Aedes aegypti and Ae. albopictus are important vectors of human diseases such as dengue, chikungunya, and zika. Among these diseases, dengue is the most rapidly spreading mosquito-borne viral disease that is endemic in more than a hundred and twenty countries in the world including Sri Lanka. Among several factors, the age structure of the dengue vector population has been identified as one of the most sensitive parameters that influence the epidemiology of the disease.
Based on experimental observations the average extrinsic incubation period (EIP), the incubation period of the dengue virus within the mosquito’s body before it is transmitted to the human body is 8–12 days [1, 2, 3, 4}. Joy et al.  have stated that wild populations with 10% − 27% of females that can survive more than the EIP are more likely to establish dengue virus transmission within that area. Hence, knowing the age structure of the wild populations in an area is vital in decision making especially in vector control programmes.
As there is no specific antiviral vaccine against dengue, efficient and environmentally friendly vector control measures are needed to prevent the disease from spread and outbreaks throughout the world. Introduction of artificially infected Ae. aegypti females with life-shortening Wolbachia pipienis (wMelPop) into the field has been introduced as one of the most promising dengue mosquito eradication technique in the world due to failures in traditional vector control measures. Wolbachia has the ability either to block the dengue virus inside the mosquito body and/or decrease the female mosquito life span than the EIP of the dengue virus. This environmentally friendly method of vector control has been successfully practiced in several areas in the world especially in Australia . Knowledge of the age of the vector population is important before designing and releasing wMelPop infected individuals into natural populations to determine the effectiveness of this method.
Morphological, biochemical, and molecular-based age grading techniques have been developed in determining the age of female mosquitoes. However, the use of both morphological and biochemical methods have become questionable due to their inability and inaccuracy in measuring the age of mosquitoes older than the EIP. The transcriptional age-grading technique has now been identified as the most accurate and precise approach in determining the chronological age of mosquitoes [3, 6, 7].
Quantification of expression levels of genes that show a variation in its expression with the age of the female mosquito is the basis of this technique. The transcription scores of these age-responsive genes of laboratory-reared mosquitoes of known ages are then feed into a multivariate calibration model which could be later used in age predictions of field/wild individuals. The mosquitoes used in the construction of the calibration model must be from the same mosquito strain of the field population where the researcher is planning to apply the technique . The analysis of age responsive genes during these studies have shown that this technique accurately detects the age of An. gambiae [8, 9, 10] and Ae. aegypti [2, 3, 4, 11, 12] older than 15 days, that is more than the EIP period. Trials, using mosquitoes reared in field cages, have concluded that the gene expression profiles of Ae. aegypti female mosquitoes could determine the age with an accuracy of ± 5 days of the actual age [11, 13, 2].
The orthologues of the eight age responsive genes i.e. Ae- 4274, Ae-4679, Ae 4916, Ae-6639, Ae-7471, Ae-8505, Ae-12750, Ae-15848 selected from Drosophila melanogaster have initially been used to predict the age of female Ae. aegypti mosquitoes under both laboratory and field conditions [6, 14, 15, 16]. According to mosquito transcriptional age grading studies CG-8505/Ae- 8505/AAEL003259 (Pupal cuticle protein 78E putative) and SCP-1/Ae.-15848/AAEL008844 (calcium binding protein, putative) displayed the largest and significant decrease in expression levels with the age of female mosquitoes while expression levels of fizzy/Ae-4274/AAEL014025 (fizzy cell cycle/ cell physiology, putative) significantly increased with the age. Hence these three genes; Ae- 8505 and Ae.-15848 and Ae-4274 have been identified as the most informative age-responsive genes that could be used in the transcriptional profiling of mosquitoes [2, 11, 13]. Cook et al.  have identified Ae- 8505 Ae-15848 and Ae-4274 genes as the most reliable age responsive genes and recommended to use these three genes for future age determination studies. The gene RsP17 (40S ribosomal protein s17) that showed an insignificant variation with the age has been exclusively used as the reference gene in normalizing the samples in these studies. Further, the expression of these genes has are independent of blood feeding, egg laying, digestion, and reproductive status of the mosquitoes [6, 8, 17].
However, this approach needs further validation and optimization based on the geographical region, as the mosquito populations may have sequence polymorphism which only affects the reliability of gene expression analysis. Therefore, Cook et al.  suggested creating separate models for mosquitoes in different geographical regions. Further, fluctuation in the environmental parameters such as temperature, humidity, photoperiod, etc. was also to be considered as these might affect the transcriptional abundance of age responsive genes . Hence, models constructed considering all or majority of these limiting factors will increase the accuracy and precision of the transcriptional age grading method.
At present dengue has become one of the major causes of hospitalization and deaths in Sri Lanka. The year 2017 reported the largest dengue outbreak (≈ 186,000 cases) in the country which is around 4.3 times an increase in the average number of cases that have been reported from 2010 to the 2016 period . Control of dengue vector populations in Sri Lanka is primarily based on the application of adulticides and larvicides. However, most of these programmes have been challenged due to the development of insecticide resistance by both the dengue vectors [19, 20]. Hence, the Sri Lankan government has planned to release wMelPop infected dengue vectors into the field . However, so far no attempts have been made in Sri Lanka to determine the chronological age and age structure of any of the dengue vectors which is essential before implementing this programme. Both primary and secondary dengue vectors, Ae. aegypti and Ae. albopictus are found in Sri Lanka. Although Ae. albopictus was considered as a rural secondary dengue vector in the past has now invaded urban areas as well and has become the most dominant Aedes species in most of the areas in the country . However, so far none of the research in the world has focused on determining the age structure of Ae. albopictus populations.
Hence, the proposed work aimed to construct multivariate calibration models using the transcriptional abundance of three age responsive genes; Ae15848, Ae8505, and Ae4274 to determine the age structure of Ae. aegypti and Ae. albopictus female mosquito strains from Sri Lanka and to investigate the influence of temperature on the expression levels of these age responsive genes of Ae. aegypti females.