Our copepod cultures

Today I will present you our copepod cultures. Some of them have been decades with us.

Oithona davisae 10x objective
Paracartia grani 5x objective
Centropages typicus 10x objective

The nauplii of the three species are also different:

These are a nauplii of Oithona davisae. They are usually pointy towards the end and have two spines in the posterior part that are almost touching by the end.

Paracartia grani nauplii are more “rectangular-looking” and the spines are open towards the sides, never touching by the end.

Paracartia grani 5x objective

In the case of Centropages typicus there are a long and a short two parallel spines in the posterior part.

Plankton and art

Those who have seen a plankton sample cannot deny its inherent beauty. From the perfect symmetry of diatoms to the complexity of some radiolarians, we can find an infinite range of shapes and colors that have been a source of inspiration for many artists, ancient and modern. Perhaps we find the maximum expression of the representation of plankton in painting. Particularly, toward the end of the 19th century and the beginning of the 20th, a series of artists exceptionally illustrated the plankton. Among these, I would undoubtedly highlight Erns Haeckel (1834-1919), who drew marine creatures, and in particular plankton, with a detail and beauty that had never seen before (Figure 1).

Figure 1. Drawing of Radiolaria by Erns Haeckel

If we talk about artists and plankton, I cannot fail to mention Miquel Alcaraz, my former advisor and friend, who recently disappeared, leaving us without a great scientist and painter of plankton (Figure 2).

Figure 2. Centropages typicus developmental stages painted by Miquel Alcaraz

Plankton have also inspired artistic creations, which are not purely descriptive. There is a long tradition of naturalists and researchers who devoted their time, around the 19th century, to create true masterpieces of art and monuments to patience, slowly placing under the microscope different species of diatoms in the correct position to create compositions that, often, remind church rose windows (Figure 3).

Figure 3. Compositions with diatoms. Left J.D. Möller, right Eduard Thum

Many sculptors also find an endless source of inspiration in planktonic creatures. For example, Mara Haseltine’s glass figures represent tintinnids, radiolarians, and other plankton organisms in a very creative way. Louise Hibbert also represents plankton, among other creatures, in her sculptures. If we move from art to artistic merchandizing, it is not difficult to find pendants inspired in plankton, usually made with 3D printing and coated with more or less precious metals. I leave you in Figure 4 a collection of all these creations (without putting commercial names or sales websites, which would not be appropriate).

Figure 4. Different sculptures and works of art inspired by plankton. Above, glass works by Mara Haseltine; middle, sculptures by Louise Hibbert; below, commercial pendants.

Cinema is closely related to painting and sculpture and is another of the world-renowned arts. We already met the Phronima in a previous post (https://wordpress.com/view/planktonocean.wordpress.com), which surely inspired the creature in the movie Alien, by Ridley Scott. However, it does not end there, from the misnamed character Plankton (it is actually a copepod) of SpongeBob animation to the short cartoon series Plankton Invasion, or the Golisopod from Pokemon (inspired by an isopod) we have representatives of plankton everywhere (Figure 5). Returning to the descriptive art, within the audiovisual medium, I would highlight the videos of Plankton Chronicles (www.planktonchronicles.org) or those of the YouTube channel from our research group (www.youtube.com/c/ZooplanktonEcologyGroupICM).

Figure 5. Plankton in cartoons.

The literary works on plankton are also extensive, and there is something to suit all tastes, more or less scientific. However, if we are talking purely about fictional literature, we should not be surprised that plankton has also inspired some other books. As an example, we could cite the book Medusa by Sergio Rossi, where some marine biologists want to prevent a voracious species of jellyfish from ending the world’s fisheries, or the Fifth day by Frank Schatzing, where lobsters full of toxic dinoflagellates explode in the faces of humans who wanted to eat them. Maybe a little far-fetched, don’t you think?

Although it sounds unbelievable, there are also architectural structures based on plankton. The diatom-house designed in Germany is an example. The Korean building that simulate the cycle of elements in the planktonic food web is another (Figure 6). There is also a team of architects called the Plankton Group; however, I do notsee much of a connection between this latter building and these creatures.

