RESUMO
Chemical insecticides remain the main strategy to combat mosquito-borne diseases, but the growing threat of insecticide resistance prompts the urgent need to develop alternative, ecofriendly, and sustainable vector control tools. Entomopathogenic fungi can overcome insecticide resistance and represent promising biocontrol tools for the control of mosquitoes. However, insects have evolved robust defense mechanisms against infection. Better understanding of mosquito defenses against fungal infection is critical for improvement of fungal efficacy. Here, we show that as the pathogenic fungus Beauveria bassiana penetrates into the host hemocoel, mosquitoes increase expression of the let-7 and miR-100 microRNAs (miRNAs). Both miRNAs translocate into fungal hyphae to specifically silence the virulence-related genes sec2p and C6TF, encoding a Rab guanine nucleotide exchange factor and a Zn(II)2Cys6 transcription factor, respectively. Inversely, expression of a let-7 sponge (anti-let-7) or a miR-100 sponge (anti-miR-100) in the fungus efficiently sequesters the corresponding translocated host miRNA. Notably, B. bassiana strains expressing anti-let-7 and anti-miR-100 are markedly more virulent to mosquitoes. Our findings reveal an insect defense strategy that employs miRNAs to induce cross-kingdom silencing of pathogen virulence-related genes, conferring resistance to infection.
Assuntos
Anopheles/genética , Beauveria/genética , Perfilação da Expressão Gênica/métodos , Resistência a Inseticidas/genética , MicroRNAs/genética , Animais , Anopheles/microbiologia , Sequência de Bases , Beauveria/patogenicidade , Feminino , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno/genética , Hifas/genética , Hifas/patogenicidade , Mutação , Homologia de Sequência do Ácido Nucleico , Esporos Fúngicos/genética , Esporos Fúngicos/patogenicidade , Virulência/genéticaRESUMO
Juvenile hormone (JH) receptors, methoprene-tolerant (Met) and Germ-cell expressed (Gce), transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with PAS-B and basic helix-loop-helix domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of fat body cells of the larval when the JH titer is low and JH induces Met nuclear import. Hsp83 was accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuated JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Choque Térmico/metabolismo , Hormônios Juvenis/metabolismo , Metoprene/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Animais , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Proteínas de Choque Térmico/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Oxidative phosphorylation, essential for energy metabolism and linked to the regulation of longevity, involves mitochondrial and nuclear genes. The functions of these genes and their evolutionary rate covariation (ERC) have been extensively studied, but little is known about whether other nuclear genes not targeted to mitochondria evolutionarily and functionally interact with mitochondrial genes. Here we systematically examined the ERC of mitochondrial and nuclear benchmarking universal single-copy ortholog (BUSCO) genes from 472 insects, identifying 75 non-mitochondria-targeted nuclear genes. We found that the uncharacterized gene CG11837-a putative ortholog of human DIMT1-regulates insect lifespan, as its knockdown reduces median lifespan in five diverse insect species and Caenorhabditis elegans, whereas its overexpression extends median lifespans in fruit flies and C. elegans and enhances oxidative phosphorylation gene activity. Additionally, DIMT1 overexpression protects human cells from cellular senescence. Together, these data provide insights into the ERC of mito-nuclear genes and suggest that CG11837 may regulate longevity across animals.
