Field investigation combined with modeling uncovers the ecological heterogeneity of Aedes albopictus habitats for strategically improving systematic management during urbanization.

BACKGROUND: Aedes albopictus is an invasive vector of serious Aedes-borne diseases of global concern. Habitat management remains a critical factor for establishing a cost-effective systematic strategy for sustainable vector control. However, the community-based characteristics of Ae. albopictus habitats in complex urbanization ecosystems are still not well understood. METHODS: A large-scale investigation of aquatic habitats, involving 12 sites selected as representative of four land use categories at three urbanization levels, was performed in Guangzhou, China during 2015-2017. The characteristics and dynamics of these Ae. albopictus habitats were assessed using habitat-type composition, habitat preference, diversity indexes and the Route index (RI), and the temporal patterns of these indexes were evaluated by locally weighted scatterplot smoothing models. The associations of RI with urbanization levels, land use categories and climatic variables were inferred using generalized additive mixed models. RESULTS: A total of 1994 potential habitats and 474 Ae. albopictus-positive habitats were inspected. The majority of these habitats were container-type habitats, with Ae. albopictus showing a particularly higher habitat preference for plastic containers, metal containers and ceramic vessels. Unexpectedly, some non-container-type habitats, especially ornamental ponds and surface water, were found to have fairly high Ae. albopictus positivity rates. Regarding habitats, the land use category residential and rural in Jiangpu (Conghua District, Guangzhou) had the highest number of Ae. albopictus habitats with the highest positive rates. The type diversity of total habitats (H-total) showed a quick increase from February to April and peaked in April, while the H-total of positive habitats (H-positive) and RIs peaked in May. RIs mainly increased with the monthly average daily mean temperature and monthly cumulative rainfall. We also observed the accumulation of diapause eggs in the winter and diapause termination in the following March. CONCLUSIONS: Ecological heterogeneity of habitat preferences of Ae. albopictus was demonstrated in four land use categories at three urbanization levels. The results reveal diversified habitat-type compositions and significant seasonal variations, indicating an ongoing adaptation of Ae. albopictus to the urbanization ecosystem. H-positivity and RIs were inferred as affected by climatic variables and diapause behavior of Ae. albopictus, suggesting that an effective control of overwintering diapause eggs is crucial. Our findings lay a foundation for establishing a stratified systematic management strategy of Ae. albopictus habitats in cities that is expected to complement and improve community-based interventions and sustainable vector management.

Development and evaluation of an efficient and real-time monitoring system for the vector mosquitoes, Aedes albopictus and Culex quinquefasciatus.

BACKGROUND: The surveillance of vector mosquitoes is essential for prevention and control of mosquito-borne diseases. In this study, we developed an internet-based vector mosquito monitor, MS-300, and evaluated its efficiency for the capture of the important vector mosquitoes, Aedes albopictus and Culex quinquefasciatus, in laboratory and field trials. METHODOLOGY/PRINCIPAL FINDINGS: The linear sizes of adult Ae. albopictus and Cx. quinquefasciatus were measured and an infrared window was designed based on these data. A device to specifically attract these two species and automatically transmit the number of captured mosquitoes to the internet was developed. The efficiency of the device in capturing the two species was tested in laboratory, semi-field and open field trials. The efficiency results for MS-300 for catching and identifying Ae. albopictus in laboratory mosquito-net cages were 98.5% and 99.3%, and 95.8% and 98.6%, respectively, for Cx. quinquefasciatus. In a wire-gauze screened house in semi-field trials, the efficiencies of MS-300 baited with a lure in catching Ae. albopictus and Cx. quinquefasciatus were 54.2% and 51.3%, respectively, which were significantly higher than 4% and 4.2% without the lure. The real-time monitoring data revealed two daily activity peaks for Ae. albopictus (8:00-10:00 and 17:00-19:00), and one peak for Cx. quinquefasciatus (20:00-24:00). During a 98-day surveillance trial in the field, totals of 1,118 Ae. albopictus and 2,302 Cx. quinquefasciatus were captured by MS-300. There is a close correlation between the number of captured mosquitoes and the temperature in the field, and a positive correlation in the species composition of the captured samples among the mosquitoes using MS-300, BioGents Sentinel traps and human landing catches. CONCLUSIONS/SIGNIFICANCE: The data support the conclusion that MS-300 can specifically and efficiently capture Ae. albopictus and Cx. quinquefasciatus, and monitor their density automatically in real-time. Therefore, MS-300 has potential for use as a surveillance tool for prevention and control of vector mosquitoes.

