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1.
Genetics ; 224(2)2023 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-37036394

RESUMEN

The advent of CRISPR/Cas9-mediated genome editing has expanded the range of animals amenable to targeted genetic analysis. This has accelerated research in animals not traditionally studied using molecular genetics. However, studying genes essential for reproduction or survival in such animals remains challenging, as they lack the tools that aid genetic analysis in traditional genetic model organisms. We recently introduced the use of distinguishably marked knock-in pairs (DMKPs) as a strategy for rapid and reliable genotyping in such species. Here we show that DMKPs also facilitate the maintenance and study of mutations that cannot be maintained in a homozygous state, a group which includes recessive lethal and sterile mutations. Using DMKPs, we disrupt the zero population growth locus in Drosophila melanogaster and in the dengue vector mosquito Aedes aegypti. In both species, DMKPs enable the maintenance of zero population growth mutant strains and the reliable recovery of zero population growth mutant animals. Male and female gonad development is disrupted in fly and mosquito zero population growth mutants, rendering both sexes sterile. In Ae. aegypti, zero population growth mutant males remain capable of inducing a mating refractory period in wild-type females and of competing with wild-type males for mates, properties compatible with zero population growth serving as a target in mosquito population suppression strategies. DMKP is readily generalizable to other species amenable to CRISPR/Cas9-mediated gene targeting, and should facilitate the study of sterile and lethal mutations in multiple organisms not traditionally studied using molecular genetics.


Asunto(s)
Aedes , Infertilidad , Animales , Masculino , Femenino , Drosophila melanogaster/genética , Mosquitos Vectores , Reproducción/genética , Aedes/genética
2.
Neuron ; 111(6): 874-887.e8, 2023 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-36640768

RESUMEN

To reproduce and to transmit disease, female mosquitoes must obtain blood meals and locate appropriate sites for egg laying (oviposition). While distinct sensory cues drive each behavior, humidity contributes to both. Here, we identify the mosquito's humidity sensors (hygrosensors). Using generalizable approaches designed to simplify genetic analysis in non-traditional model organisms, we demonstrate that the ionotropic receptor Ir93a mediates mosquito hygrosensation as well as thermosensation. We further show that Ir93a-dependent sensors drive human host proximity detection and blood-feeding behavior, consistent with the overlapping short-range heat and humidity gradients these targets generate. After blood feeding, gravid females require Ir93a to seek high humidity associated with preferred egg-laying sites. Reliance on Ir93a-dependent sensors to promote blood feeding and locate potential oviposition sites is shared between the malaria vector Anopheles gambiae and arbovirus vector Aedes aegypti. These Ir93a-dependent systems represent potential targets for efforts to control these human disease vectors.


Asunto(s)
Anopheles , Malaria , Animales , Humanos , Femenino , Oviposición , Humedad , Mosquitos Vectores , Conducta Alimentaria
3.
Science ; 367(6478): 681-684, 2020 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-32029627

RESUMEN

Mosquitoes transmit pathogens that kill >700,000 people annually. These insects use body heat to locate and feed on warm-blooded hosts, but the molecular basis of such behavior is unknown. Here, we identify ionotropic receptor IR21a, a receptor conserved throughout insects, as a key mediator of heat seeking in the malaria vector Anopheles gambiae Although Ir21a mediates heat avoidance in Drosophila, we find it drives heat seeking and heat-stimulated blood feeding in Anopheles At a cellular level, Ir21a is essential for the detection of cooling, suggesting that during evolution mosquito heat seeking relied on cooling-mediated repulsion. Our data indicate that the evolution of blood feeding in Anopheles involves repurposing an ancestral thermoreceptor from non-blood-feeding Diptera.


