江西省口岸区域白纹伊蚊特异性病毒Aedes albopictus anphevirus基因组序列研究

目的昆虫特异性病毒在白纹伊蚊中与致病性传染病病原体如登革病毒、基孔肯雅病毒和寨卡病毒等的传播和复制有着密切联系,本文对其基因组序列进行研究,为研发新型虫媒病毒防治策略提供理论基础。方法本研究利用二代测序技术,对江西省3个口岸区域(南昌昌北机场、九江城西港和赣州陆港)采集的野外白纹伊蚊种群携带的昆虫特异性病毒进行高通量测序。结果3个白纹伊蚊种群中广泛存在昆虫特异性病毒Aedes albopictus anphevirus(AealbAV)。利用MEGAHIT软件对这些病毒reads进行组装,得到2株AealbAV全基因组序列,与美国株MW147277.1高度同源(相似度均大于98%),基因登录号分别为九江株OR715784,赣州株OR729834,南昌株仅拼接出部分序列。结论AealbAV在江西白纹伊蚊种群中普遍存在,其全基因组研究为口岸区域利用昆虫特异性病毒进行白纹伊蚊综合防治提供了理论基础。

Modelling the probability of presence of Aedes aegypti and Aedes albopictus in Iran until 2070

Objective:To determine the suitable ecological habitats of Aedes(Ae.)aegypti and Ae.albopictus in Iran due to climate change by the 2070s.Methods:All data relating to the spatial distribution of Ae.aegypti and Ae.albopictus worldwide,which indicated the geographical coordinates of the collection sites of these mosquitoes,were extracted from online scientific websites and entered into an Excel file.The effect of climatic and environmental variables on these mosquitoes was evaluated using the MaxEnt model in the current and future climatic conditions in the 2030s,2050s,and 2070s.Results:The most suitable areas for the establishment of Ae.aegypti are located in the southern and northern coastal areas of Iran,based on the model outputs.The modelling result for suitable ecological niches of Ae.albopictus shows that in the current climatic conditions,the southern half of Iran from east to west,and parts of the northern coasts are prone to the presence of this species.In the future,some regions,such as Gilan and Golestan provinces,will have more potential to exist/establish Ae.albopictus.Also,according to the different climate change scenarios,suitable habitats for this species will gradually change to the northwest and west of the country.The temperature of the wettest season of the year(Bio8)and average annual temperature(Bio1)were the most effective factors in predicting the model for Ae.aegypti and Ae.albopictus,respectively.Conclusions:It is required to focus on entomological studies using different collection methods in the vulnerable areas of Iran.The future modelling results can also be used for long-term planning to prevent the entry and establishment of these invasive Aedes vectors in the country.

Toxicity of several botanical essential oils and their combinations against females of Aedes albopictus(Skuse) and Anopheles minimus(Theobald): Oviposition deterrent, ovicidal and adulticidal efficacies

Objective: To investigate the efficacies of 12 essential oil(EO) formulations from three Zingiberaceae plants(Alpinia galanga, Curcuma zedoaria, and Zingiber cassumunar) individually and in combination with an augmenting Eucalyptus globulus(E. globulus) EO against females of Aedes albopictus(Ae. albopictus) and Anopheles minimus(An. minimus). Methods: These formulations were evaluated for their ovicidal, oviposition deterrent and adulticidal activities against Ae. albopictus and An. minimus by a topical method, a double-choice method and a WHO susceptibility test, respectively. Results: It was found that all formulations of Zingiberaceae plants EOs augmented with E. globulus EO were more effective in oviposition deterrent, ovicidal, and adulticidal activities against the two mosquito species than all of the formulations used without E. globulus EO. Their oviposition deterrent, ovicidal and adulticidal activities were equivalent to those of 10% w/v cypermethrin. In contrast, 70% v/v ethyl alcohol as a control alone was not effective at all. The highest synergistic effect in effective repellency against Ae. albopictus was achieved by 5% Alpinia galanga EO + 5% E. globulus EO and against An. minimus was 5% Zingiber cassumunar EO + 5% E. globulus EO. Moreover, the highest synergistic effects in ovicidal activities against Ae. albopictus and An. minimus were achieved by 10% Zingiber cassumunar EO + 10% E. globulus EO and 5% Curcuma zedoaria EO + 5% E. globulus EO, respectively. For the adulticidal activities, the highest synergistic effect against two mosquitoes was achieved by 5% Curcuma zedoaria EO + 5% E. globulus EO. Conclusions: These results suggest that Zingiberaceae plant EOs augmented with E. globulus EO have a high potential to be developed into oviposition deterrent, ovicidal, and adulticidal agents for controlling populations of Ae. albopictus and An. minimus.

