Ae. albopictus was collected from Guangzhou (geographical coordinates: N23°07′, E113°16′) and cultivated up to the 20th generation in the Beijing Institute of Microbiology and Epidemiology. Mosquitoes were reared at 25 ± 1°C and 75 ± 5% relative humidity (RH), under a 14:10 light/dark (L/D) photoperiod, and adult mosquitoes were fed a 10% glucose solution daily.

Zika virus SZ01 strain (GenBank: KU866423) was deposited in the Microbial Culture Collection Center of the Beijing Institute of Microbiology and Epidemiology. The virus was isolated from the blood of a Chinese returning from Samoa in 2016 and has been passaged six times in C6/36 cells. The virus titer used in this study was 1.24 × 10⁷ plaque-forming units per ml (PFU/ml), and the virus was stored at −80°C.

We evaluated the effect of temperature variations on the life cycle and transmission efficacy of Ae. albopictus experimentally using three temperature regimes by programmable incubators (Shanghai Yiheng, MGC-350BP-2). The constant temperature has two groups, 23°C and 31°C, and they are the mean minimum and maximum temperatures of seasons with high mosquito-borne virus epidemics in Guangdong Province, China, in the recent 5 years (Cai et al., 2020). The daily temperature range (DTR) fluctuated between the maximum and minimum temperatures according to the daily temperature fluctuation pattern, reaching the minimum temperature at 1:00 and the maximum temperature at 13:00 (Figure 1). We programmed a photoperiod of 14:10 (L:D) h cycle, with alternations occurring at 6:00 a.m. and 8:00 p.m. and RH maintained at 75 ± 5% across all experiments. We monitored RH, air, and water temperature using a hygrometer.

After emergence, about 2,500 female mosquitoes that were 4–6 days old were deprived of sucrose solution for 24 h before being offered the infectious blood meal. The blood meal is prepared by mixing the Zika virus suspension with fresh mouse blood in a ratio of 1:1. The virus blood meal was retained at 37°C by a multifunctional insect blood supply device (Hemotek, PS6A Power Unit) and supplied to the mosquitoes for 1 h. Fully engorged females were then transferred to a single culture tube and maintained at two constant temperatures of 23 and 31°C and a DTR of 23–31°C, respectively, until 4 days post-infection (dpi), when oviposition was finished.

The female mosquitoes from three temperature groups that have completed egg laying in the culture tube are anesthetized with CO₂, from which the total RNA is extracted to determine Zika virus infection. The egg paper of the virus-positive female mosquitoes was held in desiccators at 25 ± 1°C and 90% RH with a daily photoperiod of 14:10 L:D for 6 days to permit embryonation. The egg paper was then placed at the corresponding temperature for hatching, and when the offspring mosquitoes emerged, they were anesthetized. The whole mosquito RNA was extracted, and qRT-PCR was used to detect whether each progeny mosquito carried the Zika virus.

The virus was quantified by qRT-PCR using the GoTaq^® Probe 1-Step RT-qPCR System (Promega A6120). The PCR primers included a pair of universal primers and probe (Pyke et al., 2014), F: 5'-AAGTTTGCATGCTCCAAGAAAAT-3', R: 5'-CAGCATTATCCGGTACTCCA-3', and probe: 5' FAM-ACCGGGAAGAGCATCCAGCCAGA-3' BHQ1. The reaction condition is 45°C for 15 min; 95°C for 10 min; and 40 cycles of 95°C for 15 s, and 60°C for 60 s.

Three groups, i.e., 23, 31, and 23–31°C with DTR conditions, were set in the experiment. Each group contains 50 fully engorged females that were placed in a feeding tube individually. Filter paper placed in a small container with water was supplied to the mosquitoes for oviposition 4 days after the blood meal. The eggs laid by each female mosquito were counted 4 days after the filter paper was placed, and then held in desiccators at 25 ± 1°C and 90% RH with a daily photoperiod of 14:10 L:D for 6 days to permit embryonation. The developed eggs were incubated in three different temperature conditions described previously to hatch. The total number of larvae, pupae, and adult mosquitoes in each group was counted. The larvae development time was defined as the time from the second day of hatching to the day of pupation. The hatching rate, pupation rate, and emergence rate were calculated as follows:

The data analysis was performed using the Graphpad Prism 8.02 software, and the normality of residuals was tested by the Anderson–Darling (A2^*) test. The survival curve of each group was compared by the log-rank (Mantel–Cox) test with Bonferroni correction. The number of mosquito eggs was analyzed by one-way ANOVA with Holm–Sidak's multiple comparisons test. The life cycle (hatching rate, pupation rate, and emergence rate) and IR of parental and progeny were analyzed by the chi-square test. Kruskal–Wallis test with Dunn's multiple comparisons test was used to analyze the development time and Zika virus viral load of the mosquitoes.

