Biology subjects

Pitts, R. J.

Publications and source records attributed to Pitts, R. J..

5 recordsLinked to original sources

bioRxiv2025

The co-receptors Orco and Ir8a are required for coordinated expression of chemosensory genes in the antennae of the yellow fever mosquito, Aedes aegypti.

Olfaction has been extensively studied in the yellow fever mosquito, Aedes aegypti. This species uses its sense of smell to find blood hosts and other resources, contributing to its impact as a vector for human pathogens. Two major families of protein-coding genes, the odorant receptors (Ors) and the ionotropic receptors (Irs), provide the mosquito with sensitivities to distinct classes of volatile compounds in the antennae. Individual tuning receptors in both families require co-receptors for functionality, Orco for all Ors, and Ir8a for many Irs, especially ones that are involved in carboxylic acid detection. In Drosophila melanogaster, disruptions of Orco or Ir8a impair receptor function, tuning receptor expression, and membrane localization, leading to general anosmia. We reasoned that Orco and Ir8a might also be important for coordinated chemosensory receptor expression in the antennal sensory neurons of Ae. aegypti. To test this, we performed RNAseq and differential expression analysis in wild type versus Orco-/- and Ir8a-/- mutant adult female antennae. Our analyses revealed Or and Ir tuning receptors are broadly under-expressed in Orco-/- mutants, while a subset of tuning Irs are under-expressed in Ir8a mutants. Other chemosensory and non-chemosensory genes are also dysregulated in these mutants. Further, we identify differentially expressed transcription factors including homologs of the Drosophila melanogaster Mip120 gene. These data suggest a previously unknown pleiotropic role for the Orco and Ir8a co-receptors in the coordination of expression of chemosensory receptors within the antennae of Ae. aegypti by participating in a feedback loop involving amos and members of the MMB/dREAM complex. Simple SummaryMosquitoes have an exquisite olfactory system, with which they locate bloodmeal hosts, nectar sources, and sites for egg-laying. Elucidating the mechanisms that underlie the perception of and response to chemicals in the environment is crucial for our understanding of the biology of mosquitoes that are vectors of many deadly pathogens. Olfaction is primarily mediated by large families of odorant receptors and ionotropic receptors, collectively encoding more than 200 tuning receptors, each of which recognizes one or more volatile odorants. As ligand-gated ion channels, tuning receptors of each family form complexes with the Odorant receptor co-receptor (Orco), or one of few Ionotropic receptor co-receptors (Irco), respectively. In this study, we evaluated the hypothesis that co-receptors are necessary for tuning receptor expression. To this end, we compared antennal transcriptomes of wild type adult female Aedes aegypti with Orco-/- and Ir8a-/- mutant strains. We show that the tuning receptor transcripts in the antennae are broadly dysregulated in both mutants. We discuss two possible explanations for these observations and suggest ways this knowledge can be applied to vector control strategies. O_FIG View larger version (K): org.highwire.dtl.DTLVardef@c04846org.highwire.dtl.DTLVardef@1c91514org.highwire.dtl.DTLVardef@871596org.highwire.dtl.DTLVardef@9a617d_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

molecular biology↗

bioRxiv2022

Functional conservation of Anopheline linalool receptors through 100 million years of evolution

Insects rely on olfactory receptors to detect and respond to diverse environmental chemical cues. Detection of semiochemicals by these receptors modulates insect behavior and has a direct impact on species fitness. Volatile organic compounds (VOCs) are released by animals and plants and can provide contextual cues that a blood meal host or nectar source is present. One such VOC is linalool, an enantiomeric monoterpene, that is emitted from plants and bacteria species. This compound exists in nature as one of two possible stereoisomers, (R)-(-)-linalool or (S)-(+)-linalool. In this study, we use a heterologous expression system to demonstrate differential responsiveness of a pair of Anopheline odorant receptors (Ors) to enantiomers of linalool. The mosquitoes Anopheles gambiae and Anopheles stephensi encode single copies of Or29 and Or53, which are expressed in the labella of An. gambiae. (S)-(+)-linalool activates Or29 orthologs with a higher potency than (R)-(-)-linalool, while the converse is observed for Or53 orthologs. The conservation of these receptors across a broad range of Anopheline species suggests they may function in the discrimination of linalool stereoisomers, thereby influencing the chemical ecology of mosquitoes. One potential application of this knowledge would be in the design of novel attractants or repellents to be used in integrated pest management practices.

