RESUMEN
Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile insect technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.
Asunto(s)
Anopheles , Infertilidad Masculina , Malaria , Humanos , Animales , Masculino , Femenino , Anopheles/genética , Control de Mosquitos/métodos , Mosquitos Vectores/genética , Semen , ARN Guía de Sistemas CRISPR-Cas , Infertilidad Masculina/genética , Mutagénesis , Células GerminativasRESUMEN
Anopheles gambiae and its sibling species Anopheles coluzzii are the most efficient vectors of the malaria parasite Plasmodium falciparum. When females of these species feed on an infected human host, oogenesis and parasite development proceed concurrently, but interactions between these processes are not fully understood. Using multiple natural P. falciparum isolates from Burkina Faso, we show that in both vectors, impairing steroid hormone signaling to disrupt oogenesis leads to accelerated oocyst growth and in a manner that appears to depend on both parasite and mosquito genotype. Consistently, we find that egg numbers are negatively linked to oocyst size, a metric for the rate of oocyst development. Oocyst growth rates are also strongly accelerated in females that are in a pre-gravid state, i.e. that fail to develop eggs after an initial blood meal. Overall, these findings advance our understanding of mosquito-parasite interactions that influence P. falciparum development in malaria-endemic regions.
Asunto(s)
Anopheles , Malaria Falciparum , Malaria , Animales , Femenino , Humanos , Plasmodium falciparum , Anopheles/parasitología , Mosquitos Vectores , Interacciones Huésped-Parásitos , Malaria Falciparum/parasitología , Malaria/parasitología , OocistosRESUMEN
Females from many mosquito species feed on blood to acquire nutrients for egg development. The oogenetic cycle has been characterized in the arboviral vector Aedes aegypti, where after a bloodmeal, the lipid transporter lipophorin (Lp) shuttles lipids from the midgut and fat body to the ovaries, and a yolk precursor protein, vitellogenin (Vg), is deposited into the oocyte by receptor-mediated endocytosis. Our understanding of how the roles of these two nutrient transporters are mutually coordinated is however limited in this and other mosquito species. Here, we demonstrate that in the malaria mosquito Anopheles gambiae, Lp and Vg are reciprocally regulated in a timely manner to optimize egg development and ensure fertility. Defective lipid transport via Lp knockdown triggers abortive ovarian follicle development, leading to misregulation of Vg and aberrant yolk granules. Conversely, depletion of Vg causes an upregulation of Lp in the fat body in a manner that appears to be at least partially dependent on target of rapamycin (TOR) signaling, resulting in excess lipid accumulation in the developing follicles. Embryos deposited by Vg-depleted mothers are completely inviable, and are arrested early during development, likely due to severely reduced amino acid levels and protein synthesis. Our findings demonstrate that the mutual regulation of these two nutrient transporters is essential to safeguard fertility by ensuring correct nutrient balance in the developing oocyte, and validate Vg and Lp as two potential candidates for mosquito control.
Asunto(s)
Aedes , Anopheles , Malaria , Femenino , Animales , Anopheles/genética , Mosquitos Vectores/genética , Vitelogeninas/genética , Vitelogeninas/metabolismo , Proteínas del Huevo/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Fertilidad/genética , Lípidos , Aedes/genética , Aedes/metabolismoRESUMEN
Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile Insect Technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.
RESUMEN
Insects, unlike vertebrates, are widely believed to lack male-biased sex steroid hormones1. In the malaria mosquito Anopheles gambiae, the ecdysteroid 20-hydroxyecdysone (20E) appears to have evolved to both control egg development when synthesized by females2 and to induce mating refractoriness when sexually transferred by males3. Because egg development and mating are essential reproductive traits, understanding how Anopheles females integrate these hormonal signals can spur the design of new malaria control programs. Here we reveal that these reproductive functions are regulated by distinct sex steroids through a sophisticated network of ecdysteroid-activating/inactivating enzymes. We identify a male-specific oxidized ecdysteroid, 3-dehydro-20E (3D20E), which safeguards paternity by turning off female sexual receptivity following its sexual transfer and activation by dephosphorylation. Notably, 3D20E transfer also induces expression of a reproductive gene that preserves egg development during Plasmodium infection, ensuring fitness of infected females. Female-derived 20E does not trigger sexual refractoriness but instead licenses oviposition in mated individuals once a 20E-inhibiting kinase is repressed. Identifying this male-specific insect steroid hormone and its roles in regulating female sexual receptivity, fertility and interactions with Plasmodium parasites suggests the possibility for reducing the reproductive success of malaria-transmitting mosquitoes.
