Yande Dong

Identifying the Main Mosquito Species in China Based on DNA Barcoding

Mosquitoes are insects of the Diptera, Nematocera, and Culicidae families, some species of which are important disease vectors. Identifying mosquito species based on morphological characteristics is difficult, particularly the identification of specimens collected in the field as part of disease surveillance programs. Because of this difficulty, we constructed DNA barcodes of the cytochrome c oxidase subunit 1, the COI gene, for the more common mosquito species in China, including the major disease vectors. A total of 404 mosquito specimens were collected and assigned to 15 genera and 122 species and subspecies on the basis of morphological characteristics. Individuals of the same species grouped closely together in a Neighborhood-Joining tree based on COI sequence similarity, regardless of collection site. COI gene sequence divergence was approximately 30 times higher for species in the same genus than for members of the same species. Divergence in over 98% of congeneric species ranged from 2.3% to 21.8%, whereas divergence in conspecific individuals ranged from 0% to 1.67%. Cryptic species may be common and a few pseudogenes were detected.

Culex pipiens quinquefasciatus : a potential vector to transmit Zika virus

Zika virus (ZIKV) has become a threat to global health since the outbreak in Brazil in 2015. Although ZIKV is generally considered an Aedes-transmitted pathogen, new evidence has shown that parts of the virus closely resemble Culex-transmitted viruses. Therefore, it is important to evaluate the competence of Culex species for ZIKV to understand their potential as vectors. In this study, female Culex pipiens quinquefasciatus were orally exposed to ZIKV. Mosquito midguts, salivary glands and ovaries were tested for ZIKV to measure infection and dissemination at 2, 4, 6, 8, 12, 16 and 18 days post exposure (pe). In addition, saliva was collected from mosquitoes after infection and infant mice were bitten by infected mosquitoes to measure the transmission ability of Cx. p. quinquefasciatus. The results showed that the peak time of virus appearance in the salivary glands was day 8 pe, with 90% infection rate and an estimated virus titer of 3.92±0.49 lg RNA copies/mL. Eight of the nine infant mice had positive brains after being bitten by infected mosquitoes, which meant that Cx. p. quinquefasciatus could be infected with and transmit ZIKV following oral infection. These laboratory results clearly demonstrate the potential role of Cx. p. quinquefasciatus as a vector of ZIKV in China. Because there are quite different vector management strategies required to control Aedes (Stegomyia) species and Cx. p. quinquefasciatus, an integrated approach may be required should a Zika epidemic occur.

Relationship between insecticide resistance and kdr mutations in the dengue vector Aedes aegypti in Southern China

BackgroundAedes aegypti is an important vector for dengue virus and thus has been targeted with pyrethroid insecticides in many areas of the world. As such, resistance has been detected to several of these insecticides, including in China, but the mechanisms of the resistance are not well understood in this country.MethodsUsing the World Health Organization larval mosquito bioassay, five field populations of Aedes aegypti from Southern China were characterized for their resistance to cypermethrin and cyhalothrin. RNA extraction with PCR amplification, cloning and sequencing of the sodium channel gene was followed by comparisons of susceptible and wild mosquito strains Additionally, genomic DNA was used for Allele-specific PCR (AS-PCR) genotyping of the sodium channel genes to detect S989P, V1016G and F1534C mutations and allow for correlation analysis of resistance expression for the different mutations.ResultsAll wild strains expressed resistance to cypermethrin and cyhalothrin and the resistance expression between the two insecticides was highly correlated suggesting cross-resistance between these two pyrethroids. The AS-PCR technique effectively distinguished individual genotypes for all three mutations. Among the five wild strains tested, two strains carried all three mutations. Although the S989P and V1016G mutations were positively correlated to resistance expression of both pyrethroids, the F1534C mutation was negatively correlated.ConclusionsOur methodology proved highly reliable and will aid future detection of kdr mutations. The three sodium channel mutations were common in the Ae. aegypti strains sampled from Southern China. The V1016G mutation appears to be the most important kdr mutation in Ae. aegypti strains in Southern China.

