Oviposition attractants can enhance both the surveillance and control of container-breeding Aedes mosquitoes. Although green tea has been reported to attract Ae. aegypti, the specific bioactive botanical compounds responsible remain unidentified. We evaluated teas across fermentation stages (non-fermented, fully fermented, and post-fermented) at 5 g/L for gravid Ae. albopictus using dual-choice assays. The most attractive tea (Liu-pao) was further tested across concentrations (0.5–5 g/L) and infusion ages (1–28 days). Autoclaved vs. non-autoclaved infusions assessed microbial contributions, while headspace volatiles were analyzed via HS-SPME-GC-MS. Individual compounds (cedrol and linalool oxide) were bioassayed. Only Liu-pao tea consistently attracted Ae. albopictus (OAI ≥ 0.3), peaking at 5 g/L after 7–14 days (OAIs 0.73/0.67). A 0.5 g/L infusion aged 21–28 days also attracted Ae. aegypti (OAIs 0.89/0.63). Autoclaving did not reduce attraction, confirming volatile compounds, not live microbe-mediated effects. Cedrol (1 mg/L) elicited moderate attraction (OAI 0.29) in Ae. albopictus, while linalool oxide was inactive. Post-fermented Liu-pao tea acts as a potent botanical attractant for Aedes mosquitoes. Cedrol contributes partially, but whole tea infusions outperform single compounds. These findings support developing multi-volatile “attract-and-kill” strategies targeting gravid mosquitoes.

Background Oviposition site selection is an important component of vector mosquito reproductive biology. The Asian Tiger mosquito, Aedes albopictus, is a major and important vector of arboviruses including Dengue. Previous studies documented the preference of gravid females for small, dark-colored water containers as oviposition sites, which they sought during the twilight period (dusk) of their locomotor activity. Vision plays an important role in this behavior, and factors such as the shape, size, and color of the container, light intensity, polarization, spectrum, and other visual cues guide the search for suitable oviposition sites, but the mechanistic factors driving this behavior are unclear. Methodology/Principal findings We blindfolded adult female compound eyes and observed the effects of a lack of vision on the ability to discriminate and utilize preferred oviposition sites. Furthermore, the transcriptomes of blindfolded mosquitoes were screened to identify genes with vision-sensitive expression profiles and gene-editing was used to create non-functional mutations in two of them, rhodopsin-like (mutation designated ‘rho-l△807’) and kynurenine hydroxylase (mutation designated ‘khw’). Behavioral tests of both mutant and control strains revealed that the rho-l△807 mutant mosquitoes had a significant decrease in their ability to search for preferred oviposition sites that correlated with a reduced ability to recognize long-wavelength red light. The khw mutant mosquitoes also had a reduced ability to identify preferred oviposition sites that correlated with reductions in their ability to respond to variations in daily brightness and their ability to discriminate among different color options of the containers and background monochromatic light. Conclusions/Significance This study underscores the importance of visual cues in the oviposition site selection behavior of adult female Ae. albopictus. We demonstrate that wild-type rho-l and kh gene products play a crucial role in this behavior, as mutants exhibit altered sensitivity or recognition of light intensity and substrate colors.

IntroductionAedes albopictus is an aggressive invasive mosquito species widely distributed around the world, and it is also a known vector of arboviruses. Virus metagenomics and RNA interference (RNAi) are important in studying the biology and antiviral defense of Ae. albopictus. However, the virome and potential transmission of plant viruses by Ae. albopictus remain understudied. MethodsMosquito samples of Ae. albopictus were collected from Guangzhou, China, and small RNA sequencing was performed. Raw data were filtered, and virus-associated contigs were generated using VirusDetect. The small RNA profiles were analyzed, and maximum-likelihood phylogenetic trees were constructed. ResultsThe small RNA sequencing of pooled Ae. albopictus revealed the presence of five known viruses, including Wenzhou sobemo-like virus 4, mosquito nodavirus, Aedes flavivirus, Hubei chryso-like virus 1, and Tobacco rattle virus RNA1. Additionally, 21 new viruses that had not been previously reported were identified. The mapping of reads and contig assembly provided insights into the viral diversity and genomic characteristics of these viruses. Field survey confirmed the detection of the identified viruses in Ae. albopictus collected from Guangzhou. DiscussionThe comprehensive analysis of the virus metagenomics of Ae. albopictus in this study sheds light on the diversity and prevalence of viruses in mosquito populations. The presence of known and novel viruses highlights the need for continued surveillance and investigation into their potential impact on public health. The findings also emphasize the importance of understanding the virome and potential transmission of plant viruses by Ae. albopictus. ConclusionThis study provides valuable insights into the virome of Ae. albopictus and its potential role as a vector for both known and novel viruses. Further research is needed to expand the sample size, explore additional viruses, and investigate the implications for public health.