Figure 6. Left diatom house. Right building inspired by the cycle of the elements

I do not know much about the relationship between plankton and music or dance. Even so, there is a music group called Insect surfers, which released the song Plankton Dance in 2014. However, the truth is that it becomes difficult to find a relationship between that song and plankton.

Finally, even though it is not one of the seven arts yet, we cannot deny that knowing how to cook is quite an art. Well, there are a few dishes with plankton, and they are becoming fashionable as an haute cuisine ingredient. From the traditional Chinese marinated jellyfish to the sophistication of dishes with marine phytoplankton (freeze-dried algae cultures sold at exorbitant prices) promoted by the Spanish chef Angel León, we have a variety of dishes using plankton as ingredients that we can enjoy. I had myself a Calanussoup at a meeting in Germany. I admit, however, it was rather tasteless. 

Plankton are also being used in the supplements, such as Omega 3, Spirulina, etc. 

In summary, either because we enjoy art, movies, or simply cooking with plankton, it is undeniable these creatures have entered into our lives.

Microplankton sample, 11/20/2022

I leave you with a selection of pictures from the last plankton sample (2 Km offshore Barcelona, 5 meters depth, November 20, 2022). You can find the movies here:

https://www.youtube.com/watch?v=P0KFxcoq_9A for a summary of the relevant species

https://www.youtube.com/watch?v=eYfxSVCLrC4 for Acantharia

https://www.youtube.com/watch?v=6dNyRKlIk4g for a beautiful Amoeba

https://www.youtube.com/watch?v=hRdBe37iO50 for Ceratium spp.

A brief history of plankton discovery

In this post, I will give you a few glimpses of how were the early plankton-human interactions and how we managed to study these creatures. As you will see, there was already evidence from ancient Greece that there were strange beings, including plankton organisms, that did not fit into any known classification. However,perhaps the greatest work was done by a series of amateur and professional naturalists from the 16th to the 19th centuries. Their discoveries were not always published as books but were often contained in correspondence they maintained with recognized institutions, such as the Royal Society of London. Even though many valuable records are certainly lost, a good part of this correspondence is still available, and we have evidence of the exciting progress of those people who had a whole new world to discover. You will see that I give special emphasis to the first illustrations of plankton because, as the popular saying dictates, many times a picture is worth a thousand words.

Multicellular plankton

Logically, the first recorded members of plankton were possibly jellyfish and other larger organisms, particularly those that lived attached to other animals for daily consumption, such as fish. Already in the 4th century BC, the great philosopher and naturalist Aristotle first identified a parasitic copepod. He classified it, like jellyfish and other soft-bodied organisms, such as sponges and ascidians, under the name Zoophyta, something between animals and plants, and that classification lasted for hundreds of years. However, we owe the first image of a parasitic copepod (Figure 1) to Gillaume Rondelet, a French zoologist born in 1507.

Figure 1. Below, on the right and above the tuna’s gills, you can see what could be a parasitic copepod. Illustration by Rondelet (1554).

The free-living copepods had to wait slightly longer to be discovered; in 1688, Stephan Blankaart drew the first recorded free-living (freshwater) copepod (Figure 2). Carl Linnaeus (father of modern taxonomy, 1707-1778) named them Monoculus and classified them as insects. As insects, they remained for many years until the beginning of the 19th century, when Jean-Baptiste Lamarck classified them as crustaceans, along with water fleas, amphipods and isopods.

Figure 2. The first illustration of a free-living copepod, left. Possibly of the genus Cyclops from a sample of freshwater. Stephan Blankaart (1688).

Out of curiosity, the first illustration of a free-living marine copepod corresponds to Gunnerus (1770), a Norwegian zoologist who identified Calanus finmarchicus (rather numerous and important species on the Norwegian coast), although he called it Monoculus finmarchicus (Figure 3).

Figure 3. The first illustration of a free-living marine copepod. Ernst Gunnerus (1770).

Unicellular plankton

We owe the discovery of protists to Antonie van Leeuwenhoek, who with his rudimentary microscope first saw infusoria and other planktonic organisms. Between 1674 and 1716, this Dutchman, recognized as the father of microbiology, described several species of protozoa, mostly ciliates (infusoria, Figure 4), among other planktonic creatures.