Asunto(s)
Anopheles , Ecdisteroides , Malaria , Conducta Sexual Animal , Animales , Anopheles/enzimología , Anopheles/parasitología , Anopheles/fisiología , Ecdisteroides/biosíntesis , Ecdisteroides/metabolismo , Femenino , Fertilidad , Humanos , Malaria/parasitología , Malaria/prevención & control , Malaria/transmisión , Masculino , Mosquitos Vectores/parasitología , Oviposición , Fosforilación , PlasmodiumRESUMEN
The spread of insecticide resistance in Anopheles mosquitoes and drug resistance in Plasmodium parasites is contributing to a global resurgence of malaria, making the generation of control tools that can overcome these roadblocks an urgent public health priority. We recently showed that the transmission of Plasmodium falciparum parasites can be efficiently blocked when exposing Anopheles gambiae females to antimalarials deposited on a treated surface, with no negative consequences on major components of mosquito fitness. Here, we demonstrate this approach can overcome the hurdles of insecticide resistance in mosquitoes and drug resistant in parasites. We show that the transmission-blocking efficacy of mosquito-targeted antimalarials is maintained when field-derived, insecticide resistant Anopheles are exposed to the potent cytochrome b inhibitor atovaquone, demonstrating that this drug escapes insecticide resistance mechanisms that could potentially interfere with its function. Moreover, this approach prevents transmission of field-derived, artemisinin resistant P. falciparum parasites (Kelch13 C580Y mutant), proving that this strategy could be used to prevent the spread of parasite mutations that induce resistance to front-line antimalarials. Atovaquone is also highly effective at limiting parasite development when ingested by mosquitoes in sugar solutions, including in ongoing infections. These data support the use of mosquito-targeted antimalarials as a promising tool to complement and extend the efficacy of current malaria control interventions.
Asunto(s)
Anopheles , Antimaláricos , Malaria Falciparum , Malaria , Plasmodium , Animales , Anopheles/parasitología , Antimaláricos/farmacología , Atovacuona/farmacología , Femenino , Malaria/parasitología , Malaria/prevención & control , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Malaria Falciparum/prevención & control , Plasmodium falciparum/genéticaRESUMEN
Interactions between the Anopheles mosquito vector and Plasmodium parasites shape how malaria is transmitted in endemic regions. The long association of these two organisms has led to evolutionary processes that minimize fitness costs of infection and benefit both players through shared nutrient resources, parasite immune suppression, and mosquito tolerance to infection. In this review we explore recent data describing how Plasmodium falciparum, the deadliest malaria parasite, associates with one of its most important natural mosquito hosts, Anopheles gambiae, and we discuss the implications of these findings for parasite transmission and vector control strategies currently in development.
Asunto(s)
Anopheles , Malaria Falciparum , Malaria , Plasmodium , Animales , Anopheles/parasitología , Interacciones Huésped-Parásitos , Malaria/parasitología , Malaria Falciparum/parasitología , Mosquitos Vectores/parasitología , Plasmodium falciparumRESUMEN
Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite's extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R0, and find the average R0 is higher (range: 10.1%-12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions.