Bio-Synthesis of Silver Nanoparticles from Leaf Extract of <em>Pongamia pinnata</em>as an Effective Larvicide on Dengue Vector <em>Aedes albopictus</em>(Skuse) (Diptera: Culicidae)

The frequent cases of dengue incidences are leading cause of illness and death in urban areas and Aedes albopictus (Skuse) is a primary vector for Dengue transmission in India. Synthesis of leaf mediated silver nanoparticles especially with Pongamia pinnata is a potential substitute for the existing organophosphorus insecticides like Tenophos, malathion and fenthion etc., for mosquito control programme. The nanoparticles were characterized by UV-visible absorption spectrum, X-Ray Diffraction (XRD), Fourier Transform Infrared (FTIR) and Transmission Electron Microscopy (TEM). Analysis of TEM showed that the synthesized silver nanoparticles are in spherical shape with average size of 20 nm. Further the XRD analysis confirms the nano-crystalline phase of silver with face centred cubic (FCC) crystal structure. GC-MS analysis elucidated the presence of two active ingredients, such as 9-Octadecenoic acid (Z) and n-hexadecanoic acid, which are the prominent substances considered as larvicide. Larvae were exposed to varying concentrations of plant extracts and synthesized silver nanoparticles for 24 hours. From the results, it is found that plant extracts showed moderate larvicidal effects but, the synthesized silver nanoparticles had found to be toxic to larvae at LC50 (0.25 ppm) and LC90 (1 ppm).

Role of Aedes aegypti and Aedes albopictus during the 2011 dengue fever epidemics in Hanoi, Vietnam

Objective: To record the human cases of dengue fever(DF) and investigate the Aedes mosquito species circulating during the Hanoi 2011 DF epidemics. Methods: 24 different outbreak points were recorded in 8 districts between August and December 2011. Results: 140 patients were hospitalized following dengue diagnostic with a predominance of males(59.3%) and the 15-34 age class. Only DENV-1(11.27%) and DENV-2(88.73%) serotypes were detected in human samples. Mosquito sampling performed in and around patients households revealed the predominance of Aedes aegypti(95.15%) versus Aedes albopictus(4.85%). There is a positive correlation between the population density of Aedes aegypti and the number of human cases and duration of outbreaks. Conclusions: This was not observed for Aedes albopictus. 3 pools of Aedes aegypti were positive with dengue virus, two with DENV-1 and one with DENV-2.

Larvicidal efficacy of crude and fractionated extracts of Dracaena loureiri Gagnep against Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus mosquito vectors

Objective:To evaluate the larvicidal efficacy of crude and fractionated extracts of Dracaena loureiri endocarp against Aedes aegypti,Aedes albopictus,Culex quinquefasciatus,and Anopheles minimus mosquitos.Methods:Larvicidal activity was tested according to World Health Organization standard protocol.The third-stage larvae of each mosquito species were exposed to various concentrations of Dracaena loureiri crude extract and six groups of Dracaena loureiri fractionated extracts(RC-DT 009-014).Larval mortality rates were observed after 24 h and48 h of exposure.Then,a computerized probit analysis of the mortality data was performed to determine lethal concentration 50(LC_(50))and lethal concentration 90 values.Results:Anopheles minimus larvae(24-h LC_(50)77.88 mg/L)had the highest susceptibility to crude extract,whereas others(Aedes aegypti,24-h LC_(50)224.73 mg/L;Aedes albopictus,24-h LC_(50)261.75 mg/L;and Culex quinquefasciatus,24-h LC_(50)282.86 mg/L)were significantly less susceptible.The most effective groups of fractionated extracts were RC-DT 012 and RC-DT 013.The mosquito species most susceptible to fractionated extracts was Culex quinquefasciatus,with 24-h LC_(50)values of 0.66 and 0.94 mg/L for RC-DT 012 and RC-DT 013,respectively.Conclusions:The larvicidal activity of fractionated extracts is more effective than that of crude extract against all tested mosquito species.For the most effective alternative larvicide,purification and a phytochemical constituent analysis must be performed.

