Jinbao Gu

Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution

Significance Aedes albopictus is a highly adaptive species that thrives worldwide in tropical and temperate zones. From its origin in Asia, it has established itself on every continent except Antarctica. This expansion, coupled with its ability to vector the epidemic human diseases dengue and Chikungunya fevers, make it a significant global public health threat. A complete genome sequence and transcriptome data were obtained for the Ae. albopictus Foshan strain, a colony derived from mosquitoes from its historical origin. The large genome (1,967 Mb) comprises an abundance of repetitive DNA classes and expansions of the numbers of gene family members involved in insecticide resistance, diapause, sex determination, immunity, and olfaction. This large genome repertory and plasticity may contribute to its success as an invasive species. The Asian tiger mosquito, Aedes albopictus, is a highly successful invasive species that transmits a number of human viral diseases, including dengue and Chikungunya fevers. This species has a large genome with significant population-based size variation. The complete genome sequence was determined for the Foshan strain, an established laboratory colony derived from wild mosquitoes from southeastern China, a region within the historical range of the origin of the species. The genome comprises 1,967 Mb, the largest mosquito genome sequenced to date, and its size results principally from an abundance of repetitive DNA classes. In addition, expansions of the numbers of members in gene families involved in insecticide-resistance mechanisms, diapause, sex determination, immunity, and olfaction also contribute to the larger size. Portions of integrated flavivirus-like genomes support a shared evolutionary history of association of these viruses with their vector. The large genome repertory may contribute to the adaptability and success of Ae. albopictus as an invasive species.

Development of an Efficient Recombinant Mosquito Densovirus-Mediated RNA Interference System and Its Preliminary Application in Mosquito Control

The Aedes aegypti densovirus (AeDNV) has potential as a delivery vector for foreign nucleic acids into mosquito cells. In this study, we investigated the ability of plasmids containing recombinant viral transducing genome to induce RNA interference (RNAi) effects in C6/C36 cells. We then evaluated the efficiency of a recombinant AeDNV vector to induce RNAi in Aedes albopictus larvae. We found that the expression of V-ATPase was inhibited by up to 90% at 96 h post-transfection in transfected C6/C36 cells. In addition, the bioinsecticidal activities of various RNAi-expressing AeDNV vectors used to infect Ae. albopictus larvae were also tested. We found that when Ae. albopictus larvae were infected with recombinant AeDNV, expression of V-ATPase was downregulated by nearly 70% compared to controls. Furthermore, the median survival time bioassays demonstrated that recombinant AeDNV caused more serious pathogenic effects than the wild type virus. This is the first report showing that recombinant virus plasmid and corresponding recombinant AeDNV can be used as an effective in vitro and in vivo RNAi delivery system, respectively.

Competence of Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus Mosquitoes as Zika Virus Vectors, China.

In China, the prevention and control of Zika virus disease has been a public health threat since the first imported case was reported in February 2016. To determine the vector competence of potential vector mosquito species, we experimentally infected Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus mosquitoes and determined infection rates, dissemination rates, and transmission rates. We found the highest vector competence for the imported Zika virus in Ae. aegypti mosquitoes, some susceptibility of Ae. albopictus mosquitoes, but no transmission ability for Cx. quinquefasciatus mosquitoes. Considering that, in China, Ae. albopictus mosquitoes are widely distributed but Ae. aegypti mosquito distribution is limited, Ae. albopictus mosquitoes are a potential primary vector for Zika virus and should be targeted in vector control strategies.

