I’m sorry. I had to go there. You didn’t really expect me not to, did you?
The reason, of course, is that today we got our first settled and metamorphosed Pisaster stars! We were doing our normal Monday water change when I noticed a teensy orange speck on the bottom of one of the jars. I used my beat-up old paintbrush to remove the tiny dot to a dish, put it under the dissecting scope, and saw this:
From this picture it’s a little hard to see what’s going on. The entire body has contracted a lot, from a 2.5-mm larva to about 1/4 of the original size as a 600-µm juvenile, and become much more opaque. There are tube feet and spines as well as some remnants of larval body (the soft bits at the bottom of the animal) at this in-between larvenile stage.
Here’s a picture of a fully metamorphosed little star:
I expect we’ll be seeing more tiny orange dots on the bottoms and sides of the jars in the next several weeks. At some point we will have to figure out what they eat and provide it for them. But at least we know we’re able to get them through the larval phase.
Just for kicks, here are some pictures of where we grow the larvae and how we do the twice-weekly water changes.
Today is the six-month birthday of my baby urchins! Six months ago to the day these little guys were zygotes, and six-months-plus-one-day ago their parents were roaming the intertidal. They grow up so fast!
And lastly, that little shmoo-type thing that I found in the plankton yesterday has revealed itself to be. . . an anemone!
One of the things I like best about cnidarians is the beautiful transparency of their bodies. I love how you can see fluid circulating through the tentacles. Gorgeous, isn’t it?
California is being slammed by a very intense El Niño event, and the effects are being felt up and down the coast. Seawater temperatures here in Santa Cruz have been in the 15-16°C since late May, and in the past week have shot up to 18.5°C. While Californians have their fingers crossed that El Niño will bring drought-relieving rain this winter, I’m also concerned about how it is affecting marine life.
On a whim, I decided this morning to take a look at what’s going on in the local marine plankton. I grabbed a plankton net with a mesh size of 165 µm (we call a net with this mesh size a “zooplankton net”) and headed out to the end of the wharf. The water is a milky greenish aqua color, which the Monterey Bay Aquarium says is due to a bloom of a type of phytoplankton called coccolithophores. I’ve never seen living coccolithophores before, as they are usually not common in Monterey Bay. Besides, they are really small and don’t often get caught in the type of plankton net that I deploy. So while I didn’t really think I’d catch any coccolithophores, it is always fun looking at plankton. Given the warm water and lack of productive upwelling this season, I didn’t know what to expect.
When the water around here is this color, it usually means that phytoplankton are not very abundant. And sure enough, when I pulled up the net it wasn’t very brown and didn’t have that certain smell of diatoms, which were extremely thick earlier in the season. In fact, earlier this month the Central and Northern California Ocean Observing System (CeNCOOS) detected high levels of both the toxin domoic acid and the diatom, Pseudo-nitzschia, that produces it. But in today’s sample I didn’t see a single diatom and only a few dinoflagellates. It’s conditions like this–warm, nutrient-depleted water–that the coccolithophores like.
One of the best things about examining a plankton sample is that you never know what you’ll find. Despite the lack of phytoplankton in the water, my sample was chock full of interesting zooplankters. In addition to the usual copepods (probably the most abundant animals in the world) and their larvae, there were larval polychaete worms and molluscs, medusae of multiple species, and assorted other goodies.
In the video clip below you can see the familiar baby-urchin-learning-how-to-walk, as well as a better view of the polychaete. Note the conspicuous segmentation and chaetae (bristles) that the animal splays out when disturbed or, in this case, gently squashed under a cover slip.
The little worm looks like it’s dancing! Sometimes you can see its four eyes.
This creature is called a cyphonautes larva. It is the sexually produced pelagic propagule of a benthic bryozoan colony, most likely Membranipora membranacea. If it looks like a swimming triangle, well, that’s exactly what it is.
Goodie #4:
This living lava lamp is very enigmatic. I called it a shmoo-type thing and was so intrigued that I isolated it into a separate dish for further observation. I was delighted to see that, a few minutes later, it had settled and metamorphosed into this:
It has eight stubby little tentacles and an obvious cnidarian appearance. I think it is a little anemone, but only time will tell.
