Use of transcriptional age grading technique to determine the chronological age of Sri Lankan Aedes aegypti and Aedes albopictus females.

BACKGROUND: Aedes aegypti and Ae. albopictus are important vectors of human diseases such as dengue, chikungunya, and zika. In Sri Lanka, they have been responsible for transmitting dengue virus. One of the most important parameters influencing the likelihood of arbovirus transmission is the age structure of the mosquito population. However, mosquito age is difficult to measure with accuracy. This study aims to construct multivariate calibration models using the transcriptional abundance of three age-responsive genes: Ae15848 (calcium-binding protein), Ae8505 (structural component of cuticle), and Ae4274 (fizzy cell cycle/cell division cycle 20). METHODS: The transcriptional age-grading technique was applied to determine the chronological age of Ae. aegypti and Ae. albopictus female mosquito populations from Sri Lanka using the age-responsive genes Ae15848, Ae8505, and Ae4274. Furthermore, Ae. aegypti samples obtained from colonies reared at two temperatures (23 and 27 °C) were used to investigate the influence of temperature on this age-grading technique. Expression levels of these three genes were quantified using reverse transcription qualitative PCR (qRT-PCR), and results were normalized against the housekeeping gene ribosomal gene S17 (RpS17). RESULTS: The expression of Ae15848 and Ae8505 decreased with the age of mosquitoes and showed the most significant and consistent change while expression of Ae4274 increased with age. The multivariate calibration models showed > 80% correlation between expression of these age-responsive genes and the age of female mosquitoes at both temperatures. At 27 °C the accuracy of age predictions using the models was 2.19 (± 1.66) days and 2.58 (± 2.06) days for Ae. aegypti and Ae. albopictus females, respectively. The accuracy of the model for Ae. aegypti at 23 °C was 3.42 (± 2.74) days. CONCLUSIONS: An adult rearing temperature difference of 4 °C (23-27 °C) did not significantly affect the age predictions. The calibration models created during this study could be successfully used to estimate the age of wild Ae. aegypti and Ae. albopictus mosquitoes from Sri Lanka.

DNA barcoding of morphologically characterized mosquitoes belonging to the subfamily Culicinae from Sri Lanka.

BACKGROUND: Vectors of mosquito-borne diseases in Sri Lanka, except for malaria, belong to the subfamily Culicinae, which includes nearly 84% of the mosquito fauna of the country. Hence, accurate and precise species identification of culicine mosquitoes is a crucial factor in implementing effective vector control strategies. During the present study, a combined effort using morphology and DNA barcoding was made to characterize mosquitoes of the subfamily Culicinae for the first time from nine districts of Sri Lanka. Cytochrome c oxidase subunit 1 (cox1) gene from the mitochondrial genome and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization. RESULTS: According to morphological identification, the field collected adult mosquitoes belonged to 5 genera and 14 species, i.e. Aedes aegypti, Ae. albopictus, Ae. pallidostriatus, Aedes sp. 1, Armigeres sp. 1, Culex bitaeniorhynchus, Cx. fuscocephala, Cx. gelidus, Cx. pseudovishnui, Cx. quinquefasciatus, Cx. tritaeniorhynchus, Cx. whitmorei, Mansonia uniformis and Mimomyia chamberlaini. Molecular analyses of 62 cox1 and 36 ITS2 sequences were exclusively comparable with the morphological identifications of all the species except for Ae. pallidostriatus and Aedes sp. 1. Although the species identification of Armigeres sp. 1 specimens using morphological features was not possible during this study, DNA barcodes of the specimens matched 100% with the publicly available Ar. subalbatus sequences, giving their species status. Analysis of all the cox1 sequences (14 clades supported by strong bootstrap value in the Neighbor-Joining tree and interspecific distances of > 3%) showed the presence of 14 different species. This is the first available DNA sequence in the GenBank records for morphologically identified Ae. pallidostriatus. Aedes sp. 1 could not be identified morphologically or by publicly available sequences. Aedes aegypti, Ae. albopictus and all Culex species reported during the current study are vectors of human diseases. All these vector species showed comparatively high diversity. CONCLUSIONS: The current study reflects the significance of integrated systematic approach and use of cox1 and ITS genetic markers in mosquito taxonomy. Results of DNA barcoding were comparable with morphological identifications and, more importantly, DNA barcoding could accurately identify the species in the instances where the traditional morphological identification failed due to indistinguishable characters of damaged specimens and the presence of subspecies.