RESUMO
As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ('dead') Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.
Assuntos
Aedes , Animais , Humanos , Proteínas Hedgehog/metabolismo , Mosquitos Vetores/genética , RNA/metabolismo , Ativação Transcricional , Sistemas CRISPR-CasRESUMO
Insects naturally harbor a broad range of selfish agents that can manipulate their reproduction and development, often leading to host sex ratio distortion. Such effects directly benefit the spread of the selfish agents. These agents include two broad groups: bacterial symbionts and selfish chromosomes. Recent studies have made steady progress in uncovering the cellular targets of these agents and their effector genes. Here we highlight what is known about the targeted developmental processes, developmental timing, and effector genes expressed by several selfish agents. It is now becoming apparent that: (1) the genetic toolkits used by these agents to induce a given reproductive manipulation are simple, (2) these agents target sex-specific cellular processes very early in development, and (3) in some cases, similar processes are targeted. Knowledge of the molecular underpinnings of these systems will help to solve long-standing puzzles and provide new tools for controlling insect pests.
Assuntos
Reprodução , Razão de Masculinidade , Animais , Bactérias , Cromossomos , Feminino , Insetos/genética , MasculinoRESUMO
Day length (photoperiod) and temperature oscillate daily and seasonally and are important cues for season-dependent behavior. Larval diapause of the parasitoid Nasonia vitripennis is maternally induced following a certain number of days (switch point) of a given critical photoperiod (CPP). Both the switch point and the CPP follow a latitudinal cline in European N. vitripennis populations. We previously showed that allelic frequencies of the clock gene period correlate with this diapause induction cline. Here we report that circadian expression of four clock genes-period (per), cryptochrome-2 (cry-2), clock (clk), and cycle (cyc)-oscillates as a function of photoperiod and latitude of origin in wasps from populations from the extremes of the cline. Expression amplitudes are lower in northern wasps, indicating a weaker, more plastic clock. Northern wasps also have a later onset of activity and longer free-running rhythms under constant conditions. RNA interference of per caused speeding up of the circadian clock, changed the expression of other clock genes, and delayed diapause in both southern and northern wasps. These results point toward adaptive latitudinal clock gene expression differences and to a key role of per in the timing of photoperiodic diapause induction of N. vitripennis.
Assuntos
Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Ritmo Circadiano , Diapausa de Inseto , Vespas/metabolismo , Animais , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fotoperíodo , Interferência de RNA , Vespas/genéticaRESUMO
UNLABELLED: A sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. Here we show that the Hofbauer-Buchner eyelets differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)- and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, we show that the large LNvs interact with subsets of "evening cells" to adjust the timing of the evening peak of activity in a day length-dependent manner. Our work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms. SIGNIFICANCE STATEMENT: In animals, circadian clocks have evolved to orchestrate the timing of behavior and metabolism. Consistent timing requires the entrainment these clocks to the solar day, a process that is critical for an organism's health. Light cycles are the most important external cue for the entrainment of circadian clocks, and the circadian system uses multiple photoreceptors to link timekeeping to the light/dark cycle. How light information from these photorecptors is integrated into the circadian clock neuron network to support entrainment is not understood. Our results establish that input from the HB eyelets differentially impacts the physiology of neuronal subgroups. This input pathway, together with input from the compound eyes, precisely times the activity of flies under long summer days. Our results provide a mechanistic model of light transduction and integration into the circadian system, identifying new and unexpected network motifs within the circadian clock neuron network.
