The New Internal Transcribed Spacer 2 Diagnostic Tool Clarifies the Taxonomic Position and Geographic Distribution of the North American Malaria Vector Anopheles Punctipennis

Abstract

Background: The malaria mosquito Anopheles punctipennis Say, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for An. punctipennis. An. punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. Results: In this study, we collected and sequenced ITS2 from 276 An. punctipennis specimens collected from the eastern and midwestern United States. Our analysis demonstrated a consistent sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In addition to the ITS2 sequence-based molecular tool, we developed a simple and robust Restriction Length Polymorphism approach for species identification. An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. Conclusions: This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.