This beautiful object is a radiolarian, a type of marine amoeba. The main part of the cell is concentrated towards the center and pseudopodia are extended along the skeletal spines, which, in addition to making the cell an unpleasant mouthful, also aid in buoyancy. This one was rather large, measuring about 2 mm across. I saw many of these in today’s sample.
All in all I spent a very enjoyable morning collecting and looking at plankton. I didn’t see any coccolithophores, but I’m thinking that I probably should go out again with a finer-meshed net to see if I can catch them. And to see what will happen with the zooplankton if the phytoplankton remain scarce for the rest of the season.
Although the last thing that any of us marine invertebrate biologists want to see again is a wasted sea star, the syndrome has once again been making its presence felt at the marine lab. It has been almost two years since I documented the initial outbreak, and while nobody is convinced that it has entirely run its course, most of us, myself included, had thought that perhaps the first wave had passed. Then, back in March of this year, I saw one of my stars doing this:
Those large white blotches on the aboral surface are open wounds, or lesions, through which some of the animal’s innards are protruding. The arm towards the top of the photo has also begun dissolving, literally wasting away into the environment. The lesions eat right through the epidermis, liberating the skeletal ossicles that lie underneath it; I’ve circled two of them on the right side of the photo and there are two more at the bottom.
The discovery of this wasting animal was alarming and for a while I held my breath whenever I check on stars at the lab, but after several weeks of not seeing any additional sick animals I relaxed my guard and concluded the incident was a one-off. So imagine my horror to walk in this morning and see this in one of my tables:
Sea stars generally don’t just lie on their aboral surfaces, and this animal was making no attempt to right itself. See how the margin between the arms is a little wavy? That isn’t normal, either, and shows that the animal’s ability to regulate its internal water content has been compromised. And while bat stars routinely scavenge by extruding their stomachs through the mouth and digesting whatever it comes into contact with, they don’t leave the stomach hanging outside the body when they aren’t feeding.
All of which gave me a bad feeling in the pit of my own stomach, which only got worse when I turned the animal over:
The animal appears deflated and has small lesions all over its aboral surface. I was feeling a little deflated myself when I saw this. With stars it can be difficult to determine just how alive (or how dead) an individual is. This one didn’t fall to pieces when I picked it up, which didn’t exactly surprise me because Patiria is less prone to losing its arms via autotomy than the Pisaster species (ochre, short-spined, and jewel stars) and Pycnopodia helianthoides (sunflower star), in whom one of the symptoms of wasting syndrome is a violent ripping off of one’s own arms. I suppose this makes the whole episode marginally less horrific than when I saw my Pisaster stars wasting, or maybe I’ve become jaded.
In any case, I had to decide what to do with this sick star. It was in a table with half a dozen other bat stars, so whatever it was exposed to or was itself exuding has already been spread to the others. I couldn’t leave it there to rot in place, but neither did I want to throw it away if it was still somewhat alive. I turned the animal so it was oral-side-up again and left it alone to see what would happen. If it righted itself I’d assume it was more or less alive and isolate it in a quarantine tank; if it didn’t, then all hope was lost and it could be tossed. When I was ready to leave the lab several hours later, it was in the exact same position. Verdict: dead.
So, why now? I’ve been thinking about this, and here’s what I came up with. The densovirus that has been linked to sea star wasting syndrome is always around in the environment. Like other opportunistic pathogens it doesn’t usually cause a problem until a host organism becomes stressed or compromised. For the past two years we’ve been aware of wasting events up and down the coast, which wiped out the most vulnerable individuals. Animals with resistance, however, were able to survive. The survivors may have been weakened, though, and the mild El Niño of 2014 and the much stronger one we have now in 2015 have resulted in water temperatures much higher than normal. I haven’t plotted the data yet, but in June and July the water temperature has been hovering at 15-16°C, with jumps this week up to 18.5°C over the past couple of days. These warmer temperatures can be very stressful to animals, which may be just what the densovirus needed to “announce [its] presence with authority” (that’s a quote from my favorite baseball movie, Bull Durham). Outbreaks of wasting syndrome are probably caused by a combination of factors: population density of the host animal, presence of the densovirus, overall health of the host, water temperature, water chemistry, and others I haven’t thought of. We are certainly not close to a complete understanding of this phenomenon.
At this point I don’t have many stars left in my collection. I hope I get to keep them.
