| Background:Aedes albopictus (Skuse), also known as the Asian tiger mosquito,is a competent vector for the transmission of many blood-borne epidemics such as dengue fever, Zika virus, West Nile virus infections and Chikungunya fever, and it is also the most bio-invasion capacity mosquito. Aedes albopictusis the major transmission vector of dengue fever in China. In the absence of specific antiviral therapy, controlling of transmission of DENV by vector management is the sole method available for reducing dengue-associated morbidity. Though the current large-scale use of pesticides and breeding grounds clean-up methods can reduce the density of Aedes albopictus to some extent, while it can not effectively reduce the spread of dengue virus at low level of risk. Therefore, to find a new means of controlling is an important step for developing and implementing appropriate strategies to control mosquito vector population.In temperate and subtropical populations of Aedes albopictus, exposuring to short-day lengths cause the adult female to oviposit diapause eggs in which the pharate larvae enters diapause.The rapid range expansion of Aedes albopictus, at least partially,due to its ability to enter diapause, a specific type of dormancy that significantly improves survival during winter or other unfavorable conditions. Diapause eggs of Aedes albopictushave strong resistance of dry, cold and other resilience. In both cases the species was imported as dormant eggs through used tires trade, Aedes albopictus population achieve rapid expansion, colonization and a wide range of pathogens such as dengue virus spread one of the important biological basis. Research on diapause in Aedes albopictus mosquito vector control will explore new ways to provide a scientific basis. In this study, the optimization of diapause induction and identification systemof wild strains and Foshan strainsof Aedes albopictus were established, and we also preliminarly investigate the ecological background of diapause in wild populations of Aedes albopictus.Chapter I Optimization of diapause induction and identification systems in Aedes albopictusObjective To improve Aedes albopictus synchronization system and to observe egg embryo development. Meanwhile, we aim to optimizing Aedes albopictus diapause induction system, diapause determination system and preliminary observating of mosquito eggs diapause rate at different days after spawning.Methods1. Establishment of the induced system for synchronous egglaying in Aedes albopictus with two ways:1, a dark colored cup was lined with unbleached bamboo paper and was filled halfway with deionized water; 2, a transparent plastic bowl was lined with bleached paper and was filled halfway with deionized water. Synchronous dynamic observation and comparing the wild strain and Foshan strain of Ae. Albopictus under different synchronous egglaying conditionsonthe effect of synchronous spawning, fecundity.2. Aedes albopictus eggs embryo development was observed, periods corresponding to the Aedes albopictus eggs embryo developmental stages were observed, and other mosquito egg embryo developmental stages were compared.3. Wild strain and Foshan strains were induced diapause under short photoperiod (L:D=8:16h) (Light:Dark cycle=8:16h) from the fourth instar larvae, then optimization of diapause induction system inAedes albopictus were conducted.4. Three diapause determination way (yeast powder, food slurry and ascorbic acid) were used to quantify the diapause response of additional reserved embryos that are 7-20 days old. Diapause incidence of the three methods was compared, and optimization the diapausedecision system for Aedes albopictus were conducted.5. Two diapause determination way (yeast powder and food slurry) were used to quantify the diapause response of additional reserved embryos that are 7-20 days old,30days old,45days old and 50 days old, four different spawning days diapause egg diapause rate decision, and changes in the percentage of diapause in two ways were observed.6. SPSS 13.0 were used for descriptive statistics analysis, chi square test were used between group differences, inspection standard a of 0.05, multiple samples of multiple comparison with calibration standard test:a’=2α/k(k-1). Graphpad 5.01 software were used for mapping.Results1. Establishment of the induced system for synchronous egglaying in Aedes albopictus, the egglaying amounts of the wild strain increased from 3.4% to 51% in the first day post oviposition (dpov), and rose from 30.2% to 97% within 3 dpov. The egglaying period reduced from 18 d to 7 d. For Foshan strain, 1 dpov egglaying amounts increased from 40% to 94%, and 3 dpov egglaying amounts increased from 84% to 99.7%. The egglaying period reduced from 13 d to 5d.2. Embryonic