*4.5. GC Analysis*

The GC analysis was carried out using an Agilent 6890N GC system Agilent 5975 (SEM Ltd., Istanbul, Turkey). The FID detector temperature was 300 ◦C. To obtain the same elution order with GC-MS, simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms. The analysis results are given in Table 1.

Identification of the essential oil components was carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of *n*-alkanes [25,26]. Computer matching against commercial (Wiley GC/MS Library, MassFinder Software 4.0) and in-house "Ba¸ser Library of Essential Oil Constituents" which includes over 3200 genuine compounds with MS and retention data from pure standard compounds and components of known oils as.

#### *4.6. Insects*

*Aedes aegypti* used in these studies were from a laboratory colony maintained at the Mosquito and Fly Research Unit, Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, Florida since 1952. We received the eggs and stored them in our laboratory until needed. Mosquitoes were reared to the adult stage by feeding the larvae on a larval diet of 2% slurry of 3:2 beef liver powder (now Foods, Bloomingdale, Illinois) and Brewer's yeast (Lewis Laboratories Ltd., Westport, CT, USA). The eggs were hatched and reared to the pupal stage in an environment-controlled room at a temperature of 27 ◦C ± 2 ◦C and 60 ± 10% RH in a photoperiod regimen of 12:12 (L: D) h. The adults were fed on cotton pads moistened with a 10% sucrose solution placed on the top of screens of 4-L cages.

#### *4.7. Mosquito Biting Bioassay*

Bioassays were conducted using a six-celled in vitro Klun and Debboun (K & D) module bioassay system developed by Klun et al. [27] for quantitative evaluation of biting deterrent properties of compounds. The K & D system consists of a six-well reservoir with each of the 4 × 3 cm wells containing 6 mL of feeding solution. We used the CPDA-1± ATP solution instead of human blood [22]. CPDA-1 and ATP preparations were freshly made on the day of the test and contained a green fluorescent tracer dye (fluorescent water-based tracer "Green"; www.blacklightworld.com) that allowed for the identification of mosquitoes that were fed on the solution. The squashed mosquitoes were observed under black light (FEIT, BPESL15T/BLB 13W 120VAC 60Hz 200mA, Ul#E170906) for feeding. DEET (97% purity *N*,*N*-diethyl-3-methylbenzamide) was used as a positive control (Sigma-Aldrich Co., St. Louis, MO, USA) and ethanol (Fisher Scientific Chemical Co. Fairlawn, NJ, USA) was used as solvent control. Stock and dilutions of all extracts and DEET were prepared in ethanol. All essential oils were evaluated at dosages of 10 μg/cm2 and DEET along with the pure compounds was tested at a concentration of 25 nmol/cm2.

The temperature of the solution was maintained at 37 ◦C by using a circulatory bath. The test compounds and controls were randomly applied to six 4 × 3 cm marked portions of nylon organdy strip, which was positioned over the six, membrane-covered wells. A six-celled K & D module

containing five 6–15-day-old females per cell was positioned over the six wells, trap doors were opened and mosquitoes allowed access for 3 minutes, after which they were collected back into the module. Mosquitoes were squashed and the presence of green dye (or not) in the gut was used as an indicator of feeding. A replicate consisted of six treatments: four samples, DEET, and ethanol as solvent control. Five replicates were conducted per day using new batches of mosquitoes in each replication.