In the spring and early summer, beekeeping is really easy. The nectar is flowing and the bees are busy and happy because there’s plenty of food for everybody. The colonies build up quickly and, if a beekeeper isn’t diligent, throw swarms when the bees feel they are too crowded. There’s a certain amount of good-natured competition among beekeepers for swarms but around here there are enough to go around.
The hives at my house face directly east into a wild canyon, where they forage on blackberry, coffeeberry, and poison oak in addition to the gardens and ubiquitous eucalypts in the neighborhood. It’s a pretty prime location for the bees, as they wake up as soon as the sun rises over the lip of the canyon and are shaded from the afternoon sun. Someday I’d like to do a pollen analysis of our honey and determine exactly what the bees are feeding on; it would be very interesting to see how that changes through the season.
All through the spring I spent time on the landing at the top of the stairs near the hives, writing in my nature journal or drawing. I’d sit with my back against the fence, notebook on my lap and binoculars at my side, and watch birds flying past at eye level. Because of the nectar flow the bees were mellow and pretty much ignored me, even when they were foraging in the coffeeberry bush a mere meter or so away from my head. Sometimes they even landed on me, treating me as just another surface on which to take a brief rest in their busy day.
Have you ever just sat next to a bush that’s buzzing with bees? It’s one of the more joyful and pleasant things about springtime, in my opinion, and I recommend it highly.
However, all good things must come to an end, and this holds for the nectar flow as much as for anything else. This year we had a very strong nectar flow early in the season, starting in late January and continuing until, well, some time before today. I had suspected that the spring bonanza would be short and intense, with flowers putting all of their energy into heavy nectar production early in the year while there was still some water in the ground, and it seems I was right.
When the nectar dries up, bees and beekeepers enter a time called the dearth. We beekeepers can detect the onset of the dearth in a couple of ways: (1) the hives get lighter as the bees begin to eat through their honey stores; and (2) the bees get irritable because they’re not finding much forage. While beekeepers in the springtime boast about being able to tend their hives naked, nobody would dare do so in the late summer or autumn. It turns out that right now our hives are sending us mixed signals. They are still putting up honey, at least some of them are, and they’re getting pissy.
This afternoon I went outside to my usual spot on the landing to draw for a bit. It was very pleasant there for about 20 minutes, then a single guard bee decided that This Must Not Be. I’ve noticed that bees don’t seem to like dark hair, of which I have quite a lot, possibly because it makes them think “Bear!” It doesn’t matter whether my air is pinned up or flying loose, the bees find it, get tangled in it, and try to sting my head. That’s no fun for any of us. Anyway, this persistent guard bee got it into her tiny brain that I was not to be tolerated, and she kept buzzing around my head. The buzz of an angry bee sounds different from the gentle hum of a happy bee and I was alarmed immediately. She made her point and I fell in line. I packed up my supplies and left, but the diligent guard bee followed me all the way back to the house. At that point she decided that she’d done her duty and let me escape.
This defensive behavior will only get worse as we move into autumn. Even if the bees have enough honey stored to last through the winter, they will react to the shortening days of late July and August by refusing to continue feeding their drone brothers and more aggressively defending their hives. There will be no more lounging on the landing for me until next spring.
Today is Monday, which means Scott and I changed the water for our Pisaster larvae. I should have taken some pictures to show you how we do it. Maybe next time.
The largest and most developed larvae are now 2.2-2.5 mm long, not including the long brachiolar arms, which is about as big as they’re going to get. They are still eating and developing their juvenile rudiments. Unlike the sea urchin’s pluteus larva, these brachiolaria larvae lack any kind of skeletal structure and are entirely squishy–they bend and flex along any axis and can scrunch into surprisingly tiny balls. Those long arms are flexible as well, and sometimes the larvae swim around with their arms tucked or rolled up. I haven’t been able to catch them in the act with the camera, but both Scott and I have seen the larvae react to encountering a surface by flipping the long arms around as though doing the backstroke.
If a picture is worth a thousand words, then how much is a video worth?
The weird alien-like effect is enhanced by the dark background I film them against. Except for their guts and the tips of their arms, the larvae are entirely transparent, which makes it difficult to photograph them. The black velvet that I use as a background, combined with lighting from an oblique angle, maximizes contrast and makes the transparent bodies more visible. The little illuminated “stars” in the background are actually part of the texture of the velvet.