Figure 4. Drawings of different infusoria and other organisms made by Antonie van Leeuwenhoek 1702.

He did not pay much attention to planktonic algae, and although he surely saw them, the first diatom (not quite planktonic) was described by an English gentleman, probably Charles King, in 1703 in a note sent to the Royal Society of London. From here, many naturalists devoted themselves to classifying, observing, and drawing protozoa (for example O.F. Müller has a detailed description with drawings of the behavior of tintinnids dating from 1779. Of course, the maximum expression of art depicting these beautiful creatures can be found in the drawings of the German Ernst Haeckel (Figure 5). This artist and scientist suggested in 1866 that all those animals (including bacteria) that he saw under the microscope should constitute by themselves a third independent animal kingdom called Protista (the first or primordial).

Figure 5. Illustrations of different protozoa by Ernst Haeckel.

Plankton research

Many years passed from the discovery and classification of planktonic organisms to the beginning of research into their role in the oceans. The first studies, purely descriptive of the diversity of life forms, were linked to large expeditions, such as those of Captain James C. Cook between 1768 and 1780 in the Pacific Ocean. In those expeditions, there is evidence, for example, of spots of the cyanobacterium Trychodesmium on the surface of the ocean. However, the first nets specifically designed to collect plankton were probably used by the French naturalists Francois Péron and Charles-Alexandre Lesueur during an expedition to Australia from 1801 to 1804. Additionally, during the long voyage of the HMS Beagle (1831- 1836), Charles Darwin used nets to collect samples of plankton. However, we own the creation of modern oceanography to the Challenger expedition (1872 to 1876), as it was the first organized specifically to collect data from the marine environment, including temperature, water chemistry, bottom geology, currents, and marine life. The HMS Challenger was equipped with laboratories and microscopes, as well as a team of six scientists.

In 1887, the physiologist Victor Hensen introduced the term plankton to describe all those animals that drifted in water currents. He was also the first to propose that perhaps marine life was not nourished by what flowed into rivers but by microscopic primary producers. Through his studies, he perfected existing plankton net designs and created one that was truly quantitative and is still used today (the Hensen net). From the analysis of his samples, he came to the erroneous conclusion that plankton are homogeneously distributed in the ocean and that there are too few of them to sustain fisheries. Facts that were refuted immediately afterward. The study of the distribution and abundance of the different plankton groups continued during the late 19th and early 20th centuries with names such as Marie Lebour (specialist in diatoms and dinoflagellates, 1876-1971), Alister Hardy (creator of the continuous plankton capture system, CPR, 1896-1985), Sheina Marshall (a pioneer in the study of copepod feeding, particularly Calanus sp. (Figure 6), 1896-1977), Hans Utermöhl (inventor of the sedimentation chambers named after him, 1896-1984), to name a few of them. The result of those studies is the discovery of daily patterns of vertical migration, for example. In 1817, the French naturalist George Cuvier made observations of the vertical migration of zooplankton, although he did so in a lake, and his study lasted only one day. More complete, marine and long-lasting were the investigations of the German Carl Chun, in 1888, on vertical migration.

Figure 6. The marine copepod Calanus hyperboreus.

Although there were hints of their behavior, what those microscopic beasts actually did in the sea was still mostly a mystery, and we had to wait until the 20th century for estimates of, for example, primary production, first with variations in oxygen measurements under light and dark conditions, and afterward using radioactive carbon. Similarly, the estimation of zooplankton production was established as a technique well into the 20th century. In 1963, Ramon Margalef established the basis for understanding the structure of the ecosystem and the relevance of ecosystem maturity in species succession. By the end of the 20th century, MR Landry and RP Hassett devised a method to estimate the impact of microzooplankton feeding on phytoplankton in seas and oceans. At approximately the same time, F Azam, T Fenchel and other collaborators conceptualized the operation of the microbial network and introduced the term microbial loop, which takes into account the tremendous importance of dissolved organic compounds and the key role of bacteria in the marine food web. All these milestones have been decisive in positioning us into current knowledge. However, although we have come a long way and have modern techniques and disciplines at our fingertips, such as satellite imaging, sequencing, genomics, and gene expression, we are still in the infancy of fully understanding (and predicting) the function of the different plankton groups in the marine food web.

