Tony Nolan

Stable germline transformation of the malaria mosquito Anopheles stephensi

Anopheline mosquito species are obligatory vectors for human malaria, an infectious disease that affects hundreds of millions of people living in tropical and subtropical countries. The lack of a suitable gene transfer technology for these mosquitoes has hampered the molecular genetic analysis of their physiology, including the molecular interactions between the vector and the malaria parasite. Here we show that a transposon, based on the Minos element and bearing exogenous DNA, can integrate efficiently and stably into the germ line of the human malaria vector Anopheles stephensi , through a transposase-mediated process.

A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae

Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.

Bee Venom Phospholipase Inhibits Malaria Parasite Development in Transgenic Mosquitoes

Malaria kills millions of people every year, and new control measures are urgently needed. The recent demonstration that (effector) genes can be introduced into the mosquito germ line to diminish their ability to transmit the malaria parasite offers new hope toward the fight of the disease (Ito, J., Ghosh, A., Moreira, L. A., Wimmer, E. A. & Jacobs-Lorena, M. (2002)Nature, 417, 452–455). Because of the high selection pressure that an effector gene imposes on the parasite population, development of resistant strains is likely to occur. In search of additional antiparasitic effector genes, we have generated transgenicAnopheles stephensi mosquitoes that express the bee venom phospholipase A2 (PLA2) gene from the gut-specific and blood-inducible Anopheles gambiaecarboxypeptidase (AgCP) promoter. Northern blot analysis indicated that the PLA2 mRNA is specifically expressed in the guts of transgenic mosquitoes with peak expression at ∼4 h after blood ingestion. Western blot and immunofluorescence analyses detected PLA2 protein in the midgut epithelia of transgenic mosquitoes from 8 to 24 h after a blood meal. Importantly, transgene expression reducedPlasmodium berghei oocyst formation by 87% on average and greatly impaired transmission of the parasite to naive mice. The results indicate that PLA2 may be used as an additional effector gene to block the development of the malaria parasite in mosquitoes.

A comprehensive gene expression atlas of sex- and tissue-specificity in the malaria vector, Anopheles gambiae

BackgroundThe mosquito, Anopheles gambiae, is the primary vector of human malaria, a disease responsible for millions of deaths each year. To improve strategies for controlling transmission of the causative parasite, Plasmodium falciparum, we require a thorough understanding of the developmental mechanisms, physiological processes and evolutionary pressures affecting life-history traits in the mosquito. Identifying genes expressed in particular tissues or involved in specific biological processes is an essential part of this process.ResultsIn this study, we present transcription profiles for ~82% of annotated Anopheles genes in dissected adult male and female tissues. The sensitivity afforded by examining dissected tissues found gene activity in an additional 20% of the genome that is undetected when using whole-animal samples. The somatic and reproductive tissues we examined each displayed patterns of sexually dimorphic and tissue-specific expression. By comparing expression profiles with Drosophila melanogaster we also assessed which genes are well conserved within the Diptera versus those that are more recently evolved.ConclusionsOur expression atlas and associated publicly available database, the MozAtlas (http://www.tissue-atlas.org), provides information on the relative strength and specificity of gene expression in several somatic and reproductive tissues, isolated from a single strain grown under uniform conditions. The data will serve as a reference for other mosquito researchers by providing a simple method for identifying where genes are expressed in the adult, however, in addition our resource will also provide insights into the evolutionary diversity associated with gene expression levels among species.

The post-transcriptional gene silencing machinery functions independently of DNA methylation to repress a LINE1-like retrotransposon in Neurospora crassa

Post-transcriptional gene silencing (PTGS) involving small interfering RNA (siRNA)-directed degradation of RNA transcripts and transcriptional silencing via DNA methylation have each been proposed as mechanisms of genome defence against invading nucleic acids, such as transposons and viruses. Furthermore, recent data from plants indicates that many transposons are silenced via a combination of the two mechanisms, and siRNAs can direct methylation of transposon sequences. We investigated the contribution of DNA methylation and the PTGS pathway to transposon control in the filamentous fungus Neurospora crassa. We found that repression of the LINE1-like transposon, Tad, requires the Argonaute protein QDE2 and Dicer, each of which are required for transgene-induced PTGS (quelling) in N.crassa. Interestingly, unlike quelling, the RNA-dependent RNA polymerase QDE1 and the RecQ DNA helicase QDE3 were not required for Tad control, suggesting the existence of specialized silencing pathways for diverse kinds of repetitive elements. In contrast, Tad elements were not significantly methylated and the DIM2 DNA methyltransferase, responsible for all known DNA methylation in Neurospora, had no effect on Tad control. Thus, an RNAi-related transposon silencing mechanism operates during the vegetative phase of N.crassa that is independent of DNA methylation, highlighting a major difference between this organism and other methylation-proficient species.

Transcription Regulation of Sex-Biased Genes during Ontogeny in the Malaria Vector Anopheles gambiae

In Anopheles gambiae, sex-regulated genes are responsible for controlling gender dimorphism and are therefore crucial in determining the ability of female mosquitoes to transmit human malaria. The identification and functional characterization of these genes will shed light on the sexual development and maturation of mosquitoes and provide useful targets for genetic control measures aimed at reducing mosquito fertility and/or distorting the sex ratio. We conducted a genome wide transcriptional analysis of sex-regulated genes from early developmental stages through adulthood combined with functional screening of novel gonadal genes. Our results demonstrate that the male-biased genes undergo a major transcription turnover starting from larval stages to adulthood. The male biased genes at the adult stage include a significant high number of unique sequences compared to the rest of the genome. This is in contrast to female-biased genes that are much more conserved and are mainly activated during late developmental stages. The high frequency of unique sequences would indicate that male-biased genes evolve more rapidly than the rest of the genome. This finding is particularly intriguing because A. gambiae is a strictly female monogamous species suggesting that driving forces in addition to sperm competition must account for the rapid evolution of male-biased genes. We have also identified and functionally characterized a number of previously unknown A. gambiae testis- and ovary-specific genes. Two of these genes, zero population growth and a suppressor of defective silencing 3 domain of the histone deacetylase co-repressor complex, were shown to play a key role in gonad development.