miRNA Genes of an Invasive Vector Mosquito, Aedes albopictus

Aedes albopictus, a vector of Dengue and Chikungunya viruses, is a robust invasive species in both tropical and temperate environments. MicroRNAs (miRNAs) regulate gene expression and biological processes including embryonic development, innate immunity and infection. While a number of miRNAs have been discovered in some mosquitoes, no comprehensive effort has been made to characterize them from different developmental stages from a single species. Systematic analysis of miRNAs in Ae. albopictus will improve our understanding of its basic biology and inform novel strategies to prevent virus transmission. Between 10–14 million Illumina sequencing reads per sample were obtained from embryos, larvae, pupae, adult males, sugar-fed and blood-fed adult females. A total of 119 miRNA genes represented by 215 miRNA or miRNA star (miRNA*) sequences were identified, 15 of which are novel. Eleven, two, and two of the newly-discovered miRNA genes appear specific to Aedes, Culicinae, and Culicidae, respectively. A number of miRNAs accumulate predominantly in one or two developmental stages and the large number that showed differences in abundance following a blood meal likely are important in blood-induced mosquito biology. Gene Ontology (GO) analysis of the targets of all Ae. albopictus miRNAs provides a useful starting point for the study of their functions in mosquitoes. This study is the first systematic analysis of miRNAs based on deep-sequencing of small RNA samples of all developmental stages of a mosquito species. A number of miRNAs are related to specific physiological states, most notably, pre- and post-blood feeding. The distribution of lineage-specific miRNAs is consistent with mosquito phylogeny and the presence of a number of Aedes-specific miRNAs likely reflects the divergence between the Aedes and Culex genera.

Functional characterization of three MicroRNAs of the Asian Tiger Mosquito, Aedes albopictus

BackgroundTemporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes.MethodsmiRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes.ResultsThe knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults.ConclusionThis study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector.

Molecular and functional characterization of odorant-binding protein genes in an invasive vector mosquito, Aedes albopictus.

Aedes albopictus is a major vector of dengue and Chikungunya viruses. Olfaction plays a vital role in guiding mosquito behaviors and contributes to their ability to transmit pathogens. Odorant-binding proteins (OBPs) are abundant in insect olfactory tissues and involved in the first step of odorant reception. While comprehensive descriptions are available of OBPs from Aedes aegypti, Culex quinquefasciatus and Anopheles gambiae, only a few genes from Ae. albopictus have been reported. In this study, twenty-one putative AalbOBP genes were cloned using their homologues in Ae. aegypti to query an Ae. albopictus partial genome sequence. Two antenna-specific OBPs, AalbOBP37 and AalbOBP39, display a remarkable similarity in their overall folding and binding pockets, according to molecular modeling. Binding affinity assays indicated that AalbOBP37 and AalbOBP39 had overlapping ligand affinities and are affected in different pH condition. Electroantennagrams (EAG) and behavioral tests show that these two genes were involved in olfactory reception. An improved understanding of the Ae. albopictus OBPs is expected to contribute to the development of more efficient and environmentally-friendly mosquito control strategies.

A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus

The Aedes aegypti densovirus (AeDNV) has previously shown potential in mosquito control. To improve its efficacy as a biopesticide, the gene for an excitatory insect-specific toxin from Buthus martensii Karsch (BmK IT1) was inserted into the AeDNV genome and cloned into pUCA plasmid. The coding sequence for green fluorescent protein was ligated to the C-terminus of the BmK IT1 gene as a screening marker. Recombinant and helper plasmids were cotransfected into C6/36 cells; wild-type viruses were the controls. The recombinant viruses were identified and quantified by real-time polymerase chain reaction and exposed to Ae. albopictus larvae for the evaluation of its bioinsecticidal activity. LT(50) and LD(50) bioassays showed that the recombinant AeDNV had stronger and faster pathogenic effects on Ae. albopictus than the wild-type virus. This is the first report on the recombinant AeDNA containing the insect-specific toxin, BmK IT1, which may be used to develop a novel type of insecticide.

Comparative evaluation of the efficiency of the BG-Sentinel trap, CDC light trap and Mosquito-oviposition trap for the surveillance of vector mosquitoes

