This quote by Charles Darwin, made on his journey with the ship the Beagle, describes exactly what people feel when they look at a zooplankton sample for the first time. Darwin here describes one of the first or perhaps the first use of a net to sample plankton. Plankton nets are still widely used in zooplankton research, and actually the nets have changed very little after almost 200 years. In the meantime, however, there are many more tools available for zooplankton research! How can you study zooplankton?

Zooplankton research started in the 17th century after the invention of the microscope; first the compound microscopes of Jan Swammerdam and Robert Hooke and then the stronger magnifying single lens microscope of Anthoni van Leeuwenhoek. As early as 1674 van Leeuwenhoek discovered protists and other minuscule aquatic organisms in a lake. He described plankton until just before his death, the last being the veliger larvae of oysters, in 1722. Later, zoologist Martinus Slabber described larvae of invertebrates and all kinds of other plankton in “Natuurkundige Verlustingen” (1778). Below a selection of plates from this work. What can you recognize?

Later pioneers of plankton research were Johannes Müller, who described many planktonic larvae, and his student Ernst Haeckel, famous for his voluminous works with beautiful plates of various organisms. With the discoveries by the British expedition with HMS Challenger from 1873 – 1876 came the realisation that plankton was widespread and even existed in the deep sea, until then considered lifeless. The material from the Challenger Expedition was distributed to many experts, including Ernst Haeckel. It was Victor Hensen who introduced the term “plankton”, and Ernst Haeckel who introduced the term “holoplankton” for organisms that live their whole life in the plankton and “meroplankton” for organisms that also live part of their life on the bottom.

On the RRS Discovery expedition of 1925-1927 Sir Alister Hardy introduced an innovative device, the Continuous Plankton Recorder (CPR), which could be towed behind a ship and automatically collected plankton on a roll of plankton gauze. The CPR is still widely used with many commercial ships equipped with it. The CPR data make an important contribution to the knowledge on the distribution, composition and occurrence of zooplankton all over the world. The data is freely available to anyone.

Zooplankton research, like research on other groups of organisms, is very diverse. Some examples of research directions:

• Biodiversity and biogeography: only 10% of the world’s zooplankton species have probably been discovered, some 7,000 species, and some 70,000 are still undiscovered.
• Consequences of human induced changes to the environment such as climate change and eutrophication. This can have major consequences for how many and which types of plankton can live somewhere, which in turn can affect animals that depend on this plankton for their food.
• Fisheries research; for example, research into the presence of fish larvae in order to estimate how healthy a fish population is.
• Taxonomy and evolution; for example, studying the relationship between different populations of zooplankton and the factors that influence it.
• Distribution of invasive species; for example, to predict whether an invasive species may cause problems by competing with or eating other species.

I myself have carried out research into various aspects of zooplankton. For my PhD thesis I investigated the possible consequences of the introduction of the American comb jellyfish Mnemiopsis leidyi in Dutch waters, and whether there will be long-term changes in the distribution and composition of jellyfish in the Netherlands. Later, I conducted more applied research into the possible impact of sustainable energy production by Blue Energy on zooplankton. See my personal website for more information.

Many different techniques have been devised for sampling zooplankton. In essence, these often come down to the same thing, filtering a certain amount of water with plankton through a filter with openings small enough to retain the plankton you want, but letting the plankton and other particles you don’t want pass through, after which you collect the remaining concentrated plankton. For this you can use, for example, plankton nets with larger or smaller openings or mesh sizes. Below are a number of photos of plankton gauze of different mesh sizes.

Drie verschillende maaswijdtes plankton gaas gefotografeerd onder de microscoop; 100, 25 en 5 micron.

You can see here that the smaller the mesh size, the smaller the openings in the network. However, the amount of filtering surface of the net is also becoming less and less as thread diameter does not decrease at a similar rate. Nets with a smaller mesh size therefore clog a lot faster than nets with a large mesh size because they block more smaller plankton but also because the filtering surface is getting smaller.

Not all plankton is equally common; of the smallest plankton such as protists and rotifers you may find hundreds to thousands per litre, copepods about one to ten per litre and fish larvae and jellyfish one per thousand litres.  To sample the smallest plankton you need to filter much less water and a few litres is enough, while for fish larvae you need to filter an entire swimming pool to get a good idea of how many and which species of fish larvae occur somewhere. Various kinds of nets and other devices have been developed for this purpose. Here are a few examples.

