miR-2940-1 is involved in the circadian regulation of oviposition in Aedes albopictus.

The vast majority of all global species have circadian rhythm cycles that allow them to adapt to natural environments. These regular rhythms are regulated by core clock genes and recent studies have also implicated roles for microRNAs in this regulation. Oviposition is an important circadian behavior in the reproductive cycle of insect vectors of diseases, and little is known about the rhythm or its regulation in mosquitoes. Aedes albopictus is a diurnal mosquito that transmits arboviruses and is the major cause of outbreaks of dengue fever in China. We analyzed the oviposition rhythm patterns of A. albopictus under different light/dark conditions and show that the mosquitoes have an oviposition peak between zeitgeber time 9 (ZT 9) and ZT 12. Furthermore, the antagomir-mediated knockdown of expression of the microRNA miR-2940-1 affected the oviposition rhythm of A. albopictus. These data support the conclusion that miR-2940-1 is involved in the regulation of oviposition rhythm in A. albopictus and provide a foundation for using oviposition rhythms as a new target for vector mosquito control.

Modelling the dynamic basic reproduction number of dengue based on MOI of Aedes albopictus derived from a multi-site field investigation in Guangzhou, a subtropical region.

BACKGROUND: More than half of the global population lives in areas at risk of dengue (DENV) transmission. Developing an efficient risk prediction system can help curb dengue outbreaks, but multiple variables, including mosquito-based surveillance indicators, still constrain our understanding. Mosquito or oviposition positive index (MOI) has been utilized in field surveillance to monitor the wild population density of Aedes albopictus in Guangzhou since 2005. METHODS: Based on the mosquito surveillance data using Mosq-ovitrap collection and human landing collection (HLC) launched at 12 sites in Guangzhou from 2015 to 2017, we established a MOI-based model of the basic dengue reproduction number (R0) using the classical Ross-Macdonald framework combined with a linear mixed-effects model. RESULTS: During the survey period, the mean MOI and adult mosquito density index (ADI) using HLC for Ae. albopictus were 12.96 ± 17.78 and 16.79 ± 55.92, respectively. The R0 estimated from the daily ADI (ADID) showed a significant seasonal variation. A 10-unit increase in MOI was associated with 1.08-fold (95% CI 1.05, 1.11) ADID and an increase of 0.14 (95% CI 0.05, 0.23) in the logarithmic transformation of R0. MOI-based R0 of dengue varied by month and average monthly temperature. During the active period of Ae. albopictus from April to November in Guangzhou region, a high risk of dengue outbreak was predicted by the MOI-based R0 model, especially from August to October, with the predicted R0 > 1. Meanwhile, from December to March, the estimates of MOI-based R0 were < 1. CONCLUSIONS: The present study enriched our knowledge about mosquito-based surveillance indicators and indicated that the MOI of Ae. albopictus could be valuable for application in estimating the R0 of dengue using a statistical model. The MOI-based R0 model prediction of the risk of dengue transmission varied by month and temperature in Guangzhou. Our findings lay a foundation for further development of a complex efficient dengue risk prediction system.

The dynamics of deltamethrin resistance evolution in Aedes albopictus has an impact on fitness and dengue virus type-2 vectorial capacity.

