Sorting plankton is a bit like a game of Where’s Waldo, except that Waldo is moving and translucent, and the entire background scenery is moving along with him.

In my case, the Waldos I am looking for are appendicularians. I separate them from the commotion of the background plankton by their distinctive shape and motion. They are easily confused with the transparent rod-shaped body of chaetognaths (“arrow worms”)—but have a more pronounced, football-shaped head—and the sinusoidal wriggling of a nematode—yet less fitful. Their motion can be hard to detect amidst the darts and jolts of the ever-abundant calanoid copepods.

Some days my sample (collected from the net in the figure below) is filled with so many Waldos I cannot possibly pipette them all. Some days I can sort for hours and never find a single one. Usually it is one extreme or the other: no goldilocks plankton here.

Conducting plankton tows in the Charleston Marina with my salty dog, Zephyr.

My task for this term is establishing cultures of appendicularians at our lab on the main campus of the University of Oregon in Eugene—60 miles from the ocean and 120 miles from the collection site. It is rather daunting, particularly since my appendicularians are smaller than copepods—barely visible even when backlit and examined by the squinting, trained eye. Their life cycle is about six days, depending on temperature. Scientifically speaking, they progresses from external fertilization of the egg to embryogenesis to organogenesis to metamorphosis to somatic growth to maturation and reproduction. Less scientifically, they grow from an egg to a little tadpole to a bigger tadpole to a tadpole with a disproportionally large head (yellow for females, blue for males) and then, once her and his heads fills with eggs and sperm, their gamete-brains explode and a new generation begins.

I have yet to raise appendicularians through their full life cycle. For the time being, my efforts are focused on keeping adults alive inland for a few days at a time, which necessitates to a lot of driving back and forth between Eugene and the coast. On the days when hours of scanning yields only Waldo-less samples, I wonder: is it too late to study copepods?

Hello Oregon Sea Grant Community– I’m Keats Conley, a 2014-2015 Robert E. Malouf Marine Studies Scholar. The blog below shares some recent reflections on my work with appendicularians.

April 2014: Here, in a small tourist town on the south coast of France, I am hunched over a dissecting microscope, wire-tipped dissecting probe in hand. The wire is finer than dental floss. I am using it to break my ancestors’ spines.

 I use the term “spine” loosely.

Appendicularians are a “Urochordate”, one of the three subphyla of the phylum Chordata, along with Vertebrata and Cephalochordata. Cephalochordata is a rather obscure group of small, soft, fish-like creatures called lancelets. Vertebrata includes true fish, hagfish, humans and Labradoodles alike. Urochordates are therefore a sister group to us vertebrates. We are much more closely related to appendicularians than we are to, say, the bivalve oysters we so enjoy shucking and shooting. A great deal of research has compared mammalian and Urochordata genomes to provide information on, among other things, the evolutionary origin of the vertebrate immune system, the eye lens, and the central nervous system.

Appendicularians look like a millimeter-sized translucent tadpole. Under the microscope, they appear equal parts alien and human embryo. They have a football-shaped head (the “trunk”) and a tail, which writhes wildly. I break their spines so that they will hold still long enough for me to take a photograph, which I can later use to measure their size.

The term “appendicularian” refers to the appendices of the animal, their houses. As described in a scientific paper: “The house is secreted as a rudiment by the oikoplastic epithelium, a specialized single-layered organ that covers the trunk of the animal.” In other words, they grow their house from their head. The “house” is a spherical or ellipsoidal structure made of mucus. It is secreted, and then the animal bangs its head up and down to inflate the house with water. The animal then swims inside and sits in the house, with the house roof tucked under its appendicularian “chin.” The house is made of rectangular mucus filaments that function like a spider web.[1] Structurally, its architecture is kaleidoscopically intricate, but its function is straightforward: to capture and concentrate prey particles, such as bacteria and small algae, from the surrounding seawater. The house concentrates prey up to a thousand times that of the surrounding seawater, and then the appendicularian sucks up its thick prey soup as if through a straw.

As I alternate between spine- and camera-snapping, I don’t need to follow any particular protocol. (I do try and move swiftly). In my home lab back in Oregon, a fellow Ph.D. student one building distant works with two-inch zebrafish (Danio rerio) and must adhere to procedures outlined by the University of Oregon’s Animal Care Services, the organization “responsible for administering all activities related to the care and use of animals.” An animal, in this case, is implicitly considered equivalent to a vertebrate. And as we know, although appendicularians coexist with zebrafish in the kingdom Animalia, the two occupy separate subphyla within the phylum Chordata. When I called Animal Care Services to inquire whether any particular care procedure must be followed for research on appendicularians, I was reassured that, no, Animal Care Services oversees supervision of only live vertebrates, as well as some charismatic, seemingly intelligent invertebrate mollusks, such as octopuses. But, I protested, appendicularians are a sister-group to vertebrates. A sister-group. Just the same, I am free to do what I wish with my small, sister house-builders.

The summer after my spring of spine-breaking, I served as a teaching assistant for a marine invertebrate zoology class at the Oregon Institute of Marine Biology. In a lecture on the difference between “anadromous” and “catadromous”, the professor showed a photo of the rainbow trout Oncorhynchus mykiss. In small font, the caption read: “Vertebrates are just invertebrates that happen to have backbones.”

Appendicularians don’t have a spine. They have a notochord. A notochord is a flexible, thin-walled tube, found in the embryos of all chordates. Notochords were advantageous to primitive fish-ancestors because they provided a rigid structure for muscle attachment, yet were flexible enough to allow more movement than, for example, a hard chitinous exoskeleton. In humans, the notochord of the developing embryo is a precursor that will eventually become the central nervous system, including the spinal cord and vertebrae. But in appendicularians, the notochord just stays as a notochord. They have a simple, spineless tail. And a head that builds houses.

References:

Spada, F., Steen, H., Troedsson, C., Kallesøe, T., Spriet, E., Mann, M., & Thompson, E. M. (2001). Molecular patterning of the oikoplastic epithelium of the larvacean tunicate Oikopleura dioica. Journal of Biological Chemistry,276(23), 20624-20632.

Footnotes:

[1] The next time you look at a spider web, notice that it is made up entirely of rectangles. This is because a rectangle is the shape that catches the most bugs with the minimum amount of web material. After all, spider silk is energetically costly to produce. Appendicularians employ this same strategy of catching prey using rectangular-mesh nets, except their thread is mucus rather than silk.