalopod body that doesn’t get paid so much attention to, especially by us neurobiologist types: the ink.

Fossil squid with preserved ink sac. Ink sacs are often easily visable in coleoid cephalopod fossils. (via Maitri on Flickr - click through for original.)

Most coleoid cephalopods (that is, all the living cephalopods excluding nautiluses and a few deep-water octopuses) produce ink. This ink is composed chiefly of melanin, which is a dark brown pigment that is found throughout the animal kingdom. Humans have used cephalopod ink for a variety of purposes, including writing, drawing, dying, and cooking (the fact that both a dark brown color of ink and a genus of cuttlefish are both named Sepia is not coincidence.) In fact, you can buy tubs of cephalopod ink online.

Cephalopods use their ink for a different purpose, though; it helps them get away from sticky situations. When severely startled, cephalopods will release ink from their ink sac, which is pushed out of their funnel with a jet of water (which usually also jets the cephalopod in the opposite direction away from the perceved danger.) The resulting cloud of ink could serve many functions; it could conceal the escaping cephalopod’s location from the predator, serve as a false target for the predator (who would attack the dark ink instead of pursuing its prey,) frighten the predator, or even trick the predator’s sensory systems into thinking it had already caught something (I’ll explain this last one at the end of the post.)

One neat property of cephalopod ink, though, has nothing to do with how predators perceive it, but rather how cephalopods perceive it.

When one squid in a shoal inks (“inking” being the action of expelling ink into the water) the rest of the shoal can certainly see what has happened and be alerted to the immanent danger that way. In addition to this, though, it has been hypothesized that squid can chemically sense the ink in the water, which would give them another way to keep abreast of squid-predator interactions going on around them.

One study that found evidence for this hypothesis (which is actually a part of a series of studies in this line of research) was done by Gilly and Lucero (1991) at Hopkins Marine Station in California. These investigators restrained squid (loligo opalescens) by attaching their dorsal mantle to a platform with cyanoacrylate glue (the same stuff that Super Glue is made of,) and then used a pipette to place small amounts of various substances onto a chemoreceptive organ located behind the squid’s eye.

Photograph and photomicrograph of squid olfactory organ - from Gilly and Lucero (1992)

They recorded the activity of the squid with a video camera, and everything was done remotely, so that the movement of the experimenter’s would not upset the squid and cause extra escape-like behavior. Escape-like behavior was measured in terms of the pressure inside the squid’s mantle, which was recorded via a tiny pressure transducer inserted inside the squid’s mantle. One of their records is shown here – the spikes in pressure reflect jets of water being expelled from the squid’s mantle, as it presumably attempts to escape from the chemical stimuli that signal some sort of danger in the environment.

They found that pipetting ink from an animal of the same species of the test animal onto the olfactory organ caused jetting. Furthermore, they found that a specific component of squid ink, L-DOPA (which is a precursor of melanin, the main pigment in squid ink) caused jetting just as much as did whole ink. On this basis, the authors concluded that L-DOPA is used as a sort of chemical alarm signal between L. opalescens individuals. (I should note that some of the authors cited in this post write that squid ink is actually a cue, not a signal, as a signal results in an action on the part of the receiver that benefits both the receiver and the sender of the signal. Escape responses by squid in response to the ink of conspecifics do not fit this definition.)

A more recent study by Wood, Pennoyer, and Derby (2008) looked at the responses of Caribbean reef squid (Sepioteuthis sepioidea) to squid ink preparations. This species of squid has ink that hangs together in a mucous-ey glob in the water, forming what the authors call a “psuedomorph”, or false animal shape that confuses predators.

Each squid was tested by placing a small amount of an ink preparation into its aquarium and videotaping its behavior during and after this event. The authors used a scoring system to determine how defensively each squid behaved during each test, with points on a scale of defensiveness being awarded for behaviors like jetting, specific postures that are used to hide from predators, and certain color changes that are known to signal alarm. A higher score on this measure of defensiveness indicated that the squid was “alarmed” (or something like that) during the test. Below is a video showing on of their tests, produced by New Scientist:

The authors found that S. sepioidea responsed with alarm to fresh squid ink placed in their aquarium. The ink worked to cause alarm responses even after it had been frozen, albeit not as well – the authors noted that it changed in consistency, and dispersed much more quickly. Ink that was placed into an adjacent aquarium (meaning the squid could see it, but could definitely not chemically sense it) worked very well to stimulate escape behavior. This argues that one of the stimuli that this species of squid uses to respond to ink is its appearance. What about chemoreception, though?