Asunto(s)
Anopheles/fisiología , Temperatura Corporal , Evolución Molecular , Conducta de Búsqueda de Hospedador/fisiología , Calor , Receptores Ionotrópicos de Glutamato/fisiología , Termorreceptores/fisiología , Animales , Anopheles/genética , Sangre , Femenino , Ratones , Mutación , Receptores Ionotrópicos de Glutamato/genética
4.
Neuron ; 101(4): 738-747.e3, 2019 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-30654923

RESUMEN

Thermosensation is critical for avoiding thermal extremes and regulating body temperature. While thermosensors activated by noxious temperatures respond to hot or cold, many innocuous thermosensors exhibit robust baseline activity and lack discrete temperature thresholds, suggesting they are not simply warm and cool detectors. Here, we investigate how the aristal Cold Cells encode innocuous temperatures in Drosophila. We find they are not cold sensors but cooling-activated and warming-inhibited phasic thermosensors that operate similarly at warm and cool temperatures; we propose renaming them "Cooling Cells." Unexpectedly, Cooling Cell thermosensing does not require the previously reported Brivido Transient Receptor Potential (TRP) channels. Instead, three Ionotropic Receptors (IRs), IR21a, IR25a, and IR93a, specify both the unique structure of Cooling Cell cilia endings and their thermosensitivity. Behaviorally, Cooling Cells promote both warm and cool avoidance. These findings reveal a morphogenetic role for IRs and demonstrate the central role of phasic thermosensing in innocuous thermosensation. VIDEO ABSTRACT.


Asunto(s)
Proteínas de Drosophila/metabolismo , Neurogénesis , Receptores Ionotrópicos de Glutamato/metabolismo , Células Receptoras Sensoriales/metabolismo , Sensación Térmica , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Receptores Ionotrópicos de Glutamato/genética , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/fisiología , Termotolerancia
5.
Elife ; 52016 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-27126188

RESUMEN

Animals rely on highly sensitive thermoreceptors to seek out optimal temperatures, but the molecular mechanisms of thermosensing are not well understood. The Dorsal Organ Cool Cells (DOCCs) of the Drosophila larva are a set of exceptionally thermosensitive neurons critical for larval cool avoidance. Here, we show that DOCC cool-sensing is mediated by Ionotropic Receptors (IRs), a family of sensory receptors widely studied in invertebrate chemical sensing. We find that two IRs, IR21a and IR25a, are required to mediate DOCC responses to cooling and are required for cool avoidance behavior. Furthermore, we find that ectopic expression of IR21a can confer cool-responsiveness in an Ir25a-dependent manner, suggesting an instructive role for IR21a in thermosensing. Together, these data show that IR family receptors can function together to mediate thermosensation of exquisite sensitivity.


Asunto(s)
Frío , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Drosophila/efectos de la radiación , Receptores Ionotrópicos de Glutamato/metabolismo , Sensación Térmica , Animales , Conducta Animal
6.
Curr Biol ; 25(8): 1063-8, 2015 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-25866391

RESUMEN

Ambient light affects multiple physiological functions and behaviors, such as circadian rhythms, sleep-wake activities, and development, from flies to mammals. Mammals exhibit a higher body temperature when exposed to acute light compared to when they are exposed to the dark, but the underlying mechanisms are largely unknown. The body temperature of small ectotherms, such as Drosophila, relies on the temperature of their surrounding environment, and these animals exhibit a robust temperature preference behavior. Here, we demonstrate that Drosophila prefer a ∼1° higher temperature when exposed to acute light rather than the dark. This acute light response, light-dependent temperature preference (LDTP), was observed regardless of the time of day, suggesting that LDTP is regulated separately from the circadian clock. However, screening of eye and circadian clock mutants suggests that the circadian clock neurons posterior dorsal neurons 1 (DN1(p)s) and Pigment-Dispersing Factor Receptor (PDFR) play a role in LDTP. To further investigate the role of DN1(p)s in LDTP, PDFR in DN1(p)s was knocked down, resulting in an abnormal LDTP. The phenotype of the pdfr mutant was rescued sufficiently by expressing PDFR in DN1(p)s, indicating that PDFR in DN1(p)s is responsible for LDTP. These results suggest that light positively influences temperature preference via the circadian clock neurons, DN1(p)s, which may result from the integration of light and temperature information. Given that both Drosophila and mammals respond to acute light by increasing their body temperature, the effect of acute light on temperature regulation may be conserved evolutionarily between flies and humans.