The initial signals governing sex determination vary widely among insects. Here we show that Armigeres subalbatus M factor (AsuMf), a male-specific duplication of an autosomal gene of the Drosophila behaviour/human splicing (DBHS) gene family, is the potential primary signal for sex determination in the human filariasis vector mosquito, Ar. subalbatus. Our results show that AsuMf satisfies two fundamental requirements of an M factor: male-specific expression and early embryonic expression. Ablations of AsuMf result in a shift from male- to female-specific splicing of doublesex and fruitless, leading to feminization of males both in morphology and general transcription profile. These data support the conclusion that AsuMf is essential for male development in Ar. subalbatus and reveal a male-determining factor that is derived from duplication and subsequent neofunctionalization of a member of the conserved DBHS family.

Aedes albopictus is one of the most invasive insect species in the world and an effective vector for many important arboviruses. We reported previously that Ae . albopictus Nix ( AalNix ) is the male-determining factor of this species. However, whether AalNix alone is sufficient to initiate male development is unknown. Transgenic lines that express each of the three AalNix isoforms from the native promoter were obtained using piggyBac transformation. We verified the stable expression of AalNix isoforms in the transgenic lines and confirm that one isoform, AalNix3&4 , is sufficient to convert females into fertile males (pseudo-males) that are indistinguishable from wild-type males. We also established a stable sex-converted female mosquito strain, AalNix3&4 -♂4-pseudo-male. The pseudo-male mosquitoes can fly and mate normally with wild-type female, although their mating competitiveness is lower than wild-type. This work further clarifies the role of AalNix in the sex determination pathway and will facilitate the development of Ae . albopictus control strategies that rely on male-only releases such as SIT and sex-ratio distortion.

Zika virus (ZIKV) may cause severe microcephaly in newborn babies and Guillain-Barré syndrome in some adults. In recent decades, its range has expanded in 86 countries. There are two ecologically and evolutionarily district cycles: urban cycle and sylvatic cycle. This work aimed to estimate the urban and sylvatic cycle areas of ZIKV throughout the world. The occurrence records of vectors, non-human primate hosts, and ZIKV were collected. We chose historical climate data, predicted vectors distribution, human population density, and elevation data as the variables to fit the maximum entropy model (MaxEnt). Current risk area and future prediction were performed with global climate models (GCMs) and shared socioeconomic pathways (SSPs). Predicting the ZIKV risk area would help tailor related control strategies. The results indicated that 16.6% of the world’s landmass (except Antarctica) is a risk area in the urban cycle. Approximately 6.22 billion people (78.69% of the global population) live in the risk area, with the vast majority in South Asia, tropical Africa, South America, North America, and countries around the Mediterranean Sea. Future climate change decreases the risk area of ZIKV. This study also suggested that the sylvatic cycle happened between the Tropic of Cancer and the Tropic of Capricorn. The overlap region of the urban and sylvatic cycles could be hotpots that ZIKV spill from the sylvatic to the urban cycle. It is indicated that long-term passenger screening, mosquito surveillance, and control are necessary.