Susceptibility and interactions between Aedes mosquitoes and Zika viruses.

Zika virus disease is caused by Zika virus infection, as transmitted by Aedes spp. mosquitoes. Many of the Zika virus strains isolated from patients display different pathogenicities toward humans. The vector mosquitoes for Zika virus are mainly of the Aedes genus, especially Aedes aegypti and Aedes albopictus. However, susceptibility and interactions between Aedes spp. mosquitoes and Zika viruses remain unclear. In this study, we chose two Zika virus strains (FSS13025 and PRVABC59) with different abilities to infect the primary vector mosquitoes Ae. aegypti and Ae. albopictus. The transcriptomes and small RNA profiles of infected and uninfected mosquitoes were comparatively analyzed, and differentially expressed genes were functionally examined using RNA interference. According to the results, the susceptibility of PRVABC59 was higher than that of FSS13025 in Aedes vector mosquitoes, and Ae. aegypti was more susceptible to Zika virus than was Ae. albopictus. For PRVABC59 infection, specific differential expression profiles correlated with Ae. aegypti and Ae. albopictus, and susceptibility was significantly affected when three targeted genes were successfully knocked down. Compared with PRVABC59, infection of Ae. albopictus with FSS13025 generated more 21-nt virus small interference RNA. It can be concluded that the susceptibility of vector Aedes spp. mosquitoes to Zika viruses varies and that the interactions between mosquitoes and Zika virus correlate with susceptibility.

Vertical transmission of zika virus in Aedes albopictus.

BACKGROUND: Zika virus (ZIKV) is an arthropod-borne flavivirus transmitted by Aedes mosquitoes. Aedes albopictus is an important vector of ZIKV worldwide. To date, most experiments have focused on the vertical transmission of ZIKV in Ae. aegypti, while studies on Ae. albopictus are very limited. To explore vertical transmission in Ae. albopictus, a series of laboratory studies were carried out. METHODOLOGY/PRINCIPAL FINDINGS: In this study, Ae. albopictus were blood-fed with ZIKV-infectious blood, and the ovaries and offspring viral infection rates were analyzed by reverse transcription PCR (RT-PCR), real-time reverse transcription PCR (RT-qPCR) and immunohistochemistry (IHC). ZIKV was detected in the ovaries and oviposited eggs in two gonotrophic cycles. The minimum filial egg infection rates in two gonotrophic cycles were 2.06% and 0.69%, and the effective population transmission rate was 1.87%. The hatching, pupation, and emergence rates of infected offspring were not significantly different from those of uninfected offspring, indicating that ZIKV did not prevent the offspring from completing the growth and development process. ZIKV was detected in three of thirteen C57BL/6 suckling mice bitten by ZIKV-positive F1 females, and the viremia persisted for at least seven days. CONCLUSIONS/SIGNIFICANCE: ZIKV can be vertically transmitted in Ae. albopictus via transovarial transmission. The vertical transmission rates in F1 eggs and adults were 2.06% and 1.87%, respectively. Even though the vertical transmission rates were low, the female mosquitoes infected via the congenital route horizontally transmitted ZIKV to suckling mice through bloodsucking. This is the first experimental evidence of offspring with vertically transmitted ZIKV initiating new horizontal transmission. The present study deepens the understanding of the vertical transmission of flaviviruses in Aedes mosquitoes and sheds light on the prevention and control of mosquito-borne diseases.

Differentiation of Long Non-Coding RNA and mRNA Expression Profiles in Male and Female Aedes albopictus.