To capture this video I shrunk the larvae’s universe into a single drop of water on a depression slide. This means they couldn’t swim too far out of the field of view and would have to bump into each other. Don’t worry, though, after the photo shoot I returned the larvae to one of the jars and let them swim away. They’ll be just fine.
Earlier this week an acquaintance asked me about the development of sand dollars, specifically if it is anything like that of sea urchins. It just so happens that sea urchins and sand dollars, while not in the same taxonomic family, are in the same class, the Echinoidea. As close kin, they share a similar larval form, the pluteus larva, and undergo more or less the same development. If you’re satisfied with the short answer, you can stop reading here.
Interested in the details? Then read on!
In my first year of graduate school I took a course in comparative invertebrate embryology through the University of Washington up at the Friday Harbor Lab in Puget Sound. It was a blast! We spent mornings in lecture and afternoons in the field and/or in the lab observing and drawing embryos and larvae. At night we would lie on our bellies on the dock and shine a light over the water, scooping up the critters that rose to the surface. It was in this class that I did my first studies of larval development and fell in love with the echinopluteus. In that class we studied several echinoid species, but the only one I was able to follow all the way through to metamorphosis was the sand dollar Dendraster excentricus.
Even a casual beachcomber has likely encountered the naked tests of dead sand dollars, but I’d bet that most people haven’t seen them alive. The bare test is white, while in life the animal is a fuzzy grayish-purple color. As their name indicates, they live on sandy bottoms in the shallow subtidal, where they prop themselves in the sand and catch particles of food that fall on them. This photo below is of the sand dollar exhibit at the Monterey Bay Aquarium. My friend, Chris Mah, is the owner of the most excellent Echinoblog and has explained how sand dollars really are sea urchins. Check it out for some good information from a real-life echinodermologist (yes, I just made up that word).
But the original question I was asked to address is about the development of sand dollars as it compares to that of sea urchins. Given that these animals share the same larval form, you would probably not be surprised to learn that their overall development is very similar. Just to remind myself of exactly how similar, I dug through my old embryology notebook and took pictures of some of my drawings. Keep in mind that these drawings were intended to document my observations, not to be pretty or artistic. However, they may be useful as comparisons with the photos of sea urchin larvae that I’ve been taking over the years.
According to my notes, to speed up development we cultured these larvae at room temperature so we could get through the entire larval period in the 5-week course. I didn’t record the temperature, but would guess it to be about 17ºC. At this temperature it took the Dendraster larvae only two days to get to the early pluteus stage, complete with functioning gut and skeletal rods (see drawing on right).
In contrast, the Strongylocentrotus larvae that I started this past January were still forming their guts at the ripe old age of 2 days. You’ll have to take my word for that, as I didn’t take pictures. I do have a photo of the embryos when they were 1 day old and had just hatched:
1-day-old embryos of S. purpuratus 21 January 2015 Photo credit: Allison J. Gong
By the age of 7 days, the Dendraster larvae already had three pairs of fully developed arms (the anteriolateral, postoral, and posterodorsal arms), with the fourth and final pair (the preoral arms) just beginning to form:
After a few weeks the Dendraster larvae had grown all four pairs of their arms as well as their juvenile rudiment on the left side of the gut. The individual I drew has a fully formed rudiment with five tube feet (labelled ‘podia’ in the drawing) and an additional waviness to the ciliated band (shaded in the drawing). My guess at the time was that this individual was developmentally competent, or ready to settle out of the plankton and metamorphose.
When I was in Friday Harbor I was lucky to see one of my Dendraster larvae undergo metamorphosis more or less as I was watching under a microscope. I wish I’d had the set-up to take microscope pictures, because it was an amazing phenomenon to observe. I did make one last drawing of the newly metamorphosed and benthic tiny sand dollar and its discarded larval skeletal rods:
I’m sure that I drew what I saw, but looking at this drawing with more experience I wonder if the tube feet really looked like that. Oh well. One of the last things we did as a class was “graduate” our remaining larvae off the dock and wish them luck as we released them into the real world.
