Effects of Guangzhou seasonal climate change on the development of Aedes albopictus and its susceptibility to DENV-2

The susceptibility of Asian tiger mosquitoes to DENV-2 in different seasons was observed in simulated field environments as a reference to design dengue fever control strategies in Guangzhou. The life table experiments of mosquitoes in four seasons were carried out in the field. The susceptibility of Ae. albopictus to dengue virus was observed in both environments in Guangzhou in summer and winter. Ae. albopictus was infected with dengue virus by oral feeding. On day 7 and 14 after infection, the viral load in the head, ovary, and midgut of the mosquito was detected using real-time fluorescent quantitative PCR. Immune-associated gene expression in infected mosquitoes was performed using quantitative real-time reverse transcriptase PCR. The hatching rate and pupation rate of Ae. albopictus larvae in different seasons differed significantly. The winter hatching rate of larvae was lower than that in summer, and the incubation time was longer than in summer. In the winter field environment, Ae. albopictus still underwent basic growth and development processes. Mosquitoes in the simulated field environment were more susceptible to DENV-2 than those in the simulated laboratory environment. In the midgut, viral RNA levels on day 7 in summer were higher than those on day 7 in winter (F = 14.459, P = 0.01); ovarian viral RNA levels on day 7 in summer were higher than those on day 7 in winter (F = 8.656, P < 0.001), but there was no significant difference in the viral load at other time points (P > 0.05). Dicer-2 mRNA expression on day 7 in winter was 4.071 times than that on day 7 in summer: the viral load and Dicer-2 expression correlated moderately. Ae. albopictus could still develop and transmit dengue virus in winter in Guangzhou. Mosquitoes under simulated field conditions were more susceptible to DENV-2 than those under simulated laboratory conditions.

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The data underlying the results presented in the study are available from (include the name of the third party significantly. The winter hatching rate of larvae was lower than that in summer, and the 38 incubation time was longer than in summer. In the winter field environment, Ae. 39 albopictus still underwent basic growth and development processes. Mosquitoes in the 40 simulated field environment were more susceptible to DENV-2 than those in the 41 simulated laboratory environment. In the midgut, viral RNA levels on day 7 in summer 42 were higher than those on day 7 in winter (F = 14.459, P = 0.01); ovarian viral RNA 43 levels on day 7 in summer were higher than those on day 7 in winter (F = 8.656, P < 44 0.001), but there was no significant difference in the viral load at other time points (P > 45 0.05). Dicer-2 mRNA expression on day 7 in winter was 4.071 times than that on day 46 adicionar detalhes da localidade 47 albopictus could still develop and transmit dengue virus in winter in Guangzhou. 48 Mosquitoes under simulated field conditions were more susceptible to DENV-2 than 49 those under simulated laboratory conditions. population expansion, dengue virus transmission, and disease outbreak prevention. We 56 found that Ae. albopictus could still develop in winter in Guangzhou. In addition, the viral 57 load of mosquitoes in the simulated winter environment was lower than that in summer 58 on the 7th day after infection; however, the difference in the viral load of mosquitoes in 59 winter and summer on the 14th day after infection was not statistically significant. 60 Mosquitoes in simulated field conditions were more susceptible to DENV-2 than those in 61 simulated laboratory conditions. The results suggested that Ae. albopictus retains the 62 ability to transmit dengue virus in winter in Guangzhou, and that improved dengue With the acceleration of climate warming, globalization, and urbanization, the 71 epidemic scope of dengue fever is expanding worldwide [1]. Dengue fever is a 72 worldwide public health concern, with approximately 390 million people affected Culicidae) in Guangzhou. Ae. albopictus larvae could develop and emerge and the 108 adults could survive and produce eggs in early winter in Guangzhou. The major impact 109 of changes in ambient temperature, relative humidity, and light intensity was on the egg 110 hatching rates, adult survival time, and egg mass production, rather than on pupation or 111 adult emergence rates [13].       Larvae were brought back to semi-field setting and reared in microcosms where life-193 table experiments were conducted. Emerged adults were allowed mating freely. This 194 mix will reduce the bias due to differences in larval source and inbreeding [13].

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Mosquitoes were reared until F3 eggs in the field condition. F3 eggs were used for 196 first round of life-table experiments for three reasons, to allow for get enough eggs 197 within one day, to allow for field mosquitoes to adapt the new environment and 198 mouse blood. We did not observe bottlenecks or significant loss of mosquitoes during 199 this process [13]. The laboratory strains of Ae. albopictus was reared under standard Southern Medical University. The site was the same as previously reported [13].  Newly emerged adults were used in the life-table studies with protocols similar to that 228 described in a previous study [13]. Briefly, 30 female and 30 male adult mosquitoes in each cage were counted daily. The water temperature and light intensity were 238 measured using HOBO® data loggers [13]. The survival and fecundity of the adult 239 mosquitoes were observed at about 18:00 hours every day.

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The field environment was set in the garden of Baiyun District, Guangzhou. The garden 241 was closed and managed by special personnel to avoid interference by irrelevant human 242 factors. The environment for raising Ae. albopictus in the field comprised mosquito 243 nets placed in the pavilions with sunshade roofs and ventilation around. The plants in 244 the garden are luxuriant, which was conducive to the breeding of Ae. albopictus [13]. Guangzhou. The larvae were fed with small fish diet and the adults were fed with 10% 252 glucose solution. Two groups of experiments were carried out in the field and laboratory.