Marine Zooplankton Ecology Group Youtube Channel

In the following link, you will find our Zooplankton Ecology Group Youtube Channel. It has many videos of the different organisms we found in our samples, including metazoans and protozoans. You also will find a video on how to make your home-made plankton net. I leave you a few links to the newest and the most popular ones. I hope you enjoy it.

https://www.youtube.com/c/ZooplanktonEcologyGroupICM?app=desktop

The ciliate that met a diatom: a case of symbiosis in plankton 

Sometimes, very different organisms get together and form a successful consortium. Lichens, for instance are a symbiotic consortium between and algae and a fungus. In the plankton we have similar relationships. For instance, a ciliate and an alga, once in a while stop being grazer and prey and work together for a better success. This is the case of the ciliate Eutintinnus sp. and the diatom Chaetoceros (usually Chaetoceros tetrastichon or C. dadayi). The ciliate gets protected by the long spines of the Chaetoceros and this, at its turn, gets to travel for free. It is very rare to find both species alone. 

https://www.facebook.com/ZooplanktonEcology/videos/1298394470910283

The marine food web

Here you have a marine food web graphic representation using some of my drawings. Feel free to use it for teaching, although I would ask to mention the source and authorship.

The little blurry dots represent virus and bacteria. Given they feed at all levels of the food web I positioned them all around. I hope you like it. Phytoplankton are at the base of the food web, together with some small mixotrophs. Both are grazed by larger heterotrophic and mixotrophic protists, such as ciliates and dinoflagellates, but also by appendicularians. Copepods are one step above; although they graze on both large and small protists I kept them on an upper position on the food web. Above them, we have krill, whales and jellyfish. On top of that we have fish, and at the highest level of the food web we found sharks, although humans would be a perhaps more realistic end point for the food web.

The role of bacteria and algae in the balance of life in the sea (and on land)

Is it true that half of the oxygen we breathe is produced in the sea?

The cycle of life inevitably involves creation and destruction. Organisms are born, grow, reproduce (with some luck), and die. In nature, death means an increase in the survival probabilities of someone else. Plankton are no exception to these basic rules of life. In fact, the members of the plankton take these rules to such an extreme that many times their communities (especially those in nutrient-poor waters) are maintained by recycling excretions and the remains of corpses. This recycling allows life to continue, although not eternally; a small allochthonous input of nutrients is always needed from time to time to reinvigorate the ecosystem.

Those in charge of doing the tremendous task of recycling in the water column are the bacteria. Tiny, but numerous, bacteria have colonized all the ecosystems of the planet, both aquatic and terrestrial. Without them the corpses and excrement would pile up. Taking into account the years we have lived in the planet, without the activity of bacteria we could already walk, or even climb, on a sea of ​​corpses. Bacteria carry out complex reactions in which organic matter is broken down into inorganic matter that can be used by the primary producers, the phytoplankton. They also contribute to the cycle of many chemical elements, such as sulfur, iron, etc.

Photomicrograph of bacteria and protists taken with an epifluorescence microscope

The algae and photosynthetic bacteria that compose the phytoplankton are responsible for assimilating the inorganic salts produced by the action of bacteria (especially nitrates and other forms of nitrogen, phosphates, and silicates) and incorporating them into living matter. They also capture CO2 (+ H2O) and turn it into organic matter with the help of the sun’s energy. In this process, called photosynthesis, they also release oxygen; a by-product that has a bad habit of rusting things, but that is essential for our lives. Regarding oxygen, it is quite common to read on outreach websites or the press, and even in some scientific publications, that marine algae contribute to approximately half of the oxygen we breathe. Although this is true on a geological scale of millions of years, it is not correct on a day-to-day basis. It is true that phytoplankton, throughout the life of the planet, has contributed to accumulating perhaps more than half of the oxygen in the atmosphere, and that without its photosynthetic activity, animals (at least as the we know) had not colonized the earth’s surface. It is also true that phytoplankton contribute approximately 50% of primary production (photosynthesis), and that they are responsible for more or less half of the oxygen produced on the planet each day. However, this oxygen produced in seas and oceans is mostly consumed within them by the different micro- and macro-organisms that live there. In other words, only a very small percentage reaches the atmosphere. But for now, we shouldn’t worry, it doesn’t seem like the oxygen in the atmosphere is going to run out in the immediate future.