The biology and demographic parameters of Aedes albopictus in northern peninsular Malaysia

Objective:To generate life table characteristics for the dengue vector Aedes albopictus(A. albopictus) under uncontrolled conditions,incorporating both the aquatic and the adult stages. Methods:Ten females derived from wild pupae were allowed to fully blood-feed on restrained mice.774 eggs were hatched in seasoned water.F1 larvae were followed for development until their F2 counterparts emerged as adults.Some population parameters were monitored(Fl) or estimated(F2).Results:A.albopictus exhibited increased fecundity and egg hatch success. Immature development was quick.Immature survival was high,with lowest rate in the pupal stage.Adult emergence was about 81%and sex ratio was close to 1:1.Generational mortality(K) was about 28%.A high proportion of females completed a reproductive cycle and the obtained parity rate was predicted to lead to higher fecundity in the next generation.Conclusios:It can be concluded that natural A.albopictus populations in Penang seem largely determined by quick development in combination with low immature loss and increased oviposition.

Larvicidal activities of hydro-ethanolic extracts of three Cameroonian medicinal plants against Aedes albopictus

Objective: To investigate the larvicidal activity of Syzygium guineense(Myrtaceae)(S. guineense), Monodora myristica and Zanthoxylum heitzii(Rutaceae)(Z. heitzii) extracts against Aedes albopictus(Ae. albopictus).Methods: The larvicidal activity of the hydro-ethanolic extracts from these plant species was assessed at three different concentrations(50, 100 and 200 mg/L) on first-instar of Ae. albopictus larvae in comparison with untreated controls. Mortality rate was recorded daily for a period of 12 days. The values of LC_(50) and lethal time killing 50% of the tested individuals(LT_(50)) were calculated using the log-probit analysis.Results: The root extract of S. guineense exhibited the best activity with 100% mortality after 8 days of treatment at 200 mg/L, followed by the fruit extract of Z. heitzii with83.33% mortality at the same concentration. Nonetheless, larvae were most susceptible to the fruit extract of Z. heitzii both in terms of LC_(50)(39.89 mg/L) and LT_(50)(145.68 h). A statistically significant difference between the control and the group treated at 200 mg/L was noticed in all the extracts.Conclusions: The present study shows that the hydro-ethanolic extracts of S. guineense,Monodora myristica and Z. heitzii tested have significant larvicidal activity. These preliminary results are of great interest and some of these plant species can be proposed for the formulation of new bioinsecticides to control Ae. albopictus populations.

Susceptibility of Aedes albopictus from dengue outbreak areas to temephos and Bacillus thuringiensis subsp.israelensis

