Community Research and Development Information Service - CORDIS

Identification of shifts in the composition of rotifer populations using DGGE

In this project, the change of rotifer composition during cultures experiments had to be monitored. Next to these data, rotifer samples from European commercial hatcheries had to be identified to biotype level and the composition of the rotifer culture had to be determined.

In order to be able to genetically analyse rotifer samples from hatcheries, the storage of rotifer samples from a hatchery is the easiest in ethanol. Therefore, during the project a successful DNA extraction protocol for ethanol preserved samples was designed. Now, ethanol preserved rotifer samples can be extracted and PCR is amplified with a success ratio of 95%. It is important that good quality ethanol is used, i.e. analytical grade ethanol (99°C). Within the sample a >90% ethanol has to be achieved for optimal sample preservation.

This protocol can as well be used to store samples for studies on systematics or for ecological research.

Samples containing many individual rotifers of possibly different biotypes can be collected directly from their culture medium or from natural habitats. This means that in the sample there is also a left-over of the feeds used in hatcheries (algae, bakers' yeast, culture selco) or other zooplankton present. After total DNA extraction, a fragment of the mitochondrial 16S rDNA gene is amplified with Brachionus specific primers. The 16S amplicons might consist in a mix of different genotypes of 16S fragment according to the different B. sp. biotypes that are present in that sample. The use of the SSCP (Single Strand Conformation Polymorphism) and the DGGE (Denaturing Gradient gel Electrophoresis) technique enables the detection of which biotypes are present in the rotifer sample. The SSCP technique was not able to resolve all Brachionus sp. biotypes, while the DGGE technique fully resolves all biodiversity. This technique has a 100% discriminatory power. Until now, the DGGE technique proved to be able to detect all rotifer biotypes that are abundantly present in a sample.

The DDGE technique allows a fast detection of all rotifer biotypes from the B. plicatilis complex. It can also be used to document a shift in the composition of a mixed rotifer population. In the purpose of this project, it served very well to follow up hatchery cultures in a quick way. The technique can also be used to follow the seasonal shifts of a rotifer population in a natural habitat. As several samples can be loaded on 1 DGGE gel, the technique allows very cheap processing of rotifer samples. However, the DGGE technique still needs a substantial amount of rotifers to provide a visible line on the gel.

In contrast, the microsatellite technique is able to amplify the DNA coming from 1 rotifer or 1 resting egg, on the condition that this is a B. plicatilis sensu strictu. This technique can be used especially in situations where very few material is available, e.g. resting eggs from a natural habitat.

Reported by

Ghent University
Rozier 44
9000 Ghent
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