Asunto(s)
Relojes Circadianos/fisiología , Proteínas de Drosophila/metabolismo , Luz , Neuronas/citología , Receptores Acoplados a Proteínas G/metabolismo , Núcleo Supraquiasmático/citología , Temperatura , Animales , Ritmo Circadiano/fisiología , Drosophila , Neuronas/metabolismo
7.
Nature ; 500(7464): 580-4, 2013 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-23925112

RESUMEN

Behavioural responses to temperature are critical for survival, and animals from insects to humans show strong preferences for specific temperatures. Preferred temperature selection promotes avoidance of adverse thermal environments in the short term and maintenance of optimal body temperatures over the long term, but its molecular and cellular basis is largely unknown. Recent studies have generated conflicting views of thermal preference in Drosophila, attributing importance to either internal or peripheral warmth sensors. Here we reconcile these views by showing that thermal preference is not a singular response, but involves multiple systems relevant in different contexts. We found previously that the transient receptor potential channel TRPA1 acts internally to control the slowly developing preference response of flies exposed to a shallow thermal gradient. We now find that the rapid response of flies exposed to a steep warmth gradient does not require TRPA1; rather, the gustatory receptor GR28B(D) drives this behaviour through peripheral thermosensors. Gustatory receptors are a large gene family, widely studied in insect gustation and olfaction, and are implicated in host-seeking by insect disease vectors, but have not previously been implicated in thermosensation. At the molecular level, GR28B(D) misexpression confers thermosensitivity upon diverse cell types, suggesting that it is a warmth sensor. These data reveal a new type of thermosensory molecule and uncover a functional distinction between peripheral and internal warmth sensors in this tiny ectotherm reminiscent of thermoregulatory systems in larger, endothermic animals. The use of multiple, distinct molecules to respond to a given temperature, as observed here, may facilitate independent tuning of an animal's distinct thermosensory responses.


Asunto(s)
Reacción de Prevención/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Calor , Receptores de Superficie Celular/metabolismo , Gusto , Sensación Térmica/fisiología , Animales , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Canales Iónicos , Receptores de Superficie Celular/genética , Transducción de Señal , Olfato , Canal Catiónico TRPA1 , Canales Catiónicos TRPC/deficiencia , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Termorreceptores/citología , Termorreceptores/fisiología , Sensación Térmica/genética , Factores de Tiempo
8.
Nature ; 481(7379): 76-80, 2011 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-22139422

RESUMEN

Discriminating among sensory stimuli is critical for animal survival. This discrimination is particularly essential when evaluating whether a stimulus is noxious or innocuous. From insects to humans, transient receptor potential (TRP) channels are key transducers of thermal, chemical and other sensory cues. Many TRPs are multimodal receptors that respond to diverse stimuli, but how animals distinguish sensory inputs activating the same TRP is largely unknown. Here we determine how stimuli activating Drosophila TRPA1 are discriminated. Although Drosophila TRPA1 responds to both noxious chemicals and innocuous warming, we find that TRPA1-expressing chemosensory neurons respond to chemicals but not warmth, a specificity conferred by a chemosensory-specific TRPA1 isoform with reduced thermosensitivity compared to the previously described isoform. At the molecular level, this reduction results from a unique region that robustly reduces the channel's thermosensitivity. Cell-type segregation of TRPA1 activity is critical: when the thermosensory isoform is expressed in chemosensors, flies respond to innocuous warming with regurgitation, a nocifensive response. TRPA1 isoform diversity is conserved in malaria mosquitoes, indicating that similar mechanisms may allow discrimination of host-derived warmth--an attractant--from chemical repellents. These findings indicate that reducing thermosensitivity can be critical for TRP channel functional diversification, facilitating their use in contexts in which thermal sensitivity can be maladaptive.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Calor , Canales Catiónicos TRPC/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Culicidae/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Humanos , Repelentes de Insectos/farmacología , Canales Iónicos , Datos de Secuencia Molecular , Oocitos , Especificidad de Órganos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células Receptoras Sensoriales/metabolismo , Alineación de Secuencia , Transducción de Señal , Canal Catiónico TRPA1 , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/genética , Xenopus laevis
9.
PLoS One ; 6(8): e24343, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21909389

