Cell fusing agent virus isolated from Aag2 cells does not vertically transmit in Aedes aegypti via artificial infection.

BACKGROUND: Cell fusing agent virus (CFAV) was the first insect-specific virus to be characterized, and has been reported to negatively influence the growth of arboviruses such as dengue, Zika, and La Cross, making it a promising biocontrol agent for mosquito-borne disease prevention. Aedes aegypti Aag2 cells were naturally infected with CFAV. However, the ability of this virus to stably colonize an Ae. aegypti population via artificial infection and how it influences the vector competence of this mosquito have yet to be demonstrated. METHODS: CFAV used in this study was harvested from Aag2 cells and its complete genome sequence was obtained by polymerase chain reaction and rapid amplification of complementary DNA ends, followed by Sanger sequencing. Phylogenetic analysis of newly identified CFAV sequences and other sequences retrieved from GenBank was performed. CFAV stock was inoculated into Ae. aegypti by intrathoracic injection, the survival of parental mosquitoes was monitored and CFAV copies in the whole bodies, ovaries, and carcasses of the injected F0 generation and in the whole bodies of the F1 generation on different days were examined by reverse transcription-quantitative polymerase chain reaction. RESULTS: The virus harvested from Aag2 cells comprised a mixture of three CFAV strains. All genome sequences of CFAV derived from Aag2 cells clustered into one clade but were far from those isolated or identified from Ae. aegypti. Aag2-derived CFAV efficiently replicated in the mosquito body and did not attenuate the survival of Ae. aegypti. However, the viral load in the ovarian tissues was much lower than that in other tissues and the virus could not passage to the offspring by vertical transmission. CONCLUSIONS: The results of this study demonstrate that Aag2-derived CFAV was not vertically transmitted in Ae. aegypti and provide valuable information on the colonization of mosquitoes by this virus.

Potential Global Distribution of the Invasive Mosquito Aedes koreicus under a Changing Climate.

Invasive alien species are a growing threat to natural systems, the economy, and human health. Active surveillance and responses that readily suppress newly established colonies are effective actions to mitigate the noxious consequences of biological invasions. Aedes (Hulecoeteomyia) koreicus (Edwards), a mosquito species native to East Asia, has spread to parts of Europe and Central Asia since 2008. In the last decade, Ae. koreicus has been shown to be a competent vector for chikungunya virus and Dirofilaria immitis. However, information about the current and potential distribution of Ae. koreicus is limited. Therefore, to understand the changes in their global distribution and to contribute to the monitoring and control of Ae. koreicus, in this study, the MaxEnt model was used to predict and analyze the current suitable distribution area of Ae. koreicus in the world to provide effective information.

ROS-mediated TCA cycle is greatly related to the UV resistance of Bacillus thuringiensis.

Bacillus thuringiensis (Bt) is a popular and environment-friendly biopesticide. However, similar to other microbial pesticides, Bt is limited by ultraviolet (UV) radiation during its application, which greatly reduces its toxicity and persistence. To further know the mechanism of Bt against UV radiation, metabolomic profiles between Bt LLP29 and its UV-resistant mutant LLP29-M19 were compared, analyzed, and annotated in this study, and then a total of 61 metabolites with different abundances were detected. With P < 0.05 as the standard, a total of 12 metabolic pathways were enriched, including the TCA cycle. According to the result of RT-qPCR, the expression levels of the TCA cycle key genes in Bt LL29-M19, such as icd1 citZ, citB, sdhA, sdhB, sdhC, fumA, and mdh, were found down-regulated for 85.58%, 37.02%, 70.87%, 85.97%, 76.33%, 83.15%, 87.28%, and 35.77% than those in Bt LLP29. It was consistent with the down-regulation trend of the TCA cycle key enzymes activity in Bt LLP29-M19. Consistently, the enzyme activities of ICDH, SDH, and PDH in LLP29-M19 were detected 86.28%, 43.93%, and 83.03% lower than those in Bt LLP29. It was revealed that the reduced TCA cycle was required for Bt UV radiation resistance, which was also demonstrated by the addition of inhibitors furfural and malonic acid, respectively. Based on the result of RT-qPCR, the gene transcription levels of the main reactive oxygen species (ROS) generation pathways were down-regulated, such as EMP, however, the activity of the main degrading enzymes was up-regulated, which showed the reduction of ROS generation rate was a way for the TCA cycle to regulate the anti-ultraviolet resistance of Bt. All of these provide solid evidence for reprogramming metabolomics to strengthen Bt UV radiation resistance.