Assuntos
Ritmo Circadiano/fisiologia , Atividade Motora/fisiologia , Neurônios/fisiologia , Fotoperíodo , Células Fotorreceptoras de Invertebrados/fisiologia , Sono/fisiologia , Animais , Animais Geneticamente Modificados , Antígenos CD4/genética , Antígenos CD4/metabolismo , Cálcio/metabolismo , Ritmo Circadiano/genética , Criptocromos , AMP Cíclico/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histamina/farmacologia , Luz , Atividade Motora/genética , Rede Nervosa/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Interferência de RNA/fisiologia , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X2/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Sono/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vias Visuais/fisiologiaRESUMO
Each year, hundreds of millions of people are infected with arboviruses such as dengue, yellow fever, chikungunya, and Zika, which are all primarily spread by the notorious mosquito Aedes aegypti. Traditional control measures have proven insuficient, necessitating innovations. In response, here we generate a next generation CRISPR-based precision-guided sterile insect technique (pgSIT) for Aedes aegypti that disrupts genes essential for sex determination and fertility, producing predominantly sterile males that can be deployed at any life stage. Using mathematical models and empirical testing, we demonstrate that released pgSIT males can effectively compete with, suppress, and eliminate caged mosquito populations. This versatile species-specific platform has the potential for field deployment to control wild populations, safely curtailing disease transmission.
RESUMO
Escalating vector disease burdens pose significant global health risks, as such innovative tools for targeting mosquitoes are critical. CRISPR-Cas technologies have played a crucial role in developing powerful tools for genome manipulation in various eukaryotic organisms. Although considerable efforts have focused on utilizing class II type II CRISPR-Cas9 systems for DNA targeting, these modalities are unable to target RNA molecules, limiting their utility against RNA viruses. Recently, the Cas13 family has emerged as an efficient tool for RNA targeting; however, the application of this technique in mosquitoes, particularly Aedes aegypti, has yet to be fully realized. In this study, we engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 and its potent collateral activity. REAPER remains concealed within the mosquito until an infectious blood meal is uptaken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13-mediated RNA targeting significantly reduces viral replication and viral prevalence of infection, and its promiscuous collateral activity can even kill infected mosquitoes within a few days. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.
Assuntos
Culicidae , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes , Mosquitos Vetores/genética , RNA Viral/genética , Antivirais/farmacologiaRESUMO
The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Microinjeções , Interferência de RNA , Vespas/genética , Animais , Cruzamentos Genéticos , Feminino , Testes Genéticos , Masculino , Mutação/genética , Agulhas , Pupa/genética , Análise de SobrevidaRESUMO
Numerous plants and animals harbor selfish B chromosomes that "drive" or transmit themselves at super-Mendelian frequencies, despite long-term fitness costs to the organism. Currently, it is unknown how B chromosome drive is mediated, and whether B-gene expression plays a role. We used modern sequencing technologies to analyze the fine-scale sequence composition and expression of paternal sex ratio (PSR), a B chromosome in the jewel wasp Nasonia vitripennis. PSR causes female-to-male conversion by destroying the sperm's hereditary material in young embryos to drive. Using RNA interference, we demonstrate that testis-specific expression of a PSR-linked gene, named haploidizer, facilitates this genome elimination-and-sex conversion effect. haploidizer encodes a putative protein with a DNA binding domain, suggesting a functional link with the sperm-derived chromatin.
Assuntos
Cromossomos , Evolução Molecular , Expressão Gênica , Genoma , Animais , Mapeamento Cromossômico , Biologia Computacional/métodos , Feminino , Genômica/métodos , Hibridização in Situ Fluorescente , Masculino , Anotação de Sequência Molecular , Interferência de RNA , Testículo/metabolismoRESUMO
B chromosomes are enigmatic heritable elements found in the genomes of numerous plant and animal species. Contrary to their broad distribution, most B chromosomes are non-essential. For this reason, they are regarded as genome parasites. In order to be stably transmitted through generations, many B chromosomes exhibit the ability to "drive", i.e., they transmit themselves at super-Mendelian frequencies to progeny through directed interactions with the cell division apparatus. To date, very little is understood mechanistically about how B chromosomes drive, although a likely scenario is that expression of B chromosome sequences plays a role. Here, we highlight a handful of previously identified B chromosome sequences, many of which are repetitive and non-coding in nature, that have been shown to be expressed at the transcriptional level. We speculate on how each type of expressed sequence could participate in B chromosome drive based on known functions of RNA in general chromatin- and chromosome-related processes. We also raise some challenges to functionally testing these possible roles, a goal that will be required to more fully understand whether and how B chromosomes interact with components of the cell for drive and transmission.