Objective:To monitor the current duration of the application rates in vector programme and the level of Aedes albopictus larvae susceptibility from three selected areas in northeast district of Penang on two selected larvicides,temephos and Bacillus thuringiensis subsp.israelensis(Bti) which are commonly used by Penang Health Department for vector control.Methods:The mosquito larvae were tested against two types of larvicides:(1) temephos(Abate&#174;) with diagnostic dosage(0.012 mg/L) and operational dosage(1 mg/L) and(2)Bacillus thuringiensis subsp.israelensis(Vecto Bac&#174;WG) with operational dosage ranging from 6 000 international toxic units per L to 24 000 international toxic unit per L.A total of 20 late third and early forth instar larvae were selected and transferred into paper cup sized 300 m L using wide-mouthed pipette.The larvae were distributed into each 300 m L paper cup containing 50 m L of aged tap water.The experiment was replicated five times for each concentration tested.Each test was repeated three times.The mortality was recorded after 24 h of exposure and recorded lethal time was based on 2 h for temephos and 6 h for Bti.The control consisted of ethanol for temephos and only seasoned water for Bti.Results:The result showed that Aedes albopictus from Flat Hamna,Kampung Sungai Gelugor and Kampung Tanjung Tokong were still susceptible to Bti and temephos.However,higher lethal time and resistance ratio were detected in strain from Flat Hamna which was a known dengue hot spot area in northeast of Penang.Conclusions:The application of temephos and Bti in vector control activity in these selected localities is still relevant in the control of Aedes larvae populations.

Cloning and sequence analysis of β-actin gene from Aedes albopictus (Diptera: Culicidae)

Objective： To obtain the complete β-actin gene from Aedes albopictus. Methods： Total RNA was extracted from C6/36 cells. Degenerate primers were designed based on the β-actin sequences of An. gambiae, Ae. aegypti, Cx. pipiens pallens and D. melanogaster. By RT-PCR, the product was amplified, purified, cloned into the pGT vector and sequenced. The β-actin sequence was aligned and phylogenetically analyzed by the BLAST program and the CLUSTAL W program. Results： A sequence of 1132 bp including an open reading frame of 1131 bp was obtained （GenBank DQ657949）. The deduced protein had 376 amino acids. Aligned to SWISS-PROT, it exhibited a high level of identity with β-actins from Anopheles, Drosophila and Culex at the amino acid sequence level. Phylogenetic analysis indicated that Ae. albopictus β-acfin was much more homologous with invertebrate β- actin than with vertebrate β-actin. Conclusion： The gene may be used as the internal control in the experiments of Ae. albopictus.

Bioefficacy of crude extract of Cyperus aromaticus(Family:Cyperaceae)cultured cells,against Aedes aegypti and Aedes albopictus mosquitoes

Objective:To evaluate the growth inhibition activity of the crude extract of Cyperus aromaticus(C.aromaticus)cultured cells against the 3rd instar larvae of Aedes aegypti(Linn.)and Aedes albopictus Skuse(Ae.albopictus)under laboratory conditions,and determine the sublethal effects(EI_(50))of the crude extract of C.aromaticus cultured cells on some biological and morphological parameters of both Aedes mosquito species during two generations as well.Methods:The cell suspension cultures of C.aromaticus were activated from five callus lines(P4,Pa,Z1,Z6 and Ml)derived from the root explants of in vitro plantlets.The cultured cells were extracted in chloroform and used as plant material for the present study.For detection of juvenile hormone III.the crude extracts were analyzed by HPLC.Then the crude extracts of the three C.aromaticus cultured cell lines which contained varied amounts of juvenile hormone III[high level(P4 cell line),medium level(Z1 cell line)and low level(Ml cell line)]were tested against Aedes mosquito species.Laboratory evaluation was performed against late third instar larvae of the Vector Control Research Unit strains of Ae.aegypti and Ae.albopictus using the standard WHO method.The effects of EI_(50)of the C.aromaticus cultured P4 cells on fecundity,fertility,growth period,sex ratio,adult size and longevity of Aedes mosquitoes were assessed.Results:Bioassay tests presented the remarkable growth inhibition activity of the crude extracts of C.aromaticus cultured cells against the two Aedes mosquitoes.Between the two mosquito species,Ae.albopictus was more susceptible to the crude extracts with lower EI_(50)values.EI_(50)of the crude extract of C.aromaticus cultured cells(P4)increased the sterility indices in the parental generation females in both Aedes mosquito species.A significant delay in the pupal formation and adult emergence were observed in the parental generation of the both mosquito species.The sex ratio of the adult population either parental or F1 generation of the Aedes mosquito species was not significantly affected by the EI_(50)dosage of the crude extract of C.aromaticus cultured P4 cells.A significant decrease in the wing length of the treated adult(female and male)of Aedes aegypti as well as the treated female of Ae.albopictus were observed.Longevity of the adult female of the parental generation of both Aedes mosquitoes as well as females of F1 generation of Ae.albopictus were significantly decreased.Conclusions:The present study revealed the potential of the crude extract of C.aromaticus cultured cells in controlling vector mosquito populations in the effort to reduce the transmission of vector borne diseases.