Assuntos
Caenorhabditis elegans , Evolução Molecular , Longevidade , Animais , Longevidade/genética , Humanos , Caenorhabditis elegans/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Fosforilação Oxidativa , Insetos/genética , Genoma de Inseto/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Senescência Celular/genéticaRESUMO
Anopheles mosquitoes are the primary vectors for the transmission of malaria parasites, which poses a devastating burden on global public health and welfare. The recent invasion of Anopheles stephensi in Africa has made malaria eradication more challenging due to its outdoor biting behavior and widespread resistance to insecticides. To address this issue, we developed a new approach for mosquito larvae control using gut microbiota-mediated RNA interference (RNAi). We engineered a mosquito symbiotic gut bacterium, Serratia fonticola, by deleting its RNase III gene to produce double-stranded RNAs (dsRNAs) in the mosquito larval gut. We found that the engineered S. fonticola strains can stably colonize mosquito larval guts and produce dsRNAs dsMet or dsEcR to activate RNAi and effectively suppress the expression of methoprene-tolerant gene Met and ecdysone receptor gene EcR, which encode receptors for juvenile hormone and ecdysone pathways in mosquitoes, respectively. Importantly, the engineered S. fonticola strains markedly inhibit the development of A. stephensi larvae and leads to a high mortality, providing an effective dsRNA delivery system for silencing genes in insects and a novel RNAi-mediated pest control strategy. Collectively, our symbiont-mediated RNAi (smRNAi) approach offers an innovative and sustainable method for controlling mosquito larvae and provides a promising strategy for combating malaria. IMPORTANCE Mosquitoes are vectors for various diseases, imposing a significant threat to public health globally. The recent invasion of A. stephensi in Africa has made malaria eradication more challenging due to its outdoor biting behavior and widespread resistance to insecticides. RNA interference (RNAi) is a promising approach that uses dsRNA to silence specific genes in pests. This study presents the use of a gut symbiotic bacterium, Serratia fonticola, as an efficient delivery system of dsRNA for RNAi-mediated pest control. The knockout of RNase III, a dsRNA-specific endonuclease gene, in S. fonticola using CRISPR-Cas9 led to efficient dsRNA production. Engineered strains of S. fonticola can colonize the mosquito larval gut and effectively suppress the expression of two critical genes, Met and EcR, which inhibit mosquito development and cause high mortality in mosquito larvae. This study highlights the potential of exploring the mosquito microbiota as a source of dsRNA for RNAi-based pest control.
Assuntos
Anopheles , Inseticidas , Malária , Animais , Interferência de RNA , Anopheles/genética , Anopheles/parasitologia , Larva/genética , Ribonuclease III/metabolismo , Mosquitos Vetores/genética , RNA de Cadeia Dupla , Malária/prevenção & controleRESUMO
The growing threat of insecticide resistance prompts the urgent need to develop additional tools for mosquito control. Entomopathogenic fungi provide an eco-friendly alternative to chemical insecticides. One limitation to the use of mycoinsecticides is their relatively low virulence. Here, we report an approach for suppressing mosquito immunity and increasing fungal virulence. We engineered Beauveria bassiana to express Aedes immunosuppressive microRNAs (miRNAs) to induce host RNA interference (RNAi) immune responses. We show that engineered strains can produce and deliver the miRNAs into host cells to activate cross-kingdom RNAi during infection and suppress mosquito immunity by targeting multiple host genes, thereby dramatically increasing fungal virulence against Aedes aegypti and Galleria mellonella larvae. Importantly, expressing host miRNAs also significantly increases fungal virulence against insecticide-resistant mosquitoes, creating potential for insecticide-resistance management. This pathogen-mediated RNAi (pmRNAi)-based approach provides an innovative strategy to enhance the efficacy of fungal insecticides and eliminate the likelihood of resistance development.
Assuntos
Aedes , Beauveria , Inseticidas , MicroRNAs , Animais , Inseticidas/farmacologia , Interferência de RNA , MicroRNAs/genética , Controle de Mosquitos , Aedes/genética , Beauveria/genéticaRESUMO
Anopheles mating is initiated by the swarming of males at dusk followed by females flying into the swarm. Here, we show that mosquito swarming and mating are coordinately guided by clock genes, light, and temperature. Transcriptome analysis shows up-regulation of the clock genes period (per) and timeless (tim) in the head of field-caught swarming Anopheles coluzzii males. Knockdown of per and tim expression affects Anopheles gambiae s.s. and Anopheles stephensi male mating in the laboratory, and it reduces male An. coluzzii swarming and mating under semifield conditions. Light and temperature affect mosquito mating, possibly by modulating per and/or tim expression. Moreover, the desaturase gene desat1 is up-regulated and rhythmically expressed in the heads of swarming males and regulates the production of cuticular hydrocarbons, including heptacosane, which stimulates mating activity.