The authors produced “melanin-free ink” by centrifuging fresh ink to remove all of the melanin-containing granules in it. They reasoned that this ink was just link the whole ink chemically, except that it did not contain the specific chemical that made it opaque (melanin). They found that the squids did not respond to this ink that they could not see. These results point to the use of vision exclusively in S. sepioidea in responding to other squid’s ink, apparently conclusively.

Frustratingly, they don’t, really. It would be easy enough to chalk this result up to species differences – one species can chemically sense ink, and the other species cannot. These results, however, don’t say enough to make this claim (although there may exist other research that answers this question.)

In a paper by Lucero, Farrington, and Gilly (1994), squid (L. opalescens) ink was analyzed for the presence of L-DOPA and dopamine (they found it, but that’s not the reason I mention it.) They found that, in seawater, L-DOPA and dopamine are rapidly degraded via oxidation reactions, which would certainly dampen any effects they would have on the behavior of squid swimming in that water. They also found that the L-DOPA and dopamine in squid ink did not degrade this rapidly – these preparations behaved as if they were being protected by some sort of antioxidant contained within squid ink. While the authors used ascorbic acid (a small, soluble molecule) to replicate this effect, it’s possible that any anti-oxidant activity in the squid ink is provided by a protein (or another large, centrifuge-seperable molecule.) When Wood et al. prepared their “melanin-free ink”, they may also have removed some component of the ink that is essential for its activity as a chemical signal (for a hypothetical example, a protein that prevents that oxidation of L-DOPA and dopamine in the vicinity of the ink blob.) They may even have done something that eliminated the L-DOPA and dopamine altogether – they provide not chemical analysis of their ink preparations, and so it’s hard to know. The authors acknowledge that this is a shortcoming of their work in their paper, so there’s been no oversight on their part – it just would have been nice if they’d done a bit more in the way of quantifying the chemistry of the preparations they were using. Oh well – it’s something for the next round of studies, I guess.

I mentioned that squid ink might trick predators into “thinking” they had already caught the squid and were eating it, a trick called phagomimicry. This is because squid ink (and the exudates that other molluscs exude under stress) contains, among other things, a full complement of free amino acids – these are chemicals that predators taste when they eat flesh. If a predator gets a mouthful of ink, if they can sense the amino acids that normally tell them that they’re eating flesh, they may behave as if they have already caught and/or eaten their prey, and give up pursuit.

Thanks for reading!

W. F. Gilly and Mary T. Lucero (1992). Behavioral Responses to Chemical Stimulation of the Olfactory Organ of the Squid, Loligo opalescens Journal of Experimental Biology

WOOD, J., PENNOYER, K., & DERBY, C. (2008). Ink is a conspecific alarm cue in the Caribbean reef squid, Sepioteuthis sepioidea Journal of Experimental Marine Biology and Ecology, 367 (1), 11-16 DOI: 10.1016/j.jembe.2008.08.004

Lucero, M., Farrington, H., & Gilly, W. (1994). Quantification of l-Dopa and Dopamine in Squid Ink: Implications for Chemoreception Biological Bulletin, 187 (1) DOI: 10.2307/1542165

I’m working on a post at the moment that should be up within the next few days, but I needed to get this up right now. Encephalon #83, hosted at Providentia, needs submissions – it’s set to be posted tomorrow, so get your submissions in while you can! Get them to Dr. Vitelli via that link or on twitter @rvitelli . Also, if you have any Circus of the Spineless submissions, hit me up on twitter @Cephalover or leave it them a comment.

Life is busy, isn’t it? Between final exams, applying to graduate schools, learning to play bluegrass, work, and scrambling to orchestrate a local response to the National Portrait Gallery’s suppression of AIDS-related art, I’ve gotten distracted from what really matters and let half of December go by with only a single measly post. But you, dear reader, are forever in my mind, speaking to me in an unforgiving yet encouraging tone of your hunger for cephalopod science! Well, dearest reader, I’m afraid that I’ll be a disappointment to you tonight, as well (although I have other tentacle-y matters to talk about, as you’ll see.)

I like cephalopod neuroscience. It seems so fertile, so ready to be studied. This blog was founded so that I would have a way to systematize my learning about the cephalopod nervous system. Besides my own wandering mind, I ran into a problem with that premise: there’s just not a whole lot of cephalopod neuroscience. The biggest fields of study (from what I have found) is ecology, which is probably because there’s much more money in being able to catch and eat squid than in knowing how their brains work. To try to get back to my roots, I’m going to try to use the winter break (during which I should get 1-2 hours of writing time/day) to cover what’s going on in cephalopod neuroscience these days, and write some more in-depth posts on functional neuroanatomy in cephalopods (can you tell that I’m a huge nerd yet?)

