After the teneral period, adults become more active and mate. Because periodical cicadas are superabundant, unmated teneral adults are relatively easy to obtain. Although these insects are difficult to maintain in the laboratory, they may be maintained and manipulated under seminatural conditions in outdoor cages containing living, woody LY2109761 vegetation. When males and females are confined in cages and given no choice of mates, they will engage in hybrid matings, and hybrid eggs and nymphs are viable. We developed a PCR-based method that makes use of specific primers and known Magicicada haplotype differences to detect rare mtDNA haplotypes in experimentally-crossed cicadas. We tested this method on mtDNA mixtures made by combining, in different proportions, the DNA of wild-caught individuals of known species, and we then used these primers to investigate the possibility of paternal leakage in reciprocal crosses of M. septendecim with M. cassini and M. septendecula. To ensure that the primers were amplifying mitochondrial COI and not nonfunctional nuclear copies of mitochondrial genes, the amplified paternal and maternal mtDNA from representative sequences of hybrid mixed-haplotype eggnest extractions were compared to the original COI sequence. All of the sequences exactly corresponded to the original COI sequence of the proper primer set and none of the species-specific primer sets produced sequence that had errors that suggested multiple templates. Furthermore, we found no stop codons in any of the sequences, and all speciesspecific base substitutions were silent, suggesting that our sequences belong to functional genes. We were careful to avoid contamination that would lead to false positive results. The best evidence against heterospecific contamination was that sample DNA from homospecific crosses amplified with homospecific primers but never with heterospecific primers. Homospecific contamination would be undetectable because contaminant DNA would be identical to sample DNA; however, this would not SCH772984 change our current conclusions about paternal leakage because we cannot detect paternal mtDNA in homospecific crosses for the same reason. Contaminant DNA is most likely to be amplified when there is little or no target DNA. Every set of PCR reactions included a negative control and none of these control reactions ever showed any sign of amplified DNA. A final pair of controls included DNA from non-hybrid adults of both species involved in each cross. This adult DNA was tested with both homo- and hetero-specific primers. These controls were included in every PCR set and were visualized on the same gels as the experimental samples. Failure of heterospecific primers and success of the homospecific primers assured us that the correct primers had been added to all reaction mixtures and that contamination was not present. Our results suggest that paternal leakage occurs in hybrid Magicicada.