Background Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses primarily transmitted by Aedes mosquitoes. Armigeres subalbatus is an emerging and widely distributed mosquito, and ZIKV has been detected and isolated from it. However, it is not clear whether Ar. s ubalbatus could be a vector for ZIKV and DENV or not. In this study, we investigated the infection and transmission of Ar. subalbatus to ZIKV and DENV. Methods A line of Ar. subalbatus was isolated from Guangdong, China, and further identified by the mitochondrial cytochrome oxidase subunit 1 ( COI ) gene. The adults of Ar. subalbatus were fed with blood meal containing ZIKV or DENV-2. At 4, 7, 10, 14, and 21 days post-inoculation (dpi), the infections of ZIKV or DENV-2 in the midguts, ovaries and salivary glands were detected and quantified by RT-PCR and RT-qPCR. To assess the transmissibility, suckling mice were exposed to bites of ZIKV-infected mosquitoes, and ZIKV was detected in brain tissue by RT-qPCR and plaque assays. Furthermore, the larvae of Ar. subalbatus were reared in artificial urine containing ZIKV or DENV-2. The infection rates and viral titers of larvae and adults were analyzed by RT-PCR and RT-qPCR, and the viral distribution in larval tissues was observed by immunohistochemistry. Chi-square test and one-way ANOVA analysis were used for assessing the infection rate and viral titer in varied tissues and different time points, respectively. Results Following oral inoculation, ZIKV but not DENV-2 could be detected in Ar. subalbatus midguts at 4 dpi, ovaries at 7 dpi and salivary glands at 10 dpi. The highest infection rate (IR) of ZIKV was 27.8% in midgut at 7 dpi, 9.7% in ovary and 5.6% in salivary gland at 21 dpi. Eight days after being bitten by ZIKV-positive mosquitoes, ZIKV was detected in three brain tissues out of four suckling mice exposed to bites. ZIKV could be detected in the larvae reared in artificial urine contained ZIKV at a high concentration of 10 5 pfu/ml and various tissues of adults with a low infection rate (0.70–1.35%). ZIKV could be observed in anal papillae and midgut of larvae at 4 dpi under laboratory conditions. Conclusions ZIKV but not DENV-2 can infect Ar. subalbatus by blood meal and artificial urine, and the infected mosquitoes can transmit ZIKV to suckling mice by bite. From these findings, we can conclude that the Ar. subalbatus isolated from Guangdong province, China, is a potential vector for ZIKV and should therefore be considered in vector control programs to prevent and control of Zika virus disease. Graphical Abstract

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.

蚊虫是一类重要的媒介生物,雌蚊可以通过吸血传播多种疾病,如疟疾、登革热、流行性乙型脑炎等.蚊虫嗅觉发生和雌雄性别的决定对蚊虫的生存繁衍、疾病传播至关重要,往往也是控制蚊虫种群密度、防治蚊媒传染病的关键所在.本文综述了媒介蚊虫的嗅觉发生和性别决定的基本功能、作用机制及研究进展,以期为蚊虫种群密度控制、蚊媒传染病防治等提供参考依据.

BACKGROUND Aedes albopictus is the primary vector of mosquito-borne diseases, including dengue and chikungunya, in China. The management of vector mosquitoes is the primary strategy for the control of such infectious diseases. The gravid Ae. albopictus prefers to skip-oviposit its eggs into different small water containers, and the management of these breeding places is critical for mosquito control. Bacillus thuringiensis subspecies. israelensis (Bti) is a useful biological larvicide, but the effective period of the currently available commercial product is relatively short. This study aimed to develop a long-lasting formulation of Bti to control the dengue vector mosquito Ae. albopictus. RESULTS Water-soluble polyethylene glycols and water-insoluble hexadecanol were mixed with Bti to develop the long-lasting formulation, Bti-BLOCK, based on the solid dispersion technique. The controlled release of Bti-BLOCK and its effect on Ae. albopictus were assayed in the laboratory and in the field. The results showed that Bti toxins were slowly released from Bti-BLOCK into the water and maintained at an effective dose for at least six months. Bti-BLOCK caused high mortality during the immature stage (>90%) and achieved full inhibition during pupation (100%), and the efficacy lasted at least twelve weeks in the laboratory and six weeks in the field. Furthermore, we confirmed an 89% reduction in Ae. albopictus density and reduction in the R0 of dengue to a low-risk level after six months of open-field interventions. CONCLUSIONS We developed a long-lasting biological larvicide, Bti-BLOCK, which displayed very good efficacy in the control of the dengue vector mosquito Ae. albopictus. This article is protected by copyright. All rights reserved.

Background: The use of oviposition attractants is a promising approach to control populations of mosquito vectors at the source; however, the knowledge on oviposition stimulation in Aedes albopictus is very limited. In recent years, tea bags have been proven to attract Aedes aegypti to lay eggs, but the sources of tea are wide and varied; moreover, the factors and mechanisms in tea bags that attract mosquitoes remain unclear. Methods : The effects of three kinds of Chinese tea, nonfermented, fully fermented and postfermented, were comparatively evaluated for their ability to attract mosquito oviposition at dose of 5g/L, 1.5g/L and 0.5g/L, respectively. The attractants in the Liu-pao tea infusion were isolated with gas chromatography–mass spectrometry (GC-MS). Results : Among the three types of fermented teas, the Guangxi Liu-pao tea infusion was a successful oviposition attractant for Ae. albopictus at all the dose tested, and the average OAIs of 5 g/L Liu-pao tea soaked for 7 d and 14 d were 0.73 and 0.67, respectively. Further, in one month of soaking 0.5g/L Liu-Pao tea, the average oviposition activity indexes (OAIs) for Ae.aegypti of soaking for 21 d and 28 d were 0.89 and 0.63, respectively. Functional analyses showed that the compound cedrol at 1 mg/L tested for Ae. albopictus , the average OAI was 0.38. Conclusion: Liu-pao tea can effectively attract Ae. albopictus to lay eggs, and cedrol is one of the ingredients attracting Aedes mosquitoes to lay eggs. These results shed light on the development of Aedes mosquito oviposition attractants and will help to develop innovative monitoring and prevention strategies for Aedes -borne diseases.