To evaluate the effect of different living conditions on mosquitoes, the survival curve of Ae. albopictus, which were reared under constant temperatures at 23 and 31°C or DTR condition after Zika virus infection, was monitored. The survival rate of Ae. albopictus is the lowest at 31°C and significantly lower than the other two groups (log-rank, P < 0.0001; Figure 2). The survival rate of Ae. albopictus in the DTR group is highest, but there was no significant difference between the 23°C and DTR groups (log-rank, P = 0.0665). The total number of eggs that each mosquito laid was also counted to evaluate the effect of temperature on mosquito reproduction. Results showed that mosquitoes in the DTR group laid more eggs than that in 31°C (36.5 ± 19.8 vs. 24.2 ± 15.1). These results indicated that fluctuating temperature was more beneficial for mosquitoes' survival and maintenance.

The development time was the longest in the low-temperature group at 23°C, with a median of 23.5 days, which was significantly longer than the other two groups (Figure 3). The high-temperature group had the lowest hatching rate (46.5%), which was significantly lower than the other two groups (P < 0.001) but had the highest pupation rate (significantly higher than the DTR group, P < 0.0001), emergence rate (significantly higher than the other two groups, P < 0.01, Table 1), and the shortest development time (median: 8 days). The DTR group exhibited the opposite profile of the high-temperature group, with the highest hatching rate and the lowest pupation rate (significantly lower than the other two groups) and emergence rate.

For a more comprehensive description of the effect of temperature on the transmission efficacy of the Zika virus by Ae. albopictus, Zika virus IR and viral RNA load in the parental females (F0) and the progeny (F1) from virus-positive F0 females were tested.

The IR and the Zika virus RNA copy in F0 females from three temperature groups (23, 31, and 23–31°C DTR) are presented in Figure 4. For the IR of F0 females, there was no significant difference in the three temperature groups. However, the Zika virus RNA copy in the three groups was significantly different. The lowest load was detected at 23°C with a median of 3.86 log₁₀ RNA copies/μl, and it was significantly lower than that in 31°C (median: 6.32 log₁₀ RNA copies/μl) and DTR (median: 5.28 log₁₀ RNA copies/μl) groups. Thus, temperature had no effect on virus invasion to the midgut but had a significant effect on virus replication and proliferation. Interestingly, IR and Zika virus load in F1 female mosquitoes were similar between the three temperatures.

The results showed that, in the constant temperature condition, the survival rate of Ae. albopictus in the high-temperature group was lower than that in the low-temperature group, which is similar to the results of other studies on the survival rate of Aedes aegypti (Delatte et al., 2009; Onyango et al., 2020). Although the survival rate differs among mosquito species, the trend of temperature affecting survival rate similar to the present study was still found in Culex and Anopheles mosquitoes (Aytekin et al., 2009; Faiman et al., 2017; Barreaux et al., 2018; Spanoudis et al., 2019). Spanoudis et al. (2019) explored the survival rate of Culex mosquitoes under a series of constant and fluctuating temperature conditions and found that the survival rate was highest at a constant temperature of 25°C or a DTR with an average of 25°C. In this study, the survival rate of Ae. albopictus under DTR condition, whose average temperature was 27°C, is similar to that of 23°C and significantly higher than that of 31°C. The comparison of Ae. albopictus survival under a constant temperature of 27°C and a DTR condition with an average of 27°C would be performed in the future to explore how fluctuating temperature affects the longevity of mosquitoes.

At the high temperature, the number of eggs laid by Ae. albopictus was lower. The same result was found in Anopheles, Culex, and Ae. aegypti (Carrington et al., 2013a; Marinho et al., 2016; Abouzied, 2017), whose reproductive ability declined at high temperatures. In addition, it was found in Culex paleus that no eggs were oviposited by females at 32.5°C. Interestingly, a study about the oviposition of Ae. albopictus recorded no eggs laid at 20°C (Ezeakacha and Yee, 2019). The relationship between temperature and fecundity could be explained in two ways. First, mosquitoes need longer gonotrophic cycle lengths at low temperatures. Second, fecundity-size relationships suggest that females from lower larval rearing temperatures would be larger and more fecund, whereas those from higher larval rearing temperatures would be smaller and less fecund (Sibly, 1994; Garrad et al., 2016). It appears likely that the effect of larval rearing temperature could be carried over through female size to affect fecundity. In this experiment, the pupation rate and emergence rate of Ae. albopictus were the highest at 31°C, and the mosquito larvae growth duration was the longest at 23°C.