molecular biology↗

bioRxiv2022

PrimedSherlock: A tool for rapid design of highly specific CRISPR-Cas12 crRNAs

BackgroundCRISPR-Cas based diagnostic assays provide a portable solution which bridges the benefits of qRT-PCR and serological assays in terms of portability, specificity and ease of use. CRISPR-Cas assays are rapidly fieldable, specific and have been rigorously validated against a number of targets, including HIV and vector-borne pathogens. Recently, CRISPR-Cas12 and CRISPR-Cas13 diagnostic assays have been granted FDA approval for the detection of SARS-CoV-2. A critical step in utilizing this technology requires the design of highly-specific and efficient CRISPR RNAs (crRNAs) and isothermal primers. This process involves intensive manual curation and stringent parameters for design in order to minimize off-target detection while also preserving detection across divergent strains. As such, a single, streamlined bioinformatics platform for rapidly designing crRNAs for use with the CRISPR-Cas12 platform is needed. Here we offer PrimedSherlock, an automated, computer guided process for selecting highly-specific crRNAs and primers for targets of interest. ResultsUtilizing PrimedSherlock and publicly available databases, crRNAs were designed against a selection of Flavivirus genomes, including West Nile, Zika and all four serotypes of Dengue. Using outputs from PrimedSherlock in concert with both wildtype A.s Cas12a and Alt-R Cas12a Ultra nucleases, we demonstrated sensitive detection of nucleic acids of each respective arbovirus in in-vitro fluorescence assays. Moreover, primer and crRNA combinations facilitated the detection of their intended targets with minimal off-target background noise. ConclusionsPrimedSherlock is a novel crRNA design tool, specific for CRISPR-Cas12 diagnostic platforms. It allows for the rapid identification of highly conserved crRNA targets from user-provided primer pairs or PrimedRPA output files. Initial testing of crRNAs against arboviruses of medical importance demonstrated a robust ability to distinguish multiple strains by exploiting polymorphisms within otherwise highly conserved genomic regions. As a freely-accessible software package, PrimedSherlock could significantly increase the efficiency of CRISPR-Cas12 diagnostics. Conceptually, the portability of detection kits could also be enhanced when coupled with isothermal amplification technologies.

molecular biology↗

bioRxiv2021

Mutagenesis of the Ammonium Transporter AcAmt Reveals a Reproductive Role and a Novel Ammonia-Sensing Mechanism in the Malaria Vector Mosquito Anopheles coluzzii

Anopheline mosquitoes are the sole vectors of malaria and rely on olfactory cues for host seeking in which ammonia derived from human sweat plays an essential role. To investigate the function of the Anopheles coluzzii ammonium transporter (AcAmt) in the mosquito olfactory system, we generated an AcAmt null mutant line using CRISPR/Cas9. AcAmt-/- mutants displayed a series of novel phenotypes compared with wild-type mosquitoes including significantly lower insemination rates during mating and increased mortality during eclosion. Furthermore, AcAmt-/- males showed significantly lower sugar consumption while AcAmt-/- females and pupae displayed significantly higher ammonia levels than their wild-type counterparts. Surprisingly, in contrast to previous studies in Drosophila that revealed that the mutation of the ammonium transporter (DmAmt) induces a dramatic reduction of ammonia responses in antennal coeloconic sensilla, no significant differences were observed across a range of peripheral sensory neuron responses to ammonia and other odorants between wild-type and AcAmt-/- females. Taken together, these data support the existence of a unique ammonia-sensing mechanism in mosquitoes and that the ammonium transporter may be an important molecular target for vector control. Key MessagesO_LIMutagenesis of An. coluzzii ammonium transporter AcAmt followed by comprehensive electrophysiological investigation suggest a novel ammonia-sensing pathway in Anopheles mosquitoes. C_LIO_LIAcAmt-/- mutants displayed significant deficiencies in reproduction and eclosion, which are likely due to elevated ammonia levels and reduced ability of sugar feeding. C_LIO_LIAn. coluzzii coeloconic sensilla primarily detect amines and acids. C_LI

neuroscience↗

bioRxiv2020

Mosquito indolergic receptors belong to an ancient and functionally conserved Dipteran gene lineage

Diptera is a megadiverse group of flies with sophisticated chemical detection systems, which exploits an incredible variety of ecological niches. Among the vast array of odorants in natural environments, indoles stand out as playing crucial roles in mediating fly behavior. In mosquitoes, indolic compounds are detected by an ancient class of conserved indolergic Odorant Receptors (indolORs). In this study, we have identified a set of 92 putative indolOR genes encoded in the genomes of Nematoceran and Brachyceran flies, resolved their phylogenetic relationships, and defined conserved elements in their gene structures. Further, we have quantified indolOR transcript abundance in the antennae of the housefly, Musca domestica, and have characterized MdomOR30a as a skatole receptor using a heterologous expression system. The presence of indolORs in species operating in different ecological contexts suggests that indoles act as pleiotropic signals for resource exploitation at multiple developmental stages. Further characterization of indolORs will impact our understanding of insect chemical ecology and will provide targets for the development of novel odor-based tools that can be integrated into existing vector surveillance and control programs.

evolutionary biology↗