Assuntos
Anopheles/fisiologia , Proteínas CLOCK/fisiologia , Voo Animal , Interação Gene-Ambiente , Proteínas Circadianas Period/fisiologia , Feromônios/biossíntese , Comportamento Sexual Animal , Animais , Anopheles/genética , Proteínas CLOCK/genética , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Luz , Masculino , Proteínas Circadianas Period/genética , Temperatura , TranscriptomaRESUMO
Mosquito-transmitted diseases account for about 500 000 deaths every year. Blocking these pathogens in the mosquito vector before they are transmitted to humans is an effective strategy to prevent mosquito-borne diseases. Like most higher organisms, mosquitoes harbor a highly diverse and dynamic microbial flora that can be explored for prevention of pathogen transmission. Here we review the structure and function of the mosquito microbiota, including bacteria, fungi, and viruses, and discuss the potential of using components of the microbiota to thwart pathogen transmission.
Assuntos
Culicidae/microbiologia , Culicidae/virologia , Microbiota/fisiologia , Mosquitos Vetores/microbiologia , Mosquitos Vetores/virologia , Doenças Transmitidas por Vetores/prevenção & controle , Animais , Fenômenos Fisiológicos Bacterianos , Fungos/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Doenças Transmitidas por Vetores/microbiologia , Doenças Transmitidas por Vetores/parasitologia , Doenças Transmitidas por Vetores/virologia , Fenômenos Fisiológicos ViraisRESUMO
Insecticidal fungi represent a promising alternative to chemical pesticides for disease vector control. Here, we show that the pathogenic fungus Beauveria bassiana exports a microRNA-like RNA (bba-milR1) that hijacks the host RNA-interference machinery in mosquito cells by binding to Argonaute 1 (AGO1). bba-milR1 is highly expressed during fungal penetration of the mosquito integument, and suppresses host immunity by silencing expression of the mosquito Toll receptor ligand Spätzle 4 (Spz4). Later, upon entering the hemocoel, bba-milR1 expression is decreased, which avoids induction of the host proteinase CLIPB9 that activates the melanization response. Thus, our results indicate that the pathogen deploys a cross-kingdom small-RNA effector that attenuates host immunity and facilitates infection.
Assuntos
Beauveria/imunologia , Interações Hospedeiro-Patógeno/imunologia , MicroRNAs/metabolismo , Mosquitos Vetores/imunologia , Mosquitos Vetores/microbiologia , Animais , Anopheles/imunologia , Anopheles/microbiologia , Beauveria/patogenicidade , Feminino , Perfilação da Expressão Gênica , Sistema Imunitário/imunologia , Infecções , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Malária/imunologia , Controle Biológico de Vetores , Interferência de RNA , RNA de Cadeia DuplaRESUMO
Beauveria bassiana is an environmentally friendly alternative to chemical insecticides against various agricultural insect pests and vectors of human diseases. However, its application has been limited due to slow kill and sensitivity to abiotic stresses. Understanding of the molecular pathogenesis and physiological characteristics would facilitate improvement of the fungal performance. Loss-of-function mutagenesis is the most powerful tool to characterize gene functions, but it is hampered by the low rate of homologous recombination and the limited availability of selectable markers. Here, by combining the use of uridine auxotrophy as recipient and donor DNAs harboring auxotrophic complementation gene ura5 as a selectable marker with the blastospore-based transformation system, we established a highly efficient, low false-positive background and cost-effective CRISPR/Cas9-mediated gene editing system in B. bassiana. This system has been demonstrated as a simple and powerful tool for targeted gene knock-out and/or knock-in in B. bassiana in a single gene disruption. We further demonstrated that our system allows simultaneous disruption of multiple genes via homology-directed repair in a single transformation. This technology will allow us to study functionally redundant genes and holds significant potential to greatly accelerate functional genomics studies of B. bassiana.