BACKGROUND: Worldwide invasion and expansion of Aedes albopictus, an important vector of dengue, chikungunya, and Zika viruses, has become a serious concern in global public health. Chemical insecticides are the primary means currently available to control the mosquito populations. However, long-term and large-scale use of insecticides has selected for resistance in the mosquito that is accompanied by a genetic load that impacts fitness. RESULTS: A number of laboratory strains representing different resistance mechanisms were isolated and identified from laboratory-derived, deltamethrin-resistant Ae. albopictus recovered in previous work. Resistance levels and fitness costs of the strains were evaluated and compared to characterize the evolution of the resistance genotypes and phenotypes. The heterozygous F1534S mutation (1534F/S) in the voltage gated sodium channel (vgsc) gene product (VGSC), first detected in early stages of resistance evolution, not only confers high-level resistance, but also produces no significant fitness costs, leading to the rapid spread of resistance in the population. This is followed by the increase in frequency of homozygous F1534S (1534S/S) mosquitoes that have significant fitness disadvantages, prompting the emergence of an unlinked I1532T mutation with fewer side effects and a mating advantage better adapted to the selection and reproductive pressures imposed in the experiments. Metabolic resistance with no significant fitness cost and mediating a high-tolerance resistance phenotype may play a dominant role in the subsequent evolution of resistance. The different resistant strains had similar vector competence for dengue virus type-2 (DENV-2). Furthermore, a comparative analysis of vectorial capacity revealed that increased survival due to deltamethrin resistance balanced the negative fitness cost effects and contributed to the risk of dengue virus (DENV) transmission by resistant populations. The progressive evolution of resistance results in mosquitoes with both target-site insensitivity and metabolic resistance with lower fitness costs, which further leads to resistant populations with both high resistance levels and vectorial capacity. CONCLUSIONS: This study reveals a possible mechanism for the evolution of deltamethrin resistance in Aedes albopictus. These findings will help guide practical strategies for insecticide use, resistance management and the prevention and control of mosquito-borne disease.

Discovery, Optimization, and Evaluation of Novel N-(Benzimidazol-5-yl)-1,3,4-thiadiazol-2-amine Analogues as Potent STAT3 Inhibitors for Cancer Treatment.

Signal transducer and activator of transcription 3 (STAT3) is an attractive target for cancer therapy. However, identifying potent and selective STAT3 small-molecule inhibitors with drug-like properties remains challenging. Based on a scaffold combination strategy, compounds with a novel N-(benzimidazol-5-yl)-1,3,4-thiadiazol-2-amine scaffold were designed and their inhibition of the interleukin-6 (IL-6)/JAK/STAT3 pathway was tested in HEK-Blue IL-6 reporter cells. After optimization of lead compound 12, compound 40 was identified as a selective STAT3 inhibitor that directly binds the SH2 domain to inhibit STAT3 phosphorylation, translocation, and downstream gene transcription. Compound 40 exhibited antiproliferative activities against STAT3-overactivated DU145 (IC50 value = 2.97 µM) and MDA-MB-231 (IC50 value = 3.26 µM) cancer cells and induced cell cycle arrest and apoptosis. In the DU145 xenograft model, compound 40 showed in vivo antitumor efficacy following intraperitoneal administration, with a tumor growth inhibition rate of 65.3% at 50 mg/kg, indicating promise for further development.

Chlorogenic Acid Induced Neuroblastoma Cells Differentiation via the ACAT1-TPK1-PDH Pathway.

BACKGROUND: Chlorogenic acid (CHA) has been shown to have substantial biological activities, including anti-inflammatory, antioxidant, and antitumor effects. However, the pharmacological role of CHA in neuroblastoma has not yet been assessed. Neuroblastoma is a type of cancer that develops in undifferentiated sympathetic ganglion cells. This study aims to assess the antitumor activity of CHA against neuroblastoma and reveal its mechanism of action in cell differentiation. METHODS: Be(2)-M17 and SH-SY5Y neuroblastoma cells were used to confirm the differentiation phenotype. Subcutaneous and orthotopic xenograft mouse models were also used to evaluate the antitumor activity of CHA. Seahorse assays and metabolomic analyses were further performed to investigate the roles of CHA and its target ACAT1 in mitochondrial metabolism. RESULTS: CHA induced the differentiation of Be(2)-M17 and SH-SY5Y neuroblastoma cells in vivo and in vitro. The knockdown of mitochondrial ACAT1, which was inhibited by CHA, also resulted in differentiation characteristics in vivo and in vitro. A metabolomic analysis revealed that thiamine metabolism was involved in the differentiation of neuroblastoma cells. CONCLUSIONS: These results provide evidence that CHA shows good antitumor activity against neuroblastoma via the induction of differentiation, by which the ACAT1-TPK1-PDH pathway is involved. CHA is a potential drug candidate for neuroblastoma therapy.

Metagenomic analysis reveals the virome profiles of Aedes albopictus in Guangzhou, China.

