Roles of a newly lethal cuticular structural protein, AaCPR100A, and its upstream interaction protein, G12-like, in Aedes aegypti.

Mosquitoes form a vital group of vector insects, which can transmit various diseases and filarial worms. The cuticle is a critical structure that protects mosquitoes from adverse environmental conditions and penetration resistance. Thus, cuticle proteins can be used as potential targets for controlling the mosquito population. In the present study, we found that AaCPR100A is a structural protein in the soft cuticle, which has flexibility and elasticity allowing insects to move or fly freely, of Aedes aegypti. RNA interference (RNAi) of AaCPR100A caused high mortality in Aedes aegypti larvae and adults and significantly decreased the egg hatching rate. Transmission electron microscopy (TEM) analysis revealed that the larval microstructure had no recognizable endocuticle in AaCPR100A-deficient mosquitoes. A yeast two-hybrid assay was performed to screen proteins interacting with AaCPR100A. We verified that the G12-like protein had the strongest interaction with AaCPR100A using yeast two-hybrid and GST pull-down assays. Knockdown of G12-like transcription resulted in high mortality in Ae. aegypti larvae, but not in adults. Interestingly, RNAi of G12-like rescued the high mortality of adults caused by decreased AaCPR100A expression. Additionally, adults treated with G12-like dsRNA were found to be sensitive to low temperature, and their eggshell formation and hatching were decreased. Overall, our results demonstrated that G12-like may interacts with AaCPR100A, and both G12-like and AaCPR100A are involved in Ae. aegypti cuticle development and eggshell formation. AaCPR100A and G12-like can thus be considered newly potential targets for controlling the Ae. aegypti mosquito.

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.

Pleiotropic Odorant-Binding Proteins Promote Aedes aegypti Reproduction and Flavivirus Transmission.

Insect odorant-binding proteins (OBPs) are small soluble proteins that have been assigned roles in olfaction, but their other potential functions have not been extensively explored. Using CRISPR/Cas9-mediated disruption of Aedes aegypti Obp10 and Obp22, we demonstrate the pleiotropic contribution of these proteins to multiple processes that are essential for vectorial capacity. Mutant mosquitoes have impaired host-seeking and oviposition behavior, reproduction, and arbovirus transmission. Here, we show that Obp22 is linked to the male-determining sex locus (M) on chromosome 1 and is involved in male reproduction, likely by mediating the development of spermatozoa. Although OBP10 and OBP22 are not involved in flavivirus replication, abolition of these proteins significantly reduces transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. These results extend our current understanding of the role of insect OBPs in insect reproduction and transmission of human pathogens, making them essential determinants of vectorial capacity. IMPORTANCE Aedes aegypti is the major vector for many arthropod-borne viral diseases, such as dengue, Zika, and chikungunya viruses. Previous studies suggested that odorant-binding proteins (OBPs) may have diverse physiological functions beyond the olfactory system in mosquitoes; however, these hypothesized functions have not yet been demonstrated. Here, we have used CRISPR/Cas9-based genome editing to functionally delete (knock out) Obp10 and Obp22 in Aedes aegypti. We showed that disruption of Obp10 or Obp22 significantly impairs female and male reproductive capacity by adversely affecting blood feeding, oviposition, fecundity and fertility, and the development of spermatozoa. We also showed that disruption of Obp10 or Obp22 significantly reduces the transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. Thus, our study is not only significant in understanding the functions of OBPs in mosquito biology, but also shows that OBPs may represent potent flavivirus transmission-blocking targets. Our study is in this regard particularly timely and important from a translational and public health perspective.

Brown marmorated stink bug, Halyomorpha halys (Stål), genome: putative underpinnings of polyphagy, insecticide resistance potential and biology of a top worldwide pest.

BACKGROUND: Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species' feeding and habitat traits, defining potential targets for pest management strategies. RESULTS: Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys' capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. CONCLUSIONS: Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.

Nix is a male-determining factor in the Asian tiger mosquito Aedes albopictus.