Let’s see, let’s see, what’s going on in the news… Oh, right:

Scientists and Rice University and Wood’s Hole Marine Biological Laboratory have been awarded a 6 million dollar grant to develop metamaterials (does that word bother anybody else?) that will allow the US Department of Defense to emulate the color-changing ability of cephalopods. The article I linked makes sure to mention that the grant is worth 6 million dollars, because that sounds like a lot of money – and it is. The relative cost of this project, though, is put into perspective when we consider that it’s only 6.25% of the cost of a single one of the 2,443 F-35 Jets the Department of Defense plans to buy over the course of its Joint Strike Fighter Program, and a mere 0.0009% of the Department’s yearly budget. It also approximately twice the National Park Service’s yearly budget. Not that I think the US doesn’t have its fiscal priorities straight. *rolls eyes*

Anyways, to move along, Dr. Margaret McFall-Ngai (known for her work on the Vibrio/Euprymna symbiosis) gave a great interview on the topic over at the Mother Nature Network. It’s definitely worth a read and a watch (as it even has video!)

On the west coast, squid fishermen (and fisherwomen, presumably, although I don’t know of an applicable gender neutral job title) have been very successful – so successful, in fact, that they’ve reached the State government’s catch limit for the first time since it was instated eight years ago and will not be allowed to catch squid for the rest of the season (until April.) It’s a good time to be in the squid business, I guess.

Thanks for reading!

I’m back on my mission of keeping you on the cutting edge of cephalopod-related video content online! Today’s selections all feature incirrate octopods, doing what they do best: looking incredibly weird as they slink around. The first two videos (by Tapio Kuiri and Bouju1, respectively) show hunting behavior, with some great interbrachial web shots. The last one, I have no source on, but if you like seeing writhing tentacles set to Chinese (I think) music and narration, it’ll be right up your alley! (If somebody can translate/give some background on the video, we’d all be very appreciative.)

Thanks for reading/watching!

I think everybody should check out the Challenger Reports, especially Zoology Report #44: Report on the Cephalopoda (graciously prepared and maintained by Dr. David Bossard) to read their species descriptions and see some beautiful illustrations like this:

The Challenger Expedition was one of the great early oceanographic expeditions; it’s worth reading if you’re into history or marine biology or both!

Thanks for reading!

A new edition of Encephalon is up at Bora’s Blog Around the Clock – and it’s a good one!  Topics include: infanticide, premature ejaculation, strawberry syrup, tortoise cognition, and the pseudo-reification of psychiatric problems through brain scans.  Psych and neuro bloggers are a weird bunch, I guess.

Anyways, head on over, check it out, comment around, and I’ll see you next month!  Again, let me know if you would like to host a future edition (email me at mike (dot) lisieski (at) gmail (dot) com, or PM me on twitter @Cephalover.)

Next month the carnival is at Providentia; so go ahead and send those submissions over to the good Dr. Vitelli.

I always sneer a little bit when species are described as “new”. Obviously, few species are anything like “new” – really we mean “newly discovered by science.” Anyways, the big news is that a previously undescribed species of squid was discovered by an IUCN-affiliated scientist from a sample taken in the southern Indian Ocean. A formal description is forthcoming, and you can bet I’ll cover it as soon as it comes out.

In the mean time, though, I wanted to bring you some more information about the family of squid that this guy belongs to (the Chiroteuthid family.) It turns out that I’m having some trouble digging up information on these species, as they’re relatively understudied – in my searches, though, I came across something totally unrelated (and totally awesome) that I just have to share with the internet. So, I’ll have to leave you wanting that primer on Chiroteuthid biology (I’ll probably get to it after finals) for this blurb on a very unusual octopod.

In 2004, Mark Norman, Renata Boucher, and Eric Hochberg published a description of a previously unknown species of octopod from several male specimens that was gathered in the western Pacific Ocean. They placed it in its own genus, calling it Galeoctopus lateralis. In most respects, this guy appears to be pretty typical for a deep-water octopod – in one respect, though, he’s strikingly different. See if you can tell what’s unique about this guy from this drawing of his body plan:

From Norman, Boucher, and Hochberg (2004)

Note the conspicuously short arm – more precisely, the third arm on the right side. This is the arm that contains the ligula, the organ that male octopuses use to inseminate females. Let’s take a closer look at this:

From Norman, Boucher, and Hochberg (2004)

It’s a bit hard to see in this photo, but on the oral side of the third tentacle (that is, the side that usually faces inwards, towards the mouth) there is a small opening. Inside this opening are tiny “teeth-like lugs”, which the authors suggest that males use to remove the sperm bulbs that previous males have left inside of the female they are mating with, thereby increasing the competitiveness of their own sperm. This is a pretty standard evolutionary strategy – if you prevent the other guy’s sperm from doing their thing, your own sperm (and thus your genes) have a better chance of successfully being incorporated into the next generation of your species.