Asunto(s)
Malaria Falciparum/transmisión , Mosquitos Vectores/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Animales , Anopheles/parasitología , Conducta Alimentaria , Femenino , Periodo de Incubación de Enfermedades InfecciosasRESUMEN
Anopheles mosquitoes have transmitted Plasmodium parasites for millions of years, yet it remains unclear whether they suffer fitness costs to infection. Here we report that the fecundity of virgin and mated females of two important vectors-Anopheles gambiae and Anopheles stephensi-is not affected by infection with Plasmodium falciparum, demonstrating that these human malaria parasites do not inflict this reproductive cost on their natural mosquito hosts. Additionally, parasite development is not impacted by mating status. However, in field studies using different P. falciparum isolates in Anopheles coluzzii, we find that Mating-Induced Stimulator of Oogenesis (MISO), a female reproductive gene strongly induced after mating by the sexual transfer of the steroid hormone 20-hydroxyecdysone (20E), protects females from incurring fecundity costs to infection. MISO-silenced females produce fewer eggs as they become increasingly infected with P. falciparum, while parasite development is not impacted by this gene silencing. Interestingly, previous work had shown that sexual transfer of 20E has specifically evolved in Cellia species of the Anopheles genus, driving the co-adaptation of MISO. Our data therefore suggest that evolution of male-female sexual interactions may have promoted Anopheles tolerance to P. falciparum infection in the Cellia subgenus, which comprises the most important malaria vectors.
Asunto(s)
Anopheles/genética , Interacciones Huésped-Parásitos/genética , Plasmodium falciparum/genética , Animales , Anopheles/parasitología , Ecdisterona/genética , Ecdisterona/metabolismo , Femenino , Fertilidad/genética , Expresión Génica , Hormonas/fisiología , Malaria/parasitología , Malaria Falciparum/parasitología , Masculino , Mosquitos Vectores/genética , Oogénesis , Plasmodium falciparum/patogenicidad , Reproducción/fisiologíaRESUMEN
The reproductive fitness of the Anopheles gambiae mosquito represents a promising target to prevent malaria transmission. The ecdysteroid hormone 20-hydroxyecdysone (20E), transferred from male to female during copulation, is key to An. gambiae reproductive success as it licenses females to oviposit eggs developed after blood feeding. Here we show that 20E-triggered oviposition in these mosquitoes is regulated by the stress- and immune-responsive c-Jun N-terminal kinase (JNK). The heads of mated females exhibit a transcriptional signature reminiscent of a JNK-dependent wounding response, while mating-or injection of virgins with exogenous 20E-selectively activates JNK in the same tissue. RNAi-mediated depletion of JNK pathway components inhibits oviposition in mated females, whereas JNK activation by silencing the JNK phosphatase puckered induces egg laying in virgins. Together, these data identify JNK as a potential conduit linking stress responses and reproductive success in the most important vector of malaria.
Asunto(s)
Anopheles/fisiología , Sistema de Señalización de MAP Quinasas/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Mosquitos Vectores/fisiología , Oviposición/genética , Animales , Copulación/efectos de los fármacos , Ecdisterona/farmacología , Femenino , Malaria/parasitología , Malaria/transmisión , Masculino , Proteína Quinasa 8 Activada por Mitógenos/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Plasmodium , Interferencia de ARNRESUMEN
Wolbachia, an endosymbiotic alpha-proteobacterium commonly found in insects, can inhibit the transmission of human pathogens by mosquitoes. Biocontrol programs are underway using Aedes aegypti mosquitoes trans-infected with a non-natural Wolbachia strain to reduce dengue virus transmission. Less is known about the impact of Wolbachia on the biology and vectorial capacity of Anopheles mosquitoes, the vectors of malaria parasites. A naturally occurring strain of Wolbachia, wAnga, infects populations of the major malaria vectors Anopheles gambiae and Anopheles coluzzii in Burkina Faso. Previous studies found wAnga infection was negatively correlated with Plasmodium infection in the mosquito and wAnga influenced mosquito egg-laying behavior. Here, we investigate wAnga in natural populations of An. coluzzii and its interactions with other resident microbiota using targeted 16S sequencing. Though we find no major differences in microbiota composition associated with wAnga infection, we do find several taxa that correlate with the presence or absence of wAnga in female mosquitoes following oviposition, with the caveat that we could not rule out batch effects due to the unanticipated impact of wAnga on oviposition timing. These data suggest wAnga may influence or interact with the Anopheles microbiota, which may contribute to the impact of wAnga on Anopheles biology and vectorial capacity.