Assuntos
Cromossomos/genética , Animais , Cromossomos/metabolismo , Elementos de DNA Transponíveis , Evolução Molecular , Fases de Leitura Aberta , Sequências Repetitivas de Ácido NucleicoRESUMO
Many physiological processes of living organisms show circadian rhythms, governed by an endogenous clock. This clock has a genetic basis and is entrained by external cues, such as light and temperature. Other physiological processes exhibit seasonal rhythms, that are also responsive to light and temperature. We previously reported a natural latitudinal cline of photoperiodic diapause induction in the parasitic wasp Nasonia vitripennis in Europe and a correlated haplotype frequency for the circadian clock gene period (per). To evaluate if this correlation is reflected in circadian behaviour, we investigated the circadian locomotor activity of seven populations from the cline. We found that the proportion of rhythmic males was higher than females in constant darkness, and that mating decreased rhythmicity of both sexes. Only for virgin females, the free running period (τ) increased weakly with latitude. Wasps from the most southern locality had an overall shorter free running rhythm and earlier onset, peak, and offset of activity during the 24 h period, than wasps from the northernmost locality. We evaluated this variation in rhythmicity as a function of period haplotype frequencies in the populations and discussed its functional significance in the context of local adaptation.
RESUMO
The circadian clock is a timekeeper but also helps adapt physiology to the outside world. This is because an essential feature of clocks is their ability to adjust (entrain) to the environment, with light being the most important signal. Whereas cryptochrome-mediated entrainment is well understood in Drosophila, integration of light information via the visual system lacks a neuronal or molecular mechanism. Here, we show that a single photoreceptor subtype is essential for long-day adaptation. These cells activate key circadian neurons, namely the large ventral-lateral neurons (lLNvs), which release the neuropeptide pigment-dispersing factor (PDF). RNAi and rescue experiments show that PDF from these cells is necessary and sufficient for delaying the timing of the evening (E) activity in long-day conditions. This contrasts to PDF that derives from the small ventral-lateral neurons (sLNvs), which are essential for constant darkness (DD) rhythmicity. Using a cell-specific CRISPR/Cas9 assay, we show that lLNv-derived PDF directly interacts with neurons important for E activity timing. Interestingly, this pathway is specific for long-day adaptation and appears to be dispensable in equinox or DD conditions. The results therefore indicate that external cues cause a rearrangement of neuronal hierarchy, which contributes to behavioral plasticity.
Assuntos
Relógios Circadianos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Neurônios/fisiologia , Neuropeptídeos/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Neuropeptídeos/metabolismo , Interferência de RNARESUMO
The genus Drosophila contains over 2,000 species that, stemming from a common ancestor in the Old World Tropics, populate today very different environments [1, 2] (reviewed in [3]). We found significant differences in the activity pattern of Drosophila species belonging to the holarctic virilis group, i.e., D. ezoana and D. littoralis, collected in Northern Europe, compared to that of the cosmopolitan D. melanogaster, collected close to the equator. These behavioral differences might have been of adaptive significance for colonizing high-latitude habitats and hence adjust to long photoperiods. Most interestingly, the flies' locomotor activity correlates with the neurochemistry of their circadian clock network, which differs between low and high latitude for the expression pattern of the blue light photopigment cryptochrome (CRY) and the neuropeptide Pigment-dispersing factor (PDF) [4-6]. In D. melanogaster, CRY and PDF are known to modulate the timing of activity and to maintain robust rhythmicity under constant conditions [7-11]. We could partly simulate the rhythmic behavior of the high-latitude virilis group species by mimicking their CRY/PDF expression patterns in a laboratory strain of D. melanogaster. We therefore suggest that these alterations in the CRY/PDF clock neurochemistry might have allowed the virilis group species to colonize high-latitude environments.