Aedes albopictus mosquitoes used in this study have been collected from Foshan, Guangdong Province, China, since 1981. Colony maintenance was conducted under standard insectary conditions (constant 27 ± 1°C, 70–80% relative humidity, and a 16 h:8 h light–dark photoperiod). Eggs in de-chlorinated water were hatched to larvae. One hundred larvae per liter of water were fed turtle food with 0.1 g each day for the first 4 days and then 0.2 g each day until they pupated. Pupae were transferred to cages (20 cm × 20 cm × 35 cm). After emergence, adults were provided with 10% glucose solution or defibrinated sheep blood (Solarbio, Beijing, China) for egg production.

Dengue virus 2 (New Guinea C, GenBank: AF038403.1) was provided by the Key Laboratory of Tropical Disease Control of Sun Yat-sen University (Guangzhou, China). C6/36 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and maintained at 28°C. Cells grown in a 75-cm² culture flask were inoculated with DENV-2 at a multiplicity of infection (MOI) of 1. After gentle shaking for 15 min, the culture flask was incubated at 37°C, 5% CO₂ for 2 days until obvious cytopathic effects. The supernatant was harvested after centrifugation at 1,500 × g for 5 min, separated into 0.5-mL aliquots, and frozen at -80°C.

Dengue virus 2 titer was determined by 50% tissue culture infective dose (TCID₅₀) (Richard et al., 2015). Briefly, C6/36 cells were seeded in a 96-well microtiter plate at a density of 10⁵ cells per well and cultured at 28°C for 24 h. The control solution (RPMI-1640 containing 2% FBS) and 10-fold viral dilutions (10^-1–10^-11) were inoculated into C6/36 cells in eight wells. The microtiter plate was placed at 37°C, 5% CO₂ for 2 h, and then the culture per well was replaced with RPMI-1640 supplemented with 2% FBS. The plate was incubated at 37°C, 5% CO₂ until the cytopathic effect did not continue to increase. Results were calculated according to the Karber method (Ramakrishnan, 2016).

Mosquito infection was conducted in a Biological Safety Level 2 lab. Two days before infection, frozen DENV-2 stock was passaged once more through the C6/36 cells. The titer of the fresh virus was 7.375–7.875 log₁₀TCID₅₀/mL. The DENV-2 supernatant was collected and mixed with defibrinated sheep blood at a ratio of 2:1. The blood meal was maintained at 37°C for 30 min and transferred into a Hemotek blood reservoir unit (Discovery Workshops, Lancashire, United Kingdom). Five- to seven-day-old female Ae. albopictus mosquitoes were glucose starved for 12–24 h and allowed to feed on the infectious blood meal for 30 min. After anesthesia with CO₂, fully engorged mosquitoes were removed and placed into 250-mL paper cups covered with gauze (10 mosquitoes/cup). Mosquitoes were placed in different HP400GS incubators (Ruihua, Wuhan, China) precisely set at 18, 23, 28, and 32°C. Another group of mosquitoes was placed at the fluctuating temperature (28°C for 14 h, 23°C for 2 h, and 18°C for 8 h). The control group was set for each temperature. DENV-2 stock solution was replaced with RPMI-1640 containing 2% FBS in the control group, the remaining steps were the same as mosquitoes engorged with the infectious blood meal. All treatments were maintained at 80% relative humidity and 16 h:8 h (light:dark) photoperiod, and all mosquitoes were fed 10% glucose solution.

The midgut, ovaries, and salivary glands of each mosquito from the abovementioned temperatures were dissected and detected at 0, 5, 10, and 15 days post-infection (dpi). The sample size collected from each temperature condition was 3–6 mosquitoes at 0 dpi and 10–16 mosquitoes at each additional time point. The experiment was independently repeated three to five times.