近年来,随着高通量测序技术和生物信息学分析技术的成熟和不断革新,蚊虫基因组学、转录组学、小RNA组学也获得了快速发展.迄今为止,已有包括白纹伊蚊、埃及伊蚊、冈比按蚊在内约22种媒介蚊虫的基因组被解析报道.不同蚊种的基因组大小差异很大,且与基因组中的重复序列的多少呈正相关;蚊基因组的解析和比较基因组的分析有助于探索蚊基因组的结构和功能;转录组的研究为蚊虫嗅觉、性别决定、胚胎发育等相关基因的研究提供了有效手段;小RNA组的研究揭示了miRNA和piRNA在蚊媒抗病毒免疫通路中具有重要作用.综上所述,蚊虫组学研究为防治媒介蚊虫和蚊媒传染病病提供了理论基础和数据支撑.

BACKGROUND:The Asian tiger mosquito, Aedes albopictus, an increasingly relevant arboviral vector, has spread worldwide. However, currently available tools are limited in terms of effective monitoring of vector populations and accurate determination of the extent of viral transmission, especially before and during outbreaks. Therefore, it is essential to develop novel monitoring and surveillance tools, particularly those that target adult mosquitoes and enhance the trapping efficiency for Ae. albopictus.METHODS:A variety of human body odorants associated with different types of mosquito olfactory receptors were selected, and their attractiveness to Ae. albopictus was tested by a four-arm olfactometer. The optimal compatibility and proportion of the odorants, Mix-5, was observed via orthogonal design analyses. The attractiveness of Mix-5 to Ae. albopictus in the laboratory was assessed using Mosq-ovitraps and Electric Mosquito Killers. In the field, the effectiveness of generic BG-Lure, Mix-5 and a control treatment was compared with a baited Biogents Sentinel trap (BGS-trap) using a Latin square design.RESULTS:In the olfactometer experiments, the attractiveness of the selected candidate compounds at varying dilutions was poor when the individual compounds were used alone. The optimal combination, Mix-5, was generated based on orthogonal design analyses. In the laboratory, the average numbers of female Ae. albopictus mosquitoes attracted by the synthetic odorant blend Mix-5 were 27.00 and 27.50, compared with 12.00 and 14.83 for the control, when using Mosq-ovitraps and Electric Mosquito Killers, respectively. In the field, the average number of Ae. albopictus female mosquitoes trapped by Mix-5 was 9.67 females/trap, whereas the average numbers for BG-Lure and the control were 7.78 and 4.47, respectively. The lure also played an important role in attracting Culex quinquefasciatus mosquitoes, and the average numbers of Cx. quinquefasciatus female mosquitoes attracted by Mix-5, BG-Lure and the control were 18.78, 25.11 and 12.22, respectively.CONCLUSIONS:A human odor-based bait blend was developed and exhibited enhanced effectiveness at attracting Ae. albopictus This blend can be used to monitor and trap dengue vector mosquitoes in Chinese cities.

Interactions between bacterial microbiota and mosquitoes play an important role in mosquitoes' capacity to transmit pathogens. However, microbiota assemblages within mosquitoes and the impact of microbiota in environments on mosquito development and survival remain unclear. This study examined microbiota assemblages and the effects of aquatic environment microbiota on the larval development of the Aedes albopictus mosquito, an important dengue virus vector. Life table studies have found that reducing bacterial load in natural aquatic habitats through water filtering and treatment with antibiotics significantly reduced the larva-to-adult emergence rate. This finding was consistent in two types of larval habitats examined-discarded tires and flowerpots, suggesting that bacteria play a crucial role in larval development. Pyrosequencing of the bacterial 16S rRNA gene was used to determine the diversity of bacterial communities in larval habitats and the resulting numbers of mosquitoes under both laboratory and field conditions. The microbiota profiling identified common shared bacteria among samples from different years; further studies are needed to determine whether these bacteria represent a core microbiota. The highest microbiota diversity was found in aquatic habitats, followed by mosquito larvae, and the lowest in adult mosquitoes. Mosquito larvae ingested their bacterial microbiota and nutrients from aquatic habitats of high microbiota diversity. Taken together, the results support the observation that Ae. albopictus larvae are able to utilize diverse bacteria from aquatic habitats and that live bacteria from aquatic habitats play an important role in larval mosquito development and survival. These findings provide new insights into bacteria's role in mosquito larval ecology.

