The patterns of random amplified fragments and molecular karyotypes of 12 isolates of anuran trypanosomes continuously cultured in vitro were compared by random amplified polymorphic DNA (RAPD) analysis and pulsed field gradient gel electrophoresis (PFGE). The time interval between preparation of two series of samples was one year. Changes were not observed in the number and size of sharp, amplified fragments of DNA samples from both series examined with the ten primers used. Likewise, changes in the molecular karyotypes were not detected between the two samples of these isolates. These results suggest that the molecular karyotype and the RAPD patterns of the anuran trypanosomes remain stable after being cultured continuously in vitro for one year.
Trichogramma dendrolimi, T. ostriniae, T. confusum and T. evanescens are the four most commonly occurring Trichogramma species with overlapping distribution in China. They are the most frequently used egg parasitoids for biological control of lepidopterous crop pests in China. It is difficult to differentiate Trichogramma species because of their small size and lack of differences in morphological characters. Different molecular markers were employed to molecularly characterize and differentiate these species, including direct amplification of the internally transcribed spacer 2 (ITS2) of ribosomal DNA by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and species-specific primers. The results showed that direct amplification of ITS2 could not clearly discriminate these species, but they could be differentiated using RFLP pattern obtained with endonucleases EcoRI and HindIII. The banding pattern produced by RAPD is irreproducible so it is not a suitable way to identify Trichogramma species. Finally, the species-specific primers designed based on ITS2 sequences could unequivocally distinguish the four species. The species-specific primer-based protocol proved to be the most convenient and time saving method for the identification of Trichogramma species by creating a unique PCR product, which can be used in surveying natural populations of Trichogramma species. This is the first report of the prompt identification of the four most commonly occurring Trichogramma species in China.