Marine algae

Bioluminescent plankton

Those that are lucky enough to have seen a bioluminescent beach say it is a fantastic spectacle, like few on Earth. The sparks of blue or green light that accompany the water in the winding breaking of the waves captivate young and old alike. I, unfortunately, have never seen it, although I have seen occasional sparks in the night water, and I have experienced the fun of stirring some bioluminescent protozoan culture. Just for the record: do not do that; they die after stirring them if you do it too often or too energetically.

Despite the low presence of bioluminescence on the coast – it only occurs on some beaches and from time to time – it is a well-studied and relatively widespread phenomenon among sea creatures. Even the U.S. military has been investigating its usefulness in detecting enemy submarines. What matters to us here, however, is not the military applications of this curiosity of nature but its origin and causes.

Which organisms emit light?

There are many types of bioluminescent organisms (i.e., they emit light by biological mechanisms), both marine and terrestrial: bacteria, protists, jellyfish and ctenophores, squid, worms, mollusks, crustaceans, echinoderms, fish, etc. However, what interests us are those belonging to plankton. That is, mostly unicellular protozoa and algae (especially dinoflagellates), some jellyfish and ctenophores, and some crustaceans, mainly from the copepod group.

The dinoflagellate Noctiluca scintillans is responsible for many of the bioluminescent beach shows worldwide. Photo by Albert Calbet.

Why do they do it?

Concerning plankton, the main reason for bioluminescence is to prevent predation. Imagine that you are a copepod, and suddenly you notice the suction of a fish’s mouth. Although you may have time to escape, and you are skilled enough to propel yourself at high speed, if you can also confuse your predator by releasing a flash of light so that it cannot follow you, the success of your escape will be significantly improved. Other organisms in terrestrial groups, such as insects, use luminescence to attract mates. There are also abyssal fish that emit light so that curious prey approach them like fools that go to the light and end up in their mouths. In this case, the light comes from symbiotic bacteria grown in different fish cavities. 

Marine bioluminescent bacteria are a curious case. Many groups of bacteria are symbionts of higher organisms, such as cephalopods or fish, where they perform functions of attracting prey (as we have mentioned) or repelling predators, or even to communicate. However, there are also free-living bioluminescent bacteria. Until recently, it was believed that bioluminescence was linked to the density of these organisms and was a response to high population densities. It has recently been shown that bioluminescence can also occur in individual bacteria. In these cases, it is supposed that emitting light is a mechanism for attracting predators, especially in places where nutrients are scarce. When ingested, the bacteria, which are also resistant to digestion, find a richer environment inside their host and increase their chances of survival.

How do they do it?

Bioluminescence occurs when the enzyme luciferase reacts with oxygen and a protein called luciferin. The process is as follows: Oxygen oxidizes luciferin with the help of luciferase and, using the energy stored in the ATP, they produce energy in the form of light and water as a by-product. Sounds complex, doesn’t it? But even a bacterium can do it!

 
The copepod Pleuromamma abdominalis emits flashes of light when it feels in danger. Drawing from Miquel Alcaraz

Today we will sample plankton

Those of you who follow my posts will know that plankton comprise many organisms of different sizes, from viruses about 0.00001 mm to large jellyfish up to a meter in length. It is logical, then, to think that there should be different systems for sampling plankton according to the size of the group of interest. Broadly speaking, however, we can cluster sampling or capture methods into two groups: those focused on microscopic unicellular organisms and those used by larger organisms, such as multicellular zooplankton.