Mosquito-borne viral diseases represent a major challenge to human public health. As natural vectors of arboviruses, mosquitoes can be infected by a virus, but they have evolved multiple mechanisms to tolerate constant infection and restrict viral replication via their antiviral immune system. In a state of continuous infection, a mosquito can transmit an arbovirus while obtaining a blood meal from a mammalian host. During infection, the virus is mainly inhibited through a small RNA-mediated interference mechanism. Within mosquitoes, the invaded viruses are recognized based on pathogen-associated molecular patterns, leading to the production of cytokines. These cytokines in turn bind pattern recognition receptors and activate Toll, IMD and other immune signalling pathways to expand the immune response and induce antiviral activity via immune effectors. Interestingly, the gut microbiota and Wolbachia also play a role in mosquito antiviral immunity, which is very similar to acquired immunity. This review describes the advances made in understanding various aspects of mosquito antiviral immune molecular mechanisms in detail and explores some of the unresolved issues related to the mosquito immune system.

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in G1 pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used in Ae. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.

BACKGROUND:Aedes albopictus is a globally invasive mosquito and a major vector of arboviruses, including dengue, Zika and Chikungunya. Olfactory-related behaviors, particularly host-seeking, offer opportunities to disrupt the disease-transmission process. A better understanding of odorant receptors (ORs) may assist in explaining host selection and location, and contribute to novel strategy of vector control.METHODS:Based on previous prediction of 158 putative odorant receptors by Ae. albopictus genome analysis, 29 AalORs were selected for tissue-specific expression profiles in the present study. AalOrco (AalOR7), AalOR10 and AalOR88, highly expressed in female olfactory tissues, were chosen for further structure predictions as well as functional validation including calcium imaging assay in human embryonic kidney (HEK293) cells and RNA interference assay in Ae. albopictus. We also conducted electrophysiological and behavioral assays in mosquitoes after RNA interference of the three genes to determine their roles in host-seeking.RESULTS:The results support previous conclusions that individual conventional (ORXs) and Orco can form heteromeric complexes to recognize odorants and respond to components of human volatiles in HEK293 cells. The reduction of AalOrco transcript levels led to a significant decrease in host-seeking and confusion in host preference. In contrast, AalOR10 and AalOR88 knockdown mosquitoes showed no significant behavioral differences compared with controls. The functions of conventional ORs at least AalOR10 and AalOR88 are abolished with inhibited expression of the Orco gene orthologs, along with the concomitant relevant olfactory behavior.CONCLUSIONS:Combining structural and functional data, we conclude that the product of the Orco gene in this mosquito is crucial for transmitting olfactory signaling and conventional ORs contribute directly to odorant recognition. Our results provide insight into the linkage between odorant receptors and host-seeking in this important vector species.

OBJECTIVE:To clone and identify olfactory receptor odorant receptor 7 (OR7) gene of Aedes albopictus and analyze its expression profile and calcium regulation function.METHOD:RT-PCR was used to amplify the olfactory receptor OR7 gene of Ae. albopictus and OR7 expression was detected in different tissues and organs. The coding sequence of OR7 gene was cloned in eukaryotic expression vector pME18s, which was then transfected into HEK293 cells. The calcium callback function in response to odor molecule stimulation was analyzed by calcium imaging technique.RESULTS:The OR7 gene of Ae. albopictus was cloned and sequence analysis showed that its coding region was 1395 bp. RT-PCR detected OR7 expression in the larvae, pupae and adult mosquitoes, especially in female mosquitos. Preliminary analysis of calcium callback function demonstrated the specific regulation of calcium absorption by OR7 in response to odor molecule stimulation.CONCLUSION:The OR7 gene of Ae. albopictus has been cloned successfully. OR7 is highly expressed in female mosquitos and is capable of specific recognition of the odor molecules.