The misidentification of morphologically similar mosquito species could lead to the false incrimination of a vector and/or the misappropriation of scarce resources for vector control. With numerous species of Culex
) sharing a similar geographical distribution and an unreliable morphological key, Guatemala is one such location where a molecular assay for the identification of Culex
species would contribute greatly to entomological and arbovirus ecology studies. We have developed a multiplex PCR-RFLP assay based on the internal transcribed spacer regions of the rDNA gene array to identify common Culex
) and Culex
) mosquitoes in Guatemala. Of the 16 Culex
) and two Culex
) species present in Guatemala, our assay consistently and reliably identified seven of these species, including species from which arboviruses have been isolated or detected in Latin America: Cx. chidesteri, Cx. interrogator
, Cx. nigripalpus, Cx. quinquefasciatus,
and Cx. thriambus
. Morphologically similar species and/or species that key to the same couplet in Clark-Gil and Darsie,10
such as Cx. quinquefasciatus
and Cx. interrogator
, Cx. lactator
and Cx. coronator
, and Cx. chidesteri
and Cx. nigripalpus
, can be confirmed by this molecular assay. Consequently, this multiplex PCR-RFLP assay makes significant headway towards the correct identification of Culex
species in Guatemala.
In addition to the correct identification of target species, no amplification was obtained from at least six additional non-target species of Culex (Cux.) indigenous to Guatemala: Cx. declarator, Cx. inflictus, Cx. mollis, Cx. pinarocampa, Cx. restuans, and Cx. stigmatosoma. A confirmatory restriction digest was designed to differentiate Cx. nigripalpus from Cx. thriambus.
Additional field specimens from outside Guatemala were not tested; however, primers for Cx. coronator
(Mississippi), Cx. nigripalpus
(Florida), and Cx. quinquefasciatus
(California) were designed from sequences of specimens collected in the continental United States. In comparing our sequences with those available in GenBank, priming site sequences for CXFOR and NIGR4 were identical to Cx. nigripalpus
ITS sequences from a previous study (AF521664 and AF521662 and AF520974),35
and intraspecific ITS sequence variability was minor, with only one or two nucleotide differences at the priming site evident in some other ITS clones (e.g., U33023 and U33024 from the same specimen in Florida). Successful priming and amplification from these specimens is expected. Each of these Cx. nigripalpus
sequences also lacked the NcoI
restriction site at position 108 of the CXFOR-NIGR4 amplicon, confirming the use of the RFLP to separate these Cx. nigripalpus
from Cx. thriambus
. Furthermore, ITS amplicons from the two additional Cx. thriambus
field specimens were each digested by NcoI
as expected, whereas ITS amplicons from Cx. nigripalpus
field specimens were not digested by NcoI
. Similarly for Cx. quinquefasciatus
, the priming site sequences for CXFOR and QUIN2 were identical between Cx. quinquefasciatus
from California (this study), and Cx. quinquefasciatus
from South Africa (DQ341108). Consistent amplification of ITS from Guatemalan Cx. quinquefasciatus
field specimens in this study shows that these primers are broadly applicable across the geographic range of these species.
It is unknown whether our Cx. lactator primers distinguish between the two Cx. (Phc.) species known to occur in Guatemala, Cx. corniger and Cx. lactator. Similarly, we do not know if our Cx. coronator primers are species or complex specific. The Cx. coronator complex includes Cx. coronator, Cx. usquatus, and Cx. ousqua. Adding additional species would require development of additional multiplex assays. Therefore, future work should include the design of additional molecular diagnostics covering similar or related species such as members of the Cx. coronator complex and Cx. mollis versus Cx. declarator.
The sensitivity of this assay resulted in amplification of less than one nanogram of DNA template from each species in mixed pools. Therefore, this assay can be used to confirm the identification of individual field specimens and to verify species composition in virus-positive mosquito pools. Assay validation also used field specimens stored dry on silica desiccant for 2 to 12 months before DNA extraction and voucher specimens stored in 95% ethanol. Successful and consistent PCR amplification was obtained from DNA extracted by standard salt extraction, the Qiagen blood and tissue kit, and from specimen legs sonicated in TE buffer. These results show the use of this assay across a variety of specimen collection, storage, and DNA preparation methods.
In conclusion, we have developed a novel multiplexed PCR-RFLP assay to identify seven species of Culex (Cux.) and Culex (Phc.) in Guatemala. It is our expectation that this diagnostic will serve as a valuable tool for Culex mosquito identification in Guatemala and neighboring countries in providing the ability to confirm the identity of morphologically similar species, and obtain the identity of otherwise badly damaged and unidentifiable specimens. With the inclusion of confirmed and potential arbovirus vectors, this assay will serve as an instrumental role in future ecological studies of Culex mosquitoes and arbovirus surveillance and control efforts in Guatemala.