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:Mosquitoes with aggressive biting behavior are important disease vectors threatening public health. Armigeres subalbatus, as an emerging arbovirus and filarial disease vector, exhibits aggressive host-seeking behavior and unique breeding preference for contaminated water. However, the molecular mechanisms underlying these biological characteristics remain poorly understood. This study aimed to generate a high-quality genome assembly and characterize the genetic basis of vector competence and environmental adaptation in Ar. subalbatus. METHODS:We sequenced and assembled the Ar. subalbatus genome using Oxford Nanopore long-read sequencing, Illumina short-read sequencing, and Hi-C technology. Comparative genomic analysis was performed to identify gene families related to detoxification, diapause, innate immunity, and sex determination. Gene structure analysis focused on the male-determining factor and its evolutionary relationships with other mosquito vectors. RESULTS:The genome assembly consists of three chromosomes, with a total size of 1.33 Gbp and an N50 of 430.15 Mbp (GenBank assembly: GCA_024139115.2), displaying 99.4% Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness. We identified the gene structure of the male-determining factor (AsuMf) and characterized its evolutionary relationship with other mosquito vectors. The analysis revealed expanded detoxification-related gene families including cytochrome P450s, which may facilitate adaptation to contaminated breeding sites. We characterized 566 putative diapause-related genes that could potentially contribute to geographical expansion, 334 innate immune genes, and 1673 endogenous viral elements, indicating complex virus-host interactions throughout evolution. CONCLUSIONS:Our study provides insights into the molecular basis of vector competence and adaptation in Ar. subalbatus. The expanded detoxification gene families may enable the species to survive in polluted environments, while the identified diapause-related genes could explain its geographical expansion capabilities. These findings establish a foundation for developing novel vector control strategies targeting this emerging disease vector.

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.

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.

Mosquito densoviruses (MDVs) are mosquito-specific viruses that are recommended as mosquito bio-control agents. The MDV Aedes aegypti densovirus (AeDNV) is a good candidate for controlling mosquitoes. However, the slow activity restricts their widespread use for vector control. In this study, we introduced the Bacillus thuringiensis (Bti) toxin Cry11Aa domain II loop α8 and Cyt1Aa loop β6-αE peptides into the AeDNV genome to improve its mosquitocidal efficiency; protein expression was confirmed using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Recombinant plasmids were transfected into mosquito C6/36 cell lines, and the expression of specific peptides was detected through RT-PCR. A toxicity bioassay against the first instar Aedes albopictus larvae revealed that the pathogenic activity of recombinant AeDNV was significantly higher and faster than the wild-type (wt) viruses, and mortality increased in a dose-dependent manner. The recombinant viruses were genetically stable and displayed growth phenotype and virus proliferation ability, similar to wild-type AeDNV. Our novel results offer further insights by combining two mosquitocidal pathogens to improve viral toxicity for mosquito control.