Asunto(s)
Anopheles/microbiología , Anopheles/fisiología , Interacciones Microbiota-Huesped/fisiología , Insectos Vectores , Wolbachia , Animales , Burkina Faso , Vectores de Enfermedades , Femenino , Malaria/transmisión , Control de Mosquitos/métodos , Oviposición , Enfermedades Transmitidas por Vectores/transmisiónRESUMEN
Transmission of malaria parasites occurs when a female Anopheles mosquito feeds on an infected host to acquire nutrients for egg development. How parasites are affected by oogenetic processes, principally orchestrated by the steroid hormone 20-hydroxyecdysone (20E), remains largely unknown. Here we show that Plasmodium falciparum development is intimately but not competitively linked to processes shaping Anopheles gambiae reproduction. We unveil a 20E-mediated positive correlation between egg and oocyst numbers; impairing oogenesis by multiple 20E manipulations decreases parasite intensities. These manipulations, however, accelerate Plasmodium growth rates, allowing sporozoites to become infectious sooner. Parasites exploit mosquito lipids for faster growth, but they do so without further affecting egg development. These results suggest that P. falciparum has adopted a non-competitive evolutionary strategy of resource exploitation to optimize transmission while minimizing fitness costs to its mosquito vector. Our findings have profound implications for currently proposed control strategies aimed at suppressing mosquito populations.
Asunto(s)
Ecdisterona/metabolismo , Interacciones Huésped-Parásitos/fisiología , Malaria Falciparum/parasitología , Animales , Anopheles/parasitología , Culicidae , Ecdisterona/fisiología , Femenino , Células HEK293 , Humanos , Insectos Vectores , Malaria/parasitología , Ratones , Mosquitos Vectores , Células 3T3 NIH , Oogénesis/fisiología , Plasmodium/metabolismo , Plasmodium falciparum , Esporozoítos , Esteroides/metabolismoRESUMEN
Human pathogens that are transmitted by insects are a global problem, particularly those vectored by mosquitoes; for example, malaria parasites transmitted by Anopheles species, and viruses such as dengue, Zika and chikungunya that are carried by Aedes mosquitoes. Over the past 15 years, the prevalence of malaria has been substantially reduced and virus outbreaks have been contained by controlling mosquito vectors using insecticide-based approaches. However, disease control is now threatened by alarming rates of insecticide resistance in insect populations, prompting the need to develop a new generation of specific strategies that can reduce vector-mediated transmission. Here, we review how increased knowledge in insect biology and insect-pathogen interactions is stimulating new concepts and tools for vector control. We focus on strategies that either interfere with the development of pathogens within their vectors or directly impact insect survival, including enhancement of vector-mediated immune control, manipulation of the insect microbiome, or use of powerful new genetic tools such as CRISPR-Cas systems to edit vector genomes. Finally, we offer a perspective on the implementation hurdles as well as the knowledge gaps that must be filled in the coming years to safely realize the potential of these novel strategies to eliminate the scourge of vector-borne disease.
Asunto(s)
Control de Enfermedades Transmisibles/métodos , Enfermedades Transmisibles/transmisión , Brotes de Enfermedades/prevención & control , Control de Insectos/métodos , Mosquitos Vectores/microbiología , Mosquitos Vectores/parasitología , Aedes/virología , Animales , Anopheles/microbiología , Anopheles/parasitología , Agentes de Control Biológico/farmacología , Culex/parasitología , Culex/virología , Humanos , Resistencia a los Insecticidas , Insecticidas/farmacología , Wolbachia/patogenicidadRESUMEN
BACKGROUND: Malaria parasites, transmitted by the bite of an anopheline mosquito, pose an immense public health burden on many tropical and subtropical regions. The most important malaria vectors in sub-Saharan Africa are mosquitoes of the Anopheles gambiae complex including An. gambiae (sensu stricto). Given the increasing rates of insecticide resistance in these mosquitoes, alternative control strategies based on the release of genetically modified males are being evaluated to stop transmission by these disease vectors. These strategies rely on the mating competitiveness of release males, however currently there is no method to determine male mating success without sacrificing the female. Interestingly, unlike other insects, during mating An. gambiae males transfer their male accessory glands (MAGs) seminal secretions as a coagulated mating plug which is deposited in the female atrium. RESULTS: Here we exploit this male reproductive feature and validate the use of a MAG-specific promoter to fluorescently label the mating plug and visualize the occurrence of insemination in vivo. We used the promoter region of the major mating plug protein, Plugin, to control the expression of a Plugin-tdTomato (PluTo) fusion protein, hypothesizing that this fusion protein could be incorporated into the plug for sexual transfer to the female. Anopheles gambiae PluTo transgenic males showed strong red fluorescence specifically in the MAGs and with a pattern closely matching endogenous Plugin expression. Moreover, the fusion protein was integrated into the mating plug and transferred to the female atrium during mating where it could be visualized microscopically in vivo without sacrificing the female. PluTo males were equally as competitive at mating as wild type males, and females mated to these males did not show any reduction in reproductive fitness. CONCLUSION: The validation of the first MAG-specific promoter in transgenic An. gambiae facilitates the live detection of successful insemination hours after copulation has occurred. This provides a valuable tool for the assessment of male mating competitiveness not only in laboratory experiments but also in semi-field and field studies aimed at testing the feasibility of releasing genetically modified mosquitoes for disease control.