The initial signal that governs sex determination is highly variable among insects. A homolog of Nix, the male-determining factor in Aedes aegypti, was previously found in the Asian tiger mosquito Ae. albopictus. Here we show that the Ae. albopictus Nix (AalNix) is more complex in gene structure and splice isoforms than its Ae. aegypti homolog (AaeNix). AalNix shows a similar transcription profile compared to AaeNix. CRISPR/Cas9-mediated knockouts of AalNix in vivo and in the Ae. albopictus C6/36 cells lead to a shift of dsx and fru splicing towards the female isoforms. G0 knockout males showed feminization and deformities including feminized antennae, absence or partial absence of gonocoxites, gonostyli, testes and accessory glands, and the formation of ovaries. Despite ~70 MY of divergence, Nix functions as a conserved male-determining factor in the two most important arboviral vectors, namely Ae. aegypti and Ae. albopictus.

Guy1, a Y-linked embryonic signal, regulates dosage compensation in Anopheles stephensi by increasing X gene expression.

We previously showed that Guy1, a primary signal expressed from the Y chromosome, is a strong candidate for a male-determining factor that confers female-specific lethality in Anopheles stephensi (Criscione et al., 2016). Here, we present evidence that Guy1 increases X gene expression in Guy1-transgenic females from two independent lines, providing a mechanism underlying the Guy1-conferred female lethality. The median level gene expression (MGE) of X-linked genes is significantly higher than autosomal genes in Guy1-transgenic females while there is no significant difference in MGE between X and autosomal genes in wild-type females. Furthermore, Guy1 significantly upregulates at least 40% of the 996 genes across the X chromosome in transgenic females. Guy1-conferred female-specific lethality is remarkably stable and completely penetrant. These findings indicate that Guy1 regulates dosage compensation in An. stephensi and components of dosage compensation may be explored to develop novel strategies to control mosquito-borne diseases.

Pure early zygotic genes in the Asian malaria mosquito Anopheles stephensi.

BACKGROUND: The Asian malaria mosquito, Anopheles stephensi, is a major urban malaria vector in the Middle East and on the Indian subcontinent. Early zygotic transcription, which marks the maternal-to-zygotic transition, has not been systematically studied in An. stephensi or any other Anopheles mosquitoes. Improved understanding of early embryonic gene expression in An. stephensi will facilitate genetic and evolutionary studies and help with the development of novel control strategies for this important disease vector. RESULTS: We obtained RNA-seq data in biological triplicates from four early An. stephensi embryonic time points. Using these data, we identified 70 and 153 pure early zygotic genes (pEZGs) under stringent and relaxed conditions, respectively. We show that these pEZGs are enriched in functional groups related to DNA-binding transcription regulators, cell cycle modulators, proteases, transport, and cellular metabolism. On average these pEZGs are shorter and have less introns than other An. stephensi genes. Some of the pEZGs may arise de novo while others have clear non-pEZG paralogs. There is no or very limited overlap between An. stephensi pEZGs and Drosophila melanogaster or Aedes aegypti pEZGs. Interestingly, the upstream region of An. stephensi pEZGs lack significant enrichment of a previously reported TAGteam/VBRGGTA motif found in the regulatory region of pEZGs in D. melanogaster and Ae. aegypti. However, a GT-rich motif was found in An. stephensi pEZGs instead. CONCLUSIONS: We have identified a number of pEZGs whose predicted functions and structures are consistent with their collective roles in the degradation of maternally deposited components, activation of the zygotic genome, cell division, and metabolism. The pEZGs appear to rapidly turn over within the Dipteran order and even within the Culicidae family. These pEZGs, and the shared regulatory motif, could provide the promoter or regulatory sequences to drive gene expression in the syncytial or early cellular blastoderm, a period when the developing embryo is accessible to genetic manipulation. In addition, these molecular resources may be used to achieve sex separation of mosquitoes for sterile insect technique.

Functional analysis of the promoter of an early zygotic gene KLC2 in Aedes aegypti.

BACKGROUND: Aedes aegypti is an important mosquito vector that transmits arboviruses that cause devastating diseases including Zika, dengue fever, yellow fever and chikungunya. Improved understanding of gene regulation in the early development of Ae. aegypti will facilitate genetic studies and help the development of novel control strategies of this important disease vector. RESULTS: In this study, we demonstrated through transgenic assays that the promoter of an endogenous early zygotic gene KLC2 could drive gene expression in the syncytial blastoderm and early cellular blastoderm, which is a stage that the developing germline and the rest of embryo are accessible to genetic manipulation. An unexpected expression of the reporter gene in transgenic male testes was also observed. Further analysis confirmed the expression of the endogenous KLC2 in the testes, which was not detected in the previous RNA sequencing data. CONCLUSIONS: Our finding provided a new promoter element that can be used in future genetic studies and applications in Ae. aegypti. Moreover, our transgenic reporter assays showed that cautions are needed when interpreting RNA sequencing data as transient or tissue-specific transcription may go undetected by RNAseq.

Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes.

Y chromosomes control essential male functions in many species, including sex determination and fertility. However, because of obstacles posed by repeat-rich heterochromatin, knowledge of Y chromosome sequences is limited to a handful of model organisms, constraining our understanding of Y biology across the tree of life. Here, we leverage long single-molecule sequencing to determine the content and structure of the nonrecombining Y chromosome of the primary African malaria mosquito, Anopheles gambiae We find that the An. gambiae Y consists almost entirely of a few massively amplified, tandemly arrayed repeats, some of which can recombine with similar repeats on the X chromosome. Sex-specific genome resequencing in a recent species radiation, the An. gambiae complex, revealed rapid sequence turnover within An. gambiae and among species. Exploiting 52 sex-specific An. gambiae RNA-Seq datasets representing all developmental stages, we identified a small repertoire of Y-linked genes that lack X gametologs and are not Y-linked in any other species except An. gambiae, with the notable exception of YG2, a candidate male-determining gene. YG2 is the only gene conserved and exclusive to the Y in all species examined, yet sequence similarity to YG2 is not detectable in the genome of a more distant mosquito relative, suggesting rapid evolution of Y chromosome genes in this highly dynamic genus of malaria vectors. The extensive characterization of the An. gambiae Y provides a long-awaited foundation for studying male mosquito biology, and will inform novel mosquito control strategies based on the manipulation of Y chromosomes.

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

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.

Next-generation sequencing reveals recent horizontal transfer of a DNA transposon between divergent mosquitoes.

Horizontal transfer of genetic material between complex organisms often involves transposable elements (TEs). For example, a DNA transposon mariner has been shown to undergo horizontal transfer between different orders of insects and between different phyla of animals. Here we report the discovery and characterization of an ITmD37D transposon, MJ1, in Anopheles sinensis. We show that some MJ1 elements in Aedes aegypti and An. sinensis contain intact open reading frames and share nearly 99% nucleotide identity over the entire transposon, which is unexpectedly high given that these two genera had diverged 145-200 million years ago. Chromosomal hybridization and TE-display showed that MJ1 copy number is low in An. sinensis. Among 24 mosquito species surveyed, MJ1 is only found in Ae. aegypti and the hyrcanus group of anopheline mosquitoes to which An. sinensis belongs. Phylogenetic analysis is consistent with horizontal transfer and provides the basis for inference of its timing and direction. Although report of horizontal transfer of DNA transposons between higher eukaryotes is accumulating, our analysis is one of a small number of cases in which horizontal transfer of nearly identical TEs among highly divergent species has been thoroughly investigated and strongly supported. Horizontal transfer involving mosquitoes is of particular interest because there are ongoing investigations of the possibility of spreading pathogen-resistant genes into mosquito populations to control malaria and other infectious diseases. The initial indication of horizontal transfer of MJ1 came from comparisons between a 0.4x coverage An. sinensis 454 sequence database and available TEs in mosquito genomes. Therefore we have shown that it is feasible to use low coverage sequencing to systematically uncover horizontal transfer events. Expanding such efforts across a wide range of species will generate novel insights into the relative frequency of horizontal transfer of different TEs and provide the evolutionary context of these lateral transfer events.

Genome sequence of Aedes aegypti, a major arbovirus vector.

We present a draft sequence of the genome of Aedes aegypti, the primary vector for yellow fever and dengue fever, which at approximately 1376 million base pairs is about 5 times the size of the genome of the malaria vector Anopheles gambiae. Nearly 50% of the Ae. aegypti genome consists of transposable elements. These contribute to a factor of approximately 4 to 6 increase in average gene length and in sizes of intergenic regions relative to An. gambiae and Drosophila melanogaster. Nonetheless, chromosomal synteny is generally maintained among all three insects, although conservation of orthologous gene order is higher (by a factor of approximately 2) between the mosquito species than between either of them and the fruit fly. An increase in genes encoding odorant binding, cytochrome P450, and cuticle domains relative to An. gambiae suggests that members of these protein families underpin some of the biological differences between the two mosquito species.