Larvicidal activity of Xenorhabdus and Photorhabdus bacteria against Aedes aegypti and Aedes albopictus

Objective: To evaluate the efficacy of symbiotic bacteria, Xenorhabdus indica, Xenorhabdus stockiae, Photorhabdus luminescens subsp. akhurstii and Photorhabdus luminescens subsp. hainanensis as a larvicide against Aedes aegypti and Aedes albopictus. Methods: Larvae(L3-L4) of Aedes aegypti and Aedes albopictus were given 2 m L of a suspension 107-108 CFU/m L of each symbiotic bacterium. Distilled water and Escherichia coli ATCC襅25922 were used as the control. The mortality rate of the larval mosquitoes was observed at 24, 48, 72 and 96 h. The experiment was performed in triplicates. Results: The larvae of both Aedes species started to die at 24 h exposure. Aedes aegypti showed the highest mortality rate(87%-99%), 96 h after exposure to Xenorhabdus stockiae(b NBP22.2_TH). The mortality rate of Aedes albopictus was between 82% and 96% at 96 h after exposure to Xenorhabdus indica(b KK26.2_TH). Low effectiveness of distilled water and Escherichia coli ATCC襅25922 were observed in both Aedes larvae, with a mortality rate of 2% to 12%. Conclusions: The study confirms the oral toxicity of Xenorhabdus and Photorhabdus bacteria against Aedes spp. Xenorhabdus stockiae and Xenorhabdus indica may be an alternative agent for control Aedes spp. This is basic information for further study on the mechanism of action on Aedes larvae or application to control mosquito larvae in the community.

Modified technique of Wolbachia removal from Malaysian Aedes albopictus

Objective:To develop an artificial and modified Wolbaehia removal technique using tetracycline from naturally Wolbachia infected Aedes albopictus(Ae.albopictus)so as to be able to produce generations of Wolbaehia free offsprings.Methods:In this study,seven different tetracycline treatment methods were conducted to obtain the best removal method.Four methods focused on larvae tetracycline treatment,one method on both larvae and adult tetracycline treatment and the last two methods on adult mosquito sucrose treatment.Results:All larval tetracycline treatments resulted in either high larvae mortality,sterile F_o adult mosquitoes or unsuccessful Wolbaehia removal.Treatment of both larvae and adults resulted in reduced larvae mortality,successful Wolbachia removal but slow mosquito fecundity.As for the adult treatment,1.0 mg/mL as previously published was not aisle to completely remove Wolbaehia in F,generation whereas 1.25 mg/mL successfully removed Wolbachia from F,and F,mosquitoes in 2 weeks.Conclusions:This method is different from the previously published methods as it provides an improved Watbachia removal technique from Ae.albopictus with high egg hatchability.low larvae mortality,increased fecundity and better Wolbaehia removal rate.

四种植物提取物对白纹伊蚊(Aedes albopictus)驱避作用研究

研究了紫茎泽兰等四种植物提取物对白纹伊蚊成蚊的驱避活性,同时采用GC-MS定性分析了四种提取物的化学成分。结果发现在1.5 mg·cm^(-2)的涂抹剂量下,紫茎泽兰、南天竹果、南天竹叶以及竹叶花椒叶提取物对白纹伊蚊的100%保护时间分别为3.63 h、3.125 h、2.94 h和1.49 h。由此可知,白纹伊蚊成蚊驱避效果最好的是紫茎泽兰提取物,效果最差的是竹叶花椒叶提取物。研究结果表明,紫茎泽兰提取物对白纹伊蚊成蚊有较好的驱避作用,可作为一种驱蚊植物材料开发利用。

