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Screening Daphnia for parasites

Procedure to find and identify epibionts and parasites in Daphnia and zooplankton samples.

This is a fool-proof guide to find and identify parasites and epibionts in zooplankton samples. Most steps are trivial, but to be complete I outline them anyway.

Daphnia

Some diseases can be spotted even in the natural habitat. Note the reddish D. magna. This female is infected with Spirobacillus.

Take a fresh plankton sample. Only live material can be used. Most fixatives will destroy the material and make identification of the parasites and epibionts impossible. There are some fixatives for certain groups of parasites, but they are usually not useful for fixation of large samples (check the more specialised literature on these groups). Fresh samples can be kept for several days in the cold (4 to 10 degrees C) before examination. Make sure the samples are not to dense. If mortality occurs in a sample before examination, check the dead animals as well. Infected hosts often die first under stressful conditions (Note: parasites in dead hosts are often very difficult to find and identify, because various scavengers penetrate into the cadaver).

Step 1: Screening large numbers of Daphnia

Daphnia

Infected Daphnia are often (but not always) very conspicuous. They can be easily seen by eye in a large mass sample. Some of the more rare parasites which occur only in very low prevalences, e.g. the bacterium Spirobacillus, might be only discovered in this way. It is helpful to view the Daphniaagainst a dark background (e.g. black cardboard below a glass beaker with the light shining from top). Some parasitized Daphnia appear strongly coloured under these conditions. E.g. many microsporidian parasites which infect the ovaries and fat cells make the internal parts of the host shine white. Further, hold the glass beaker against some light (window, desk-lamp, ... ) to check for parasites which alter the transparency of the hosts. Some parasitized Daphnia appear black under these conditions. Pick out all Daphnia which appear 'odd' in the sample. The same procedure can be repeated using a dissecting microscope. Place 50 to 100 Daphnia in a petridish and suck off the water to restrict their movement. Place them under a dissecting (stereo) microscope and view them with light from the top (with dark background) and/or with light coming through the bottom. Many infections can be easily seen in this way (Note: most gut infections will still remain unrecognised). Pick out odd looking animals. Note that some parasites are seen only in one or the other way, while others are seen under both top and bottom light sources. For example, white bacterial disease makes the fat cells of their hosts shine bright white (with a touch of green) when the light comes from the top, but is invisible when the light shines through the animal. Therefore, it is recommended to view the hosts both ways.

Step 2: Looking at a smaller number of hosts

Daphnia

Using the right back ground when screening samples can make a big difference.

It is typical for most parasites to be found only in larger hosts. Therefore I concentrate usually on adult hosts and normally on females. Ten adult females is a convenient number of hosts to investigate at a time. I place them on a microscopic slide and search them with high magnification (25, 50 or 100 times) for epibionts. Some features of epibionts can only be studied under these conditions. For example the peritriche ciliates of the genus Epistylis detach from the surface of their hosts as soon as they are squashed with a cover slip. For other peritriche ciliates it is important to study their movements. For example you can use a needle to irritate the ciliates (just touch them carefully) to test for they reaction. For example, Vorticella contracts its stalk, Epistylis does not.

Step 3: Dissecting the host

Daphnia

The same Daphnia seen under three light conditions. Left: light from top; right: light from below; center: light from below and from top. Note the difference in the visibility of the parasite (White Fat Cell Disease).

For identification of most parasites it is necessary to dissect the host. This allows one to detect diseases that are still in an early phase of infection. Methods for dissection vary from person to person and you might find your own way of doing it. The essential point is to get the gut out of the host and to separate the different tissues of the host such that they can be studied under high magnification. The most simple (and gruesome) way to dissect a host is to use two dissecting needles. Hold the daphniid by sticking one needle through the centre of the body. Insert the other needle into the head about where the antennae insert. Pull the head off. Usually the gut will go with either the head or stay in the body. Remove the remaining tissue from the gut and set it aside. Place the antennae, the furca and the carapace separately from the internal body parts. Cover with a cover slip and suck dry, but be careful not to destroy the gut. With some experience you may dissect 10 or more Daphnia on one microscopic slide. To find and identify epibionts search the furca, the antennae and the carapace under low magnification (100 to 200 x). For the parasites you need 400 times magnification and phase contrast. Start searching the internal tissues for parasites. Heavy infections with microsporidians are very quickly seen. The entire view is filled with spores. Early infections are only seen as a few spores floating around. If you have no idea how the parasite might look, watch out for any small particles (2 to 10 micrometers) of regular shape. Usually they come in large masses (Note: spores come seldom alone. Ignore single objects which look like spores. There are many interesting objects floating around in a Daphnia). Often parasite spores come in characteristic clusters. It takes a bit of time to distinguish host tissue from parasites. Sometimes it is helpful to look through a number of hosts to get a picture of what is normal and what is parasite material in a host. There are a number of parasites which disappear under the microscope. Many bacterial disease are caused by very small coccoid bacteria which are nearly invisible. In some cases a host looks bright red (Spirobacillus infection) or bright white (White bacterial disease) but after dissection of the host nothing is found. It needs some more sophisticated fixing and high magnification (1000 times) to get of good picture of these bugs. However, with some experience one can identify them from the phenotype of the infected host. The gut of a Daphnia might host various parasites, but also contains the food, which can obscure the picture. The most common gut parasites are microsporidians which infect gut epithelium cells. The clusters of the parasites can be recognised easily. The spores of these microsporidians are usually very small (2 - 3 microns) and are expelled with the gut content. Other gut parasites are amoebae (they move about in fresh preparations) and the haplosporidium Caullerya.

Using this three step procedure, you should be able to find most parasites unless they are very rare and do not produce strong effects on the phenotype of the host. Have fun!