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Precise regulation of Type I interferon signaling is crucial for combating infection and cancer while avoiding autoimmunity. Type I interferon signaling is negatively regulated by USP18. USP18 cleaves ISG15, an interferon-induced ubiquitin-like modification, via its canonical catalytic function, and inhibits Type I interferon receptor activity through its scaffold role. USP18 loss-of-function dramatically impacts immune regulation, pathogen susceptibility, and tumor growth. However, prior studies have reached conflicting conclusions regarding the relative importance of catalytic versus scaffold function. Here, we develop biochemical and cellular methods to systematically define the physiological role of USP18. By comparing a patient-derived mutation impairing scaffold function (I60N) to a mutation disrupting catalytic activity (C64S), we demonstrate that scaffold function is critical for cancer cell vulnerability to Type I interferon. Surprisingly, we discovered that human USP18 exhibits minimal catalytic activity, in stark contrast to mouse USP18. These findings resolve human USP18's mechanism-of-action and enable USP18-targeted therapeutics., Competing Interests: VJ, HW, CWL, DRH, KL, ECR, MY, ZKJ, EC, YX, JS, BY, AG, PL, ACG, VF, PDW, and FW are current or former employees of Pfizer and may own Pfizer stock. BMK and APF receive research funding from Pfizer. This work was supported by Pfizer, and APF and BMK are funded by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant nr - 2018-I2M-2-002)., (© 2024 Pfizer Inc.)

Male and female Aedes aegypti mosquitoes survive by feeding on floral nectar for metabolic energy, but females require blood protein, obtained from biting a host, for egg development. Although males exclusively derive energy from nectar sugars, females must select the meal that best matches their present metabolic and reproductive needs. In females, blood and nectar promote independent feeding behaviors with distinct sensory appendages, meal sizes, digestive tract targets, and metabolic fates. Understanding how male and female mosquitoes recognize, locate, and metabolize nutrients is essential for characterizing the survival and reproductive capabilities of this mosquito. Here, we provide an introduction to blood versus nectar feeding and methods to quantify nectar and blood meal sizes in individual Ae. aegypti mosquitoes. Precise quantification of meal size is crucial for ensuring consistency in assays that record events downstream of feeding behavior, including host attraction or fecundity., (© 2022 Cold Spring Harbor Laboratory Press.)

Both male and female mosquitoes consume sugar-rich nectar meals required for metabolic energy, but only females consume protein-rich blood meals, which are required for egg development. The size of each meal consumed has subsequent effects on behavior and reproduction; therefore, precise quantification is an important aspect of mosquito feeding behavior studies. This protocol describes a high-throughput, end-point assay to quantify meal volumes ingested by individual mosquitoes. The addition of a fluorescent dye to the meal allows for meal size quantification. Individual mosquitoes that have been fed this meal are homogenized in 96-well plates, and the fluorescence levels are measured with a plate reader. This protocol can also be adapted to determine if alteration of meal composition affects the ingested meal volume, if mosquito strain or genotype dictates consumption, or if meals are derived from multiple sources., (© 2022 Cold Spring Harbor Laboratory Press.)

Blood-feeding mosquitoes survive by feeding on nectar for metabolic energy but require a blood meal to develop eggs. Aedes aegypti females must accurately discriminate blood and nectar because each meal promotes mutually exclusive feeding programs with distinct sensory appendages, meal sizes, digestive tract targets, and metabolic fates. We investigated the syringe-like blood-feeding appendage, the stylet, and discovered that sexually dimorphic stylet neurons taste blood. Using pan-neuronal calcium imaging, we found that blood is detected by four functionally distinct stylet neuron classes, each tuned to specific blood components associated with diverse taste qualities. Stylet neurons are insensitive to nectar-specific sugars and respond to glucose only in the presence of additional blood components. The distinction between blood and nectar is therefore encoded in specialized neurons at the very first level of sensory detection in mosquitoes. This innate ability to recognize blood is the basis of vector-borne disease transmission to millions of people worldwide., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females. Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito. These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

The gut microbiota affects tissue physiology, metabolism, and function of both the immune and nervous systems. We found that intrinsic enteric-associated neurons (iEANs) in mice are functionally adapted to the intestinal segment they occupy; ileal and colonic neurons are more responsive to microbial colonization than duodenal neurons. Specifically, a microbially responsive subset of viscerofugal CART + neurons, enriched in the ileum and colon, modulated feeding and glucose metabolism. These CART + neurons send axons to the prevertebral ganglia and are polysynaptically connected to the liver and pancreas. Microbiota depletion led to NLRP6- and caspase 11-dependent loss of CART + neurons and impaired glucose regulation. Hence, iEAN subsets appear to be capable of regulating blood glucose levels independently from the central nervous system., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)