It has been reported that increasing temperature lessens the mosquito gonotrophic cycle, thereby altering mosquito development and reproductive capacity (Afrane et al., 2006; Lardeux et al., 2008). This is due to the accelerated digestion of blood meal by mosquitoes at high temperatures, and mosquitoes obtained nutrients for rapid development and a series of reproductions (Sy et al., 2014). In this study, the mosquito midgut from each group was dissected on 3 dpi to examine blood digestion speed. It was found that the blood meal at 31°C and DTR had been basically digested and the blood meal in the midgut was invisible, while the blood meal in the midgut at 23°C was still visible (Figure 5), thus confirming this explanation.

For transovarial transmission, studies have confirmed that Ae. aegypti and Ae. albopictus can transmit the Zika virus through eggs (Li et al., 2017). In this study, we found that the IR at low temperature (23°C) was lower than that of 31°C and DTR. This is similar to the results of other studies on the effect of temperature on mosquito infection with viruses (Liu et al., 2017; Tesla et al., 2018; Blagrove et al., 2020). Low temperature reduces the ability of the virus to break through the mosquito midgut barrier, resulting in lower IRs and limiting virus invasion and propagation in mosquitoes. Under high temperatures, the IR of mosquitoes increases but so does the mortality rate, which also diminishes the vector competence of mosquitoes. In contrast, there are no significant differences in IR among those three temperature groups. Carrington et al. reported that DTR decreases IR but did not influence the transmission of DENV (Lambrechts et al., 2011; Carrington et al., 2013b,c).

The development time of Ae. albopictus is linearly related to temperature. Mosquito larvae take a longer time to develop into pupae at low temperatures, the development time required at high temperatures is significantly reduced compared to low temperatures, and similar results were found on the development time of Culex (Sibly, 1994; Afrane et al., 2006; Garrad et al., 2016). In this study, the development time of mosquitoes in the DTR was significantly shorter than the low-temperature group and longer than the high-temperature group. Richardson et al. found that fluctuating temperature had no effect on the development time of Ae. aegypti (Lardeux et al., 2008). However, there are also findings that the range of fluctuating temperatures is one of the factors affecting the development time of mosquitoes. When the temperature range of DTR is small (≤ 8°C), mosquito development time is not different between DTR and constant temperature, but when the temperature fluctuation range of the DTR group is 15°C, the large range of DTR will accelerate the development speed of mosquitoes (Sy et al., 2014).

Previous studies have shown that fluctuating temperatures are more accurate to investigate the effects of temperature on the mosquito life cycle (Bradshaw, 1980; Murdock et al., 2012a,b, 2013) because mosquitoes in the field live in fluctuating diurnal temperatures, not the constant temperature in the laboratory. While the results of this study showed that, compared to the constant temperature, DTR had complex effects on the biological parameters of mosquitoes. For example, in the DTR condition, the survival rate, number of eggs laid, hatching rate, pupation rate, and emergence rate of the Ae. albopictus infected with ZIKV were similar to the low-temperature group and different from the high-temperature group, while the development time and the viral load were similar to the high-temperature group and different from the low-temperature group. Our results implicated that not only temperature but also temperature pattern influence mosquito development, survival, and arbovirus transmission in a complicated way. Those findings may contribute to model construction that predicts vector-borne disease epidemics.

It should be noted that all conclusions of how temperature and temperature pattern influence the life cycle of Ae. albopictus in this study was based on the data from mosquitoes exposed to the Zika virus. Despite arboviruses generally not being highly pathogenic to mosquitoes (Patrican, 1985; Moncayo et al., 2000; Ciota et al., 2011), they sometimes alter mosquito biology depending on virus-mosquito species. For example, the chikungunya virus infection reduced the life span and shortened the time before the egg laying of Ae. albopictus Reunion strain but not the Mayotte strain (Martin et al., 2010). More interestingly, among the mosquitoes that were exposed to virus infection, those viral RNA-positive mosquitoes lived longer than that of virus negative mosquitoes (Ciota et al., 2011; Maciel-de-Freitas et al., 2011). It is possible that not only temperature pattern but also the interaction of temperature and virus infection contributed to differences in survival, fecundity, and development of Ae. albopictus between constant and fluctuating temperature conditions. However, our findings undoubtedly demonstrated that temperature significantly influences the survival of and virus transmission by mosquitoes under arbovirus infection status and suggest that vector competence evaluation should be conducted under fluctuating conditions for a more precise result.