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The mosquitoes were provided with 10% glucose daily, and, every three days, a mouse 254 was placed in each cage for approximately 4 hours to blood-feed the mosquitoes, the 255 mice were confined to a wooden board, in order to oviposition of female mosquitoes.  for 15 min, the culture flask was incubated at 37 °C and 5% CO2 for 2 days until obvious cytopathic effects were observed, Generally, the culture temperature of C6/36 cells is 268 28°C, but for the benefit of virus proliferation, we adopt the culture temperature of 37°269 C.
[53]. The supernatant was harvested after centrifugation at 1,500 × g for 5 min, 270 separated into 0.5-mL aliquots, and frozen at 80 °C. The Dengue virus 2 titer was 271 determined using the 50% tissue culture infective dose (TCID50) method [60].         Guangzhou strain in spring was lower than that in summer (P < 0.008). The pupation 445 rate of the laboratory strain in spring was lower than that in summer and autumn (P < 446 0.008). The pupation rate of the laboratory strain in winter was lower than that in 447 autumn (P < 0.008).  Table ).  Table, Fig 2).

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The survival of the Guangzhou strain and laboratory strain in the field environment is 479 shown in Fig 3(A), and that in the laboratory environment is shown in Fig 3(B). The   Table 2.  Table 2. There was no significant difference in the emergence rate of the 513 laboratory strains in the field over the four seasons (P > 0.008). In the laboratory, the 514 emergence rate of the Guangzhou strain in spring and winter was lower than that in 515 summer and autumn (P < 0.008).

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Analysis of oviposition of mosquitoes showed that adult mosquitoes could normally 517 suck blood and lay eggs in the laboratory and outdoor environment in summer, but 518 sometimes they did not suck blood and lay eggs in field environment in winter. The The group with the least number of eggs laid was the winter field environment group, 523 total four groups, two of the groups did not lay eggs, but the total number of eggs laid 524 in another two of the groups were 51 and 230, respectively (S4 Table,

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The survival time of female adults was longer than that of male adults (F = 8.291, P < 545 0.05). There was a significant difference in survival time between male and female Ae. 546 albopictus in summer and winter (F = 5.535, 9.407, all P < 0.05) ( Table 3).    (Table 4).

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The average relative humidity of outdoor environment in summer was 79.0 ± 8.8%.

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Summer is a rainy season in Guangzhou; therefore, the relative humidity of the outdoor 569 environment in summer was higher than that of the laboratory environment. The 570 average relative humidity in winter was 73.9 ± 11.3%, the results showed that the 571 relative humidity of Guangzhou in winter changed greatly, and the average relative 572 humidity was still higher than that of the laboratory environment.  Table 5 and Fig 7A.  The results showed that there were significant differences in the infection rate of the 589 ovary at 7 dpi under different experimental conditions (χ 2 = 672, P < 0. 05). The 590 infection rate of head was also statistically different (χ 2 = 608, P < 0. 05). The infection 591 rate of the ovary and head at 7 dpi under simulated field conditions was higher than that 592 under laboratory conditions. At 14 dpi, as shown in Table 5 and Fig 7B,  Under the same experimental conditions, the infection rate of the same tissue at 599 different time points is shown in Fig 7(C, D). Under laboratory conditions, the infection 600 rates of the ovary and head at 7 dpi were 44.0% and 56.0%, respectively, and at 14 dpi were 90.7% and 85%, respectively. The chi squared test showed that the ovarian 602 infection rate at 7 dpi was lower than that at 14 dpi (χ 2 = 629, P < 0. 05), and the head 603 infection rate at 7 dpi was lower than that at 14 dpi (χ 2 = 8.608, P < 0. 05). Under 604 simulated field conditions, the infection rates of the ovary, midgut, and head were all 605 above 88%. There was no significant difference in the infection rates of the same tissue 606 between 7 and 14 dpi (P > 0.05). The results showed that mosquitoes in simulated field 607 environment were more susceptible to DENV-2 than those in the simulated laboratory 608 environment.

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The infection rates of the same tissue in different seasons are shown in Fig 7 (E, F). The 611 infection rates of the ovary and head at 7 dpi in summer and winter were higher than 612 80%. The infection rate of the midgut was 99.0% in summer, but only 66% in winter.

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The infection rate of the same tissue at 7 dpi was tested using the chi squared test. The  same season is shown in Fig 8A and Table. 6. There was no significant difference in the 627 RNA copy number of mosquitoes infected with dengue virus in the ovaries, head, and 628 midgut tissues at 7 and 14 dpi (P > 0.05). The results of variance tests showed that the 629 mean copy number of virus RNA in the midgut at 7 dpi in summer was higher than that 630 at 7 dpi in winter (F = 14.459, P P < 0.05). The mean copy number of virus RNA in the 631 ovary at 7 dpi in summer was also higher than that at 7 dpi in winter (F =8.656, P < 632 0.001); however, there was no significant difference in the number of virus copies at 633 other time points (P > 0.05) (Fig 8B). seasons was comparable to that in the most suitable living environment (the laboratory 705 environment), This is consistent with our previous report [13]. In this study, the average 706 temperature in winter was 18.2 °C, and the pupation rate of Guangzhou was 96%. The 707 average temperatures in summer and autumn were 28.8 and 22.9 °C respectively, and 708 the pupation rates were 96% and 95% respectively. Thus, the pupation rate remains at 709 a high level under different temperature conditions in the field.  In addition, we found that the average copy number of dengue virus RNA in the 827 midgut and ovary of mosquitoes at 7 dpi in summer was higher than that at 7 dpi in 828 winter. We speculated that the mechanism might be related to the regulation of 829 immune-related genes in different seasonal environments. Our results showed that the 830 expression of Dicer2 at 7 dpi in winter was significantly higher than that at 7 dpi in 831 summer, and the difference was statistically significant. These results were consistent with our results.

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There is a defect in our study: the use of head samples to determine potential  In this study, it was found that Ae. albopictus could still develop in winter in Guangzhou.

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The results of the present study suggest that Ae. albopictus still has the ability to