Metavirome of 31 tick species provides a compendium of 1,801 RNA virus genomes.

The increasing prevalence and expanding distribution of tick-borne viruses globally have raised health concerns, but the full repertoire of the tick virome has not been assessed. We sequenced the meta-transcriptomes of 31 different tick species in the Ixodidae and Argasidae families from across mainland China, and identified 724 RNA viruses with distinctive virome compositions among genera. A total of 1,801 assembled and complete or nearly complete viral genomes revealed an extensive diversity of genome architectures of tick-associated viruses, highlighting ticks as a reservoir of RNA viruses. We examined the phylogenies of different virus families to investigate virome evolution and found that the most diverse tick-associated viruses are positive-strand RNA virus families that demonstrate more ancient divergence than other arboviruses. Tick-specific viruses are often associated with only a few tick species, whereas virus clades that can infect vertebrates are found in a wider range of tick species. We hypothesize that tick viruses can exhibit both 'specialist' and 'generalist' evolutionary trends. We hope that our virome dataset will enable much-needed research on vertebrate-pathogenic tick-associated viruses.

Population structure, dispersion patterns and genetic diversity of two major invasive and commensal zoonotic disease hosts (Rattus norvegicus and Rattus tanezumi) from the southeastern coast of China.

Background: The invasive brownrat (Rattus norvegicus) and the Oriental rats (Rattus tanezumi) are common commensal murid that are important hosts for rodent-borne diseases in southeast Asia. Understanding their population structure and genetic diversity is essential to uncover their invasion biology and distribution dynamics that are essential for controlling rodent-borne diseases. Methods: TA total of 103 R. norvegicus and 85 R. tanezumi were collected from 13 to 9 coastal areas of six provincial monitoring sentinel sites, respectivelyto assess patterns in their microsatellite loci and their mitochondrial coxl gene region. Results: Eleven sampled populations of R. norvegicus were divided into two major clusters by region. The observed heterozygosity values of all regional populations were smaller than expected genetic diversity heterozygosity values and deviated from Hardy-Weinberg equilibrium Nine sample populations of R. tanezumi were divided into three clusters; two that included sample from Hainan and Fujian provinces, and one that included samples from the other provinces and cities. The genetic diversity of R. tanezumi was highest in samples from Jiangsu and Guangdong provinces. Conclusion: The data in this paper confirm the two invasive rodent species from the southeastern coastal region of China may have relied on maritime transport to spread from the southern region of China to the Yangtze River basin. R. tanezumi may then hanve migrated unidirectionally, along the southeastern provinces of China towards the north, while R. norvegicus spread in a complex and multidirectional manner in Hainan, Fujian, Zhejiang and Jiangsu Provinces of the country.

Large-Scale Comparative Analyses of Tick Genomes Elucidate Their Genetic Diversity and Vector Capacities.

Among arthropod vectors, ticks transmit the most diverse human and animal pathogens, leading to an increasing number of new challenges worldwide. Here we sequenced and assembled high-quality genomes of six ixodid tick species and further resequenced 678 tick specimens to understand three key aspects of ticks: genetic diversity, population structure, and pathogen distribution. We explored the genetic basis common to ticks, including heme and hemoglobin digestion, iron metabolism, and reactive oxygen species, and unveiled for the first time that genetic structure and pathogen composition in different tick species are mainly shaped by ecological and geographic factors. We further identified species-specific determinants associated with different host ranges, life cycles, and distributions. The findings of this study are an invaluable resource for research and control of ticks and tick-borne diseases.

Function of Aedes aegypti galectin-6 in modulation of Cry11Aa toxicity.

Galectins are a family of ß-galactoside binding proteins, and insect galectins play a role in immune responses and may also affect Cry toxin activity. In this study, we aimed to further understand the function and molecular mechanism of Aedes aegypti galectin-6 in modulation of Cry11Aa toxicity. A. aegypti galectin-6 was cloned, and the recombinant galectin-6 was expressed and purified. Bioassays indicated that galectin-6 could reduce the toxicity of Cry11Aa, protecting A. aegypti larvae. To determine interactions among galectin-6, Cry11Aa and putative toxin receptors, Octet Red System, western blotting, far-western blotting and ELISA assays were performed. Octet Red System showed that galectin-6 bound to BBMVs of A. aegypti larvae with lower affinity than that of Cry11Aa. Western blotting and far-western blotting analyses demonstrated that galectin-6 bound to A. aegypti ALP1 and APN2 as well as to BBMVs, consistent with the results of ELISA and protein docking simulations. However, galectin-6 did not bind to Cadherin in far-western blotting or ELISA assay, though the protein docking simulations suggested their binding potential. These findings support the conclusion that galectin-6 may block Cry11Aa from binding to ALP1 and APN2 due to structural similarity, which might decrease the mosquitocidal toxicity of Cry11Aa.