development of Aedes albopictus was divided into five stages:blastoderm formation & gastrulation, germ band extension, germ band retraction, dorsal closure and the end. Compared to other mosquito species, Aedes albopictus embryo development time more slower than Aedes aegypti and Anophelesalbitarsis, approximately 12 HAE was completely formed gastrulation,60 HAE, the egg embryo development is complete, the abdominal segment significantly.3. Optimization of diapause induction system in wild strains and foshan strains of Aedes albopictus, diapause incidence of each biological replicate of Foshan strain ranged from86.60%～0.76% and diapause incidence of each biological replicate of wild strain ranged from 88.64%～97.21%.4. Using yeast powder, food slurry and ascorbic acid three diapause determination to quantify the diapause response of 7-20 days oldadditional reserved embryos of wild strain, found that there was no statistical significance between food slurry and yeast powder under different photoperiods. Between ascorbic acid and yeast powder, food slurry diapause incidence rate differences were statistically significant (x2=42.13, p<0.001; x2=9.7, p=0.008).5. Using yeast powder, food slurry and ascorbic acid three diapause determination to quantify the diapause response of 7-20 days oldadditional reserved embryosof Foshan strain, found that there was no statistical significance between food slurry and yeast powder under different photoperiods. Between ascorbic acid and yeast powder, food slurrydiapause incidence rate differences were statistically significant(x2=65.11,P<0.011; x2=50.28,p<0.001).6. Using yeast powder, food slurry two diapause determination to quantify the diapause responseof 7-20 and 30 days oldadditional reserved embryos,found that there was no statistical significance between food slurry and yeast powder. But for 45 and 50 days oldadditional reserved embryos, two method of diapause incidence rate differences were statistically significant (x2=35.72,p<0.001; x2==16.81, p<0.001). Using yeast powderto quantify the diapause response, diapause incidence was lower than food slurry.7. Using food slurry to quantify the diapause response of 7-20,30,45,50 days oldadditional reserved embryos, found that the diapause incidence of mosquito eggs in four different days showed significant difference. After further statisticalmethod of comparison, found that there was no statistical difference between 7～20,30d days old additional reserved embryos, and 7-20 and 30days old additional reserved embryos showed significant differences in diapause rate between 45,50 days old additional reserved embryos, respectively. The same results were also presented for the use of slurry food as a method of diapause determination.Conclusion1. Wild strain and Foshan strain of Aedes albopictus using black cup as trap egg containers and unbleached bamboo paper as oviposition conditions could get significantly improved synchronization oviposition effect. Aedes albopictusembryonic developed slower than other mosquito species1. Wild strains and foshan strains of Aedes albopictus can be induced to oviposit diapause eggs under short photoperiod (L:D= 8:16h),21℃ and approximately 80% relative humidity. Diapause incidence of each biological replicate of Foshanstrain ranged from86.60%～97.21%.2. Both yeast powder and food slurry can be used to determined diapause eggs.3.7-20 and 30 days oldadditional reserved embryos under short-day lengths are in diapause stage.4. Diapause incidence using yeast power method is lower than using food slurry when detesting 45,50 days old additional reserved embryos.Chapter Ⅱ Preliminary investigation on the ecological background of diapause in wild populations of Aedes albopictusObjective The investigation of mosquito density seasonal fluctuation and the diapause background in Guangzhou.Methods1.50 mosquito oviposition trap fill one third with deionized water and placed a bleached paper on the thereon bottom, were placed in six District, Guangzhou. In each monitoring point 25-30 meters away from laying a mosquito oviposition, four days after collecting mosquito oviposition trap, the number of observations to mosquitoes and spawning positive have been observed, calculate as mosquito oviposition indexs (MOI).2. Mosquito population density has been investigate using human induced method in the study areas. Select the shade as detect locations, Monitors exposed side of the lower leg, recorded number of females within 30min rest captured by electric suction blower. Record the time when to start and end, location, temperature, and humidity. Calculate as mosquito density index (MDI).3. Larval developmental sites survey has been observed in every month. Detecting human using a portable global positioning system(GPS) device (GARMIN Corporation, Taibei, Taiwan), along the monitoring path, uniform pace, recording all larval developmental sites,a container was recorded as positive for Ae. albopictus when at least one immature of either species was observed. Calculate as the route index (RI).4. The variance analysis, Pearson correlation, Spearman rank correlation and multiple linear regression analysis were used to explore different urbanization area and meteorological factors of MOI, MDI and RI, and analysis the relationship between MOI, MDI and RI.5. Eggs were collected from mosquito oviposition trap:recycling eggs from mosquito oviposition trap from February 2015-January 2016 placed in the wild monthly. The eggs are immersed in water for 10 days, then centrifuged after removing the supernatant. Eggs was obtain and counted under a stereomicroscope.6. Eggs were collected from positive container:January 2016 and February in positive container (in the past month were positive, in January and February no positive) using the medial eyebrow brush to obtain.7. Yeast powder were used to determining diapause eggs collected from mosquito oviposition trap and positive container.8. SPSS 13.0 were used for statistical analysis, standard alpha is 0.05. T test and variance analysis were used to measure the difference between the data sets, and the LSD was used to compare the difference between the two two groups. Graphpad 5.01 software were used for mapping.Results1. Mosquito oviposition index peak of four kinds of ecological environment (sites, parks, residential areas and schools)have occurred in May and June, in which the school mosquito oviposition index reached the highest median in June, up to 55%.2. The four kinds of ecological environment(Site, parks, residential areas and schools)of MDI peak time is slightly different, which site MDI peak appeared in May and June, park MDI peak appeared in the May to July, residential MDI in larger from May to October, school MDI peak appeared in May. Site MDI median of the larger in May and June, respectively,25/person/h and 20/ person/h.3. Site, parks, residential areas and schools of the peak of RI appeared slightly different, and the residents of the district and the school’s RI peak appeared in May, the park’s RI in September and October, the site of the RI large in March to September.4. There were significant differences (F=4.818, p=0.02) in the RI value between the three sides of the central city, suburban and rural areas. Two two comparison showed that RI was significantly higher in the rural areas than in the central urban area and the suburbs.5. MOI had positive correlation with the highest average temperature, monthly mean temperature, monthly lowest air temperature, mean monthly precipitation and average sunshine time length and MDI showed a positive correlation with the highest average temperature, monthly mean temperature, monthly minimum temperature and monthly average sunshine length, and RI only have a positive correlation with monthly average sunshine hours.6. Correlation analysis showed that between ADI and RI, between ADI and MOI has a strong positive correlation (r=0.818, p= 0.001, r= 0.930, p< 0.001), and between RI and MOI has strong positive correlation (r= 0.634,p= 0.027).7. Eggs obtained from mosquitoes ovitraps:Yuexiu District 44 grains, Tianhe District 43 grains, Baiyun District 214 grains,244 grains Luogang District, Huadu District 169 grains, Conghua District 137 grains, totally 851 grains. In July in Tianhe District found a grain of diapause eggs, in August in the Luogang District found one, Huadu District found two, in September in the Yuexiu District found two, Luogang District found one, Huadu District found one; in November in Huadu found one; in December in Huadu also found only one. Lastly 9 diapause eggs were determined using Yeast Powder.8. The January 2016 and February wild breeding places of Aedes albopictus eggs collected each won 69 and 82. Use yeast powder way diapause diapause eggs after the determination is determined as 12.Conclusion1. RI, MDI and MOI of four ecological environment (site, park, residential area and school) showed different growth and decline trend with the change of seasons.2. Month the highest average temperature and monthly mean temperature, monthly lowest air temperature, mean monthly precipitation and average sunshine time long Aedes albopictus eggs have a certain impact, while the rainfall and the correlation of mosquito density is not high, only average sunshine time is long and the number of larva.3. There is a strong correlation between MOI, MDI and RI.4. Wild strains of Aedes albopictus in Guangzhou in diapause and quiescence a combination of ways to spend the cold winter. |
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