Asunto(s)
Anopheles/genética , Malaria/prevención & control , Control de Mosquitos/métodos , Mosquitos Vectores/genética , Procesos de Determinación del Sexo , Animales , Animales Modificados Genéticamente , Anopheles/fisiología , Copulación , Femenino , Humanos , Malaria/transmisión , Masculino , Mosquitos Vectores/fisiologíaRESUMEN
The maternally inherited alpha-proteobacterium Wolbachia has been proposed as a tool to block transmission of devastating mosquito-borne infectious diseases like dengue and malaria. Here we study the reproductive manipulations induced by a recently identified Wolbachia strain that stably infects natural mosquito populations of a major malaria vector, Anopheles coluzzii, in Burkina Faso. We determine that these infections significantly accelerate egg laying but do not induce cytoplasmic incompatibility or sex-ratio distortion, two parasitic reproductive phenotypes that facilitate the spread of other Wolbachia strains within insect hosts. Analysis of 221 blood-fed A. coluzzii females collected from houses shows a negative correlation between the presence of Plasmodium parasites and Wolbachia infection. A mathematical model incorporating these results predicts that infection with these endosymbionts may reduce malaria prevalence in human populations. These data suggest that Wolbachia may be an important player in malaria transmission dynamics in Sub-Saharan Africa.
Asunto(s)
Anopheles/fisiología , Malaria/transmisión , Mosquitos Vectores/fisiología , Oviposición/fisiología , Plasmodium/crecimiento & desarrollo , Wolbachia/fisiología , Animales , Anopheles/microbiología , Anopheles/parasitología , Burkina Faso/epidemiología , ADN Bacteriano/aislamiento & purificación , Transmisión de Enfermedad Infecciosa/prevención & control , Femenino , Interacciones Huésped-Patógeno , Larva/crecimiento & desarrollo , Malaria/epidemiología , Masculino , Herencia Materna , Modelos Biológicos , Mosquitos Vectores/microbiología , Mosquitos Vectores/parasitología , Plasmodium/patogenicidad , Prevalencia , ARN Ribosómico 16S/genética , Simbiosis/fisiología , Wolbachia/aislamiento & purificaciónRESUMEN
Studying the reproductive strategies of insect species that transmit diseases to humans can identify new exploitable targets for the development of vector control methods. Here we describe shared characteristics and individual features of the reproductive biology of three major disease vectors: Anopheles gambiae, Aedes aegypti and Glossina morsitans. Current studies are identifying i) species-specific molecular cascades that determine female monandrous behavior, ii) core aspects of egg development that could be disrupted for controlling natural populations, and iii) the increasingly apparent role of resident microbiota in shaping reproductive success and disease transmission potential. The recent completion of multiple genome sequencing projects is allowing comparative genomics studies that not only increase our knowledge of reproductive processes but also facilitate the identification of novel targets for vector control.