With my most recent batch of S. purpuratus larvae, I began seeing competence at 45 days post-fertilization. The first bona fide juvenile urchin didn’t begin crawling around on tube feet until 50 days:
The next logical question would be: Why do the sand dollars develop so much more quickly than the urchins? I don’t have a definitive answer for that. Since that class in Friday Harbor I haven’t had another chance to study sand dollars, but in my experience my most recent cohort of sea urchins progressed through development at the normal pace for the species in this location. Some species just take longer than others, and the differences could be due to any number or combination of factors: water temperature, genetics, presence or absence of settling cues, water chemistry, and so on. The take-home message, if you’ve managed to read this far, is that yes, sand dollars and sea urchins undergo pretty much the same development. It’s the same as the short answer at the top of the post, but wasn’t it fun getting there the long way?
Remember those little urchins I brought into the world back in January? Well, they’re doing well, for the most part. About a month ago I took about 250 of them, measured them, and divided them into three feeding treatments: one group I left on the coralline rocks they all cut their teeth on, one group is eating the green alga Ulva, and the third group is eating the kelp Macrocystis pyrifera. My plan is to keep the groups on these foods and monitor growth and survival.
After one month it appears that mortality and growth are not related. I have lost more urchins from the Macrocystis treatment than from the other two, and yet those that have survived this far have grown quite a bit. A month of the experiment gives me exactly two data points, which may over time indicate the beginning of a trend but for now are entirely meaningless. I’ll have to wait at least another month to see if what’s happening now continues.
However, I also took pictures of the urchins, and some of them are getting so pretty! I’m curious to see if the two macroalgal diets (Macrocystis and Ulva) affect the color of the urchins as they grow. Of course, color is very subjective and I can’t duplicate the exact lighting conditions when I take microscope pictures of different subjects, so at this point they all look the same no matter which food they’ve been eating.
My most colorful urchin at the moment is a little guy from the Ulva food treatment. Its test diameter is only about 4 mm, but its color is very vibrant:
In addition to the five distinct reddish-purple bands on the body, I like that this urchin has so much color on its spines. This individual looks like it may skip the green stage that urchins of this species go through and go straight to purple.
Some people are energized by constant activity, feeding on and drawing strength from the buzz of never-ending stimulation. Although I sometimes wish I were, I am not one of them. I am, according to any personality test I’ve ever taken, the quintessential introvert. I am much more comfortable in small groups than large crowds, prefer not to be the center of attention, and (most importantly, I think) need to spend time alone to recharge my emotional and psychological batteries.
This is especially true when I’ve been doing a lot of teaching. I love teaching and gladly give every bit of my energy and passion to my students, but it takes a lot out of me. All of the time spent being “on” in a classroom needs to be balanced with time away from people. Sometimes this takes the form of holing up in the lab and preparing for the next day’s class, but when I’m lucky it means me going off into the outdoors by myself.
Alone time with Nature is balm to my soul. So far this year I’ve made 25 trips to the intertidal, most of them by myself. The spring and summer low tides which occur in the early mornings, are the ones I love the most. There’s something magical about being in the field as the sun appears over the horizon, when the ocean is calm and the winds haven’t picked up yet. One of the things I like most about the early morning low tides is that most people are still in bed and I get the intertidal to myself, where I can poke around at my own pace and allow my attention to wander to whatever catches my eye.
The other morning I was up the coast a bit and this is what caught my eye:
I couldn’t remember the last time I’d seen one of these crabs, but the name popped right into my head. Over the years I’ve learned to trust my gut instinct when an animal’s name just shows up like that, especially with the marine invertebrates. So when I got back to the lab I looked it up and, yep, that was it. But in the meantime I was woolgathering, following mental threads of images of these crabs from books, as the more I thought about it the more convinced I was that I never had actually seen one in the field. I still think that this individual may be the first one I’ve ever seen alive in its natural habitat.
All in all it was a glorious morning, warm even. I had to shed a couple of layers when the sun came out. See how flat the water is?
All of this isn’t to say that I don’t like sharing the intertidal with friends. I do enjoy taking people with me, and there are a few people whose company I would welcome any time. They know who they are, I think. But for the most part I don’t mind going out by myself (except for the one time there was a guy, obviously digging illegally for clams, who gave me a longer-than-necessary look as I walked past him on the beach) and actually enjoy it. So don’t feel too sorry for me when I can’t find someone to go with me, and realize that if I invite you to join me then it’s because I really want to spend time with you.