The authors hypothesize that this unique structure is complementary to an equally unique bit of anatomy that is found in females of the species, an enlarged muscular appendage of the oviduct:

Male Galeoctopus may use the mouth-like transverse groove of the muscular ligula to grip and rupture the sperm bulbs of previous suitors… The muscular flange on the distal oviducts of the female may be related to a vigorous mating process, these muscles potentially anchoring the oviducts during copulation to prevent them tearing free from the visceral wall.

Wow.

NORMAN, M. (2004). THE SHARKCLUB OCTOPUS, GALEOCTOPUS LATERALIS, A NEW GENUS AND SPECIES OF DEEP-WATER OCTOPUS FROM THE WESTERN PACIFIC OCEAN (CEPHALOPODA: OCTOPODIDAE) Journal Molluscan Studies, 70 (3), 247-256 DOI: 10.1093/mollus/70.3.247

First order of business: I got to feed a giant Pacific octopus (GPO) a scallop today! By hand! It was pretty incredible. Thanks to the good folks at the Aquarium of Niagara (particularly Dan, the Director of Exhibits,) I got to see the nuts and bolts of the octopus enclosure and interact with Twister, their resident octopus (who is a female, if anybody was wondering.) It was a great time. One of the most interesting was seeing how they keep her from escaping – the top of the tank is open, but has 2-foot high walls around it that are covered in astroturf-like plastic material. Although Twister doesn’t seem to mind the feel of the stuff, her suckers can’t grip it and so she can’t pull herself out. Neat, right?

I’d also like to plug their Pepsi Refresh project, while I’m at it. They want to win a $50,000 grant to build a new coral reef exhibit, with the goal of increasing public awareness of the importance and precariousness of reef health in light of climate changes. This aquarium does a lot to educate the public in the region (I’ve hardly gone there without seeing a school field trip,) and so it would be money well spent. To help the Aquarium of Niagara win this grant, please head over to their page and vote for them! You can sign in with your facebook account or create an account with them – it only takes a second. You can vote once per day, and can even do it with your cell phone by texting their code, 102344 to Pepsi (73774). Please do all you can to help make this exhibit a reality!

Moving on: a friend just brought a new video game to my attention: Octodad. From the official website:

Octodad is a third person adventure game about destruction, deception, and fatherhood. The player controls Octodad, a dapper octopus masquerading as a human, as he goes about a day of his life. His existence is a constant struggle, as he must master mundane tasks with his unwieldy boneless tentacles while simultaneously keeping his cephalopodian nature a secret from his human family.

They have a free download on their website. I’m currently downloading it – I’ll post an update another day when I’ve had a chance to play it. The concept, though, is golden.

Thanks for reading!

One of the projects that The Southern Fried Science Network and friends supported during the Donor’s Choose initiative, “A Look Inside: Squid Dissection” aimed to get squid dissection kits for a class from Wicklund Elementary School in California to use in their biology lessons. I’m happy to say that this project is fully funded, and Mr. L’s class will get their squids (and get to dissect them, too!) Here’s the thank-you letter he sent:

Thank you all for supporting my class and helping to support my project. It is so hard in this day to teach Science in fun and creative ways. My students are eagerly waiting to do this project. They have been very excited about this since I mentioned it to them. I can’t wait to tell them that this project is now a go!

Your donation will help my students gain the practical knowledge needed to conduct a real science experiment.

On behalf of my students, thank you very much!

You should be proud of yourselves, dear generous donors (readers or not.) It’s a good day when a kid learns about a squid.

To recap the last post on the Euprymna/Vibrio symbiosis: Euprymna scopoles (also known as the Bobtail squid) is a tiny species of squid that has two light organs in the underside of its mantle. Vibrio fischeri is a species of bacteria, of which some varieties can live inside of the bobtail squid’s light organs. These bacteria produce light, which the squid uses for camoflauge.

The story of how the two evolved together to make the working symbiosis is long, complex, and as of now incomplete – scientists are still piecing together all of the many adaptations that allow these two species to live together. I’ll try to bring both of us, dear reader, at least one step closer to making sense of it in this post.