CTLGA9 Interacts with ALP1 and APN Receptors To Modulate Cry11Aa Toxicity in Aedes aegypti.

The mosquito Aedes aegypti is associated with the spread of many viral diseases in humans, including Dengue virus (DENVs), Yellow fever virus (YFV), Zika virus (ZIKV), and Chikungunya virus (CHIKV). Bacillus thuringiensis (Bt) is widely used as a biopesticide, which produces Cry toxins for mosquito control. The Cry toxins bind mainly to important receptors, including alkaline phosphatase (ALP) and aminopeptidase-N (APN). This work investigated the function of a C-type lectin, CTLGA9, in A. aegypti in response to Cry toxins. Our results showed by far-western blot and ELISA methods that the CTLTGA9 protein interacted with brush border membrane vesicles (BBMVs) of A. aegypti larvae and with ALP1, APN, and Cry11Aa proteins. Furthermore, molecular docking showed overlapping binding sites in ALP1 and APN for binding to Cry11Aa and CTLGA9. The toxicity assays further demonstrated that CTLGA9 inhibited the larvicidal activity of Cry toxins. According to the results of molecular docking, CTLGA9 may compete with Cry11Aa for binding to ALP1 and APN receptors and thus decreases the mosquitocidal toxicity of Cry11Aa. Our results provide further insights into better understanding the mechanism of Cry toxins and help improve the Cry toxicity for mosquito control.

Cloning, expression and activity of ATP-binding protein in Bacillus thuringiensis toxicity modulation against Aedes aegypti.

BACKGROUND: Bacillus thuringiensis israelensis (Bti) is a widely used mosquitocidal microbial pesticide due to its high toxicity. ATP-binding proteins (ABP) are prevalently detected in insects and are related to reaction against Bti toxins. However, the function of ABP in mosquito biocontrol is little known, especially in Aedes aegypti. Therefore, this study aimed to clarify the function of ABP in Ae. aegypti against Bti toxin. RESULTS: Aedes aegypti ABP (GenBank: XM_001661856.2) was cloned, expressed and purified in this study. Far-western blotting and ELISA were also carried out to confirm the interaction between ABP and Cry11Aa. A bioassay of Cry11Aa was performed both in the presence and absence of ABP, which showed that the mortality of Ae. aegypti is increased with an increase in ABP. CONCLUSIONS: Our results suggest that ABP in Ae. aegypti can modulate the toxicity of Cry11Aa toxin to mosquitoes by binding to Bti toxin. This could not only enrich the mechanism of Bt toxin, but also provide more data for the biocontrol of this transmission vector.

Is Genetic Continuity Between Anopheles sinensis (Diptera: Culicidae) and its Sibling Species Due to Gene Introgression or Incomplete Speciation?

The Anopheles mosquito Hyrcanus Group is widely distributed geographically across both Palearctic and Oriental regions and comprises 26 valid species. Although the species Anopheles sinensis Wiedemann (1828) is the most common in China and has a low potential vector rank, it has nevertheless long been thought to be an important natural malaria vector within the middle and lower reaches of the Yangtze River. A number of previous research studies have found evidence to support the occurrence of natural hybridization between An. sinensis and Anopheles kleini Rueda, 2005 (a competent malaria vector). We, therefore, collected a sample series of An. sinensis and morphologically similar species across China and undertook ribosomal and mitochondrial DNA analyses in order to assess genetic differentiation (Fst) and gene flow (Nm) amongst different groups. This enabled us to evaluate divergence times between morphologically similar species using the cytochrome oxidase I (COI) gene. The results of this study reveal significant genetic similarities between An. sinensis, An. kleini, and Anopheles belenrae Rueda, 2005 and therefore imply that correct molecular identifications will require additional molecular markers. As results also reveal the presence of gene flow between these three species, their taxonomic status will require further work. Data suggest that An. kleini is the most basal of the three species, while An. sinensis and An. belenrae share the closest genetic relationship.

C-Type Lectin-20 Interacts with ALP1 Receptor to Reduce Cry Toxicity in Aedes aegypti.