When I moved to the coast these many years ago and started poking around in the local intertidal, I became entranced with little animals called staurozoans. I can’t claim to have been to every intertidal site in the area, but I’ve been to several of them and I personally know the staurozoans to occur at only two sites: Carmel Point (I’ve seen them there once) and Franklin Point (I used to see them there fairly regularly). In 2007 I went out to Franklin Point every month that had a negative low tide during daylight hours to monitor the abundance and size of the staurozoans; heck, once I even went out in the dark armed with a headlamp and a friend who was supposed to watch my back but instead fell asleep against the cliff. The staurozoans were easy to find that year and occurred in large numbers.
I used to be able to find the staurozoans in one particular area on the north side of Franklin Point where the water continually swashes back and forth.
The staurozoans would always be attached to algae, often perfectly matching the color of their substrate. I remember seeing two versions, one a reddish brown and the other a vibrant bottle green color, of the same species of Haliclystus.
In March of this year I saw a lot of small staurozoans when I braved the afternoon winds at Franklin Point. The conditions were pretty horrid, with the water all churned up and murky so I couldn’t take any pictures, but I was happy to see my little guys because it meant they were there. I hadn’t seen them for a few years before this past spring and was beginning to doubt my search image. Huzzah for validating my gut feeling! I may have whooped and done the happy dance in my hip boots that afternoon.
Fast forward almost three months and three additional trips out to Franklin Point before I found a staurozoan this morning. One. And it was only about 0.5 cm tall, the same size that they were in March. And it was brown, the same color as most of the algae out there. Because they live where the water is constantly moving it’s really hard to photograph them in situ. This is the best I could do:
It’s hard to appreciate from this photo just how beautiful these animals are. They are very animated, swaying in the current and although they are attached they can slowly creep over surfaces or even detach, somersault around, and re-attach. Back in the day when I used to find them frequently I brought some back to the lab to observe them more closely. I could get them to feed, but they never lasted more than about a week in captivity.
So, what exactly are staurozoans? They are cnidarians, kin to sea anemones, hydroids, Velella velella, and jellies. Their common name is stalked jellies, and for a long time biologists considered them to be closely related to the jellies in the cnidarian class Scyphozoa. However, recent studies of the genetics of staurozoans have caused taxonomists to elevate these creatures to their own class, the Staurozoa.
Not much is known about the ecology of Haliclystus in California, probably because they are so damn difficult to find in the field. I have one or maybe two more trips out to Franklin Point this summer before we lose the minus tides for the season; hopefully they will still be there. I’d love to get some better pictures of them to show my students this fall. Wish me luck!
I did a quick search, and there doesn’t seem to be a collective noun for sea stars. I’m going to remedy that by declaring “constellation” to be the official term for a group of sea stars. And by “official” I mean that’s the term I’m going to use. Who knows, maybe it’ll take.
In any case, I certainly have a constellation of sea star larvae in each of my jars. Today I pipetted a lot of them into a bowl, and they look pretty cool all swimming together, like strange alien spaceships. What do you think?
The largest of the larvae are over 2 mm long now, and the brachiolar arms have grown much longer. They have three adhesive papillae on the ventral side of the anterior projection and well-formed juvenile rudiments, where the water vascular system is forming. They’re much too big to fit under the compound scope, so the only way to get pictures of the entire body is through the dissecting scope:
In the above photo you are looking at the larva’s ventral surface, so the animal’s left side on the right side of the photo, and vice versa. If you squint you might be able to convince yourself that you see a small whitish bleb on the left side of the stomach; that’s the rudiment. Since it doesn’t make much sense under this magnification, I removed this individual to a slide and put it under the compound scope. It doesn’t fit in the field of view, so I took pictures of each half of the body. If I were clever with photo editing software I’d be able to mesh these photos into a single image. Alas….
This gives you a better view of the juvenile rudiment on the animal’s left. Those three roundish blobs are tube feet! I think it’s likely that at some point in the not-too-distant future the larvae will be competent, which means they’d be physiologically and anatomically capable of metamorphosis. It seems to me that they are still developing very quickly, and with seawater temperatures consistent at 15-16°C I don’t expect that to change. So far, so good!
Edit 4 July 2015: Look at what my online friend Becca can do! She was able to merge my photos into a single image. Now you can see the entire body! Thanks, Becca!
Composite image of brachiolaria larva of Pisaster ochraceus, age 31 days 3 July 2015