The Asia tiger mosquito (Aedes albopictus) is an important vector of arboviruses, and females can transmit pathogens such as the dengue, zika, and chikungunya viruses. Understanding sex-related differences in this mosquito is fundamental for vector control. However, there are no reports of systematic characterization of long non-coding RNAs (lncRNAs) in male and female Ae. albopictus. To investigate the roles of coding and non-coding RNAs in both sexes of Ae. albopictus, RNA sequencing was performed on male and female samples. The results showed 305 differentially expressed protein-coding genes (DEGs) between males and females, of which 198 were highly expressed in males and 125 were highly expressed in females. Sex-associated gene ontology terms were enriched. Analysis with the FEELnc software identified 2,623 novel lncRNAs, of which 26 showed male high expression and 11 showed female high expression. Quantitative real-time PCR of randomly selected DEGs and lncRNAs supported the validity of the RNA sequencing results. Knocking down male high-expressed gene AALF000433 in male adults reduced the egg hatching rate. This study provides valuable data on sex-specific expression of protein-coding genes and lncRNAs in adult Ae. albopictus, which will guide further studies aimed at understanding sex development and determination mechanisms in this species.

A Novel Densovirus Isolated From the Asian Tiger Mosquito Displays Varied Pathogenicity Depending on Its Host Species.

Mosquito-borne viral diseases (MBVDs) continue to pose a significant global public health burden. Mosquito control remains a core intervention strategy in integrated mosquito management (IMM) programs to reduce the transmission of MBVDs. Mosquito densoviruses (MDVs) are mosquito-specific entomopathogenic viruses, and their attractive biological and pathogenic characteristics make MDVs potential biological control agents as alternatives to traditional chemical pesticides. However, different viral strains vary greatly in their pathogenicity against different mosquito species, which poses an obstacle for the wide application of MDVs in vector control. In this study, a novel MDV, Aedes albopictus densovirus-7 (AalDV-7), was isolated from field-collected Aedes albopictus in the dengue-endemic area of Guangzhou, China. The complete 4,048 nt genome of AalDV-7 was cloned and sequenced, and the transcription and translation of three open reading frames (ORFs) were characterized. Phylogenetic analysis indicated that AalDV-7 clustered with MDVs mostly isolated from indigenous mosquitoes. The pathogenicity of AalDV-7 to A. albopictus, Aedes aegypti, and Culex quinquefasciatus larvae was completely different, and the median lethal dose (LD50) of AalDV-7 in A. albopictus which was 109.48 genome equivalents per ml (geq/ml) was 12 and 46 times lower than those in A. aegypti (1010.56 geq/ml) and C. quinquefasciatus (1011.15 geq/ml). Furthermore, the median lethal time (LT50) value in A. albopictus (7.72 days) was 25% and 26% shorter than those in A. aegypti (10.24 days) and C. quinquefasciatus (10.42 days) at a titer of 1011 geq/ml. Furthermore, the mortality of AalDV-7-infected mosquitoes increased in a dose-dependent manner, and the highest mortality was found in A. albopictus larvae exposed to 1011 geq/ml AalDV-7 (82.00%). Sublethal effects analysis also showed that AalDV-7 infection significantly decreased pupation and emergence rates. The 1st-2nd instar larvae of all three mosquito species showed a near 100% infection rate, and the highest relative vial titer (305.97 ± 67.57 geq/ng) was observed in the 1st-2nd instar larvae of C. quinquefasciatus. These pathogenic characteristics make AalDV-7 a potential MBVDs control agent in China, whereas its negligible pathogenicity and high infection rate and viral dose in vivo make it a good candidate for gene delivery vectors in C. quinquefasciatus gene function analysis. In conclusion, the continuous discovery and isolation of new MDVs enrich the pool of mosquito entomopathogenic viruses and provide a variety of choices for optimal MDVs or combinations of MDVs to target certain mosquitoes.

Development of large-scale mosquito densovirus production by in vivo methods.