RESUMEN
Wolbachia are maternally transmitted intracellular bacteria that invade insect populations by manipulating their reproduction and immunity and thus limiting the spread of numerous human pathogens. Experimental Wolbachia infections can reduce Plasmodium numbers in Anopheles mosquitoes in the laboratory, however, natural Wolbachia infections in field anophelines have never been reported. Here we show evidence of Wolbachia infections in Anopheles gambiae in Burkina Faso, West Africa. Sequencing of the 16S rRNA gene identified Wolbachia sequences in both female and male germlines across two seasons, and determined that these sequences are vertically transmitted from mother to offspring. Whole-genome sequencing of positive samples suggests that the genetic material identified in An. gambiae belongs to a novel Wolbachia strain, related to but distinct from strains infecting other arthropods. The evidence of Wolbachia infections in natural Anopheles populations promotes further investigations on the possible use of natural Wolbachia-Anopheles associations to limit malaria transmission.
Asunto(s)
Anopheles/microbiología , Wolbachia/aislamiento & purificación , Animales , Burkina Faso , Femenino , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/transmisión , Masculino , Control de Mosquitos , Filogenia , ARN Ribosómico 16S/genética , Wolbachia/genéticaRESUMEN
Anopheles gambiae mosquitoes are major African vectors of malaria, a disease that kills more than 600,000 people every year. Given the spread of insecticide resistance in natural mosquito populations, alternative vector control strategies aimed at reducing the reproductive success of mosquitoes are being promoted. Unlike many other insects, An. gambiae females mate a single time in their lives and must use sperm stored in the sperm storage organ, the spermatheca, to fertilize a lifetime's supply of eggs. Maintenance of sperm viability during storage is therefore crucial to the reproductive capacity of these mosquitoes. However, to date, no information is available on the factors and mechanisms ensuring sperm functionality in the spermatheca. Here we identify cellular components and molecular mechanisms used by An. gambiae females to maximize their fertility. Pathways of energy metabolism, cellular transport, and oxidative stress are strongly regulated by mating in the spermatheca. We identify the mating-induced heme peroxidase (HPX) 15 as an important factor in long-term fertility, and demonstrate that its function is required during multiple gonotrophic cycles. We find that HPX15 induction is regulated by sexually transferred 20-hydroxy-ecdysone (20E), a steroid hormone that is produced by the male accessory glands and transferred during copulation, and that expression of this peroxidase is mediated via the 20E nuclear receptor. To our knowledge, our findings provide the first evidence of the mechanisms regulating fertility in Anopheles, and identify HPX15 as a target for vector control.
Asunto(s)
Estructuras Animales/enzimología , Anopheles/enzimología , Proteínas de Insectos/metabolismo , Peroxidasa/metabolismo , Conducta Sexual Animal , Espermatozoides/enzimología , Estructuras Animales/citología , Estructuras Animales/efectos de los fármacos , Estructuras Animales/ultraestructura , Animales , Anopheles/efectos de los fármacos , Anopheles/genética , Ecdisona/farmacología , Activación Enzimática/efectos de los fármacos , Femenino , Fertilidad/efectos de los fármacos , Fertilidad/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hemo/metabolismo , Proteínas de Insectos/genética , Masculino , Peroxidasa/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Conducta Sexual Animal/efectos de los fármacos , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Espermatozoides/ultraestructura , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND: Caenorhabditis elegans provides a genetically tractable model organism to investigate the network of genes involved in fat metabolism and how regulation is perturbed to produce the complex phenotype of obesity. C. elegans possess the full range of desaturases, including the Δ9 desaturases expressed by fat-5, fat-6 and fat-7. They regulate the biosynthesis of monounsaturated fatty acids, used for the synthesis of lipids including phospholipids, triglycerides and cholesteryl esters. RESULTS: Liquid chromatography mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to define the metabolome of all the possible knock-outs for the Δ9 desaturases, including for the first time intact lipids. Despite the genes having similar enzymatic roles, excellent discrimination was achievable for all single and viable double mutants highlighting the distinctive roles of fat-6 and fat-7, both expressing steroyl-CoA desaturases. The metabolomic changes extend to aqueous metabolites demonstrating the influence Δ9 desaturases have on regulating global metabolism and highlighting how comprehensive metabolomics is more discriminatory than classically used dyes for fat staining. CONCLUSIONS: The propagation of metabolic changes across the network of metabolism demonstrates that modification of the Δ9 desaturases places C.elegans into a catabolic state compared with wildtype controls.