Submitted to: Journal of Invertebrate Pathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/7/2002
Publication Date: 11/19/2002
Citation: Valles, S.M., Oi, D.H., Perera, O.P., Williams, D.F. 2002. DETECTION OF THELOHANIA SOLENOPSAE (MICROSPORIDIA: THELOHANIIDAE) IN SOLENOPSIS INVICTA (HYMENOPTERA: FORMICIDAE) BY MUTLIPLEX PCR. Journal of Invertebrate Pathology. 81(3):196-201. Interpretive Summary: The red imported fire ant was introduced into the United States in the 1930s and currently infests about 300 million acres. It causes significant economic losses in livestock and agricultural production and poses a serious threat to human health. Annually, 50% of people in infested areas are stung, occasionally with fatal consequences. A promising biological control agent of the red imported fire ant is Thelohania solenopsae, an intracellular parasite. Development of Thelohania solenopsae as a biological control agent will depend on elucidation of the life cycle. Scientists at the Center for Medical, Agricultural and Veterinary Entomology in Gainesville, FL, have developed a molecular technique (PCR) capable of detecting Thelohania solenopsae at all stages of its development. This method is a significant improvement upon existing methods and will aid the discovery of the life cycle and its subsequent dissemination.
Technical Abstract: Oligonucleotide primer pairs were designed to unique areas of the small subunit (16S) rRNA gene of Thelohania solenopsae and a region of the Gp-9 gene of Solenopsis invicta. Multiplex and single primer PCR resulted in highly sensitive and specific detection of T. solenopsae infection of S. invicta. The T. solenopsae-specific primer pair only amplified DNA from T. solenopsae and T. solenopsae-infected S. invicta. This primer pair did not produce any amplification products from DNA preparations from uninfected S. invicta, 7 additional species of microsporidia (including Vairimorpha invicta), or Mattesia spp. The Gp-9-specific primers recognized and amplified DNA from S. invicta, S. richteri, S. geminata, and the invicta/richteri hybrid, but not from T. solenopsae, and, as such, served as a positive control verifying successful DNA preparation. Multiplex PCR detected T. solenopsae in worker fire ants infected with as few as 5,000 spores. Furthermore, multiplex PCR detected T. solenopsae in all developmental stages of S. invicta. However, detection could be made more sensitive by using only the T. solenopsae-specific primer pair; ants infected with as few as 10 spores were able to be discerned. Multiplex PCR detection of T. solenopsae offers the advantages of a positive control, a single PCR reaction, detection of all developmental stages, and increased sensitivity and specificity compared with microscopy.