Introduction: Aedes 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. Methods: Mosquito 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. Results: The 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. Discussion: The 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. Conclusion: This 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.

Mosquito densovirus significantly 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 confirmed vector responsible for dengue epidemics in Guangzhou, China. Mosquito densoviruses (MDVs) are pathogenic mosquito-specific 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 immunofluorescence 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 quantified 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, significant reductions in DENV-2 titers in supernatant at 120 hpi, and an apparent decrease in DENV-2-positive cells at 96 and 120 hpi were observed. In vivo, DENV-2 in the ovaries and salivary glands was first detected at 10 dpi in both monoinfected and superinfected Ae. albopictus females, while MDV superinfection 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 MDV-infected 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.

A DBHS family member regulates male determination in the filariasis vector Armigeres subalbatus.

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.

Clock genes regulate mating activity rhythms in the vector mosquitoes, Aedes albopictus and Culex quinquefasciatus.

BACKGROUND: Endogenous circadian rhythms result from genetically-encoded molecular clocks, whose components and downstream output factors cooperate to generate cyclic changes in activity. Mating is an important activity of mosquitoes, however, the key aspects of mating rhythm patterns and their regulatory mechanisms in two vector mosquito species, Aedes albopictus and Culex quinquefasciatus, remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: We determined and compared the diel mating activity rhythms of these two mosquito species and discovered that Ae. albopictus had mating peaks in the light/dark transition periods (ZT0-3 and ZT9-12), while Cx. quinquefasciatus only had a mating peak at ZT12-15. Knockouts of the clock (clk) orthologous genes (Aalclk and Cxqclk) resulted in phase delay or phase reversal of the mating peaks in Ae. albopictus and Cx. quinquefasciatus, respectively. In addition, the temporal expression pattern of the desaturase orthologous genes, desat1, in both mosquito species was also different in respective wild-type strains and showed phase changes similar to the mating rhythms in clk mutant strains. Inhibition of desat1 expression resulted in decreased mating activity in male mosquitoes of both species but not females. In addition, desat1 regulated cuticular hydrocarbons' synthesis in both species. Silencing desat1 in male Ae. albopictus resulted in decreases of nonadecane and tricosane, which promoted mating, with concomitant increases of heptacosane, which inhibited mating. Silencing desat1 in male Cx. quinquefasciatus also resulted in decreases of tricosane, which promoted mating. CONCLUSIONS/SIGNIFICANCE: These results suggest that Aalclk and Cxqclk have significant roles in the mating activity rhythms in both Ae. albopictus and Cx. quinquefasciatus by regulating the temporal expression of the desat1 gene under LD cycles, which affects sex pheromone synthesis and mating. This work provides insights into the molecular regulatory mechanism of distinct mating rhythm of Ae. albopictus and Cx. quinquefasciatus and may provide a basis for the control of these two important vector mosquitoes.

Virome and nrEVEome diversity of Aedes albopictus mosquitoes from La Reunion Island and China.

BACKGROUND: Aedes albopictus is a public health threat for its worldwide spread and ability to transmit arboviruses. Understanding mechanisms of mosquito immunity can provide new tools to control arbovirus spread. The genomes of Aedes mosquitoes contain hundreds of nonretroviral endogenous viral elements (nrEVEs), which are enriched in piRNA clusters and produce piRNAs, with the potential to target cognate viruses. Recently, one nrEVE was shown to limit cognate viral infection through nrEVE-derived piRNAs. These findings suggest that nrEVEs constitute an archive of past viral infection and that the landscape of viral integrations may be variable across populations depending on their viral exposure. METHODS: We used bioinformatics and molecular approaches to identify known and novel (i.e. absent in the reference genome) viral integrations in the genome of wild collected Aedes albopictus mosquitoes and characterize their virome. RESULTS: We showed that the landscape of viral integrations is dynamic with seven novel viral integrations being characterized, but does not correlate with the virome, which includes both viral species known and unknown to infect mosquitoes. However, the small RNA coverage profile of nrEVEs and the viral genomic contigs we identified confirmed an interaction among these elements and the piRNA and siRNA pathways in mosquitoes. CONCLUSIONS: Mosquitoes nrEVEs have been recently described as a new form of heritable, sequence-specific mechanism of antiviral immunity. Our results contribute to understanding the dynamic distribution of nrEVEs in the genomes of wild Ae. albopictus and their interaction with mosquito viruses.