Insecticide resistance status and biochemical mechanisms involved in Aedes mosquitoes: A scoping review

Mosquitoes belonging to the genus Aedes pose a significant threat to human health on a global scenario due to their role in transmission of dengue,chikungunya,zika,and yellow fever.In absence of specific medications and vaccines against these diseases,disease prevention relies on vector control.However,in today’s world,vector control is facing major challenges due to the onset of insecticide resistance in mosquitoes.There are four main mechanisms of insecticide resistance,namely,behavioral resistance,reduced penetration/cuticular resistance,metabolic detoxification,and target site resistance;however,the latter two mechanisms have been studied widely in Aedes mosquitoes.Insecticide resistance in Aedes mosquitoes is widespread throughout the world.This review compiles the degree of insecticide resistance/susceptibility prevailing among different field populations of Aedes mosquitoes worldwide.In addition,the review has detailed the mechanisms providing the resistance phenomenon observed in nature in Aedes mosquitoes.

Climate change and Aedes albopictus risks in China:current impact and future projection

Background Future distribution of dengue risk is usually predicted based on predicted climate changes using general circulation models(GCMs).However,it is difficult to validate the GCM results and assess the uncertainty of the predictions.The observed changes in climate may be very different from the GCM results.We aim to utilize trends in observed climate dynamics to predict future risks of Aedes albopictus in China.Methods We collected Ae.albopictus surveillance data and observed climate records from 80 meteorological stations from 1970 to 2021.We analyzed the trends in climate change in China and made predictions on future climate for the years 2050 and 2080 based on trend analyses.We analyzed the relationship between climatic variables and the prevalence of Ae.albopictus in different months/seasons.We built a classification tree model(based on the average of 999 runs of classification and regression tree analyses)to predict the monthly/seasonal Ae.albopictus distribution based on the average climate from 1970 to 2000 and assessed the contributions of different climatic variables to the Ae.albopictus distribution.Using these models,we projected the future distributions of Ae.albopictus for 2050 and 2080.Results The study included Ae.albopictus surveillance from 259 sites in China found that winter to early spring(November–February)temperatures were strongly correlated with Ae.albopictus prevalence(prediction accuracy ranges 93.0–98.8%)—the higher the temperature the higher the prevalence,while precipitation in summer(June–September)was important predictor for Ae.albopictus prevalence.The machine learning tree models predicted the current prevalence of Ae.albopictus with high levels of agreement(accuracy>90%and Kappa agreement>80%for all 12 months).Overall,winter temperature contributed the most to Ae.albopictus distribution,followed by summer precipitation.An increase in temperature was observed from 1970 to 2021 in most places in China,and annual change rates varied substantially from-0.22℃/year to 0.58℃/year among sites,with the largest increase in temperature occurring from February to April(an annual increase of 1.4–4.7℃ in monthly mean,0.6–4.0℃ in monthly minimum,and 1.3–4.3℃ in monthly maximum temperature)and the smallest in November and December.Temperature increases were lower in the tropics/subtropics(1.5–2.3℃ from February–April)compared to the high-latitude areas(2.6–4.6℃ from February–April).The projected temperatures in 2050 and 2080 by this study were approximately 1–1.5℃ higher than those projected by GCMs.The estimated current Ae.albopictus risk distribution had a northern boundary of north-central China and the southern edge of northeastern China,with a risk period of June–September.The projected future Ae.albopictus risks in 2050 and 2080 cover nearly all of China,with an expanded risk period of April–October.The current at-risk population was estimated to be 960 million and the future at-risk population was projected to be 1.2 billion.Conclusions The magnitude of climate change in China is likely to surpass GCM predictions.Future dengue risks will expand to cover nearly all of China if current climate trends continue.