An Anopheles gambiae salivary gland promoter analysis in Drosophila melanogaster and Anopheles stephensi.

Regulatory regions driving gene expression in specific target organs of the African malaria vector Anopheles gambiae are of critical relevance for studies on Plasmodium–Anopheles interactions as well as to devise strategies for blocking malaria parasite development in the mosquito. In order to identify an appropriate salivary gland promoter we analysed the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland‐specific genes AgApy and D7r4. An 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic Anopheles stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal‐rather than in the distal‐lateral lobes of female glands. Surprisingly, a promoter fragment from the D7r4 gene conferred strong tissue‐specific expression in Drosophila melanogaster but only low transcription levels in transgenic An. stephensi. These results imply a certain conservation of gland‐specific control elements between the fruit fly and the mosquito suggesting that an increased degree of complexity, probably connected to the evolution of haematophagy, underlies the regulation of tissue‐specific expression in mosquito female salivary glands.

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

Significance Interest in male mosquitoes has been motivated by the potential to develop novel vector control strategies, exploiting the fact that males do not feed on blood or transmit diseases, such as malaria. However, genetic studies of male Anopheles mosquitoes have been impeded by the lack of molecular characterization of the Y chromosome. Here we show that the Anopheles gambiae Y chromosome contains a very small repertoire of genes, with massively amplified tandem arrays of a small number of satellites and transposable elements constituting the vast majority of the sequence. These genes and repeats evolve rapidly, bringing about remodeling of the Y, even among closely related species. Our study provides a long-awaited foundation for studying mosquito Y chromosome biology and evolution. 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.

The RNA-dependent RNA polymerase essential for post-transcriptional gene silencing in Neurospora crassa interacts with replication protein A

Post-transcriptional gene silencing (PTGS) pathways play a role in genome defence and have been extensively studied, yet how repetitive elements in the genome are identified is still unclear. It has been suggested that they may produce aberrant transcripts (aRNA) that are converted by an RNA-dependent RNA polymerase (RdRP) into double-stranded RNA (dsRNA), the essential intermediate of PTGS. However, how RdRP enzymes recognize aberrant transcripts remains a key question. Here we show that in Neurospora crassa the RdRP QDE-1 interacts with Replication Protein A (RPA), part of the DNA replication machinery. We show that both QDE-1 and RPA are nuclear proteins and that QDE-1 is specifically recruited onto the repetitive transgenic loci. We speculate that this localization of QDE-1 could allow the in situ production of dsRNA using transgenic nascent transcripts as templates, as in other systems. Supporting a link between the two proteins, we found that the accumulation of short interfering RNAs (siRNAs), the hallmark of silencing, is dependent on an ongoing DNA synthesis. The interaction between QDE-1 and RPA is important since it should guide further studies aimed at understanding the specificity of the RdRP and it provides for the first time a potential link between a PTGS component and the DNA replication machinery.

Analysis of Two Novel Midgut-Specific Promoters Driving Transgene Expression in Anopheles stephensi Mosquitoes

Background Tissue-specific promoters controlling the expression of transgenes in Anopheles mosquitoes represent a valuable tool both for studying the interaction between these malaria vectors and the Plasmodium parasites they transmit and for novel malaria control strategies based on developing Plasmodium-refractory mosquitoes by expressing anti-parasitic genes. With this aim we have studied the promoter regions of two genes from the most important malaria vector, Anopheles gambiae, whose expression is strongly induced upon blood feeding. Results We analysed the A. gambiae Antryp1 and G12 genes, which we have shown to be midgut-specific and maximally expressed at 24 hours post-bloodmeal (PBM). Antryp1, required for bloodmeal digestion, encodes one member of a family of 7 trypsin genes. The G12 gene, of unknown function, was previously identified in our laboratory in a screen for genes induced in response to a bloodmeal. We fused 1.1 kb of the upstream regions containing the putative promoter of these genes to reporter genes and transformed these into the Indian malaria vector A. stephensi to see if we could recapitulate the expression pattern of the endogenous genes. Both the Antryp1 and G12 upstream regions were able to drive female-predominant, midgut-specific expression in transgenic mosquitoes. Expression of the Antryp1-driven reporter in transgenic A. stephensi lines was low, undetectable by northern blot analysis, and failed to fully match the induction kinetics of the endogenous Antryp1 gene in A. gambiae. This incomplete conservation of expression suggests either subtle differences in the transcriptional machinery between A. stephensi and A. gambiae or that the upstream region chosen lacked all the control elements. In contrast, the G12 upstream region was able to faithfully reproduce the expression profile of the endogenous A. gambiae gene, showing female midgut specificity in the adult mosquito and massive induction PBM, peaking at 24 hours. Conclusions Our studies on two putative blood-meal induced, midgut-specific promoters validate the use of G12 upstream regulatory regions to drive targeted transgene expression coinciding spatially and temporally with pre-sporogonic stages of Plasmodium parasites in the mosquito, offering the possibility of manipulating vector competence or performing functional studies on vector-parasite interactions.