Small unicellular plankton sampling tools

This type of plankton, which includes viruses, bacteria, algae, and protozoa, is usually caught in conjunction with the surrounding water. For this purpose, we have more or less sophisticated devices, from a simple bucket to hydrographic bottles that allow us to take plankton of discrete depths. A hydrographic bottle is nothing more than a cylinder, usually made of PVC or methacrylate, with two airtight lids that can be closed when the bottle is at the desired depth. The most commonly used hydrographic bottle design is called Niskin, by Shale Niskin, who invented it in 1966, but there are others like the Nansen, Van Dorn, and so on. These bottles are usually mounted on a structure called rosette, which surrounds devices that allow us to observe, among other things, in real-time the depth of the bottles, the temperature and salinity of the water, and the fluorescence emitted by the algae that live there. This set of devices is called CTD (conductivity, temperature, and depth) and is essential in any oceanographic cruise. We can also collect seawater with the organisms accompanying it with suction pumps that go down to the depth that interests us. All of these systems are only for sampling microscopic plankton. Copepods, fish larvae, or large crustaceans would be misrepresented if collected by a bottle because their abundance is low, and also because they tend to escape when they detect the bottle, pump, or bucket.

Figure. Hydrographic bottles: A) Van Dorn bottles held by the author in a campaign in the Antarctic Ocean. B) Niskin bottles mounted on a rosette around a CTD. Photos Albert Calbet.
 

Zooplankton fishing nets

In the title of this section, you will see that I have used the word fishing. The fact is that the mechanisms for catching large zooplankton do not differ much from those used for fish. We use plankton nets, which are usually conical. Depending on our target group, the nets are wider or narrower, longer or shorter, and have a larger or smaller diameter mesh pore. To give you an idea, nets with a pore mesh size of 20 µm (0.02 mm) allow us to capture large phytoplankton and microzooplankton, those of 200 µm (0.2 mm) would be indicated by adult copepods, and those of 0.5 to a few millimeters of pore-size would be used for krill, fish larvae, etc. Depending on the interest and net type, towing can be done vertically, horizontally, or obliquely.

The simple structure I have described to you is the most common and corresponds to nets such as the Juday Bogorov, the WP2, the Bongo, etc., but there are more complex and mechanized nets (for example, the Bioness, or the LHPR, abbreviation of Longhurst Hardy Plankton Recorder) that allow us to fish with different mesh sizes at the same time, sample different depths, and at the same time having a record of the depth and physicochemical parameters of the water. Some devices incorporate video cameras that give images or recordings of everything that enters their field of vision, but of course, they do not capture organisms.

Because of their fragility, certain plankton organisms are complicated to catch intact with a plankton net. This would be the case with gelatinous plankton. If we want to obtain living and perfect specimens of certain species of gelatinous plankton, we have no choice but to get wet and take them out of their environment one by one and very carefully.

Figure. Fishing nets. A) double WP2 net. B) Bongo nets. C) LHPR. Photos Albert Calbet

Preservation of samples

We already have our plankton samples, so what do we do now? As for the capturing devices, the processing of the samples will also depend on the group to study. The samples can generally be viewed life or preserved for later study. Preservation can be with chemical reagents, such as formaldehyde or Lugol’s solution (basically a modified iodine tincture), by freezing at -20 or -80ºC (depending on the analysis we want to do), or by filtration and drying, or filtration and extraction of pigments in acetone, ethanol, etc. Also, sometimes we need combinations of several of the mentioned techniques, such as fixing, sample filtering, and subsequent freezing. This last technique is used when we want to observe unicellular organisms in an epifluorescence microscope.

Sample analysis

If we want to see and classify plankton, the most practical is to use stereoscopic magnifiers or microscopes. However, special machines allow us to process samples faster but lose fine taxonomic resolution compared to a good human specialist. For example, for bacteria we can use the flow cytometer; for algae and microzooplankton, the FlowCam; and for larger zooplankton, the Zooscan. All these devices share the same principle. The sample passes through a tube or is placed on a plate, is stimulated by a light source (laser or normal light), and the emission of photons or images is captured by specialized video or photo cameras. These images or light emissions are often processed by complex computer programs to estimate abundance and primary classification of what is in the water.However, new techniques for biochemical and molecular analysis of samples are gaining popularity recently. These techniques are up-and-coming and can give us an idea of ​​the diversity and some physiological processes in the water. Yet, we are still far from being able to provi