Susceptibility of Aedes flavopictus miyarai and Aedes galloisi mosquito species in Japan to dengue type 2 virus

Objective: To evaluate the potential of local mosquitoes to act as vectors for dengue transmission in Japan.Methods: Serotype 2 Th NH28/93 was used to test the dengue susceptibility profiles of Aedes flavopictus miyarai(Ae. f. miyarai), Aedes galloisi(Ae. galloisi) and Aedes albopictus(Ae.albopictus), which were collected in Japan. We used Aedes aegypti from Thailand as a positive control. The mosquitoes were infected with the virus intrathoracically or orally. At 10 or 14 days post infection, the mosquitoes were dissected and total RNA was extracted from their abdomens, thoraxes, heads and legs. Mosquito susceptibility to dengue virus was evaluated using RT-PCR with dengue virus-specific primers. Differences in the infection and mortality rates of the different mosquito species were tested using Fisher's exact probability test.Results: The infection rates for dengue virus administered intrathoracically to Ae. f. miyarai,Ae. galloisi and Aedes aegypti mosquitoes were identical by RT-PCR on Day 10 post infection.All of the body parts we tested were RT-PCR-positive for dengue virus. For the orally administered virus, the infection rates in the different body parts of the Ae. f. miyarai mosquitoes were slightly higher than those of Ae. albopictus mosquitoes, but were similar to the control mosquitoes(P > 0.05). The mortality rates for Ae. f. miyarai and Ae. albopictus mosquitoes were similar(P = 0.19). Our data indicated that dengue virus was able to replicate and disseminate to secondary infection sites in all of the four mosquito species(Japanese and Thai).Conclusions: Ae. albopictus is a well-known candidate for dengue transmission in Japan. However, our data suggest that Ae. f. miyarai from Ishigaki Island(near Okinawa Island) and Ae. galloisi from Hokkaido(Northern Japan) should also be regarded as potential vectors for dengue transmission in these regions. Further studies on these mosquitoes should be conducted.

Mosquito densovirus signifcantly reduces the vector susceptibility to dengue virus serotype 2 in Aedes albopictus mosquitoes(Diptera:Culicidae)

Background Dengue virus(DENV)is a major public health threat,with Aedes albopictus being the confrmed vector responsible for dengue epidemics in Guangzhou,China.Mosquito densoviruses(MDVs)are pathogenic mosquitospecifc viruses,and a novel MDV was previously isolated from Ae.albopictus in Guangzhou.This study aims to determine the prevalence of MDVs in wild Ae.albopictus populations and investigate their potential interactions with DENV and impact on vector susceptibility for DENV.Methods The prevalence of MDV in wild mosquitoes in China was investigated using open access sequencing data and PCR detection in Ae.albopictus in Guangzhou.The viral infection rate and titers in MDV-persistent C6/36 cells were evaluated at 12,24,48,72,96,and 120 h post infection(hpi)by indirect immunofuorescence assay(IFA)and real time quantitative PCR(RT-qPCR).The midgut infection rate(MIR),dissemination rate(DR),and salivary gland infection rate(SGIR)in various tissues of MDV-infected mosquitoes were detected and quantifed at 0,5,10,and 15 days post infection(dpi)by RT-PCR and RT-qPCR.The chi-square test evaluated dengue virus serotype 2(DENV-2)and Aedes aegypti densovirus(AaeDV)infection rates and related indices in mosquitoes,while Tukey’s LSD and t-tests compared viral titers in C6/36 cells and tissues over time.Results The results revealed a relatively wide distribution of MDVs in Aedes,Culex,and Anopheles mosquitoes in China and an over 68%positive rate.In vitro,signifcant reductions in DENV-2 titers in supernatant at 120 hpi,and an appar‑ent decrease in DENV-2-positive cells at 96 and 120 hpi were observed.In vivo,DENV-2 in the ovaries and salivary glands was frst detected at 10 dpi in both monoinfected and superinfected Ae.albopictus females,while MDV super‑infection with DENV-2 suppressed the salivary gland infection rate at 15 dpi.DENV-2 titer in the ovary and salivary glands of Ae.albopictus was reduced in superinfected mosquitoes at 15 dpi.Conclusions MDVs is widespread in natural mosquito populations,and replication of DENV-2 is suppressed in MDVinfected Ae.albopictus,thus reducing vector susceptibility to DENV-2.Our study supports the hypothesis that MDVs may contribute to reducing transmission of DENV and provides an alternative strategy for mosquito-transmitted disease control.

