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Title: RIBOSOMAL DNA ITS2 SEQUENCES DIFFERENTIATE SIX SPECIES IN THE ANOPHELES (ANOPHELES) CRUCIANS COMPLEX (DIPTERA: CULICIDAE)

Author
item WILKERSON, RICHARD - WRAIR
item Reinert, John
item LI, CONG - WRAIR

Submitted to: Journal of Medical Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/12/2003
Publication Date: 6/1/2004
Citation: Wilkerson, R.C., Reinert, J.F., Li, C. 2004. Ribosomal dna its2 sequences differentiate six species in the anopheles (anopheles) crucians complex (diptera: culicidae). Journal of Medical Entomology. 41(3):392-401.

Interpretive Summary: Mosquitoes suck blood and transmit disease agents that cause sickness and death in animals and humans. Before efforts to control mosquitoes can be made, however, accurate identification of the species involved is required. This information, which is based on microscopic features of the external anatomy of the mosquito, is used to ensure that insecticides, biological control agents, and other interventions are applied in an effective and safe manner. ARS scientists at the Center for Medical, Agricultural, and Veterinary Entomology in Gainesville, FL and scientists at the Walter Reed Army Institute of Research in Silver Spring, MD are working continuously to develop new and improved techniques for mosquito identification. The information resulting from this research allows animal and public health specialists and pest/vector control personnel to identify mosquito species accurately and quickly, and to apply mosquito control measures in a safe and timely manner.

Technical Abstract: Anopheles (Anopheles) crucians Wiedemann (sensu lato) was investigated for the presence of cryptic species using rDNA ITS2 sequences. This complex of species presently contains the named species An. crucians, An. bradleyi King, and An. georgianus King. Adult female mosquitoes were collected at 28 sites in Alabama, Florida, Georgia, North Carolina, Mississippi, and Louisiana resulting in 245 progeny broods. Species were identified using preliminary morphological characters and then the ITS2 was amplified from all broods. The result was 5 distinct sizes of amplification product and, based on morphological characters, one of the size classes was suspected to consist of 2 species. All 6 putative species were then sequenced, five directly, and the sixth, because of extreme intragenomic size variability, cloned. The ITS2 sequences were markedly distinct for all 6 species. Species designations and ITS2 sequence lengths (base pairs in parentheses) were: A (461), B (1000+), C (204), D (293), E (195), and An. bradleyi (208). Species B showed both large intraspecific and intragenomic sequence variability and is distinguished by having the longest ITS2 found so far in an Anopheles. Phylogenetic analysis using parsimony of 201 alignable bases gave good support for the relationship of species A and E, and moderate support for the clade that includes species A, E, and D, and for the sister group relationship of An. bradleyi and species C. Based on these data we found that all species could be identified with PCR using a mixture of 4 primers in a single reaction. Members of this complex were often found in sympatry, with the adults of five species collected at a single site in central Florida.