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The Solenopsis venom protein 2 transcript was amplified, sequenced, probed, and analyzed from Solenopsis invicta x Solenopsis richteri hybrid ant colonies (hybrids) collected from across Tennessee to determine the extent of introgression of each parent allele (Solenopsis invicta venom protein 2 [Soli2] and Solenopsis richteri venom protein 2 [Solr2]). Chemotaxonomic analyses of venom alkaloids and cuticular hydrocarbons were used to categorize hybrid colonies and their relative relatedness to each parent species. Hybrid colonies were chosen randomly from each chemotaxonomic hybridization category, including “very near S. richteri,” “near S. richteri,” “near S. invicta,” and “very near S. invicta.” Lateral flow immunoassays for detection of the Soli2 and Solr2 venom proteins were largely in agreement with the chemotaxonomic analyses for the very near S. richteri (100% Solr2) and very near S. invicta (80% Soli2, 20% Soli2 + Solr2 detected in the sample) groups, while Soli2 and Solr2 were reported in 60% and 40% in the near S. invicta and near S. richteri chemotaxonomic groups. Analysis of transcripts from the hybrid colonies revealed a sequence with 100% identity to Soli2 (GenBank Accession L09560) and three unique sequences, which we identify as Solenopsis hybrid venom protein 2 (Solh2; GenBank Accession MT150127), Solenopsis hybrid truncated venom protein 2 (Solh2Tr97; Genbank Accession MT150129), and Solenopsis richteri venom protein 2, D to A change at position 69 (Solr2A69; GenBank Accession MT150128). The predicted open reading frame for Solh2 and Solh2Tr97 revealed sequences unique to hybrid ants, with Solh2Tr97an alternatively spliced form. A third unique sequence, Solr2A69, is likely the correct sequence for Solr2, which appears to have been published previously with a sequencing error (GenBank Accession P35776).

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