Aedes aegypti is a crucial vector for human diseases, such as yellow fever, dengue, chikungunya, and Zika viruses. Today, a major challenge throughout the globe is the insufficient availability of antiviral drugs and vaccines against arboviruses, and toxins produced by Bacillus thuringiensis (Bt) are still used as biological agents for mosquito control. The use of Cry toxins to kill insects mainly depends on the interaction between Cry toxins and important toxin receptors, such as alkaline phosphatase (ALP). In this study, we investigated the function of A. aegypti C-type lectin-20 (CTL-20) in the tolerance of Cry toxins. We showed that recombinant CTL-20 protein interacted with both Cry11Aa and ALP1 by the Far-Western blot and ELISA methods, and CTL-20 bound to A. aegypti larval brush border membrane vesicles (BBMVs). Binding affinity of CTL-20 to ALP1 was higher than that of Cry11Aa to ALP1. Furthermore, the survival rate of A. aegypti larvae fed with Cry11Aa toxin mixed with recombinant CTL-20 fusion protein was significantly increased compared with that of the control larvae fed with Cry11Aa mixed with thioredoxin. Our novel results suggest that midgut proteins like CTLs may interfere with interactions between Cry toxins and toxin receptors by binding to both Cry toxins and receptors to alter Cry toxicity.

Identification of Disease-Transmitting Mosquitoes: Development of Species-Specific Probes for DNA Chip Assay Using Mitochondrial COI and ND2 Genes and Ribosomal Internal Transcribed Spacer 2.

Mosquitoes, which transmit infectious diseases, such as malaria and dengue fever, are harmful to human health. Thus, accurate and rapid identification of vectors is a critical step for the control of mosquito-borne diseases. However, phenotypic variations in adults, lack of recognizable features of the immature, and fragility of mosquitoes make identification difficult. Molecular approaches have been widely applied to identify mosquitoes, yet these methods have been focused only on the identification of a few species. This study used sequences of two mitochondrial genes, COI and ND2, and a ribosomal gene, ITS2, to design species-specific probes. Biochips thus developed were able to provide simultaneous identification of nine important medical and veterinary species, including the immature, from genera of Aedes, Anopheles, Armigeres, and Culex. This chip was also applied to samples collected from the field. Despite its inability to resolve the close affinity species of Culex quinquefasciatus and Culex pipiens molestus, pertinent biochips are expected to be applied to a mass screening method.

Different toxicity of the novel Bacillus thuringiensis (Bacillales: Bacillaceae) strain LLP29 against Aedes albopictus and Culex quinquefasciatus (Diptera: Culicidae).

Bacillus thuringiensis (Bt) (Berliner) strain LLP29 produces a crystal protein Cyt1Aa6 toxic to mosquito vectors of human diseases. However, the susceptibility of Culex quinquefasciatus (Say) in the current study was 8.25 times higher than that of Aedes albopictus (Skuse) with this single protein Cyt1Aa6 purified from LLP29. To understand the mechanism of the novel mosquitocidal protein, the binding characteristic of brush border membrane vesicles from the two tested mosquitoes was investigated. Enzyme-linked immunosorbent assay showed that Cyt1Aa6 bound to the two mosquitoes' brush border membrane vesicles. However, the titer of Ae. albopictus was a little higher than that of Cx. quinquefasciatus, with 3.21 and 2.91, respectively. Ligand Western blot analysis showed Cyt1Aa6 toxin specifically bound to the same three proteins (i.e., 68, 54, and 26 kDa) in the two mosquitoes, but one another protein, approximately to 37 kDa, could just be detected in Cx. quinquefasciatus. However, little difference was found in the test of immunohistochemistry. Cyt1Aa6 was detected in the midguts of both mosquitoes with histopathological changes. It would of great importance to the knowledge of the novel toxin against to Cx. quinquefasciatus and Ae. albopictus.

Microbial ecology and association of Bacillus thuringiensis in chicken feces originating from feed.