The legs and wings of each mosquito were removed and washed three times in PBS. Disposable insect microneedles were used to separate the midgut, ovaries, and salivary glands of each mosquito under an anatomical lens. Tissues were washed three times in PBS droplets and then transferred to 50 μL of TRIzol (Ambion, Life Technologies, Carlsbad, CA, United States) in 1.5-mL Eppendorf tubes. Total RNA was extracted according to TRIzol manufacturer’s protocol. cDNA was synthesized using a DENV-2-specific primer (5′-TGGTCTTTCCCAGCGTCAAT-3′), and the recommendations of the GoScript^TM Reverse Transcription System (Promega, Madison, WI, United States) were followed.

Polymerase chain reaction (PCR) was used to detect DENV-2 in the tissues. A pair of primers was synthesized as described previously (forward primer: 5′-TCAATATGCTGAAACGCGCGAGAAACCG-3′; reverse primer: 5′-TTGCACCAACAGTCAATGTCTTCAGGTTC-3′) (Lanciotti et al., 1992). The target fragment was 511 bp, which was located in the partial capsid and membrane protein region. The total volume of the PCR reaction system was 25 μL, including 12.5 μL Maxima Hot Start Green PCR Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, United States), 0.5 μL of each primer (10 μM), 1 μL cDNA, and 10.5 μL RNase-free water. PCR reaction conditions were the following: 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 1 min, and 72°C for 7 min. PCR products were identified by 1% agarose gel electrophoresis, ligated with pMD18-T (Takara, Dalian, China) and confirmed by sequencing. Comparing to the tissues from the control group, the positive tissues were determined by detecting specific DENV-2 sequence.

The vector competence of Ae. albopictus for DENV-2 transmission was evaluated by infection rate (IR), dissemination rate (DR), and population transmission rate (PTR), as follows (Di Luca et al., 2016):

The amount of DENV-2 in the positive tissues of mosquitoes was further detected by absolute quantitative real-time PCR (qRT-PCR). The plasmid standard was constructed as previously described (Zhang et al., 2010). In brief, the 3′-UTR region of DENV-2 was amplified by PCR using specific primers (forward primer: 5′-TCCCTTACAAATCGCAGCAAC-3′; reverse primer: 5′-TGGTCTTTCCCAGCGTCAAT-3′). The fragment with 127 bp was cloned into pMD18-T and linearized by EcoRI. The concentration of the plasmid was 26.9 ng/μL, which was transformed into a copy number (8.71 × 10⁹/μL).

The qRT-PCR reaction mixture per well contained 10 μL SYBR^® selected master mix, 1 μL of each primer (10 μM), 2 μL cDNA or the plasmid standard, and 6 μL RNase-free water. The reaction was performed in the 7500 Real-Time PCR System as follows: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 1 min. Melting curves were given at 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. A standard curve was established by 10-fold dilutions of the plasmid standard (8.71 × 10²–8.71 × 10⁷). The result of qRT-PCR was ascertained by using non-template, negative (mosquito infected with C6/36 cells) and positive control (mosquito infected with DENV-2 at 0 dpi). Its minimum detecting amount is 87.1 copies/reaction of DENV-2. Each sample was conducted in three replicates, and the results were determined by the melting curve and cycle threshold values.

All statistical analyses were performed with SPSS 20.0 (IBM, Chicago, IL, United States). Under constant temperature, the vector competence of Ae. albopictus for DENV-2 was analyzed using logistic regression. IR, DR, and PTR were separately compared at different temperatures or different time points. P-value was corrected by Bonferroni adjustments. Chi-square (and Fisher’s exact) tests were used to determine the difference in vector competence between 28°C and the fluctuating temperature (28-23-18°C). The amounts of DENV-2 in the tissues were log-transformed and then analyzed by post hoc Tukey’s LSD tests of variance analysis for the constant-temperature groups. Student’s t-test was used to compare the DENV-2 titer of the tissues between 28°C and (28-23-18°C). P < 0.05 was considered statistically significant.