First report on co-occurrence knockdown resistance mutations and susceptibility to beta-cypermethrin in Anopheles sinensis from Jiangsu Province, China.

The increasing prevalence of insecticide resistance in Anopheles sinensis, a major vector of malaria in Jiangsu province in eastern China, threatens to compromise the successful use of insecticides in malaria control strategies. It is therefore vital to understand the insecticide resistance status of An. sinensis in the region. This study examined the nucleotide diversity of the para-sodium channel and knockdown resistance (kdr) in five field populations of adult An. sinensis mosquitoes collected in Jiangsu province, identifying the L1014F and L1014C substitutions for the first time. Competitive polymerase chain reaction (PCR) amplification of specific allele (cPASA) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) for resistance diagnosis were developed and validated. Comparing the results with direct sequencing revealed that the PCR-RFLP method was more sensitive and specific whereas the cPASA method was more convenient and suitable. The significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014C substitutions in the kdr gene provides a useful molecular marker for monitoring beta-cypermethrin resistance in natural populations of An. sinensis. Our results point to the L1014F substitution as the key mutation associated with beta-cypermethrin resistance. The high resistance and mutation frequency detected in the five populations also suggest cross-resistance with other pyrethroids may occur in An. sinensis, highlighting the need for further surveys to map insecticide resistance in China and the adoption of a rational management of insecticide application for resistance management and mosquito vector control.

Survival and replication of dengue-2 virus in diapausing eggs of Aedes albopictus (Diptera: Culicidae)

Abstract Survival and replication of dengue-2 virus (family Flaviviridae, genus Flavivirus, DENV-2) was determined in diapausing eggs of Aedes albopictus (Skuse) (Diptera: Culicidae) to evaluate the importance of this mechanism for viral maintenance during adverse climatic conditions. Infected and uninfected eggs of Ae. albopictus were induced to diapause under low temperature and short photoperiod. Virus was detected by reverse transcription-polymerase chain reaction and isolated in C6/36 cells from both diapausing eggs and nondiapausing eggs. Nucleic acid hybridization was used to monitor viral replication in diapausing eggs. DENV-2 might survive in infected diapausing eggs in a relatively quiescent status based on the absence of a replicative intermediate RNA. In contrast, the viral replicative intermediate RNA along with the replicative form RNA was detected in nondiapausing eggs. It seemed that the virus replicated more actively in nondiapausing eggs than in diapausing eggs. Infected diapausing eggs may play an important role in the maintenance of DENV during adverse climatic conditions in nature.

Pyrethroid resistance and distribution of kdr allele in Culex pipiens pallens in north China

Abstract A field survey was conducted to estimate resistance of Culex pipiens pallens, to pyrethroid insecticides in north China. We collected mosquito as egg rafts from 11 areas in Beijing and Zhangjiakou, Hebei province. Resistance to conventional pesticides, Es-bioallethrin, permethrin, deltamethrin, beta-cypermethrin, were measured using larval bioassays. The mutation kdr (L1014F) was also confirmed by sequence analysis. Our results indicate that the mosquitoes in north China still have a low or a moderate degree of resistance to pyrethroids. We then used competitive polymerase chain reaction (PCR) amplification of specific allele (cPASA) assay to monitor frequencies of kdr mutation in mosquito field populations. The kdr allele was present at frequencies of 7.9 – 37.8% in the field samples. Labeled allele-specific oligonucleotide hybridization (PCR-ASO) was performed and validated with cPASA. This proves that cPASA is an efficient molecular approach for monitoring insecticide resistance. Regression analysis indicated a significant correlation between kdr allele frequencies and the levels of resistance to knockdown resistance by permethrin, Es-bioallethrin, deltamethrin, and beta-cypermethrin. The results validate the role of the PCR-based molecular assay as a diagnostic tool in monitoring resistance to pyrethroids and also to provide useful information on population genetics of mosquito resistance to pyrethroids.

Experimental studies on comparison of the potential vector competence of four species of Culex mosquitoes in China to transmit West Nile virus.