RESUMEN

Pain is a significant medical concern and represents a major unmet clinical need. The ability to perceive and react to tissue-damaging stimuli is essential in order to maintain bodily integrity in the face of environmental danger. To prevent damage the systems that detect noxious stimuli are therefore under strict evolutionary pressure. We developed a high-throughput behavioral method to identify genes contributing to thermal nociception in the fruit fly and have reported a large-scale screen that identified the Ca²âº channel straightjacket (stj) as a conserved regulator of thermal nociception. Here we present the minimal anatomical and neuronal requirements for Drosophila to avoid noxious heat in our novel behavioral paradigm. Bioinformatics analysis of our whole genome data set revealed 23 genes implicated in Ca²âº signaling that are required for noxious heat avoidance. One of these genes, the conserved thermoreceptor TrpA1, was confirmed as a bona fide "pain" gene in both adult and larval fly nociception paradigms. The nociceptive function of TrpA1 required expression within the Drosophila nervous system, specifically within nociceptive multi-dendritic (MD) sensory neurons. Therefore, our analysis identifies the channel TRPA1 as a conserved regulator of nociception.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Nocicepción/fisiología , Canales Catiónicos TRPC/metabolismo , Temperatura , Animales , Antenas de Artrópodos/metabolismo , Reacción de Prevención , Señalización del Calcio/genética , Dendritas/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Ensayos Analíticos de Alto Rendimiento , Canales Iónicos/metabolismo , Larva/metabolismo , Canal Catiónico TRPA1
10.
Nature ; 464(7288): 597-600, 2010 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-20237474

RESUMEN

Chemical nociception, the detection of tissue-damaging chemicals, is important for animal survival and causes human pain and inflammation, but its evolutionary origins are largely unknown. Reactive electrophiles are a class of noxious compounds humans find pungent and irritating, such as allyl isothiocyanate (in wasabi) and acrolein (in cigarette smoke). Diverse animals, from insects to humans, find reactive electrophiles aversive, but whether this reflects conservation of an ancient sensory modality has been unclear. Here we identify the molecular basis of reactive electrophile detection in flies. We demonstrate that Drosophila TRPA1 (Transient receptor potential A1), the Drosophila melanogaster orthologue of the human irritant sensor, acts in gustatory chemosensors to inhibit reactive electrophile ingestion. We show that fly and mosquito TRPA1 orthologues are molecular sensors of electrophiles, using a mechanism conserved with vertebrate TRPA1s. Phylogenetic analyses indicate that invertebrate and vertebrate TRPA1s share a common ancestor that possessed critical characteristics required for electrophile detection. These findings support emergence of TRPA1-based electrophile detection in a common bilaterian ancestor, with widespread conservation throughout vertebrate and invertebrate evolution. Such conservation contrasts with the evolutionary divergence of canonical olfactory and gustatory receptors and may relate to electrophile toxicity. We propose that human pain perception relies on an ancient chemical sensor conserved across approximately 500 million years of animal evolution.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPC/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/clasificación , Drosophila melanogaster/genética , Evolución Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Canales Iónicos , Datos de Secuencia Molecular , Mutación , Filogenia , Canal Catiónico TRPA1 , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/genética , Percepción del Gusto/fisiología
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