Plankton sampling with nets can be done by lowering the net in a straight line to the bottom (vertically) and lifting it up again, or by dragging the net behind you while sailing at a constant speed (horizontally) or while slowly lifting the net up (diagonally or oblique). There are also all kinds of ways to sample only a certain depth, such as self-closing nets, plankton pumps, niskin bottles or Schindler-Patalas zooplankton traps. Besides nets, there are many other ways to collect plankton. Large or fragile animals such as jellyfish are best caught manually by scooping them off the surface with a cup, or underwater while diving or with a (remote-controlled) submarine. There is also always the simplest way: just throw a bucket of water through a sieve.

How does this work in practice? As an example below a video of a day of zooplankton sampling on the Oosterschelde and Grevelingenmeer, for my PhD research:

Fixation and preservation

For some applications plankton is analysed alive, such as when checking whether any plankton hitchhiked to a new area in ships’ ballastwater. Most often however it is not possible or practical to analyse plankton samples immediately after collection. Plankton samples deteriorate rapidly and thus have to be fixed and preserved if they are to be analysed in a lab. The most used fixation and preservation method uses formalin, a solution of formaldehyde, sometimes along with other chemicals. Formalin preserves most zooplankton taxa well, but it has the big downside that it is a toxic, carcinogenic chemical. Because of this formalin preserved samples have to be analysed in a laboratory with proper ventilation. Another downside is that formaldehyde degrades DNA, making it difficult to impossible to do any DNA-based identification. Alternative conservation solutions used are ethanol, Lugol and DESS. Freezing or freeze drying is also used if the whole sample is used for analyses and identification of organisms in the sample is not needed.

The conventional analysis of zooplankton samples is by sorting, identifying and counting the plankton using a low magnification stereomicroscope. For species-level identification a higher magnification conventional microscope is often needed to distinguish the tiny morphological details between species. See the resources page for sources to use in plankton identification.

Morphological identification allows for species-level identification in many taxa. Morphological identification of plankton samples is done by specialists experienced in taxonomy; the identification of organisms. Depending on the diversity and density of organisms in a sample a specialist needs hours to days to analyse a sample. This makes analysing plankton samples time consuming and expensive and has unfortunately lead to a lack of monitoring of zooplankton in many areas.

Novel techniques for plankton sampling

Recent technological advancements have made it possible to to sample and analyse plankton at high spatial, temporal and/or taxonomical resolution.

DNA metabarcoding

For this technique DNA from a mixed sample is extracted, sequenced and compared with a database of known DNA sequences for species to find out which species’ DNA was present in the samples. This allows for efficient and quick identification of many species, even ones that cannot be distinguished from other species by conventional means such as cryptic species and larval stages of many invertebrates. Estimating the abundance of organisms using DNA-based methods is still challenging but the accuracy of abundance estimates will likely increase with improvements in methodology. An important caveat with DNA metabarcoding is that in order to detect a species in a sample its DNA sequence data for the targeted gene region should be available. This means that it is important to have a complete well and curated reference database of barcodes.

Plankton imaging

In Plankton Imaging, images of planktonic organisms are collected, stored and classified. Advantages in camera technology, data storage and computer vision are enabling the acquisition, storage and processing of large volumes of images. Many different options are available to image plankton samples in the lab and directly in the water!

Imaging of plankton samples

Analysis of plankton samples using imaging can be done with different approaches. In flow-through systems a sample is pumped through a small transparent flow cell where a camera images each particle as it passes by. Commercially available flow through systems are available such as the FlowCam and for smaller plankton imaging flow cytometers such as the cytosense and imaging flow cytobot. There is also a great initiative to develop a low-cost open source option for flow through systems, the PlanktoScope.

An alternative approach is to scan samples using a flatbed scanner (such as the zooscan) where a sample is poured on a glass plate and scanned in the same way as you would scan a document. Using computer vision techniques individuel particles are identified on the images and classified. For the classification researchers developed an online tool called EcoTaxa where you can upload your dataset and even use data collected by others to train a classifier to classify the objects on your images. Alternatively you can develop your own analysis pipeline.

In-situ plankton imaging

With in-situ plankton imaging you image plankton directly in its natural environment. This can be done using underwater microscopes mounted on frames that are deployed from ships, or by pumping water from outside of a ship through an onboard flow cell with a camera system. In this way you can examine plankton at a high spatial and temporal resolution without having to take thousands of samples! This saves researchers a lot of time and money, and no animals need to be killed for it. Combining high resolution plankton imaging with simultaneous measurements of environmental parameters like temperature, salinity, depth and oxygen concentration allows investigation of plankton dynamics at fine scales impossible to achieve using traditional net sampling.

In-situ plankton imaging systems are generally optimised to sample a certain size range of organisms. Some examples of in-situ imaging systems are the ISIIS, CPICS, VPR and UVP . The Plankton Imager is a system that is used onboard research vessels.