A unified global genotyping framework of dengue virus serotype-1 for a stratified coordinated surveillance strategy of dengue epidemics.

BACKGROUND: Dengue is the fastest spreading arboviral disease, posing great challenges on global public health. A reproduceable and comparable global genotyping framework for contextualizing spatiotemporal epidemiological data of dengue virus (DENV) is essential for research studies and collaborative surveillance. METHODS: Targeting DENV-1 spreading prominently in recent decades, by reconciling all qualified complete E gene sequences of 5003 DENV-1 strains with epidemiological information from 78 epidemic countries/areas ranging from 1944 to 2018, we established and characterized a unified global high-resolution genotyping framework using phylogenetics, population genetics, phylogeography, and phylodynamics. RESULTS: The defined framework was discriminated with three hierarchical layers of genotype, subgenotype and clade with respective mean pairwise distances 2-6%, 0.8-2%, and ≤ 0.8%. The global epidemic patterns of DENV-1 showed strong geographic constraints representing stratified spatial-genetic epidemic pairs of Continent-Genotype, Region-Subgenotype and Nation-Clade, thereby identifying 12 epidemic regions which prospectively facilitates the region-based coordination. The increasing cross-transmission trends were also demonstrated. The traditional endemic countries such as Thailand, Vietnam and Indonesia displayed as persisting dominant source centers, while the emerging epidemic countries such as China, Australia, and the USA, where dengue outbreaks were frequently triggered by importation, showed a growing trend of DENV-1 diffusion. The probably hidden epidemics were found especially in Africa and India. Then, our framework can be utilized in an accurate stratified coordinated surveillance based on the defined viral population compositions. Thereby it is prospectively valuable for further hampering the ongoing transition process of epidemic to endemic, addressing the issue of inadequate monitoring, and warning us to be concerned about the cross-national, cross-regional, and cross-continental diffusions of dengue, which can potentially trigger large epidemics. CONCLUSIONS: The framework and its utilization in quantitatively assessing DENV-1 epidemics has laid a foundation and re-unveiled the urgency for establishing a stratified coordinated surveillance platform for blocking global spreading of dengue. This framework is also expected to bridge classical DENV-1 genotyping with genomic epidemiology and risk modeling. We will promote it to the public and update it periodically.

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

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

MAGI2-AS3 restrains proliferation, glycolysis, and triggers apoptosis in acute lymphoblastic leukemia via regulating miR-452-5p/FOXN3 pathway.

Objectives: MAGI2-AS3 is a cancer suppressor gene of multiple malignancies. Acute lymphoblastic leukemia (ALL) is an important type of leukemia that especially occurs in children. Our work evaluated the modulation of MAGI2-AS3 in ALL. Materials and Methods: qPCR and Western blotting were adopted for detection of target molecular expression. Growth and apoptosis were determined by CCK8 assay and Annexin V/PI staining. Glycolysis was detected by commercial kits. The direct binding between miR-452-5p and MAGI2-AS3 or FOXN3 was assessed by luciferase reporter assay. Tumor growth was measured in nude mice in vivo. Results: MAGI2-AS3 was down-regulated in ALL. Enforced expression of MAGI2-AS3 inhibited growth and glycolysis while promoting apoptosis of ALL cells. Moreover, MAGI2-AS3 up-regulated FOXN3 via sponging miR-452-5p. FOXN3 depletion abrogated MAGI2-AS3-mediated anti-cancer action. More importantly, MAGI2-AS3 repressed ALL cell growth in nude mice through regulation of miR-452-5p/FOXN3. Conclusion: MAGI2-AS3 inhibits ALL development via modulating miR-452-5p/FOXN3.

Armigeres subalbatus is a potential vector for Zika virus but not dengue virus.

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. subalbatus 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 105 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.

The AalNix3&4 isoform is required and sufficient to convert Aedes albopictus females into males.

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.

