Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Abstract

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed

First record of Anopheles konderi Galvão & Damasceno (Diptera: Culicidae) carrying eggs of Dermatobia hominis (Linnaeus Jr.) (Diptera: Oestridae), from Oriximiná municipality, Pará, Brazil

“…Subsequently, studies have reported that these two species differ in terms of their behavioral patterns and ITS2 (Internal Transcribed Spacer) and COI molecular markers, and also possibly as malaria vectors 11,12 . Currently, A. konderi is recognized as a distinct species from A. oswaldoi s.l., although it is a member of a group of cryptic species within the A. oswaldoi complex [11][12][13] .…”

Discriminación por morfometría geométrica de once especies de Anopheles (Nyssorhynchus) presentes en Colombia

“…Los especímenes capturados cuando "aterrizaban" sobre el cebo humano protegido o las hembras recién emergidas procedentes de series o isofamilias (22,23) se identificaron usando las claves morfológicas disponibles (2,5,6 (27).…”

“…La cercanía de A. oswaldoi con A. benarrochi B no se puede comparar en otros árboles debido a que esta última especie se reconoció recientemente (27). Su cercanía se debe a que son muy similares morfológicamente, lo que impide su separación en el estadio de adulto hembra.…”

“…Dentro del grupo Oswaldoi, las hembras de A. benarrochi B halladas en el departamento del Putumayo, Colombia, presentan características morfológicas que comparten en muchos casos con A. oswaldoi (27).…”

“…El análisis discriminante de la conformación del ala separó, sin lugar a dudas, ambas especies (figura 6B) y logró reclasificar correctamente el 87% de las hembras de A. benarrochi B y el 90% de las de A. oswaldoi. Hasta la fecha, las hembras de A. oswaldoi y A. benarrochi B sólo se pueden separar sin ambigüedades por técnicas de biología molecular, usando PCR-RFLP del ITS-2 (27). A pesar de que esta metodología separa ambas especies en el 100%, la necesidad de conservar los ejemplares en sílica-gel y los altos costos de procesamiento dificultan su aplicación a gran escala.…”