ABSTRACT To assess the risk that indigenous mosquitoes in China are capable of transmitting and sustaining West Nile virus (WNV), four important Culex mosquito species, Culex tritaeniorhynchus, Culex modestus, Culex pipiens pallens, and Culex pipiens quinquefasciatus, were allowed to feed on the artificial infectious blood meal with WNV dose of 106,8 plaque-forming unit/ml and tested ≈2 wk later to determine infection and transmission rates. The results indicated that four Culex mosquitoes were competent laboratory vectors of WNV. The infection rates and transmission rates were statistical differences among different species of mosquito (&khgr;2 = 20.620, P = 0.000; &khgr;2 = 15.020, P = 0.005, respectively). The highest infection rate and transmission rate were obtained with Cx. tritaeniorhynchus (87.5 and 74.2%, respectively).

Evaluation of lambda-cyhalothrin barrier spray on vegetation for control of Aedes albopictus in China.

Abstract Aedes albopictus is a primary nuisance pest and disease vector in China. Control of Ae. albopictus using current methods is difficult because of diverse larval breeding sites. In the present study, lambda-cyhalothrin was applied as a barrier spray to vegetation surrounding human dwellings for control of adult mosquito populations. Compared to the landing rate counts in the untreated control site, the barrier spray resulted in an 83–98% reduction of Ae. albopictus within 9 wk posttreatment. The study results indicate that lambda-cyhalothrin applied as a barrier spray to vegetation can reduce Ae. albopictus landing rates collections for more than 2 months.

Vector competence of Aedes albopictus and Aedes aegypti (Diptera: Culicidae) for DEN2-43 and New Guinea C virus strains of dengue 2 virus ☆

The vector competence of Aedes albopictus and Aedes aegypti with regard to DEN2-43 and New Guinea C (NGC) virus strains of Dengue 2 viruses was assessed and compared. The infection and dissemination rate and distribution of DEN2-43 antigens in orally infected Ae. albopictus was investigated using the reverse transcription polymerase chain reaction and an indirect immunofluorescence assay. To better understand the initial infection, dissemination and transmission of these viral strains in vector mosquitoes, Ae. albopoictus and Ae. aegypti were fed an artificial blood meal containing either the DEN2-43 or NGC strain. There was no significant difference in the infection and dissemination rates of DEN2-43 and NGC virus strains in Ae. albopictus, however, Ae. aegypti was more susceptible to infection by NGC than DEN2-43 vrius strain. Ae. albopictus mosquitoes infected with the NGC strain developed a higher percentage of midgut infections than those infected with the DEN2-43 strain (t=2.893, df=7, P=0.024). Approximately 26.7% of midgut samples were positive for the NGC antigen 5 days after infection, and 80% of mosquitoes had infected midgets after 15 days. The NGC antigen first became evident in mosquito salivary glands on Day 5, and 40% of mosquitoes had infected salivary by Day 9. In contrast, the DEN2-43 antigen first became evident in salivary glands on Day 7. The infection rate of NGC and DEN2-43 virus strains in salivary glands were similar. These results indicate that Ae. albopictus and Ae. aegypti are moderately competent vectors for the DEN2-43 virus, which could provide basic data for the epidemiology study of dengue fever in China.

Vector competence and transovarial transmission of two Aedes aegypti strains to Zika virus

Zika virus (ZIKV) has become a serious threat to global health since the outbreak in Brazil in 2015. Additional Chinese cases have continuously been reported since the first case of laboratory-confirmed ZIKV infection in China on 6 February 2016. Aedes aegypti is the most important vector for ZIKV. This study shows that two strains from China exhibit high levels of midgut infection and highly disseminated infection of salivary glands and ovaries. Both strains can transmit ZIKV to infant mice bitten by infectious mosquitoes. Moreover, the results provide the evidence of transovarial transmission of ZIKV in mosquitoes. The study indicates that the two Ae. aegypti strains are not only effective transmission vectors but also persistent survival hosts for ZIKV during unfavorable inter-epidemic periods. This function as a reservoir of infection has epidemiological implications that further enhance the risk of potential future outbreaks.