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

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 Aedes albopictus is one of the most invasive species in the world as well as the important vector for mosquito-borne diseases such as dengue fever, chikungunya fever and zika virus disease. Chemical control of mosquitoes is an effective method to control mosquito-borne diseases, however, the wide and improper application of insecticides for vector control has led to serious resistance problems. At present, there have been many reports on the resistance to pyrethroid insecticides in vector mosquitoes including deltamethrin to Aedes albopictus . However, the fitness cost and vector competence of deltamethrin resistant Aedes albopictus remain unknown. To understand the impact of insecticide resistant mosquito is of great significance for the prevention and control mosquitoes and mosquito-borne diseases. Methodology/Principal findings A laboratory resistant strain (Lab-R) of Aedes albopictus was established by deltamethrin insecticide selecting from the laboratory susceptible strain (Lab-S). The life table between the two strains were comparatively analyzed. The average development time of Lab-R and Lab-S in larvae was 9.7 days and 8.2 days ( P < 0.005), and in pupae was 2.0 days and 1.8 days respectively ( P > 0.05), indicating that deltamethrin resistance prolongs the larval development time of resistant mosquitoes. The average survival time of resistant adults was significantly shorter than that of susceptible adults, while the body weight of resistant female adults was significantly higher than that of the susceptible females. We also compared the vector competence for dengue virus type-2 (DENV-2) between the two strains via RT-qPCR. Considering the results of infection rate (IR) and virus load, there was no difference between the two strains during the early period of infection (4, 7, 10 day post infection (dpi)). However, in the later period of infection (14 dpi), IR and virus load in heads, salivary glands and ovaries of the resistant mosquitoes were significantly lower than those of the susceptible strain (IR of heads, salivary glands and ovaries: P < 0.05; virus load in heads and salivary glands: P < 0.05; virus load in ovaries: P < 0.001). And then, fourteen days after the DENV-2-infectious blood meal, females of the susceptible and resistant strains were allow to bite 5-day-old suckling mice. Both stains of mosquito can transmit DENV-2 to mice, but the onset of viremia was later in the mice biting by resistant group as well as lower virus copies in serum and brains, suggesting that the horizontal transmission of the resistant strain is lower than the susceptible strain. Meanwhile, we also detected IR of egg pools of the two strains on 14 dpi and found that the resistant strain were less capable of vertical transmission than susceptible mosquitoes. In addition, the average survival time of the resistant females infected with DENV-2 was 16 days, which was the shortest among the four groups of female mosquitoes, suggesting that deltamethrin resistance would shorten the life span of female Aedes albopictus infected with DENV-2. Conclusions/Significance As Aedes albopictus developing high resistance to deltamethrin, the resistance prolonged the growth and development of larvae, shorten the life span of adults, as well as reduced the vector competence of resistant Aedes albopictus for DENV-2. It can be concluded that the resistance to deltamethrin in Aedes albopictus is a double-edged sword, which not only endow the mosquito survive under the pressure of insecticide, but also increase the fitness cost and decrease its vector competence. However, Aedes albopictus resistant to deltamethrin can still complete the external incubation period and transmit dengue virus, which remains a potential vector for dengue virus transmission and becomes a threat to public health. Therefore, we should pay high attention for the problem of insecticide resistance so that to better prevent and control mosquito-borne diseases.

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.

Background: Aedes albopictus (Skuse) is an invasive mosquito that has become an important vector of chikungunya, dengue and Zika viruses. In the absence of specific antiviral therapy or a vaccine, vector management is the sole method available for reducing Aedes-induced disease morbidity. Determining the resistance status of Ae. albopictus to insecticides and exploring the resistance mechanisms is essential for future vector control planning. Methods: Aedes albopictus larvae and pupae were sampled from six sites (two sites each from urban, suburban and rural) in Guangzhou. The resistance bioassays were conducted against Bacillus thuringiensis israelensis (Bti): deltamethrin, propoxur and malathion for larvae; and deltamethrin, DDT, propoxur and malathion for adults. P450 monooxygenase (P450s), glutathione S-transferase (GSTs) and carboxylesterase (COEs) activities of adult mosquitoes were measured. Mutations at the knockdown resistance (kdr) gene were analyzed, and the association between kdr mutations and phenotypic resistance was tested. Results: Adult bioassays revealed varied susceptibility against DDT, deltamethrin and propoxur in the six Ae. albopictus populations. Significantly lower mortality rates were found in urban populations than suburban and rural populations. Urban mosquito populations showed resistance against DDT, deltamethrin and propoxur, while one rural population was resistant to DDT. All populations tested were susceptible to malathion. Larval bioassays results indicated that all populations of Ae. albopictus were sensitive to the larvicide Bti and malathion. Resistance to deltamethrin and propoxur was common in larval populations. The F1534S and F1534 L mutations were found to be significantly associated with deltamethrin resistance. Biochemical assays indicated elevated detoxification enzyme activities in the field mosquito populations. Conclusions: Aedes albopictus populations in Guangzhou, especially in urban areas, have developed resistance to the commonly used insecticides, primarily DDT and deltamethrin. This finding calls for resistance management and developing counter measures to mitigate the spread of resistance.

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.