Benzobis(imidazole) derivatives as STAT3 signal inhibitors with antitumor activity.

Polycyclic aromatic systems have been considered good biological probes, but some may also be good scaffolds for drug development. In this study, a series of benzobis(imidazole) derivatives were identified as STAT3 signal inhibitors, among which compound 24 showed significant inhibition of IL-6 induced JAK/STAT3 signalling pathway activation. Moreover, 24 inhibited cancer cell growth and migration, and induced cell apoptosis as well as cycle arrest in human hepatocellular carcinoma cells (HepG2) and oesophageal carcinoma cells (EC109). Compound 24 also displayed obvious antitumor activity in a mouse HepG2 cell xenograft tumor model without affecting the body weight. These results confirmed that 24 was a potential STAT3 signal inhibitor with certain antitumor activity.

Genomic Shifts, Phenotypic Clines, and Fitness Costs Associated With Cold Tolerance in the Asian Tiger Mosquito.

Climatic variation is a key driver of genetic differentiation and phenotypic traits evolution, and local adaptation to temperature is expected in widespread species. We investigated phenotypic and genomic changes in the native range of the Asian tiger mosquito, Aedes albopictus. We first refine the phylogeographic structure based on genome-wide regions (1,901 double-digest restriction-site associated DNA single nucleotide polymophisms [ddRAD SNPs]) from 41 populations. We then explore the patterns of cold adaptation using phenotypic traits measured in common garden (wing size and cold tolerance) and genotype-temperature associations at targeted candidate regions (51,706 exon-capture SNPs) from nine populations. We confirm the existence of three evolutionary lineages including clades A (Malaysia, Thailand, Cambodia, and Laos), B (China and Okinawa), and C (South Korea and Japan). We identified temperature-associated differentiation in 15 out of 221 candidate regions but none in ddRAD regions, supporting the role of directional selection in detected genes. These include genes involved in lipid metabolism and a circadian clock gene. Most outlier SNPs are differently fixed between clades A and C, whereas clade B has an intermediate pattern. Females are larger at higher latitude yet produce no more eggs, which might favor the storage of energetic reserves in colder climate. Nondiapausing eggs from temperate populations survive better to cold exposure than those from tropical populations, suggesting they are protected from freezing damages but this cold tolerance has a fitness cost in terms of egg viability. Altogether, our results provide strong evidence for the thermal adaptation of A. albopictus across its wide temperature range.

Interspecific mating bias may drive Aedes albopictus displacement of Aedes aegypti during its range expansion.

Aedes albopictus is the most invasive mosquito in the world and often displaces Ae. aegypti in regions where their populations overlap. Interspecific mating has been proposed as a possible cause for this displacement, but whether this applies across the range of their sympatry remains unclear. Aedes albopictus and Ae. aegypti collected from allopatric and sympatric areas in China were allowed to interact in cage experiments with different crosses and sex-choices. The results confirm that asymmetric interspecific mating occurs in these populations with matings between allopatric Ae. albopictus males and Ae. aegypti females being significantly higher (55.2%) than those between Ae. aegypti males and Ae. albopictus females (27.0%), and sympatric mosquitoes showed a similar but lower frequency bias, 25.7% versus 6.2%, respectively. The cross-mated females can mate second time (remate) with the respective conspecific males and the 66.7% remating success of female Ae. albopictus was significantly higher than the 9.3% of Ae. aegypti females. Furthermore, 17.8% of the matings of Ae. albopictus males exposed to mixed pools of Ae. albopictus and Ae. aegypti females and 9.3% of the matings of Ae. aegypti males with mixed Ae. aegypti and Ae. albopictus females were interspecific. The difference in the length of clasper between male Ae. albopictus (0.524 mm) and Ae. aegypti (0.409 mm) may be correlated with corresponding mates. We conclude that stronger Ae. albopictus male interspecific mating and more avid female intraspecific remating result in a satyr effect and contribute to competitive displacement of Ae. aegypti as allopatric Ae. albopictus invade during range expansion.

Recombinant Mosquito Densovirus with Bti Toxins Significantly Improves Pathogenicity against Aedes albopictus.

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 ß6-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.