BACKGROUND: Mosquito-borne diseases (MBDs) cause a significant proportion of the global infectious disease burden. Vector control remains the primary strategy available to reduce the transmission of MBDs. However, long-term, wide-scale and large-scale traditional chemical pesticide application has caused significant and increased negative effects on ecosystems and broader emerging insecticide resistance in vectors; therefore, the development of a novel alternative approach is urgently needed. Mosquito densoviruses (MDVs) are entomopathogenic viruses that exhibit a narrow host range and multiple transmission patterns, making MDVs a great potential bioinsecticide. However, the application process has been relatively stagnant over the past three decades. The major obstacle has been that viruses must be produced in mosquito cell lines; therefore, the production process is both expensive and time-consuming. METHODS: In our study, two wild-type (wt) MDVs, AaeDV and AalDV-3, and a recombinant rAaeDV-210 were used to infect the Aag2 and C6/36 mosquito cell lines and the 1st-2nd-instar and 3rd-4th-instar larvae of Ae. albopictus, Ae. aegypti and Cx. quinquefasciatus. Viral titers and yields in cells, media, larvae and rearing water and total viral yield were evaluated. Three kinds of virus displayed higher maximum virus titers in vivo than in vitro, and they displayed higher maximum viral yields in rearing water. RESULTS: The three viruses displayed higher total maximum viral yields in C6/36 cells than in Aag2 cells. The three viruses displayed higher total maximum viral yields in Aedes mosquitoes than in Culex mosquitoes. Higher viral yields were produced by 1st-2nd-instar larvae compared to 3rd-4th-instar larvae. The recombinant viruses did not display significantly lower yields than wt viruses in nearly all samples. In summary, by using 100 1st-2nd-instar Aedes mosquito larvae in 200 ml of rearing water, more than 1013 genome equivalents (geq) MDV yield can be obtained. CONCLUSIONS: Considering the lower production cost, this in vivo method has great potential for the large-scale production of MDVs. MDVs exhibit promising prospects and great potential for mosquito control in the future.

Temperature Increase Enhances Aedes albopictus Competence to Transmit Dengue Virus.

Dengue is a mosquito-borne disease that has been an epidemic in China for many years. Aedes albopictus is the dominant Aedes mosquito species and the main vector of dengue in China. Epidemiologically, dengue mainly occurs in Guangdong Province; it does not occur or rarely occurs in other areas of mainland China. This distribution may be associated with climate, mosquito density, and other factors in different regions; however, the effect of temperature on the vector competence of Ae. albopictus for dengue viruses (DENV) remains unclear. In this study, Ae. albopictus was orally infected with dengue virus 2 (DENV-2) and reared at constant temperatures (18, 23, 28, and 32°C) and a fluctuating temperature (28-23-18°C). The infection status of the midguts, ovaries, and salivary glands of each mosquito was detected by polymerase chain reaction (PCR) at 0, 5, 10, and 15 days post-infection (dpi). DENV-2 RNA copies from positive tissues were quantified by quantitative real time PCR (qRT-PCR). At 18°C, DENV-2 proliferated slowly in the midgut of Ae. albopictus, and the virus could not spread to the salivary glands. At 23 and 28°C, DENV-2 was detected in the ovaries and salivary glands at 10 dpi. The rates of infection, dissemination, population transmission, and DENV-2 copies at 28°C were higher than those at 23°C at any time point. At 32°C, the extrinsic incubation period (EIP) for DENV-2 in Ae. albopictus was only 5 dpi, and the vector competence was the highest among all the temperatures. Compared with 28°C, at 28-23-18°C, the positive rate and the amount of DENV-2 in the salivary glands were significantly lower. Therefore, temperature is an important factor affecting the vector competence of Ae. albopictus for DENV-2. Within the suitable temperature range, the replication of DENV-2 in Ae. albopictus accelerated, and the EIP was shorter with a higher temperature. Our results provide a guide for vector control and an experimental basis for differences in the spatial distribution of dengue cases.

Competence of Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus Mosquitoes as Zika Virus Vectors, China.

In China, the prevention and control of Zika virus disease has been a public health threat since the first imported case was reported in February 2016. To determine the vector competence of potential vector mosquito species, we experimentally infected Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus mosquitoes and determined infection rates, dissemination rates, and transmission rates. We found the highest vector competence for the imported Zika virus in Ae. aegypti mosquitoes, some susceptibility of Ae. albopictus mosquitoes, but no transmission ability for Cx. quinquefasciatus mosquitoes. Considering that, in China, Ae. albopictus mosquitoes are widely distributed but Ae. aegypti mosquito distribution is limited, Ae. albopictus mosquitoes are a potential primary vector for Zika virus and should be targeted in vector control strategies.