A juvenile bobtail squid. Photo by Loh Kok Sheng (click through to see his blog.)

The important thing about this symbiosis is that it is selective. The squid has these little pouches which are just perfect for bacterial growth, but only one species of bacteria is found there. This involves a number of processes of selection – for example, the squid’s immune system sends cells into the crypts of the light organs to eat up invading bacteria, and the lining of the light organs secrete antimicrobial chemicals (like nitric oxide.) Vibrio fischeri has evolved to allow it to prosper under these conditions. The first step of the symbiosis, though, involves the bacteria and the squid finding each other. The ocean is big, and there are lots of bacteria and squid in it; how do these two get together so reliably?

Nyholm and McFall-Ngai address this in a 2003 paper that examines what they call “the first site of symbiont specificity”: the mucus that coats the opening of the light organ crypts in juvenile squid. Let’s start from the beginning:

A baby bobtail squid hatches from its egg. (Awwwww! So cute!) While it developed, its bacterial partner was nowhere to be found. After it hatches, though, a colony of V. fisheri will become established in its light organs within mere hours. Nyholm and McFall-Ngai looked at the surface of the mantle where the crypts open to the seawater and found that specialized cells in this area secrete mucus in response to the seawater that it would normally encounter right after hatching. This mucus helps trap bacteria, which can then colonize the light organ. V. fisheri normally make up about 0.1% of the bacteria found in seawater, though, so in order to beat out the competition, they must have some ability to interact with the light organ in a special way. Nyholm and McFall-Ngai hypothesized that the mucus layer on the outside of the light organ was key to this specificity, and conducted a number of experiments to test this idea.

First of all, they found that if they exposed hatchling bobtail squids to seawater without V. fisheri in it, all sorts of bacteria could be found in the light organ mucus. However, when they used seawater with small amounts of V. fisheri in it (again, on the order of one-tenth of one percent of the total bacteria in the water,) the colonies that formed in the mucus were almost exclusively V. fisheri. This indicates that this mucus excretion has some role in establishing the specificity of this symbiotic relationship, in that it somehow “screened out” all of the other species of bacteria that might have taken hold in the mucus and started to multiply.

Image of a V. fisheri colony in a bobtail squid light organ, labeled with green fluorescent protein. Aa, anterior appendage; pa, posterior appendage. (from Nyholm and McFall-Ngai, 2003)

They also determined that most of the V. fisheri present when they took their measurements had been collected from the water; this is in contrast to a scenario where a few cells were captured and then multiplied. To do this, they used a chemical called nalidixic that prevents cell replication while not affecting cell growth – when exposed to this chemical, bacteria won’t divide, they will simply elongate. By looking at how long V. fisheri cells grew in the light organ mucus, the experimenters determined that the cells were growing at a low rate in the mucus – in fact, they were growing much more slowly than they do in a plain culture! Thus, it’s unlikely that a few cells were captured by the mucus and then dividing into the large colonies they found; rather, there may exist some way for V. fisheri to selectively adhere to the mucus and be efficiently collected from the water (the authors say that this is unlikely, but not completely ruled out – it seems to me a likely explanation, especially taking into account the results of a series of studies that I’ll write on soon.)

The authors than tried using killed V. fisheri, to see if there is something specific to the presence of the bacteria (for example, some component of their outer membrane) that inhibits the growth of other bacteria. They found that, although killed V. fisheri could still adhere to the light organ mucus, they did not prevent the growth of other species of non-symbiotic bacteria. This implies that the bacteria perform some active process that prevents the growth of other bacteria in the light organ and allows V. fisheri to establish its dominance there, even though the mere presence of V. fisheri bacteria doesn’t kill other kinds of bacteria.

This symbiosis, then, which occurs very quickly and very specifically, depends (as most great things do) on mucus. Somehow, V. fisheri interacts with the squid’s secretions to beat out it many competitors. Interestingly, though (and I won’t cover the methods here, for time’s sake) the authors also found that the V. fisheri that colonize the crypts initially are not necessarily able to produce luminescence. It seems that the species of bacteria is selected during the initial stages of colonization, but that later on, specific strains that are better able to produce light are selected for while those that do not produce light are expelled or die off – each stage of selection no doubt involving a complex set of signals between the squid and the bacteria.

Thanks for reading!

Nyholm, S., & McFall-Ngai, M. (2003). Dominance of Vibrio fischeri in Secreted Mucus outside the Light Organ of Euprymna scolopes: the First Site of Symbiont Specificity Applied and Environmental Microbiology, 69 (7), 3932-3937 DOI: 10.1128/AEM.69.7.3932-3937.2003