To explain the association of Bacillus thuringiensis (Bt) with animal feces, an ecological analysis in chickens was conducted by introducing a cry(-) strain marked by production of green fluorescent protein (GFP). After feeding with the tagged Bt strains, the feces of the tested chickens were collected at different times, isolated, and the morphology of Bt was observed. It was shown that Bt strain HD-73GFP in spore form could be isolated from feces of chickens for a period of 13 d, and then it disappeared thereafter. Bt could be detected only up to day 4 (but not thereafter), when chickens were fed with vegetative cells of HD-73GFP. To confirm the source of newly isolated strains, the gfp gene was examined by polymerase chain reaction (PCR), which showed that all the isolated strains harbored the marker gene. Recent data from isolation and PCR had suggested that fecal Bt strains had originated from food. Chicken tissues were thus dissected to isolate Bt strains and to investigate whether Bt could be located in vivo. Bt was located within the duodenum in spore form. Compared to the morphology of the isolated strains at different growth times, the growth rates of all the tested Bt had little changes when passing through the digestive system to the feces. Dissection of the chickens confirmed that Bt was safe for the tested animal.

Mosquito cell line C6/36 shows resistance to Cyt1Aa6.

This study aimed to investigate the resistance mechanism of C6/36 cells to Cyt1Aa6 protein under selection pressure. Receptor binding properties of Cyt1Aa6 toward sensitive and resistant C6/36 cells were investigated. More sensitive cells were detected with goat-anti-rabbit-FITC-labeled antibody, and the quantity of in vitro activated Cyt1Aa6 toxin bound to resistant cells was greatly reduced. Ligand western blot assays showed that disappearance of the 26 kDa protein and weakness of the positive bands of 68 kDa from resistant cells might lead to the resistance of C6/36 cells to Cyt1Aa6 toxin. The resistance of C6/36 cells was detected under selection in vitro-activated Cytl1Aa6 toxin. Receptor binding demonstrated that reduced Cyt1Aa6 bound to resistant cells, which might be closely related to the disappearance and weakness of some proteins. The results presented here are the first to demonstrate that Cyt1Aa protein, a uniquely characteristic toxin, induced resistance at the cellular level. It might be attributed to the change of receptors.

A differentially displayed mRNA related to resistance to Bacillus thuringiensis israelensis of Aedes albopictus selected in vitro-activated Cyt1Aa6.

We compared the differential display of Aedes albopictus cells, both resistant and susceptible to Bacillus thuringiensis israelensis (Bti), using differentially displayed reverse transcription polymerase chain reaction. We found 1 band about 200 base pairs long. After cloning and sequencing, the differentially expressed gene was similar to some partial messenger ribonucleic acid of Ae. aegypti, Culex quinquefasciatus, and Anopheles gambiae, rather than Ae. albopictus. This will be of some value for clarifying the mechanism of mosquito resistance to Bti products.

A novel mosquitocidal Bacillus thuringiensis strain LLP29 isolated from the phylloplane of Magnolia denudata.

Eleven Bacillus thuringiensis isolates were recovered from phylloplanes of Magnolia denudata, a specific source of new strains of B. thuringiensis. Among these, a new strain, LLP29, was found to be most toxic to mosquitoes based on the results of preliminary toxicity analysis. Phase contrast microscopy, mosquitocidal activity, polymerase chain reaction (PCR) analysis and parasporal inclusion were performed to learn more about the characteristics of this novel mosquitocidal isolate. The LC(50) values of LLP29 against Aedes albopictus and Culex quinquefasciatus were 0.33 and 0.04 ng of protein/ml, respectively. The cyt1 gene, which encodes the Cyt protein that is toxic to mosquitoes, was subsequently detected, cloned, sequenced and expressed in acrystalliferous Bt HD73 Cry(-). The results indicated that it might be a member of the cyt1Aa gene group. The novel strain LLP29 appears to be a new subspecies of B. thuringiensis and should prove useful in the control of mosquitoes and mosquito-borne diseases.

Cloning, expression and characterisation of a promising mosquitocidal gene.

A new mosquitocidal gene, cyt1Aa6, was isolated and cloned from the novel Bacillus thuringiensis strain LLP29, previously isolated from the phylloplane of Magnolia denudata. Nucleotide sequence analysis of cyt1Aa6 indicated that the open reading frame consisted of 750 base pairs, encoding 249 amino acid sequences with a calculated molecular weight of 27.3681 kDa and a PI value of 4.77. An homological comparison revealed that the cyt1Aa6 amino acid sequence was 99% identical with those of known Cyt1Aa proteins. In addition, the cyt1Aa6 gene was successfully expressed in Escherichia coli BL21. Bioassays on Aedes albopictus showed that Bt LLP29 and the expressed BL21 were both toxic to 3rd-instar mosquito larvae. The isolation of cyt1Aa6 provides new opportunities for selecting new strains and to obtain novel B. thuringiensis products based on its toxins.