Genetic structure of Aedes albopictus(Diptera:Culicidae)populations in China and relationship with the knockdown resistance mutations

Background Mosquito control is needed to prevent dengue fever, which is mainly spread byAedes albopictus in China. Application of insecticides is one of the main mosquito control methods;however, this approach can fail due to the knockdown resistance (kdr) gene mutation that causes decreased sensitivity to insecticides inAe. albopictus. Thekdr mutation patterns among different regions in China differ significantly. However, the underlying mechanism and factors that influencekdr mutation remain unclear. To explore the potential influence of genetic background on the development of insecticide resistance inAe. albopictus, we analyzed the genetic structure ofAe. albopictus populations in China and its correlation with majorkdr mutations.Methods We collectedAe. albopictus from 17 sites in 11 provinces (municipalities) across China from 2016 to 2021 and extracted the genomic DNA from individual adult mosquitoes. We selected eight microsatellite loci for genotyping, and based on microsatellite scores, we estimated intraspecific genetic diversity, population structure, and effective population size. The association between the intrapopulation genetic variation and F1534 mutation rate was evaluated by the Pearson correlation coefficient.Results Based on variation analysis of the microsatellite loci of 453 mosquitoes representing 17 populations throughout China, more than 90% of the variation occurred within individuals, whereas only about 9% of the variation occurred among populations, indicating that field populations ofAe. albopictus are highly polymorphic. The northern populations tended to belong to gene pool I (BJFT 60.4%, SXXA 58.4%, SDJN 56.1%, SXYC 46.8%), the eastern populations tended to belong to pool III (SH 49.5%, JZHZ 48.1%), and the southern populations tended to belong to three different gene pools. Moreover, we observed that the greater the fixation index (FST), the lower the wild-type frequency of F1534 ofVSGC.Conclusions The degree of genetic differentiation amongAe. albopictus populations in China was low. These populations were divided into three gene pools, in which the northern and eastern pools are relatively homogeneous, while the southern gene pool is heterogeneous. The possible correlation between its genetic variations andkdr mutations is also noteworthy.

A novel strategy for screening mutations in the voltage-gated sodium channel gene of Aedes albopictus based on multiplex PCR-mass spectrometry minisequencing technology

Background The current prevention and control strategy forAedes albopictus heavily relies on comprehensive management, such as environmental management and chemical control. However, the wide application of pyrethroids has facilitated the development of insecticide resistance, primarily via mutations in the voltage-gated sodium channel (VGSC) gene. This study aims to develop a novel strategy for detecting mutations in the VGSC gene inAe. albopictus using multiplex PCR-mass spectrometry (MPCR-MS) minisequencing technology.Methods We established a new strategy for detecting mutations in the VGSC gene inAe. albopictus using MPCR-MS minisequencing technology. MPCR amplification and mass probe extension (MPE) were first used, followed by single nucleotide polymorphism (SNP) typing mass spectrometry, which allows the simultaneous detection of multiple mutation sites of the VGSC gene in 96 samples ofAe. albopictus. A total of 70 wild-collectedAe. albopictus were used to evaluate the performance of the method by comparing it with other methods.Results Three target sites (1016, 1532, 1534) in the VGSC gene can be detected simultaneously by double PCR amplification combined with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, achieving a detection limit of 20 fg/μl. We applied this method to 70 wild-collectedAe. albopictus, and the obtained genotypes were consistent with the routine sequencing results, suggesting the accuracy of our method.Conclusions MPCR-MS minisequencing technology provides a sensitive and high-throughput approach toAe. albopictus VGSC gene mutation screening. Compared with conventional sequencing, this method is economical and time-saving. It is of great value for insecticide resistance surveillance in areas with a high risk of vector-borne disease.