BackgroundThe surveillance of vector mosquitoes is important for the control of mosquito-borne diseases. To identify a suitable surveillance tool for the adult dengue vector Aedes albopictus, the efficacy of the BG-Sentinel trap, CDC light trap and Mosquito-oviposition trap (MOT) on the capture of vector mosquitoes were comparatively evaluated in this study.MethodsThe capture efficiencies of the BG-Sentinel trap, CDC light trap and Mosquito-oviposition trap for common vector mosquitoes were tested in a laboratory setting, through the release-recapture method, and at two field sites of Guangzhou, China from June 2013 to May 2014. The captured mosquitoes were counted, species identified and compared among the three traps on the basis of species.ResultsIn the release-recapture experiments in a laboratory setting, the BG-Sentinel trap caught significantly more Aedes albopictus and Culex quinquefasciatus than the CDC light trap and Mosquito-ovitrap, except for Anopheles sinensis. The BG-Sentinel trap had a higher efficacy in capturing female rather than male Ae. albopictus and Cx. quinquefasciatus, but the capture in CDC light traps displayed no significant differences. In the field trial, BG-Sentinel traps collected more Aedes albopictus than CDC light traps and MOTs collected in both urban and suburban areas. The BG-Sentinel trap was more sensitive for monitoring the population density of Aedes albopictus than the CDC light trap and MOT during the peak months of the year 2013. However, on an average, CDC light traps captured significantly more Cx. quinquefasciatus than BG-Sentinel traps. The population dynamics of Cx. quinquefasciatus displayed a significant seasonal variation, with the lowest numbers in the middle of the year.ConclusionsThis study indicates that the BG-Sentinel trap is more effective than the commonly used CDC light trap and MOT in sampling adult Aedes albopictus and Culex quinquefasciatus. We recommend its use in the surveillance of dengue vector mosquitoes in China.

Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus

We previously reported that mosquito densoviruses (MDVs) are potential vectors for delivering foreign nucleic acids into mosquito cells. However, considering existing expression strategies, recombinant viruses would inevitably become replication-defective viruses and lose their ability for secondary transmission. The packaging limitations of the virion represent a barrier for the development of MDVs for viral paratransgenesis or as high-efficiency bioinsecticides. Herein, we report the development of a non-defective recombinant Aedes aegypti densovirus (AaeDV) miRNA expression system, mediated by an artificial intron, using an intronic miRNA expression strategy. We demonstrated that this recombinant vector could be used to overexpress endogenous miRNAs or to decrease endogenous miRNAs by generating antisense sponges to explore the biological functions of miRNAs. In addition, the vector could express antisense-miRNAs to induce efficient gene silencing in vivo and in vitro. The recombinant virus effectively self-replicated and retained its secondary transmission ability, similar to the wild-type virus. The recombinant virus was also genetically stable. This study demonstrated the first construction of a non-defective recombinant MDV miRNA expression system, which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of viral paratransgenesis for dengue virus control.

piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues

The Asian tiger mosquito, Aedes albopictus, is a competent vector for the majority of arboviruses. The mosquito innate immune response is a primary determinant for arthropod-borne virus transmission, and the midgut is the first barrier to pathogen transmission. Mosquito antiviral immunity is primarily mediated by the small interfering RNA pathway. However, the roles that the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway play in antiviral immunity in Ae. albopictus and its midgut still need further exploration. This study aimed to explore the profiles of both viral-derived and host-originated piRNAs in the whole body and midgut infected with Dengue virus 2 (DENV-2) in Ae. albopictus, and to elucidate gene expression profile differences of the PIWI protein family between adult females and their midguts. A deep sequencing-based method was used to identify and analyze small non-coding RNAs, especially the piRNA profiles in DENV-2-infected Ae. albopictus and its midgut. The top-ranked, differentially-expressed piRNAs were further validated using Stem-loop qRT-PCR. Bioinformatics analyses and reverse-transcription PCR (RT-PCR) methods were used to detect PIWI protein family members, and their expression profiles. DENV-2 derived piRNAs (vpiRNA, 24–30 nts) were observed in both infected Ae. albopictus and its midgut; however, only vpiRNA in the whole-body library had a weak preference for adenine at position 10 (10A) in the sense molecules as a feature of secondary piRNA. These vpiRNAs were not equally distributed, instead they were derived from a few specific regions of the genome, especially several hot spots, and displayed an obvious positive strand bias. We refer to the differentially expressed host piRNAs after DENV infection as virus-induced host endogenous piRNAs (vepiRNAs). However, we found that vepiRNAs were abundant in mosquito whole-body tissue, but deficient in the midgut. A total of eleven PIWI family genes were identified in Ae. albopictus; however, only AalPiwi5–7 and AalAgo3(1–2) were readily detected in the midgut. The characteristics of piRNAs in DENV-2-infected Ae. albopictus adult females were similar to those previously described for flavivirus infections but were not observed in the midgut. The reduced levels of vepiRNAs and incomplete expression of PIWI pathway genes in midgut samples from DENV-2-infected Ae. albopictus suggests that viral regulation of host piRNAs may not be an important factor in the midgut.